Data from: Evolutionary origin of highly repetitive plastid genomes within the clover genus (Trifolium)

AbstractBackground: Some clover species, particularly Trifolium subterraneum, have previously been reported to have highly unusual plastomes, relative to closely related legumes, enlarged with many duplications, gene losses and the presence of DNA unique to Trifolium, which may represent horizontal transfer. In order to pinpoint the evolutionary origin of this phenomenon within the genus Trifolium, we sequenced and assembled the plastomes of eight additional Trifolium species widely sampled from across the genus. Results: The Trifolium plastomes fell into two groups: those of Trifolium boissieri, T. strictum and T. glanduliferum (representing subgenus Chronosemium and subg. Trifolium section Paramesus) were tractable, assembled readily and were not unusual in the general context of Fabeae plastomes. The other Trifolium species (“core Trifolium”) proved refractory to assembly mainly because of numerous short duplications. These species form a single clade, which we call the “refractory clade” (comprising subg, Trifolium sections Lupinaster, Trifolium, Trichocephalum, Vesicastrum and Trifoliastrum). The characteristics of the refractory clade are the presence of numerous short duplications and 7-15% longer genomes than the tractable species. Molecular dating estimates that the origin of the most recent common ancestor (MRCA) of the refractory clade is approximately 13.1 million years ago (MYA). This is considerably younger than the estimated MRCA ages of Trifolium (c. 18.6 MYA) andTrifolium subg. Trifolium (16.1 MYA). Conclusions: We conclude that the unusual repetitive plastome type previously characterized in Trifolium subterraneum had a single origin within Trifolium and is characteristic of most (but not all) species of subgenus Trifolium. It appears that an ancestral plastome within Trifolium underwent an evolutionary change resulting in plastomes that either actively promoted, were permissive to, or were unable to control, duplications within the genome. The precise mechanism of this important change in the mode and tempo of plastome evolution deserves further investigation., Usage notesTrifolium_plastid_codingA concatenated matrix of 59 protein coding plastid genes. Only coding regions were used, introns and intergenic regions were excluded in the alignment, due to plastome rearrangements (see manuscript of further description). The alignment was generated using Mafft v7.053b using -auto flag and trimmed using TrimAl with the -automated1 flag. This process was pipelined using Plast2Phy (https://github.com/saemi/plast2phy).Trifolium_plastid_coding_MLML tree generated using GARLI (the config file is the ReadMe file). The model used GTR+G, based on a jModelTest search and an AIC test. 10 starts were prefromed and a 100 bootstrap replicates. Medicago truncatula is the outrgroup.

研究背景与摘要：此前有研究报道，部分三叶草属物种，尤其是地下三叶草（Trifolium subterraneum），相较于近缘豆科植物拥有极不寻常的质体基因组（plastomes）：其基因组因大量重复序列、基因丢失以及存在三叶草属特有的DNA序列而扩增，这类特有序列可能源自水平基因转移（horizontal transfer）。为明确该现象在三叶草属内的演化起源，我们对该属广泛分布的8个新增三叶草物种的质体基因组进行了测序与组装。
研究结果：三叶草属的质体基因组可分为两类：波氏三叶草（Trifolium boissieri）、直立三叶草（Trifolium strictum）和腺毛三叶草（Trifolium glanduliferum，分别代表Chronosemium亚属以及三叶草亚属（subg. Trifolium）Paramesus组）的质体基因组易于组装，在野豌豆族（Fabeae）质体基因组的整体框架下并无异常。其余三叶草物种（“核心三叶草类群”）则因存在大量短片段重复序列而难以组装。该类群构成单一演化支，我们将其命名为“难组装演化支”（refractory clade），涵盖三叶草亚属的Lupinaster组、Trifolium组、Trichocephalum组、Vesicastrum组以及Trifoliastrum组。难组装演化支的特征为存在大量短片段重复序列，且基因组长度比易组装物种长7%~15%。分子定年结果显示，难组装演化支的最近共同祖先（most recent common ancestor, MRCA）起源于约1310万年前（MYA），这一年代远晚于三叶草属整体的最近共同祖先估测年代（约1860万年前）以及三叶草亚属的最近共同祖先估测年代（1610万年前）。
研究结论：我们认为，此前在地下三叶草中发现的异常重复型质体基因组在三叶草属内仅起源一次，且是三叶草亚属多数（而非全部）物种的特征。三叶草属内的祖先质体基因组曾发生演化改变，使得其基因组要么主动促进、要么容许、要么无法控制基因组内的重复序列扩增。这种改变质体基因组演化模式与速率的关键机制，仍有待进一步研究。
使用说明：
1. Trifolium_plastid_coding：由59个质体蛋白编码基因拼接而成的矩阵。由于质体基因组存在重排现象（详见相关研究论文的详细描述），比对过程中仅保留编码区序列，剔除内含子与基因间区序列。该比对文件通过Mafft v7.053b的-auto参数生成，并使用TrimAl的-automated1参数进行修剪，整个流程通过Plast2Phy工具（https://github.com/saemi/plast2phy）以流水线方式完成。
2. Trifolium_plastid_coding_ML：使用GARLI生成的最大似然树（配置文件见ReadMe文件）。模型选用GTR+G，该选型基于jModelTest搜索结果与AIC检验确定。共进行10次初始运行与100次自展重复检验。外类群为蒺藜苜蓿（Medicago truncatula）。