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.