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Previous studies identified sex-associated variants in the follicle-stimulating hormone receptor (fshr) gene following an XX\/XY sex determination (SD) system. However, these variants could not be fully associated with sex in all samples. This suggests other genes and\/or environmental factors may be involved in the SD of this species, denoting intraspecific variation. In this study, we constructed a new high-quality genome assembly of M. cephalus. We then re-sequenced the whole genome in males and females from two divergent Mediterranean populations to ascertain whether other genetic variants could also be involved in SD. fshr gene variants showed to only partially explain SD, while a new intronic variant in the sestd1 gene appeared to be associated with SD following a ZZ\/ZW system. The presence of other putative candidate SD variants showing significant differences between the two populations suggested a regional pattern of variation in SD in the Mediterranean Sea. The incomplete association of all the identified variants also pointed to a potential role for environmental factors.<\/jats:p>","DOI":"10.3390\/ani15162445","type":"journal-article","created":{"date-parts":[[2025,8,20]],"date-time":"2025-08-20T15:53:37Z","timestamp":1755705217000},"page":"2445","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Complex Sex Determination in the Grey Mullet Mugil cephalus Suggested by Individual Whole Genome Sequence Data"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-6410-8635","authenticated-orcid":false,"given":"Mbarsid","family":"Racaku","sequence":"first","affiliation":[{"name":"Department of Comparative Biomedicine and Food Science, University of Padova, 35020 Legnaro, Padua, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5928-9361","authenticated-orcid":false,"given":"Serena","family":"Ferraresso","sequence":"additional","affiliation":[{"name":"Department of Comparative Biomedicine and Food Science, University of Padova, 35020 Legnaro, Padua, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4060-4728","authenticated-orcid":false,"given":"Massimiliano","family":"Babbucci","sequence":"additional","affiliation":[{"name":"Department of Comparative Biomedicine and Food Science, University of Padova, 35020 Legnaro, Padua, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9904-9282","authenticated-orcid":false,"given":"Andres","family":"Blanco","sequence":"additional","affiliation":[{"name":"Department of Zoology, Genetics and Physical Anthropology, Faculty of Veterinary, Universidade de Santiago de Compostela, Campus Terra, 27002 Lugo, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0615-7941","authenticated-orcid":false,"given":"Costas S.","family":"Tsigenopoulos","sequence":"additional","affiliation":[{"name":"Institute of Marine Biology, Biotechnology and Aquaculture, Hellenic Centre for Marine Research, P.O. Box 2214, 71003 Heraklion, Crete, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7518-0542","authenticated-orcid":false,"given":"Tereza","family":"Manousaki","sequence":"additional","affiliation":[{"name":"Institute of Marine Biology, Biotechnology and Aquaculture, Hellenic Centre for Marine Research, P.O. Box 2214, 71003 Heraklion, Crete, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-3550-4688","authenticated-orcid":false,"given":"Jelena","family":"Radojicic","sequence":"additional","affiliation":[{"name":"Institute of Marine Biology, Biotechnology and Aquaculture, Hellenic Centre for Marine Research, P.O. Box 2214, 71003 Heraklion, Crete, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5077-1909","authenticated-orcid":false,"given":"Vasileios","family":"Papadogiannis","sequence":"additional","affiliation":[{"name":"Institute of Marine Biology, Biotechnology and Aquaculture, Hellenic Centre for Marine Research, P.O. Box 2214, 71003 Heraklion, Crete, Greece"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8438-9305","authenticated-orcid":false,"given":"Paulino","family":"Mart\u00ednez","sequence":"additional","affiliation":[{"name":"Department of Zoology, Genetics and Physical Anthropology, Faculty of Veterinary, Universidade de Santiago de Compostela, Campus Terra, 27002 Lugo, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4351-2826","authenticated-orcid":false,"given":"Luca","family":"Bargelloni","sequence":"additional","affiliation":[{"name":"Department of Comparative Biomedicine and Food Science, University of Padova, 35020 Legnaro, Padua, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1794-5791","authenticated-orcid":false,"given":"Tomaso","family":"Patarnello","sequence":"additional","affiliation":[{"name":"Department of Comparative Biomedicine and Food Science, University of Padova, 35020 Legnaro, Padua, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,8,20]]},"reference":[{"key":"ref_1","unstructured":"Fricke, R., Eschmeyer, W.N., and Van der Laan, R. 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