【作者】 李静；

【导师】 赵之伟； 杨祝良；

【作者基本信息】 云南大学 ， 植物学， 2019， 博士

【摘要】 糙皮侧耳物种复合群Pleurotus ostreatus species complex隶属于侧耳科侧耳属,是一类广布于北半球温带和亚热带地区的腐生真菌,具有重要经济和生态价值,但对其物种划分、生物地理及种质资源研究还十分有限。该复合群物种在我国广泛栽培,并被统称为“平菇”,但由于该复合群物种形态分化滞后和形态可塑性较强,分类困难而混乱,前期研究大多因为取样不全或基于有限的基因片段,未能充分揭示该复合群的物种多样性。如何精准构建我国“平菇”家谱并快速准确地鉴别其中的物种,是生产实践中亟待解决的问题。此外,“平菇”家族中的糙皮侧耳P.ostreatus栽培较为广泛,但目前对其遗传多样性认识不深,频繁地国内外引种驯化导致其育种历史不明,有碍今后育种工作。在广泛采样的基础上,本研究基于四个基因片段对中国糙皮侧耳物种复合群真菌开展分子系统学研究,以期揭示我国“平菇”栽培菌株的分类地位;在全球广泛采样并借阅国内外馆藏标本的基础上,利用二代测序技术对糙皮侧耳物种复合群进行单拷贝直系同源核基因测序,以便重建系统发育框架并对其开展生物地理学分析;基于SSR分子标记,对我国糙皮侧耳菌株进行遗传多样性研究。主要结果如下:1.中国糙皮侧耳物种复合群的物种鉴别基于核糖体内转录间隔区(ITS)、基因编码翻译延长因子(TEF1)以及基因编码RNA聚合酶II第一大亚基和第二大亚基(RPB1和RPB2)四个基因片段,自测DNA序列1001条,对我国284份来自不同菌种保藏中心、菌种公司和野外采集的“平菇”菌株和标本进行了分子系统研究。研究结果明晰了我国栽培“平菇”的种间关系,结合形态特征将其分成7个物种,即冷杉侧耳P.abieticola、佛罗里达侧耳参照种P.cf.floridanus、刺芹侧耳(杏鲍菇)P.eryngii、糙皮侧耳P.ostreatus、卵孢侧耳P.placentodes、肺形侧耳P.pulmonarius和“白灵菇”P.tuoliensis;发现现有栽培菌株56%的学名存在误用现象;评价认为RPB2是最适合作为该复合群物种鉴定的DNA条形码。2.全球糙皮侧耳物种复合群的分子系统发育重建和系统发育种识别基于40个单拷贝直系同源核基因片段,对来自亚洲、欧洲、北美洲、南美洲和非洲的糙皮侧耳物种复合群样品进行了分子系统发育分析,以1762条自测序列构建了该物种复合群较为全面的分子系统发育框架。结果显示:40个基因的联合矩阵分析强烈支持糙皮侧耳物种复合群是一个自然的单系,共包括三个主要分支和七个亚分支;支系Ⅰ位于该复合群的最基部;支系Ⅲ是最大的支系,其中亚支系Ⅲa为本研究首次提出的独立支系,与分布在北半球的其他三个亚支系形成了姐妹群关系。根据多基因谱系一致性系统发育种识别法(GCPSR),共识别出糙皮侧耳物种复合群的20个系统发育种,其中包括7个新的系统发育种。3.糙皮侧耳物种复合群的生物地理研究基于分子钟推算和祖先分布区重建,首次开展了糙皮侧耳物种复合群的历史生物地理学研究。结果显示,糙皮侧耳物种复合群可能于始新世晚期(38Ma)起源于东亚,青藏高原的隆升和晚始新世全球气候变冷可能导致其分化为两个主要分支。糙皮侧耳物种复合群于不同地质时期可能通过白令陆桥或北大西洋陆桥从东亚传播至北美,之后由于多次扩散和隔离事件分别在新旧世界内部进行多样性分化。渐新世中期,东亚和南美之间的联系可能是通过长距离扩散实现的。中新世晚期全球气候进一步变冷变干和第四纪冰期导致的生境片断化,促进了糙皮侧耳复合群内的多样性分化。间断分布于东亚、北美、欧洲和非洲的Ⅱc亚支系中的物种可能是异域物种形成的结果。糙皮侧耳复合群早期物种生长的主要基质是松科、壳斗科等针叶树和阔叶树植物,约于6 Ma发生了向伞形科植物的转换。糙皮侧耳物种复合群的演化速率并非恒定,于中新世晚期其演化速率发生了加速。4.中国糙皮侧耳种质资源多样性基于已有佛罗里达侧耳参照种的基因组序列,开发了21个具有较高多态性的SSR位点,并在我国91个糙皮侧耳菌株中进行扩增。该研究为我国糙皮侧耳栽培菌株构建起了分子指纹,证实了我国糙皮侧耳存在品种名混乱问题,10.5%的栽培菌株被冠以不合适的名称;发现了我国糙皮侧耳野生菌株具有较高的遗传多样性,蕴含着宝贵的基因库,对栽培菌株改良具有潜在重要价值;揭示了我国糙皮侧耳栽培菌株的育种历史,现存栽培菌株有三大来源:或引种自欧洲,或由中国野生菌株驯化,或中国菌株和欧洲菌株多次杂交。为有效保护和合理开发糙皮侧耳种质资源,本研究构建了我国糙皮侧耳核心种质库。更多还原

【Abstract】 Pleurotus ostreatus species complex(the oyster mushrooms),belonging to the genus Pleurotus of the family Pleurotaceae,is saprotrophic and of significant economic and ecological importance in the Northern Hemisphere.However,previous studies of species delimitation,biogeography and germplasm resources have been limited.Although species of the P.ostreatus complex are one of the main groups of cultivated edible mushrooms in China,the inconsistent nomenclature is controversial and has led to much confusion due to phenotypic plasticity and morphological stasis.The previous molecular phylogenetic analyses based on limited sampling or gene fragments were insufficient to reveal its species diversity.How to recognize the species of the complex rapidly and accurately is an urgent task in the mushroom industry.In addition,Pleurotus ostreatus is more widely distributed in China than other oyster mushrooms,however,its genetic diversity is not well known.The cultivated strains of this species have been frequently exchanged domestically and internationally,but no detailed breeding history has been documented.With samples gathered from a broad survey in China,molecular analyses of the P.ostreatus species complex were conducted using nucleotide sequences of four-gene loci to uncover the taxonomic position of the oyster mushrooms strains.With samples collected and loaned from different herbaria of the world,nuclear single-copy orthologous gene sequencing was used to reconstruct molecular phylogenetic frame and conduct biogeographic analyses of the P.ostreatus species complex.Genetic diversity study of P.ostreatus strains based on SSR molecular marker was also conducted.The main results are summarized as follows.1.The species delimitation of the P.ostreatus species complex in ChinaMolecular identification of 284 samples gathered from different mushroom spawn preservation centers,companies,and field isolations was conducted based on 1001 newly generated and other available sequences of the internal transcribed spacer region(ITS),translation elongation factor 1-α(TEF1)and two genes that encode subunits of RNA polymerase ??(RPB1 and RPB2).Our analyses elucidated the relationships of the oyster mushrooms of China and indicated that all of the strains could be divided into seven lineages,representing one morphological species each,namely P.abieticola,P.cf.floridanus,P.eryngii var.eryngii,P.ostreatus,P.placentodes,P.pulmonarius,and P.tuoliensis.The study revealed that 56 % of all the samples were labeled with improper scientific names.Meanwhile,the suitability of the four candidate segments as DNA barcodes were evaluated.It was indicated that RPB2 was the most promising candidate segment for the recognition of the species in the P.ostreatus complex,taking into consideration the polymorphisms and other aspects of the four markers.2.The phylogenetic reconstruction and species recognition of the P.ostreatus species complex in the worldWith samples of the P.ostreatus species complex from East Asia,Europe,North and South America,and Africa,the phylogenetic analyses were carried out based on 1762 newly generated sequences of 40 nuclear single-copy orthologous genes.Here,a robust phylogeny of the P.ostreatus species complex was presented,with a full resolution of the relationships of the major clades and the species.The P.ostreatus species complex was strongly supported as monophyletic,including three major clades and seven subclades.Clade Ⅰ was the most basal lineage in the P.ostreatus species complex.Clade Ⅲ was the largest clade among three clades,and subclade Ⅲa was firstly discovered as a unique clade,sister to other three subclades in the Northern Hemisphere.Twenty phylogenetic species were recognized based on Genealogical Concordance Phylogical Species Recognition(GCPSR),with seven putatively new species.3.The biogeography of the P.ostreatus species complexThe biogeographic analyses of the P.ostreatus species complex were conducted based on molecular clock estimation and ancestral area reconstruction for the first time.The data suggested that the most recent common ancestor of the P.ostreatus species complex diversified in the late Eocene(about 38 Ma)in East Asia,and diversification of the major clades may have been triggered by the uplifts of the Qinghai-Tibet Plateau(QTP)and global cooling of the late Eocene.Species of the P.ostreatus complex migrated from the East Asia into North America across the Bering Land Bridge or the North Atlantic Land Bridge at different times during the late Oligocene,late Miocene and late Pliocene,and then diversified in the Old and New Worlds simultaneously through multiple dispersal and vicariance events.The dispersal from East Asia to South America in the middle Oligocene was probably achieved by a long-distance dispersal event.Intensification of aridity and climate cooling events in the late Miocene and Quaternary glacial probably had a significant influence on diversification patterns within the P.ostreatus species complex.The disjunctions among East Asia,Europe,North America and Africa within Clade Ⅱc are hypothesized to be a result of allopatric speciation.Plant debris from coniferious and deciduous trees,such as families Pinaceae and Fagaceae,was the earlier substrate for the P.ostreatus species complex.Substrate transitions to Apiaceae probably occurred no earlier than 6 Ma.The diversification rate is variable in the evolutionary history of the P.ostreatus species complex and one radiation occurred in the late Miocene.4.Genetic diversity of P.ostreatus in China91 strains of P.ostreatus were analysed using 21 simple sequence repeats(SSR)developed from an enriched genomic library of P.cf.floridanus genome.Among the cultivated strains,10.5 % were previously labelled with improper names,suggesting that the cultivated strains of P.ostreatus in China exist the strain name confusion.It’s indicated that wild strains harbored greater genetic diversity than the cultivated strains and speculated that wild stains may harbor a large gene pool in nature available for strain breeding.With regard to the cultivation history of P.ostreatus,the cultivated strains in China have three sources: introduction from Europe,domestication from wild strains from China,or hybridisation of the European and Chinese strains.Furthermore,a core collection of P.ostreatus with 34 strains was proposed.更多还原