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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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