Taxonomy based on the biological species concept: the case of the Rhodnius robustus complex (Hemiptera, Triatominae)

Rhodnius robustus is a paraphyletic taxon composed of different lineages, being lineages II and III currently described as R. montenegrensis and R. marabaensis. However, it has been suggested that two distinct clades may be associated with lineage II: R. montenegrensis and R. robustus II. Among the different species concepts, the biological concept is based mainly on interspecific reproductive isolation. Thus, we performed crosses between R. montenegrensis and R. marabaensis and between R. montenegrensis and R. robustus II, and we revisited and discussed taxonomically and evolutionarily all intra and interspecific crosses of R. robustus  sensu lato (s. l.).

Reciprocal experimental crosses were conducted between R. montenegrensis with R. marabaensis and R. robustus II. Furthermore, intercrosses were performed up to the fourth (F4) or fifth generation (depending on the reproductive barrier). Finally, cytogenetic analyzes were performed in the gonads to evaluate the degree of pairing of the hybrids' chromosomes.

Hybrids were obtained in all directions of the crosses. However, crosses between R. montenegrensis females and R. marabaensis males resulted in a very low number of offspring. When intercrosses were performed, the hybrids showed high fertility, possibly resulting from heterosis. Only the F4 was unviable due to hybrid collapse. In the other direction, the hybrids were completely infertile, characterizing the hybrid sterility. Hybrids from crosses between R. montenegrensis females and R. robustus II males died before reaching adulthood (characterizing the hybrid collapse) and in the other direction, no reproductive barriers were observed.

We demonstrated that, on the basis of the biological species concept, R. montenegrensis and R. marabaensis are valid species and reproductively isolated. Furthermore, we observed partial isolation between R. montenegrensis and R. robustus II, highlighting the need for integrative taxonomy studies to elucidate this clade. Finally, we revisited the literature and observed that: R. robustus s. l. is reproductively isolated from R. nasutus and R. neglectus; with the exception of specimens from Amazonas, R. prolixus does not present reproductive isolation when crossed with R. robustus (from Colombia, Peru and Venezuela); and the "intraspecific" barriers of R. robustus s. l. possibly represent different species (R. montenegrensis, R. marabaensis, and/or R. barretti).