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Several phenotypic identification problems, as well as divergences between classical and molecular taxonomy, have been reported. Furthermore, phylogenetic and phylogenomic studies demonstrated possible introgression events between\n \n R. prolixus<\/jats:italic>\n <\/jats:italic>\n and\n \n R. robustus<\/jats:italic>\n <\/jats:italic>\n . Based on the above, we revisited all the literature on hybridization involving\n \n R. prolixus<\/jats:italic>\n <\/jats:italic>\n and performed interspecific crosses between\n \n R. prolixus<\/jats:italic>\n <\/jats:italic>\n and other species of the\n \n R. prolixus<\/jats:italic>\n <\/jats:italic>\n group (\n \n R. nasutus<\/jats:italic>\n <\/jats:italic>\n ,\n \n R. neivai<\/jats:italic>\n <\/jats:italic>\n , and\n \n R. robustus<\/jats:italic>\n <\/jats:italic>\n ) to evaluate potential reproductive barriers and discuss taxonomic and evolutionary issues related to intra- and interspecific reproductive isolation. With the exception of the cross between\n \n R. prolixus<\/jats:italic>\n <\/jats:italic>\n females and\n \n R. neivai<\/jats:italic>\n <\/jats:italic>\n males, all other combinations resulted in hybrid offspring. Moreover, except for the cross between\n \n R. prolixus<\/jats:italic>\n <\/jats:italic>\n females and\n \n R. robustus<\/jats:italic>\n <\/jats:italic>\n males, all other combinations exhibited postzygotic barriers, including inviability, sterility and\/or hybrid collapse. These results indicate that, in at least one direction,\n \n R. nasutus<\/jats:italic>\n <\/jats:italic>\n ,\n \n R. neivai<\/jats:italic>\n <\/jats:italic>\n , and\n \n R. robustus<\/jats:italic>\n <\/jats:italic>\n are reproductively isolated from\n \n R. prolixus<\/jats:italic>\n <\/jats:italic>\n , confirming the specific status of the four taxa. Furthermore, based on the observed barriers, we suggest that introgression is unlikely between\n \n R. prolixus<\/jats:italic>\n <\/jats:italic>\n and\n \n R. nasutus<\/jats:italic>\n <\/jats:italic>\n , unlike\n \n R. neivai<\/jats:italic>\n <\/jats:italic>\n and\n \n R. robustus<\/jats:italic>\n <\/jats:italic>\n , which could exchange genetic material with\n \n R. prolixus<\/jats:italic>\n <\/jats:italic>\n through introgression, under natural conditions. Finally, we discuss all available literature on intra- and interspecific crosses of\n \n R. prolixus<\/jats:italic>\n <\/jats:italic>\n , demonstrating that\n \n R. pictipes<\/jats:italic>\n <\/jats:italic>\n and\n \n R. neglectus<\/jats:italic>\n <\/jats:italic>\n are also reproductively isolated from\n \n R. prolixus<\/jats:italic>\n <\/jats:italic>\n . Additionally, we highlight reproductive barriers observed between allopatric populations of\n \n R. prolixus<\/jats:italic>\n <\/jats:italic>\n , emphasizing the need for a phylogenomic study \u2013 including field-collected specimens sampled across the entire distribution of\n \n R. prolixus<\/jats:italic>\n <\/jats:italic>\n \u2013 to clarify evolutionary and taxonomic questions.\n <\/jats:p>","DOI":"10.1371\/journal.pone.0335238","type":"journal-article","created":{"date-parts":[[2025,10,31]],"date-time":"2025-10-31T17:58:32Z","timestamp":1761933512000},"page":"e0335238","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":1,"title":["More than half a century of evolutionary studies in Rhodnius prolixus St\u00e5l, 1859 (Hemiptera, Triatominae): revisiting and discussing old and new data on intra- and interspecific reproductive barriers"],"prefix":"10.1371","volume":"20","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-8333-3764","authenticated-orcid":true,"given":"Kaio Cesar Chaboli","family":"Alevi","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Amanda","family":"Ravazi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jader","family":"de Oliveira","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yago Visinho","family":"dos Reis","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Isadora de Freitas","family":"Bittinelli","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Luiza Maria Grzyb","family":"Delgado","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6301-7219","authenticated-orcid":true,"given":"Jociel Klleyton Santos","family":"Santana","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Maria Terc\u00edlia Vilela","family":"de Azeredo-Oliveira","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jo\u00e3o Aristeu","family":"da Rosa","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mauro Toledo","family":"Marrelli","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4027-9205","authenticated-orcid":true,"given":"Cleber","family":"Galv\u00e3o","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"340","published-online":{"date-parts":[[2025,10,31]]},"reference":[{"key":"pone.0335238.ref001","unstructured":"WHO. 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