Ancient environmental DNA (aeDNA) from lake sediments has yielded remarkable insights for the reconstruction of past ecosystems, including suggestions of late survival of extinct species. However, translocation and lateral inflow of DNA in sediments can potentially distort the stratigraphic signal of the DNA. Using three different approaches on two short lake sediment cores of the Yamal peninsula, West Siberia, with ages spanning only the past hundreds of years, we detect DNA and identified mitochondrial genomes of multiple mammoth and woolly rhinoceros individuals—both species that have been extinct for thousands of years on the mainland. The occurrence of clearly identifiable aeDNA of extinct Pleistocene megafauna (e.g. >400 K reads in one core) throughout these two short subsurface cores, along with specificities of sedimentology and dating, confirm that processes acting on regional scales, such as extensive permafrost thawing, can influence the aeDNA record and should be accounted for in aeDNA paleoecology.

The woolly rhinoceros (Coelodonta antiquitatis) is an iconic species of the Eurasian Pleistocene megafauna, which was abundant in Eurasia in the Pleistocene until its demise beginning approximately 10 000 years ago. Despite the early recovery of several specimens from well-known European archaeological sites, including its type specimen (Blumenbach 1799), no genomes of European populations were available so far, and all available genomic data originated exclusively from Siberian populations. Using coprolites of cave hyenas (Crocuta crocuta spelea) recovered from Middle Palaeolithic layers of two caves in Germany (Bockstein-Loch and Hohlenstein-Stadel), we isolated and enriched predator and prey DNA to assemble the first European woolly rhinoceros mitogenomes, in addition to cave hyena mitogenomes. Both coprolite samples produced copious sequences assigned to C. crocuta (27% and 59% mitogenome coverage, respectively) and woolly rhinoceros (Coelodonta antiquitatis; 27% and 81% coverage, respectively). The sequences suggested considerable DNA degradation, which may limit the conclusions to be drawn; however, the mitogenomes of European woolly rhinoceros are genetically distinct from the Siberian woolly rhinoceros, and analyses of the more complete mitogenome suggest a split of the populations potentially coinciding with the earliest fossil records of woolly rhinoceros in Europe.

In conservation science, observation-based methods are generally applied to wildlife monitoring. While useful, such approaches are often restricted to well-characterized and conspicuous species. Environmental DNA (eDNA) can complement observation-dependent surveys, as sample collection is generally less labor-intensive. Furthermore, eDNA can be used to simultaneously detect multiple taxa in various habitats. Most eDNA applications rely on short PCR product-based meta-barcoding approaches. However, such approaches may be less robust when applied to genetically uncharacterized species. Hybridization capture techniques while less sensitive than metabarcoding, can identifying divergent sequences, especially those obtained from highly degraded DNA. To assess eDNA based methods for surveillance in a conservation context, we collected samples during the dry season from randomly selected waterholes from a protected area in eastern Cambodia. We applied both hybridization capture enrichment and metabarcoding targeting mammal mitogenomes to water (N = 46) and sediment (N = 10) samples. Seventeen species were detected, including 11 mammals, three amphibians, two reptiles, and one bird. Six species overlapped between the two applied methods. Seven species were hybridization capture-specific detections, and four were metabarcoding-specific. Metabarcoding was more sensitive to abundant or large body-size species while hybridization capture provided more mitogenomic information. While both methods have some advantages over observational approaches, combining them may improve the sensitivity, number of species detected and amount of genetic information obtained from eDNA. We demonstrate that eDNA from tropical forest waterholes can be used to determine the presence of wildlife that may be difficult to detect using other observational approaches.

Climate change has a major impact on arctic and boreal terrestrial ecosystems as warming leads to northward treeline shifts, inducing consequences for heterotrophic organisms associated with the plant taxa. To unravel ecological dependencies, we address how long-term climatic changes have shaped the palaeo-ecosystems at selected sites in Siberia. We investigated sedimentary ancient DNA from five lakes spanning the last 47,000 years, using the ITS1 marker for fungi and the chloroplast P6 loop marker for vegetation metabarcoding. After bioinformatic processing with the OBItools pipeline, we obtained 706 unique fungal operational taxonomic units (OTUs) and 243 amplicon sequence variants (ASVs) for the plants. We show higher OTU numbers in dry forest tundra as well as boreal forests compared to wet southern tundra. The most abundant fungal taxa in our dataset are Pseudeurotiaceae, Mortierella, Sordariomyceta, Exophiala, Oidiodendron, Protoventuria, Candida vartiovaarae, Pseudeurotium, Gryganskiella fimbricystis, and Trichosporiella cerebriformis. The overall fungal composition is explained by the plant composition as revealed by redundancy analysis. The fungal functional groups show antagonistic relationships in their climate susceptibility. The advance of woody taxa in response to past warming led to an increase in the abundance of mycorrhizae, lichens, and parasites, while yeast and saprotroph distribution declined. We also show co-occurrences between Salicaceae, Larix, and Alnus and their associated pathogens and detect higher mycorrhizal fungus diversity with the presence of Pinaceae. Under future warming, we can expect feedback between fungus compositional and plant diversity changes which will affect forest advance and stability in arctic regions.

ABSTRACT Terrestrial mammals shape their ecosystems, and mammalian community assemblages can be important indicators of ecosystem functioning and ecosystem changes over time. Numerous taxa of terrestrial mammals are currently threatened by habitat loss and face displacement to new geographical areas or systems to which they are less suited and where they may affect the original communities. Understanding past ecosystem changes is important for predicting future responses of species assemblages to changes in their environments. Thus, ecological and evolutionary history, as well as adaptive capacity, are important predictors of future population viability. Genomic and metagenomic approaches using environmental or ancient DNA offer a wealth of information regarding genome‐wide variation of changing communities or of taxonomic groups over time, which may help explain past changes and predict future responses of communities to changes in their environment; however, to date, such studies are relatively scarce. We review studies on environmental DNA and environmental genomics of terrestrial mammals to assess the potential of such approaches regarding past, contemporary, and future terrestrial ecosystems, identify inherent challenges, and discuss potential applications. We elaborate on lessons to be learned from mammal genomics of past ecosystems and compare metabarcoding with general metagenetic and metagenomic techniques. We provide a comprehensive overview of current applications, challenges, and future potential of environmental DNA with regards to terrestrial mammals. As current major challenges regarding mammalian eDNA we identify its scarcity and patchy distribution, along with the persistent necessity of genomic reference data. While the latter are steadily increasing, the former can only be tackled by explicitly mapping the environment to gain understanding of spatial eDNA distribution. Such understanding may facilitate informed choices of sample sites and substrates and, together with new sequencing techniques, this can allow mammalian eDNA to be maximally exploited as a source of biodiversity data.

Equine herpesviruses (EHV) are a major health concern for domestic and wild equids and represent one of the most economically important disease agents of horses. Most known EHVs are transmitted directly between individuals as a result of direct exposure to exudates and aerosols. However, accumulating evidence suggests that environmental transmission may play a role including air, water, and fomites. Here, we reviewed studies on environmental stability and transmission of EHVs, which may influence viral dynamics and the use of environmental samples for monitoring EHV shedding.

In climates with seasonally limited precipitation, terrestrial animals congregate at high densities at scarce water sources. We hypothesize that viruses can exploit the recurrence of these diverse animal congregations to spread. In this study, we test the central prediction of this hypothesis — that viruses employing this transmission strategy remain stable and infectious in water. Equid herpesviruses (EHVs) were chosen as a model as they have been shown to remain stable and infectious in water for weeks under laboratory conditions. Using fecal data from wild equids from a previous study, we establish that EHVs are shed more frequently by their hosts during the dry season, increasing the probability of water source contamination with EHV. We document the presence of several strains of EHVs present in high genome copy number from the surface water and sediments of waterholes sampled across a variety of mammalian assemblages, locations, temperatures and pH. Phylogenetic analysis reveals that the different EHV strains found exhibit little divergence despite representing ancient lineages. We employed molecular approaches to show that EHVs shed remain stable in waterholes with detection decreasing with increasing temperature in sediments. Infectivity experiments using cell culture reveals that EHVs remain infectious in water derived from waterholes. The results are supportive of water as an abiotic viral vector for EHV.

Climate change has a great impact on boreal ecosystems including Siberian larch forests. As a consequence of warming, larch grow is possible in areas where climate used to be too cold, leading to a shift of the tree line into more arctic regions. Most plants co-exist in symbiosis with heterotrophic organisms surrounding their root system. In arctic ecosystems, mycorrhizal fungi are a prerequisite for plant establishment and survival because they support nutrient uptake from nutrient-poor soils and maintain the water supply. Until now, however, knowledge about the co-variation of vegetation and fungi is poor. Certainly, the understanding of dynamic changes in biotic interactions is important to understand adaptation mechanisms of ecosystems to climate change.We investigated sedimentary ancient DNA from Lake Levinson Lessing, Taymyr Peninsula (Arctic Siberia, tundra), Lake Lama, Lake Kyutyunda (both northern Siberia, tundra-taiga transition zone) and Lake Bolshoe Toko (southern Siberia, forest area) covering the last about 45.000 years using ITS primers for fungi along with the chloroplast P6 loop marker for vegetation metabarcoding. We found changes in the fungal communities that are in broad agreement with vegetation turnover. To our knowledge, this is the first broad ecological study on lake sediment cores to analyze fungal biodiversity in relation to vegetation change on millennial time scales.

The Arctic is currently experiencing dramatic ecosystem changes with immediate effects on biodiversity. Sedimentary ancient DNA is a unique and valuable source of information on ecosystem changes over a long temporal scale. Understanding these past changes may help predict the relative impacts of climate change, herbivory, and anthropogenic effects on present ecosystems. In the BiodivERsA project “Future ArcTic Ecosystems” (FATE), we aim to assess changes in past herbivore abundance over large spatial (circumarctic) and temporal (Last Glacial Maximum until today) scales using three (semi-)quantitative methods on sedimentary ancient DNA of plants, herbivores, and herbivore proxies (i.e. coprophilous fungi and parasites) – metabarcoding, hybridization capture enrichment, and droplet digital PCR (ddPCR). Metabarcoding was applied to DNA of plants and also of coprophilous fungi as proxies of herbivore abundance. This approach is an established and important tool for assessing biodiversity from recent environmental DNA; however, quantification of specific taxa may be complicated due to inherent methodological biases (e.g. amplification efficiency due to primer bias), and our current understanding of the factors affecting potential quantification by metabarcoding is still limited. Moreover, ancient DNA is highly fragmented, which may prevent PCR amplification altogether. As an alternative, target enrichment by hybridization capture is a method that does not depend on target PCR amplification and is typically not affected by DNA fragmentation. Furthermore, hybridization capture can be used to target numerous genetic markers of a vast range of highly diverse taxa. We are using hybridization capture to enrich DNA of a range of herbivore species and numerous proxy organisms. Metabarcoding and hybridization capture can be applied to a vast taxonomic range and may be used quantitatively based on relative sequencing read abundance; however, the respective read abundance may be confounded by random and systematic errors and other biases. We are therefore using an additional quantification method – ddPCR – on several selected taxa, which is taxon-specific but facilitates highly accurate quantification of template DNA molecules in a given sample. The combined taxonomic and quantitative results of these three approaches are used to generate highly resolved datasets on past vegetation and herbivores, which allows us to reconstruct past vegetation changes over large spatial (circumarctic) and temporal (Last Glacial Maximum until today) scales. Detailed inferences on herbivore abundance and reconstructing past ecological conditions may be important for ecosystem management and conservation in the face of accelerating changes in Arctic ecosystems due to global climate change.

Sedimentary ancient DNA has by now become a recognized source of information on past biodiversity change, but our understanding of its dynamics and taphonomy is still limited. While for environmental DNA in water, dedicated investigations on its provenance and degradation are being increasingly carried out, we know very little about sedimentary DNA, in particular with respect to aquatic organisms. We are therefore conducting investigations on the distribution of DNA in surface sediments and a short sediment core, with a focus on aquatic communities in the large and heterogeneous Lake Constance. Targeted organisms range from phyto- and zooplankton to fish and waterbirds. Initial results and comparison with sightings of rare species indicate that the DNA is not distributed uniformly or widely across the lake, especially for multicellular animals, but rather linked to the local presence of the organisms. This has implications for our understanding of how DNA enters the sediment and for paleoecological inferences derived from these records.

Equid herpesviruses (EHVs) are pathogens of equid and nonequid hosts that can cause disease and fatalities in captivity and in the wild. EHVs establish latent infections but can reactivate, and most EHVs are shed via the nasal passage. Therefore, nasal swabs are generally used for EHV monitoring. However, invasive sampling of wild equids is difficult. While feces is a commonly used substrate for detecting other pathogens, to our knowledge, EHVs have never been detected in feces of naturally infected equids. We systematically tested zebra feces for EHV presence by (i) establishing nested PCR conditions for fecal DNA extracts, (ii) controlling for environmental EHV contamination, and (iii) large-scale testing on a free-ranging zebra population. A dilution minimizing inhibition while maximizing viral DNA concentrations was determined in captive Grevy's zebra (Equus grevyi) fecal samples from individuals shedding EHV nasally. Sixteen of 42 fecal samples (38%) were EHV positive. To demonstrate that the EHV positivity was not a result of environmental contamination, rectal swabs of wild zebras were screened (n = 18 [Equus quagga and E. zebra]), and 50% were EHV positive, indicating that the source of EHV in feces is likely the intestinal mucosa and not postdefecation contamination. Out of 270 fecal samples of wild zebras, 26% were EHV positive. Quantitative PCRs showed that the amount of virus DNA in feces was not significantly smaller than that in other samples. In summary, fecal sampling facilitates large-scale screening and may be useful to noninvasively investigate phylogenetic EHV diversity in wild and domestic equids. IMPORTANCE Equid herpesviruses (EHVs) establish latent infections, and many EHVs are shed and transmitted via nasal discharge primarily through droplet and aerosol infection. Obtaining nasal swabs and other invasive samples from wildlife is often not possible without capture and physical restraint of individuals, which are resource intensive and a health risk for the captured animals. Fecal EHV shedding has never been demonstrated for naturally infected equids. We established the conditions for fecal EHV screening, and our results suggest that testing fecal samples is an effective noninvasive approach for monitoring acute EHV shedding in equids.

Determining species distributions can be extremely challenging but is crucial to ecological and conservation research. Environmental DNA (eDNA) approaches have shown particular promise in aquatic systems for several vertebrate and invertebrate species. For terrestrial animals, however, eDNA-based surveys are considerably more difficult due to the lack of or difficulty in obtaining appropriate sampling substrate. In water-limited ecosystem where terrestrial mammals are often forced to congregate at waterholes, water and sediment from shared water sources may be a suitable substrate for noninvasive eDNA approaches. We characterized mitochondrial DNA sequences from a broad range of terrestrial mammal species in two different African ecosystems (in Namibia and Tanzania) using eDNA isolated from native water, sediment and water filtered through glass fibre filters. A hybridization capture enrichment with RNA probes targeting the mitochondrial genomes of 38 mammal species representing the genera/families expected at the respective ecosystems was employed, and 16 species were identified, with a maximum mitogenome coverage of 99.8%. Conventional genus-specific PCRs were tested on environmental samples for two genera producing fewer positive results than hybridization capture enrichment. An experiment with mock samples using DNA from non-African mammals showed that baits covering 30% of nontarget mitogenomes produced 91% mitogenome coverage after capture. In the mock samples, over-representation of DNA of one species still allowed for the detection of DNA of other species that was at a 100-fold lower concentration. Hybridization capture enrichment of eDNA is therefore an effective method for monitoring terrestrial mammal species from shared water sources.

Time is a limited resource and how well it is allocated to competing behaviours can profoundly affect Darwinian fitness. Life history theory predicts that the amount of time allocated to vital behaviours will change with life history stage, resulting in trade-offs between competing behaviours. Moreover, a range of environmental factors can also affect activity budgets. We studied diurnal time allocation by migratory plains zebras (Equus quagga) in the Serengeti ecosystem, Tanzania, and investigated the effect of life history stage, social environment, habitat structure, and day time on time allocation to five behavioural categories (grazing, resting, vigilance, movement, other). We expected (1) increased vulnerability to predation and impeded predator detection to increase vigilance and decrease resting and grazing; (2) energetically costly life stages to increase grazing and decrease resting; and (3) increasing age in young to result in increased vigilance and grazing and decreased resting. Our findings revealed that in young zebras, resting decreased and grazing increased from the youngest to the oldest age class. Band stallions spent more time grazing and less time resting and moving than bachelors. Lactating mares devoted more time to grazing but less to resting and vigilance than other mares. Mares spent most time vigilant in the last third and stallions in the first third of the day. Adult zebras moved more, and mares were more vigilant in the woodland boundary than on short grass plains. Taken together, our study identifies intrinsic and extrinsic factors shaping time allocation decisions and trade-offs between competing behaviours in plains zebra. How well animals allocate their limited time to competing behaviours will affect their survival and reproduction. For example, energetically costly life history stages often require an increase in foraging whereas when predators threaten survival, more time should be allocated to vigilance. Increased investment of time in one behaviour requires decreased investment in another or other behaviours; thus, trade-offs in time are expected. Life history theory predicts substantial changes in the time allocated to vital behaviours between life stages. We investigated the effect of life history stage and environmental factors on the behavioural time budgets of migratory plains zebras in the Serengeti ecosystem. Our results showed that life history stage, juvenile age and environmental factors determine how time is invested in vital behaviours, and the behavioural trade-offs this entails.

Adverse environmental stimuli (stressors) activate the hypothalamic-pituitary-adrenal axis and contribute to allostatic load. This study investigates the contribution of environmental stressors and life history stage to allostatic load in a migratory population of plains zebras (Equus quagga) in the Serengeti ecosystem, in Tanzania, which experiences large local variations in aggregation. We expected higher fGCM response to the environmental stressors of feeding competition, predation pressure and unpredictable social relationships in larger than in smaller aggregations, and in animals at energetically costly life history stages. As the study was conducted during the 2016 El Niño, we did not expect food quality of forage or a lack of water to strongly affect fGCM responses in the dry season. We measured fecal glucocorticoid metabolite (fGCM) concentrations using an enzyme immunoassay (EIA) targeting 11β-hydroxyetiocholanolone and validated its reliability in captive plains zebras. Our results revealed significantly higher fGCM concentrations 1) in large aggregations than in smaller groupings, and 2) in band stallions than in bachelor males. Concentrations of fGCM were not significantly higher in females at the energetically costly life stage of late pregnancy/lactation. The higher allostatic load of stallions associated with females, than bachelor males is likely caused by social stressors. In conclusion, migratory zebras have elevated allostatic loads in large aggregations that probably result from their combined responses to increased feeding competition, predation pressure and various social stressors. Further research is required to disentangle the contribution of these stressors to allostatic load in migratory populations.

Equine Herpesviruses (EHV) are common and often latent pathogens of equids which can cause fatalities when transmitted to non-equids. Stress and elevated glucocorticoids have been associated with EHV reactivation in domestic horses, but little is known about the correlation between stress and viral reactivation in wild equids. We investigated the effect of an environmental stressor (social group restructuring following a translocation event) on EHV reactivation in captive Grévy’s zebras (Equus grevyi). A mare was translocated by road transport from Zoo Mulhouse, France, to join a resident group of three mares in Tierpark Berlin, Germany. We used an indirect sampling method to assess the frequency of EHV shedding for 14 days immediately after the translocation event (termed the ‘experimental period’). The results were compared with those from two control periods, one preceding and one subsequent to the experimental period. In addition, we measured fecal glucocorticoid metabolite (fGCM) concentrations daily in all individuals from 6 days before, to 14 days after translocation. We found significantly higher EHV shedding frequencies during the experimental period, compared to each of the two control periods. All animals showed significantly elevated fGCM concentrations, compared to fGCM levels before translocation. Finally, we found that an increase in fGCM concentration was significantly associated with an increased likelihood of EHV shedding. Although the small number of animals in the study limits the conclusions that can be drawn from the study, taken together, our results support the hypothesis that environmental stressors induce viral reactivation in wild equids. Our results suggest that potentials stressors such as group restructuring and translocation should be considered in the management of zoological collections to reduce the risk of fatal EHV infections in novel hosts. Moreover, environmental stressors may play an important role in EHV reactivation and spread in wild equid populations.

Viruses may have a dramatic impact on the health of their animal hosts. The patho-physiological mechanisms underlying viral infections in animals are, however, not well understood. It is increasingly recognized that oxidative stress may be a major physiological cost of viral infections. Here we compare three blood-based markers of oxidative status in herpes positive and negative individuals of the domestic horse (Equus ferus caballus) and of both captive and free-ranging Mongolian khulan (Equus hemionus hemionus) and plains zebra (Equus quagga). Herpes positive free-ranging animals had significantly more protein oxidative damage and lower glutathione peroxidase (antioxidant enzyme) than negative ones, providing correlative support for a link between oxidative stress and herpesvirus infection in free-living equids. Conversely, we found weak evidence for oxidative stress in herpes positive captive animals. Hence our work indicates that environment (captive versus free living) might affect the physiological response of equids to herpesvirus infection. The Mongolian khulan and the plains zebra are currently classified as near threatened by the International Union for Conservation of Nature. Thus, understanding health impacts of pathogens on these species is critical to maintaining viable captive and wild populations.

Direct collection of samples from wildlife can be difficult and sometimes impossible. Non-invasive remote sampling for the purpose of DNA extraction is a potential tool for monitoring the presence of wildlife at the individual level, and for identifying the pathogens shed by wildlife. Equine herpesviruses (EHV) are common pathogens of equids that can be fatal if transmitted to other mammals. Transmission usually occurs by nasal aerosol discharge from virus-shedding individuals. The aim of this study was to validate a simple, non-invasive method to track EHV shedding in zebras and to establish an efficient protocol for genotyping individual zebras from environmental DNA (eDNA). A commercially available horse enrichment toy was deployed in captive Grévy's, mountain, and plains zebra enclosures and swabbed after 4-24 hr. Using eDNA extracted from these swabs four EHV strains (EHV-1, EHV-7, wild ass herpesvirus and zebra herpesvirus) were detected by PCR and confirmed by sequencing, and 12 of 16 zebras present in the enclosures were identified as having interacted with the enrichment toy by mitochondrial DNA amplification and sequencing. We conclude that, when direct sampling is difficult or prohibited, non-invasive sampling of eDNA can be a useful tool to determine the genetics of individuals or populations and for detecting pathogen shedding in captive wildlife.

The social organization of giraffes (Giraffa camelopardalis) imposes a high-cost reproductive strategy on bulls, which adopt a 'roving male' tactic. Our observations on wild giraffes confirm that bulls indeed have unsynchronized rut-like periods, not unlike another tropical megaherbivore, the elephant, but on a much shorter timescale. We found profound changes in male sexual and social activities at the scale of about two weeks. This so far undescribed rutting behaviour is closely correlated with changes in androgen concentrations and appears to be driven by them. The short time scale of the changes in sexual and social activity may explain why dominance and reproductive status in male giraffe in the field seem to be unstable.