Allopatric divergence and hybridization within Cupressus chengiana (Cupressaceae), a threatened conifer in the northern Hengduan Mountains of western China

Having a comprehensive understanding of population structure, genetic differentiation and demographic history is important for the conservation and management of threatened species. High‐throughput sequencing (HTS) provides exciting opportunities to address a wide range of factors for conservation genetics. Here, we generated HTS data and identified 266,884 high‐quality single nucleotide polymorphisms from 82 individuals of Cupressus chengiana , to assess population genomics across the species' full range, comprising the Daduhe River (DDH), Minjiang River (MJR) and Bailongjiang River (BLJ) catchments in western China. admixture , principal components analysis and phylogenetic analyses indicated that each region contains a distinct lineage, with high levels of differentiation between them (DDH, MJR and BLJ lineages). MJR was newly distinguished compared to previous surveys, and evidence including coalescent simulations supported a hybrid origin of MJR during the Quaternary. Each of these three lineages should be recognized as an evolutionarily significant unit (ESU), due to isolation, differing genetic adaptations and different demographic history. Currently, each ESU faces distinct threats, and will require different conservation strategies. Our work shows that population genomic approaches using HTS can reconstruct the complex evolutionary history of threatened species in mountainous regions, and hence inform conservation efforts, and contribute to the understanding of high biodiversity in mountains.

Methods
We identified 266,884 high-quality SNPs from 82 individuals to assess population genomics of Cupressus chengiana across its full range. 

To get a high-quality reference, single-molecule real-time (SMRT) sequencing was used to obtain the full-length transcriptome of C. chengiana. A total of 82 samples were collected for RNA-seq. We used bwa-mem  to align the quality-filtered reads of each individual to the refences sequences. We used the “mpileup” command in SAMTOOLS to identify SNPs. Data were filtered with the following processes: SNPs with a mapping quality <30, a mapping depth <10, genotyping rate <50% per group, minor allele frequency (MAF) <5%, or in 5bps windows around any indel.

全面解析种群结构、遗传分化与种群历史，对濒危物种的保护与管理至关重要。高通量测序（High-throughput sequencing, HTS）为保护遗传学领域的多样研究议题提供了重要机遇。本研究针对中国西部大渡河流域（Daduhe River, DDH）、岷江河域（Minjiang River, MJR）及白龙江流域（Bailongjiang River, BLJ）的全分布区岷江柏木（Cupressus chengiana）82个个体开展高通量测序，共鉴定得到266,884个高质量单核苷酸多态性（single nucleotide polymorphism, SNP）位点，以全面评估该物种的种群基因组特征。

群体结构分析（admixture）、主成分分析（principal components analysis, PCA）及系统发育分析结果显示，三个流域分别对应一个独立演化支系，且支系间存在高度遗传分化（DDH、MJR与BLJ支系）。相较于既往调查，本研究首次明确区分出MJR支系，结合溯祖模拟的结果证实，MJR支系在第四纪时期起源于杂交事件。鉴于这三个支系彼此隔离、具备差异化的遗传适应特征与种群历史，均应被认定为进化显著单元（evolutionarily significant unit, ESU）。当前，每个进化显著单元均面临独特的生存威胁，因此需要制定针对性的保护策略。本研究表明，基于高通量测序的种群基因组学方法能够解析山地濒危物种复杂的演化历史，可为保护实践提供科学支撑，同时有助于深化对山地高生物多样性成因的理解。

## 研究方法
本研究从82个岷江柏木个体中鉴定得到266,884个高质量单核苷酸多态性位点，以评估其全分布区的种群基因组特征。

为获取高质量参考序列，本研究采用单分子实时测序（single-molecule real-time sequencing, SMRT）技术获取岷江柏木的全长转录组。共计收集82个样本用于RNA测序。我们使用bwa-mem工具将每个个体经过质量过滤的测序读段比对至参考序列。随后利用SAMTOOLS软件的"mpileup"命令鉴定单核苷酸多态性位点。数据过滤流程如下：剔除比对质量值<30、测序深度<10、每个群体的基因分型率<50%、次要等位基因频率（minor allele frequency, MAF）<5%，以及位于任意插入缺失（indel）上下游5bp窗口内的单核苷酸多态性位点。