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Orchids are typically self-compatible, and in this family self-fertilization is usually prevented by pre-pollination barriers. However, genetic self-incompatibility has been found in species of some lineages of the myophilous subtribe Pleurothallidinae. We carried out experimental pollinations to determine the mating systems of four Anathallis species, a representative genus of a large clade of this subtribe. This is one of the few clades of Pleurothallidinae not studied on this subject to date. All four species present strong self-incompatibility, reinforcing previous findings that this mating system is widespread in the Pleurothallidinae. However, the four species showed two different selfincompatibility reactions, one typical of gametophytic self-incompatibility (pollen tubes becoming irregular in their trajectory with variation in diameter and excessive deposition of callose) and the other typical of sporophytic self-incompatibility (absence of pollen grain germination). The two types of self-incompatibility reaction we found are associated with the two previous groups in previous classification systems: the species that showed a reaction similar to gametophytic self-incompatibility used to be included in Pleurothallis subgen. Specklinia sect. Muscosae and the three species that showed a reaction similar to sporophytic self-incompatibility used to be included in Pleurothallis subgen. Acuminatia. Mating system studies have the potential to help elucidate the phylogeny of groups within the Orchidaceae. We suggest that a wider survey of both mating systems and phylogeny is needed to help answer questions on the phylogeny of these groups.

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aBstract. We developed a project addressing the determination of the reproductive system through experimental pollinations of species in the major genera representing all major lineages of Pleurothallidinae in order to determine occurrence of self-incompatibility in the subtribe, in which group it has possibly appeared for the first time, and how many times it has evolved. Additionally we surveyed the floral biology of species of Octomeria, a genus with morphological characters typical of bee-pollinated flowers that was previously regarded as mellitophilous. At the moment, all but one of the species studied in selected large genera of the major lineages (Acianthera, Anathallis, Masdevallia, Octomeria, Specklinia, and Stelis) are self-incompatible. The species studied may possess complete, strong or partial self-incompatibility. We found two different sites where self-incompatibility reactions occur, the stigma and the stylar channel, and both sites were not found in the same genus except for Anathallis. In Anathallis, the two groups that differ morphologically (formerly Pleurothallis subgen. Specklinia sect. Muscosae and sect. Acuminatae) exhibit different sites of reaction. Flowers of Octomeria species produce nectar and are pollinated by both male and female Sciaridae flies, and the populations have high genetic variability similar to that found in Acianthera species. Our results indicate that self-incompatibility is a generalized widespread feature of the myophilous clade of the Pleurothallidinae and possibly evolved early in the subtribe, enabling the maintenance of high levels of genetic variability in the populations of each species in spite of pollinator behavior. Moreover, although sapromyophily (pollination by female flies by deceit) is much more common in the group, myophily s.s. (pollination by anthophilous flies seeking nectar) apparently evolved first from the plesiomorphic mellitophilous condition. In addition to the morphological synapomorphy uniting the members of the former circumscription of the subtribe (an articulation between the ovary and pedicel), our results also point to the occurrence of two biological synapomorphies in the group -self-incompatibility and myophily. Based on these differences, we suggest the splitting of Pleurothallidinae as presently recognized into two subtribes: one composed of the large, self-incompatible, myophilous clade and the other of the small, self-compatible, ornithophilous clade.rEsuMEn. Desarrollamos un proyecto dirigido a determinar, a través de polinización experimental, la autoincompatibilidad de los géneros representativos de los linajes más importantes de Pleurothallidinae, buscando el grupo donde posiblemente apareció por primera vez y cuántas veces ha ocurrido desde entonces. Adicionalmente estudiamos la biología floral de algunas especies de Octomeria, un género con características típicas de plantas con flores polinizadas por abejas, que previamente han sido conocidas como melitófilas. Hasta el momento, todas excepto una, de las especies estudiadas...