Re-analyses of relationships between size-fractioned eDNA and fish
abundance
We reanalyzed the dataset from a previous study to further examine the
effects of particle size and size fraction on eDNA-based abundance
estimation (Jo et al., 2019). The aforementioned study measured the size
distribution of T. japonicus eDNA derived from mitochondrial and
nuclear DNA. Briefly, rearing water was sequentially filtered using
different pore size filters; the particle size distributions of target
eDNA were compared among rearing temperature, fish biomass in a tank,
and target genes (mitochondrial or nuclear). Detailed information on the
experimental design, water sampling, and molecular analyses can be
accessed in Jo et al. (2019). Here, we performed linear regressions
using the dataset and assessed the variation in the relationship between
eDNA concentration and fish biomass according to eDNA size fraction. The
eDNA samples that were collected a day before the fish removal and
passed through sequential filters with 10, 3, 0.8, and 0.4 μm pore sizes
(i.e., >10, 3–10, 0.8–3, and 0.4–0.8 μm size fractions,
respectively) were compiled. The fish biomass in 200-L tanks ranged from
5.8 to 460.1 g. We then calculated two kinds of cumulative eDNA
concentrations from each size-fractioned eDNA concentration as follows:
(i) Upside-cumulative eDNA (UC) meaning the cumulative eDNA
concentration at all size fractions that can be collected by a given
pore size filter:
・UC>10 µm =
C>10 µm
・UC>3 µm =
C>10 µm + C3–10 µm
・UC>0.8 µm =
C>10 µm + C3–10 µm +
C0.8–3 µm
・UC>0.4 µm =
C>10 µm + C3–10 µm +
C0.8–3 µm + C0.4–0.8 µm
(ii) Downside-cumulative eDNA (DC) meaning the cumulative eDNA
concentration at all size fractions that can be collected by the
smallest pore size filter (0.4 µm) following pre-filtration with a given
pore size filter:
・DC>0.4 µm = C0.4–0.8 µm+ C0.8–3 µm + C3–10 µm +
C>10 µm
・DC0.4–10 µm = C0.4–0.8 µm +
C0.8–3 µm + C3–10 µm
・DC0.4–3 µm = C0.4–0.8 µm +
C0.8–3 µm
・DC0.4–0.8 µm = C0.4–0.8 µm
where CX means the concentration of target eDNA at X µm
size fraction (i.e., size-fractioned eDNA). As described above, we
performed linear regressions of size-fractioned (CX) and
both cumulative eDNA (UCX or DCX)
concentrations (log10-transformed + 1) against fish biomass
(log10-transformed) and estimated their R2values and regression slopes with 95% CIs. Additionally, the relativeR2 values were calculated to simplify the
effect of size fraction on the relationships, where theR2 values at a given size fraction were divided
by the R 2 value at >10 µm size
fraction for the size-fractioned (CX) and
upside-cumulative (UCX) eDNA and at >0.4 µm
size fraction for the downside-cumulative eDNA (DCX)
corresponding to a similar temperature level and marker type.
Moreover, we assessed the effect of eDNA size fraction on theR2 values and slopes of linear regressions,
using the three types of eDNA concentrations and linear mixed models. In
the former models, the R2 values (Fisher’s
z-transformed) were included as the dependent variable, each size
fraction (µm) was included as the fixed effect, and temperature levels
(13, 18, 23, and 28°C) and marker types were included as the random
effects. On the other hand, in the latter models, every kind of eDNA
concentration (CX, UCX, or
DCX; log10-transformed + 1) was included as the
dependent variable, fish biomass (log10-transformed), each size
fraction, and their interaction were included as the fixed effects, and
tank replicates, temperature levels, and marker types as the random
effects. In these analyses, water temperature levels and marker types
were not included as the fixed effects because these factors were not of
interest in this study.