Resolving Relationships in an Exceedingly Young Orchid Lineage Using Genotyping-by-sequencing Data

Poor morphological and molecular differentiation in recently diversified lineages is a widespread phenomenon in plants. Phylogenetic relationships within such species complexes are often difficult to resolve because of the low variability in traditional molecular loci, as well as various other biological phenomena responsible for topological incongruence such as ILS and hybridization. In this study, we employ a Genotyping-by-sequencing (GBS) approach to disentangle evolutionary relationships within a species complex belonging to the Neotropical orchid genus Cycnoches. The complex includes seven taxa distributed in Central America and the adjacent Chocó biogeographic region, nested within a clade estimated to have first diversified in the early Quaternary. Previous phylogenies inferred from a handful of loci failed to provide support for internal relationships within the complex. Our Neighbor-net and coalescent-based analyses inferred from ca. 13,000 GBS loci obtained from 31 individuals belonging to six of the seven traditionally accepted Cycnoches species provided a robustly supported network. The resulting three main clades are corroborated by morphological traits and geographical distributions. Similarly, Maximum Likelihood (ML) inferences of concatenated GBS-loci produced results comparable with those derived from coalescence and network-based methods, albeit always with poor statistical support. The low support evident in the ML phylogeny might be attributed to the abundance of uninformative GBS loci, which can account for up to 50% of the total number of loci recovered. The phylogenomic framework provided here, as well as morphological evidence and geographical patterns, suggest that the six entities previously thought to be different species might actually represent only three distinct segregates. Our study is the first to demonstrate the utility of GBS data in phylogenomic research of a very young Neotropical plant clade (~2 Ma), and it paves the way for the study of the many other species complexes that populate the species-rich orchid family.