Department of Plant Sciences, School of Biology, College of Science, University of Tehran, Tehran, Iran.
Department of Horticultural Science, Faculty of Agriculture, Tarbiat Modares University (TMU), Tehran, Iran.
PLoS One. 2020 May 7;15(5):e0232471. doi: 10.1371/journal.pone.0232471. eCollection 2020.
Many studies on Heracleum have shown poor correspondence between observed molecular clusters and established taxonomic classification amongst closely related species. This might reflect both unresolved taxonomy but perhaps also a lack of good genetic markers. This lack of appropriate and cost effective species-specific genetic markers hinders a resolved relationship for the species complex, and this in turn causes profound management challenges for a genus that contains both endemic species, with important ecological roles, and species with an invasive potential. Microsatellites are traditionally considered markers of choice for comprehensive, yet inexpensive, analyses of genetic variation, including examination of population structure, species identity, linkage map construction and cryptic speciation. In this study, we have used double digest restriction site associated DNA sequencing (ddRADseq) to develop microsatellite markers in Heracleum rechingeri. Genomic DNA from three individuals were digested with Sbf1 and Nde1 and size selected for library construction. The size-selected fragments were sequenced on an Ion Torrent sequencer and a total of 54 microsatellite sequences were bioinformatically confirmed. Twenty five loci were then tested for amplification, resulting in 19 of these being successfully amplified across eight species, comprising both the so-called thick-stemmed species (H. persicum, H. rechingeri, H. gorganicum and H. lasiopetalum), and thin-stemmed species (H. anisactis, H. pastinasifolium and H. transcaucasicum). Both Bayesian and distance-based clustering, and principal coordinate analyses clearly separated these into two groups. Surprisingly, three H. pastinacifolium populations were not separated from populations of the morphologically similar endemic species, H. anisactis, suggesting lack of genetic differentiation. Likewise, high genetic similarity was found between H. persicum and H. rechingeri populations, questioning taxonomic separation at the species level between these taxa. Further analyses are needed to re-evaluate the taxonomic significance of observed morphological variability currently applied to distinguish these sister taxa. Nevertheless, our results represent progress in the effort to develop cost-efficient molecular tools for species discrimination in this genus.
许多有关独活属的研究表明，在密切相关的物种中，观察到的分子聚类与已建立的分类学分类之间存在较差的对应关系。这可能反映了未解决的分类学问题，但也可能缺乏良好的遗传标记。缺乏适当且具有成本效益的物种特异性遗传标记阻碍了对物种复合体的明确关系，这反过来又给包含具有重要生态作用的特有物种和具有入侵潜力的物种的属带来了深远的管理挑战。微卫星传统上被认为是综合分析遗传变异的首选标记，包括种群结构、物种身份、连锁图谱构建和隐种形成的分析。在这项研究中，我们使用双酶切限制位点相关 DNA 测序（ddRADseq）在独活属中开发微卫星标记。从三个个体中提取基因组 DNA，用 Sbf1 和 Nde1 消化，并进行大小选择以构建文库。对大小选择的片段进行 Ion Torrent 测序仪测序，共生物信息学确认了 54 个微卫星序列。然后测试了 25 个位点的扩增，其中 19 个成功扩增了包括厚茎物种（独活、独活、独活和独活）和薄茎物种（独活、独活和独活）在内的 8 个物种。贝叶斯和基于距离的聚类以及主坐标分析清楚地将它们分为两组。令人惊讶的是，三个独活种群并未与形态相似的特有物种独活种群分离，表明遗传分化缺乏。同样，独活和独活种群之间发现了高度的遗传相似性，质疑这些类群在物种水平上的分类分离。需要进一步分析来重新评估目前用于区分这些姐妹种的观察到的形态变异性的分类学意义。尽管如此，我们的结果代表了在开发该属物种鉴别成本效益高的分子工具方面取得的进展。
