Phylogeography and Molecular Species Delimitation Reveal Cryptic and Incipient Speciation in Synchronous Flashing Fireflies (Coleoptera: Lampyridae) of Southeast Asia

ABSTRACT Synchronous flashing fireflies of the genus Pteroptyx are ubiquitous throughout Southeast Asia, yet, knowledge on its biodiversity and evolutionary history remains lacking. Recent studies have revealed notable population-level phylogeographic structure within the P. tener and P. bearni groups in Malaysia, suggesting that cryptic species may exist. Additionally, the close morphological and genetic affinity of the recently described species P. balingiana to P. malaccae has raised questions about its validity. In this study, we assembled the most densely sampled genetic dataset on Pteroptyx to-date to estimate a comprehensive phylogeny using mitochondrial and nuclear DNA and subsequently implemented a suite of distance-, phylogeny-, and coalescent-based species delimitation methods to characterize species boundaries within the P. tener , P. bearni , and P. balingiana/P. malaccae groups. Using a total evidence approach from multiple lines of evidence, we showed that populations of P. tener along the west coast of Peninsular Malaysia are sufficiently divergent from populations from the east coast and Borneo to warrant specific recognition, despite the absence of morphological differentiation. Conversely, divergence of P. bearni from Borneo and eastern Peninsular Malaysia, as well as P. balingiana from P. malaccae were modest and their distinction as separate species were ambiguous; consistent with incipient species in the gray zone of speciation. Overall, this study contributes to the limited but growing body of genetic work on Southeast Asian fireflies and underscores the urgent need to increase the breadth and depth of geographic, taxonomic, and genetic sampling to provide a deeper understanding of their biodiversity and evolutionary history.