Characterization of novel Trypanosoma species from birds and amphibians in the Colombian dry forest using an integrative taxonomic approach

Trypanosomatids are flagellated protozoa that infect a wide range of hosts. While Trypanosoma and Leishmania are extensively studied due to their medical importance, information regarding species infecting wildlife remains scarce. This study aimed to characterize trypanosomatids from Colombian amphibians, birds, and mammals using an integrative taxonomic approach. We sampled 46 birds, 18 reptiles and amphibians, and 124 mammals within the tropical dry forest of Tolima, Colombia. Detection was performed employing novel Hsp70 (671 bp) and 18S rRNA (1,400–1,900 bp) primers. Positive PCR products were sequenced using Oxford Nanopore Technologies for phylogenetic inference and species delimitation. Morphometric traits were measured from blood smears, and phenotypic variation was assessed via PCA and PERMANOVA. The combination of molecular markers, species delimitation, and morphometrics led to the discovery of three new species: Trypanosoma vallejoi n. sp. and Trypanosoma carranzai n. sp. in wild birds, and Trypanosoma guhli n. sp. in amphibians. This integrative framework provides a robust and cost-effective tool for trypanosomatid diagnosis in wildlife. These findings reveal a hidden diversity of trypanosomatids in Neotropical ecosystems and demonstrate the value of molecular tools in bridging wildlife surveillance with evolutionary and taxonomic research.