Environmental DNA (eDNA) extracted from water samples has recently shown potential as a valuable source of population genetic information for aquatic macroorganisms. This approach offers several potential advantages compared with conventional tissue‐based methods, including the fact that eDNA sampling is noninvasive and generally more cost‐efficient. Currently, eDNA approaches have been limited to single‐marker studies of mitochondrial DNA (mtDNA), and the relationship between eDNA haplotype composition and true haplotype composition still needs to be thoroughly verified. This will require testing of bioinformatic and statistical software to correct for erroneous sequences, as well as biases and random variation in relative sequence abundances. However, eDNA‐based population genetic methods have far‐reaching potential for both basic and applied research. In this paper, we present a brief overview of the achievements of eDNA‐based population genetics to date, and outline the prospects for future developments in the field, including the estimation of nuclear DNA (nuDNA) variation and epigenetic information. We discuss the challenges associated with eDNA samples as opposed to those of individual tissue samples and assess whether eDNA might offer additional types of information unobtainable with tissue samples. Lastly, we provide recommendations for determining whether an eDNA approach would be a useful and suitable choice in different research settings. We limit our discussion largely to contemporary aquatic systems, but the advantages, challenges, and perspectives can to a large degree be generalized to eDNA studies with a different spatial and temporal focus.

Temporal variation in eDNA signals is increasingly explored for understanding community ecology in aquatic habitats. Seasonal changes have been addressed using eDNA sampling, but very little is known regarding short‐term temporal variation that spans hours to days. To address this, we filtered marine water samples from a single coastal site in Denmark every hour for 32 h. We used metabarcoding to target both fish and broader eukaryote diversity and evaluated temporal changes in this marine community. Results revealed variation in fish species richness (15–27) and eukaryote class richness (35–64) across the 32 h of sampling, and we further evaluated sampling efforts needed to reach different levels of diversity saturation. Relative read frequency data for both fish and eukaryotes indicated a clear diel change in community composition, with different communities detected during daylight versus dark hours. The abundance signals in our data reflected biological variation rather than stochastic variation, since replicates taken at the same hour were more similar to each other than those taken at different hours. Our compositional results indicated a dynamic community, rather than a static pool of eDNA—even across a few hours. The fish data showed a daily pattern of relative species abundances, and the uncoupling of fish and broader eukaryote data suggest that variation in eDNA profiles across a single day can provide valuable information reflecting diel changes, at least for highly mobile organism groups. However, our results also point to several pitfalls in current eDNA experimental design, in which samples are taken over large areas without relative time‐consistency or short‐term replication. Our findings shed new light on short‐term variation in coastal eDNA and have wide implications for experimental study design and for incorporating temporality into project conceptualization for future aquatic biodiversity monitoring.

Over the last two decades, the use of DNA barcodes has transformed our ability to identify and assess life on our planet. Both strengths and weaknesses of the method have been exemplified through thousands of peer-reviewed scientific articles. Given the novel sequencing approaches, currently capable of generating millions of reads at low cost, we reflect on the questions: What will the future bring for DNA barcoding? Will identification of species using short, standardized fragments of DNA stand the test of time? We present reflected opinions of early career biodiversity researchers in the form of a SWOT analysis and discuss answers to these questions.

Environmental DNA (eDNA) provides a promising supplement to traditional sampling methods for population genetic inferences, but current studies have almost entirely focused on short mitochondrial markers. Here, we develop one mitochondrial and one nuclear set of target capture probes for the whale shark (Rhincodon typus) and test them on seawater samples collected in Qatar to investigate the potential of target capture for eDNA‐based population studies. The mitochondrial target capture successfully retrieved ~235× (90× − 352× per base position) coverage of the whale shark mitogenome. Using a minor allele frequency of 5%, we find 29 variable sites throughout the mitogenome, indicative of at least five contributing individuals. We also retrieved numerous mitochondrial reads from an abundant nontarget species, mackerel tuna (Euthynnus affinis), showing a clear relationship between sequence similarity to the capture probes and the number of captured reads. The nuclear target capture probes retrieved only a few reads and polymorphic variants from the whale shark, but we successfully obtained millions of reads and thousands of polymorphic variants with different allele frequencies from E. affinis. We demonstrate that target capture of complete mitochondrial genomes and thousands of nuclear loci is possible from aquatic eDNA samples. Our results highlight that careful probe design, taking into account the range of divergence between target and nontarget sequences as well as presence of nontarget species at the sampling site, is crucial to consider. eDNA sampling coupled with target capture approaches provide an efficient means with which to retrieve population genomic data from aggregating and spawning aquatic species.

Environmental DNA (eDNA) metabarcoding is increasingly being implemented as a non‐invasive and efficient approach for biodiversity research and monitoring across ecosystems. However, accurate detection of species with eDNA requires robust experimental designs as eDNA analysis carries a risk of contamination at every step of the fieldwork and laboratory processes. Several studies focus on rigorous laboratory procedures and processing of sequencing data, but surprisingly, little research investigates the process of background input of DNA in the field. For example, airborne DNA from localities outside the study area could potentially contaminate eDNA samples. Here, we use an experimental setup and eDNA metabarcoding to study the diversity and accumulation of airborne eukaryotic eDNA on exposed surfaces in the field. At two different natural locations, a coastal marine site and a terrestrial grassland site, we placed open containers each filled with 0.5 liters of water, which was then sampled at eight successive time points after exposure to the surroundings. We found an accumulation of detected species richness in the samples, which reached its maximum at the end of the experiment, 24 h after exposure. This result was consistent across both sites and across two markers (COI for eukaryotes and 12S for vertebrates). While many of the detected species were contaminants commonly found in eDNA studies, we also detected several other eukaryotic taxa. Most notable were metazoan species such as birds, fish, and insects, likely originating from airborne transport of eDNA. We also found that increasing the number of PCR cycles tended to have a positive impact on richness for the unfiltered reads but a negative impact on the richness after bioinformatic filtering. Our results add to the sparse evidence that metazoan eDNA can be transported by air, which have wide implications for eDNA research and calls for increased implementation of field control samples.