Barcoding gene (cox1 mtDNA) meta-analysis: A continental perspective for Tylodelphys species (Digenea: Diplostomidae).

The genus Tylodelphys comprises globally distributed digenean flukes with complex life cycles involving freshwater snails, fish or amphibians, and piscivorous birds. However, species delimitation remains challenging due to morphological convergence among larval stages and incomplete resolution of the life cycles. This study conducted a global meta-analysis of mitochondrial cytochrome c oxidase subunit I (cox1) sequences retrieved from GenBank and BOLD Systems to assess genetic diversity, phylogenetic relationships, and life-cycle linkage within the genus, and to explore the influence of continental isolation or host-driven diversification. Analyses of 496 sequences revealed substantial genetic diversity and recovered 29 well-supported lineages, indicating widespread cryptic speciation. Despite this diversity, only four species (Tylodelphys mashonense, Tylodelphys circibuteonis, Tylodelphys clavata, and Tylodelphys excavata) have life cycles supported by molecular data in databases that link larval and adult stages. Phylogeographic patterns showed strong continental structuring, with no evidence of haplotype sharing among regions, suggesting long-term isolation, host-associated diversification, or sampling bias. Conserved use of planorbid snails as first intermediate hosts and consistent metacercarial tissue tropism indicate evolutionary stability in key life-history traits. In contrast, variation in the second intermediate and definitive hosts suggests ecological flexibility and potential host-switching. Overall, these findings highlight a pronounced gap between cox1-barcoded species and biologically validated species and demonstrate the need to increase barcoding effort in Tylodelphys studies. This study recommends that future studies employ geographic collaborations and integrative approaches that combine multilocus data with targeted life-cycle studies to improve species delimitation and phylogeography and to clarify transmission dynamics in freshwater ecosystems.