DataSheet_1_Evolutionary and phylogenetic analyses of 11 Cerasus species based on the complete chloroplast genome.docx

The subgenus Cerasus, one of the most important groups in the genus Prunus sensu lato, comprises over 100 species; however, the taxonomic classification and phylogenetic relationships of Cerasus remain controversial. Therefore, it is necessary to reconstruct the phylogenetic tree for known Cerasus species. Here, we report the chloroplast (cp) genome sequences of 11 Cerasus species to provide insight into evolution of the plastome. The cp genomes of the 11 Cerasus species (157,571–158,830 bp) displayed a typical quadripartite circular structure. The plastomes contain 115 unique genes, including 80 protein-coding genes, four ribosomal RNAs, and 31 transfer RNAs. Twenty genes were found to be duplicated in inverted repeats as well as at the boundary. The conserved non-coding sequences showed significant divergence compared with the coding regions. We found 12 genes and 14 intergenic regions with higher nucleotide diversity and more polymorphic sites, including matK, rps16, rbcL, rps16-trnQ, petN-psbM, and trnL-trnF. During cp plastome evolution, the codon profile has been strongly biased toward the use of A/T at the third base, and leucine and isoleucine codons appear the most frequently. We identified strong purifying selection on the rpoA, cemA, atpA, and petB genes; whereas ccsA, rps19, matK, rpoC2, ycf2 and ndhI showed a signature of possible positive selection during the course of Cerasus evolution. In addition, we further analyzed the phylogenetic relationships of these species with 57 other congenic related species.Through reconstructing the Cerasus phylogeny tree, we found that true cherry is similar to the flora of China forming a distinct group, from which P. mahaleb was separated as an independent subclade. Microcerasus was genetically closer to Amygdalus, Armeniaca, and Prunus (sensu stricto) than to members of true cherry, whereas P. japonica and P. tomentosa were most closely related to P. triloba and P. pedunculata. However, P. tianshanica formed a clade with P. cerasus, P. fruticosa, P. cerasus × P. canescens ‘Gisela 6’, and P. avium as a true cherry group. These results provide new insights into the plastome evolution of Cerasus, along with potential molecular markers and candidate DNA barcodes for further phylogenetic and phylogeographic analyses of Cerasus species.

樱亚属（Cerasus）是广义李属（Prunus sensu lato）中最重要的类群之一，包含超过100个物种；然而樱亚属的分类学界定与系统发育关系仍存在争议。因此，对已知樱亚属物种开展系统发育树重建具有重要意义。本研究报道了11个樱亚属物种的叶绿体（chloroplast, cp）基因组序列，以期解析其质体基因组（plastome）的演化特征。
这11个樱亚属物种的叶绿体基因组长度介于157571~158830 bp之间，呈现典型的四分体环状结构。其质体基因组共包含115个独特基因，其中80个为蛋白编码基因、4个核糖体RNA（ribosomal RNA, rRNA）基因以及31个转运RNA（transfer RNA, tRNA）基因。另有20个基因在反向重复区域及边界处发生了重复。
相较于编码区，保守非编码序列呈现出显著的序列分化。本研究鉴定出12个基因与14个基因间区具有较高的核苷酸多样性（nucleotide diversity）与多态性位点，包括matK、rps16、rbcL、rps16-trnQ、petN-psbM以及trnL-trnF等。
在叶绿体质体基因组演化过程中，密码子使用偏好性表现为第三位碱基显著偏向A/T，且亮氨酸与异亮氨酸的密码子使用频率最高。本研究发现rpoA、cemA、atpA以及petB基因受到强烈的纯化选择（purifying selection）作用；而ccsA、rps19、matK、rpoC2、ycf2与ndhI基因则在樱亚属演化过程中呈现出潜在正选择（positive selection）的信号。
此外，本研究还结合另外57个同属近缘物种，进一步分析了本次研究的11个樱亚属物种的系统发育关系。通过重建樱亚属系统发育树，本研究发现真樱桃类群与中国分布的类群共同构成一个独立演化支，其中圆叶樱桃（Prunus mahaleb）单独形成一个亚支。微樱亚属（Microcerasus）与桃属（Amygdalus）、杏属（Armeniaca）以及狭义李属（Prunus sensu stricto）的亲缘关系较其与真樱桃类群的亲缘关系更近；而日本樱桃（Prunus japonica）与毛樱桃（Prunus tomentosa）则与三叶樱桃（Prunus triloba）、长梗樱桃（Prunus pedunculata）亲缘关系最近。但天山樱桃（Prunus tianshanica）与酸樱桃（Prunus cerasus）、灌木樱桃（Prunus fruticosa）、酸樱桃×灰毛樱桃‘吉塞拉6号’（P. cerasus × P. canescens ‘Gisela 6’）以及欧洲甜樱桃（Prunus avium）共同聚为真樱桃类群演化支。
本研究结果为樱亚属质体基因组演化提供了新的认知，同时也为后续樱亚属物种的系统发育与系统地理学（phylogeography）研究提供了潜在的分子标记（molecular marker）与候选DNA条形码（DNA barcode）。