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      Phylogenetic Analyses of Armillaria Reveal at Least 15 Phylogenetic Lineages in China, Seven of Which Are Associated with Cultivated Gastrodia elata

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          Abstract

          Fungal species of Armillaria, which can act as plant pathogens and/or symbionts of the Chinese traditional medicinal herb Gastrodia elata (“Tianma”), are ecologically and economically important and have consequently attracted the attention of mycologists. However, their taxonomy has been highly dependent on morphological characterization and mating tests. In this study, we phylogenetically analyzed Chinese Armillaria samples using the sequences of the internal transcribed spacer region, translation elongation factor-1 alpha gene and beta-tubulin gene. Our data revealed at least 15 phylogenetic lineages of Armillaria from China, of which seven were newly discovered and two were recorded from China for the first time. Fourteen Chinese biological species of Armillaria, which were previously defined based on mating tests, could be assigned to the 15 phylogenetic lineages identified herein. Seven of the 15 phylogenetic lineages were found to be disjunctively distributed in different continents of the Northern Hemisphere, while eight were revealed to be endemic to certain continents. In addition, we found that seven phylogenetic lineages of Armillaria were used for the cultivation of Tianma, only two of which had been recorded to be associated with Tianma previously. We also illustrated that G. elata f. glauca (“Brown Tianma”) and G. elata f. elata (“Red Tianma”), two cultivars of Tianma grown in different regions of China, form symbiotic relationships with different phylogenetic lineages of Armillaria. These findings should aid the development of Tianma cultivation in China.

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          Phylogenetic species recognition and species concepts in fungi.

          The operational species concept, i.e., the one used to recognize species, is contrasted to the theoretical species concept. A phylogenetic approach to recognize fungal species based on concordance of multiple gene genealogies is compared to those based on morphology and reproductive behavior. Examples where Phylogenetic Species Recognition has been applied to fungi are reviewed and concerns regarding Phylogenetic Species Recognition are discussed.
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            Phyutility: a phyloinformatics tool for trees, alignments and molecular data.

            Phyutility provides a set of phyloinformatics tools for summarizing and manipulating phylogenetic trees, manipulating molecular data and retrieving data from NCBI. Its simple command-line interface allows for easy integration into scripted analyses, and is able to handle large datasets with an integrated database. Phyutility, including source code, documentation, examples, and executables, is available at http://code.google.com/p/phyutility.
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              A multi-gene phylogeny of Clavicipitaceae (Ascomycota, Fungi): identification of localized incongruence using a combinational bootstrap approach.

              Multi-gene phylogenetic analyses were conducted to address the evolution of Clavicipitaceae (Ascomycota). Data are presented here for approximately 5900 base pairs from portions of seven loci: the nuclear ribosomal small and large subunit DNA (nrSSU and nrLSU), beta-tubulin, elongation factor 1alpha (EF-1alpha), the largest and second largest subunits of RNA polymerase II (RPB1 and RPB2), and mitochondrial ATP Synthase subunit 6 (mtATP6). These data were analyzed in a complete 66-taxon matrix and 91-taxon supermatrix that included some missing data. Separate phylogenetic analyses, with data partitioned according to genes, produced some conflicting results. The results of separate analyses from RPB1 and RPB2 are in agreement with the combined analyses that resolve a paraphyletic Clavicipitaceae comprising three well-supported clades (i.e., Clavicipitaceae clade A, B, and C), whereas the tree obtained from mtATP6 is in strong conflict with the monophyly of Clavicipitaceae clade B and the sister-group relationship of Hypocreaceae and Clavicipitaceae clade C. The distribution of relative contribution of nodal support for each gene partition was assessed using both partitioned Bremer support (PBS) values and combinational bootstrap (CB) analyses, the latter of which analyzed bootstrap proportions from all possible combinations of the seven gene partitions. These results suggest that CB analyses provide a more consistent estimate of nodal support than PBS and that combining heterogeneous gene partitions, which individually support a limited number of nodes, results in increased support for overall tree topology. Analyses of the 91-taxa supermatrix data sets revealed that some nodes were more strongly supported by increased taxon sampling. Identifying the localized incongruence of mtATP6 and analyses of complete and supermatrix data sets strengthen the evidence for rejecting the monophyly of Clavicipitaceae and much of the current subfamilial classification of the family. Although the monophyly of the grass-associated subfamily Clavicipitoideae (e.g., Claviceps, Balansia, and Epichloë) is strongly supported, the subfamily Cordycipitoideae (e.g., Cordyceps and Torrubiella) is not monophyletic. In particular, species of the genus Cordyceps, which are pathogens of arthropods and truffles, are found in all three clavicipitaceous clades. These results imply that most characters used in the current familial classification of Clavicipitaceae are not diagnostic of monophyly.
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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, CA USA )
                1932-6203
                3 May 2016
                2016
                : 11
                : 5
                : e0154794
                Affiliations
                [1 ]Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Heilongtan, Kunming 650201, China
                [2 ]College of Life Sciences, Chongqing Normal University, Chongqing, 401331, China
                [3 ]University of Chinese Academy of Sciences, No. 19A Yuquan Road, Beijing 100049, China
                [4 ]General Station of Forest Pest Control, State Forestry Administration, Shenyang 110034, China
                USDA Forest Service—RMRS, UNITED STATES
                Author notes

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

                Conceived and designed the experiments: TG ZLY. Performed the experiments: TG WQX. Analyzed the data: TG ZLY HCW. Contributed reagents/materials/analysis tools: TG HCW JZ. Wrote the paper: TG ZLY HCW.

                Article
                PONE-D-15-44992
                10.1371/journal.pone.0154794
                4854404
                27138686
                e5a27ea2-cd93-45d4-a528-3749c6ddff75
                © 2016 Guo et al

                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
                : 13 October 2015
                : 19 April 2016
                Page count
                Figures: 4, Tables: 2, Pages: 21
                Funding
                This study was financed by the National Natural Science Foundation of China (Nos. 30800006 and 31170024), the Chongqing Natural Science Foundation (No. CSTC, 2009BA5030), and the Key Laboratory for Plant Diversity and Biogeography of East Asia, KIB, Chinese Academy of Sciences (KLBB 201207). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
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                All relevant data are within the paper and its Supporting Information files. DNA accession numbers have been detailed in Table 1 of the manuscript.

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