桫椤目的叶绿体系统发育基因组学研究：解析影响系统树重建的因素

This project aims for reconstructing a comprehensive and well-supported phylogeny of Cyatheales based on the complete chloroplast (cp) genomic sequences of representative species of Cyatheales and its related groups. We focus on tackling the problems induced by confounding factors, including evolutionary rate heterogeneity, “ancient rapid radiations”, and long outgroup branches. The main research contents are as follows: (1) We will sequence the complete cp genomes, reveal the general cp genome features, and annotate the cp genes of the representatives of Cyatheales and their outgroups including Schizaeales, Salviniales, and Polypodiales, by using next generation sequencing (NGS). (2) We will discriminate the cp genes that show/do not show lineage-specific rate heterogeneity within a maximum likelihood framework by using a model comparison approach. We will further dissect and consolidate the pattern of rate heterogeneity in a Bayesian framework by using relaxed clock models. (3) For cp genes not showing rate heterogeneity, their sequences will be used to construct single-gene data sets and concatenated to create combined datasets. The datasets will then be analyzed by using maximum parsimony, maximum likelihood, and Bayesian methods to explore the phylogenetic positions of Thyrsopteridaceae, Metaxyaceae, and Hymenophyllaceae (Hymenophyllopsis), to infer the inter-familial relationships, and to examine the relationships among main clades in Cyatheales. (4) For cp genes showing rate heterogeneity, we will attempt to evaluate the effects of rate heterogeneity on the tree topology and branch support and to identify the phylogenetic artifacts that are induced by “ancient rapid radiations” and long outgroup branches. First, both “reduced consensus” and “reduced data” approaches will be employed within the maximum likelihood framework to perform the analyses. Second, two relaxed clock models will be applied within Bayesian framework: one with uncorrelated lognormally distributed branch rates and another with randomly assigned local clocks. Research results of this project may provide valuable evidences for the revision of the classification of Cyatheales.

基于桫椤目及其相关类群的叶绿体基因组全序列，在排除速率异质性、“古老快速辐射”及外类群组成的干扰后，重建桫椤目可靠的系统发育关系。内容包括：一，采用NGS测定桫椤目及其外类群代表植物的cpDNA全序列，分析其一般特征。二，用极大似然法，通过模型比较，区分进化速率存在和不存在异质性的叶绿体基因；还将利用贝叶斯法的放松分子钟模型，解析速率异质性的式样。三，对不存在速率异质性的基因，采用最简约法、极大似然法和贝叶斯法构建系统树；研究伞序蕨科、蚌沙蕨科以及拟膜蕨科（属）的系统位置，分辨桫椤目主要分支之间的关系，查明关键科之间的系统发育关系。四，对速率异质的基因，在极大似然法框架内，借助“缩减一致树”和“缩减数据”两种途径；而在贝叶斯框架内，则利用速率为非相关对数正态分布和局部分子钟的位置可随机分配的模型，探究异质速率对系统树的影响，鉴别系统发育假象。研究结果可为桫椤目分类系统的修订提供分子证据。

本项目通过测定桫椤目及其相关类群的叶绿体基因组全序列，开展了叶绿体系统发育基因组学研究，并对在系统树重建过程中影响分支结构和支持强度的关键因素进行了解析。已经完成以下研究内容并获得重要结果：（1）测定了黑桫椤、中华桫椤、大叶黑桫椤、金毛狗蕨、乌蕨、贯众、光石韦、似薄唇蕨、友水龙骨、鸟巢蕨、江南星蕨、卵叶盾蕨及光亮瘤蕨等蕨类植物的叶绿体基因组全序列，报道了这些基因组的一般特征、组织方式和变化动态。这为开展蕨类植物的叶绿体系统发育基因组学提供了重要的数据资源。（2）明确了内含子丢失和外显子拷贝数增加对重建系统发育产生的影响。以蕨类的叶绿体rps12基因为对象，在极大似然分析框架下，对其在各主要支系的非同义替换率（dN）和同义替换率（dS）进行了检验。注意到内含子丢失和外显子剂量的减少，使rps12的dN显著加快，而dS的改变相对较小。另外，还检测了选择对rpoC1基因的作用以及内含子缺失对进化速率带来的效应；发现内含子缺失对转换率、颠换率以及dN有影响。这些结果提示，内含子丢失和基因剂量改变会导致速率异质性，影响系统树重建。（3）认识到水龙骨科的叶绿体基因组并不像之前所认为的偏于保守，而是存在由插入引起的新的变动。大的插入片段导致该科的叶绿体基因组在大小上变动很大。特别是修蕨属姬茀蕨的反向重复区发生了显著扩张，并拥有一个独有的倒位。（4）基于高度分化的叶绿体基因组区域的序列重建系统发育。在盾蕨属属内以及盾蕨属和瘤蕨属属间分别鉴定出高度分化的序列，并用于系统发育基因组学分析。（5）借助基因顺序确定关键类群之间的系统关系。注意到蕨类叶绿体基因组的基因顺序分为两类；根据基因重排的分析结果，认为膜蕨目和里白目的关系更为接近。项目的研究结果发表在BMC Plant Biology、Molecular Phylogenetics and Evolution、Frontiers in Plant Science及Frontiers in Genetics等重要SCI国际学术刊物，被国内外其他研究组多次引用。