Continuous temperature monitoring for two adjacent tropical crater lakes in Mexico at 4200 m amsl shows that the lakes have rhythmic episodes of heating and cooling with a duration of ~ 30 days during the warmest months. Our results demonstrate that eDNA signals show very strong seasonal stratification during summer and mixing during autumn in a manner that closely reflects the thermal preference of fishes. Several studies have used eDNA to monitor seasonal shifts in community assemblages in river estuaries (De Souza, Godwin, Renshaw, & Larson, 2016; Stoeckle, Soboleva, & Charlop‐Powers, 2017), coastal ecosystems (Berry et al., 2019; Sigsgaard et al., 2017) and large lakes (Bista et al., 2016; Handley et al., 2019). Samples were dual‐indexed with v2 Nextera DNA indexes (Illumina). NEET Biology Question Bank for {Topic}: Students preparing for the National Eligibility cum Entrance Test (NEET) can check the question bank of Biology’s NEET Biology Question Bank for {Topic}: Students preparing for the National Eligibility cum Entrance Test (NEET) can check the question bank of Biology’s chapter Organism and Population from Unit 10 here. Fish species are arranged in order of warm to cold thermal guilds (Table, NMDS plots for each lake showing community dissimilarities detected by each sample. Within the affected range (2.5–4.5 °C), the rate of earlier development of strong stratification increased with increasing T 20–80 ( Fig. However, there is emerging evidence that within‐habitat eDNA can reflect local species richness and also peak in concentration during seasonal events (Bylemans et al., 2018; Erickson, Merkes, & Mize, 2019; Harper, Anucha, Turnbull, Bean, & Leaver, 2018; Spear et al., 2015). Working off-campus? Symbols represent the eDNA of warm‐water fish (red squares), cool‐water fish (open grey circles) and cold‐water fish (filled dark blue circles). eDNA of all species is thoroughly mixed throughout the water column. This result was confirmed by our mixed effects modelling approach to describe the distribution of fish ASV counts. Here, we tested how seasonal … These findings contribute to our overall understanding of the “ecology” of eDNA within lake ecosystems, illustrating how the strong interaction between seasonal thermal structure in lakes and thermal niches of species on very localized spatial scales influences our ability to detect species. (Source: Caissie, D., 2006, The thermal regime of rivers—A review: Freshwater Biology, v. 51, p. 1389-1406) This detrimental environmental consequence was realized in the mid 2000s and to restore the suitability of this reach for salmon spawning, the U.S. Army Corps of Engineers added a sliding gate assembly to the intake structure at Cougar Dam. Fishes select habitat due to bioenergetic requirements: this diagram shows potential habitat selection by warm‐water, cool‐water (able to inhabit all layers of the lake), and cold‐water fishes. These patterns are typical of those found in previous years during peak stratification and turnover for lakes in this region (Sichewski & Cruikshank, 1998). Thermal stratification and fish thermal preference explain vertical eDNA distributions in lakes, Freshwater Institute, Fisheries & Oceans Canada, 501 University Crescent, Department of Biology, Queen’s University, Department of Biology, Lakehead University. Raw fastq files, sample x ASV tables and the sequence composition of the ASVs are available at Dryad (doi.org/10.5061/dryad.000000022). The DNA was eluted in two elutions of 60 µl AE buffer and stored at −80°C. We adjusted for varying depth interval size and variation in the total amount of telemetry detections for each lake over the relevant time period. Thus, the seasonal cycle of lake stratification can concentrate organisms within, or isolate organisms from, certain habitats at different times of the year. Can we manage fisheries with the inherent uncertainty from eDNA? Many seed species have an embryonic dormancy phase, and generally will not sprout until this dormancy is broken. We used a variance stabilizing transformation on our sample x ASV matrix to account for uneven library size across our samples. (2016) only detected S. alpinus at the deepest sampling points in a metabarcoding study of English lakes. Thermal energy storage systems help to couple thermal energy generation and process demand in cogeneration facilities. At the same time, opposite habitat restrictions would be occurring for warm‐water fishes, resulting in the restriction of their distribution to the upper, warmer waters of lakes (McMeans et al., 2020). During stratification, the relative proportions of ASVs from each species per sample changed dramatically at different depths in the lakes (Figure 2a). Acoustic telemetry showed that S. namaycush inhabited the bottom two‐thirds of the water column during stratification, although they were less likely to occupy the deepest depths (Figure 4b red bars, median depth of telemetry detections = 7.74–11.90 m). The experiments were designed by JEL and MEC. Thermocline depths are also strongly influenced by lake clarity—specifically, the concentration of dissolved organic carbon. Our results reflect those of Handley et al., (2019), who found greater heterogeneity in community composition of samples at three depth points during summer sampling when compared with winter sampling in their study of a single deep lake (1,480 ha, depth of 44 m/64 m in two basins), and that eDNA from a cold‐water stenotherm (S. alpinus) was only detectable in midwater and deep‐water habitats. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Other taxonomic groups appeared at very low frequencies when our ASVs were matched against the NCBI database, such as bacterial, mammalian and bird taxa, but as they were not the focus of our study they were excluded. autumn turnover) result in homogenous eDNA signals for all thermal guilds of fishes throughout the water column (Figure 1b). Nonbiological nucleotides were removed (primers, indices and adapters) using cutadapt (Martin, 2011). La stratification des lacs est la tendance des lacs à former des couches thermiques séparées et distinctes par temps chaud. These included a model with only the two main effects (no interaction) for average telemetry detections for the data from a month prior to sampling, as well as models with and without the interaction term for both the week prior to sampling, as well as the day of sampling, indicating that there were not large differences in the abilities of the different temporal groupings of telemetry detections to predict S. namaycush eDNA. An important seasonal feature of many temperate lakes is stratification, where isolated layers of water are formed. Negative PCR controls were included on each plate by substituting nuclease free water (Qiagen) for DNA. Environmental DNA (eDNA) is increasingly being used to conduct biodiversity surveys, species occupancy studies, and detect endangered and invasive species (Deiner et al., 2017; Taberlet, Coissac, Pompanon, Brochmann, & Willerslev, 2012). The amount of S. namaycush DNA was four orders of magnitude less at the shallowest measurement points (1–1.5 m from the surface). By contrast, during autumn lake turnover, the fish species assemblage as detected by eDNA was homogenous throughout the water column. Thermal stratification is a natural phenomenon that occurs in lakes as a result of the thermal expansion properties of water. The experiments were analysed and the figures made by JEL. The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. These studies were essential for determining the relative contributions to the distribution and persistence of eDNA particles. Thermal stratification affects other physical and chemical factors by limiting migration and/or interacting with them in the reservoir water profile. Our findings may also apply to other monomictic, dimictic and meromictic lakes, as well as tropical and temperate oceans, which undergo periods of seasonal or permanent stratification. Typically stratified lakes show three distinct layers, the Epilimnion comprising the top warm layer, the thermocline (or Metalimnion): the middle layer, which may change depth throughout the day, and the colder Hypolimnion extending to the floor of the lake. Fish were collected and the telemetry tags implanted under the following permits: Ontario Ministry of Natural Resources and Forestry Licence to Collect Fish for Scientific Purposes #1085769 (2017), #1089495 (2018) and Lakehead University Animal Use Protocol #1464657 (renewed in 2017 and 2018). We used the following PCR chemistry with the MiFish‐U primers: 7.4 µl nuclease free water (Qiagen), 1.25 µl 10X buffer (Genscript), 1 mM MgCl2 (Thermo Fisher Scientific), 0.2 mM GeneDirex dNTPs, 0.05 mg bovine serum albumen (Thermo Fisher Scientific), 0.25 mM each primer, 1U taq (Genscript) and 2 µl DNA in a final volume of 12.5 µl. Using this approach, environmental factors can either be studied in isolation or as a multifactorial experiment in combination with a low number of other variables, while allowing for experimental replication and some control of other sources of environmental variation. We refer to the shallowest depth as sampling point one and the deepest depth as point six. Instead, information about sequences found in blank samples is displayed in Table S6. This primer pair was able to amplify and discriminate between the greatest number of fish species, prioritizing species known to exist in our study area. 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