The arboreal frogs in the genus Spinomantis (Amphibia: Mantellidae) from Madagascar: a survey of molecular, morphological and bioacoustic variation, with descriptions of four new species

We review the systematics of the Madagascar-endemic frogs of the genus Spinomantis based on DNA sequences of mitochondrial and nuclear-encoded genes from 368 individuals. We find the arboreal species in the genus, the focus of this study, to form a well-supported clade in a mitochondrial multi-gene tree, containing three subclades, here named the peraccae clade, the aglavei clade, and the massi clade. Each of these contained numerous deep mitochondrial lineages differing by at least 3.1% uncorrected pairwise distance in a fragment of the mitochondrial 16S rRNA gene. Our preferred partition distinguishes 20 mitochondrial species-level lineages in the arboreal Spinomantis clade, including seven nominal species, four new species described herein, and nine additional candidate species that require further study. The four new species described herein are delimited by a combination of several lines of evidence, including high divergence in mitochondrial DNA (> 3.5% 16S divergence to nominal species), isolated phylogenetic position, differentiation in nuclearencoded gene fragments, and morphological and bioacoustic divergence: S. chlorostictus sp. n. is a species of the peraccae clade primarily from northern Madagascar differing by color pattern and bioacoustics from S. peraccae; S. royae sp. n. and S. leonieae sp. n., belonging to the aglavei clade, are morphologically similar to S. fimbriatus but differ by color pattern and advertisement call; and S. fohitsilo sp. n. is morphologically similar to S. massi but not that species’ direct sister group, and differs by its advertisement call. Our study provides a baseline for understanding the diversity and biogeography of Spinomantis, highlights the enormous amount of yet unexplored amphibian diversity in Madagascar, and points to geographical regions and taxa where further field exploration, including the collection of voucher specimens and of bioacoustic data, is needed to complete the herpetofaunal inventory of the island.