Historical fragmentation in Atlantic Forest explains the diversification of a clade of mountaintop bromeligenous frogs (Leptodactylidae: Crossodactylodes)

The Atlantic Forest domain is a biodiversity hotspot with remarkable amphibian diversity, including over 700 species, 70% of which are endemic. Most of these endemic species have restricted geographic ranges, often confined to mountainous areas, as exemplified by the leptodactylid genus Crossodactylodes. These frogs are characterised by small body sizes, a bromeligenous habit and limited dispersal abilities, with species often restricted to their type localities. Previous studies have revealed geographically structured lineages within the genus, even when separated by short distances. Here, we focused on a clade of Crossodactylodes comprising three lineages from southeastern Brazil, inhabiting montane forest ‘islands’ distinct from surrounding lowland areas regarding vegetation structure and microclimate. We integrated genetic, geographic, morphometric and qualitative morphological data to assess species boundaries through species delimitation analyses and validation procedures. This integrative approach provided evidence supporting the recognition of one lineage as a distinct taxonomic entity, which we formally describe herein as Crossodactylodes alairi sp. nov. Additionally, we applied coalescent simulations and supervised machine-learning approaches to evaluate alternative diversification hypotheses. Our results provide strong support for fragmentation models, suggesting that divergences within the focal lineages were driven by climate-related habitat fragmentation during the Plio-Pleistocene. Given that these lineages inhabit a non-macrorefugium region of the Atlantic Forest, their evolutionary trajectories were likely shaped by survival in isolated microrefugia that offered stable and suitable microclimatic conditions amidst broader environmental changes.