DNA barcoding reveals cryptic diversity in chiggers (Trombiculidae) parasitizing small mammals across Peninsular Malaysia.

Chiggers (Acariformes: Trombiculidae) are ectoparasites of small mammals and important vectors of scrub typhus, yet their taxonomy and molecular diversity in Southeast Asia remain poorly resolved. This study represents the first DNA barcoding and phylogenetic assessment of chiggers from small mammals across Peninsular Malaysia, integrating mitochondrial (COI) and nuclear (28 S rDNA) genes. A total of 192 chiggers representing 11 morphospecies from six genera were sequenced, with tree topologies inferred using maximum likelihood and supported by multiple species delimitation methods. The COI gene discriminated five species as monophyletic, while analysis of the 28 S rDNA gene clarified phylogenetic relationships within Walchia and Leptotrombidium. Discordance between morphological and molecular boundaries in several taxa, particularly Gahrliepia fletcheri, Leptotrombidium deliense, and Walchiella oudemansi, suggests the existence of cryptic lineages and potential regional diversification. Importantly, this study provides the first molecular records for eight species, including the only barcodes from the genera Gahrliepia and Walchiella worldwide. These findings provide important resources for the molecular identification of Southeast Asian chiggers and underscore the need for integrative approaches combining morphology, molecular data, and biogeography to resolve complex species boundaries and improve vector surveillance for mite-borne diseases.