With over 25,000 species, the drivers of diversity in the Orchidaceae remain to be fully understood. Here we outline a multi-tiered sequence capture strategy aimed at capturing 100’s of loci to enable phylogenetic resolution from subtribe to subspecific levels in orchids of the tribe Diurideae. For the probe design, we mined subsets of 18 transcriptomes, to give five target sequence sets aimed at the tribe (Sets 1 & 2), subtribe (Set 3), and within subtribe levels (Sets 4 & 5). Analysis included alternative de novo and reference-guided assembly, before target sequence extraction, annotation and alignment, and application of a homology-aware k-mer block phylogenomic approach, prior to phylogenetic inference using maximum-likelihood. Our evaluation considered 87 taxa in two test datasets: 67 samples spanning the tribe, and 72samples involving 24 closely related Caladenia species. The tiered design achieved high target loci recovery (>89%), with the median number of recovered loci in Sets 1–5 as follows: 212, 219, 816, 1024, and 1009, respectively. Interestingly, as a first test of the homologous k-mer approach for targeted sequence capture data, our study revealed its potential for enabling robust phylogenetic species tree inferences. Specifically, we found matching, and in one case improved phylogenetic resolution within species complexes, compared to conventional phylogenetic analysis involving target gene extraction. Our findings indicate that a customised multi-tiered sequence capture strategy, in combination with promising yet under-utilized phylogenomic approaches, will be effective for groups where interspecific divergence is recent, but information on deeper phylogenetic relationships is also required.

Please see the full publication in Molecular Ecology Resources along with the Online Supplementary material.  See also the Readme file (Peakall MER-20-0380 Dyrad Readme.pdf) in this repository for info on the data structure.