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      Taxonomic Status, Phylogenetic Affinities and Genetic Diversity of a Presumed Extinct Genus, Paraisometrum W.T. Wang (Gesneriaceae) from the Karst Regions of Southwest China

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          Abstract

          Background

          The karst regions in South China have an abundance of endemic plants that face high extinction risks. The Chinese Gesneriaceae endemic Paraisometrum mileense ( =  Oreocharis mileensis), was presumed extinct for 100 years. After its re-discovery, the species has become one of five key plants selected by the Chinese forestry government to establish a new conservation category for plants with extremely small populations. For conservation purposes, we studied the phylogenetic and population genetic status of P. mileense at the three only known localities in Guangxi, Guizhou and Yunnan.

          Methodology/Principal Findings

          We collected 64 samples (52 species) of Oreocharis and 8 samples from three provinces of P. mileense and generated molecular phylogenies, and inferred that P. mileense represents a relatively isolated and derived taxonomic unit within Oreocharis. Phylogeographic results of 104 samples of 12 populations of P. mileense indicated that the populations in Yunnan have derived from those in Guangxi and Guizhou. Based on AFLP data, the populations were found to harbor low levels of genetic diversity ( He = 0.118), with no apparent gradient across the species’ range, a restricted gene flow and significant isolation-by-distance with limited genetic differentiation among the populations across the three provinces ( F ST = 0.207, P<0.001). The 10 populations in Yunnan were found to represent two distinct lineages residing at different altitudes and distances from villages.

          Conclusion/Significance

          The low levels of genetic diversity found in P. mileense are perhaps a consequence of severe bottlenecks in the recent past. The distribution of the genetic diversity suggests that all populations are significant for conservation. Current in situ and ex situ measures are discussed. Further conservation actions are apparently needed to fully safeguard this conservation flagship species. Our work provides a model of an integrated study for the numerous endemic species in the karst regions with extremely small populations.

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          Most cited references15

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          MRBAYES: Bayesian inference of phylogenetic trees.

          The program MRBAYES performs Bayesian inference of phylogeny using a variant of Markov chain Monte Carlo. MRBAYES, including the source code, documentation, sample data files, and an executable, is available at http://brahms.biology.rochester.edu/software.html.
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            Universal primers for amplification of three non-coding regions of chloroplast DNA.

            Six primers for the amplification of three non-coding regions of chloroplast DNA via the polymerase chain reaction (PCR) have been designed. In order to find out whether these primers were universal, we used them in an attempt to amplify DNA from various plant species. The primers worked for most species tested including algae, bryophytes, pteridophytes, gymnosperms and angiosperms. The fact that they amplify chloroplast DNA non-coding regions over a wide taxonomic range means that these primers may be used to study the population biology (in supplying markers) and evolution (inter- and probably intraspecific phylogenies) of plants.
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              AFLP: a new technique for DNA fingerprinting.

              A novel DNA fingerprinting technique called AFLP is described. The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA. The technique involves three steps: (i) restriction of the DNA and ligation of oligonucleotide adapters, (ii) selective amplification of sets of restriction fragments, and (iii) gel analysis of the amplified fragments. PCR amplification of restriction fragments is achieved by using the adapter and restriction site sequence as target sites for primer annealing. The selective amplification is achieved by the use of primers that extend into the restriction fragments, amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction sites. Using this method, sets of restriction fragments may be visualized by PCR without knowledge of nucleotide sequence. The method allows the specific co-amplification of high numbers of restriction fragments. The number of fragments that can be analyzed simultaneously, however, is dependent on the resolution of the detection system. Typically 50-100 restriction fragments are amplified and detected on denaturing polyacrylamide gels. The AFLP technique provides a novel and very powerful DNA fingerprinting technique for DNAs of any origin or complexity.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                24 September 2014
                : 9
                : 9
                : e107967
                Affiliations
                [1 ]Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan, China
                [2 ]Plant Germplasm and Genomics Center, Germplasm Bank of Wild Species, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan, China
                [3 ]Science Division, Royal Botanic Garden Edinburgh, Edinburgh, Scotland, United Kingdom
                [4 ]Guangxi Institute of Botany, Guangxi Zhuang Autonomous Region and Chinese Academy of Sciences, Guilin, Guangxi, China
                [5 ]University of the Chinese Academy of Sciences, Beijing, China
                St. Petersburg Pasteur Institute, Russian Federation
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: WHC HW MM. Performed the experiments: WHC JBY KN ZRZ. Analyzed the data: MM HW YMS. Contributed reagents/materials/analysis tools: YMS MM FW. Contributed to the writing of the manuscript: WHC MM YMS.

                Article
                PONE-D-14-17178
                10.1371/journal.pone.0107967
                4176718
                25250568
                ffdb1a83-eebd-482d-ae34-81630cd25d5f
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 16 April 2014
                : 18 August 2014
                Page count
                Pages: 15
                Funding
                This work was supported by the National Natural Science Foundation of China (grant no. 31000258, 31470306), the National Key Basic Research Program of China (no. 2014CB954100), the Open Project of the Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany (grant no. KLBB201304) and the project of Integrated Scientific Surveys to Daweishan National Park in China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Evolutionary Biology
                Evolutionary Processes
                Species Extinction
                Evolutionary Systematics
                Phylogenetics
                Plant Phylogenetics
                Population Genetics
                Haplotypes
                Genetics
                Plant Genetics
                Plant Science
                Population Biology
                Taxonomy
                Ecology and Environmental Sciences
                Conservation Science
                Science Policy
                Science Education
                Custom metadata
                The authors confirm that all data underlying the findings are fully available without restriction. All data underlying the findings in our study are freely and fully available; they have been provided as supplementary materials ( Table S3, AFLP binary matrix), and have been deposited in an appropriate public repository (Gene sequences in GenBank, with the text and Table S1 providing accession numbers). We would provide to other researchers samples of the genomic DNA used in this study. Other researchers could obtain the GPS information if they first obtain permission from the Yunnan forestry administration. Please contact Yu-Min Shui ( ymshui@ 123456mail.kib.ac.cn ) at the Kunming Institute of Botany, Chinese Academy of Sciences and, Fang Li ( bhcdzwz_217@ 123456163.com ) of the Yunnan Forestry Administration, to whom researchers should address requests for permits and information on obtaining GPS coordinates and genomic DNA samples.

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