Genotyping-by-Sequencing in a Species Complex of Australian Hummock Grasses (Triodia): Methodological Insights and Phylogenetic Resolution

Abstract

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.

A revision of the Triodia basedowii species complex and close relatives (Poaceae: Chloridoideae)

“…Largely on the basis of previous investigations of the Triodia basedowii complex and close relatives (Anderson et al 2016(Anderson et al , 2017, and in conjunction with additional morphological measurements for the current study, we found evidence to support the recognition of eight new species, four of which are largely restricted to the Pilbara region of Western Australia. A taxonomic account of the group is provided in the following section.…”

“…In cases where morphological characters between two or more species overlap, ITS sequences from Anderson et al (2016) were used to make diagnoses clearer. Genomic divergences estimated from distance-based clustering and phylogenetic branch lengths (Anderson et al 2017) were evaluated in the context of previous morphological and genetic work on the complex (Anderson et al 2016). Taxa that showed strong evidence for distinction (e.g.…”

“…and the new species T. infesta B.M. Anderson & M.D.Barrett) that were included with the complex in previous analyses of morphological and molecular data (Anderson et al 2016(Anderson et al , 2017.…”

“…using integrative taxonomy (Dayrat 2005;Padial et al 2010;Yeates et al 2011). Species in this revision, therefore, are delimited using genetic and genomic evidence from two previous studies (Anderson et al 2016(Anderson et al , 2017 in addition to morphology. As outlined in the delimitation work of Anderson et al (2016), we adopt de Queiroz's (1998Queiroz's ( , 2007 general lineage concept of species, under which species are defined as separately evolving segments of metapopulation lineages.…”

Resolving relationships in an exceedingly young orchid lineage using Genotyping-by-sequencing data

“…RADseq has also been successfully employed to tease apart the inter-and intraspecific relationships of a recently diversified clade of Heliconius butterflies (Nadeau et al, 2013). However, the extent to which such data are useful for inferring phylogenetic relationships in plant species complexes has been little studied (Anderson et al, 2017).…”