Integrating different tools to disentangle species complexes: A case study in Epidendrum (Orchidaceae)

Species delimitation remains a central problem in systematic, taxonomic and evolutionary studies. However, the precise delimitation of species depends on the criteria used to identify lineages and the specific species concept that is applied. Recently, multidisciplinary studies using different data sources have significantly improved the delimitation of species within complex taxonomic groups, leading to an integrative taxonomy. To investigate the species delimitation within the Atlantic clade of Epidendrum (subg. Amphyglottium), four different species criteria were examined (phenetic differentiation, monophyly, diagnosability, absence of genetic intermediates). Morphometrics, plastid DNA sequences and nuclear microsatellite markers were used to explore the agreement between patterns recovered and species criteria tested. The conflicts among species criteria are discussed in light of pollination ecology, patterns of gene flow, reproductive isolation mechanisms and selective pressures currently acting in deceptive orchid species. Four currently recognized species from the Atlantic clade could be delimitated, including one newly described species, Epidendrum flammeus. Three out of five species satisfied the monophyly criterion, and few diagnostic flower characters were found among species. In contrast, nuclear microsatellite data correctly assigned individuals to their respective species, even in the presence of weak reproductive isolation and extensive hybridization events reported in the literature. One important implication of this finding is that phylogenetic studies in Epidendrum spp. should make use of single- or low-copy nuclear loci instead of plastid markers, which may be true for other plant groups. The results also indicate that the generalized pollination syndrome found among species of the Atlantic clade, the different levels of gene flow observed between nuclear and plastid markers, and hybridization events are com monly observed as the main evolutionary forces within this orchid group. Finally, we discuss evolutionary processes and taxonomic limits among these species, and we highlight the need to increase the inter-disciplinary approach to investigate species limits in complex plant groups.