A multitiered sequence capture strategy spanning broad evolutionary scales: Application for phylogenetic and phylogeographic studies of orchids

Abstract

With over 25,000 species, the drivers of diversity in the Orchidaceae remain to be fully understood. Here, we outline a multitiered sequence capture strategy aimed at capturing hundreds 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 maximum likelihood and coalescence‐based phylogenetic inference. Our evaluation considered 87 taxa in two test data sets: 67 samples spanning the tribe, and 72 samples 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 customized multitiered sequence capture strategy, in combination with promising yet underutilized phylogenomic approaches, will be effective for groups where interspecific divergence is recent, but information on deeper phylogenetic relationships is also required.

Anthocyanin and Flavonol Glycoside Metabolic Pathways Underpin Floral Color Mimicry and Contrast in a Sexually Deceptive Orchid

“…It is further of interest that the genus Chiloglottis belongs to a subtribe (Drakaeinae) in which all genera are exclusively sexually deceptive (with the exception of some rare self-pollinating cases) and all fall within a well-resolved clade on a long branch of the diverse predominantly Australasian tribe the Diurideae ( Peakall et al, 2021 ). Thus, it is likely that the complex chemical and genetic basis of floral color mimicry may have been finely tuned by a long evolutionary process in these sexually deceptive orchids ( Weston et al, 2014 ; Peakall et al, 2021 ). This is in contrast with some of the well-known flower color polymorphisms found within natural populations ( Streisfeld and Rausher, 2011 ; Kellenberger et al, 2019 ), and even the flower color changes associated with key pollinator shifts that are often mediated by relatively simple genetic changes restricted to a few genes within the anthocyanin and flavonol glycoside pathways ( Wessinger and Rausher, 2012 ).…”

“…This is in contrast with some of the well-known flower color polymorphisms found within natural populations ( Streisfeld and Rausher, 2011 ; Kellenberger et al, 2019 ), and even the flower color changes associated with key pollinator shifts that are often mediated by relatively simple genetic changes restricted to a few genes within the anthocyanin and flavonol glycoside pathways ( Wessinger and Rausher, 2012 ). It will be of interest in future research to determine whether sexually deceptive lineages of more recent evolutionary origin than Chiloglottis (e.g., Caladenia , Caladeniinae; Diurideae) ( Peakall et al, 2021 ) exhibit floral color adaptions based on simple genetic changes, as one might expect at the early stages in the evolution of sexual deception.…”

“…Despite the exceptional diversity (>1850 species) of the Australian orchid flora ( Jones, 2021 ; Peakall et al, 2021 ), to the best of our knowledge only one previous study of floral anthocyanins has been published. This study, dating back to the 1970s, reported a qualitative survey of floral anthocyanins across some brightly colored and non-sexually deceptive species.…”

“…Our findings in Chiloglottis trapeziformis indicate that its floral color adaptations for sexual deception require complex tissue-specific coordinated regulation at multiple developmental stages, involving many genes and diverse pigment metabolic and cellular constituent regulating pathways. It is further of interest that the genus Chiloglottis belongs to a subtribe (Drakaeinae) in which all genera are exclusively sexually deceptive (with the exception of some rare self-pollinating cases) and all fall within a well-resolved clade on a long branch of the diverse predominantly Australasian tribe the Diurideae ( Peakall et al, 2021 ). Thus, it is likely that the complex chemical and genetic basis of floral color mimicry may have been finely tuned by a long evolutionary process in these sexually deceptive orchids ( Weston et al, 2014 ; Peakall et al, 2021 ).…”

Notes on Australasian Orchidaceae 7: Updates on the nomenclature of Sullivania (Diurideae, subtribe Drakaeinae)

“…and Caleana R.Br. are sister genera within the subtribe Drakaeinae and molecular studies have confirmed their close ancestral relationship (Miller & Clements 2014, Peakall et al 2021. Differences in their morphology (Blaxell 1972) and pollination syndromes (Cady 1965, Bower 2014 support their status as distinct genera.…”