Integrative Taxonomy Reveals a Candidate Lineage Within the Rhinolophus macrotis Group

Accurate species delimitation is fundamental yet challenging, particularly in recently diverged, phenotypically conservative taxa such as bats. The “Rhinolophus macrotis group” represents one of the most taxonomically contentious groups among horseshoe bats. During field surveys in Southwest China, we discovered an unidentified Rhinolophus sp. occurring sympatrically with R. osgoodi and R. episcopus, sharing broad morphological affinities with recognized species of the “R. macrotis group.” To explore its taxonomic status, we employed an integrative approach combining morphological, acoustic, and multi-locus genetic (mitogenomic and nuclear) data. Phenotypically, Rhinolophus sp. closely resembles R. osgoodi but can be distinguished by its divergent echolocation resting frequency. Genetically, while mitochondrial data deeply nested Rhinolophus sp. within R. osgoodi with a shallow divergence time, phylogenies based on two nuclear introns positioned it closer to two other species, R. episcopus and R. siamensis. Species delimitation based on these genetic markers revealed a pattern of mitochondrial subdivision contrasted by overly conservative nuclear signals. Such mito-nuclear discordance suggests a complex evolutionary history that complicates taxonomic assignments. Given that only three specimens of Rhinolophus sp. were available, which precludes a robust assessment of intraspecific variation, we provisionally designate it as a candidate lineage within the “R. macrotis group,” warranting future validation with additional comprehensive evidence. This study highlights the indispensable utility of integrative taxonomy in uncovering hidden diversity and provides insights into chiropteran evolutionary history.