{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,10,31]],"date-time":"2026-10-31T16:32:35Z","timestamp":1793464355659,"version":"4.5.0"},"posted":{"date-parts":[[2025,11,29]]},"group-title":"Evolutionary Biology","reference-count":97,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,11,29]],"date-time":"2025-11-29T00:00:00Z","timestamp":1764374400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/"}],"funder":[{"award":["R01-GM083300"],"award-info":[{"award-number":["R01-GM083300"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R01-HD108914"],"award-info":[{"award-number":["R01-HD108914"]}],"id":[{"id":"https:\/\/ror.org\/04byxyr05","id-type":"ROR","asserted-by":"publisher"}]},{"name":"National Cancer Institute","award":["P30-CA008748"],"award-info":[{"award-number":["P30-CA008748"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,10,28]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Multiple classes of selfish genetic elements, including transposable elements, meiotic drivers, and viruses, are suppressed by small interfering RNAs (siRNAs) that guide RNA interference (RNAi). These are interlocked within continually evolving genetic conflicts that are characterized by extremely rapid dynamics of change in both sequence and copy number. Indeed, it was shown that across\n                  <jats:italic>Drosophila<\/jats:italic>\n                  , the core RNAi machinery evolves under positive selection, and that the RNAi effector\n                  <jats:italic>AGO2<\/jats:italic>\n                  has additional copies in some species. This contrasts with the core microRNA (miRNA) machinery in\n                  <jats:italic>Drosophila<\/jats:italic>\n                  , which evolves under negative selection and maintains one-to-one orthologs not only amongst flies, but even to mammals. Here, we analyze &gt;300 long read genome assemblies of\n                  <jats:italic>Drosophila<\/jats:italic>\n                  species to generalize these attributes. Not only do we find recurrent expansion of\n                  <jats:italic>AGO2<\/jats:italic>\n                  across \u223c100 species, including lineages with ongoing amplification of AGO2, we also find dozens of species with extra copies of the other core RNAi factors,\n                  <jats:italic>r2d2<\/jats:italic>\n                  and\n                  <jats:italic>dicer-2<\/jats:italic>\n                  . In many cases, these additional RNAi factor copies co-exist, or are nested, within a lineage bearing an ancestral expansion of\n                  <jats:italic>AGO2<\/jats:italic>\n                  . Transcriptome data provide evidence that core RNAi factors, including certain lineage-specific copies, are biased for testis expression. Finally, we use small RNA data to annotate hairpin RNAs (hpRNAs) in\n                  <jats:italic>D. pseudoobscura<\/jats:italic>\n                  , one of the species with prominent amplification of RNAi factors. We find rampant\n                  <jats:italic>de novo<\/jats:italic>\n                  hpRNA loci in this species, whose siRNAs are predominantly expressed in testis. All together, these findings highlight evolutionary plasticity of the fly RNAi pathway and affirm that it is preferentially deployed in the germline of the heterogametic sex. When considered alongside abundant genetic data for recurrent sex ratio meiotic drive against the Y chromosome, these genomic data strongly imply that a fundamental role of endogenous RNAi is to control sex chromosome conflicts.\n                <\/jats:p>","DOI":"10.1101\/2025.11.28.691189","type":"posted-content","created":{"date-parts":[[2025,11,30]],"date-time":"2025-11-30T05:15:10Z","timestamp":1764479710000},"source":"Crossref","is-referenced-by-count":0,"title":["Recurrent expansion and rapid evolution of the Drosophilid RNAi pathway in testis"],"prefix":"10.64898","author":[{"given":"Kimberly","family":"Elicker","sequence":"first","affiliation":[{"name":"Laboratory of Non-Canonical Modes of Inheritance, Rockefeller University, New York, NY 10065, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Meysoon","family":"Quraishi","sequence":"additional","affiliation":[{"name":"Developmental Biology Program, Sloan Kettering Institute, New York, NY 10065, 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S.","family":"Flynt","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, University of Mississippi, University, MS 38677, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiayu","family":"Wen","sequence":"additional","affiliation":[{"name":"Division of Genome Sciences and Cancer, The John Curtin School of Medical Research The Australian National University, Canberra, Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lamia","family":"Wahba","sequence":"additional","affiliation":[{"name":"Laboratory of Non-Canonical Modes of Inheritance, Rockefeller University, New York, NY 10065, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8432-5851","authenticated-orcid":false,"given":"Eric C.","family":"Lai","sequence":"additional","affiliation":[{"name":"Developmental Biology Program, Sloan Kettering Institute, New York, NY 10065, USA"},{"name":"Louis V Gerstner, Jr 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