Investigating temporal and spatial variation of eDNA in a nearshore rocky reef environment

Environmental DNA (eDNA) is increasingly used to measure biodiversity of marine ecosystems. However, key aspects of spatial and temporal dynamics of eDNA remain unknown. Particularly, it is unclear how long eDNA signals persist locally in dynamic marine environments, since degradation rates have predominantly been quantified through mesocosm studies. To determine in situ eDNA residence times, we introduced an eDNA signal from a non-native fish into a Southern California rocky reef ecosystem, and then measured changes in both introduced and background eDNA signals over 96 hours. Foreign eDNA signal could no longer be detected 7.5 hours after introduction, far exceeding disappearance rates quantified in laboratory studies. In addition, native vertebrate eDNA signals varied greatly over the 96 hours of observation, but time of day and tidal direction did not drive this variation in community structure. Species accumulation curves showed that standard sampling protocols using 3 replicate 1 L sea water samples were insufficient to capture full diversity of local marine vertebrates, capturing only 76% of all taxa. Despite this limitation, a single eDNA sample captured greater vertbrate diversity than 18 SCUBA based underwater visual transect surveys conducted at a nearby site. There was no significant difference in species richness between temporal replicates and spatial replicates, suggesting a space for time substitution may be effective for fully capturing the diversity of local marine vertebrate communities in nearshore rocky reef environments. This result is particularly important in designing eDNA metabarcoding sampling protocols to capture local marine species diversity.

69 degradation, advection, generation, dispersion, and/or diffusion in marine systems dramatically 70 differs from laboratory experiments. However, it is unclear whether this result is generalizable to 71 all marine ecosystems, including temperate or polar ecosystems where colder water temperatures 72 could slow degradation processes.

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In dynamic aquatic ecosystems, the combination of eDNA degradation and transport can 74 result in temporal variation in eDNA signatures [12]. As such, it is essential to understand 75 temporal variation of eDNA in marine environments as well as the spatial scale of eDNA 76 variation so that proper eDNA sampling strategies can be developed and results can be properly 77 interpreted. Our present understanding of short-term temporal variation in eDNA signatures of 78 entire marine vertebrate communities is limited to a single study on an intertidal ecosystem. 79 Kelly et al. [13] found that tides did not have a strong or consistent effect on community 80 composition, but that temperature and salinity did have a significant effect, suggesting that the 81 movement of water masses-rather than tides alone-has the strongest effect on eDNA 82 signatures.  , and an extension step at 72°C for 1 min (S3 Table). Thirty-five 175 additional cycles were then carried out at an annealing temperature of 50°C using the same 176 denaturation and extension steps above, followed by a final extension at 72°C for 10 min (S3 177 Table). All PCR experiments included negative controls. We then confirmed successful PCR 178 amplification through gel electrophoresis on 2% agarose gels.

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To prepare the sequencing library, we first pooled 5 μL from each of the 3 PCR technical 180 replicates. We then purified these PCR products, removing strands less than 100 bp long, using  Table).

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To test the underlying factors shaping temporal variation in native eDNA community 219 signatures, we investigated how species communities changed in response to three variables:  Table).   Table); 264 however, only one species, Chromis punctipinnis, was detected in every sample (S1 Fig). The 265 remaining taxa detected exhibited heterogeneous patterns and were absent from one or more 266 sampling points and times; this pattern was also observed for the subset of species monitored by 267 the KFM, PISCO, and Reef Check (Figs 2 and S2). We note that the presence of a spike in 268 foreign eDNA did not reduce the detection of native taxa. The mean number of taxa detected 269 when the foreign eDNA signature was present was 21 species (σ=6) and the mean number of 270 taxa detected when the foreign eDNA signature was absent was 20 species (σ=5).

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Results from PERMANOVA found that time point accounted for the largest portion of 278 variation in vertebrate assemblages (PERMANOVA; R 2 =32%) (Fig 3). The next most important 279 sources of variation were direction of tide (PERMANOVA; R 2 =8%) and location 280 (PERMANOVA; R 2 =7%) (Fig 3). The remaining 54% of variation was unaccounted for (Fig 3).    While eDNA holds promise to improve the way that we monitor marine biodiversity, there 449 is much to be learned about the dynamics of eDNA in the natural environment. Diffusion and 450 transport of eDNA, not just degradation, impact our ability to detect taxa within the marine