Bat-associated Trypanosoma diversity, geographic extension of known clades, and predominance of T. cruzi TcIV in a conserved tropical forest of southeastern Mexico.

Trypanosomatids of the Schizotrypanum clade display high ecological and evolutionary diversity, yet their sylvatic cycles remain understudied in many Neotropical biomes. We conducted the first integrative molecular survey of Trypanosoma diversity in a conserved seasonal tropical forest in the Yucatán Peninsula, Mexico. We screened Triatoma dimidiata vectors and small mammal hosts (rodents and bats) using satellite DNA (SatDNA) for Trypanosoma cruzi detection, Miniexon PCR for T. cruzi discrete typing unit (DTU) identification, and 18 S rRNA sequencing to identify other Trypanosoma species in bats. Trypanosoma cruzi was detected by SatDNA in 81.8% of collected vectors (95% CI: 75.07–87.38%; n = 165) and in 46.6% of rodents (95% CI: 36.7–56.7%, n = 103) and in 46.2% of bats (95% CI: 38.56–53.97%; n = 171). Discrete typing unit (DTU) analysis revealed that T. cruzi DTU IV (TcIV) predominated in the conserved tropical forest, being mainly associated with vectors and rodents, whereas TcI was more frequent in bats. Phylogenetic reconstruction based on 18 S rRNA revealed multiple bat-associated lineages, including sequences related to T. wauwau, T. madeirae, and Neobat clades—reported here for the first time in Mexico. Our results underscore the role of protected forests as reservoirs of trypanosomatid diversity and the relevance of multi-host surveillance to better understand transmission dynamics. This study contributes novel insights into the ecology, biogeography, and evolutionary history of Trypanosoma spp. in Mesoamerica and supports efforts to strengthen early detection of potential zoonotic threats.