Expanding anchored hybrid enrichment to resolve both deep and shallow relationships within the spider tree of life

Background: Despite considerable effort, progress in spider molecular systematics has lagged behind many other comparable arthropod groups, thereby hindering family-level resolution, classification, and testing of important macroevolutionary hypotheses. Recently, alternative targeted sequence capture techniques have provided molecular systematics a powerful tool for resolving relationships across the Tree of Life. One of these approaches, Anchored Hybrid Enrichment (AHE), is designed to recover hundreds of unique orthologous loci from across the genome, for resolving both shallow and deep-scale evolutionary relationships within non-model systems. Herein we present a modification of the AHE approach that expands its use for application in spiders, with a particular emphasis on the infraorder Mygalomorphae. Results: Our aim was to design a set of probes that effectively capture loci informative at a diversity of phylogenetic timescales. Following identification of putative arthropod-wide loci, we utilized homologous transcriptome sequences from 17 species across all spiders to identify exon boundaries. Conserved regions with variable flanking regions were then sought across the tick genome, three published araneomorph spider genomes, and raw genomic reads of two mygalomorph taxa. Following development of the 585 target loci in the Spider Probe Kit, we applied AHE across three taxonomic depths to evaluate performance: deep-level spider family relationships (33 taxa, 327 loci); family and generic relationships within the mygalomorph family Euctenizidae (25 taxa, 403 loci); and species relationships in the North American tarantula genus Aphonopelma (83 taxa, 581 loci). At the deepest level, all three major spider lineages (the Mesothelae, Mygalomorphae, and Araneomorphae) were supported with high bootstrap support. Strong support was also found throughout the Euctenizidae, including generic relationships within the family and species relationships within the genus Aptostichus. As in the Euctenizidae, virtually identical topologies were inferred with high support throughout Aphonopelma. Conclusions: The Spider Probe Kit, the first implementation of AHE methodology in Class Arachnida, holds great promise for gathering the types and quantities of molecular data needed to accelerate an understanding of the spider Tree of Life by providing a mechanism whereby different researchers can confidently and effectively use the same loci for independent projects, yet allowing synthesis of data across independent research groups.

研究背景：尽管已付出大量研究努力，蜘蛛分子系统学的进展仍滞后于诸多其他同类节肢动物类群，这阻碍了科级分类单元的界定、系统分类，以及重要宏演化假说的验证。近年来，靶向序列捕获技术的新兴方案为分子系统学研究提供了强大工具，可用于解析生命之树各分支的演化关系。其中锚定杂交富集（Anchored Hybrid Enrichment, AHE）技术专为从全基因组中回收数百个独特的直系同源基因座而设计，可用于解析非模式生物类群内浅层次与深层次的演化关系。本文提出一种针对蜘蛛类群优化的AHE技术改良方案，尤其针对原蛛亚目（Mygalomorphae）类群进行了适配。

研究结果：本研究旨在设计一套可有效捕获不同系统发育时间尺度下信息型基因座的探针组。在鉴定出节肢动物广谱候选基因座后，我们利用覆盖所有蜘蛛类群的17个物种的同源转录组序列确定了外显子边界。随后，我们在蜱类基因组、3个已发表的新蛛亚目（Araneomorphae）蜘蛛基因组，以及2个原蛛亚目类群的原始基因组测序读段中，搜寻带有可变侧翼区的保守区域。基于蜘蛛探针组（Spider Probe Kit）的585个目标基因座开发完成后，我们通过三个分类层级的实验评估了该技术的性能：1. 蜘蛛科级深层演化关系（33个分类单元，327个基因座）；2. 真栉蛛科（Euctenizidae）科级及属级演化关系（25个分类单元，403个基因座）；3. 北美捕鸟蛛属（Aphonopelma）的物种种级演化关系（83个分类单元，581个基因座）。在最深层级的分析中，蜘蛛三大主要类群（中纺亚目Mesothelae、原蛛亚目Mygalomorphae和新蛛亚目Araneomorphae）均获得了高自举支持度。真栉蛛科类群的分析同样获得了强劲支持，包括该科内的属级关系以及Aptostichus属内的物种种级关系。与真栉蛛科的分析结果一致，捕鸟蛛属（Aphonopelma）的分析也得到了几乎完全一致的系统发育拓扑结构，且支持度较高。

研究结论：蜘蛛探针组是锚定杂交富集技术在蛛形纲（Arachnida）中的首次应用，其具备极大的应用潜力：该技术可高效获取所需类型与规模的分子数据，通过让不同研究人员能够可靠且高效地在独立研究中使用同一套基因座，同时实现不同研究团队间的数据整合，从而加速我们对蜘蛛生命之树演化关系的认知。