6. Future perspectives
The advent of eDNA quantification has offered an exciting but as yet untapped future in discovering the complex and dynamic pattern of species interactions. Implementation of eDNA analysis has thus far proved helpful in studying rapid changes in ecosystems (e.g., diversity and species interaction changes due to anthropogenic pressure; DiBattistaet al., 2020) and may also advance our understanding of the effect of habitat fragmentation, sudden natural calamities, or rapid climatic changes (Bartlett et al., 2016). Environmental DNA may even demonstrate utility in assessing how range or phenological shifts via climate change alter PAI. For example, will climate change maintain or dismantle entire networks of integrated species? We envision research into the congruence or discordance of plant flowering time and their pollinators. Certainly, the ease of collecting eDNA is a major advantage to questions requiring successive time series data (e.g., coevolution, or niche separation) and we expect this to be a major avenue for investigation in the near future.
The ease and rapidity of eDNA analysis particularly lends itself to the monitoring of invasive species (Kim et al., 2018), and here too eDNA methodology may illuminate how invasive species change complex species interactions on an ecosystem scale. While it is true that invasive species, at least initially, add to the net biodiversity of a region, will these species also add to species-interactions, weaken specialized species interactions, or break them altogether? Here, eDNA analysis may be especially important for these assessments early during colonization events, when invasive species removal and thus their impact to well-established species interactions, may be circumvented.
 Recent methodological developments to collect and extract environmental RNA (eRNA) might also be leveraged to understand changes of gene expression with physical and biological pressure (functional genomics;Tsuri et al., 2021), with possibilities of expansion into ecological epigenetics, ecosystem health, functional metagenomics, population-level inference, or even the interface of species-species interactions (e.g., Stewart & Taylor, 2020; Veilleux et al., 2021). Unlike eDNA, eRNA can go beyond species and PAI quantification, such as understanding life histories, ages, metabolic activities, physiological conditions and health of interacting organisms. Functional information of a species, population or community and their functional genes can be detected from mRNA profiling, or miRNA for studying the health of organisms. Furthermore, the short persistence time of eRNA and resulting low concentration within the environment may help to avoid false positive results and even potentially provide an estimate for the relative time of eDNA deposition and thus organismal origin (Marshall et al., 2021). However, to date detection methods for eRNA are not yet well established, lack broad validation in the field, and insufficient reference data may raise concern.
7. Conclusions
In the context of global biodiversity decline where ecosystems are under heavy stress and subjected to rapid changes, it is critical to increase our knowledge of species interactions to support the restoration and conservation of ecosystems effectively. Threats to species and ecosystem integrity are often assessed in terms of habitat loss, overharvesting, or over-predation (Kerr & Deguise, 2004). Yet, populations may also decline through successive loss of species interactions (Valiente‐Banuet et al., 2015; Simmons et al., 2020) and studying species in isolation may limit our full understanding of the changes and threats to entire ecosystem of interacting species (Roslin & Majaneva, 2016). In fact, positive and antagonistic interactions synergically work to maintain the stability, health, and function of an ecosystem, demanding a fast, reliable and non-invasive/destructive approach. Currently, eDNA-based methods exhibit accurate information about species-specificity, community dynamics and ecological networks. Although to date there remains a limited number of investigations using eDNA to critically assess and identify PAI, we propose eDNA methods to herald a revolutionary era for studying complex and cryptic ecological links in nature.