Primers and probe details showing sequences, target taxa and fragment sizes.

<p>Fragment sizes are given in base pairs including primers. All primers were designed for this study and amplify part of the Cytochrome b (<i>cyt-b</i>) gene. All regular PCRs were performed at 50°C annealing temperature and all qPCRs at 60°C annealing temperature. Probes are Minor Groove Binding (MGB) probes and have the modifications; 5′: 6-Fam (D-L-Probe), 3′: BHQ-1.</p

Greenland eDNA raw sequencing data

This contains the raw fastq files from eDNA sequencing from the study. Data is Illumina MiSeq paird-end in four libraries

Detection of a Diverse Marine Fish Fauna Using Environmental DNA from Seawater Samples

<div><p>Marine ecosystems worldwide are under threat with many fish species and populations suffering from human over-exploitation. This is greatly impacting global biodiversity, economy and human health. Intriguingly, marine fish are largely surveyed using selective and invasive methods, which are mostly limited to commercial species, and restricted to particular areas with favourable conditions. Furthermore, misidentification of species represents a major problem. Here, we investigate the potential of using metabarcoding of environmental DNA (eDNA) obtained directly from seawater samples to account for marine fish biodiversity. This eDNA approach has recently been used successfully in freshwater environments, but never in marine settings. We isolate eDNA from ½-litre seawater samples collected in a temperate marine ecosystem in Denmark. Using next-generation DNA sequencing of PCR amplicons, we obtain eDNA from 15 different fish species, including both important consumption species, as well as species rarely or never recorded by conventional monitoring. We also detect eDNA from a rare vagrant species in the area; European pilchard (<em>Sardina pilchardus</em>). Additionally, we detect four bird species. Records in national databases confirmed the occurrence of all detected species. To investigate the efficiency of the eDNA approach, we compared its performance with 9 methods conventionally used in marine fish surveys. Promisingly, eDNA covered the fish diversity better than or equal to any of the applied conventional methods. Our study demonstrates that even small samples of seawater contain eDNA from a wide range of local fish species. Finally, in order to examine the potential dispersal of eDNA in oceans, we performed an experiment addressing eDNA degradation in seawater, which shows that even small (100-bp) eDNA fragments degrades beyond detectability within days.</p> <p>Although further studies are needed to validate the eDNA approach in varying environmental conditions, our findings provide a strong proof-of-concept with great perspectives for future monitoring of marine biodiversity and resources.</p> </div

Summary of results showing sampling site and panel of fish species recovered by eDNA.

<p>Sampling locality (The Sound, Elsinore, Denmark) for this study with the three sampling sites; 1) open beach, 2) outer pier, 3) inner pier. The 15 different fish species obtained by eDNA in this study are shown with colour codes explaining in which of the three sampling sites they were found. All fish drawings by Susanne Weitemeyer ©.</p

Summary of species-specific eDNA sequences recovered in this study.

<p>All sequences are generated by pyrosequencing using Roche GS FLX 454 platform, except the 5 sequences obtained with species-specific primers (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041732#pone-0041732-t002" target="_blank">Table 2</a>), which are generated by cloning and subsequent Sanger sequencing. All sequences are full-length 100% match to the particular species only, identified by BLAST to the Genbank nucleotide database. Sequences are given without primers.</p

Number of fish species recorded by 9 different conventional survey methods and eDNA at The Sound of Elsinore, Denmark.

<p>Bars show mean number of fish species caught across surveys in 2009, 2010 and 2011 and error bars represent the standard deviation (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041732#pone.0041732.s001" target="_blank">Table S1</a>). The eDNA bar represents the total amount of fish species recorded by this method in 2011. *) Depend heavily on competent experts in fish identification. **) Only possible where seabed conditions allow it.</p

Comparing Ancient DNA Preservation in Petrous Bone and Tooth Cementum

<div><p>Large-scale genomic analyses of ancient human populations have become feasible partly due to refined sampling methods. The inner part of petrous bones and the cementum layer in teeth roots are currently recognized as the best substrates for such research. We present a comparative analysis of DNA preservation in these two substrates obtained from the same human skulls, across a range of different ages and preservation environments. Both substrates display significantly higher endogenous DNA content (average of 16.4% and 40.0% for teeth and petrous bones, respectively) than parietal skull bone (average of 2.2%). Despite sample-to-sample variation, petrous bone overall performs better than tooth cementum (p = 0.001). This difference, however, is driven largely by a cluster of viking skeletons from one particular locality, showing relatively poor molecular tooth preservation (<10% endogenous DNA). In the remaining skeletons there is no systematic difference between the two substrates. A crude preservation (good/bad) applied to each sample prior to DNA-extraction predicted the above/below 10% endogenous DNA threshold in 80% of the cases. Interestingly, we observe signficantly higher levels of cytosine to thymine deamination damage and lower proportions of mitochondrial/nuclear DNA in petrous bone compared to tooth cementum. Lastly, we show that petrous bones from ancient cremated individuals contain no measurable levels of authentic human DNA. Based on these findings we discuss the <i>pros</i> and <i>cons</i> of sampling the different elements.</p></div

Results from eDNA degradation experiment. eDNA concentration in seawater as a function of time for the two fish species; <i>Platichthys flesus</i> (circles) and <i>Gasterosteus aculeatus</i> (triangles), investigated in a 50 l aquarium.

<p>Time points with no detection of eDNA signals are shown in red. The lines show simple exponential decay models, <i>p</i><0.001 (<i>Platichthys flesus</i>) and <i>p</i><0.05 (<i>Gasterosteus aculeatus</i>). Dashed line shows the suggested detection threshold of 25 DNA molecules pr 400 ml seawater. Estimated time for eDNA to degrade beyond the detection threshold was estimated to be 0.9 days for <i>Gasterosteus aculeatus</i> and 6.7 days for <i>Platichthys flesus</i>. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041732#s4" target="_blank">Materials and Methods</a> section.</p

Endogenous DNA content and visual preservation.

<p>A complete overview of the endogenous DNA content and the visual state of preservation (as defined in the method section) for each of the non-cremated samples. Sample names: T = tooth, P = petrous bone, S = parietal (skull) bone. WP, well-preserved. PP, poorly preserved.</p

Environmental DNA and fish density.

<p>Relationship between relative eDNA read frequencies and relative biomass (A) as well as relative abundance (number of individuals) (B). Results are shown for all fish families detected using both methods across all samples. Regression lines are shown, and a linear model (on log transformed data) showed that eDNA reads correlated with biomass (p<0.0001, R² = 0.26) and number of individuals (p<0.0001, R² = 0.24).</p