Ancestral reconstruction of reproductive traits shows no tendency toward terrestriality in leptodactyline frogs

Background: Traditionally, the evolution of terrestrial reproduction in anurans from ancestors that bred in water has been accepted in the literature. Still, the existence of intermediate stages of water dependency, such as species that lay eggs close to water (e.g., in burrows) instead of in bodies of water, supports the hypothesis of an ordered and gradual evolution in the direction of a more terrestrial form of reproduction. However, this conventional view has recently been challenged for some anurans groups. Leptodactylinae frogs are a remarkable example of anurans with an outstanding diversity in terms of reproductive features, with distinct water dependency among lineages. Here, we tested the hypothesis of a gradual and ordered tendency towards terrestriality in Leptodactylinae, including the existence of obligatory intermediate stages, such as semi-terrestrial reproductive strategies. We also addressed the association between reproductive modes and the morphological and ecological features. Results: An ancestral reconstruction analysis indicated that even though shifts from aquatic to terrestrial breeding occurred throughout the history of Leptodactylus and Adenomera, shifts from terrestrial to aquatic reproduction happened at almost the same frequency. Our results also demonstrated that reproductive modes for semi-terrestrial tadpoles were not necessarily an intermediate form between aquatic and terrestrial breeds. Correlations among reproductive modes and other life-history traits suggested that tadpole environment, clutch size, nuptial spines, and egg pigmentation were co-evolving and driven by water dependency. Conclusions: Our results found no evidence of evolutionary tendencies toward terrestriality in Leptodactylinae. We found reversals from terrestrial to aquatic tadpole development and no evidence of obligatory intermediate stages, such as semi-terrestrial reproductive strategies. We also found correlations between reproductive modes and other life-history traits driven by water dependence. Aquatic reproductive modes are associated with higher clutch sizes, lentic waters, and the presence of nuptial spines and egg pigmentation.