Environmental DNA metabarcoding reveals temporal dynamics but functional stability of arthropod communities in cattle dung.

Terrestrial invertebrates are highly important for the decomposition of dung from large mammals. Mammal dung has been present in many of Earth's ecosystems for millions of years, enabling the evolution of a broad diversity of dung-associated invertebrates that process various components of the dung. Today, large herbivorous mammals are increasingly introduced to ecosystems with the aim of restoring the ecological functions formerly provided by their extinct counterparts. However, we still know little about the ecosystem functions and nutrient flows in these rewilded ecosystems, including the dynamics of dung decomposition. In fact, the succession of insect communities in dung is an area of limited research attention also outside a rewilding context. In this study, we use environmental DNA metabarcoding of dung from rewilded Galloway cattle in an experimental set-up to investigate invertebrate communities and functional dynamics over a time span of 53 days, starting from the time of deposition. We find a strong signal of successional change in community composition, including for the species that are directly dependent on dung as a resource. While several of these species were detected consistently across the sampling period, others appeared confined to either early or late successional stages. We believe that this is indicative of evolutionary adaptation to a highly dynamic resource, with species showing niche partitioning on a temporal scale. However, our results show consistently high species diversity within the functional groups that are directly dependent on dung. Our findings of such redundancy suggest functional stability of the dung-associated invertebrate community, with several species ready to fill vacant niches if other species disappear. Importantly, this might also buffer the ecosystem functions related to dung decomposition against environmental change. Interestingly, alpha diversity peaked after approximately 20-25 days in both meadow and pasture habitats, and did not decrease substantially during the experimental period, probably due to preservation of eDNA in the dung after the disappearance of visiting invertebrates, and from detection of tissue remains and cryptic life stages.

A vision for global eDNA-based monitoring in a changing world.

Environmental DNA (eDNA) has opened promising avenues for establishing standardized, cost-efficient monitoring of biodiversity. However, comprehensive and systematic implementation is urgently needed to address the current biodiversity crisis. Here, we envision a global eDNA biomonitoring scheme, which could potentially revolutionize the understanding and conservation of life on Earth.

Contrasting seasonal patterns in diet and dung-associated invertebrates of feral cattle and horses in a rewilding area.

Trophic rewilding is increasingly applied in restoration efforts, with the aim of reintroducing the ecological functions provided by large-bodied mammals and thereby promote self-regulating, biodiverse ecosystems. However, empirical evidence for the effects of megafauna introductions on the abundance and richness of other organisms such as plants and invertebrates, and the mechanisms involved still need strengthening. In this study, we use environmental DNA (eDNA) metabarcoding of dung from co-existing feral cattle and horses to assess the seasonal variation in plant diet and dung-associated arthropods and nematodes. We found consistently high diet richness of horses, with low seasonal variability, while the generally lower dietary diversity of cattle increased substantially during summer. Intriguingly, season-specific diets differed, with a greater proportion of trees in the horses' diet during winter, where cattle relied more on shrubs. Graminoids were predominantly found in the diet of horses, but were generally underrepresented compared to previous studies, possibly due to the high prevalence of forbs in the study area. Dung-associated arthropod richness was higher for cattle, largely due to a high richness of flies during summer. Several species of dung-associated arthropods were found primarily in dung from one of the two herbivores, and our data confirmed known patterns of seasonal activity. Nematode richness was constantly higher for horses, and nematode communities were markedly different between the two species. Our results demonstrate complementary effects of cattle and horses through diet differences and dung-associated invertebrate communities, enhancing our understanding of large herbivore effects on vegetation and associated biodiversity. These results are directly applicable for decision-making in rewilding projects, suggesting biodiversity-benefits by inclusion of functionally different herbivores.

Individual haplotyping of whale sharks from seawater environmental DNA.

Population genetic data can provide valuable information on the demography of a species. For rare and elusive marine megafauna, samples for generating the data are traditionally obtained from tissue biopsies, which can be logistically difficult and expensive to collect and require invasive sampling techniques. Analysis of environmental DNA (eDNA) offers an alternative, minimally invasive approach to provide important genetic information. Although eDNA approaches have been studied extensively for species detection and biodiversity monitoring in metabarcoding studies, the potential for the technique to address population-level questions remains largely unexplored. Here, we applied "eDNA haplotyping" to obtain estimates of the intraspecific genetic diversity of a whale shark (Rhincodon typus) aggregation at Ningaloo reef, Australia. Over 2 weeks, we collected seawater samples directly behind individual sharks prior to taking a tissue biopsy sample from the same animal. Our data showed a 100% match between mtDNA sequences recovered in the eDNA and tissue sample for all 28 individuals sampled. In the seawater samples, >97% of all reads were assigned to six dominant haplotypes, and a clear dominant signal (~99% of sample reads) was recovered in each sample. Our study demonstrates accurate individual-level haplotyping from seawater eDNA. When DNA from one individual clearly dominates each eDNA sample, it provides many of the same opportunities for population genetic analyses as a tissue sample, potentially removing the need for tissue sampling. Our results show that eDNA approaches for population-level analyses have the potential to supply critical demographic data for the conservation and management of marine megafauna.

Environmental DNA metabarcoding of cow dung reveals taxonomic and functional diversity of invertebrate assemblages.

Insects and other terrestrial invertebrates are declining in species richness and abundance. This includes the invertebrates associated with herbivore dung, which have been negatively affected by grazing abandonment and the progressive loss of large herbivores since the Late Pleistocene. Importantly, traditional monitoring of these invertebrates is time-consuming and requires considerable taxonomic expertise, which is becoming increasingly scarce. In this study, we investigated the potential of environmental DNA (eDNA) metabarcoding of cow dung samples for biomonitoring of dung-associated invertebrates. From eight cowpats we recovered eDNA from 12 orders, 29 families, and at least 54 species of invertebrates (mostly insects), representing several functional groups. Furthermore, species compositions differed between the three sampled habitats of dry grassland, meadow, and forest. These differences were in accordance with the species' ecology; for instance, several species known to be associated with humid conditions or lower temperatures were found only in the forest habitat. We discuss potential caveats of the method, as well as directions for future study and perspectives for implementation in research and monitoring.

Genome-scale target capture of mitochondrial and nuclear environmental DNA from water samples.

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.

Population-level inferences from environmental DNA-Current status and future perspectives.

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.

Using vertebrate environmental DNA from seawater in biomonitoring of marine habitats.

Conservation and management of marine biodiversity depends on biomonitoring of marine habitats, but current approaches are resource-intensive and require different approaches for different organisms. Environmental DNA (eDNA) extracted from water samples is an efficient and versatile approach to detecting aquatic animals. In the ocean, eDNA composition reflects local fauna at fine spatial scales, but little is known about the effectiveness of eDNA-based monitoring of marine communities at larger scales. We investigated the potential of eDNA to characterize and distinguish marine communities at large spatial scales by comparing vertebrate species composition among marine habitats in Qatar, the Arabian Gulf (also known as the Persian Gulf), based on eDNA metabarcoding of seawater samples. We conducted species accumulation analyses to estimate how much of the vertebrate diversity we detected. We obtained eDNA sequences from a diverse assemblage of marine vertebrates, spanning 191 taxa in 73 families. These included rare and endangered species and covered 36% of the bony fish genera previously recorded in the Gulf. Sites of similar habitat type were also similar in eDNA composition. The species accumulation analyses showed that the number of sample replicates was insufficient for some sampling sites but suggested that a few hundred eDNA samples could potentially capture >90% of the marine vertebrate diversity in the study area. Our results confirm that seawater samples contain habitat-characteristic molecular signatures and that eDNA monitoring can efficiently cover vertebrate diversity at scales relevant to national and regional conservation and management.

ADN Ambiental de Vertebrados Tomado del Agua Marina para Realizar Biomonitoreos de los Hábitats Marinos Resumen La conservación y el manejo de la biodiversidad marina depende del biomonitoreo de los hábitats marinos, pero las estrategias actuales requieren de muchos recursos y de diferentes estrategias para diferentes organismos. El ADN ambiental (ADNa) extraído de muestras de agua es una estrategia eficiente y versátil para detectar animales acuáticos. En el océano, la composición del ADNa refleja la fauna local a escalas espaciales finas, pero se sabe poco sobre la efectividad del monitoreo basado en el ADNa de las comunidades marinas a grandes escalas. Investigamos el potencial del ADNa para caracterizar y distinguir las comunidades marinas a escalas espaciales grandes mediante una comparación de la composición de especies de vertebrados entre los hábitats marinos de Qatar, en el Golfo Arábigo (también conocido como el Golfo Persa), con base en el meta-código de barras del ADNa extraído de muestras de agua de mar. Realizamos análisis de acumulación de especies para estimar cuánta de la diversidad de vertebrados logramos detectar. Obtuvimos secuencias de ADNa de diversos ensamblajes de vertebrados marinos, los cuales abarcaron 191 taxones de 73 familias. Estos taxones incluyeron a especies raras y en peligro de extinción y cubrieron el 36% de los géneros de peces óseos previamente registrados en el golfo. Los sitios con tipos similares de hábitat también fueron similares en cuanto a la composición del ADNa. Los análisis de acumulación de especies mostraron que el número de réplicas de muestras fue insuficiente para algunos sitios de muestreo, pero sugieren que unos cientos de muestras de ADNa podrían capturar potencialmente >90% de la diversidad de vertebrados marinos en el área de estudio. Nuestros resultados confirman que las muestras de agua marina contienen firmas moleculares características del hábitat y que el monitoreo de ADNa puede cubrir eficientemente la diversidad de vertebrados a escalas relevantes para la conservación y el manejo nacional y regional.

Environmental DNA from Seawater Samples Correlate with Trawl Catches of Subarctic, Deepwater Fishes.

Remote polar and deepwater fish faunas are under pressure from ongoing climate change and increasing fishing effort. However, these fish communities are difficult to monitor for logistic and financial reasons. Currently, monitoring of marine fishes largely relies on invasive techniques such as bottom trawling, and on official reporting of global catches, which can be unreliable. Thus, there is need for alternative and non-invasive techniques for qualitative and quantitative oceanic fish surveys. Here we report environmental DNA (eDNA) metabarcoding of seawater samples from continental slope depths in Southwest Greenland. We collected seawater samples at depths of 188-918 m and compared seawater eDNA to catch data from trawling. We used Illumina sequencing of PCR products to demonstrate that eDNA reads show equivalence to fishing catch data obtained from trawling. Twenty-six families were found with both trawling and eDNA, while three families were found only with eDNA and two families were found only with trawling. Key commercial fish species for Greenland were the most abundant species in both eDNA reads and biomass catch, and interpolation of eDNA abundances between sampling sites showed good correspondence with catch sizes. Environmental DNA sequence reads from the fish assemblages correlated with biomass and abundance data obtained from trawling. Interestingly, the Greenland shark (Somniosus microcephalus) showed high abundance of eDNA reads despite only a single specimen being caught, demonstrating the relevance of the eDNA approach for large species that can probably avoid bottom trawls in most cases. Quantitative detection of marine fish using eDNA remains to be tested further to ascertain whether this technique is able to yield credible results for routine application in fisheries. Nevertheless, our study demonstrates that eDNA reads can be used as a qualitative and quantitative proxy for marine fish assemblages in deepwater oceanic habitats. This relates directly to applied fisheries as well as to monitoring effects of ongoing climate change on marine biodiversity-especially in polar ecosystems.

Population characteristics of a large whale shark aggregation inferred from seawater environmental DNA.

Population genetics is essential for understanding and managing marine ecosystems, but sampling remains challenging. We demonstrate that high-throughput sequencing of seawater environmental DNA can provide useful estimates of genetic diversity in a whale shark (Rhincodon typus) aggregation. We recover similar mitochondrial haplotype frequencies in seawater compared to tissue samples, reliably placing the studied aggregation in a global genetic context and expanding the applications of environmental DNA to encompass population genetics of aquatic organisms.