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To address this, we conducted a controlled experiment over 1 year to assess the persistence of eDNA from an arboreal mammal across four substrates (water, sediment, sheltered nest\u2010box surfaces, and exposed nest\u2010box surfaces). Environmental DNA deposited by captive individuals was sampled repeatedly following removal of animals and analysed using both species\u2010specific qPCR and metabarcoding. We modelled degradation using quantitative decay, detection probability, and conditional decay frameworks, and applied a random breakage model to estimate theoretical fragment persistence. Decay followed first\u2010order exponential kinetics in water and on nest box surfaces but not in sediment, which exhibited high stochasticity. We observed a large difference in degradation rates between substrates, with exponential decay models revealing half\u2010lives ranging from 7.3\u2009days in water to 276\u2009days on sheltered internal surfaces. Rapid decay in water restricted detections to approximately 77\u2009days, while persistence models predicted that sheltered surfaces could retain detectable DNA for more than 4\u2009years (&gt;\u20091700\u2009days), indicating the potential for prolonged accumulation of legacy DNA in protected environments. While species\u2010specific qPCR offered higher sensitivity than metabarcoding, both methods had parallel decay patterns. Our findings indicate that substrate selection is a critical decision when designing an eDNA survey, as there is a trade\u2010off between maximising detection probability and maintaining temporal resolution.<\/jats:p>","DOI":"10.1111\/1755-0998.70205","type":"journal-article","created":{"date-parts":[[2026,9,23]],"date-time":"2026-09-23T05:17:45Z","timestamp":1790140665000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Substrate\u2010Dependent Variation in Environmental DNA Persistence and Degradation From a Small Mammal"],"prefix":"10.1111","volume":"26","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5065-4876","authenticated-orcid":false,"given":"Austin M.","family":"Guthrie","sequence":"first","affiliation":[{"name":"Trace and Environmental DNA Laboratory, School of Molecular and Life Sciences Curtin University  Perth Western Australia Australia"},{"name":"Mine Site Biomonitoring Using eDNA Research Group, School of Molecular and Life Sciences Curtin University  Perth Western Australia Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christine E.","family":"Cooper","sequence":"additional","affiliation":[{"name":"School of Molecular and Life Sciences Curtin University  Perth Western Australia Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3036-5479","authenticated-orcid":false,"given":"Philip W.","family":"Bateman","sequence":"additional","affiliation":[{"name":"Mine Site Biomonitoring Using eDNA Research Group, School of Molecular and Life Sciences Curtin University  Perth Western Australia Australia"},{"name":"Behavioural Ecology Laboratory, School of Molecular and Life Sciences Curtin University  Perth Western Australia Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1658-9927","authenticated-orcid":false,"given":"Mieke","family":"van der Heyde","sequence":"additional","affiliation":[{"name":"Trace and Environmental DNA Laboratory, School of Molecular and Life Sciences Curtin University  Perth Western Australia Australia"},{"name":"Mine Site Biomonitoring Using eDNA Research Group, School of Molecular and Life Sciences Curtin University  Perth Western Australia Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4424-3568","authenticated-orcid":false,"given":"Morten E.","family":"Allentoft","sequence":"additional","affiliation":[{"name":"Trace and Environmental DNA Laboratory, School of Molecular and Life Sciences Curtin University  Perth Western Australia Australia"},{"name":"Lundbeck Foundation GeoGenetics Centre, Globe Institute University of Copenhagen  Copenhagen Denmark"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8238-0534","authenticated-orcid":false,"given":"Paul","family":"Nevill","sequence":"additional","affiliation":[{"name":"Trace and Environmental DNA Laboratory, School of Molecular and Life Sciences Curtin University  Perth Western Australia Australia"},{"name":"Mine Site Biomonitoring Using eDNA Research Group, School of Molecular and Life Sciences Curtin University  Perth Western Australia Australia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,9,22]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598\u2010024\u201064612\u20105"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.3390\/genes10030192"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1038\/s41598\u2010023\u201027512\u20108"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.1098\/rspb.2012.1745"},{"key":"e_1_2_10_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0022\u20102836(05)80360\u20102"},{"key":"e_1_2_10_7_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365\u2010294X.2011.05261.x"},{"key":"e_1_2_10_8_1","volume-title":"FastQC: A Quality Control Tool for High Throughput Sequence Data [Computer Software]","author":"Andrews S.","year":"2010"},{"key":"e_1_2_10_9_1","doi-asserted-by":"publisher","DOI":"10.3390\/biology10121223"},{"key":"e_1_2_10_10_1","doi-asserted-by":"publisher","DOI":"10.1007\/s10592\u2010015\u20100775\u20104"},{"key":"e_1_2_10_11_1","doi-asserted-by":"publisher","DOI":"10.1021\/es404734p"},{"key":"e_1_2_10_12_1","doi-asserted-by":"publisher","DOI":"10.18637\/jss.v067.i01"},{"key":"e_1_2_10_13_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.scitotenv.2024.171810"},{"key":"e_1_2_10_14_1","doi-asserted-by":"publisher","DOI":"10.1111\/1755\u20100998.12428"},{"key":"e_1_2_10_15_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.3869"},{"key":"e_1_2_10_16_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.scitotenv.2025.179057"},{"key":"e_1_2_10_17_1","volume-title":"Hollowed Out? 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