Untangling the diversity of Trypanosomes infecting Colombian amphibians: morphometric and molecular insights

Amphibians, though widely distributed and diverse, are increasingly threatened by environmental changes and emerging diseases. Among the lesser-known yet prevalent pathogens affecting them are Trypanosoma parasites, which remain poorly studied, particularly in terms of taxonomy and molecular diversity. In Colombia, one of the most amphibian-rich countries globally, no prior studies had addressed the molecular identification of Trypanosoma species in anurans. This study aimed to strengthen the taxonomy of these parasites by integrating morphological, morphometric, and molecular analyses. A total of 434 amphibian samples from the GERPH biological collection were examined microscopically, with 92 testing positive for Trypanosoma . Thirteen morphometric and eleven categorical variables were used to characterize the parasites, leading to the identification of twelve distinct morphotypes through qualitative classification and multivariate analysis. Of the positive samples, 48.9% exhibited more than one morphotype, with morphotypes I, II, and III being the most widespread and generalist. To explore molecular diversity, DNA from 76 individuals positive by microscopy was analyzed using PCR targeting the 18S rRNA gene, yielding nine molecular lineages in 38 single-infection cases. Five morphotypes were successfully linked to molecular lineages, enabling barcode associations that facilitated the description of a new species, the expansion of the known range of Trypanosoma tungarae , and the identification of a potential species complex. This study highlights the importance of integrating morphological and molecular tools to elucidate parasite taxonomy and host-parasite relationships, underscoring the need for further research into vector ecology, molecular barcoding, and in vivo isolation methods to understand Trypanosoma diversity in amphibians.