Phylogenetic comparative methods improve the selection of characters for generic delimitations in a hyperdiverse Neotropical orchid clade.

Taxonomic delimitations are challenging because of the convergent and variable nature of phenotypic traits. This is evident in species-rich lineages, where the ancestral and derived states and their gains and losses are difficult to assess. Phylogenetic comparative methods help to evaluate the convergent evolution of a given morphological character, thus enabling the discovery of traits useful for classifications. In this study, we investigate the evolution of selected traits to test for their suitability for generic delimitations in the clade Lepanthes, one of the Neotropical species-richest groups. We evaluated every generic name proposed in the Lepanthes clade producing densely sampled phylogenies with Maximum Parsimony, Maximum Likelihood, and Bayesian approaches. Using Ancestral State Reconstructions, we then assessed 18 phenotypic characters that have been traditionally employed to diagnose genera. We propose the recognition of 14 genera based on solid morphological delimitations. Among the characters assessed, we identified 16 plesiomorphies, 12 homoplastic characters, and seven synapomorphies, the latter of which are reproductive features mostly related to the pollination by pseudocopulation and possibly correlated with rapid diversifications in Lepanthes. Furthermore, the ancestral states of some reproductive characters suggest that these traits are associated with pollination mechanisms alike promoting homoplasy. Our methodological approach enables the discovery of useful traits for generic delimitations in the Lepanthes clade and offers various other testable hypotheses on trait evolution for future research on Pleurothallidinae orchids because the phenotypic variation of some characters evaluated here also occurs in other diverse genera.

Morphological and Molecular Characterization of Orchid Fruit Development.

Efficient seed dispersal in flowering plants is enabled by the development of fruits, which can be either dehiscent or indehiscent. Dehiscent fruits open at maturity to shatter the seeds, while indehiscent fruits do not open and the seeds are dispersed in various ways. The diversity in fruit morphology and seed shattering mechanisms is enormous within the flowering plants. How these different fruit types develop and which molecular networks are driving fruit diversification is still largely unknown, despite progress in eudicot model species. The orchid family, known for its astonishing floral diversity, displays a huge variation in fruit dehiscence types, which have been poorly investigated. We undertook a combined approach to understand fruit morphology and dehiscence in different orchid species to get more insight into the molecular network that underlies orchid fruit development. We describe fruit development in detail for the epiphytic orchid species Erycina pusilla and compare it to two terrestrial orchid species: Cynorkis fastigiata and Epipactis helleborine. Our anatomical analysis provides further evidence for the split carpel model, which explains the presence of three fertile and three sterile valves in most orchid species. Interesting differences were observed in the lignification patterns of the dehiscence zones. While C. fastigiata and E. helleborine develop a lignified layer at the valve boundaries, E. pusilla fruits did not lignify at these boundaries, but formed a cuticle-like layer instead. We characterized orthologs of fruit-associated MADS-domain transcription factors and of the Arabidopsis dehiscence-related genes INDEHISCENT (IND)/HECATE 3 (HEC3), REPLUMLESS (RPL) and SPATULA (SPT)/ALCATRAZ (ALC) in E. pusilla, and found that the key players of the eudicot fruit regulatory network appear well-conserved in monocots. Protein-protein interaction studies revealed that MADS-domain complexes comprised of FRUITFULL (FUL), SEPALLATA (SEP) and AGAMOUS (AG) /SHATTERPROOF (SHP) orthologs can also be formed in E. pusilla, and that the expression of HEC3, RPL, and SPT can be associated with dehiscence zone development similar to Arabidopsis. Our expression analysis also indicates differences, however, which may underlie fruit divergence.

Pollination of Specklinia by nectar-feeding Drosophila: the first reported case of a deceptive syndrome employing aggregation pheromones in Orchidaceae.

BACKGROUND AND AIMS: The first documented observation of pollination in Pleurothallidinae was that of Endrés, who noticed that the 'viscid sepals' of Specklinia endotrachys were visited by a 'small fly'. Chase would later identify the visiting flies as being members of the genus Drosophila. This study documents and describes how species of the S. endotrachys complex are pollinated by different Drosophila species. METHODS: Specimens of Specklinia and Drosophila were collected in the field in Costa Rica and preserved in the JBL and L herbaria. Flies were photographed, filmed and observed for several days during a 2-year period and were identified by a combination of non-invasive DNA barcoding and anatomical surveys. Tissue samples of the sepals, petals and labellum of Specklinia species were observed and documented by SEM, LM and TEM. Electroantennogram experiments were carried out on Drosophila hydei using the known aggregation pheromones ethyl tiglate, methyl tiglate and isopropyl tiglate. Floral compounds were analysed by gas chromatography-mass spectometry using those same pheromones as standards. KEY RESULTS: Flowers of S. endotrachys, S. pfavii, S. remotiflora and S. spectabilis are visited and pollinated by several different but closely related Drosophila species. The flies are arrested by aggregation pheromones, including ethyl tiglate, methyl tiglate and isopropyl tiglate, released by the flowers, and to which at least D. hydei is very sensitive. Visible nectar drops on the adaxial surface of sepals are secreted by nectar-secreting stomata, encouraging male and female Drosophila to linger on the flowers for several hours at a time. The flies frequently show courtship behaviour, occasionally copulating. Several different Drosophila species can be found on a single Specklinia species. CONCLUSIONS: Species of the S. endotrachys group share a similar pollination syndrome. There seem to be no species-specific relationships between the orchids and the flies. It is not expected that Specklinia species will hybridize naturally as their populations do not overlap geographically. The combination of pheromone attraction and nectar feeding is likely to be a generalized pollination syndrome in Pleurothallidinae.