A multi-tiered sequence capture strategy spanning broad evolutionary scales: application for phylogenetic and phylogeographic studies of orchids

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.