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

Background: 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.

研究背景：此前已有研究报道，部分三叶草属（Trifolium）物种——尤其是地下三叶草（Trifolium subterraneum）——与近缘豆科植物相比，其质体基因组（plastomes）存在极为特殊的结构：基因组扩增并伴随大量重复序列、基因丢失，以及三叶草属特有的DNA序列，这类特有序列可能来源于水平基因转移（horizontal gene transfer）。为精准定位该特殊现象在三叶草属内的进化起源，我们对覆盖该属广泛类群的另外8个三叶草属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：博伊斯三叶草（Trifolium boissieri）、直立三叶草（T. strictum）和腺毛三叶草（T. glanduliferum）（分别对应 Chronosemium 亚属以及三叶草亚属 Paramesus 组）的质体基因组易于组装，在菜豆族（Fabeae）质体基因组的整体背景下并无特殊之处。其余三叶草属物种（“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。这类物种构成一个单系演化支，我们将其命名为“难组装演化支”（refractory clade），该演化支包含三叶草亚属的 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，且其基因组长度比易组装类群长7%~15%。分子定年分析显示，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算年代（约1860万年前）以及三叶草亚属的最近共同祖先年代（1610万年前）。

研究结论：我们认为，此前在地下三叶草中鉴定出的特殊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一次进化改变，使得其质体基因组要么主动促进、要么容许、抑或无法管控基因组内的重复序列产生。这种改变质体基因组进化模式与速率的关键机制，仍有待进一步研究。