Fecal DNA Metabarcoding Research Articles

Faecal DNA Metabarcoding for Diet Analysis of Endangered Fish Species, Odontobutis obscurus.

Understanding the ecological characteristics of endangered species is essential for their management and conservation. The dark sleeper (Odontobutis obscurus) is a carnivorous freshwater fish that has been designated an endangered species in South Korea because of its highly restricted habitat range and small population size. However, ecological research on prey items for this species remains severely limited globally, posing challenges for establishing conservation measures such as translocation management. In this study, the food sources and feeding strategies of O. obscurus were identified through non-invasive faecal DNA metabarcoding. A total of 24 O. obscurus specimens were collected from Sanyang Stream on Geoje Island, and faecal DNA was amplified using mitochondrial cytochrome c oxidase I (COI) primer. As a result, 13 prey items were identified, comprising fish and benthic invertebrates. Dietary metrics revealed the overall food composition and major food source of O. obscurus, and the Costello method enabled graphical diet analysis, suggesting that O. obscurus is a generalist, feeding on a wide variety of food sources. The food selectivity index suggested that O. obscurus prefers specific prey items, such as genus Misgurnus, Zacco (fish), Radix (gastropod), and Ephemera (mayfly). This molecular method facilitated the diet analysis of an endangered fish species that cannot undergo dissection, providing crucial information for their management, particularly regarding translocation efforts.

Black-throated blue warblers (Setophaga caerulescens) exhibit diet flexibility and track seasonal changes in insect availability.

Changes in leaf phenology from warming spring and autumn temperatures have lengthened the temperate zone growing "green" season and breeding window for migratory birds in North America. However, the fitness benefits of an extended breeding season will depend, in part, on whether species have sufficient dietary flexibility to accommodate seasonal changes in prey availability. We used fecal DNA metabarcoding to test the hypothesis that seasonal changes in the diets of the insectivorous, migratory black-throated blue warbler (Setophaga caerulescens) track changes in the availability of arthropod prey at the Hubbard Brook Experimental Forest, New Hampshire, USA. We examined changes across the breeding season and along an elevation gradient encompassing a 2-week difference in green season length. From 98 fecal samples, we identified 395 taxa from 17 arthropod orders; 242 were identified to species, with Cecrita guttivitta (saddled prominent moth), Theridion frondeum (eastern long-legged cobweaver), and Philodromus rufus (white-striped running crab spider) occurring at the highest frequency. We found significant differences in diet composition between survey periods and weak differences among elevation zones. Variance in diet composition was highest late in the season, and diet richness and diversity were highest early in the season. Diet composition was associated with changes in prey availability surveyed over the green season. However, several taxa occurred in diets more or less than expected relative to their frequency of occurrence from survey data, suggesting that prey selection or avoidance sometimes accompanies opportunistic foraging. This study demonstrates that black-throated blue warblers exhibit diet flexibility and track seasonal changes in prey availability, which has implications for migratory bird responses to climate-induced changes in insect communities with longer green seasons.

Evaluating the winter diet of elk using DNA metabarcoding analysis

North American elk Cervus canadensis inhabited portions of the eastern United States until extirpation in the mid‐1800s. From 2000 to 2008, 201 elk were reintroduced to the North Cumberland Wildlife Management Area (NCWMA), Tennessee. The stocking source was Elk Island National Park, Alberta Canada where there are two distinct genetic populations isolated from the north and south. This genetic structure has largely persisted in the population after translocation. Food habits were evaluated in the early stages of restoration, but the population has had approximately 20 years to adapt to the landscape, and current food habits are unknown. To assess diet composition using DNA metabarcoding, we collected fecal pellets of elk from 65 openings within the 79 318 ha NCWMA weekly from February to April of 2019. We targeted the ITS2 region of the nuclear ribosomal DNA to amplify vegetation sequences found in the internal portion of the elk feces. DNA metabarcoding of feces was linked to results from an accompanying elk population genetics study to investigate food habits between sexes from the two different genetic groups. The majority (80.298%) of sequences matched plants from 21 genera. The top genera (> 5.000%) represented were Vaccinium (15.216%), Festuca (8.446%), Rosa (6.358%), Robinia (5.793%), and Eleagnus (5.186%). Elk heavily used woody plants before and after spring green‐up (> 50% of diet). However, the quantity of forbs in their diet more than doubled after emergence in the spring. The sex‐genetic groups consumed similar vegetation in approximately proportionate amounts. Diversity analyses revealed a significant difference in plant genera sequence detection between males from the two genetic groups, although this finding is likely explained by limited sample size. NCWMA elk used a variety of forage in the winter and DNA metabarcoding analysis allows for a comprehensive analysis of food habits useful for monitoring how elk respond dietarily to habitat management.

Multiple methods of diet assessment reveal differences in Atlantic puffin diet between ages, breeding stages, and years

IntroductionAtlantic puffin (Fratercula arctica, hereafter “puffin”) reproductive success in the Gulf of Maine (GoM) has declined following a recent oceanographic regime shift that has led to rapid warming and increasingly frequent marine heatwaves. Concurrent changes in both the regional forage fish community and puffin chick diets and provisioning rates suggest that inadequate prey resources may be driving this decline. Traditional, noninvasive methods of diet assessment, however, are unable to determine seabird diet at many age classes and breeding stages.MethodsTo determine what prey GoM puffins were feeding on during two years of marine heatwave conditions, we assessed puffin diet using two complementary methods: traditional, observational methods that utilize bill-load photography and emerging methods employing fecal DNA metabarcoding. We then examined the effect of methodology, age, breeding stage, and year on puffin diet composition. ResultsWe identified a strong correlation between the composition of chick diet as estimated through traditional and emerging methods, supporting the interpretation of DNA relative read abundance as a quantitative metric of diet composition. Both methods identified the same dominant prey groups yet metabarcoding identified a greater number of species and offered higher taxonomic resolution. Additionally, metabarcoding revealed adult puffin diet during the incubation period for the first time. Although puffin adults and chicks fed on many of the same prey types, adults consumed a greater variety of taxa and consumed more low quality prey types than they provisioned chicks.DiscussionFor both age classes, diet varied both between and within years, likely reflecting changes in the local forage fish community in response to environmental variability. Puffins exploited unusual abundances of typically-uncommon prey during these two years of marine heatwave conditions, yet low puffin productivity suggests the observed dietary plasticity was not fully able to compensate for apparent prey shortages. Continued refinement of molecular methods and the interpretation of the data they provide will enable better assessments of how seabirds of diverse ages and breeding stages are adapting to changing prey communities.

Fecal DNA metabarcoding reveals the winter diet of Eurasian otter (Lutra lutra) in Northeast China

As the top predator of freshwater ecosystems, the Eurasian otter (Lutra lutra) plays a vital role in maintaining food web structure and ecosystem stability. Understanding the dietary composition and dynamics of the Eurasian otter is crucial for comprehending its ecosystem function and interspecies interactions, providing vital information for determining conservation and management strategies. Currently, DNA metabarcoding technology has been extensively applied in dietary studies as a more effective and accurate method for identifying prey species. However, there remains an insufficient amount of research on otter diet using this technology, especially in Northeast China. In this study, we analyzed the vertebrate dietary composition and spatial variation of otters during the winter in Northeast China using DNA metabarcoding technology. We confirm 106 fecal samples belonging to Eurasian otter DNA from 118 putative otters fecal samples, and then performed polymerase chain reaction (PCR) amplifications using vertebrate universal primers with the otter blocking primer (OBS1) and the human blocking primer (HomoB) to the sequence. Subsequently, high-throughput sequencing was performed to analyze the dietary composition of Eurasian otter. We identified a total of 34 prey taxa, including 31 fish, 2 frog and 1bird. Our results showed that the winter diet of the Eurasian otter was dominated by fish (60.11 %), (e.g., Cottidae, Cyprinidae), followed by Ranidae (39.88 %). Furthermore, geographical variations in dietary preferences were observed. This study confirms that Eurasian otter is a generalist piscivorous predator, and that its diet could be related to the availability of resources in the habitat. This study enriches the information on the diet of Eurasian otter, and can be used to provide a reference for the development of effective conservation strategies. At the same time, it confirms the reliability of the fecal DNA metabarcoding technique for carnivore predation analysis, and provides a new perspective for the investigation and research of otters and other mammals.

Diet of moulting Swainson's Thrushes (Catharus ustulatus) and Tennessee Warblers (Leiothlypis peregrina) at a stopover site during fall migration measured with fecal DNA metabarcoding

Moult and migration are energetically demanding and require adequate nutrition. In some species, individuals may interrupt their fall migration to moult at discrete stopover locations outside of their breeding grounds (i.e., moult-migration) leading to competing nutritional demands for moult and migration. Here, we use DNA barcoding of fecal samples to compare the diet of moulting and actively migrating (post-moult) Swainson’s Thrushes (Catharus ustulatus) and Tennessee Warblers (Leiothlypis peregrina) during their fall migration stopover at a large urban greenspace in Montreal, Canada. Diet differed according to moult status, species, and seasonality. Swainson’s Thrushes had a broad diet with frequent detections of both insects and berry-producing shrubs; while detections in Tennessee Warblers’ diets were mainly arthropods. For both species, more actively migrating individuals consumed fleshy-fruiting plants than moulting individuals. A higher proportion of moulting birds consumed arthropods compared to active migrants, due to either arthropod availability or a dietary preference for proteinaceous foods to grow feathers. Both species and moult classes consumed more native plants than non-native plants later in the season. We show the importance of managing urban greenspaces with native plants and diverse food sources that can provide for the different dietary needs of migratory birds.

Fecal DNA metabarcoding helps characterize the Canada jay's diet and confirms its reliance on stored food for winter survival and breeding.

Accurately determining the diet of wild animals can be challenging if food items are small, visible only briefly, or rendered visually unidentifiable in the digestive system. In some food caching species, an additional challenge is determining whether consumed diet items have been previously stored or are fresh. The Canada jay (Perisoreus canadensis) is a generalist resident of North American boreal and subalpine forests with anatomical and behavioural adaptations allowing it to make thousands of arboreal food caches in summer and fall that are presumably responsible for its high winter survival and late winter/early spring breeding. We used DNA fecal metabarcoding to obtain novel information on nestling diets and compiled a dataset of 662 published and unpublished direct observations or stomach contents identifications of natural foods consumed by Canada jays throughout the year. We then used detailed natural history information to make informed decisions on whether each item identified to species in the diets of winter adults and nestlings was best characterized as 'likely cached', 'likely fresh' (i.e., was available as a non-cached item when it appeared in a jay's feces or stomach), or 'either possible'. Of the 87 food items consumed by adults in the winter, 39% were classified as 'likely cached' and 6% were deemed to be 'likely fresh'. For nestlings, 29% of 125 food items identified to species were 'likely cached' and 38% were 'likely fresh'. Our results support both the indispensability of cached food for Canada jay winter survival and previous suggestions that cached food is important for late winter/early spring breeding. Our work highlights the value of combining metabarcoding, stomach contents analysis, and direct observations to determine the cached vs. non-cached origins of consumed food items and the identity of food caches, some of which could be especially vulnerable to degradation through climate change.

Evaluating Fecal DNA Metabarcoding to Estimate the Dietary Botanical Composition of Goats

Improved techniques are needed to estimate the dietary species composition of grazing animals. The fecal DNA metabarcoding technique has been proposed as a noninvasive method to determine dietary species composition. However, studies suggest that it is more effective to qualitatively estimate species composition of wild and domestic animals than for accurately quantifying diet composition. The technique, using the trnL barcode, was evaluated with 10 individually penned goats, which were fed hand-harvested plant materials that were collected fresh each morning before known amounts of each type of plant materials were fed and completely consumed by the goats. Goats were fed at 3% of their body weight on a DM basis. Broadleaf plant materials fed were Russian olive (Elaeagnus angustifolia), silver buffaloberry (Shepherdia argentea), western snowberry (Symphoricarpos occidentalis), and wormwood sage (Artemisia absinthium) plus a mixture of predominantly smooth bromegrass (Bromus inermis), which also included small amounts of other grasses such as Kentucky bluegrass (Poa pratensis) and forbs such as leafy spurge (Euphorbia esula). Estimates of the dietary composition of the goats were different (P ≤ 0.04) from those of their known dietary composition, except for wormwood sage when expressed on a crude protein basis (P = 0.98), regardless of whether known composition was expressed on a dry matter or crude protein basis. Therefore, we conclude that the fecal DNA metabarcoding technique for estimating the dietary composition of goat diets is useful qualitatively but needs further development to be a better quantitative technique.

Interplay of competition and facilitation in grazing succession by migrant Serengeti herbivores.

Competition, facilitation, and predation offer alternative explanations for successional patterns of migratory herbivores. However, these interactions are difficult to measure, leaving uncertainty about the mechanisms underlying body-size-dependent grazing-and even whether succession occurs at all. We used data from an 8-year camera-trap survey, GPS-collared herbivores, and fecal DNA metabarcoding to analyze the timing, arrival order, and interactions among migratory grazers in Serengeti National Park. Temporal grazing succession is characterized by a "push-pull" dynamic: Competitive grazing nudges zebra ahead of co-migrating wildebeest, whereas grass consumption by these large-bodied migrants attracts trailing, small-bodied gazelle that benefit from facilitation. "Natural experiments" involving intense wildfires and rainfall respectively disrupted and strengthened these effects. Our results highlight a balance between facilitative and competitive forces in co-regulating large-scale ungulate migrations.

Fecal DNA metabarcoding reveals seasonal and annual variation in willow ptarmigan diet.

Understanding spatio-temporal variation in the diet of alpine herbivores is important to predict how a changing climate will affect these species in the future. We examined the spatio-temporal variation in willow ptarmigan (Lagopus l. lagopus) diet using DNA metabarcoding of fecal pellets sampled from winter to early summer over three consecutive years. Furthermore, we assessed how snow cover and vegetation phenology affected diet variation. We also investigated sex differences in diet composition. We identified 18 important diet taxa and the genera Betula, Vaccinium and Empetrum occurred most frequently. Diet composition and richness varied within and between years. Seasonally, there was a shift from a narrow winter diet dominated by trees and dwarf shrubs to a broader spring diet with more nutritious field vegetation. This seasonal progression differed among years. The temporal variation in diet was better explained by day of year than by snow cover and vegetation phenology. Females had a more diverse diet than males, but there were no sex differences in diet composition. Our results demonstrate that metabarcoding of fecal samples provides the opportunity to assess factors affecting diet composition of species in alpine ecosystems in the context of a changing climate.

Nestling Diet of Two Sympatric Insectivorous Passerines in Different Habitats—A Metabarcoding Study

Increasing landscape transformations and urbanisation affect insectivorous bird populations in various ways such as food availability, breeding phenology, or reproductive success. Especially during the breeding season, many passerine birds rely on the availability of caterpillars as the main prey for their nestlings. Previous studies suggested that similar diet preferences of sympatric species may result in interspecific competition, as demonstrated for Blue and Great Tits in forest habitats. However, nestling diet and prey preferences in other habitats are not fully understood. Prey availability, especially caterpillars, is lower in cities than in forests, thus influencing prey choice and interspecific competition. Here we used faecal DNA metabarcoding to investigate if nestling diet composition of the two sympatric species Blue Cyanistes caeruleus and Great Tits Parus major varied among species and different habitats (forest, traditional orchards, and urban parks). Furthermore, we examined food availability by DNA barcoding of the arboreal arthropod communities among habitats and compared them to the nestling diet to infer parental prey selectivity. The study was carried out in central Germany from 2018 to 2019. Blue and Great Tits showed a diverse diet which was dominated by Lepidoptera in all habitats. Lepidopteran diet components were most similar between forest and orchard sites, as were the components with other arthropods between orchard and urban sites. Both tit species showed selectivity for the lepidopteran families Geometridae and Tortricidae in all habitats, and for Noctuidae (Lepidoptera), Tenthredinidae and Braconidae (Hymenoptera) in forest and orchard sites. As the tits showed preferences for mainly families of Lepidoptera, and Hymenoptera, our approach provides a baseline to support monitoring of these groups.

Noise pollution alters the diet composition of invertebrate consumers both in and beyond a noise-exposed grassland ecosystem.

Anthropogenic noise is ubiquitous globally. However, we know little about how the impacts of noise alter fundamental ecosystem properties, such as resource consumption by invertebrate consumers. Using experimental noise manipulation and faecal DNA metabarcoding, we assessed how the direct and cross-trophic indirect effects of noise altered the dietary richness and specializations of omnivorous grasshoppers in a grassland ecosystem. We found that the experimental noise treatment expanded grasshoppers' dietary richness and resulted in dietary generalizations in both noise-exposed and adjacent relatively quieter areas. Unexpectedly, however, these dietary changes were primarily explained by the direct effect of noise not only in the noise-exposed areas but also in the adjacent quieter areas and were relaxed by indirect effects of noise such as reduced birds and predation risk and increased grasshoppers. Our work suggests that noise pollution can be key in explaining the variation of invertebrate consumers' diets across a gradient of noise-exposed environments.

Using faecal DNA metabarcoding to determine the diet of the long-tailed bat, Chalinolobus tuberculatus

ABSTRACT Dietary analysis of nocturnal aerial insectivores such as bats is not possible by direct visual observation of predation, therefore diet is traditionally determined by analysing dietary remains in scats. Analysing DNA in scats through metabarcoding can reveal dietary items not preserved in faeces but also resolve dietary preferences to species level. DNA metabarcoding analysis of scats collected from six individual long-tailed bats (Chalinolobus tuberculatus) from Whirinaki Forest Park revealed a predominance of aquatic insects (Ephemeroptera and Trichoptera) and small-bodied moths (Lepidoptera). This contrasts with earlier studies of faecal remains in this species which revealed a strong preference for Dipterans and Coleopterans. The use of DNA metabarcoding may provide a better understanding of diets, as soft-bodied insects can be detected even when not apparent in physical examinations of scat, and thus will allow for more robust comparison of differences in diets between populations and seasons. The application of DNA metabarcoding is a simple but high-resolution tool to allow further study on dietary preference in this endangered species.

Eurasian otters prefer to prey on religious released non-native fish on the Qinghai-Tibetan Plateau.

Religious wildlife release is prevalent worldwide, especially in Asia countries. It is one of the anthropogenic pathways to cause biological invasions. Religious fish release is common on the Qinghai-Tibetan Plateau, yet few studies have assessed the influences of religious fish release on local species. In Yushu, a city on the Qinghai-Tibetan Plateau, we interviewed local people, conducted fish trap surveys in local rivers, and examined the diet of Eurasian otters Lutra lutra using a fecal DNA metabarcoding approach. We found that fish release started at least in 1980-1990s in Yushu. Tibetan residents released fish in large amounts and released fish were usually exotic commercial fish purchased from market. Despite such long-term and intensive fish release activities, released fish were few in local rivers. On the other hand, Eurasian otters mainly prey on fish and released fish accounted for ~20% of relative read abundance of prey DNA in otters' diet, indicating their high preference on released fish. Our study suggested that religious fish release may provide additional food resources for otters, whereas otters, as a top predator in local rivers, may deplete non-native fish once they were released and, therefore, reduce the probability of colonization of released fish, although further studies are required to assess otters' impact. Our study revealed otters' diet in Yushu, providing basic information for local otter management and conservation. Furthermore, it represents a case showing that native predators prey on religious released animals, implying a probable direction for controlling invasive species through native predator conservation.

Dietary analysis of three important mariculture species in South Korea using DNA metabarcoding in fecal samples

Knowledge of dietary composition is crucial for understanding the trophic ecology of a mariculture species and the available food in aquafarms, which is fundamental for assessing ecosystem health status and determining ecological response to ecosystem variation to develop a better management plan for marine farming. For suspension-feeding invertebrates, the growth and development rely heavily on the availability and quality of phytoplankton. However, their growth rate can be severely impacted by food deficiency or exposure to harmful algae blooms (HABs) and pathogenic parasites, which are major causes of mass mortality in mariculture. This study aimed to conduct a dietary analysis of three important mariculture species — Magallana gigas, Halocynthia roretzi, and Mizuhopecten yessoensis — on three farms in South Korea using DNA metabarcoding. Sequence processing and filtering generated 304 validated amplicon sequence variants from six fecal samples, taxonomically identifying 15 phyla, 18 classes, 32 orders, 39 families, 43 genera, and 46 species. The DNA recovery of Dinoflagellata was predominantly detected in all fecal samples, followed by Apicomplexa, Bacillariophyta, and Ciliophora. Of the three mariculture species, the fecal assemblage of M. gigas exhibited the highest richness and diversity, with a high read proportion of phytoplankton (specifically Dinoflagellata), while such proportions were relatively low in the M. yessoensis samples. Excessive read proportions of parasites (Apicomplexa) were detected in the fecal samples of two H. roretzi, indicating a possible link to as substantial decrease in H. roretzi production observed in South Korea. A total of 27 key species were detected in six fecal samples, including a high read proportion of Prorocentrum triestinum, which is responsible for HABs in Korean coastal waters. These results emphasize the potential of fecal DNA metabarcoding in the genetic monitoring of aquaculture farms/ecosystems and could be a useful reference for develop effective management plans to guarantee sustainable mariculture production.

Bats are sentinels for invasive pest surveillance based on DNA metabarcoding

Loss of crop yield due to non-native invasive pests is a global phenomenon with profound population, biodiversity, ecosystem, and economic impacts. The polyphagous and migratory fall armyworm (FAW) Spodoptera frugiperda (Lepidoptera, Noctuidae) has become a major invasive pest around the globe in the past decade, posing a great threat to global food security. Early warning and timely detection of this pest and its associated agricultural risks are essential for preventing, intervening, controlling, and mitigating invasion threats, which could reduce crop losses and economic damage. However, one key challenge is how to effectively and accurately monitor pest invasion as soon as possible prior to their establishment. Here, we aimed to determine the effectiveness of four different methods for identifying the presence of FAW, namely DNA metabarcoding, field survey, light trap, and sex pheromone trap. We collected fecal samples of six bat species in the FAW colonization area, including Rhinolophus pearsonii, Rhinolophus rex, Ia io, Myotis altarium, Myotis pilosus, and Myotis chinensis. We then utilized bat fecal samples to monitor FAW through DNA metabarcoding during its early establishment stages and compared this approach to three traditional surveillance methods. The results revealed interspecific differences in the occurrence frequency of FAW in different bat species. Furthermore, bat fecal DNA metabarcoding successfully monitored the occurrence of this pest nearly one month in advance compared with traditional approaches, which coincides with the adult migration time of larval counterparts found in the field based on effective accumulated temperature calculations. Our results showed that DNA metabarcoding dietary analysis of generalist insectivorous bats can be an effective, noninvasive, and economically viable tool for invasion pests monitoring to inform management decisions.

Sympatric rodents in a desert shrubland differ in arthropod consumption

The foraging ecology of sympatric mammals within xeric habitats is poorly understood. In this study, we surveyed the diets of two sympatric woodrat species, Neotoma bryanti, N. lepida and their hybrids in a desert shrubland during the dry season using stable isotope analyses of hair and arthropod DNA metabarcoding of feces. We found that woodrats regardless of their ancestry fed on similar plants during the same season across multiple years. However, stable isotope analyses and arthropod metabarcoding were consistent with greater arthropod ingestion by N. bryanti compared to N. lepida or hybrids. These results suggest that while the diets of these sympatric woodrats largely overlap during the dry season in terms of the plant components, they differ in terms of arthropod ingestion. This arthropod feeding may serve to reduce interspecific competition or act as dietary supplementation during nutritionally challenging periods. These results demonstrate that integrating DNA metabarcoding approaches with stable isotope tracers enables a more rigorous evaluation of animal foraging behavior.

Echolocating bats prefer a high risk-high gain foraging strategy to increase prey profitability.

Predators that target multiple prey types are predicted to switch foraging modes according to prey profitability to increase energy returns in dynamic environments. Here, we use bat-borne tags and DNA metabarcoding of feces to test the hypothesis that greater mouse-eared bats make immediate foraging decisions based on prey profitability and changes in the environment. We show that these bats use two foraging strategies with similar average nightly captures of 25 small, aerial insects and 29 large, ground-dwelling insects per bat, but with much higher capture success in the air (76%) vs ground (30%). However, owing to the 3-20 times larger ground prey, 85% of the nightly food acquisition comes from ground prey despite the 2.5 times higher failure rates. We find that most bats use the same foraging strategy on a given night suggesting that bats adapt their hunting behavior to weather and ground conditions. We conclude that these bats use high risk-high gain gleaning of ground prey as a primary foraging tactic, but switch to aerial hunting when environmental changes reduce the profitability of ground prey, showing that prey switching matched to environmental dynamics plays a key role in covering the energy intake even in specialized predators.

Food webs reveal coexistence mechanisms and community organization in carnivores

Globally, massive carnivore guild extirpations have led to trophic downgrading and compromised ecosystem services. However, the complexity of multi-carnivore food webs complicates accurate identification of species interactions and community organization. Here, we used fecal DNA metabarcoding to investigate three communities that together encompass eight large- and meso-carnivore species and their 44 prey taxa of the Qinghai-Tibet Plateau (QTP), one of the last places on Earth that still harbors intact carnivore assemblages. Quantitative food-web analyses revealed pronounced interspecific variations in the carnivores' prey compositions and dietary partitioning both between and within guilds. Additionally, body masses of the carnivores and their prey exhibited consistent hump-shaped correlations across communities. Overall, differences in prey diversity, size category, and proportional utilization among the carnivore species result in trophic niche segregation that likely promotes carnivore coexistence in the harsh QTP environment. Network structure analyses detected significant modularity in all food webs but nestedness in only one. Furthermore, network characterization identified pikas (Ochotona spp.), bharal (Pseudois nayaur), and domestic yak (Bos grunniens) as potential keystone prey across the areas. Our results paint a holistic and detailed picture of the QTP carnivore assemblages' trophic networks and demonstrate that the combined use of the molecular dietary approach and network analysis can generate structural insights into carnivore coexistence and can identify functionally important species in complex communities. Such knowledge can help safeguard carnivore guild integrity and enhance community resilience to environmental perturbations in the sensitive QTP ecosystems.

Variation in adult and pup wolf diets at natal den sites is influenced by forest composition and configuration.

Although wolves are wide-ranging generalist carnivores throughout their life cycle, during the pup-rearing season wolf activity is focused on natal den sites where pup survival depends upon pack members provisioning food. Because prey availability is influenced by habitat quality within the home range, we investigated the relative importance of prey species for adults and pups and further examined the relationship between habitat characteristics, wolf diet, and litter size on Prince of Wales Island (POW) in Southeast Alaska. During 2012-2020, we detected 13 active den sites within the home ranges of nine wolf packs. We estimated minimum pup counts using motion-detecting cameras and individual genotypes from noninvasive samples (hair: n=322; scat: n=227) and quantified wolf diet composition using fecal DNA metabarcoding (n=538). We assessed habitat composition, configuration, and connectivity within denning and annual home ranges estimated using wolf GPS-collar data. Contrary to expectations, wolves had a more constricted diet during denning season (April 15-July 31), and within this season pups had a narrower dietary niche (species richness [S]=4) focused more on deer (relative frequency of occurrence [O/I]=0.924) than adults (S=15; deer O/I=0.591). Litter size had a positive relationship with the relative frequency of deer in a wolf pack's diet. Wolf consumption of deer was positively associated with the proportion of young-growth forest (≤25 years old) within denning and annual home ranges. High levels of vegetation patch interspersion, and the density of closed logging roads were also important predictors, suggesting these habitat qualities were influential for increasing the availability of deer to wolves. Our results contrast with previous research indicating wolf pup diets included more alternate prey (i.e., beaver) than adults and emphasize the importance of deer to wolf viability on POW, especially during denning season.