Taxonomy And Biogeography Of Ophiocordyceps Sinensis And Its Relatives In Southeastern Qinghai-Tibetan Plateau

Speciation and differentiation of fungi are closely related to the comprehensive factors such as the evolutionary mechanism of the species itself,paleogeography and climate.The Qinghai-Tibet Plateau(QTP)experienced complex process characterized by poly-stages,nonuniform velocity and nonuniformity,followed by glacial and interglacial periods in the Quaternary.The great geological and climatic changes have taken place.Thus,it has gathered a rich flora of ancient Gondwana,Laurasia and the local endemic species,which made it become one of the world's species diversity centers.In the genus Ophiocordyceps,only 6 species(O.emeiensis,O.kurijimeaensis,O.lanpingensis,O.laojunshanensis,O.liangshanensis and O.sinensis)have been reported from the QTP.In contrast with its rich biodiversity,the inconclusive and insufficient understanding the diversity of the genus directly affects the comprehensive evaluation of Ophiocordyceps resources in this area.This study was aimed to investigate the species diversity and differentiation characteristics of Ophiocordyceps in the Qinghai-Tibetan Plateau.The purpose was to uncover the speciation and dispersal evolutionary mechanism of this group at the specific QTP geographical environmental conditions.The main results were as follows.1.Species diversity of O.sinensis and its allies in the Qinghai-Tibet Plateau:A total of 13 Ophiocordyceps species were found.Besides the previously reported 6 species,seven new species were described based on morphyologies and molecular phylogeny:O.albastromata,O.alpina,O.amara,O.bambusicola,O.fusca,O.megala and O.nigristromata.A monophyletic group were identified based on the phylogeny construction of these Ophiocordyceps species and another 347 samples in Ophiocordyceps by using five-loci(SSU,LSU,tef-la,rpbl and rpb2),We defined this single ancestral origin monophylic group as the QTP Core Clade.Whatsmore,based on the phylogeny,there were other six Ophiocordyceps species clastered to this core clade but distributed outside of the QTP.They were O.macroacicularis(Japan),O.xuefengensis(Hunan,China),O.illustris(West and South Africa),O.robertsii(Australia and New Zealand)and O.karstii(Guizhou,China and Sapa,Vietnam),and O.wenshanensis(Yunnan,China).2.Speciation and differentiation of the QTP Core Clade in correlation with the QTP uplifts:The divergence time of Ophiocordyceps with emphasis on the QTP Core Clade was estimated using a Bayesian method by using five-loci(SSU,LSU,tef-1?,rpb1 and rpb2)phylogeny based on four fossils calibrations.The divergence of the main clade occurred at 28.89-17.61 Ma.Meanwhile,dating analyses of the host insects was caculated by using mitochondrial total 13 protein encoding genes,and three calibrations were used as time makers.It supported that the divergence of the Thitarodes,the main host genus of O.sinensis,happened at 25.26 Ma.The divergence time of the Ophiocordyceps and their host insects was consistent with the second QTP uplifting movement in the Oligocene and Miocene(25-17 Ma).The strong consistency between the two indicated that the uplift of the QTP should have effected on,or even played an important role in the speciation and differentiation of the Ophiocordyceps distributed in the QTP.The subsequent differentiation of Ophiocordyceps species was also connected with the third and forth Qinghai-Tibet movements.3.Origin of the QTP Core Clade:Based on the distribution patterns of 18 species in the QTP Core Clade,it was deduced that southeast Hengduan Mountains was the origin center of the QTP Core Clade.With geological uplift and climate change,local differentiation occurs at the HMs,mainly caused temperate-subtropical climate types of Ophiocordyceps.And the latter,due to the geological uplift of the QTP,significant differences had formed in altitude and climate temperature around the QTP.These provided rich geographical and ecological climate conditions for the diversity and differentiation of the Ophiocordyceps species.The distributions of this group were gradually spreaded out of the QTP.After long-term selection and adaptation,their phylogenetic structures were highly consistent with the species distributed in the QTP,but their distribution were become local endeminc without the QTP.4.Origin and genetic differention of Ophiocordyceps sinensis:Biogeography of O.sinensis was carried out based on a large-scale sampling.ITS was screened as molecular markers for phylogeographic analyses of O.sinensis.111 haplotypes of ITS were identified among the 948 samples representing 96 populations.The high genetic diversity was revealed in O.sinensis.Eight phylogenetic clades were identified.Both southeastern Tibet and northwestern Yunnan are the centers of genetic diversity and genetic differentiation of the fungus.At least three geographically distributed patterns instead of the single latitude divergence feature were revealed corresponded to the intraspecific genetic differentiation of this fungus.The ancestor of O.sinensis might have originated from the the uniform palo-land of south Tibet and northwest of Yunnan in the late Miocene(-6 Ma),then it was affected by the uplift of the plateau,passively spreaded to the plateau,and gradually evolved into a unique group that was adapted to the high altitude and low temperature climate in the QTP.We systematically reported the Ophiocordyceps species in the QTP,investigated the species diversity and their phylogenetic structures.It was deduced that the monophyletic characteristics of Ophiocordyceps sinensis and its allies was originated in the paleo-land of the southeast Hengduan Mountains.The differentiation and biogeographical characteristics were deeply discussed among and within Ophiocordyceps species in the QTP.It was suggestted that the differentiations of the Ophiocordyceps were strong correlated to the geological uplift movements in this area.It is speculated that the geographical change caused the spread and dispersal around the QTP.The Ophiocordyceps colonized and diversified at different climates and temperatures conditions.Therefore,the species diversity and evolutionary mechanism of Ophiocordyceps sinensis and its allies was revealed in the process of historical evolution of QTP.