Genetic, morphological, and bioacoustic assessments of species limits in the greater African roundleaf bats, Macronycteris (Hipposideridae)

Accurate delimitation of species boundaries is crucial for uncovering evolutionary history and guiding conservation efforts in systematically complex groups. We evaluated species limits within the genus Macronycteris, large African roundleaf bats, using genetic, morphological, and bioacoustic data. Although mitochondrial DNA supported four reciprocally monophyletic species, nuclear introns revealed incomplete lineage sorting and limited genetic differentiation, suggesting recent diversification. Species delimitation analyses based on cytochrome b indicated up to eight putative lineages, whereas multi-locus analyses supported the five currently recognized species. Morphometric analyses of >300 specimens revealed significant interspecific differences in external and cranial traits, with Macronycteris gigas being markedly larger than congeners. Insular species were smaller than mainland forms, consistent with the island rule. Unlike most bats, all species exhibited strong male-biased sexual size dimorphism, but the extent of dimorphism did not follow Rensch’s rule. Bioacoustic analyses differentiated species, with echolocation frequencies varying significantly across species and localities. Discriminant analyses of cranial and acoustic traits achieved high classification accuracy, reinforcing phenotypic distinctions among genetically similar taxa. Our results highlight a case of phenotypic and ecological divergence outpacing molecular differentiation in a recent bat radiation and provide a robust framework for species delimitation in Macronycteris, with implications for taxonomy, biogeography, and conservation.