Anderson Benjamin M, Thiele Kevin R, Krauss Siegfried L, Barrett Matthew D
School of Plant Biology, The University of Western Australia, Crawley, Western Australia, Australia.
Kings Park and Botanic Garden, Botanic Gardens and Parks Authority, Kings Park, Western Australia, Australia.
PLoS One. 2017 Jan 30;12(1):e0171053. doi: 10.1371/journal.pone.0171053. eCollection 2017.
Next-generation sequencing is becoming increasingly accessible to researchers asking biosystematic questions, but current best practice in both choosing a specific approach and effectively analysing the resulting data set is still being explored. We present a case study for the use of genotyping-by-sequencing (GBS) to resolve relationships in a species complex of Australian arid and semi-arid grasses (Triodia R.Br.), highlighting our solutions to methodological challenges in the use of GBS data. We merged overlapping paired-end reads then optimised locus assembly in the program PyRAD to generate GBS data sets for phylogenetic and distance-based analyses. In addition to traditional concatenation analyses in RAxML, we also demonstrate the novel use of summary species tree analyses (taking gene trees as input) with GBS loci. We found that while species tree analyses were relatively robust to variation in PyRAD assembly parameters, our RAxML analyses resulted in well-supported but conflicting topologies under different assembly settings. Despite this conflict, multiple clades in the complex were consistently supported as distinct across analyses. Our GBS data assembly and analyses improve the resolution of taxa and phylogenetic relationships in the Triodia basedowii complex compared to our previous study based on Sanger sequencing of nuclear (ITS/ETS) and chloroplast (rps16-trnK spacer) markers. The genomic results also partly support previous evidence for hybridization between species in the complex. Our methodological insights for analysing GBS data will assist researchers using similar data to resolve phylogenetic relationships within species complexes.
下一代测序技术对于研究生物系统学问题的研究人员来说越来越容易获得，但目前在选择特定方法和有效分析所得数据集方面的最佳实践仍在探索中。我们展示了一个使用简化基因组测序（GBS）来解析澳大利亚干旱和半干旱草类（Triodia R.Br.）物种复合体中亲缘关系的案例研究，突出了我们在使用GBS数据时应对方法学挑战的解决方案。我们合并了重叠的双端读数，然后在PyRAD程序中优化位点组装，以生成用于系统发育和基于距离分析的GBS数据集。除了在RAxML中进行传统的串联分析外，我们还展示了将GBS位点用于总结物种树分析（以基因树作为输入）的新用途。我们发现，虽然物种树分析对PyRAD组装参数的变化相对稳健，但我们的RAxML分析在不同的组装设置下产生了支持良好但相互冲突的拓扑结构。尽管存在这种冲突，但在多个分析中，该复合体中的多个分支始终被支持为不同的。与我们之前基于核（ITS/ETS）和叶绿体（rps16-trnK间隔区）标记的桑格测序研究相比，我们的GBS数据组装和分析提高了基于owii复合体的Triodia类群和系统发育关系的分辨率。基因组结果也部分支持了该复合体中物种间杂交的先前证据。我们分析GBS数据的方法学见解将有助于使用类似数据来解析物种复合体中系统发育关系的研究人员。
