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Particularly, gene misregulation in interspecific hybrids can inform about what genes and pathways underlie hybrid dysfunction. In <jats:italic>Drosophila<\/jats:italic> hybrids, how regulatory evolution impacts different reproductive tissues remains understudied. Here, we generate a new genome assembly and annotation in <jats:italic>Drosophila willistoni<\/jats:italic> and analyse the patterns of transcriptome divergence between two allopatrically evolved <jats:italic>D. willistoni<\/jats:italic> subspecies, their male sterile and female fertile hybrid progeny across testis, male accessory gland, and ovary. Patterns of transcriptome divergence and modes of regulatory evolution were tissue\u2010specific. Despite no indication for cell\u2010type differences in hybrid testis, this tissue exhibited the largest magnitude of expression differentiation between subspecies and between parentals and hybrids. No evidence for anomalous dosage compensation in hybrid male tissues was detected nor was a differential role for the neo\u2010 and the ancestral arms of the <jats:italic>D. willistoni X<\/jats:italic> chromosome. Compared to the autosomes, the <jats:italic>X<\/jats:italic> chromosome appeared enriched for transgressively expressed genes in testis despite being the least differentiated in expression between subspecies. Evidence for fine genome clustering of transgressively expressed genes suggests a role of chromatin structure on hybrid gene misregulation. Lastly, transgressively expressed genes in the testis of the sterile male progeny were enriched for GO terms not typically associated with sperm function, instead hinting at anomalous development of the reproductive tissue. Our thorough tissue\u2010level portrait of transcriptome differentiation between recently diverged <jats:italic>D. willistoni<\/jats:italic> subspecies and their hybrids provides a more nuanced view of early regulatory changes during speciation.<\/jats:p>","DOI":"10.1111\/mec.16941","type":"journal-article","created":{"date-parts":[[2023,3,31]],"date-time":"2023-03-31T14:30:37Z","timestamp":1680273037000},"page":"3605-3623","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["Gene expression differentiation in the reproductive tissues of <i>Drosophila willistoni<\/i> subspecies and their hybrids"],"prefix":"10.1111","volume":"32","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3585-3129","authenticated-orcid":false,"given":"Jos\u00e9 M.","family":"Ranz","sequence":"first","affiliation":[{"name":"Department of Ecology and Evolutionary Biology University of California Irvine  California 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