Integrating restriction site-associated DNA sequencing (RAD-seq) with morphological cladistic analysis clarifies evolutionary relationships among major species groups of bee orchids

Abstract

Background and AimsBee orchids (Ophrys) have become the most popular model system for studying reproduction via insect-mediated pseudo-copulation and for exploring the consequent, putatively adaptive, evolutionary radiations. However, despite intensive past research, both the phylogenetic structure and species diversity within the genus remain highly contentious. Here, we integrate next-generation sequencing and morphological cladistic techniques to clarify the phylogeny of the genus.MethodsAt least two accessions of each of the ten species groups previously circumscribed from large-scale cloned nuclear ribosomal internal transcibed spacer (nrITS) sequencing were subjected to restriction site-associated sequencing (RAD-seq). The resulting matrix of 4159 single nucleotide polymorphisms (SNPs) for 34 accessions was used to construct an unrooted network and a rooted maximum likelihood phylogeny. A parallel morphological cladistic matrix of 43 characters generated both polymorphic and non-polymorphic sets of parsimony trees before being mapped across the RAD-seq topology.Key ResultsRAD-seq data strongly support the monophyly of nine out of ten groups previously circumscribed using nrITS and resolve three major clades; in contrast, supposed microspecies are barely distinguishable. Strong incongruence separated the RAD-seq trees from both the morphological trees and traditional classifications; mapping of the morphological characters across the RAD-seq topology rendered them far more homoplastic.ConclusionsThe comparatively high level of morphological homoplasy reflects extensive convergence, whereas the derived placement of the fusca group is attributed to paedomorphic simplification. The phenotype of the most recent common ancestor of the extant lineages is inferred, but it post-dates the majority of the character-state changes that typify the genus. RAD-seq may represent the high-water mark of the contribution of molecular phylogenetics to understanding evolution within Ophrys; further progress will require large-scale population-level studies that integrate phenotypic and genotypic data in a cogent conceptual framework.

RAD-Seq Data Point to a Distinct Split in Liriodendron (Magnoliaceae) and Obvious East–West Genetic Divergence in L. chinense

“…For example, RAD-Seq has been used to identify agronomically important genes in breeding for crops such as maize [24] and rice [25]. The results of RAD-Seq can be used to infer the origin of the arctic-alpine genus Cassiope (Ericaceae) [26] and to clarify the evolutionary relationships in bee orchids [27], and it can also provide a robust phylogenetic hypothesis for variously related taxa of Ohomopterus ground beetles [28], unambiguously resolved phylogenetic relationships among recalcitrant taxa of Paragorgia [29], and clear relationships of the Nyssa sylvatica complex [30]. In the present study, we applied RAD-Seq to investigate the genetic structure within Liriodendron and explored the phylogenetic relationships within this intercontinental, discontinuous genus.…”

Conservation genomics of an Australian orchid complex with implications for the taxonomic and conservation status ofCorybas dowlingii

“…Hence, these approaches provide critical genomic data to evaluate taxonomic concepts in species complexes and to inform the development of effective conservation strategies for threatened species. Reduced representation library sequencing approaches, such as RADseq, ddRADseq and genotypingby-sequencing (GBS), have proven to be powerful tools for disentangling complex relationships and delimiting taxonomic boundaries in orchids (Ahrens et al 2017;Bateman et al 2018;Brandrud et al 2019a). Here, we present a study using more than 13,000 SNPs to unravel the complex relationships in the C. aconitiflorus complex containing three morphologically similar orchid species endemic to Australia.…”

“…Recent advances in high-throughput DNA sequencing and bioinformatics offer powerful genomic approaches to resolve complex inter-and intraspecific relationships at unprecedented resolution, facilitating the re-assessment of taxonomic concepts in species complexes and the conservation status of rare and threatened species (Ahrens et al 2017;Bateman et al 2018;Coates et al 2018;Cozzolino et al 2019;Taylor and Larson 2019).…”

“…DNA fragments of a certain size range are selected as subset for library preparation and then subjected to high throughput sequencing (Miller et al 2007;Baird et al 2008;Peterson et al 2012) (Miller et al 2007;Lemmon and Lemmon 2013). RADseq has been successfully used to clarify inter-and intraspecific relationships and to assess taxonomic delimitation in species complexes, including in orchids (Brandrud et al, 2019a, b;Wagner et al 2013;Jones et al 2013;Eaton and Ree 2013;Escudero et al 2014;Takahashi et al 2014;Mort et al 2015;Herrera and Shank 2016;Beheregaray et al 2017;Bateman et al 2018;Hipp et al 2018).…”

Different filtering strategies of genotyping‐by‐sequencing data provide complementary resolutions of species boundaries and relationships in a clade of sexually deceptive orchids

“…Thus, this complex represents an ideal group for testing the application of NGS-based multilocus analyses for inference of relationships and species delimitation. The RADseq method has very recently been applied to the phylogeny of Ophrys at the level of the~10 main lineages, confirming the suitability of NGS methods and approaches for phylogenetic purposes in taxonomically complex groups (Bateman et al, 2018). However, only one attempt to use multilocus NGS approaches has previously been made at the within-species-complex level at the transition zone between species and population levels.…”