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Since the discovery of European flounder (Platichthys flesus) in the freshwater environment of the isolated Lake Pulmanki in the 1970s, the evolutionary origin and reproductive strategy of this euryhaline fish has remained a mystery. Using otolith chemistry and whole-genome resequencing, we tested the hypothesis that flounders in Lake Pulmanki constitute a freshwater-adapted lineage, distinct from their marine ancestors. Otoliths confirmed that most of the flounders caught in Lake Pulmanki were born and lived exclusively in freshwater, providing the first evidence for a fully freshwater-confined life cycle in Platichthys. Unexpectedly, most individuals in nearby fjords were also born in freshwater. While the genome-wide differentiation among marine- and freshwater-born flounders was weak, 77 SNPs across 18 chromosomes were significantly associated with habitat-use type, mapping to 36 genes. Moreover, outlier loci show strong interchromosomal linkage disequilibrium, suggesting divergent selection for allelic combinations associated with reproductive habitat use. This pattern is consistent with a genetic basis for early habitat use differences involving epistatic interactions among alleles in different chromosomes, pointing to a genetic adaptation for freshwater reproduction in Lake Pulmanki.<\/jats:p>","DOI":"10.1093\/evolut\/qpag165","type":"journal-article","created":{"date-parts":[[2026,9,21]],"date-time":"2026-09-21T11:08:33Z","timestamp":1789988913000},"source":"Crossref","is-referenced-by-count":0,"title":["Evolution of freshwater reproduction in a marine fish: Evidence from genetics and otolith microchemistry"],"prefix":"10.1093","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7405-5907","authenticated-orcid":false,"given":"Thais","family":"Ferreira-Ara\u00fajo","sequence":"first","affiliation":[{"name":"Ecological Genetics Research Unit, Organismal and Evolutionary Biology Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki , Helsinki ,","place":["Finland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Petri","family":"Kemppainen","sequence":"additional","affiliation":[{"name":"Ecological Genetics Research Unit, Organismal and Evolutionary Biology Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki , Helsinki ,","place":["Finland"]},{"name":"Area of Ecology and Biodiversity, School of Biological Sciences , Kadoorie Biological Sciences Building, Pokfulam Road, The University of Hong Kong , SAR","place":["Hong Kong"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Susanne E","family":"Tanner","sequence":"additional","affiliation":[{"name":"University of Lisbon MARE\u2014Marine and Environmental Sciences Centre & ARNET\u2014Aquatic Research Network Associated Laboratory, Department of Biology, Faculty of Sciences, , Lisbon ,","place":["Portugal"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9843-9465","authenticated-orcid":false,"given":"Patrick","family":"Reis-Santos","sequence":"additional","affiliation":[{"name":"Southern Seas Ecology Laboratories, School of Biological Sciences, Adelaide University ,","place":["Australia"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7680-2240","authenticated-orcid":false,"given":"Bronwyn M","family":"Gillanders","sequence":"additional","affiliation":[{"name":"Southern Seas Ecology Laboratories, School of Biological Sciences, Adelaide University ,","place":["Australia"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kimmo K","family":"Kahilainen","sequence":"additional","affiliation":[{"name":"Lammi Biological Station, University of Helsinki, Lammi ,","place":["Finland"]},{"name":"Kilpisj\u00e4rvi Biological Station, University of Helsinki, Kilpisj\u00e4rvi ,","place":["Finland"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Juha","family":"Meril\u00e4","sequence":"additional","affiliation":[{"name":"Ecological Genetics Research Unit, Organismal and Evolutionary Biology Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki , Helsinki ,","place":["Finland"]},{"name":"Area of Ecology and Biodiversity, School of Biological Sciences , Kadoorie Biological Sciences Building, Pokfulam Road, The University of Hong Kong , SAR","place":["Hong Kong"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paolo","family":"Momigliano","sequence":"additional","affiliation":[{"name":"Area of Ecology and Biodiversity, School of Biological Sciences , Kadoorie Biological Sciences Building, Pokfulam Road, The University of Hong Kong , SAR","place":["Hong Kong"]},{"name":"Swire Institute of Marine Science, The University of Hong Kong , SAR ,","place":["Hong Kong"]},{"name":"Tuscia University Department of Ecological and Biological Sciences, , Viterbo ,","place":["Italy"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"286","published-online":{"date-parts":[[2026,9,22]]},"reference":[{"issue":"2","key":"2026101610372093200_bib1","doi-asserted-by":"publisher","first-page":"388","DOI":"10.1093\/icb\/icac071","article-title":"Freshwater colonization, adaptation, and genomic divergence in threespine stickleback","volume":"62","author":"Aguirre","year":"2022","journal-title":"Integrative and Comparative Biology"},{"issue":"1","key":"2026101610372093200_bib2","doi-asserted-by":"publisher","first-page":"89","DOI":"10.1007\/s10750-004-8337-7","article-title":"Diversity and distributions of circumpolar fresh- and brackish-water Mysis (Crustacea: Mysida): Descriptions of M. relicta Lov\u00e9n, 1862, M. salemaai n. 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