The family of pantropical spiral gingers (Costaceae Nakai; c. 125 spp.) can be used as a model to enhance our understanding of the mechanisms underlying Neotropical diversity. Costaceae has higher taxonomic diversity in South and Central America (c. 72 Neotropical species, c. 30 African, c. 23 Southeast Asian), particularly due to a radiation of Neotropical species of the genus Costus L. (c. 57 spp.). However, a well-supported phylogeny of the Neotropical spiral gingers including thorough sampling of proposed species encompassing their full morphologic and geographic variation is lacking, partly due to poor resolution recovered in previous analyses using a small sampling of loci. Here we use a phylogenomic approach to estimate the phylogeny of a sample of Neotropical Costus species using a targeted enrichment approach. Baits were designed to capture conserved elements' variable at the species level using available genomic sequences of Costus species and relatives. We obtained 832 loci (generating 791,954 aligned base pairs and 31,142 parsimony informative sites) for samples that encompassed the geographical and/or morphological diversity of some recognized species. Higher support values that improve the results of previous studies were obtained when including all the available loci, even those producing unresolved gene trees and having a low proportion of variable sites. Concatenation and coalescent-based species trees methods converge in almost the same topology suggesting a robust estimation of the relationships, even under the high levels of gene tree conflict presented here. The bait set design here presented made inferring a robust phylogeny to test taxonomic hypotheses possible and will improve our understanding of the origins of the charismatic diversity of the Neotropical spiral gingers.

Selection together with variation in floral traits can act to mold floral form, often driven by a plant’s predominant or most effective pollinators. To investigate the evolution of traits associated with pollination, we developed a phylogenetic framework for evaluating tempo and mode of pollination shifts across the genus Costus L., known for its evolutionary toggle between traits related to bee and bird pollination. Using a target enrichment approach, we obtained 957 loci for 171 accessions to expand the phylogenetic sampling of Neotropical Costus. In addition, we performed whole genome resequencing for a subset of 20 closely related species with contrasting pollination syndromes. For each of these 20 genomes, a high-quality assembled transcriptome was used as reference for consensus calling of candidate loci hypothesized to be associated with pollination-related traits of interest. To test for the role these candidate genes may play in evolutionary shifts in pollinators, signatures of selection were estimated as dN/dS across the identified candidate loci. We obtained a well-resolved phylogeny for Neotropical Costus despite conflict among gene trees that provide evidence of incomplete lineage sorting and/or reticulation. The overall topology and the network of genome-wide single nucleotide polymorphisms (SNPs) indicate that multiple shifts in pollination strategy have occurred across Costus, while also suggesting the presence of previously undetected signatures of hybridization between distantly related taxa. Traits related to pollination syndromes are strongly correlated and have been gained and lost in concert several times throughout the evolution of the genus. The presence of bract appendages is correlated with two traits associated with defenses against herbivory. Although labellum shape is strongly correlated with overall pollination syndrome, we found no significant impact of labellum shape on diversification rates. Evidence suggests an interplay of pollination success with other selective pressures shaping the evolution of the Costus inflorescence. Although most of the loci used for phylogenetic inference appear to be under purifying selection, many candidate genes associated with functional traits show evidence of being under positive selection. Together these results indicate an interplay of phylogenetic history with adaptive evolution leading to the diversification of pollination-associated traits in Neotropical Costus.