Reproductive barriers confirm absence of hybrid speciation and introgression in the Rhodnius pictipes and R....

The diversification of Rhodnius species has been primarily attributed to vicariance events. Phylogenomic studies have proposed various evolutionary hypotheses - such as incomplete lineage sorting, hybrid speciation, and introgression - to explain the different tree topologies observed for Rhodniini tribe. To assess reproductive compatibility and identify potential reproductive barriers, we conducted experimental crosses between R. pictipes and R. brethesi (R. pictipes group) and between R. ecuadoriensis and R. colombiensis (R. pallescens group). Hybrids were obtained in only one direction for both crosses (R. colombiensis ♀ × R. ecuadoriensis ♂ and R. brethesi ♀ × R. pictipes ♂), and hatching rates were very low. In the R. pallescens group, all hybrid nymphs died during the first nymphal stages (hybrid inviability). Similarly, in the R. pictipes group, 96.67% of hybrid nymphs failed to reach adulthood, with only a single hybrid male surviving to the adult stage, confirming hybrid inviability. Cytogenetic analysis of this hybrid revealed multiple chromosome pairing errors, indicating hybrid sterility. These findings confirm that the parental species represent distinct and valid taxa. More importantly, they strongly suggest that the hypotheses of introgression and hybrid speciation are unlikely, underscoring the necessity of integrative approaches to better understand the evolutionary processes shaping the Rhodniini.