In this study, we analyse a species complex in Epidendrum, a mega-diverse Neotropical orchid genus, that is formed by the 11 Brazilian species of the E. difforme group. Although this group (c. 100 taxa) exhibits relatively high levels of floral variation, the Brazilian species are similar, making delimitation problematic. Here we combine molecular (phylogenetics), morphological (geometric morphometrics), genome size and cytogenetic (chromosome counts and CMA/DAPI staining) data to investigate circumscription of these species. Our results were interpreted by looking for congruence of the results as a means to delimit species. The studied taxa appear to be monophyletic, and karyotypically all analysed accessions were 2n = 40. Their 1C values vary from 1.99 ± 07 pg to 2.84 ± 0.12 pg. We did not find evidence for recent polyploidy or dysploidy and, apparently, these phenomena have not been important in the evolution of this species complex. On the other hand, we found high levels of polymorphism for CMA/DAPI banding, and variation in genome size appears to be positively correlated with latitude. Geometric morphometrics indicate that E. sanchezii and E. anatipedium/E. amarajiense are distinct from the remaining species, and three groups of other species can be separated using canonical variables analysis (CVA). Variation in lip shape, genome size and heterochromatin patterns of the taxa are not fully congruent with the phylogenetic analysis, but our results allowed us to delimit with full confidence four species: E. amapense, E. anatipedium (including E. amarajiense), E. pseudodifforme (including E. campaccii and E. thiagoi) and E. sanchezii. Four others will be tentatively maintained but need further study. Our results indicate that it will be necessary to reassess many of the species complexes in the genus using a similar multidisciplinary perspective to evaluate the number of taxa that should be recognized.
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