Discussion
Dynamics of the state, transport, and fate of eDNA can have serious
implications for determining species presence in molecular detection
studies and can result in false positive or false negative results
(Darling and Mahon, 2011; Schultz & Lance, 2015). Such errors can have
unwelcome consequences when management actions are implemented or
withheld as a result (Darling and Mahon, 2011). For example, failing to
detect a nascent invasive species’ population may delay rapid response
and eradication efforts, or failure to detect the presence of a rare
native species may result in a failure to implement conservation
measures. If eDNA is to be utilized to detect terrestrial species, the
ecology of terrestrially deposited aboveground eDNA must be carefully
documented so that sampling and survey methods maximize detection
probability and avoid false positive results. To this end, we explored
the ecology of aboveground terrestrial eDNA, which is elsewhere poorly
documented. To accomplish this, we made a slurry from Halyomorpha
halys excrement to allow us to ourselves deposit eDNA on surfaces for
experimentation. In doing so, our results were not contingent onH. halys specimens in the field or its behavior, as its only
contribution to the study was eDNA in various states (i.e.
intracellular, intraorganellar, and extracellular). We found no
significant difference in the amount of intracellular eDNA collected
among filter pore sizes from 1 to 10 µm, and while extracellular eDNA
may be collected with 0.2 µm filters, its rapid degradation from
aboveground surface substrates make it an unreliable source of eDNA to
determine species presence. Furthermore, rainfall has a dramatic
influence on the persistence of aboveground terrestrial eDNA as even
mild rainfall will remove most eDNA that otherwise could have been
available for collection. These results provide three critical insights
for using eDNA in surveys of aboveground terrestrial ecosystems.
First, while we did not see a significant difference between filter pore
sizes in our experiments, there was a trend towards an inverse
relationship between eDNA copy number and filter pore size, potentially
due to greater capture of free-floating nuclei in addition to intact
cells (insect nuclei are ~4–10 µm in diameter; Price &
Ratcliffe, 1974). The increased capture of nuclei (or mitochondria if
the target is a mtDNA locus) could extend the detectability window of
this source of DNA since the persistence of organelles within the
environment would be in addition to the persistence time of intact
cells. This outcome may or may not be desirable if the goal of the
survey is to detect species that were recently present within a
terrestrial ecosystem. There is however a more practical consideration
to the choice of pore size for sampling terrestrial species. Under field
conditions, smaller pore sizes restrict the volume of solution that can
be filtered before filter saturation, which likely decreases the
sensitivity of the survey or requires a greater number of samples (and
expense) to achieve similar levels of detection (Schultz & Lance,
2015). Valentin et al. (2018, 2020) found that 10 µm filters were more
practical for field sampling of aboveground terrestrial eDNA within
agricultural and forested ecosystems than smaller pore sizes, as greater
volumes of solution could be filtered before filter saturation occurred.
Our results echo Turner et al. (2014) in that the choice of filter pore
size will reflect a trade-off between the eDNA yield per volume filtered
and total filtering capacity, which can affect detection of rare species
(Schultz & Lance, 2015). Given that animal cells generally average
10-20 µm in diameter (Price & Ratcliffe, 1974; Guertin & Sabatini,
2005), our results should be highly generalizable to numerous
invertebrate and vertebrate species of interest.
Second, our results suggest that while collecting aboveground
terrestrial eDNA, emphasis should be placed on intracellular rather than
extracellular eDNA due to the extremely short retention time of
extracellular eDNA in terrestrial settings. Interestingly, this result
ran counter to our initial expectation. We assumed the fragment size of
our target DNA region (i.e., 96 bp of ITS1 nuclear DNA) was sufficiently
small that it would be retained within the environment for an
exceedingly long time, much like in soils (Andersen et al., 2012), thus
being a concern when estimating rates of false positives (i.e. for
recent presence) during eDNA surveys. Rather, the brief persistence of
extracellular eDNA within aboveground terrestrial environments means it
is likely to be sufficiently degraded prior to its collection from the
field. However, high-frequency surveys targeting extracellular eDNA may
be possible for discerning occupancy of a species when fine temporal
grain sampling is the survey objective (surveys that track occupancy
over days or weeks). Ultimately, the specific research objective will
dictate whether intracellular or extracellular eDNA is the more suitable
target DNA state.
The decay rates of eDNA we document may be accelerated or decelerated
depending on seasonality and location. Our study was conducted in
mid-summer at temperate latitude (40.49° latitude), which dictated UV
exposure over the course of a day post-deposition. Had these experiments
been conducted in a more equatorial region, then eDNA may have decayed
more rapidly due to elevated solar radiation levels. Perhaps more
interestingly, we observed substantial degradation of intracellular eDNA
in full shade, which we assumed would have resulted in a slower decay
rate than what we observed. This result could be due to reflected UV
(i.e. , albedo) (Lenoble, 2000) indicating that environments with
a high albedo may see decay rates closer to full solar exposure
regardless of whether they are shaded (J. Turner & Parisi, 2018).
Environments with very high albedo (i.e. snow-covered environments; J.
Turner & Parisi, 2018) may see higher than expected degradation rates
if estimating decay solely from UV exposure, or assuming low levels of
UV exposure due to latitudinal position and/or weather, and not
accounting for albedo. While we did not record temperature data, there
remains a question of how temperature interacts with UV to determine
terrestrial aboveground eDNA decay rates. Several studies have
documented the effect of temperature on eDNA decay, though to our
knowledge all such studies took place within aquatic environments
(Eichmiller, Best, & Sorensen, 2016; Jo, Murakami, Yamamoto, Masuda, et
al., 2019; Tsuji, Ushio, Sakurai, Minamoto, et al., 2017; Strickler et
al., 2015). eDNA within aquatic ecosystems is largely suspected to
degrade from microbial and enzymatic activity (Eichmiller et al., 2016;
Jo et al., 2019; Tsuji et al., 2017; Strickler et al., 2015), which is
unlikely to be as dominant a degradation force in aboveground
terrestrial ecosystems as desiccation of cells is rapid. Nonetheless,
differences in ambient temperature and humidity are a clear next step in
understanding the ecology of aboveground terrestrial eDNA, especially as
they interact with UV exposure. Seasonal and locational differences in
UV exposure deserve further study as they will affect decisions for
deployment such as the sampling window and the frequency of site
visitations to carry out terrestrial eDNA surveys.
Finally, even a small amount of rain or mist drastically reduces the
quantity of eDNA present on aboveground terrestrial surfaces. Without
accounting for weather preceding an eDNA survey, the results of such
surveys will likely produce an abundance of false negative results. We
found eDNA was better retained on textured vegetation surfaces over
smooth ones during mild weather conditions (i.e. misty rain). Yet, no
matter what the intensity of rain, so long as a sufficient quantity fell
(220 ml in our trials), a near-complete removal of eDNA results. This
result is corroborated by the findings of Staley et al. (2018), who
found that eDNA derived from aboveground terrestrial species can be
sampled within nearby waterways right after heavy rainfall events. eDNA
removed from surfaces due to rain will thus also make its way into the
soil, which can then be used to collect eDNA derived from aboveground
terrestrial species (e.g. Buxton et al., 2018; Kucherenko et al., 2018;
Leempoel et al., 2019; Sales et al., 2019; Walker et al., 2017; Staley,
Chuong, Hill, Grabuski, et al., 2018). However, the life cycle of eDNA
as it moves from aboveground vegetation into the soil column, and even
deep into the subsoil (Andersen et al. 2012), remains poorly understood
and warrants further exploration of the transport of eDNA through the
soil column.
Here we report novel insights into the ecology of aboveground
terrestrial eDNA and highlight several dynamics that are key to
designing and deploying a terrestrial eDNA survey. By better
understanding these processes, surveyors can account for environmental
influences, such as rainfall and UV on detection dynamics, to develop
best practice approaches that mitigate erroneous results from
terrestrial eDNA surveys. When combined with laboratory best practices,
like multi-level controls (Harper, Buxton, Reese, Bruce et al., 2019),
such efforts allow for the development of robust survey frameworks for
species-specific and community-level terrestrial eDNA surveys.