Phylogenetic analyses were conducted on six plastid markers and ITS from 43 accessions representing all eight currently recognized Hexalectris species with the goals of testing species circumscriptions and determining interspecific relationships. The monophyly of H. warnockii, H. grandiflora, H. brevicaulis, and H. nitida, plus the H. spicata species complex, are strongly supported. The remaining species are not monophyletic, prompting the recircumscription of H. spicata s. 1. as H. spicata s. s. and H. arizonica, H. revoluta s. 1. as H. revoluta s. s. and H. colemanii, and H. fallax as a synonym of H. parviflora. Four major morphologically distinct lineages exist within the genus: H. warnockii, H. grandiflora, H. brevicaulis, and the H. spicata species complex. Within the latter, only H. spicata s. s. and H. nitida are strongly supported as sister species among all trees. The positions of H. revoluta s. s. and H. parviflora remain unresolved due to incongruence in the plastid trees when psbA is included or excluded, while the positions of H. colemanii and H. arizonica are unclear due to incongruence between plastid and ITS topologies. The latter incongruence suggests that either or both species are of hybrid origin or that the ITS topology has been influenced by incomplete lineage sorting.
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