The relation between eDNA concentration and biomass, and the temporal changes of eDNA concentration in <i>Hydrilla verticillata</i> and <i>Egeria densa</i> under single-species conditions.

<p>The means of eDNA concentration were calculated for each water bag (A, B). The error bars indicate ±1 standard deviation. Dotted lines (C, F), dashed lines (D, G), and solid lines (E, H) show the three conditions with low, middle, and high biomass, respectively. Six symbols in each graph indicate the six replicates for each condition. Note that biomass of some samples with a 4 cm-fragment is larger than those with two 4 cm-fragments.</p

The location of 21 ponds in the field survey.

<p>The numbers correspond to the site IDs in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156217#pone.0156217.t001" target="_blank">Table 1</a>. Closed and open circles indicate that the presence of <i>Hydrilla verticillata</i> that was or was not reported in the past, respectively. The number in parentheses shows the eDNA concentration (copies L^-1). This map was constructed using Fundamental Geospatial Data published by the Geographical Survey Institute, Japan.</p

The relationship between eDNA concentration and biomass, and the temporal changes of eDNA concentration in <i>Hydrilla verticillata</i> and <i>Egeria densa</i> under two-species conditions.

<p>The mean eDNA concentrations were calculated for each water bag (A, B). The error bars indicate ±1 standard deviation. Dotted lines (C,H), dashed lines (D, G), and solid lines (E, F) show the three conditions with low, middle, and high biomass, respectively. Six symbols in each graph indicate the six replicates for each condition.</p

Results of generalized linear mixed models analyzing the effects of biomass and the number of experimental days on the eDNA concentration in the aquarium experiments for <i>Hydrilla verticillata</i> and <i>Egeria densa</i>.

<p>The results of random effect were shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156217#pone.0156217.s004" target="_blank">S2 Table</a>.</p

The correlation coefficients among six eDNA sequential samples of the temporal survey.

<p>The correlation coefficients among six eDNA sequential samples of the temporal survey.</p

The schematic drawing of the sampling point for temporal survey and the results.

<p>(a) The schematic drawing of the sampling point for temporal survey conducted at the pier of Maizuru Fisheries Station, Kyoto University. The surface and sea floor water samples were collected at three (A, B, and C) points where the water depth is 4, 6, and 8 m, respectively. (b) The temporal change of observed numbers of <i>C</i>. <i>pacifica</i> in the visual survey and the eDNA concentration in each surface sample. (c) Temporal change in observed numbers of <i>C</i>. <i>pacifica</i> in the visual survey and eDNA concentration in each sea floor sample. Note that the observed numbers of individuals were presented as the average of 10 days and that the same observation data were presented in (b) and (c) for better visibility.</p

The results of the spatial survey.

<p>(a) The estimated abundance of <i>C</i>. <i>pacifica</i> at the day of survey based on the visual survey. (b) The concentration of <i>C</i>. <i>pacifica</i> eDNA at the surface and (c) on the sea floor.</p

The results of the tank experiments.

<p>(a) Time dependent change of eDNA concentration (copies L^-1) in the tank experiment. (b) Relative eDNA concentration, i.e., the concentration at time <i>t</i> divided by the initial concentration in the tank, represents the combined exponential degradation in all three tanks. The exponential regression curve for the eDNA degradation was y = 0.998e^-0.0335x (where x and y mean time in hour and relative DNA concentration at time x, respectively).</p

The comparison between eDNA concentrations of surface and sea floor samples.

<p>The eDNA concentrations in the sea floor were significantly higher than that of surface samples (Wilcoxon signed rank test, <i>P</i> = 0.00051).</p

Observed fish biomass using echo sounder.

<p>Vertical bar on the cruise track (gray line) indicates local s_a values (i.e., fish biomass observed using quantitative echo sounder), which is the integrated s_v of a water column with a cross-sectional area of 1 m². This figure is depicted according to s_a extracted every 80-m intervals. Note that this figure shows a summary of field observation using echo sounder. We used s_v values rather than s_a values as index of fish biomass in regression analyses (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149786#pone.0149786.s001" target="_blank">S1 Fig</a>).</p