Phylogeny and morphological evolution of the Neotropical genus Cuspidaria (Bignonieae, Bignoniaceae): Combining high-throughput sequencing and targeted loci data

The combined use of high-throughput sequencing (HTS) with targeted loci data offers an excellent opportunity to reconstruct robust and comprehensive phylogenies at fine taxonomic scales using a hybrid approach. In this study, we infer the phylogeny of Cuspidaria (Bignonieae, Bignoniaceae), a diverse clade of lianas and shrubs centered in South America's wet and dry forests. We used HTS to obtain complete or nearly complete plastome sequences of seven individuals of Cuspidaria selected to represent the main clades in the genus. This sampling strategy aimed to reconstruct relationships at deeper nodes (i.e., backbone). We also used targeted loci data obtained through Sanger sequencing to obtain sequences of two chloroplast markers (ndhF, rpl32-trnL) and one nuclear marker (PepC) for multiple individuals of 18 out of the 21 species of Cuspidaria recognized in the most recent treatment of the genus; only C. emmonsii, C. lachnaea, and C. simplicifolia were not sampled. This broad sampling strategy aimed to test the monophyly of individual species and reconstruct fine-scale interspecific relationships within the genus. Both datasets were analyzed separately and combined using Bayesian inference and maximum likelihood approaches. The combined dataset includes 65 individuals representing 18 previously recognized species of Cuspidaria plus outgroup taxa. The analysis of the combined dataset recovered a monophyletic Cuspidaria, excluding C. bracteata, which was nested within the outgroup and is best treated elsewhere. Ancestral character state reconstructions provided novel insights into the evolution of morphology and identified multiple putative morphological synapomorphies for key lineages of Cuspidaria. Namely, anthers curved forward was reconstructed as a potential synapomorphy for the Cuspidaria clade, whereas interpetiolar gland fields in stems and inflorescence, subulate or inconspicuous prophylls of the axillary buds, biternate leaves, actinodromous venation, calyx cupulate or spathaceous, calyx apices dentate or irregularly lobed, fruits with winged valves, and fruit midrib limited by two longitudinal ridges were reconstructed as potential synapomorphies of different clades within the genus. Other traits such as habit, tertiary venation, inflorescence type, corolla color, number of ovule series, and pollen unit were highly labile. As circumscribed here, Cuspidaria is composed of eight main clades supported by molecular data and morphological synapomorphies: Sideropogon, Tetrastichella, Cinerea, Paracarpaea, Cremastus, Blepharitheca, Saldanhaea, and Cuspidaria s.str. clades. All species are monophyletic, except for C. pulchra and C. sceptrum, which form a species complex and are best treated as a single taxon. The necessary taxonomic changes are proposed, leading to the recognition of 19 species in Cuspidaria.