Data from: Sticky genomes: using NGS evidence to test hybrid speciation hypotheses

Hypotheses of hybrid origin are common. Here we use next generation sequencing to test a hybrid hypothesis for a non-model insect with a large genome. We compared a putative hybrid triploid stick insect species (Acanthoxyla geisovii) with its putative paternal diploid taxon (Clitarchus hookeri), a relationship that provides clear predictions for the relative genetic diversity within each genome. The parental taxon is expected to have comparatively low allelic diversity that is nested within the diversity of the hybrid daughter genome. The scale of genome sequencing required was conveniently achieved by extracting mRNA and sequencing cDNA to examine expressed allelic diversity. This allowed us to test hybrid-progenitor relationships among non-model organisms with large genomes and different ploidy levels. Examination of thousands of independent loci avoids potential problems produced by the silencing of parts of one or other of the parental genomes, a phenomenon sometimes associated with the process of stabilisation of a hybrid genome. Transcript assembles were assessed for evidence of paralogs and/or alternative splice variants before proceeding. Comparison of transcript assemblies was not an appropriate measure of genetic variability, but by mapping reads back to clusters derived from each species we determined levels of allelic diversity. We found greater cDNA sequence diversity among alleles in the putative hybrid species (Acanthoxyla geisovii) than the non-hybrid. The allelic diversity within the putative paternal species (Clitachus hookeri) nested within the hybrid-daughter genome, supports the current view of a hybrid-progenitor relationship for these stick insect species. Next generation sequencing technology provides opportunities for testing evolutionary hypotheses with non-model organisms, including, as here, genomes that are large due to polyploidy

杂交起源假说较为普遍。本研究借助下一代测序技术（next generation sequencing），针对一个拥有大基因组的非模式昆虫（non-model insect）验证其杂交起源假说。我们将推定的杂交起源三倍体竹节虫物种（*Acanthoxyla geisovii*）与其推定的父系二倍体类群（*Clitarchus hookeri*）进行对比，该类群关系可为两个基因组内的相对遗传多样性提供明确的预测依据。预期父本类群的等位基因多样性（allelic diversity）相对较低，且该多样性嵌套于杂交子代基因组的多样性之中。
所需规模的基因组测序可通过提取mRNA并对cDNA进行测序以检测表达型等位基因多样性来便捷实现。这使得我们能够针对拥有大基因组及不同倍性水平（ploidy levels）的非模式生物（non-model organism），验证其杂交-祖先类群关系。对数千个独立基因座（loci，单数为locus）的检测，可规避因某一亲本基因组部分片段发生基因沉默而产生的潜在问题——该现象有时与杂交基因组的稳定化过程相关。
在开展后续分析前，我们先对转录本组装结果进行评估，以排查旁系同源基因（paralogs）及/或可变剪接变体（alternative splice variants）的存在证据。直接对比转录本组装结果并非衡量遗传变异的合适方式，但通过将测序读段（reads）比对到源自每个物种的聚类簇中，我们得以确定等位基因多样性水平。
我们发现，推定的杂交物种（*Acanthoxyla geisovii*）的等位基因间cDNA序列多样性高于非杂交亲本类群。推定父本类群（*Clitachus hookeri*）内的等位基因多样性嵌套于杂交子代基因组中，这支持了当前关于这些竹节虫物种存在杂交-祖先类群关系的观点。下一代测序技术为利用非模式生物验证演化假说提供了契机，正如本研究所示，该技术可应用于因多倍性（polyploidy）而拥有大基因组的物种。