2. Methods
In order to understand the state-of-the-art in eDNA to study PAI and enable an effective discussion of this application, we qualitatively reviewed studies incorporating eDNA methods into PAI research (targeted and metabarcoding approaches) by searching the literature published between 2009 to 2021 using Google Scholar (
https://scholar.google.com/) with the following search conditions: 1) “[eDNA] AND [plant-animal interactions]”; 2) “[eDNA] AND [pollination]”; 3) “[pollen metabarcoding]”; 4) “[eDNA] AND [herbivory]”; 5) “[herbivory metabarcoding]”; and 6) “[fecal metabarcoding]”. The content of the top 100 publications on Google Scholar were verified manually. We selected papers that worked on non-destructive eDNA-based methods for representation. The selected publications were then evaluated to understand the potential advantages and limitations of eDNA-based methods in the study of PAI.
3. Why use eDNA-based methods for studying PAI?
Conventional methods such as field observation, histological and biochemical analysis, various camera, malaise or pitfall traps, etc., have proved their utility for identifying and expanding knowledge on PAI across numerous species groups, research questions, and intended outcomes (ecological, or evolutionary fundamental knowledge, agricultural/horticultural production, conservation management and action plans). Yet historically, most studies on PAI generally focus on pairs of species (Herrera and Pellmyr, 2009), and thus the ecologically complex interactions between species groups remain less understood (Luna & Dáttilo, 2021). Indeed, numerous animal species are interconnected with plants, they may co-exist or not, but still have potential impacts on each other across their network (Luna & Dáttilo, 2021). These interactions are dynamic processes and thus their subsequent observation is often difficult using discrete means of data collection.
In direct comparison with conventional methods for example, DNA metabarcoding has greater ability to detect closely related taxa (Macgregor et al., 2019), is time-efficient and cost-effective, whereas the application of conventional methods may be difficult for large-scale sampling. Additionally, conventional methods may be unable to resolve diverse yet morphologically conserved groups (e.g., Nematodes; Derycke et al., 2010), particularly cryptic species (Sheth and Thaker, 2017). Thus, implementation of molecular methods may help us to understand how evolutionary mechanisms shape different PAI and assemble related species. Studying PAI would therefore frequently require sampling methods that provide broad spatiotemporal inference, involving entire species groups.
Indeed, DNA-based methods offer broad output with the capability of identifying multiple PAI simultaneously, and the ease at which DNA is collected and analysed also affords multiple sampling events for an integrative approach (Evans and Kitson, 2020). More direct DNA-based methods (e.g., metabarcoding of gut contents, or bulk samples - contain several organisms from different taxonomic group together, e.g, Kick-Net sampling or insect trap, that amalgamate entire organisms into a single sample, Taberlet et al., 2018) have already proved useful in elucidating complex species and trophic interactions (García-Robledo et al., 2013). For instance, direct DNA analysis from gut content or bulk samples have illuminated different nodes across various food webs, and reconstructed the trophic links in terrestrial (Wirta et al. 2014, 2015a, 2015b, 2016; Gogarten et al., 2020), aquatic (Leray et al., 2012; Leray et al., 2015) and often inaccessible environments, such as deep-sea beds, hydrothermal vents, and cold-seeps (Olsen et al., 2014). Several reviews to date have summarized the history, achievements, and current applications of studying species interaction using direct DNA-based methods across multiple fields (Symondson, 2002; Valentini et al., 2009; Pompanon et al., 2012; Clare, 2014; Kress et al., 2015; Evans et al., 2016). Still direct DNA-based methods with tissue, bulk, and gut content samples can be destructive in nature, sometimes requiring the sacrifice of focal organisms which is not ideal or practical for species of conservation concern. Both conventional and direct DNA based methods further (generally) focus on animal interactions with different plants, whereas the converse (plant interactions with multiple animal species) remain less understood.
Novel sampling techniques such as the collection of DNA from soil, water or air environments (eDNA) offers a method for studying PAI with the potential capability for identifying multiple PAI simultaneously, while also facilitating a conservation-friendly, non-invasive and non-destructive (including non-target taxa) alternative. In fact, advancements including DNA collections the surface of organisms (e.g., DNA from leaf surfaces; Valentin et al., 2020), preventing the scarification or sacrifice of organisms, further highlights the non-destructive advantages of eDNA for elucidating complex PAI