Introduction
Environmental DNA (eDNA) is the total pool of DNA isolated from
environmental samples (Pawlowski et al., 2020). Macroorganisms such as
fish are believed to produce eDNA in the form of epidermis, mucus, and
feces into their outer environments (Barnes & Turner, 2016;
Rodriguez-Ezpeleta et al., 2021). Recently, the eDNA-based monitoring of
species distribution, abundance, and composition has been developed to
target a variety of taxa in aquatic and terrestrial environments (e.g.,
Ficetola et al., 2008; Yamanaka & Minamoto, 2016; Ushio et al., 2018;
Valentin et al., 2020). The target eDNA, which is detected by a
polymerase chain reaction (PCR), allows to evaluate species presence and
relative abundance, making eDNA analysis a non-disruptive,
cost-effective, and high-sensitivity monitoring tool compared with
traditional capture-based surveys (e.g., Thomsen et al., 2012; Miya et
al., 2015; Jo et al., 2020a; Lopes et al., 2021). Although there is
potential for eDNA-based biomonitoring to become an essential approach
for biodiversity and ecosystem conservation, knowledge on the production
source and persistence state of eDNA, as well as its transport and
degradation, is lacking (Barnes & Turner, 2016; Harrison et al., 2019;
Jo et al., 2021a). The lack of or scarcity in understanding the
characteristics and dynamics of eDNA causes uncertainty in eDNA-based
species inferences, often resulting in ecological interpretation
difficulty (Hansen et al., 2018).
Specifically, the estimation of species abundance is a significant
challenge in eDNA analysis (Roussel et al., 2015; Harper et al., 2018).
Although the eDNA concentration shows a positive correlation with target
species abundance for multiple taxa (Takahara et al., 2012; Pilliod et
al., 2013; Uthicke et al., 2018; Wu et al., 2018; Ponce et al., 2021),
in natural environments, these relationships are weakened compared with
those in controlled laboratory conditions (Yates et al., 2019). Numerous
environmental factors complicate the diffusion, retention, and
degradation of eDNA in natural environments (e.g., temperature
fluctuation, water chemistry, and hydrogeographic conditions), which
renders eDNA quantification and its relationship with species abundance
in the field unclear. A few studies previously addressed this challenge
by correcting the eDNA concentration with a river flow (e.g., eDNA
flux), mathematically modeling the processes of eDNA transport and
degradation (Carraro et al., 2018; Levi et al., 2019; Fukaya et al.,
2021). Nevertheless, the practical application of eDNA-based abundance
estimation with high reliability in natural environments is still in its
infancy.
Given that eDNA degradation significantly depends on the complex
interactions between the eDNA state and multiple abiotic factors (Jo &
Minamoto, 2021), the cellular and molecular structures of eDNA function
as important factors to better understand the relationship between eDNA
concentration and species abundance. Furthermore, a recent review
discussed the benefit of targeting various types of eDNA beyond short
mitochondrial DNA fragments (nuclear eDNA, longer eDNA fragments, and
larger eDNA particles) for reliable species detection and accurate
abundance estimation (Jo et al., 2021). Larger eDNA particles (eDNA
detected in more significant size fractions) can more frequently possess
longer eDNA fragments (Jo et al., 2020b) that persist for a shorter
duration in water because of the inflow of degraded eDNA from larger to
smaller size fractions (Jo et al., 2019). This finding suggests that the
larger eDNA particles, likely derived from intra-cellular DNA (e.g.,
cell and tissue fragments), are released and assumed to be fresher and
more precise biological signals than their smaller counterparts (e.g.,
organelle and extra-cellular DNA) (Jo et al., 2019; 2021). Therefore,
the larger eDNA particles collected using a larger pore filter may
accurately imitate the neighboring species abundance.
Conversely, we suspected that the larger eDNA particles do not
continuously improve the species abundance estimation accuracy. Although
much of macrobial eDNA is concentrated in a 1–10-µm size fraction
(Turner et al., 2014; Jo et al., 2019; Zhao et al., 2021), it exists in
water at various size fractions (<0.2 to >180 μm
in diameter). Among them, eDNA particles collected by a filter with a
considerable pore size (tens or hundreds of micrometers, e.g., tissue
aggregation and scale) are clumped and distributed heterogeneously in
water (Furlan et al., 2016). The stochasticity of retrieving such
“huge” eDNA particles in a water sample could worsen the relationship
between eDNA concentration and species abundance. Hence, the eDNA
particle size and size fraction may associate nonlinearly with the
relationship between eDNA concentration and species abundance.
Additionally, it is believed that longer eDNA fragments are degraded
rapidly (e.g., Jo et al., 2017; Shogren et al., 2018), whereas agreement
is not usually reached in previous studies (e.g., Bylemans et al.,
2018). The longer eDNA fragments can persist for a shorter duration in
water owing to its rapid degradation (Jo et al., 2017). Given its
characteristics, the longer eDNA fragments also reflect more recent
biological information than the shorter fragments. However, compared
with shorter fragments, longer eDNA fragments are expected to be less
frequently detected, specifically in natural environments (Jo et al.,
2021b). The stochasticity and heterogeneity of longer eDNA fragments
lead to higher variances of eDNA yields across replicated samplings,
showing their poor applicability for accurate abundance estimation.
Altogether, it should be evaluated how target marker length and particle
size of target eDNA could influence the relationship between eDNA
concentration and species abundance.
This study compared the relationships between eDNA concentration and
species abundance among different eDNA size fractions and target marker
lengths and proposed the characteristics of eDNA suitable for accurate
abundance estimation. To address the issue, an aquarium experiment was
performed using zebrafish (Danio rerio ). We reared the fish in
experimental tanks with different individual densities, filtered the
rearing water samples using other pore size filters, and quantified eDNA
concentrations targeting different fragment lengths. In this study, we
focused on the R2 values and slopes of the
linear regressions as the parameters showing the relationship between
eDNA concentration and species abundance; the former parameter
represented the accuracy of the relationship, and the latter the
sensitivity of eDNA concentration in response to changes in species
abundance (Eichmiller et al., 2016). Furthermore, we reanalyzed the
dataset from a previous study that measured the particle size
distribution of Japanese jack mackerel (Trachurus japonicus ) eDNA
(Jo et al., 2019), and assessed the importance of the eDNA particle size
and size fraction for the abundance estimation performance.