DataSheet_2_Plastome Evolution and Phylogeny of Orchidaceae, With 24 New Sequences.pdf

In order to understand the evolution of the orchid plastome, we annotated and compared 124 complete plastomes of Orchidaceae representing all the major lineages in their structures, gene contents, gene rearrangements, and IR contractions/expansions. Forty-two of these plastomes were generated from the corresponding author's laboratory, and 24 plastomes—including nine genera (Amitostigma, Bulbophyllum, Dactylorhiza, Dipodium, Galearis, Gymnadenia, Hetaeria, Oreorchis, and Sedirea)—are new in this study. All orchid plastomes, except Aphyllorchis montana, Epipogium aphyllum, and Gastrodia elata, have a quadripartite structure consisting of a large single copy (LSC), two inverted repeats (IRs), and a small single copy (SSC) region. The IR region was completely lost in the A. montana and G. elata plastomes. The SSC is lost in the E. aphyllum plastome. The smallest plastome size was 19,047 bp, in E. roseum, and the largest plastome size was 178,131 bp, in Cypripedium formosanum. The small plastome sizes are primarily the result of gene losses associated with mycoheterotrophic habitats, while the large plastome sizes are due to the expansion of noncoding regions. The minimal number of common genes among orchid plastomes to maintain minimal plastome activity was 15, including the three subunits of rpl (14, 16, and 36), seven subunits of rps (2, 3, 4, 7, 8, 11, and 14), three subunits of rrn (5, 16, and 23), trnC-GCA, and clpP genes. Three stages of gene loss were observed among the orchid plastomes. The first was ndh gene loss, which is widespread in Apostasioideae, Vanilloideae, Cypripedioideae, and Epidendroideae, but rare in the Orchidoideae. The second stage was the loss of photosynthetic genes (atp, pet, psa, and psb) and rpo gene subunits, which are restricted to Aphyllorchis, Hetaeria, Hexalectris, and some species of Corallorhiza and Neottia. The third stage was gene loss related to prokaryotic gene expression (rpl, rps, trn, and others), which was observed in Epipogium, Gastrodia, Lecanorchis, and Rhizanthella. In addition, an intermediate stage between the second and third stage was observed in Cyrtosia (Vanilloideae). The majority of intron losses are associated with the loss of their corresponding genes. In some orchid taxa, however, introns have been lost in rpl16, rps16, and clpP(2) without their corresponding gene being lost. A total of 104 gene rearrangements were counted when comparing 116 orchid plastomes. Among them, many were concentrated near the IRa/b-SSC junction area. The plastome phylogeny of 124 orchid species confirmed the relationship of {Apostasioideae [Vanilloideae (Cypripedioideae (Orchidoideae, Epidendroideae))]} at the subfamily level and the phylogenetic relationships of 17 tribes were also established. Molecular clock analysis based on the whole plastome sequences suggested that Orchidaceae diverged from its sister family 99.2 mya, and the estimated divergence times of five subfamilies are as follows: Apostasioideae (79.91 mya), Vanilloideae (69.84 mya), Cypripedioideae (64.97 mya), Orchidoideae (59.16 mya), and Epidendroideae (59.16 mya). We also released the first nuclear ribosomal (nr) DNA unit (18S-ITS1-5.8S-ITS2-28S-NTS-ETS) sequences for the 42 species of Orchidaceae. Finally, the phylogenetic tree based on the nrDNA unit sequences is compared to the tree based on the 42 identical plastome sequences, and the differences between the two datasets are discussed in this paper.

为解析兰科（Orchidaceae）质体基因组（plastome）的演化历程，本研究对涵盖所有主要支系的124个完整兰科质体基因组进行注释与比较分析，涉及基因组结构、基因组成、基因重排及反向重复区（IR）收缩/扩张特征。其中42个质体基因组由通讯作者实验室完成测序，本研究首次报道24个质体基因组，涉及9个属（无柱兰属Amitostigma、石豆兰属Bulbophyllum、手参属Dactylorhiza、Dipodium属、玉凤花属Galearis、Gymnadenia属、阔蕊兰属Hetaeria、Oreorchis属、Sedirea属）。除Aphyllorchis montana、Epipogium aphyllum和天麻Gastrodia elata外，所有兰科质体基因组均具备四分体结构，包含一个大单拷贝区（LSC）、两个反向重复区（IRs）以及一个小单拷贝区（SSC）。A. montana与G. elata的质体基因组完全丢失了IR区，而E. aphyllum的质体基因组则丢失了SSC区。本研究中最小的质体基因组为19047 bp，存在于E. roseum中；最大的质体基因组为178131 bp，存在于台湾杓兰Cypripedium formosanum中。质体基因组尺寸偏小主要源于与菌异养生境相关的基因丢失，而尺寸偏大则归因于非编码区的扩张。维持质体基因组基本活性所需的兰科质体基因组共有的最少基因共15个，包括核糖体大亚基基因rpl的3个亚基（rpl14、rpl16、rpl36）、核糖体小亚基基因rps的7个亚基（rps2、rps3、rps4、rps7、rps8、rps11、rps14）、核糖体RNA基因rrn的3个亚基（rrn5、rrn16、rrn23）、trnC-GCA基因以及clpP基因。兰科质体基因组的基因丢失可分为三个阶段：第一阶段为NADH脱氢酶（ndh）基因丢失，该现象在拟兰亚科Apostasioideae、香荚兰亚科Vanilloideae、杓兰亚科Cypripedioideae和树兰亚科Epidendroideae中广泛存在，但在兰亚科Orchidoideae中较为罕见；第二阶段为光合相关基因（atp、pet、psa、psb）以及RNA聚合酶（rpo）基因亚基的丢失，该类丢失仅局限于无叶兰属Aphyllorchis、阔蕊兰属Hetaeria、Hexalectris属以及部分珊瑚兰属Corallorhiza和鸟巢兰属Neottia物种；第三阶段为与原核型基因表达相关的基因（rpl、rps、trn等）丢失，该现象见于隐柱兰属Epipogium、天麻属Gastrodia、Lecanorchis属以及Rhizanthella属。此外，肉果兰属Cyrtosia（香荚兰亚科）呈现出介于第二与第三阶段之间的中间过渡型基因丢失模式。多数内含子丢失与其对应基因的丢失相关，但部分兰科类群中，rpl16、rps16以及clpP(2)的内含子发生丢失时，其对应的基因并未丢失。通过对116个兰科质体基因组的比较，共鉴定到104处基因重排事件，其中多数集中于IRa/b-SSC连接区域附近。基于124个兰科物种质体基因组构建的系统发育树，确认了亚科水平的系统发育关系为{拟兰亚科 [香荚兰亚科 (杓兰亚科 (兰亚科，树兰亚科))]}，同时明确了17个族的系统发育关系。基于全质体基因组序列的分子钟分析显示，兰科与其姐妹类群的分化时间约为99.2百万年前（mya），五个亚科的估计分化时间分别为：拟兰亚科（79.91 mya）、香荚兰亚科（69.84 mya）、杓兰亚科（64.97 mya）、兰亚科（59.16 mya）以及树兰亚科（59.16 mya）。本研究还首次发布了42个兰科物种的核核糖体（nr）DNA单元序列（18S-ITS1-5.8S-ITS2-28S-NTS-ETS）。最后，本研究将基于nrDNA单元序列构建的系统发育树与基于42个同源质体基因组序列构建的系统发育树进行了比较，并讨论了两个数据集之间的差异。