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Measuring the level of genetic drift using effective population size (<jats:italic>N<\/jats:italic><jats:sub>e<\/jats:sub>) is highly useful for management. Single\u2010cohort genetic <jats:italic>N<\/jats:italic><jats:sub>e<\/jats:sub> estimators approximate the number of breeders in one season (<jats:italic>N<\/jats:italic><jats:sub>b<\/jats:sub>): a value &lt;\u2009100 signals likely inbreeding depression. Per\u2010generation <jats:italic>N<\/jats:italic><jats:sub>e<\/jats:sub>\u2009&lt;\u20091000 estimated from multiple cohort signals reduced adaptive potential. Natural populations rarely meet assumptions of <jats:italic>N<\/jats:italic><jats:sub>e<\/jats:sub>\u2010estimation, so interpreting estimates is challenging. Macquarie perch is an endangered Australian freshwater fish threatened by severely reduced range, habitat loss, and fragmentation. To counteract low <jats:italic>N<\/jats:italic><jats:sub>e<\/jats:sub>, augmented gene flow is being implemented in several populations. In the Murrumbidgee River, unknown effects of water management on among\u2010site connectivity impede the design of effective interventions. Using DArT SNPs for 328 Murrumbidgee individuals sampled across several sites and years with different flow conditions, we assessed population structure, site isolation, heterozygosity, inbreeding, and <jats:italic>N<\/jats:italic><jats:sub>e<\/jats:sub>. We tested for inbreeding depression, assessed genetic diversity and dispersal, and evaluated whether individuals translocated from Cataract Reservoir to the Murrumbidgee River bred, and interbred with local fish. We found strong genetic structure, indicating complete or partial isolation of river fragments. This structure violates assumptions of <jats:italic>N<\/jats:italic><jats:sub>e<\/jats:sub> estimation, resulting in strongly downwardly biased <jats:italic>N<\/jats:italic><jats:sub>b<\/jats:sub> estimates unless assessed per\u2010site, highlighting the necessity to account for population structure while estimating <jats:italic>N<\/jats:italic><jats:sub>e<\/jats:sub>. Inbreeding depression was not detected, but with low <jats:italic>N<\/jats:italic><jats:sub>b<\/jats:sub> at each site, inbreeding and inbreeding depression are likely. These results flagged the necessity to address within\u2010river population connectivity through flow management and genetic mixing through translocations among sites and from other populations. Three detected genetically diverse offspring of a translocated Cataract fish and a local parent indicated that genetic mixing is in progress. Including admixed individuals in estimates yielded lower <jats:italic>N<\/jats:italic><jats:sub>e<\/jats:sub> but higher heterozygosity, suggesting heterozygosity is a preferable indicator of genetic augmentation.<\/jats:p>","DOI":"10.1111\/eva.70019","type":"journal-article","created":{"date-parts":[[2024,10,12]],"date-time":"2024-10-12T07:40:21Z","timestamp":1728718821000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Immediate Genetic Augmentation and Enhanced Habitat Connectivity Are Required to Secure the Future of an Iconic Endangered Freshwater Fish Population"],"prefix":"10.1111","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9455-4124","authenticated-orcid":false,"given":"Alexandra","family":"Pavlova","sequence":"first","affiliation":[{"name":"Wildlife Genetic Management Group, School of Biological Sciences Monash University  Melbourne Victoria Australia"},{"name":"School of Biological Sciences Monash University  Melbourne Victoria Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Luke","family":"Pearce","sequence":"additional","affiliation":[{"name":"NSW Department of Primary Industries  Albury New South Wales Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Felicity","family":"Sturgiss","sequence":"additional","affiliation":[{"name":"NSW Local Land Services, South East Local Land Services  Braidwood New South Wales Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Erin","family":"Lake","sequence":"additional","affiliation":[{"name":"NSW Department of Primary Industries, Department of Regional NSW  Nowra New South Wales Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8139-7059","authenticated-orcid":false,"given":"Paul","family":"Sunnucks","sequence":"additional","affiliation":[{"name":"Wildlife Genetic Management Group, School of Biological Sciences Monash University  Melbourne Victoria Australia"},{"name":"School of Biological Sciences Monash University  Melbourne Victoria Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mark","family":"Lintermans","sequence":"additional","affiliation":[{"name":"Fish Fondler Pty Ltd  Bungendore New South Wales Australia"},{"name":"Centre for Applied Water Science, Institute for Applied Ecology University of Canberra  Canberra Australian Capital Territory Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2024,10,12]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.1890\/13-1328.1"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.1111\/mec.12770"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1071\/MF12245"},{"key":"e_1_2_10_5_1","volume-title":"Potential Barriers to Upstream Migration of Macquarie Perch to Spawning Areas in the Cotter River, ACT","author":"Broadhurst B. 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