Data from: Hybridization relics complicate barcode-based identification of species in earthworms

Introgressive hybridization results in mito-nuclear discordance which could obscure the delimitation of closely related taxa. Although such events are increasingly reported, they have been poorly studied in earthworms. Here, we propose a method for investigating the degree of introgressive hybridization between three taxa of the Allolobophora chlorotica aggregate within two field populations (N = 67 and N = 105) using a reference dataset including published DNA barcoding and microsatellite data of all known A. chlorotica lineages (N = 85). For this, we used both molecular phylogenetic and population genetic approaches. The test of correspondence between mitochondrial cytochrome c oxidase I (COI) lineages and clusters of nuclear microsatellite genotypes allowed individuals to be sorted in three categories (matching, admixed and non-matching) and additional markers (mitochondrial NADH dehydrogenase subunit 1, nuclear Histone 3 and Internal transcribed Spacer Region 2) were used for phylogenetic reconstructions in order to check assignments. Although 15 admixed individuals were observed, no early-generation hybrids were detected within the two populations. Interestingly, 14 non-matching individuals (i.e. with a mtDNA haplotype that did not correspond to their nuclear cluster) were detected, a pattern that would result after multiple generations of unidirectional hybridization of female from one taxon to male of the other taxon. Because earthworms are simultaneous hermaphrodites, these events of unidirectional hybridization suggest sterility of the male function in several crosses and highlight that some individuals can be misidentified if reliance is placed on COI barcodes alone. These findings could improve the use of these barcodes in earthworms for species delineation.渐渗杂交可引发线粒体-核基因冲突，该现象可能会模糊近缘类群的分类界定。尽管此类事件的报道日益增多，但在蚯蚓中相关研究却相对匮乏。本研究针对两个野外种群（样本量分别为N=67与N=105）中的绿色异唇蚓复合群（Allolobophora chlorotica aggregate）内的3个类群，提出了一种探究其渐渗杂交程度的方法，所用参考数据集涵盖已发表的所有已知A. chlorotica支系的DNA条形码（DNA barcoding）与微卫星（microsatellite）数据（总样本量N=85）。研究采用了分子系统发育分析与群体遗传学分析两种手段：通过检验线粒体细胞色素c氧化酶亚基I（COI）支系与核微卫星基因型聚类簇的对应关系，可将个体划分为匹配型、混杂型与非匹配型三类；同时借助额外分子标记——线粒体烟酰胺腺嘌呤二核苷酸脱氢酶亚基1（NADH dehydrogenase subunit 1, ND1）、核组蛋白3（Histone 3）以及内部转录间隔区2（Internal transcribed Spacer Region 2, ITS2）——开展系统发育重建，以验证个体分类结果的可靠性。尽管共观测到15个混杂个体，但未在两个种群中发现早期世代杂交个体。值得注意的是，本次研究共检测到14个非匹配型个体，即其线粒体DNA单倍型与核基因聚类结果不匹配，该模式符合一个类群的雌性与另一个类群的雄性经过多代单向杂交后产生的结果。由于蚯蚓为雌雄同体生物，此类单向杂交事件暗示部分杂交组合的雄性功能不育，同时也表明若仅依赖COI条形码进行物种鉴定，可能会导致部分个体被错误鉴定。本研究结果可为DNA条形码在蚯蚓物种界定中的应用提供优化参考与思路。