DIETARY ANALYSIS: MAMMALS

BATS

Painter M. L., Chambers C. L., Siders M., Doucett R. R., Whitaker Jr J. O., Phillips D. L. (2009): Diet of spotted bats (Euderma maculatum) in Arizona as indicated by fecal analysis and stable isotopes. Canadian Journal of Zoology 87: 865-875.
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We assessed diet of spotted bats (Euderma maculatum (J.A. Allen, 1891)) by visual analysis of bat feces and stable carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope analysis of bat feces, wing, hair, and insect prey. We collected 33 fecal samples from spotted bats and trapped 3755 insects where bats foraged. Lepidopterans averaged 99.6% of feces by volume; other insects were not a major component of diet. The δ¹³C and δ¹⁵N values of bat feces were similar to those of moths from families Noctuidae (N), Lasiocampidae (L), and Geometridae (G), but differed from Arctiidae (A) and Sphingidae (S). We used a mixing model to reconstruct diet; three families (N, L, G) represented the majority (88%–100%) of the diet with A + S representing 0%–12%. Although we compared δ¹³C and δ¹⁵N values of wing, hair, and feces of spotted bats, feces best represented recent diet; wing and hair were more enriched than feces by 3‰ and 6‰, respectively. This pattern was consistent with that reported for other bat species. We suggest that spotted bats persist across a wide latitudinal gradient partly because they can forage on a variety of noctuid, geometrid, and lasiocampid moths. Using visual fecal inspection with stable isotope analysis provided information on families of moths consumed by an uncommon bat species.

Bohmann K., Monadjem A., Lehmkuhl Noer C., Rasmussen M., Zeale M. R., Clare E., Jones G., Willerslev E., Gilbert M. T. P. (2011): Molecular diet analysis of two African free-tailed bats (Molossidae) using high throughput sequencing. Plos One 6: e21441.
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Given the diversity of prey consumed by insectivorous bats, it is difficult to discern the composition of their diet using morphological or conventional PCR-based analyses of their faeces. We demonstrate the use of a powerful alternate tool, the use of the Roche FLX sequencing platform to deep-sequence uniquely 5′ tagged insect-generic barcode cytochrome c oxidase I (COI) fragments, that were PCR amplified from faecal pellets of two free-tailed bat species Chaerephon pumilus and Mops condylurus (family: Molossidae). Although the analyses were challenged by the paucity of southern African insect COI sequences in the GenBank and BOLD databases, similarity to existing collections allowed the preliminary identification of 25 prey families from six orders of insects within the diet of C. pumilus, and 24 families from seven orders within the diet of M. condylurus. Insects identified to families within the orders Lepidoptera and Diptera were widely present among the faecal samples analysed. The two families that were observed most frequently were Noctuidae and Nymphalidae (Lepidoptera). Species-level analysis of the data was accomplished using novel bioinformatics techniques for the identification of molecular operational taxonomic units (MOTU). Based on these analyses, our data provide little evidence of resource partitioning between sympatric M. condylurus and C. pumilus in the Simunye region of Swaziland at the time of year when the samples were collected, although as more complete databases against which to compare the sequences are generated this may have to be re-evaluated.

Vesterinen E. J., Lilley T., Laine V. N., Wahlberg N. (2013): Next generation sequencing of fecal DNA reveals the dietary diversity of the widespread insectivorous predator Daubenton’s bat (Myotis daubentonii) in Southwestern Finland. Plos One 8: e82168.
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Understanding predator-prey dynamics is a fundamental task in the evaluation of the adaptive capacities of species. However, direct observations or morphological identification of fecal remains do not offer an effective way to study the dietary ecology of elusive species, such as nocturnal insectivorous bats. However, recent advances in molecular techniques have opened a new method for identifying prey species from fecal samples. In this study, we amplified species-specific mitochondrial COI fragments from fecal DNA extractions from 34 individual Daubenton’s bats (Myotis daubentonii) collected between 2008 and 2010 from southwestern Finland. Altogether, 128 different species of prey were identified based on a comprehensive local DNA reference library. In our study area, Daubenton’s bats feed most frequently on insects of the orders Diptera (found in the diet of 94% individuals), Trichoptera (69%) and Lepidoptera (63%). The most frequent dipteran family in the diet was Chironomidae, which was found in 31 of 34 individuals. Most common prey species were chironomids Microtendipes pedellus (found in 50% of bats), Glyptotendipes cauliginellus (44%), and Procladius ferrugineus (41%). For the first time, an accurate species level list of the diet of the insectivorous Daubenton’s bat (Myotis daubentonii) in Finland is presented. We report a generally applicable method for describing the arthropod diet of vertebrate predators. We compare public databases to a national database to highlight the importance of a local reference database.

Bobrowiec P. E. D., Lemes M. R., Gribel R. (2015): Prey preference of the common vampire bat (Desmodus rotundus, Chiroptera) using molecular analysis. Journal of Mammalogy 96: 54-63.
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Morphological identification of prey fragments in vampire bat feces is impossible because of an exclusively blood-based diet. Therefore, studies of their foraging ecology require innovative approaches. We investigated the diet of Desmodus rotundus using a PCR–restriction fragment length polymorphism (RFLP) molecular method by amplifying the cytochrome b mitochondrial gene (380bp) from DNA fecal samples collected from captive bats fed with blood from chickens, cattle, pigs, dogs, and humans—the 5 most frequently attacked prey species in rural areas of the Brazilian Amazonia. The prey preference of the vampire bat was investigated in 18 riverine villages, where the availability of domestic animals to bats was quantified. Prey DNA amplified from fecal samples exhibited no visible signals of vampire bat DNA. A PCR–RFLP flowchart and a combination of 2 DNA restriction enzymes allowed the direct identification of prey to species level. The enzymes’ restriction profile did not overlap with those of vampire bats or wild mammal and avian species. Chickens were the most attacked prey species (61.4% of the identifications, n = 27), but pigs were highly preferred in relation to prey availability. This suggests a preference for mammalian blood in D. rotundus diet, with chickens exploited as a secondary food source. No wild vertebrate species was identified in the fecal samples, indicating that vampire bats are selectively feeding on the blood of domesticated animals, probably because they are more predictable and easily accessed resources.

Aizpurua O., Budinski I., Georgiakakis P., Gopalakrishnan S., Ibañez C., Mata V., Rebelo H., Russo D., Szodoray‐Parádi F., Zhelyazkova V., Zrncic V. (2018): Agriculture shapes the trophic niche of a bat preying on multiple pest arthropods across Europe: evidence from DNA metabarcoding. Molecular Ecology 27: 815-825.
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The interaction between agricultural production and wildlife can shape, and even condition, the functioning of both systems. In this study, we i) explored the degree to which a widespread European bat, namely the common bent‐wing bat Miniopterus schreibersii, consumes crop‐damaging insects at a continental scale, and ii) tested whether its dietary niche is shaped by the extension and type of agricultural fields. We employed a dual‐primer DNA metabarcoding approach to characterize arthropod 16S and COI DNA sequences within bat faecal pellets collected across 16 Southern European localities, to first characterize the bat species’ dietary niche, second measure the incidence of agricultural pests across their ranges and third assess whether geographical dietary variation responds to climatic, landscape diversity, agriculture type and vegetation productivity factors. We detected 12 arthropod orders, among which lepidopterans were predominant. We identified >200 species, 44 of which are known to cause agricultural damage. Pest species were detected at all but one sampling site and in 94% of the analysed samples. Furthermore, the dietary diversity of M. schreibersii exhibited a negative linear relation with the area of intensive agricultural fields, thus suggesting crops restrict the dietary niche of bats to prey taxa associated with agricultural production within their foraging range. Overall, our results imply that M. schreibersii might be a valuable asset for biological pest suppression in a variety of agricultural productions and highlight the dynamic interplay between wildlife and agricultural systems.

Arrizabalaga‐Escudero A., Clare E. L., Salsamendi E., Alberdi A., Garin I., Aihartza J., Goiti U. (2018): Assessing niche partitioning of co‐occurring sibling bat species by DNA metabarcoding. Molecular Ecology 27: 1273-1283.
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Niche partitioning through foraging is a mechanism likely involved in facilitating the coexistence of ecologically similar and co‐occurring animal species by separating their use of resources. Yet, this mechanism is not well understood in flying insectivorous animals. This is particularly true of bats, where many ecologically similar or cryptic species coexist. The detailed analysis of the foraging niche in sympatric, cryptic sibling species provides an excellent framework to disentangle the role of specific niche factors likely involved in facilitating coexistence. We used DNA metabarcoding to determine the prey species consumed by a population of sympatric sibling Rhinolophus euryale and Rhinolophus mehelyi whose use of habitat in both sympatric and allopatric ranges has been well established through radio tracking. Although some subtle dietary differences exist in prey species composition, the diet of both bats greatly overlapped (O_jk = 0.83) due to the consumption of the same common and widespread moths. Those dietary differences we did detect might be related to divergences in prey availabilities among foraging habitats, which prior radio tracking on the same population showed are differentially used and selected when both species co‐occur. This minor dietary segregation in sympatry may be the result of foraging on the same prey‐types and could contribute to reduce potential competitive interactions (e.g., for prey, acoustic space). Our results highlight the need to evaluate the spatial niche dimension in mediating the co‐occurrence of similar insectivorous bat species, a niche factor likely involved in processes of bat species coexistence.

Lim V. C., Ramli R., Bhassu S., Wilson J. J. (2018): Pollination implications of the diverse diet of tropical nectar-feeding bats roosting in an urban cave. PeerJ 6: e4572.
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Intense landscaping often alters the plant composition in urban areas. Knowing which plant species that pollinators are visiting in urban areas is necessary for understanding how landscaping impacts biodiversity and associated ecosystem services. The cave nectar bat, Eonycteris spelaea, is an important pollinator for many plants and is often recorded in human-dominated habitats. Previous studies of the diet of E. spelaea relied on morphological identification of pollen grains found in faeces and on the body of bats and by necessity disregarded other forms of digested plant material present in the faeces (i.e., plant juice and remnants). The main objective of this study was to examine the diet of the nectarivorous bat, E. spelaea, roosting in an urban cave at Batu Caves, Peninsular Malaysia by identifying the plant material present in the faeces of bats using DNA metabarcoding. Faeces were collected under the roost of E. spelaea once a week from December 2015 to March 2016. Plant DNA was extracted from the faeces, Polymerase chain reaction (PCR) amplified at ITS2 and rbcL regions and mass sequenced. The resultant plant operational taxonomic units were searched against NCBI GenBank for identification. A total of 55 species of plants were detected from faeces of E. spelaea including Artocarpus heterophyllus, Duabanga grandiflora and Musa spp. which are likely to be important food resources for the cave nectar bat. Many native plant species that had not been reported in previous dietary studies of E. spelaea were detected in this study including Bauhinia strychnoidea and Urophyllum leucophlaeum, suggesting that E. spelaea remains a crucial pollinator for these plants even in highly disturbed habitats. The detection of many introduced plant species in the bat faeces indicates that E. spelaea are exploiting them, particularly Xanthostemon chrysanthus, as food resources in urban area. Commercial food crops were detected from all of the faecal samples, suggesting that E. spelaea feed predominantly on the crops particularly jackfruit and banana and play a significant role in pollination of economically important plants. Ferns and figs were also detected in the faeces of E. spelaea suggesting future research avenues to determine whether the ‘specialised nectarivorous’ E. spelaea feed opportunistically on other parts of plants.

Kemp J., López-Baucells A., Rocha R., Wangensteen O. S., Andriatafika Z., Nair A., Cabeza M. (2019): Bats as potential suppressors of multiple agricultural pests: a case study from Madagascar. Agriculture, Ecosystems & Environment 269: 88-96.
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The conversion of natural habitats to agriculture is one of the main drivers of biotic change. Madagascar is no exception and land-use change, mostly driven by slash-and-burn agriculture, is impacting the island’s exceptional biodiversity. Although most species are negatively affected by agricultural expansion, some, such as synanthropic bats, are capable of exploring newly available resources and benefit from man-made agricultural ecosystems. As bats are known predators of agricultural pests it seems possible that Malagasy bats may be preferentially foraging within agricultural areas and therefore provide important pest suppression services. To investigate the potential role of bats as pest suppressors, we conducted acoustic surveys of insectivorous bats in and around Ranomafana National Park, Madagascar, during November and December 2015. We surveyed five landcover types: irrigated rice, hillside rice, secondary vegetation, forest fragment and continuous forest. 9569 bat passes from a regional assemblage of 19 species were recorded. In parallel, we collected faeces from the six most common bat species to detect insect pest species in their diet using DNA metabarcoding. Total bat activity was higher over rice fields when compared to forest and bats belonging to the open space and edge space sonotypes were the most benefited by the conversion of forest to hillside and irrigated rice. Two economically important rice pests were detected in the faecal samples collected – the paddy swarming armyworm Spodoptera mauritia was detected in Mops leucogaster samples while the grass webworm Herpetogramma licarsisalis was detected from Mormopterus jugularis and Miniopterus majori samples. Other crops pests detected included the sugarcane cicada Yanga guttulata, the macadamia nut-borer Thaumatotibia batrachopa and the sober tabby Ericeia inangulata (a pest of citrus fruits). Samples from all bat species also contained reads from important insect disease vectors. In light of our results we argue that Malagasy insectivorous bats have the potential to suppress agricultural pests. It is important to retain and maximise Malagasy bat populations as they may contribute to higher agricultural yields and promote sustainable livelihoods.

Kolkert H., Andrew R., Smith R., Rader R., Reid N. (2020): Insectivorous bats selectively source moths and eat mostly pest insects on dryland and irrigated cotton farms. Ecology and Evolution 10: 371-388.
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Insectivorous bats are efficient predators of pest arthropods in agroecosystems. This pest control service has been estimated to be worth billions of dollars to agriculture globally. However, few studies have explicitly investigated the composition and abundance of dietary prey items consumed or assessed the ratio of pest and beneficial arthropods, making it difficult to evaluate the quality of the pest control service provided. In this study, we used metabarcoding to identify the prey items eaten by insectivorous bats over the cotton-growing season in an intensive cropping region in northern New South Wales, Australia. We found that seven species of insectivorous bat (n = 58) consumed 728 prey species, 13 of which represented around 50% of total prey abundance consumed. Importantly, the identified prey items included major arthropod pests, comprising 65% of prey relative abundance and 13% of prey species recorded. Significant cotton pests such as Helicoverpa punctigera (Australian bollworm) and Achyra affinitalis (cotton webspinner) were detected in at least 76% of bat fecal samples, with Teleogryllus oceanicus (field crickets), Helicoverpa armigera (cotton bollworm), and Crocidosema plebejana (cotton tipworm) detected in 55% of bat fecal samples. Our results indicate that insectivorous bats are selective predators that exploit a narrow selection of preferred pest taxa and potentially play an important role in controlling lepidopteran pests on cotton farms. Our study provides crucial information for farmers to determine the service or disservice provided by insectivorous bats in relation to crops, for on-farm decision making.

Bell K. L., Batchelor K. L., Bradford M., McKeown A., Macdonald S. L., Westcott D. (2021): Optimisation of a pollen DNA metabarcoding method for diet analysis of flying-foxes (Pteropus spp.). Australian Journal of Zoology 68: 273-284.
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Determining the diet of flying-foxes can increase understanding of how they function as pollinators and seed dispersers, as well as managing any negative impacts of large roosts. Traditional methods for diet analysis are time consuming, and not feasible to conduct for hundreds of animals. In this study, we optimised a method for diet analysis, based on DNA metabarcoding of environmental DNA (eDNA) from pollen and other plant parts in the faeces. We found that existing eDNA metabarcoding protocols are suitable, with the most useful results being obtained using a commercial food DNA extraction kit, and sequencing 350–450 base pairs of a DNA barcode from the internally transcribed spacer region (ITS2), with ~550 base pairs of the chloroplast rubisco large subunit (rbcL) as a secondary DNA barcode. A list of forage plants was generated for the little red flying-fox (Pteropus scapulatus), the black flying-fox (Pteropus alecto) and the spectacled flying-fox (Pteropus conspicillatus) from our collection sites across Queensland. The diets were determined to comprise predominantly Myrtaceae species, particularly those in the genera Eucalyptus, Melaleuca and Corymbia. With more plant genomes becoming publicly available in the future, there are likely to be further applications of eDNA methods in understanding the role of flying-foxes as pollinators and seed dispersers.

Heim O., Puisto A. I., Sääksjärvi I., Fukui D., Vesterinen E. J. (2021): Dietary analysis reveals differences in the prey use of two sympatric bat species. Ecology and Evolution 11: 18651-18661.
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One mechanism for morphologically similar and sympatric species to avoid competition and facilitate coexistence is to feed on different prey items within different microhabitats. In the current study, we investigated and compared the diet of the two most common and similar-sized bat species in Japan—Murina ussuriensis (Ognev, 1913) and Myotis ikonnikovi (Ognev, 1912)—to gain more knowledge about the degree of overlap in their diet and their foraging behavior. We found that both bat species consumed prey from the orders of Lepidoptera and Diptera most frequently, while the proportion of Dipterans was higher in the diet of M. ikonnikovi. Furthermore, we found a higher prey diversity in the diet of M. ikonnikovi compared to that of M. ussuriensis that might indicate that the former is a more generalist predator than the latter. In contrast, the diet of M. ussuriensis contained many Lepidopteran families. The higher probability of prey items likely captured via gleaning to occur in the diet of M. ussuriensis in contrast to M. ikonnikovi indicates that M. ussuriensis might switch between aerial-hawking and gleaning modes of foraging behavior. We encourage further studies across various types of habitats and seasons to investigate the flexibility of the diet composition and foraging behavior of these two bat species.

Hughes M. J., de Torrez E. C. B., Ober H. K. (2021): Big bats binge bad bugs: Variation in crop pest consumption by common bat species. Agriculture, Ecosystems & Environment 314: 107414.
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As generalist predators, insectivorous bats exploit fluctuations in prey distribution and abundance. A more nuanced understanding of the influence of bats on arthropod pests requires documentation of the pest species bats consume and of the conditions associated with variation in rates of pest consumption. Here, we used high-throughput metabarcoding of DNA extracted from bat feces to investigate diets of 180 bats representing three Vespertilionidae species common to the southeastern US, a region dominated by agriculture and pine plantations. We detected 23 species of agricultural pests in bat diets, including pests responsible for severe economic damage, such as Helicoverpa zea, Spodoptera frugiperda, Chloridea virescens and Chrysodeixis includens. Incidence of pest consumption was high: 61% of all bats had consumed at least one agricultural pest species, with each bat consuming an average of 1.7 pest species. The likelihood of consumption of pests to row crops and the average size of pests consumed varied by bat species, with a large foliage-roosting species (Lasiurus seminolus) consuming a greater variety of pest species and pest species larger in size than smaller crevice, cavity, and cave roosting bat species (Nycticeus humeralis, Myotis austroriparius). Likelihood of pest consumption also varied among sampling periods (season) and among bats of different sizes (as reflected by wing length and mass). Overall, likelihood of pest consumption was higher in the late summer season than during spring or early summer, and higher among larger bats than smaller bats. Bat characteristics and seasonality were generally more effective than geographic features and weather conditions in predicting pest consumption patterns. Strategies for enhancing pest consumption services by bats in agroecosystems should strive to maintain and enhance diverse bat populations on a landscape scale by protecting and augmenting roost structures appropriate for each species. Our finding of widespread pest consumption by bats contributes to mounting evidence worldwide of the important role bats play in agricultural systems and highlights the value of incorporating bat conservation into integrated pest management programs globally.

O’Rourke D. R., Mangan M. T., Mangan K. E., Bokulich N. A., MacManes M. D., Foster J. T. (2021): Lord of the Diptera (and moths and a spider): Molecular diet analyses and foraging ecology of Indiana bats in Illinois. Frontiers in Ecology and Evolution 9: 623655.
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Effective management of endangered or threatened wildlife requires an understanding of how foraging habitats are used by those populations. Molecular diet analysis of fecal samples offers a cost-effective and non-invasive method to investigate how diets of wild populations vary with respect to spatial and temporal factors. For the federally endangered Indiana bat (Myotis sodalis), documenting its preferred food sources can provide critical information to promote effective conservation of this federally endangered species. Using cytochrome oxidase I amplicon sequence data from Indiana bat guano samples collected at two roosting areas in Cypress Creek National Wildlife Refuge, we found that dipteran taxa (i.e., flies) associated with riparian habitats were the most frequently detected taxon and represented the majority of the sequence diversity among the arthropods sampled. A select few arthropods from other taxa—especially spiders—are also likely important to Indiana bat diets in this refuge. A supervised learning analysis of diet components suggest only a small fraction of the frequently detected taxa are important contributors to spatial and temporal variation. Overall, these data depict the Indiana bat as a generalist consumer whose diet includes some prey items associated with particular seasonal or spatial components, along with other taxa repeatedly consumed throughout the entire foraging season. These molecular diet analyses suggest that protecting foraging resources specifically associated with the riparian habitat of Cypress Creek National Wildlife Refuge is essential to promote effective Indiana bat conservation.

Schattanek P., Riccabona S. A., Rennstam Rubbmark O., Traugott M. (2021): Detection of prey DNA in bat feces: Effects of time since feeding, meal size, and prey identity. Environmental DNA 3: 959-969.
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Molecular analyses of feces are widely used to study the feeding ecology of bats. However, little is known about how detectability of prey DNA from bat feces is influenced by gut retention time, mass of prey eaten, or prey identity, which hampers the interpretation of field data. Here, we address these knowledge gaps by conducting a feeding experiment in which different insect species and meal sizes were offered to two bat species, Myotis daubentonii and Pipistrellus nathusii. The feeding regime consisted of three main and three side prey species fed in different masses, whereupon feces were collected over 72 h and examined for prey DNA using a species-specific multiplex PCR system. DNA of all three main prey species was detectable from feces produced within 1 h after consumption of a meal. Prey detectability decreased over the following 10–20 h, and after 40 h only spurious detections occurred. For both prey and bats, species identity affected detectability: for example, mealworms were detected for a longer time post-feeding compared to wax moths/house flies, and the latter were detected for a shorter time in M. daubentonii than in P. nathusii. While DNA from all three side prey species was detectable, signals were weak and detections limited to the first 20 h after consumption. These findings indicate that detectability of prey DNA from bat feces is foremost affected by the mass of prey consumed and time post-feeding, whereas it does not strongly differ between prey species. To increase the detection of prey ingested in low mass, we suggest collecting fecal samples soon after bats return from foraging, as detectability is highest right after feeding occurs, and detections largely depict the previous foraging bout (with detection windows usually <24 h).

Tournayre O., Leuchtmann M., Galan M., Trillat M., Piry S., Pinaud D., Filippi-Codaccioni O., Pontier D., Charbonnel N. (2021): eDNA metabarcoding reveals a core and secondary diets of the greater horseshoe bat with strong spatio‐temporal plasticity. Environmental DNA 3: 277-296.
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Divoll T. J., Brown V. A., McCracken G. F., O’Keefe J. M. (2022): Prey size is more representative than prey taxa when measuring dietary overlap in sympatric forest bats. Environmental DNA 4: 1407-1419.
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In temperate forests, insectivorous bats need to use variable hunting strategies as prey availability fluctuates during the growing season. At sites with variable amounts of forested habitat, sympatric bat species may exhibit high dietary overlap, but mechanisms of coexistence are unknown. We analyzed the diets of sympatric Myotis septentrionalis and Myotis sodalis in two different landscapes in central Indiana, USA: a riparian agricultural site and a managed forest site, aiming to assess interspecific dietary overlap. We collected fecal samples from 166 Myotis over four maternity seasons and used DNA metabarcoding to assess niche overlap by operational taxonomic units (OTUs, n = 708) and prey-size taxa (n = 79). Analyzing diets by prey-size classes, as opposed to only species, provided an alternative interpretation of dietary richness and overlap that may more accurately represent predator perceptions of prey. With overall prey sizes ranging from 2–36 mm, M. septentrionalis (n = 88) consumed larger prey (10.0 ± 5.5 mm, μ ± σ) than M. sodalis (n = 78; 8.9 ± 5.0 mm). Myotis sodalis had a more diverse diet (547 OTUs) compared to M. septentrionalis (453 OTUs) despite the smaller sample size. Of the 708 OTUs detected, 41% (292) were common across bat species. We found greater dietary overlap between species within each site than within either species across sites, suggesting both species mostly consume whatever insects are available on the landscape. Flexible hunting strategies may allow these species to coexist by consuming different sizes of prey at different rates in the tradeoff between travel cost and profitability. Prey size is an important measure for dietary overlap and resource partitioning in bats that few studies have yet considered, with implications for linking predators, prey, and habitats.

Maslo B., Mau R. L., Kerwin K., McDonough R., McHale E., Foster J. T. (2022): Bats provide a critical ecosystem service by consuming a large diversity of agricultural pest insects. Agriculture, Ecosystems & Environment 324: 107722.
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Biodiversity directly influences the delivery of multiple ecosystem services, most notably within agriculture. Projected future global demands for food, fiber and bioenergy will require enhancement of agricultural productivity, but favoring biodiversity-based ecosystem services generally remains underutilized in agricultural practice. In addition, agricultural intensification is a key driver of biodiversity loss. A significant obstacle preventing the adoption of ecologically sensitive practices is a lack of knowledge of the species delivering the services. Insectivorous bats have long been suggested to regulate insect pest populations and may be a critical component of biodiversity-based ecosystem services. Bats may also serve as agents of insect pest surveillance through environmental DNA (eDNA) monitoring approaches. However, the biological and economic importance of bats to agriculture remains under-quantified. Here we catalogued the dietary niche of two North American bats, little brown bat (Myotis lucifugus) and big brown bat (Eptesicus fuscus), through DNA metabarcoding of guano collected from seven roosting sites over a 26-week period. We measured the frequency of occurrence of known pest species in guano samples, compared interspecific differences in diet, and examined seasonal patterns in prey selection. Overall, we detected 653 unique prey species, 160 of which were known agricultural pests or disease vectors. Species diversity of prey species consumed varied by bat species and across the season, with big brown bats accounting for the majority of arthropod diversity detected. However, little brown bats consumed relatively more aquatic insects than big brown bats, suggesting that increased bat species richness in a landscape can amplify their net pest regulation service. Further, we hypothesized that detection probabilities of target insect pests would be higher in guano samples than in conventional survey methods. Multi-survey occupancy modeling revealed significantly lower detectability in bat guano than in conventional monitoring traps, however, highlighting important tradeoffs in selection of survey methods. Overall, the results presented here contribute to a growing evidence base supporting the role bats play in the provisioning of biodiversity-based ecosystem services.

O’Rourke D., Rouillard N. P., Parise K. L., Foster J. T. (2022): Spatial and temporal variation in New Hampshire bat diets. Scientific Reports 12: 14334.
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Insectivorous bats consume a diverse array of arthropod prey, with diets varying by bat species, sampling location, and season. North American bat diets remain incompletely described, which is concerning at a time when many bat and insect populations appear to be declining. Understanding the variability in foraging is thus an essential component for effective bat conservation. To comprehensively evaluate local foraging, we assessed the spatial and temporal variability in prey consumed by the little brown bat, Myotis lucifugus, in New Hampshire, USA. We collected bat guano samples from 20 sites over 2 years and analyzed sequence data for 899 of these samples using a molecular metabarcoding approach targeting the cytochrome oxidase I subunit (COI) gene. Some prey items were broadly shared across locations and sampling dates, with the most frequently detected arthropod orders broadly similar to previous morphological and molecular analyses; at least one representative sequence variant was assigned to Coleoptera in 92% of samples, with other frequently detected orders including Diptera (73%), Lepidoptera (65%), Trichoptera (38%), and Ephemeroptera (32%). More specifically, two turf and forest pests were routinely detected: white grubs in the genus Phyllophaga (50%), and the Asiatic Garden beetle, Maladera castanea (36%). Despite the prevalence of a few taxa shared among many samples and distinct seasonal peaks in consumption of specific arthropods, diet composition varied both temporally and spatially. However, species richness did not strongly vary indicating consumption of a broad diversity of taxa throughout the summer. These data characterize little brown bats as flexible foragers adept at consuming a broad array of locally available prey resources.

CARNIVORES

Corbett L. K. (1989): Assessing the diet of dingoes from feces: a comparison of 3 methods. The Journal of Wildlife Management 2: 343-346.
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I assessed diet of the dingo (Canis familiaris dingo) from 2,495 fecal samples using frequency of occurrence of prey types, relative weight of remains of prey types, and biomass of ingested prey types. There were no significant differences between methods in ranking prey types provided that ≥70 fecal samples/month were analyzed. The widely used frequency method that is used to understand what and relatively how much is eaten is justified in diet studies of dingoes and other carnivores. Only the biomass ingested method allows an evaluation of the biomass or numbers of particular prey species that are eaten. For dingoes, and probably also for other carnivores, objective adjustments need to be applied to account for differences in size between juvenile and adult large prey species and for differences in the proportion of each age class eaten.

Munro R. H. M., Nielsen S. E., Price M. H., Stenhouse G. B., Boyce M. S. (2006): Seasonal and diel patterns of grizzly bear diet and activity in west-central Alberta. Journal of Mammalogy 87: 1112-1121.
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Seasonal food habits and activity patterns were examined for grizzly bears (Ursus arctos) in west-central Alberta, Canada, to better understand habitat requirements in a threatened population. Food habits were based on an analysis of 665 feces collected from 18 grizzly bears between April and October 2001–2003. Trends in the use of foods were comparable to those of other central Rocky Mountain populations, with minor differences likely reflecting regional habitat and forage availability. Five activities (bedding, sweet vetch digging, insect feeding, frugivory, and ungulate kills) were identified for each of 1,032 field-visited global positioning system radiotelemetry locations from 9 female grizzly bears. We predicted the probability of each activity during relevant periods by time of day (crepuscular, diurnal, and nocturnal) and habitat. Selection ratios were used to assess which habitat and time periods were selected. Activity patterns changed considerably over a 24-h period, with foraging activities occurring mostly during diurnal and crepuscular periods and bedding at night. Habitats were important predictors of activity. Forested areas were selected for bedding areas, whereas digging, insect-foraging, and frugivory activities were associated with herbaceous, recently disturbed forest and open-canopy forests. We suggest that researchers consider behavior and time of day in analyses of habitat selection to improve explanations of habitat use and mechanisms of selection.

Prugh L. R., Arthur S. M., Ritland C. E. (2008): Use of faecal genotyping to determine individual diet. Wildlife Biology 14: 318-330.
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Faecal genotyping has been proposed as a method to examine the diets of individuals, but this application has been virtually unexplored by wildlife biologists. We used faecal genotyping and conventional scat analysis to determine the diets of 42 coyotes Canis latrans belonging to nine social groups in Alaska. We use rarefaction to examine the effect of scat sample size on the accuracy and precision of individual diets, and we simulate diets from scats to determine how diet richness and evenness affect sample size requirements. We then demonstrate the utility of this technique by examining variation in diet among individual coyotes and social groups in relation to prey availability. Estimates of diet diversity and composition were highly variable when <10 scats were used to construct the diet. Diets simulated with a uniform (i.e. even) distribution of prey items required generally smaller sample sizes to estimate diet diversity and richness than diets with exponentially distributed items; however, items in actual scats were exponentially distributed. We found moderate dietary variability among individuals in our study area, and diet overlap was higher among coyotes within social groups than between groups. As predicted by optimal foraging theory, the niche widths of all coyote groups expanded as their primary prey (the snowshoe hare Lepus americanus) became scarce during our three-year study. Despite increased niche width, diet overlap among groups remained constant, suggesting that coyotes selected differing alternative prey. Spatiotemporal variation in snowshoe hare availability explained 70% of the variation in hare consumption among groups, indicating that variation in local prey availability may be the primary cause of diet variation among coyotes. Although faecal genotyping can be used to address ecological questions at the individual level, studies should be designed specifically for this purpose so that sufficient numbers of faeces can be obtained.

Tollit D. J., Schulze A. D., Trites A. W., Olesiuk P. F., Crockford S. J., Gelatt T. S., Ream R. R., Miller K. M. (2009): Development and application of DNA techniques for validating and improving pinniped diet estimates. Ecological Applications 19: 889-905.
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Polymerase chain reaction techniques were developed and applied to identify DNA from >40 species of prey contained in fecal (scat) soft‐part matrix collected at terrestrial sites used by Steller sea lions (Eumetopias jubatus) in British Columbia and the eastern Aleutian Islands, Alaska. Sixty percent more fish and cephalopod prey were identified by morphological analyses of hard parts compared with DNA analysis of soft parts (hard parts identified higher relative proportions of Ammodytes sp., Cottidae, and certain Gadidae). DNA identified 213 prey occurrences, of which 75 (35%) were undetected by hard parts (mainly Salmonidae, Pleuronectidae, Elasmobranchii, and Cephalopoda), and thereby increased species occurrences by 22% overall and species richness in 44% of cases (when comparing 110 scats that amplified prey DNA). Prey composition was identical within only 20% of scats. Overall, diet composition derived from both identification techniques combined did not differ significantly from hard‐part identification alone, suggesting that past scat‐based diet studies have not missed major dietary components. However, significant differences in relative diet contributions across scats (as identified using the two techniques separately) reflect passage rate differences between hard and soft digested material and highlight certain hypothesized limitations in conventional morphological‐based methods (e.g., differences in resistance to digestion, hard part regurgitation, partial and secondary prey consumption), as well as potential technical issues (e.g., resolution of primer efficiency and sensitivity and scat subsampling protocols). DNA analysis of salmon occurrence (from scat soft‐part matrix and 238 archived salmon hard parts) provided species‐level taxonomic resolution that could not be obtained by morphological identification and showed that Steller sea lions were primarily consuming pink (Oncorhynchus gorbuscha) and chum (Oncorhynchus keta) salmon. Notably, DNA from Atlantic salmon (Salmo salar) that likely originated from a distant fish farm was also detected in two scats from one site in the eastern Aleutian Islands. Overall, molecular techniques are valuable for identifying prey in the fecal remains of marine predators. Combining DNA and hard‐part identification will effectively alleviate certain predicted biases and will ultimately enhance measures of diet richness, fisheries interactions (especially salmon‐related ones), and the ecological role of pinnipeds and other marine predators, to the benefit of marine wildlife conservationists and fisheries managers.

O’Meara D. B., Sheehy E., Turner P. D., O’Mahony D., Harrington A. P., Denman H., Lawton C., MacPherson J., O’Reilly C. (2014): Non-invasive multi-species monitoring: real-time PCR detection of small mammal and squirrel prey DNA in pine marten (Martes martes) scats. Acta Theriologica 59: 111-117.
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DNA identification of mammal species occurring in the diet of a predator is potentially a useful approach to remotely monitor the distribution of multiple species. This is important in Ireland, where it has been shown that the combined presence of the introduced bank vole and greater white-toothed shrew impact the distribution of the indigenous small mammals, the wood mouse and pygmy shrew. Direct monitoring of these species and their interactions requires trapping, a labour-intensive and costly approach. In this study, we applied an indirect method by genetically testing the presence of small mammals in pine marten scats collected during the National Pine Marten Survey (2005–2007) to map their distribution. We also included additional scats to investigate if less common prey items, the red squirrel and grey squirrel, could also be detected. This study demonstrates that all target species were genetically detected from pine marten scats. This strategy could be implemented as a monitoring programme for indigenous and introduced mammal species.

Kavčič I., Adamič M., Kaczensky P., Krofel M., Kobal M., Jerina K. (2015): Fast food bears: brown bear diet in a human-dominated landscape with intensive supplemental feeding. Wildlife Biology 21: wlb.00013.
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Distribution, quantity and quality of food resources affect the diet and several other life-history traits of large mammals. Supplemental feeding of wildlife has high potential for influencing the behaviour and diet of opportunistic omnivores, such as bears. Supplemental feeding of brown bears Ursus arctos is a common practice in several European countries, but the effects of this controversial and expensive management measure on bear diet and behaviour are poorly understood. We analysed 714 brown bear scats collected throughout the year in three regions of Slovenia with different densities of supplemental feeding sites. Supplemental food was the most important food category in the bear diet and represented 34% of the annual estimated dietary energy content (maize: 22%, livestock carrion: 12%). The proportion of supplemental food in the diet varied with season and region, being highest in spring and in the region with the highest density of feeding sites. However, considerable seasonal changes in bear diet, despite year-round access to supplemental food, suggest that bears prefer high-energy natural food sources, particularly insects, fruits, and hard mast, when available. Despite high availability and use of supplemental food, human—bear conflicts are frequent in Slovenia. In addition, evidence from earlier studies suggests that changes in diet and foraging behaviour due to supplemental feeding may affect several aspects of bear biology and in some cases increase the probability of human—bear conflicts. Thus, we caution against promoting unconditional supplemental feeding as a measure to prevent or reduce human—bear conflicts.

Emami-Khoyi A., Hartley D. A., Paterson A. M., Boren L. J., Cruickshank R. H., Ross J. G., Murphy E. C., Else T. A. (2016): Identifying prey items from New Zealand fur seal (Arctocephalus forsteri) faeces using massive parallel sequencing. Conservation Genetics Resources 8: 343-352.
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The New Zealand fur seal (Arctocephalus forsteri) is one of many pinniped species that has shown a remarkable recovery from the brink of extinction after cessation of commercial sealing during the eighteenth and nineteenth centuries. It is commonly believed that this species competes with recreational and commercial fisheries. We identified prey items using massive parallel sequencing from New Zealand fur seal faecal samples that were collected throughout the species distribution. The data support generalist feeding behaviour for this species. The diet composition showed significant geographical and inter-seasonal variation. As many as 46 species of fish and 18 species of cephalopod were identified from a single colony. The data suggest cartilaginous species (sharks, rays, and skates) constitute an important part of the New Zealand fur seal diet. Approximately 10 % of the species identified in the seal diet were of significant commercial value, which indicates some qualitative food competition between New Zealand fur seals and commercial fisheries in exploiting marine species.

Berry T. E., Osterrieder S. K., Murray D. C., Coghlan M. L., Richardson A. J., Grealy A. K., Stat M., Bejder L., Bunce M. (2017): DNA metabarcoding for diet analysis and biodiversity: A case study using the endangered Australian sea lion (Neophoca cinerea). Ecology and Evolution 7: 5435-5453.
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The analysis of apex predator diet has the ability to deliver valuable insights into ecosystem health, and the potential impacts a predator might have on commercially relevant species. The Australian sea lion (Neophoca cinerea) is an endemic apex predator and one of the world’s most endangered pinnipeds. Given that prey availability is vital to the survival of top predators, this study set out to understand what dietary information DNA metabarcoding could yield from 36 sea lion scats collected across 1,500 km of its distribution in southwest Western Australia. A combination of PCR assays were designed to target a variety of potential sea lion prey, including mammals, fish, crustaceans, cephalopods, and birds. Over 1.2 million metabarcodes identified six classes from three phyla, together representing over 80 taxa. The results confirm that the Australian sea lion is a wide‐ranging opportunistic predator that consumes an array of mainly demersal fauna. Further, the important commercial species Sepioteuthis australis (southern calamari squid) and Panulirus cygnus (western rock lobster) were detected, but were present in <25% of samples. Some of the taxa identified, such as fish, sharks and rays, clarify previous knowledge of sea lion prey, and some, such as eel taxa and two gastropod species, represent new dietary insights. Even with modest sample sizes, a spatial analysis of taxa and operational taxonomic units found within the scat shows significant differences in diet between many of the sample locations and identifies the primary taxa that are driving this variance. This study provides new insights into the diet of this endangered predator and confirms the efficacy of DNA metabarcoding of scat as a noninvasive tool to more broadly define regional biodiversity.

Hui T. C., Morita Y., Kobayashi Y., Mitani Y., Miyashita K. (2017): Dietary analysis of harbour seals (Phoca vitulina) from faecal samples and overlap with fisheries in Erimo, Japan. Marine Ecology 38: e12431.
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The number of harbour seals in Japan has been rebounding since protection began in the mid‐1980s. With the increase in seal numbers, increased conflict with fisheries has occurred through depredation and the belief that seals compete with fisheries for prey. However, competition can only be determined if the prey species and quantities seals consume over time are known. We studied the diet of harbour seals in Erimo, site of the largest population of harbour seals in Japan, from 2011 to 2012 and assessed the degree of prey overlap with local fisheries. We used both hard parts and DNA techniques to identify prey items in seal scats, and compared these results to local fisheries data. A total of 46 prey occurrences was detected by both methods, of which 17 matched between techniques at least to the family level. Hard parts methods identified five incidences of prey undetected by DNA methods in five scats (one incidence per scat). DNA methods identified 24 additional prey occurrences in 13 scats, for which no hard part evidence for that prey had been found. This more than doubled the total number of prey occurrences across the 15 scats compared. Overall, the most frequently occurring harbour seal prey were walleye pollock (Theragra chalcogramma), sculpins and snailfishes. In contrast, the top three groups targeted by fisheries were codfishes, salmon and invertebrates. Many species common in the harbour seal’s diet such as snailfishes and blennies were not targeted by fisheries. Fishes such as greenlings, sculpins, rockfishes and Japanese anchovy (Engraulis japonicus) were common in the diet of harbour seals, but made up a very small proportion of fisheries catches in Erimo. The importance of other prey species varied between seasons. Sculpins, greenlings and forage fishes were the top three prey groups for harbour seals in spring by percent modified frequency of occurrence, whereas the most important target groups by percentage mass caught by fisheries were codfishes, cephalopods and other invertebrates. Sculpins and rockfishes remained as important prey items for harbour seals in summer and autumn, in addition to codfishes, snailfishes and blennies. In contrast, the main groups caught by fisheries in summer were other fishes, particularly Japanese amberjack (Seriola quinqueradiata) and various species of sharks and eels, and invertebrates. By autumn, Erimo fisheries had focussed on catching salmon. Salmon were taken by harbour seals as well during this period, but at a relatively lower frequency compared to other prey groups. The results of our study show that although harbour seals consumed several of the prey species targeted by fisheries, the relative importance of these species to seals and fisheries and the seasons in which they were targeted were different.

Forin-Wiart M. A., Poulle M. L., Piry S., Cosson J. F., Larose C., Galan M. (2018): Evaluating metabarcoding to analyse diet composition of species foraging in anthropogenic landscapes using Ion Torrent and Illumina sequencing. Scientific Reports 8: 17091.
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DNA metabarcoding of faecal samples is being successfully used to study the foraging niche of species. We assessed the ability of two benchtop high-throughput sequencing (HTS) platforms, to identify a large taxonomic array of food items from domestic cats Felis silvestris catus, including prey and human-related food taxa (pet food and leftovers leaving undetectable solid remains in faeces). Scats from a captive feeding trial (n = 41) and from free-ranging individuals (n = 326) were collected and analysed using a cytb mini-barcode in independent PCR replicates on the Ion PGM and the MiSeq platforms. Outputs from MiSeq were more sensitive and reproducible than those from Ion PGM due to a higher sequencing depth and sequence quality on MiSeq. DNA from intact prey taxa was detected more often (82% of the expected occurrences) than DNA from pet food (54%) and raw fish and meat (31%). We assumed that this variability was linked to different degree of DNA degradation: The Ion PGM detected significantly less human-linked food, birds, field voles, murids and shrews in the field-collected samples than the MiSeq platform. Pooling the replicates from both platforms and filtering the data allowed identification of at least one food item in 87.4% of the field-collected samples. Our DNA metabarcoding approach identified 29 prey taxa, of which 25 to species level (90% of items) including 9 rodents, 3 insectivores, 12 birds and 1 reptile and 33 human-related food taxa of which 23 were identified to genus level (75% of items). Our results demonstrate that using HTS platforms such as MiSeq, which provide reads of sufficiently high quantity and quality, with sufficient numbers of technical replicates, is a robust and non-invasive approach for further dietary studies on animals foraging on a wide range of food items in anthropogenic landscapes.

Reisinger R. R., Landman M., Mgibantaka N., Smale M. J., Bester M. N., De Bruyn P. N., Pistorius P. A. (2018): Overlap and temporal variation in the diets of sympatric Antarctic and Subantarctic fur seals (Arctocephalus spp.) at Marion Island, Prince Edward Islands. Polar Research 37: 1451142.
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Antarctic (Arctocephalus gazella) and Subantarctic (A. tropicalis) fur seals are important predators in the Southern Ocean. Marion Island (southern Indian Ocean) hosts the largest sympatric breeding populations of these two species. Environmental and population changes here over two decades may have influenced their diet and trophic interactions. To quantify diet, we analysed prey remains in scat samples from Antarctic (661 scats) and Subantarctic (750 scats) fur seals collected at Marion Island (2006–2010). We assessed diet composition over time and calculated dietary overlap. The diet of both species was dominated by fish prey (98.2% and 99.4% of prey items), mainly myctophids. Antarctic fur seals consumed small numbers of penguins, cephalopods and crustaceans. In Subantarctic fur seal scats, crustaceans and cephalopods were rare and penguin remains were absent. The diets of the two species overlapped substantially (Pianka’s index = 0.98), however, small but significant differences in the relative proportions of prey were evident. Seasonal and annual diet changes suggest that their diet is similarly influenced by patterns of local prey availability and abundance. Despite substantial changes in the population size and trajectory of Antarctic and Subantarctic fur seals – which would be expected to influence trophic interactions between them – comparing our data to those from earlier studies (1989–2000) did not reveal significant long-term dietary changes in either species.

Schwarz D., Spitzer S. M., Thomas A. C., Kohnert C. M., Keates T. R., Acevedo‐Gutiérrez A. (2018): Large‐scale molecular diet analysis in a generalist marine mammal reveals male preference for prey of conservation concern. Ecology and Evolution 8: 9889-9905.
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Sex‐specific diet information is important in the determination of predator impacts on prey populations. Unfortunately, the diet of males and females can be difficult to describe, particularly when they are marine predators. We combined two molecular techniques to describe haul‐out use and prey preferences of male and female harbor seals (Phoca vitulina) from Comox and Cowichan Bay (Canada) during 2012–2013. DNA metabarcoding quantified the diet proportions comprised of prey species in harbor seal scat, and qPCR determined the sex of the individual that deposited each scat. Using 287 female and 260 male samples, we compared the monthly sex ratio with GLMs and analyzed prey consumption relative to sex, season, site, and year with PERMANOVA. The sex ratio between monthly samples differed widely in both years (range = 12%–79% males) and showed different patterns at each haul‐out site. Male and female diet differed across both years and sites: Females consumed a high proportion of demersal fish species while males consumed more salmonid species. Diet composition was related to both sex and season (PERMANOVA: R² = 27%, p < 0.001; R² = 24%, p < 0.001, respectively) and their interaction (PERMANOVA: R² = 11%, p < 0.001). Diet differences between males and females were consistent across site and year, suggesting fundamental foraging differences, including that males may have a larger impact on salmonids than females. Our novel combination of techniques allowed for both prey taxonomic and spatiotemporal resolution unprecedented in marine predators.

Smith J. A., Thomas A. C., Levi T., Wang Y., Wilmers C. C. (2018): Human activity reduces niche partitioning among three widespread mesocarnivores. Oikos 127: 890-901.
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Anthropogenic disturbances can constrain the realized niche space of wildlife by inducing avoidance behaviors and altering community dynamics. Human activity might contribute to reduced partitioning of niche space by carnivores that consume similar resources, both by promoting tolerant species while also altering behavior of species (e.g. activity patterns). We investigated the influence of anthropogenic disturbance on habitat and dietary niche breadth and overlap among competing carnivores, and explored if altered resource partitioning could be explained by human‐induced activity shifts. To describe the diets of coyotes, bobcat, and gray foxes, we designed a citizen science program to collect carnivore scat samples in low‐ (‘wildland’) and high‐ (‘interface’) human‐use open space preserves, and obtained diet estimates using a DNA metabarcoding approach. Habitat use was determined at scat locations. We found that coyotes expanded habitat and dietary niche breadth in interface preserves, whereas bobcats and foxes narrowed both niche breadth measures. High human use was related to increased dietary niche overlap among all mesocarnivore pairs, increased coyote habitat overlap with bobcats and foxes, and a small reduction in habitat overlap between bobcats and foxes. The strongest increase in diet overlap was among coyotes and foxes, which was smaller in magnitude than their habitat overlap increase. Finally, coyote scats were more likely to contain nocturnal prey in interface preserves, whereas foxes appeared to reduce consumption of nocturnal prey. Our results suggest that dominant and generalist mesocarnivores may encroach on the niche space of subordinate mesocarnivores in areas with high human activity, and that patterns in resource use may be related to human‐induced activity shifts.

Momeni S., Malekian M., Hemami M. R. (2019): Molecular versus morphological approaches to diet analysis of the caracal (Caracal caracal). Mammalia 83: 586-592.
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Diet analysis is an essential part in understanding the biology of a species and functioning of ecosystems. Traditional morphological identification of undigested remains in the scats and molecular analyses of prey species’ DNA have previously been used to assess diet. In the present study, caracal diet in the Abbasabad Wildlife Refuge, Central Iran, was investigated using both molecular and morphological methods. We collected 22 scat samples from caracal dens in the region. Feces were washed on sieves and their remaining components were morphologically identified. We also targeted a 307-base pair fragment of the cytochrome b gene to amplify and sequence the species’ DNA. Morphological analyses revealed that 76% of the diet comprised rodent species. We identified a total of nine prey taxa using the molecular method, including six rodents, one hare, one hedgehog and one wild goat. There was a general agreement between the molecular and morphological results; however, molecular methods tended to allow a better identification of the prey species. Therefore, the DNA-based approach acts as a valuable complement to current morphological methods in the study of a rare felid’s diet when no hair reference library exists.

Nawaz M. A., Valentini A., Khan N. K., Miquel C., Taberlet P., Swenson J. E. (2019): Diet of the brown bear in Himalaya: combining classical and molecular genetic techniques. Plos One 14: e0225698.
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The ecological requirements of brown bears are poorly known in the Himalaya region, which complicates conservation efforts. We documented the diet of the Himalayan brown bear (Ursus arctos isabellinus) by combining classical scat analysis and a newly developed molecular genetic technique (the trnL approach), in Deosai National Park, Pakistan. Brown bears consumed over 50 plant species, invertebrates, ungulates, and several rodents. Eight plant families; Poaceae, Polygonaceae, Cyperaceae, Apiaceae, Asteraceae, Caryophyllaceae, Lamiaceae, and Rubiaceae were commonly eaten with graminoids comprising the bulk of the diet. Golden marmots comprised the major mammalian biomass in the park, and were also the main meat source for bears. Animal matter, making 36% of dietary content, contributed half of the digestible energy, due to its higher nutritious value. We did not find a significant temporal pattern in diet, perhaps because the availability of the major diet (graminoids) did not change over the foraging period. Male brown bears were more carnivorous than females, probably because of their larger size, which requires higher energy and also makes them more efficient in capturing marmots. Frequencies of three plant species were also significantly higher in male brown bears; Bistorta affinis, Carex diluta, and Carex sp. Diet of the brown bear differed significantly between the park and surrounding valleys. In valleys, diet consisted predominantly of graminoids and crops, whereas the park provided more nutritious and diverse foodThe estimated digestible energy available to brown bears in Deosai was the lowest documented among brown bear populations, due to the lack of fruits and a relatively lower meat content. The low nutritious diet and high cost of metabolism in a high-altitude environment, probably explains the very low reproductive potential of this population.

Nelms S. E., Parry H. E., Bennett K. A., Galloway T. S., Godley B. J., Santillo D., Lindeque P. K. (2019): What goes in, must come out: combining scat‐based molecular diet analysis and quantification of ingested microplastics in a marine top predator. Methods in Ecology and Evolution 10: 1712-1722.
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Microplastics (plastic particles <5 mm in size) are highly available for ingestion by a wide range of organisms, either through direct consumption or indirectly, via trophic transfer, from prey to predator. The latter is a poorly understood, but potentially major, route of microplastic ingestion for marine top predators. We developed a novel and effective methodology pipeline to investigate dietary exposure of wild top predators (grey seals; Halichoerus grypus) to microplastics, by combining scat‐based molecular techniques with a microplastic isolation method. We employed DNA metabarcoding, a rapid method of biodiversity assessment, to garner detailed information on prey composition from scats, and investigated the potential relationship between diet and microplastic burden. Outcomes of the method development process and results of both diet composition from metabarcoding analysis and detection of microplastics are presented. Importantly, the pipeline performed well and initial results suggest the frequency of microplastics detected in seal scats may be related to the type of prey consumed. Our non‐invasive, data‐rich approach maximizes time and resource–efficiency, while minimizing costs and sample volumes required for analysis. This pipeline could be used to underpin a much‐needed increase in understanding of the relationship between diet composition and rates of microplastic ingestion in high trophic level species.

Werhahn G., Kusi N., Li X., Chen C., Zhi L., Lázaro Martín R., Sillero-Zubiri C., Macdonald D. W. (2019): Himalayan wolf foraging ecology and the importance of wild prey. Global Ecology and Conservation 20: e00780.
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Carnivore predation on livestock and game species leads to human-carnivore conflict. Thus, understanding the foraging ecology of threatened carnivores is important for conservation planning. We explore the summer diet of the Himalayan wolf, and of sympatric carnivores, based on the analysis of 257 field collected and genetically confirmed scat samples collected across three study areas in the Himalayas of Nepal (Humla, Dolpa, and Kanchenjunga Conservation Area) and two study areas on the Tibetan Plateau of China (Zhaqing and Namsai Township). We compared the prey species consumed to the relative availability of wild and domestic prey species. Himalayan wolves tend to select wild over domestic prey, smaller (e.g. Tibetan gazelle, Procapra picticaudata) over larger sized wild ungulates (e.g. White-lipped deer, Cervus albirostris), and plains-dwelling (Tibetan gazelle) over cliff-dwelling ungulates (naur, Pseudois nayaur). Tibetan gazelle was consistently selected for by the Himalayan wolf and smaller mammals such as Himalayan marmot (Marmota himalayana), woolly hare (Lepus oiostolus) and pikas (Ochotona spp.) are important supplementary food resources. Himalayan wolves avoided livestock which showed a seasonal high abundance, that exceeded many-fold the abundance of wild prey species during the summer study period. Given this seasonally high livestock abundance, depredation by Himalayan wolves is inevitable and a major conservation concern. Habitat encroachment and depletion of wild prey populations are important drivers of this conflict. But we found that livestock was avoided when wild prey was available, a finding that can direct conservation. We conclude that the protection of Himalayan wolves, and other sympatric carnivores can be enhanced by a) securing healthy wild prey populations (ungulates and small mammals) through setting aside wildlife habitat refuges, and b) more sustainable livestock herding including reduced livestock loads and improved herding practices and protection.

Buglione M., Petrelli S., Troiano C., Notomista T., Rivieccio E., Fulgione D. (2020): The diet of otters (Lutra lutra) on the Agri river system, one of the most important presence sites in Italy: a molecular approach. PeerJ 8: e9606.
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The Eurasian otter (Lutra lutra) underwent a strong decline in large areas of the Central-Western part of its distribution range, during the second half of the twentieth century. In Italy, only residual fragmented nuclei survive in the Central-Southern part of the peninsula. Nowadays, the otter is one of the most endangered mammals in Italy, and increasing the knowledge about the ecology of this species is a key step in defining fitting management strategies. Here we provide information about the diet of otter on the Agri river system, one of the most important presence sites in Italy, to understand both the species’ food requirements and the impact on fish communities. DNA metabarcoding and High Throughput Sequencing were used on DNA extracted from spraints. We amplified DNA with a primer set for vertebrates, focusing efforts on the bulk of the otter’s diet (fishes and amphibians). Our findings showed that the diet of the otter was dominated by cyprinids (97.77%, and 99.14% of fishes), while amphibians represented 0.85% of the sequences analyzed. Results are in general accordance with previous studies based on morphological characterization; however, molecular analyses allow the resolving of some morphological uncertainties. Although the study area offers a very wide range of available prey, the diet of the otters shows marked selectivity. We highlighted a variation in prey consumed, in accordance with the typology of water system (i.e., river, lake, tributary). Some of the preys found in the diet were alien species introduced by man for sport fishing. Our findings could help define strategies useful for the conservation of the otter population in Southern Italy, suggesting management actions directed at avoiding fish community alterations through illegal stockings without severe controls on their taxonomic status. These introductions could result in a general reduction in the diversity of the otter’s preys, affecting its predatory behavior.

Cáceres-Martínez C. H., Montano L. R. S., Acevedo A. A., González-Maya J. F. (2020): Diet of Andean bears in Tamá National Natural Park, Colombia. Ursus 2020: 1-11.
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Diet is among the most important and basic ecological aspects of a species. Information on diet composition, seasonality, and spatial variation is necessary for adequate ecological understanding and therefore conservation. Andean bears (Tremarctos ornatus) have been poorly studied across their distribution, and especially in Colombia; therefore, most ecological aspects remain unknown. The objective of this work was to assess Andean bears’ diet and seasonality in order to contribute to the knowledge, management, and conservation of the species in northeastern Colombia. Between June 2012 and May 2015, 83 linear transects were surveyed at least once in 35 localities in Tamá National Natural Park in search of fresh bear scats. Transects covered all land-cover types in the park proportionately. We collected 148 scats for diet analyses, and compared all items found with reference material. We assessed diet contents in terms of frequency of occurrence and compared across seasons, elevation, and land-cover types. Overall, plant items showed the greatest frequency of occurrence in the scats (96.8%), followed by mammals (6.7%) and insects (6.7%); we found this pattern for all land-cover types and all elevation ranges. We found seasonal variation and differences in use of some food items. We found the largest niche breadth for the dry season (B = 7.10) compared with the wet season (B = 4.14). Plant items were more frequently consumed during both dry and rainy seasons (0.78 ± 0.16 and 0.82 ± 0.11, respectively) and bromeliads were the most important food item during both seasons (0.32 and 0.46). This variation probably is related to the structure, phenology, and composition of vegetation in the area. More data are needed for estimating foraging preferences and their relationship with food availability and other aspects related to behavior and individual resource use.

Gil-Sánchez J. M., Herrera-Sánchez F. J., Rodríguez-Siles J., Sáez J. M., Díaz-Portero M. Á., Arredondo Á. et al. (2020): Feeding specialization of honey badgers in the Sahara Desert: a trial of life in a hard environment. Diversity 12: 59.
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The honey badger (Mellivora capensis) is a medium-sized carnivore distributed throughout Africa to the Arabian Peninsula, Iran, Turkmenistan, and India. However, available information on its ecology is very scarce. We studied its feeding ecology in the remote north-western Sahara Desert, based on the contents of 125 fecal samples collected during large scale surveys. Samples were confirmed to belong to honey badgers by camera trapping and genetic analyses. Barely 18 prey species were detected. The diet primarily consisted of spiny-tailed lizards Uromastyx nigriventris and U. dispar (72% of volume in scats). Secondary prey items were arthropods (14%), small mammals (8%), other reptiles (4%), and eggs (0.8%). Some small geographic and temporal differences were related to the consumption of beetle larvae and rodents as alternative prey. Camera trapping and distance sampling surveys showed that diel activities did not overlap between honey badgers and spiny-tailed lizards, suggesting that badgers primarily dig lizards out of their burrows when inactive. Consumption of spiny lizards by other sympatric meso-carnivores was < 6.1% of occurrence (223 analyzed scats); the honey badger behaved as a trophic specialist in the Sahara, probably thanks to exclusive anatomical adaptations for digging. We discuss the role of this circumstance minimizing the exploitative competition, which could allow the survival of this large mustelid in this low productive and highly competitive environment.

Sonsthagen S. A., Jay C. V., Cornman R. S., Fischbach A. S., Grebmeier J. M., Talbot S. L. (2020): DNA metabarcoding of feces to infer summer diet of Pacific walruses. Marine Mammal Science 36: 1196-1211.
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Environmental conditions in the Chukchi Sea are changing rapidly and may alter the abundance and distribution of marine species and their benthic prey. We used a metabarcoding approach to identify potentially important prey taxa from Pacific walrus (Odobenus rosmarus divergens) fecal samples (n = 87). Bivalvia was the most dominant class of prey (66% of all normalized counts) and occurred in 98% of the samples. Polychaeta and Gastropoda occurred in 70% and 62% of the samples, respectively. The remaining nine invertebrate classes comprised <21% of all normalized counts. The common occurrence of these three prey classes is consistent with examinations of walrus stomach contents. Despite these consistencies, biases in the metabarcoding approach to determine diet from feces have been highlighted in other studies and require further study, in addition to biases that may have arisen from our opportunistic sampling. However, this noninvasive approach provides accurate identification of prey taxa from degraded samples and could yield much-needed information on shifts in walrus diet in a rapidly changing Arctic.

Dudenhoeffer M., Roth J. D., Johnson L. K., Petersen S. D. (2021): Arctic fox winter dietary response to damped lemming cycles estimated from fecal DNA. Journal of Mammalogy 102: 1455-1465.
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Climate-caused changes in prey abundance may alter predator–prey dynamics in the Arctic food web. Lemmings (Dicrostonyx spp.) are important prey for Arctic foxes (Vulpes lagopus) and their annual population fluctuations drive fox reproduction, creating strongly linked predator–prey population cycles. Winter diet directly impacts Arctic fox reproductive success, but winter prey diversity on the tundra is low. Strategies such as using the marine environment to scavenge seals have allowed Arctic foxes to persist during years of low lemming abundance. However, warming winters have decreased snowpack quality, preventing lemmings from reaching their previous high abundances, which may reduce their impact on predator dynamics. We investigated Arctic fox dietary response to lemming abundance by reconstructing Arctic fox winter diet in the low Arctic. Next-generation sequencing of fecal DNA, from samples (n = 627) collected at dens in winters of 2011–2018, identified prey both from terrestrial and marine environments. Despite lemming cycle damping, Arctic foxes still increased lemming consumption during years of higher lemming abundance. Alternative prey such as marine resources were consumed more during years of low lemming abundance, with up to 45% of samples containing marine resources in low lemming years. In addition, Arctic foxes consumed high proportions of meadow voles (Microtus pennsylvanicus), which may represent a new alternative prey, suggesting climate change may be creating new foraging opportunities. Changes in prey abundance illustrate how climate-caused disturbances are altering Arctic food-web dynamics. Dietary flexibility and availability of alternative prey may become increasingly important for Arctic predators as the Arctic continues to change.

Nørgaard L., Olesen C. R., Trøjelsgaard K., Pertoldi C., Nielsen J. L., Taberlet P., Ruiz-González A., De Barba M., Iacolina L. (2021): eDNA metabarcoding for biodiversity assessment, generalist predators as sampling assistants. Scientific Reports 11: 6820.
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With an accelerating negative impact of anthropogenic actions on natural ecosystems, non-invasive biodiversity assessments are becoming increasingly crucial. As a consequence, the interest in the application of environmental DNA (eDNA) survey techniques has increased. The use of eDNA extracted from faeces from generalist predators, have recently been described as “biodiversity capsules” and suggested as a complementary tool for improving current biodiversity assessments. In this study, using faecal samples from two generalist omnivore species, the Eurasian badger and the red fox, we evaluated the applicability of eDNA metabarcoding in determining dietary composition, compared to macroscopic diet identification techniques. Subsequently, we used the dietary information obtained to assess its contribution to biodiversity assessments. Compared to classic macroscopic techniques, we found that eDNA metabarcoding detected more taxa, at higher taxonomic resolution, and proved to be an important technique to verify the species identification of the predator from field collected faeces. Furthermore, we showed how dietary analyses complemented field observations in describing biodiversity by identifying consumed flora and fauna that went unnoticed during field observations. While diet analysis approaches could not substitute field observations entirely, we suggest that their integration with other methods might overcome intrinsic limitations of single techniques in future biodiversity surveys.

Quéméré E., Aucourd M., Troispoux V., Brosse S., Murienne J., Covain R., Le Bail P. Y., Olivier J., Tysklind N., Galan M. (2021): Unraveling the dietary diversity of Neotropical top predators using scat DNA metabarcoding: A case study on the elusive Giant Otter. Environmental DNA 3: 889-900.
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Large carnivores play a pivotal regulating role in maintaining healthy and balanced ecosystems; however, most of them are rare and elusive, and knowledge about their resource consumption is scarce. Traditional methods based on morphological identification of undigested remains are labor intensive and often not sufficiently accurate, leading to errors and biased ecological inferences. Here, we developed a multi-marker DNA metabarcoding approach to analyze the dietary diversity of giant otters (Pteronura brasiliensis) from fecal DNA while controlling predator species identity. We combined two mitochondrial markers, 12S rRNA and cytochrome c oxidase 1 (COI) gene, that target the full range of potential vertebrate and invertebrate prey. We compiled a local reference database of DNA barcodes for most potentially ingested fish, which were used to evaluate the specificity of the metabarcoding primers in silico. Most prey are identified at the species level (>90%) and the dietary profiles provided independently by the two markers are highly similar, whether in terms of list of prey or frequency of occurrences, hence validating the approach. We detected a higher number of rare fish prey with the 12S primers that amplified solely Teleost species while the degenerate COI primers revealed non-fish prey (e.g., amphibians, snakes, birds, and earthworms) and confirmed predator species identity. This study demonstrated that scat DNA metabarcoding is particularly useful to provide in-depth information on elusive carnivorous dietary profile. Our methodology opens up new opportunities to understand how top carnivores diet cope with the effects of anthropogenic alteration of ecosystems and identify conflicts with humans and livestock.

Thompson E. R., Driscoll D. A., Venn S. E., Geary W. L., Ritchie E. G. (2022): Interspecific variation in the diet of a native apex predator and invasive mesopredator in an alpine ecosystem. Austral Ecology 4: 1260-1270.
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Carnivores have key ecological roles in structuring and regulating ecosystems through their impacts on prey populations. When apex- and meso-predators co-occur in ecosystems, there is the potential for complex interspecific interactions and trophic dynamics that can affect the composition and functioning of ecological communities. Investigating the diet of sympatric carnivores can allow us to better understand their ecological roles (e.g. potential suppression of herbivores) or impacts (e.g. predation of threatened species). Australia’s alpine region provides an ideal system in which to explore spatial and temporal variation in predator and prey interactions, using the dingo (Canis dingo) and invasive red fox (Vulpes vulpes) diet. We examined the diet of dingoes and foxes across three different mountains and seasons in Victoria’s alpine region, using macroscopic scat analysis. There was little diet overlap between the two carnivores, with foxes having a broader diet than dingoes. Dingoes primarily consumed larger mammal species, including invasive sambar deer (Cervus unicolor, 44%), and the native common wombat (Vombatus ursinus, 34%), whereas foxes typically consumed smaller mammals, including the native bush rat (Rattus fuscipes, 55%), and the invasive European rabbit (Oryctolagus cuniculus, 15%). Dingoes consumed more than thirty times the volume of large invasive mammals (predominantly sambar deer) than did foxes. Foxes consumed close to 15 times as many critical weight range individuals per scat than dingoes. Only one threatened critical weight range mammal species was identified within scats, the broad-toothed rat (Mastacomys fuscus), found within five fox scats. Our results suggest that the introduction of novel prey may alter predator–predator interactions by causing a reduction in the dietary overlap. Therefore, in the context of integrated wildlife management and biodiversity conservation, any control of novel, invasive prey populations needs to consider possible flow on effects to apex- and meso-predator diets and potential secondary impacts on native prey.

CETACEANS

Smith S. C., Whitehead H. (2000): The diet of Galapagos sperm whales Physeter macrocephalus as indicated by fecal sample analysis. Marine Mammal Science 16: 315-325.
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Fecal samples were collected while following sperm whales (Physeter macrocephalus) off the Galápagos Islands, Ecuador. They contained 133 upper beaks and 164 lower beaks of cephalopods. Analysis of the lower beaks suggests that the sperm whales fed primarily on three genera of cephalopods; Histioteutbis (62%), Ancistrocbeirus (16%), and Octopoteutbis (7%). The beak dimensions indicate that the cephalopods ranged in mantle length from 5 to 54 cm and in mass from 12 to 650 g. Fecal samples varied significantly between five study years and over different parts of the study area, but the number of beaks collected per sample did not correlate significantly with defecation rate (a measure of feeding success). Using beak material from fecal samples gives a biased estimate of sperm whale diet, reducing the frequencies of very small and very large cephalopods. However, all other available methods of assessing sperm whale diet also possess biases.

Ford M. J., Hempelmann J., Hanson M. B., Ayres K. L., Baird R. W., Emmons C. K., Lundin J. I., Schorr G. S., Wasser S. K., Park L. K. (2016): Estimation of a killer whale (Orcinus orca) population’s diet using sequencing analysis of DNA from feces. Plos One 11: e0144956.
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Estimating diet composition is important for understanding interactions between predators and prey and thus illuminating ecosystem function. The diet of many species, however, is difficult to observe directly. Genetic analysis of fecal material collected in the field is therefore a useful tool for gaining insight into wild animal diets. In this study, we used high-throughput DNA sequencing to quantitatively estimate the diet composition of an endangered population of wild killer whales (Orcinus orca) in their summer range in the Salish Sea. We combined 175 fecal samples collected between May and September from five years between 2006 and 2011 into 13 sample groups. Two known DNA composition control groups were also created. Each group was sequenced at a ~330bp segment of the 16s gene in the mitochondrial genome using an Illumina MiSeq sequencing system. After several quality controls steps, 4,987,107 individual sequences were aligned to a custom sequence database containing 19 potential fish prey species and the most likely species of each fecal-derived sequence was determined. Based on these alignments, salmonids made up >98.6% of the total sequences and thus of the inferred diet. Of the six salmonid species, Chinook salmon made up 79.5% of the sequences, followed by coho salmon (15%). Over all years, a clear pattern emerged with Chinook salmon dominating the estimated diet early in the summer, and coho salmon contributing an average of >40% of the diet in late summer. Sockeye salmon appeared to be occasionally important, at >18% in some sample groups. Non-salmonids were rarely observed. Our results are consistent with earlier results based on surface prey remains, and confirm the importance of Chinook salmon in this population’s summer diet.

Carroll E. L., Gallego R., Sewell M. A., Zeldis J., Ranjard L., Ross H. A., Tooman L. K., O’Rorke R., Newcomb R. D., Constantine R. (2019): Multi-locus DNA metabarcoding of zooplankton communities and scat reveal trophic interactions of a generalist predator. Scientific Reports 9: 281.
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To understand the ecosystem dynamics that underpin the year-round presence of a large generalist consumer, the Bryde’s whale (Balaenoptera edeni brydei), we use a DNA metabarcoding approach and systematic zooplankton surveys to investigate seasonal and regional changes in zooplankton communities and if whale diet reflects such changes. Twenty-four zooplankton community samples were collected from three regions throughout the Hauraki Gulf, New Zealand, over two temperature regimes (warm and cool seasons), as well as 20 samples of opportunistically collected Bryde’s whale scat. Multi-locus DNA barcode libraries were constructed from 18S and COI gene fragments, representing a trade-off between identification and resolution of metazoan taxa. Zooplankton community OTU occurrence and relative read abundance showed regional and seasonal differences based on permutational analyses of variance in both DNA barcodes, with significant changes in biodiversity indices linked to season in COI only. In contrast, we did not find evidence that Bryde’s whale diet shows seasonal or regional trends, but instead indicated clear prey preferences for krill-like crustaceans, copepods, salps and ray-finned fishes independent of prey availability. The year-round presence of Bryde’s whales in the Hauraki Gulf is likely associated with the patterns of distribution and abundance of these key prey items.

Hanson M. B., Emmons C. K., Ford M. J., Everett M., Parsons K., Park L. K., Hempelmann J., Van Doornik D. M., Schorr G. S., Jacobsen J. K., Sears M. F. (2021): Endangered predators and endangered prey: Seasonal diet of Southern Resident killer whales. Plos One 16: e0247031.
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Understanding diet is critical for conservation of endangered predators. Southern Resident killer whales (SRKW) (Orcinus orca) are an endangered population occurring primarily along the outer coast and inland waters of Washington and British Columbia. Insufficient prey has been identified as a factor limiting their recovery, so a clear understanding of their seasonal diet is a high conservation priority. Previous studies have shown that their summer diet in inland waters consists primarily of Chinook salmon (Oncorhynchus tshawytscha), despite that species’ rarity compared to some other salmonids. During other times of the year, when occurrence patterns include other portions of their range, their diet remains largely unknown. To address this data gap, we collected feces and prey remains from October to May 2004–2017 in both the Salish Sea and outer coast waters. Using visual and genetic species identification for prey remains and genetic approaches for fecal samples, we characterized the diet of the SRKWs in fall, winter, and spring. Chinook salmon were identified as an important prey item year-round, averaging ~50% of their diet in the fall, increasing to 70–80% in the mid-winter/early spring, and increasing to nearly 100% in the spring. Other salmon species and non-salmonid fishes, also made substantial dietary contributions. The relatively high species diversity in winter suggested a possible lack of Chinook salmon, probably due to seasonally lower densities, based on SRKW’s proclivity to selectively consume this species in other seasons. A wide diversity of Chinook salmon stocks were consumed, many of which are also at risk. Although outer coast Chinook samples included 14 stocks, four rivers systems accounted for over 90% of samples, predominantly the Columbia River. Increasing the abundance of Chinook salmon stocks that inhabit the whales’ winter range may be an effective conservation strategy for this population.

Günther B., Jourdain E., Rubincam L., Karoliussen R., Cox S. L., Arnaud Haond S. (2022): Feces DNA analyses track the rehabilitation of a free-ranging beluga whale. Scientific Reports 12: 6412.
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Following the sudden appearance, and subsequent efforts to support the survival of a beluga whale (Delphinapterus leucas) speculated to have been previously trained off the coast of Norway, we investigate the animal’s ability to readapt to life in the wild. Dietary DNA (dDNA) analysis was used to assess diet throughout this rehabilitation process, and during a return to unassisted foraging and self-feeding. Metabarcoding of feces collected throughout this process, confirmed the diversification of the beluga whale’s diet to local prey. These findings are indicative of improved foraging behavior, and the ability of this individual to resume wild foraging following a period of dependency in managed care. New insight of digestion rates, and the time window during which prey detection through dDNA analysis is appropriate was also obtained. Beyond the case study presented here, we demonstrate the power of dDNA analysis as a non-intrusive tool to assess the diet of large mammals and track progress adapting to life in the wild following release from captivity and rehabilitation programs.

ELEPHANTS

Vogel S. M., Blumenthal S. A., de Boer W. F., Masake M., Newton I., Songhurst A. C., McCulloch G., Stronza A., Henley M. D., Coulson T. (2020): Timing of dietary switching by savannah elephants in relation to crop consumption. Biological Conservation 249: 108703.
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Tree and grass quality on the African savannah shows seasonal variation, driving mixed-feeding herbivores to switch between browsing and grazing. During this switch, crop consumption could be an attractive alternative to browsing. We analysed elephant diet variability in the Okavango Delta, Botswana, using faecal stable isotope ratios of carbon (δ^13C) and frequencies of elephant crop consumption, to determine the extent to which crop consumption relates to this potential switch. Although elephants did increase their relative grass consumption in the wet season, browse dominated the annual diet. After February, the proportion of grass in the diet dropped considerably, and continued decreasing through April when farmers reported most crop consumption. Generalized Linear Models revealed that the occurrence of elephant crop consumption increased with the proportion of grass consumed and with decreasing grass quality. The proportion of grass in elephant faeces increased with increasing crop consumption intensity. As crop consumption could also be related to nutrient deficiencies in elephant diet, we calculated the total dietary input of nutrients to reveal potential deficiencies. Elephant diet contained insufficient levels of sodium year-round, and insufficient phosphorus from February to July. As the latter coincides with the timing of crop consumption, we consider our results an indication that phosphorus –and potentially sodium – deficiencies, could play a role in elephant dietary choices, including crop consuming behaviour. Further experimental research is required to show whether supplying elephants with supplementary phosphorus and sodium sources could reduce this micro-nutrient deficiency, and could play a role in reducing elephant crop consumption.

Liyanage D. J., Fernando P., Dayawansa P. N., Janaka H. K., Pastorini J. (2021): The elephant at the dump: how does garbage consumption impact Asian elephants?. Mammalian Biology 101: 1089-1097.
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We studied garbage consumption by Asian elephants at the Uddakandara garbage dump in southern Sri Lanka. Garbage at the dump was classified under six categories and quantified using a grid overlay. Elephants visiting the dump were individually identified by morphological criteria and items and quantities consumed by them were determined by focal animal sampling. Dung of elephants that did not consume garbage and those from the dump were compared quantitatively and dung constituents assessed by washing through three layered sieves. A total of 17 individual elephants visited the garbage dump during the study period, all of who were males. The observed sexual bias could be related to behavioural differences between the sexes. Elephants mostly consumed ‘fruits and vegetables’ and ‘prepared food’, possibly due to their higher palatability and nutritional value. Ingestion of polythene was incidental and associated with consuming prepared food. Proportions of the six categories in elephant diet and garbage piles were significantly different, indicating that elephants were highly selective when feeding. Elephant arrivals increased in response to unloading of garbage, suggesting attraction to fresh garbage. Dung analysis found that garbage consumption did not change the quantity and constituents of dung, except for the presence of anthropogenic items. As consumed anthropogenic items were regularly excreted, retention and obstruction of the alimentary tract are unlikely in elephants. Elephants feeding on garbage had better body condition than non-garbage consuming elephants, indicating that garbage provided better nutrition than natural food and was not detrimental to their health.

Meyer J. M., Honig N., Hadly E. A. (2022): Diet DNA reveals novel African Forest elephant ecology on the grasslands of the Congo Basin. Environmental DNA 4: 846-867.
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Elephants are essential ecological engineers, creating and maintaining landscape structure and ecosystem function. The recently distinguished and critically endangered forest elephant is currently classified as a selective, non-destructive frugivorous browser that maintains forest diversity, while the savanna elephant is a mixed feeder, often pushing over trees while maintaining grasslands. The presence and diets of forest elephants on grasslands and the potential maintenance of these systems remain largely unexplored. In the ecotone between the Guinea-Congolian forest and Sudanian-Guinean savanna ecosystems in Garamba National Park, DRC, we investigated forest elephant diet selection as a function of sex, age, and habitat using diet DNA (dDNA) metabarcoding of non-invasively collected dung. GPS collar data were used to determine annual habitat use. Dietary niche partitioning was assessed among megaherbivores in the grasslands. Fecal samples represented the diet of individuals within each habitat, providing valuable insight into the plant biodiversity. Ecological patterns of diet were also revealed using a taxonomically free exact sequence variance approach, highlighting useability in a poorly characterized region. In the early wet season, these typically frugivorous forest elephants were consuming mostly grasses in both the woodland and grassland habitats and showing no sexual dimorphism in diet selection when in the same habitats. However, males were greater risk-takers, entering the human-altered landscape to forage on fruit. The forest elephants play a distinctive role within this tropical grassland when compared to other megaherbivores and utilize the unique ecosystem throughout the year. This elephant population is exhibiting behavioral plasticity and shifting their gardening efforts to a novel resource in the grasslands as opposed to their standard role in the forests, which is key to understanding their impact as ecosystem drivers within this landscape. This shift in behavior may result in this recovering elephant population playing a functional role in the restoration and maintenance of these grasslands.

HEDGEHOGS, SHREWS, AND DESMANS

Brown D. S., Burger R., Cole N., Vencatasamy D., Clare E. L., Montazam A., Symondson W. O. C. (2014): Dietary competition between the alien Asian Musk Shrew (Suncus murinus) and a re‐introduced population of Telfair’s Skink (Leiolopisma telfairii). Molecular Ecology 23: 3695-3705.
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Re-introduction of rare species to parts of their historical range is becoming increasingly important as a conservation strategy. Telfair’s Skinks (Leiolopisma telfairii), once widespread on Mauritius, were until recently found only on Round Island. There it is vulnerable to stochastic events, including the introduction of alien predators that may either prey upon it or compete for food resources. Consequently, skinks have been introduced to Ile aux Aigrettes, another small Mauritian island that has been cleared of rats. However, the island has been invaded by Asian Musk Shrews (Suncus murinus), a commensal species spread by man well beyond its natural Asian range. Our aim was to use next-generation sequencing to analyse the diets of the shrews and skinks to look for niche competition. DNA was extracted from skink faeces and from the stomach contents of shrews. Application of shrew- and skink-specific primers revealed no mutual predation. The DNA was then amplified using general invertebrate primers with tags to identify individual predators, and then sequenced by 454 pyrosequencing. 119 prey MOTUs (molecular taxonomic units) were isolated, although none could be identified to species. Seeding of cladograms with known sequences allowed higher taxonomic assignments in some cases. Although most MOTUs were not shared by shrews and skinks, Pianka’s niche overlap test showed significant prey overlap, suggesting potentially strong competition where food resources are limited. These results suggest that removal of the shrews from the island should remain a priority.

Gillet F., Tiouchichine M. L., Galan M., Blan F., Némoz M., Aulagnier S., Michaux J. R. (2015): A new method to identify the endangered Pyrenean desman (Galemys pyrenaicus) and to study its diet, using next generation sequencing from faeces. Mammalian Biology 80: 505-509.
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The Pyrenean desman (Galemys pyrenaicus) is a small endangered semi-aquatic mammal endemic to the Pyrenean Mountains and to the northern half of the Iberian Peninsula whose ecology and biology are still poorly known. The aim of this study was to identify Pyrenean desman faeces and to analyze its diet from this material using next-generation sequencing methods. We amplified and sequenced a small DNA minibarcode (133 bp) of the COI gene in twenty-four putative faeces samples of Pyrenean desman and successfully identified the species in 16 samples. Other identified species were mammals, birds and amphibians, evidencing the potential application of our methods to a larger panel of taxa. In the Pyrenean desman faeces, we were able to identify nineteen prey species with a positive match (more than 98% of identity with a reference sequence) and eleven putative prey species with lower identity scores (90–96%). The nineteen species belonged to four orders and eleven families among which Trichoptera and Hydropsychidae were the most frequent, respectively. Future improvements could be obtained by extending the reference DNA sequence collection to reach precise identifications over the Desman’s range and by increasing the sampling to gain a better knowledge of the local diet of this endangered species. Such information is of great importance to propose the best management measures for its conservation.

Biffi M., Laffaille P., Jabiol J., André A., Gillet F., Lamothe S., Michaux J. R., Buisson L. (2017): Comparison of diet and prey selectivity of the Pyrenean desman and the Eurasian water shrew using next-generation sequencing methods. Mammalian Biology 87: 176-184.
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In this study, the interactions between two semi-aquatic mammals, the endangered Pyrenean desman Galemys pyrenaicus and the Eurasian water shrew Neomysfodiens, were investigated through the analysis of their summer diet using next-generation sequencing methods, combined with analyses of prey selectivity and trophic overlap. The diet of these predators was highly diverse including 194 and 205 genera for G. pyrenaicus and N. fodiens respectively. Overall, both species exhibited rather non-selective foraging strategies as the most frequently consumed invertebrates were also the most frequent and abundant in the streams. This supported a generalist foraging behaviour for G. pyrenaicus and N. fodiens in the study area. The Pianka index (0.4) indicated a significant but moderate dietary overlap as G. pyrenaicus mostly relied on prey with aquatic stages whereas prey of N. fodiens were mainly terrestrial. Moreover, no difference in G. pyrenaicus prey consumption was found in presence or absence of N. fodiens. A differential use of trophic resources through mechanisms such as plastic feeding behaviour or differences in foraging micro-habitat are likely to facilitate the coexistence between these two mammal species.

Esnaola A., Arrizabalaga-Escudero A., González-Esteban J., Elosegi A., Aihartza J. (2018): Determining diet from faeces: Selection of metabarcoding primers for the insectivore Pyrenean desman (Galemys pyrenaicus). Plos One 13: e0208986.
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Molecular techniques allow non-invasive dietary studies from faeces, providing an invaluable tool to unveil ecological requirements of endangered or elusive species. They contribute to progress on important issues such as genomics, population genetics, dietary studies or reproductive analyses, essential knowledge for conservation biology. Nevertheless, these techniques require general methods to be tailored to the specific research objectives, as well as to substrate- and species-specific constraints. In this pilot study we test a range of available primers to optimise diet analysis from metabarcoding of faeces of a generalist aquatic insectivore, the endangered Pyrenean desman (Galemys pyrenaicus, É. Geoffroy Saint-Hilaire, 1811, Talpidae), as a step to improve the knowledge of the conservation biology of this species. Twenty-four faeces were collected in the field, DNA was extracted from them, and fragments of the standard barcode region (COI) were PCR amplified by using five primer sets (Brandon-Mong, Gillet, Leray, Meusnier and Zeale). PCR outputs were sequenced on the Illumina MiSeq platform, sequences were processed, clustered into OTUs (Operational Taxonomic Units) using UPARSE algorithm and BLASTed against the NCBI database. Although all primer sets successfully amplified their target fragments, they differed considerably in the amounts of sequence reads, rough OTUs, and taxonomically assigned OTUs. Primer sets consistently identified a few abundant prey taxa, probably representing the staple food of the Pyrenean desman. However, they differed in the less common prey groups. Overall, the combination of Gillet and Zeale primer sets were most cost-effective to identify the widest taxonomic range of prey as well as the desman itself, which could be further improved stepwise by adding sequentially the outputs of Leray, Brandon-Mong and Meusnier primers. These results are relevant for the conservation biology of this endangered species as they allow a better characterization of its food and habitat requirements.

Hawlitschek O., Fernandez-Gonzalez A., Balmori-de la Puente A., Castresana J. (2018): A pipeline for metabarcoding and diet analysis from fecal samples developed for a small semi-aquatic mammal. Plos One 13: e0201763.
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Metabarcoding allows the genetic analysis of pooled samples of various sources. It is becoming popular in the study of animal diet, especially because it allows the analysis of the composition of feces without the need of handling animals. In this work, we studied the diet of the Pyrenean desman (Galemys pyrenaicus), a small semi-aquatic mammal endemic to the Iberian Peninsula and the Pyrenees, by sequencing COI minibarcodes from feces using next-generation sequencing techniques. For the identification of assembled sequences, we employed a tree-based identification method that used a reference tree of sequences of freshwater organisms. The comparison of freshly collected fecal samples and older samples showed that fresh samples produced significantly more sequencing reads. They also rendered more operational taxonomical units (OTUs), but not significantly. Our analyses of 41 samples identified 224 OTUs corresponding to species of the reference tree. Ephemeroptera, Diptera excl. Chironomidae, and Chironomidae were the most highly represented groups in terms of reads as well as samples. Other groups of freshwater organisms detected were Plecoptera, Trichoptera, Neuropteroida, Coleoptera, Crustacea, and Annelida. Our results are largely in line with previous morphological and genetic studies on the diet of the Pyrenean desman, but allowed the identification of a higher diversity of OTUs in each sample. Additionally, the bioinformatic pipeline we developed for deep sequencing of fecal samples will enable the quantitative analysis of the diet of this and other species, which can be highly useful to determine their ecological requirements.

Esnaola A., Larrañaga A., González-Esteban J., Elosegi A., Aihartza J. (2023): Using biological traits to assess diet selection: the case of the Pyrenean desman. Journal of Mammalogy 104: 1205–1215.
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Traditionally, researchers have assessed diet selection by comparing consumed versus available taxa. However, taxonomic assignment is probably irrelevant for predators, who likely base their selection on characteristics including prey size, habitat, or behavior. Here, we use an aquatic insectivore, the threatened Pyrenean Desman (Galemys pyrenaicus), as a model species to assess whether biological traits help unravel the criteria driving food and habitat preferences. We reanalyzed data from a previous taxonomy-based study of prey selection in two contrasting streams, one with excellent conservation status and the other affected by diversion for hydropower and forestry. Available and consumed prey were characterized according to nine biological traits, and diet selection was estimated by comparing availability—measured from Surber net samples, and consumption—analyzed by metabarcoding desman feces. Traits offered a biologically coherent image of diet and almost identical selection patterns in both streams, depicting a highly specialized rheophilic predator. Desmans positively selected prey with a preference for fast flow and boulder substrate, indicating their preferred riffle habitat. On the other hand, they positively selected prey with larger but not the largest potential size, living in the water column or the litter, and not inside sediments. They also chose agile prey, swimmers or prey attached to the substrate, prey with high body flexibility, and prey living exposed and clustered in groups. Overall, our results offer a picture of desman diet preference and point to biological traits as being better than taxonomic identity to describe the diet preference of consumers.

MARSUPIALS

Windley H. R., Wallis I. R., DeGabriel J. L., Moore B. D., Johnson C. N., Foley W. J. (2013): A faecal index of diet quality that predicts reproductive success in a marsupial folivore. Oecologia 173: 203-212.
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Estimating the nutritional value of a herbivore’s diet is difficult because it requires knowing what the animal eats, the relative quality of each component and how these components interact in relation to animal physiology. Current methods are cumbersome and rely on many assumptions that are hard to evaluate. We describe a new method for estimating relative diet quality directly from faeces that avoids the problems inherent in other methods. We combine this method with near infrared reflectance spectroscopy (NIRS) to analyse many samples and thus provide a technique with immense value in ecological studies. The method stems from the correlation between the concentrations of dietary and faecal nitrogen in herbivores eating a tannin-free diet, but a weaker relationship in browsers that ingest substantial amounts of tannins, which form complexes with proteins. These complexes reduce the availability of nitrogen and may increase faecal nitrogen concentrations. Using the tannin-binding compound, polyethylene glycol, we showed that tannin-bound nitrogen is a significant and variable part of faecal nitrogen in wild common brushtail possums (Trichosurus vulpecula). We developed a technique to measure faecal available nitrogen and found that it predicted the reproductive success of female brushtail possums in northern Australia. Faecal available nitrogen combined with NIRS provides a powerful tool for estimating the relative nutritional value of the diets of browsing herbivores in many ecological systems. It is a better indicator of diet quality than other commonly used single-nutrient measures such as faecal nitrogen and foliage analysis paired with observed feeding behaviour.

Fancourt B. A., Hawkins C. E., Nicol S. C. (2018): Mechanisms of climate-change-induced species decline: spatial, temporal and long-term variation in the diet of an endangered marsupial carnivore, the eastern quoll. Wildlife Research 45: 737-750.
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Climate change is having significant impacts on species worldwide. The endangered eastern quoll (Dasyurus viverrinus) has recently undergone rapid and severe population decline, with no sign of recovery. Spatially and temporally-explicit weather modelling suggests a prolonged period of unfavourable weather conditions during 2001–03 as the proximate cause of decline. However, the mechanisms of this weather-induced decline are not currently understood. The aim of this study was to investigate the hypothesis that changing weather conditions have altered the availability of key prey species, potentially contributing to the species’ decline. We analysed 229 scats collected from 125 individual wild quolls across four sites between July 2011 and May 2012. Variation in dietary composition and niche breadth was compared across sites and seasons. We also compared contemporary dietary composition and niche breadth to historic dietary studies performed before the species’ decline, to identify any key changes in dietary composition over time. Dietary composition and niche breadth were similar across sites but differed between seasons. Dietary niche contracted during winter (July) and early spring (September) when insect larvae formed the bulk of quoll diet, rendering the species vulnerable to weather-related fluctuations in food availability at that time. Large differences were also evident between current and historic dietary composition, with a marked shift from insect larvae to mammals, predominantly due to a reduction in corbie (Oncopera intricata) and southern armyworm (Persectania ewingii) moth larvae. Quoll abundance appears positively related to corbie larva abundance during winter, and both quoll and corbie larva abundance appear negatively related to winter rainfall. The lower contribution of insects at sites with low quoll densities suggests that insects represent an important food item for eastern quolls during winter, when dietary niche is narrowest and energy demands are highest. Our findings suggest that weather-induced fluctuations in quoll abundance, including the significant statewide decline during 2001–03, are potentially driven by weather-induced fluctuations in corbie larva abundance. Continued deterioration in climatic suitability with recent and predicted climate change could further threaten eastern quolls through reductions in the availability and stability of reliable food sources at critical life-history stages when dietary options are already limited.

Melo G. L., Santos S. A., Sponchiado J., Cáceres N. C., Lessa L. G. (2019): Feeding ecology of the marsupial Thylamys macrurus (Olfers 1818) (Mammalia, Didelphidae) in woodland patches of Cerrado, central-western Brazil. Mammalia 83: 41-48.
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Knowledge about the feeding ecology of didelphid marsupials remains incipient, especially in environments with marked seasonality in resource supply, such as in the Cerrado. We analysed the diet composition of Thylamys macrurus in Cerrado patches in central-western Brazil. We also evaluated seasonal and sexual variation in the species’ diet, as well as if the accumulation of fat in the tail shows a seasonal pattern. We obtained 87 captures of 68 T. macrurus specimens and 29 faecal samples. Six categories of food items (Hymenoptera, Isoptera, Coleoptera, Arachnida, Blattodea and seeds) were identified. Arthropods were recorded in all samples and seeds in 10.3%. The species showed seasonal variation in diet, consuming primarily of arthropods throughout the year but incorporating a higher proportion of fruits during the wet season. Males and females also differed in diet composition; males consumed a wider range of items in the dry season. Although more detailed analyses are still needed to better understand the T. macrurus food consumption pattern, our results indicate a possible relationship between dietary variation and local availability of resources, suggesting an opportunistic foraging pattern. The accumulation of fat in the tail also presents a seasonal pattern, with a larger tail diameter in the dry season.

Camp A., Croxford A. E., Ford C. S., Baumann U., Clements P. R., Hiendleder S., Woolford L., Netzel G., Boardman W. S., Fletcher M. T., Wilkinson M. J. (2020): Dual-locus DNA metabarcoding reveals southern hairy-nosed wombats (Lasiorhinus latifrons Owen) have a summer diet dominated by toxic invasive plants. Plos One 15: e0229390.
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Habitat degradation and summer droughts severely restrict feeding options for the endangered southern hairy-nosed wombat (SHNW; Lasiorhinus latifrons). We reconstructed SHNW summer diets by DNA metabarcoding from feces. We initially validated rbcL and ndhJ diet reconstructions using autopsied and captive animals. Subsequent diet reconstructions of wild wombats broadly reflected vegetative ground cover, implying local rather than long-range foraging. Diets were all dominated by alien invasives. Chemical analysis of alien food revealed Carrichtera annua contains high levels of glucosinolates. Clinical examination (7 animals) and autopsy (12 animals) revealed that the most degraded site also contained most individuals showing signs of glucosinolate poisoning. We infer that dietary poisoning through the ingestion of alien invasives may have contributed to the recent population crashes in the region. In floristically diverse sites, individuals appear to be able to manage glucosinolate intake by avoidance or episodic feeding but this strategy is less tractable in the most degraded sites. We conclude that recovery of the most affected populations may require effective Carrichtera management and interim supplementary feeding. More generally, we argue that protection against population decline by poisoning in territorial herbivores requires knowledge of their diet and of those food plants containing toxic principles.

Sobek C. J., Walker F. M. (2020): Square salads: exploring the diet of the southern hairy-nosed wombat (Lasiorhinus latifrons) via DNA metabarcoding. Australian Mammalogy 43: 104-109.
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Southern hairy-nosed wombats (Lasiorhinus latifrons) are large semi-fossorial marsupials that inhabit semiarid areas of southern South Australia and southeast Western Australia. Despite their size and prominence on the landscape, little is published about diet and foraging preferences. Further, it is unclear whether this wombat species’ diet overlaps with sympatric western grey kangaroos (Macropus fuliginosus), which are locally abundant across the southern hairy-nosed wombat’s distribution. Competition for food resources may be an added stressor in Murraylands populations where kangaroos are plentiful. Here, we identified diet items and dietary overlap of southern hairy-nosed wombats and western grey kangaroos. We accomplished this by opportunistically sampling fresh faecal pellets from both species at Brookfield Conservation Park in April 2017. We performed DNA metabarcoding via Illumina next generation sequencing of the faecal DNA using the plant ITS2 gene. We identified 10 genera in the wombat diet and 20 in the diet of kangaroos; diets of both species included native and introduced genera. Eight genera were shared between both marsupials. This work highlights the capabilities of non-invasive genetic sampling in concert with DNA metabarcoding to elucidate diet and dietary overlap between species.

Herath A. P., Wat K. K., Banks P. B., McArthur C. (2021): Animal personality drives individual dietary specialisation across multiple dimensions in a mammalian herbivore. Functional Ecology 35: 2253-2265.
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Animal personality influences how individuals perceive and react to different stimuli, such as food quality and predation risk, thereby shaping foraging behaviour. As a consequence, animal personality is predicted to influence the diet and dietary specialisation of individuals within a population, but animal personality and dietary specialisation have yet to be linked. Here, we tested whether individual diet and dietary specialisation are a function of animal personality, using an arboreal herbivore, the common brushtail possum, as a model species. We hypothesised that proactive individuals have more diverse foraging opportunities than reactive individuals. We therefore predicted that proactive animals (more exploratory, bold and more active) would be less specialised, with a broader and higher quality diet than their reactive counterparts. We quantified the personality traits and dietary niche using proportional similarity index and specialisation categories of possums (n = 30) in a population flanking native eucalypt woodland and residential gardens in suburban Sydney, Australia. We found that personality traits were related to the breadth and quality of the realised diets of individual possums. As predicted, proactive individuals had a broad, high-quality diet with less individual dietary specialisation than reactive individuals. Highly exploratory individuals had a more diverse diet than less exploratory individuals, and bold individuals were more likely than shy individuals to consume plants on the ground. Our study demonstrates an important link between animal personality and individual dietary specialisation. The finding that the personality of individuals is associated with different diet choices within the same landscape is fundamentally significant. First, personality as a driver of niche partitioning likely reduces within-species competition and hence could contribute to adaptive capacity. Second, the strength of ecological processes arising from interactions (e.g. predator–prey and plant–herbivore interactions) could differ among individuals according to their personality traits. Our findings are also relevant for effectively managing both threatened native and invasive species. Management strategies will be improved by incorporating knowledge of individual traits and their ecological consequences, plus the ecological context of food- and fear-scapes.

MONOTREMES

Hawke T., Bino G., Shackleton M. E., Ross A. K., Kingsford R. T. (2022): Using DNA metabarcoding as a novel approach for analysis of platypus diet. Scientific Reports 12: 2247.
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Platypuses (Ornithorhynchus anatinus) forage for macroinvertebrate prey exclusively in freshwater habitats. Because food material in their faeces is well digested and mostly unidentifiable, previous dietary studies have relied on cheek pouch assessments and stable isotope analysis. Given DNA metabarcoding can identify species composition from only fragments of genetic material, we investigated its effectiveness in analysing the diet of platypuses, and to assess variation across seasons and sexes. Of the 18 orders and 60 families identified, Ephemeroptera and Diptera were the most prevalent orders, detected in 100% of samples, followed by Trichoptera, Pulmonata, and Odonata (86.21% of samples). Caenidae and Chironomidae were the most common families. Diptera had a high average DNA read, suggesting it is an important dietary component that may have been underestimated in previous studies. We found no variation in diet between sexes and only minimal changes between seasons. DNA metabarcoding proved to be a highly useful tool for assessing platypus diet, improving prey identification compared to cheek pouch analysis, which can underestimate soft-bodied organisms, and stable isotope analysis which cannot distinguish all taxa isotopically. This will be a useful tool for investigating how platypus prey diversity is impacted by habitat degradation as a result of anthropogenic stressors.

PRIMATES

Bogart S. L., Pruetz J. D. (2011): Insectivory of savanna chimpanzees (Pan troglodytes verus) at Fongoli, Senegal. American Journal of Physical Anthropology 145: 11-20.
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Little is known about the behavior of chimpanzees living in savanna-woodlands, although they are of particular interest to anthropologists for the insight they can provide regarding the ecological pressures affecting early hominins living in similar habitats. Fongoli, Senegal, is the first site where savanna chimpanzees have been habituated for observational data collection and is the hottest and driest site where such observation of chimpanzees occurs today. Previously, indirect evidence suggested these chimpanzees consumed termites throughout the year, an unusual occurrence for western and eastern chimpanzees. Although meat eating by chimpanzees continues to receive much attention, their use of invertebrate prey has received less emphasis in scenarios of hominin evolution. Here, we further examine the invertebrate diet of Fongoli chimpanzees using direct observational methods and accounting for potential environmental influences. Termite feeding positively correlated with high temperatures. Fongoli chimpanzees spend more time obtaining termites than any other chimpanzee population studied, and this extensive insectivory contributes to the list of distinctive behaviors they display relative to chimpanzees living in more forested habitats. We suggest that savanna chimpanzees at Fongoli differ significantly from chimpanzees elsewhere as a result of the selective pressures characterizing their harsh environment, and this contrast provides an example of a viable referential model for better understanding human evolution. Specifically, our results support the hypotheses that invertebrate prey may have figured more prominently into the diet of early hominins in similar habitats, especially given that invertebrates are an important source of protein and other essential nutrients in a highly seasonal environment.

Blumenthal S. A., Chritz K. L., Rothman J. M., Cerling T. E. (2012): Detecting intraannual dietary variability in wild mountain gorillas by stable isotope analysis of feces. Proceedings of the National Academy of Sciences 109: 21277-21282.
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We use stable isotope ratios in feces of wild mountain gorillas (Gorilla beringei) to test the hypothesis that diet shifts within a single year, as measured by dry mass intake, can be recovered. Isotopic separation of staple foods indicates that intraannual changes in the isotopic composition of feces reflect shifts in diet. Fruits are isotopically distinct compared with other staple foods, and peaks in fecal δ¹³C values are interpreted as periods of increased fruit feeding. Bayesian mixing model results demonstrate that, although the timing of these diet shifts match observational data, the modeled increase in proportional fruit feeding does not capture the full shift. Variation in the isotopic and nutritional composition of gorilla foods is largely independent, highlighting the difficulty for estimating nutritional intake with stable isotopes. Our results demonstrate the potential value of fecal sampling for quantifying short-term, intraindividual dietary variability in primates and other animals with high temporal resolution even when the diet is composed of C₃ plants.

Quéméré E., Hibert F., Miquel C., Lhuillier E., Rasolondraibe E., Champeau J., Rabarivola C., Nusbaumer L., Chatelain C., Gautier L., Ranirison P., Crouau-Roy B., Taberlet P., Chikhi L. (2013): A DNA metabarcoding study of a primate dietary diversity and plasticity across its entire fragmented range. Plos One 8: e58971.
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In tropical regions, most primary ecosystems have been replaced by mosaic landscapes in which species must cope with a large shift in the distribution of their habitat and associated food resources. Primates are particularly vulnerable to habitat modifications. Most species persist in small fragments surrounded by complex human-mediated matrices whose structure and connectivity may strongly influence their dispersal and feeding behavior. Behavioral plasticity appears to be a crucial parameter governing the ability of organisms to exploit the resources offered by new matrix habitats and thus to persist in fragmented habitats. In this study, we were interested in the dietary plasticity of the golden-crowned sifaka (Propithecus tattersalli), an endangered species of lemur, found only in the Daraina region in north-eastern Madagascar. We used a DNA-based approach combining the barcoding concept and Illumina next-generation sequencing to (i) describe the species diet across its entire range and (ii) evaluate the influence of landscape heterogeneity on diet diversity and composition. Faeces from 96 individuals were sampled across the entire species range and their contents were analyzed using the trnL metabarcoding approach. In parallel, we built a large DNA reference database based on a checklist of the plant species of the Daraina region. Our results suggest that golden-crowned sifakas exhibit remarkable dietary diversity with at least 130 plant species belonging to 80 genera and 49 different families. We highlighted an influence of both habitat type and openness on diet composition suggesting a high flexibility of foraging strategies. Moreover, we observed the presence of numerous cultivated and naturalized plants in the faeces of groups living in forest edge areas. Overall, our findings support our initial expectation that P. tattersalli is able to cope with the current level of alteration of the landscape and confirm our previous results on the distribution and the dispersal ability of this species.

Hamad I., Delaporte E., Raoult D., Bittar F. (2014): Detection of termites and other insects consumed by African great apes using molecular fecal analysis. Scientific Reports 4: 4478.
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The consumption of insects by apes has previously been reported based on direct observations and/or trail signs in feces. However, DNA-based diet analyses may have the potential to reveal trophic links for these wild species. Herein, we analyzed the insect-diet diversity of 9 feces obtained from three species of African great apes, gorilla (Gorilla gorilla gorilla), chimpanzee (Pan troglodytes) and bonobo (Pan paniscus), using two mitochondrial amplifications for arthropods. A total of 1056 clones were sequenced for Cyt-b and COI gene libraries, which contained 50 and 56 operational taxonomic units (OTUs), respectively. BLAST research revealed that the OTUs belonged to 32 families from 5 orders (Diptera, Isoptera, Lepidoptera, Coleoptera and Orthoptera). While ants were not detected by this method, the consumption of flies, beetles, moths, mosquitoes and termites was evident in these samples. Our findings indicate that molecular techniques can be used to analyze insect food items in wild animals.

Phillips C., Lancelotti C. (2014): Chimpanzee diet: phytolithic analysis of feces. American Journal of Primatology 76: 757-773.
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Most primate populations remain unobservable; therefore, researchers depend on the analyses of indirect evidence encountered at a study‐site in order to understand their behavioral ecology. Diet can be determined through the analyses of scats or feeding remains encountered on‐site. This allows aspects of their dietary repertoire to be established, which has implications both for conservation efforts (by locating food resources), and for understanding the evolution of hominin diet (if used as referential models). Macroscopic inspection of fecal samples is a common method applied to ascertain a primate population’s diet. However, new approaches are required to identify food‐items unrecognizable at this level. We applied a dry ash extraction method to fecal samples (N = 50) collected from 10 adult chimpanzees in Kanyawara, Kibale National Park, Uganda and also to plant parts (N = 66) from 34 species known to be included in the diet of this community of apes. We recovered phytoliths in 26 of the 34 plant species. Fifteen phytolith morphotypes were only detected in 14 plant species (termed “distinct” phytoliths). We used these distinct phytoliths to identify plant foods (i.e., that they were associated with) in fecal samples. We then validated findings by checking if the 10 chimpanzees had eaten parts of these plants ∼24 hr prior to fecal sample collection; six plant species associated with five distinct phytoliths had been eaten. Finally, we compared plant foods identified in fecal samples from phytolith analyses with plants that had been identified from macroscopic inspection of the same fecal samples. Findings from phytolith analyses corroborate with those from macroscopic inspection by expanding the total number of plant species identified per fecal sample (i.e., we identified certain plant parts that remained unrecognizable at macroscopic level). This study highlights the potential of phytolith analyses of feces to increase our knowledgebase of the dietary repertoire of primate populations.

Carvalho J. S., Vicente L., Marques T. A. (2015): Chimpanzee (Pan troglodytes verus) diet composition and food availability in a human-modified landscape at Lagoas de Cufada Natural Park, Guinea-Bissau. International Journal of Primatology 36: 802-822.
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Nonhuman primate populations are facing widespread conversion of their habitat to human-modified landscapes dominated by agriculture, in which cultivated species may constitute alternative food resources, particularly during periods of wild food scarcity. We assessed intraseasonal variation in dietary composition and diversity of the western chimpanzee (Pan troglodytes verus) at Lagoas de Cufada Natural Park, Guinea-Bissau, a forest–savannah mosaic disturbed by humans, in relation to food availability. We also investigated spatial variation in dietary composition. We collected phenological data from March 2011 to February 2012 by sampling focal plant taxa and conducted macroscopic analyses of fecal samples and feeding remains during the dry season (February–May and October–December) of 2011. More fruits were available in the dry than in the wet season, and ripe fruit availability peaked in the late dry season. Chimpanzees showed a fruit-based diet composed of 31 identified plant species. Fecal samples (N = 210) were dominated by wild species (82 % volume), while cultivated species were rare (0.9 % volume; 17 % volume of unidentified species). The consumption of fruit species increased with ripe fruit availability, but a few wild fruit species were selected disproportionately to their overall availability. There was no association between dietary composition and distances among fecal samples, suggesting that chimpanzees have access to and largely use the same set of plant species over the entire study area. Moreover, the proximity to agricultural areas did not influence dietary composition. Our findings highlight that chimpanzees in this human-modified landscape still rely mostly on the consumption of wild fruit species and rarely include cultivated foods in their diet. Overall, our study underscores the importance of knowledge of feeding ecology to understand better the effects of anthropogenic habitat modification on primate diet and distribution as well as the limits to their persistence in the expanding human-dominated agricultural landscapes across their range.

Phillips C. A., O’Connell T. C. (2016): Fecal carbon and nitrogen isotopic analysis as an indicator of diet in Kanyawara chimpanzees, Kibale National Park, Uganda. American Journal of Physical Anthropology 161: 685-697.
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The shorter‐term overview from feces provides scope to investigate dietary fluctuations. We assess the correlation of stable isotopic fecal values with recorded seasonal diet of 10 adult chimpanzees (P. t. schweinfurthii) of the Kanyawara community (Kibale National Park, Uganda) and whether fecal nitrogen levels (%N) indicate a change in crude protein intake. We recorded food eaten by each ape and collected both concurrent fecal samples (N = 115) and plant foods eaten by this community (N = 64). We compared fecal δ¹³C and δ¹⁵N values (also %N) with: (a) plant values; (b) feeding data; and (c) food‐items found macroscopically in the fecal samples. Interspecies and intraspecies differences in plant and fecal isotope values (and %N) as well as seasonality in diet were determined using parametric and nonparametric tests. No difference in plant δ¹³C and δ¹⁵N values was found at intraspecies or interspecies level. Fecal isotope values reflected a diet of C₃ plants from evergreen forest vegetation. Seasonal differences in δ¹³C and δ¹⁵N corresponded with aspects of feeding and fecal macroscopic data, but only at community level. A change in crude protein intake was not indicated from %N content. This study further validates the use of staple isotope analyses of primate feces to provide a dietary overview, revealing seasonal differences at community level; however, conclusive results may be limited for individuals when using short sampling periods. Further study of variables that influence fecal %N content is also suggested to interpret crude protein intake.

Piel A. K., Strampelli P., Greathead E., Hernandez-Aguilar R. A., Moore J., Stewart F. A. (2017): The diet of open-habitat chimpanzees (Pan troglodytes schweinfurthii) in the Issa valley, western Tanzania. Journal of Human Evolution 112: 57-69.
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Comparative data on the diets of extant primates inform hypotheses about hominin resource use. Historically, data describing chimpanzee diets stem primarily from forest-dwelling communities, and we lack comparative data from chimpanzees that live in mosaic habitats that more closely resemble those reconstructed for Plio-Pleistocene hominins. We present data on the diet of a partially-habituated community of open habitat chimpanzees (Pan troglodytes schweinfurthii) from the Issa valley, western Tanzania, collected over a four-year period. Based mostly on macroscopic faecal analysis, Issa chimpanzees consumed a minimum of 69 plant species. There was no relationship between plant consumption and either fruit availability or feeding tree density; the most frequently consumed plant species were found in riverine forests, with woodland species consumed more frequently during the late dry season. We conclude by contextualising these findings with those of other open-habitat chimpanzee sites, and also by discussing how our results contribute towards reconstructions of early hominin exploitation of mosaic landscapes.

Koops K., Wrangham R. W., Cumberlidge N., Fitzgerald M. A., van Leeuwen K. L., Rothman J. M., Matsuzawa T. (2019): Crab-fishing by chimpanzees in the Nimba Mountains, Guinea. Journal of Human Evolution 133: 230-241.
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The significance of aquatic food resources for hominins is poorly understood, despite evidence of consumption as early as 1.95 million years ago (Ma). Here we present the first evidence of a non-human ape habitually catching and consuming aquatic crabs. Chimpanzees (Pan troglodytes verus) in the rainforest of the Nimba Mountains (Guinea) consumed freshwater crabs year-round, irrespective of rainfall or ripe fruit availability. Parties of females and offspring fished for crabs more than predicted and for longer durations than adult males. Across months, crab-fishing was negatively correlated with ant-dipping, suggesting a similar nutritional role. These findings contribute to our understanding of aquatic faunivory among hominins. First, aquatic faunivory can occur in closed forests in addition to open wetlands. Second, aquatic fauna could have been a staple part of some hominin diets, rather than merely a fallback food. Third, the habitual consumption of aquatic fauna could have been especially important for females and their immature offspring. In addition to providing small amounts of essential fatty acids, crabs might also be eaten for their micronutrients such as sodium and calcium, especially by females and young individuals who may have limited access to meat.

Abwe E. E., Morgan B. J., Doudja R., Kentatchime F., Mba F., Dadjo A., Venditti D. M., Mitchell M. W., Fosso B., Mounga A., Fotso R. C., Gonder M. K. (2020): Dietary ecology of the nigeria–cameroon chimpanzee (Pan troglodytes ellioti). International Journal of Primatology 41: 81-104.
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Examining the diets of primate populations inhabiting different habitat types could be useful in understanding local adaptation and divergence between these populations. In Cameroon, the Nigeria–Cameroon chimpanzee (Pan troglodytes ellioti) is subdivided into two genetically distinct populations that occupy different habitat types; one occurs in forests to the west and the other in a forest–woodland–savanna mosaic (ecotone) in the center of the country. We used macroscopic fecal sample assessment to investigate dietary differences in relation to monthly fruit availability among P. t. ellioti communities in both habitat types: at Ebo Forest (rainforest) and Mbam and Djerem National Park (ecotone). We collected data simultaneously across three sites: Bekob and Njuma (rainforest) and Ganga (ecotone) from January 2016 to December 2017. In the dry season, fruits were the most important dietary component at Bekob and Njuma, while nonfruit plant material (leaves, pith, and bark) were most important at Ganga. In the wet season, the proportion of fruits in the diet was greatest at Ganga, while nonfruit plant material dominated chimpanzee diets at Bekob and Njuma. Chimpanzees at Bekob ate more fruits from introduced and secondary forest plant species than those at Njuma and Ganga, especially during periods of fruit scarcity. Animal consumption was higher at Ganga and was inversely associated with fruit consumption. These observations link habitat diversity to differences in feeding ecology among genetically distinct populations of P. t. ellioti. We speculate that dietary differences reflect broader socioecological variation between these populations, and collectively, that these factors promote intraspecific divergence.

Matthews J. K., Ridley A., Kaplin B. A., Grueter C. C. (2020): A comparison of fecal sampling and direct feeding observations for quantifying the diet of a frugivorous primate. Current Zoology 66: 333-343.
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Both observational and indirect evidence are widely used to determine the diets of wild animals. Direct observations are often assumed to provide the most comprehensive reflection of diet, but many wild animals are logistically challenging to observe. Despite the regular use of observational and indirect methods for inferring diet in wild animals, they have rarely been compared in detail for the same study population. Over 12 months this study assessed the congruence of methods in estimating the diet of a montane community of eastern chimpanzees Pan troglodytes schweinfurthii in Nyungwe National Park, Rwanda using observational scan samples and macroscopic fecal inspection. The assessment of the number of food species consumed each month was comparable between methods, but the estimation of the composition of items in the diet differed significantly. Most notably, the fecal samples significantly underestimated the consumption of flowers, and certain fruit species, which based on direct behavioral observations were seasonally consumed at very high rates. Conversely, direct observations underestimated the consumption of leaves and pith in comparison to results present in the fecal samples. These results suggest that combining methods where possible is most useful for accurate monitoring of dietary trends, particularly for species that experience significant seasonal shifts in their diet.

Osman N. A., Abdul-Latiff M. A. B., Mohd-Ridwan A. R., Yaakop S., Nor S. M., Md-Zain B. M. (2020): Diet composition of the wild stump-tailed macaque (Macaca arctoides) in Perlis State Park, Peninsular Malaysia, using a chloroplast tRNL DNA metabarcoding approach: A preliminary study. Animals 10: 2215.
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Understanding dietary diversity is a fundamental task in the study of stump-tailed macaque, Macaca arctoides in its natural habitat. However, direct feeding observation and morphological identification using fecal samples are not effective and nearly impossible to obtain in natural habitats because this species is sensitive to human presence. As ecological methods are challenging and time-consuming, DNA metabarcoding offers a more powerful assessment of the diet. We used a chloroplast tRNL DNA metabarcoding approach to identify the diversity of plants consumed by free-ranging M. arctoides in the Malaysia–Thailand border region located in Perlis State Park, Peninsular Malaysia. DNA was extracted from three fecal samples, and chloroplast tRNL DNA was amplified and sequenced using the Illumina MiniSeq platform. Sequences were analyzed using the CLC Genomic Workbench software. A total of 145 plant species from 46 families were successfully identified as being consumed by M. arctoides. The most abundant species were yellow saraca, Saraca thaipingensis (11.70%), common fig, Ficus carica (9.33%), aramata, Clathrotropis brachypetala (5.90%), sea fig, Ficus superba (5.44%), and envireira, Malmea dielsiana (1.70%). However, Clathrotropis and Malmea are not considered Malaysian trees because of limited data available from Malaysian plant DNA. Our study is the first to identify plant taxa up to the species level consumed by stump-tailed macaques based on a DNA metabarcoding approach. This result provides an important understanding on diet of wild M. arctoides that only reside in Perlis State Park, Malaysia.

Uwimbabazi M., Raubenheimer D., Tweheyo M., Basuta G. I., Conklin‐Brittain N. L., Wrangham R. W., Rothman J. M. (2021): Nutritional geometry of female chimpanzees (Pan troglodytes). American Journal of Primatology 83: e23269.
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Primate foraging is influenced by the spatial and temporal distribution of foods, which may facilitate or constrain optimal nutrient intakes. Chimpanzees are frugivorous primates that mainly subsist on ripe fruit that is typically low in available protein (AP) and high in easily digestible carbohydrates. Because chimpanzees prefer ripe fruit and often eat it in large quantities compared with other foods, we hypothesized that protein intake would be tightly regulated while non-protein energy (NPE) would vary with fruit intake. To test this hypothesis, we conducted all-day follows on female chimpanzees, recorded all types of food consumed (i.e., drupes, figs, and non-fruit foods), estimated the nutritional contributions of these foods to daily NPE and AP intake and investigated how the ratio of NPE to AP varied due to changes in the types of foods consumed. Although the proportions of drupes, figs, and non-fruit foods varied in their diets, female chimpanzees maintained a relatively stable intake of AP while intake of NPE varied depending on the daily diet, demonstrating that like other frugivorous primates studied to date, chimpanzees prioritize protein. The mean daily ratio of NPE to AP was 7:1, which is similar to that of other frugivorous primates studied. Our results support the hypothesis that frugivorous animals may generally prioritize AP, while maximizing NPE intake within that constraint, and could shed light on aspects of human dietary evolution.

Brun L., Schneider J., Carrió E. M., Dongre P., Taberlet P., Fumagalli L. (2022): Focal vs. fecal: Seasonal variation in the diet of wild vervet monkeys from observational and DNA metabarcoding data. Ecology and Evolution 12: e9358.
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Assessing the diet of wild animals reveals valuable information about their ecology and trophic relationships that may help elucidate dynamic interactions in ecosystems and forecast responses to environmental changes. Advances in molecular biology provide valuable research tools in this field. However, comparative empirical research is still required to highlight strengths and potential biases of different approaches. Therefore, this study compares environmental DNA and observational methods for the same study population and sampling duration. We employed DNA metabarcoding assays targeting plant and arthropod diet items in 823 fecal samples collected over 12 months in a wild population of an omnivorous primate, the vervet monkey (Chlorocebus pygerythrus). DNA metabarcoding data were subsequently compared to direct observations. We observed the same seasonal patterns of plant consumption with both methods; however, DNA metabarcoding showed considerably greater taxonomic coverage and resolution compared to observations, mostly due to the construction of a local plant DNA database. We found a strong effect of season on variation in plant consumption largely shaped by the dry and wet seasons. The seasonal effect on arthropod consumption was weaker, but feeding on arthropods was more frequent in spring and summer, showing overall that vervets adapt their diet according to available resources. The DNA metabarcoding assay outperformed also direct observations of arthropod consumption in both taxonomic coverage and resolution. Combining traditional techniques and DNA metabarcoding data can therefore not only provide enhanced assessments of complex diets and trophic interactions to the benefit of wildlife conservationists and managers but also opens new perspectives for behavioral ecologists studying whether diet variation in social species is induced by environmental differences or might reflect selective foraging behaviors.

Yoshikawa M., Ogawa H., Koganezawa M., Idani G. I. (2022): Seasonal food changes and feeding behaviour adaptations of savanna chimpanzees at Nguye in Ugalla, Tanzania. Primates 63: 585-601.
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We studied the feeding strategies of savanna chimpanzees (Pan troglodytes) at Nguye in Ugalla, Western Tanzania (05°13’S, 30°28’E). Among the driest most open chimpanzee habitats, Ugalla is covered mainly by woodlands. We analysed undigested contents in chimpanzee faeces, and conducted a vegetation survey and a 1-year phenology survey every 2 weeks. The fruits of some trees with the highest biomass had high appearance rates in faeces (e.g. Parinari curatellifolia and Diplorhynchus condylocarpon). Herbaceous Aframomum mala fruits grew in large patches in savanna woodland near forest edges along rivers and had the highest appearance frequency over the longest seasonal period in faeces. Other species with higher appearance rates in faeces for long seasonal periods included Grewia mollis at the forest edge and Thespesia garckeana growing on termite mounds at the forest edge. These two tree species had low biomass. Thus, savanna chimpanzees fed on some tree foods with higher biomass, herbaceous fruits instead of scarcer tree fruits, and fruits at forest edges and in forests which occupy a small portion of the study area, in addition to woodlands which occupy a large proportion. The forest edge and interior run continuously for long distances along rivers. Forest occupies 2% of this area, but chimpanzees can continuously obtain food by moving along riverine forest. To compensate for fruit scarcity in the non-fruiting (early rainy) season, chimpanzees ate fibrous, low-quality plant parts. Chimpanzees formed smaller parties when ripe fruits and unripe legumes were scarcer. Using these feeding strategies, chimpanzees adapted to savanna woodlands.

Schneider J., Brun L., Taberlet P., Fumagalli L., van de Waal E. (2023): Molecular assessment of dietary variation in neighbouring primate groups. Methods in Ecology and Evolution 14: 1925-1936.
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Facing rapid environmental changes and anthropogenic habitat destruction, animal behavioural plasticity becomes an adaptive potential that needs to be considered in conservation strategies along with, for example, genetic diversity. Here, we evaluate to what extent non-invasive environmental DNA (eDNA) methods may contribute to the assessment of intraspecies behavioural plasticity in terms of foraging behaviour. We analysed DNA metabarcoding data for plant components in the diet of four neighbouring groups of wild vervet monkeys Chlorocebus pygerythrus to identify intergroup variation (IGV). The faecal samples considered for the analyses were limited to the summer season to minimise the impact of seasonality. Each sample was attributed by observation to individuals with known life history data. A plant survey was conducted in each group home range during the study period to account for environmental variation. We observed mixed results when testing whether IGV in plant consumption was greater than intragroup variation, indicating that the influence of social dynamics must be considered. Intragroup variation was positively correlated with group size. We observed IGV in diet composition among all groups as well as in some pairwise comparisons. We found significant dietary differences between two group pairs when considering only adult females. Lastly, we observed IGV in foraging of specific plants that were not explained by their distribution, suggesting behavioural differences in selectivity between groups. Our study system and organism, being a highly social and non-threatened primate species, with constant gene flow and overlapping territories between groups, provides an ideal model to evaluate the usage of eDNA-based methods to better understand the impact of social factors on IGV. Our results highlight the need to consider social and demographic factors, the impact of which remains complicated to disentangle from environmental factors. However, we emphasise the great potential for studying social groups using eDNA and that such studies are needed to better understand intraspecific behavioural plasticity in wild populations.

RABBITS, HARES, AND PIKAS

Aryal A., Brunton D., Ji W., Yadav H. K., Adhikari B., Raubenheimer D. (2012): Diet and habitat use of hispid hare Caprolagus hispidus in Shuklaphanta Wildlife Reserve, Nepal. Mammal Study 37: 147-154.
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Hispid hare Caprolagus hispidus is one of the less studied endangered small mammal species in the world. Hispid hare distribution includes the tropical grassland ecosystem in Nepal. Grassland fire is one of the management regimes used in this region and its impact on biodiversity is controversial. We investigated the diet and habitat use of hispid hare before and after a grassland fire at Shuklaphanta Wildlife Reserve (SWR) in Nepal. Fecal pellets were used for micro-histological analysis to understand hispid hare diet. We laid out sampling plots in areas where we encountered hispid hare sign and recorded habitat and vegetation information. We also looked for signs of hare presence along systematically positioned transect lines and used these data to assess the population status of hispid hare. Population density of hispid hare was 5.76 individuals/km² and we estimated a population size of 219 ± 40 hispid hare within the 41 km² grasslands of SWR. Hispid hare primarily used tall grassland habitat. Nineteen plant species were identified in hispid hare pellets with Saccharum spontaneum and Imperata cylindrica having the highest frequency of occurrence. There were no significant differences in the distribution of plant species in the pellets before and after the fire; however a significantly higher diversity of plants were recorded in hispid hare diet after the fire. We recommend a change to the timing of grass burning to either before or after the hispid hare breeding season to reduce the direct (burning, destruction of nests) and indirect (increased risk of predation) negative effects of such grassland management on hare populations. Population management strategies and a field based conservation captive breeding program should be implemented immediately to maintain a viable population of hispid hare in SWR.

Freschi P., Fascetti S., Musto M., Mallia E., Blasi A. C., Cosentino C., Paolino R. (2014): Diet of the Apennine hare in a southern Italy Regional Park. European Journal of Wildlife Research 60: 423-430.
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In this study, we examined the annual diet composition of Lepus corsicanus in two different sites within a southern Italy Regional Park. Vegetation of site 1 was composed of a mixed scrub forest (Viburno–Quercetum ilicis s.l.), a ripisilva (Roso sempervirentis–Populetum nigrae), some thermophilous scrubs (Pruno–Rhamnetalia alaterni), and a Pinus halepensis reforested area. Site 2 comprised a mixed-oak forest (Centaureo–Quercetum pubescentis s.l.) with meadows and arable lands. Micro-histological analysis of faecal samples revealed that hares utilised 70 different species of plants during the year, indicating the capability of the Apennine hare of exploiting a wide variety of vegetation. Herbaceous plants (Hemicryptophytes, particularly graminoid grasses, and Geophytes) predominated in the diet. Brachypodium sylvaticum (9.44 %) and Allium subhirsutum (8.28 %) were the major contributors to the diet in sites 2 and 1, respectively. Other taxa found most often in the diet were Trifolium pratense (site 1: 8.19 %; site 2: 5.80 %) and Prunus spinosa (site 1: 7.03 %; site 2: 4.10 %). Significant differences were found between sites in terms of diet richness, diversity, and evenness. Nevertheless, both the similarity indices (Morisita–Horn: 0.79; Sørensen: 0.87) showed that the food composition of the hare’s diet was broadly the same in both sites. Some qualitative and quantitative differences between sites were due to the availability or consumption of some plant species and evidenced that the Apennine hare can modify its trophic niche in order to adapt its dietary requirements to the availability of food.

Lush L., Ward A. I., Wheeler P. (2017): Dietary niche partitioning between sympatric brown hares and rabbits. Journal of Zoology 303: 36-45.
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Coexistence of ecologically similar species is sustained by niche partitioning, a fundamental element of which is diet. Overlapping of resource requirements between sympatric species can create interspecific competitive or facilitative effects on the foraging behaviour of herbivores. Brown hares (Lepus europaeus) and European rabbits (Oryctolagus cuniculus) are similar in size, morphology, feeding type and occupy the same habitats, but direct evidence of competition for resources between them is lacking. Both species are widespread and simultaneously pests and species of conservation concern in different parts of their range. We investigated dietary overlap of brown hares and European rabbits in pastures in relation to pasture management and hare and rabbit abundance. Grasses were the predominant component in both hare and rabbit diets with high overlap of plant species. Both rabbits and hares showed some selectivity for particular plants with evidence of consistent selection for Phleum spp. and relative avoidance of Poa spp. However, differences in the smaller components of hare and rabbit diet resulted in significant differences in diet overall. There was no evidence that higher relative density of one species led to dietary shifts but pasture management affected the diet of both species. Nutritional composition of diets of both species also differed between cattle and sheep pastures with higher fibre, ash and fat in the former. Our data provide no evidence of competitive exclusion between rabbits and hares on the basis of diet, but suggest that the effects of livestock on their respective diets may influence indirect competition in favour of rabbits over hares.

Buglione M., Maselli V., Rippa D., de Filippo G., Trapanese M., Fulgione D. (2018): A pilot study on the application of DNA metabarcoding for non-invasive diet analysis in the Italian hare. Mammalian Biology 88: 31-42.
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The Italian hare is endemic to central and southern Italy and currently designated as vulnerable by the International Union for the Conservation of Nature. Despite the worrying status, few studies have explored its ecology. In this scenario, improving knowledge of food preferences is of primary importance. Here, we present a non-invasive pilot study set to analyse the diet composition of the Italian hare from southern Italy, starting from fecal pellets, and using, for the first time on this species, DNA metabarcoding and next generation sequencing. The findings indicated that this approach provides reliable qualitative and semi-quantitative information, allowing the characterization of the hare diet and its seasonal variation using 22 fecal samples. In a single experiment, through time and cost-effective screening of multiple DNA metabarcodes, we detected a broad diversity of plants (99 taxa). Unlike traditional methods, this approach can identify items that leave no solid remains or that simply are lacking in diagnostic taxonomic features. Moreover, it starts from small amounts of input fecal material, useful in investigation on elusive and vulnerable species. The major guideline for future applications is to use a barcode short enough to allow amplification of environmental degraded DNA and the selection of the universal primers for PCR amplification. The results should be useful to define management actions for conservation of endemic Italian hare.

Rizzardini G., Fascetti S., Pietri C., Riga F., Cosentino C., Freschi P. (2019): Feeding preferences in dry season of the Italian hare (Lepus corsicanus) in two sites of Corsica. European Journal of Wildlife Research 65: 43.
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In this research, the diet composition and feeding selection of the Italian hare were evaluated and compared in two sites of Haute-Corse region localised in the territories of Tallone and Aleria. The present study is the first considering feeding selection of Lepus corsicanus. The considered period ranged from June to October. Quadrat method was used to assess plant frequency, while diet composition was determined by microhistological analysis of faecal pellets collected monthly. Grasses represented the basis of the diet, with frequencies around 50% in both study areas, followed by non-leguminous forbs with an incidence of 29% in Aleria and over 31% in Tallone. Leguminous forbs and shrubs complemented its diet. Poaceae resulted to be the most preferred and selected family in the diet in both sites. In the diet, we observed 79 species, but only a few of them were in percentages greater than 5%. The most utilised species in the diet were Brachypodium sylvaticum, Briza maxima, and Trifolium angustifolium in Aleria and Digitaria sanguinalis, Briza maxima, and Daucus carota in Tallone. Our study evidenced that in the considered areas, characterised also in the dry period by wide plant diversity, the Italian hare behaved as generalist. Significant differences in the diet composition and in the diversity index between the two sites showed the adaptability of the Italian hare to different habitats and the influence of the vegetation on feeding habits of the species.

Buglione M., De Filippo G., Conti P., Fulgione D. (2022): Eating in an extreme environment: diet of the European hare (Lepus europaeus) on Vesuvius. The European Zoological Journal 89: 1201-1214.
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The European hare (Lepus europaeus) is cosmopolitan species, living in a variety of habitats and showing a diversified diet, that has been described mainly from agricultural meadows and crops, with little information available for extreme environments. Here, we describe, for the first time, the diet of the European hare from Mount Vesuvius, using DNA metabarcoding and high-throughput sequencing on DNA extracted from faecal pellets, a proxy for a population living in a volcanic environment. The DNA from pellets was first genetically assigned to European hare using high-resolution melting analysis. The diet of the hare on Vesuvius is mainly composed of herbaceous species belonging to Fabaceae (86.26% of total diet). The most frequent plant items ingested by the species are Galega officinalis and Lupinus angustifolius (67.10% of total diet), although these are detected only sporadically in the study area. Indeed, the spectrum of available plants also includes other easily accessible wild (i.e. Lolium sp., Bromus sp., Rumex sp.) and cultivated (i.e. Solanum lycopersicum, Cucumis melo, Pisum sativum) plant items, found only in traces in the diet of the hares. Our contribution adds information on the trophic ecology of the European hare, exploring its ability to live in an extreme environment. This could be useful to set a management strategy for conservation of the species, which is ecologically relevant on Vesuvius as prey for birds and mammals, as well as a vegetation modulator via selective grazing by endozoochory. Furthermore, our study represents the latest information on the diet of the hare living in an environment that no longer exists: an extensive fire destroyed about 80% of the woody area after our sampling. The post-fire regrowth is transforming the original environment and consequently the trophic availability for the European hare.

RODENTS

Gil-Delgado J. A., Mira O., Viñals A., Gómez J., Banyuls N., Vives-Ferrándiz C. (2010): Diet of the garden dormouse (Eliomys quercinus Linnaeus 1766) in orange groves: seasonal variation and use of available resources. Mammalia 74: 147-151.
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The aim of this study was to describe the feeding habits of the garden dormouse Eliomys quercinus in orange groves of eastern Spain. The study area was an extensive orange grove near Sagunto (Valencia, Spain). This study combines two different non-invasive methods, the study of food store remains and the macroscopic analysis of feces. A total of 527 feces and the food remains of 172 food stores were recollected from nest boxes. Each feces was classified into five categories according to its characteristics: arthropods, gastropods, oranges, plants, and others. Throughout the year, the garden dormouse consumed all these types of food. The most consumed prey in summer and autumn were insects. However, in winter and spring orange fruit was the principal food type. Seasonal variation of orange, arthropods, gastropods, and plant matter consumption occurred. Among animal prey types, insects were the most consumed group and large-sized species were preferred. Vertebrate preys were found in food stores. The annual variation of the garden dormouse diet owing to the seasonal availability of resources in an agricultural environment revealed its feeding behavior plasticity.

Juškaitis R., Baltrūnaitė L. (2013): Feeding on the edge: the diet of the hazel dormouse Muscardinus avellanarius (Linnaeus 1758) on the northern periphery of its distributional range. Mammalia 77: 149-155.
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Although the hazel dormouse Muscardinus avellanarius is considered both a highly specialized and a threatened mammal species in Europe, it is relatively common and widespread in Lithuania, situated on the northern periphery of its distributional range. We studied dormouse diet over the entire activity season using microscopic analysis of feces to gain a better understanding of its ecology. Our results confirm that the hazel dormouse is indeed a selective feeder, always showing a preference for the reproductive parts of plants. However, the plant species used by dormice in Lithuania are different from those used in other parts of the dormouse range (e.g., England). In Lithuania, the main dormouse food sources are the inflorescence of willow, oak, and spruce in spring; the berries of honeysuckle, raspberry, and glossy buckthorn in summer; and the berries of glossy buckthorn, oak acorns, and hazelnuts in autumn. Only in early summer, the proportion of food of animal origin is high. The berries of glossy buckthorn are very important to dormouse for feeding and accumulation of fat reserves in autumn. The adaptability of dormice to feed on local plants, as well as the sufficient diversity of food plants in their habitats, allows these animals to be relatively common and widespread on the northern periphery of their range.

Latinne A., Galan M., Waengsothorn S., Rojanadilok P., Eiamampai K., Sribuarod K., Michaux J. (2014): Diet analysis of Leopoldamys neilli, a cave-dwelling rodent in Southeast Asia, using next-generation sequencing from feces. Journal of Cave and Karst Studies 76: 139-145.
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Leopoldamys neilli is a Murinae rodent endemic to limestone karst of Thailand and the Lao PDR, but its ecology and the reasons of its endemism to karst are still totally unknown. The aim of this pilot study was to examine the plant composition of the diet of L. neilli at the level of order and family using DNA for molecular identification and to compare it with two other forest-dwelling Leopoldamys species, L. herberti and L. sabanus. A 202bp fragment of the rbcL gene was amplified and sequenced for twenty-three fecal samples of the three species using 454 pyrosequencing. We successfully identified a total of seventeen orders and twenty-one plant families, corresponding to thirty-three putative species, in the feces of these three Leopoldamys species. Solanaceae were the most common plants in the diet of L. neilli regardless of the region and sampling season, and they were also present in feces of both L. herberti and L. sabanus. The Araceae, Fabaceae, and Apocynaceae families were also identified in feces of L. neilli collected in various regions of Thailand and at different seasons. Plants of the Oleaceae family are consumed by both L. herberti and L. sabanus but were not found in the diet of L. neilli. Further improvements of the study, such as the use of additional genes, the creation of a reference collection, the microhistological examination of plant fragments to determine which parts of the plant are consumed, and the analysis of the animal diet of Leopoldamys are suggested to enhance the quality and accuracy of the results obtained.

Iwanowicz D. D., Vandergast A. G., Cornman R. S., Adams C. R., Kohn J. R., Fisher R. N., Brehme C. S. (2016): Metabarcoding of fecal samples to determine herbivore diets: a case study of the endangered Pacific pocket mouse. Plos One 11: e0165366.
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Understanding the diet of an endangered species illuminates the animal’s ecology, habitat requirements, and conservation needs. However, direct observation of diet can be difficult, particularly for small, nocturnal animals such as the Pacific pocket mouse (Heteromyidae: Perognathus longimembris pacificus). Very little is known of the dietary habits of this federally endangered rodent, hindering management and restoration efforts. We used a metabarcoding approach to identify source plants in fecal samples (N = 52) from the three remaining populations known. The internal transcribed spacers (ITS) of the nuclear ribosomal loci were sequenced following the Illumina MiSeq amplicon strategy and processed reads were mapped to reference databases. We evaluated a range of threshold mapping criteria and found the best-performing setting generally recovered two distinct mock communities in proportions similar to expectation. We tested our method on captive animals fed a known diet and recovered almost all plant sources, but found substantial heterogeneity among fecal pellets collected from the same individual at the same time. Observed richness did not increase with pooling of pellets from the same individual. In field-collected samples, we identified 4–14 plant genera in individual samples and 74 genera overall, but over 50 percent of reads mapped to just six species in five genera. We simulated the effects of sequencing error, variable read length, and chimera formation to infer taxon-specific rates of misassignment for the local flora, which were generally low with some exceptions. Richness at the species and genus levels did not reach a clear asymptote, suggesting that diet breadth remained underestimated in the current pool of samples. Large numbers of scat samples are therefore needed to make inferences about diet and resource selection in future studies of the Pacific pocket mouse. We conclude that our minimally invasive method is promising for determining herbivore diets given a library of sequences from local plants.

Leitner B. M., Leitner P. (2017): Diet of the Mohave ground squirrel (Xerospermophilus mohavensis) in relation to season and rainfall. Western North American Naturalist 77: 1-13.
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The Mohave ground squirrel (Xerospermophilus mohavensis) is endemic to the western Mojave Desert of California. It is listed as threatened under the California Endangered Species Act, yet there is little published information on its habitat requirements. We studied the diet of Mohave ground squirrels at 4 sites in desert scrub habitat in Inyo County, California, primarily by microhistological analysis of 754 samples of fecal pellets collected from live-trapped animals. Over all sites and seasons, shrub foliage was the largest component of the diet (39.8% relative density) and mainly derived from several taxa of Chenopodiaceae: winterfat (Krascheninnikovia lanata), spiny hopsage (Grayia spinosa), and saltbushes (Atriplex spp.). Forb leaves were next in importance (34.1% relative density), especially from Fabaceae (Astragalus and Lupinus), Polemoniaceae (Gilia and Linanthus), and Asteraceae. Flowers, pollen, and seeds were also major components (20.3% relative density). Leaves composed nearly all of the diet in spring, whereas pollen, flowers, and seeds made up about a third of the diet in summer. Following dry winters when annual forbs were limited, Mohave ground squirrels depended primarily on foliage from perennial shrubs and forbs. Following wet winters when spring annuals were abundant and most plant species flowered and set seed prolifically, squirrels consumed a high proportion of leaves plus flowers, pollen, and seeds of annual forbs. Mohave ground squirrels reproduced only after winter rainfall >80 mm that resulted in a standing crop of herbaceous annuals ≥100 kg · ha^-1. Mohave ground squirrels consumed very little of the nonnative annual plant biomass present on our study sites (Erodium, Salsola, Bromus, and Schismus contributed <3% overall to the diet). Conservation implications include the following: (1) priority should be given to protecting habitats supporting preferred perennial forage plants, including winterfat and spiny hopsage; (2) habitats with an understory dominated by native annual forbs have higher value than those dominated by nonnative plants, especially annual grasses; and (3) if climate change results in lower and less regular winter precipitation, suitable habitat for Mohave ground squirrels may be reduced and fragmented in the drier portions of the geographic range.

Sato J. J., Shimada T., Kyogoku D., Komura T., Uemura S., Saitoh T., Isagi Y. (2018): Dietary niche partitioning between sympatric wood mouse species (Muridae: Apodemus) revealed by DNA meta-barcoding analysis. Journal of Mammalogy 99: 952-964.
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Plant diets of 2 sympatric species of wood mice in Japan (Apodemus argenteus and A. speciosus) were determined by DNA meta-barcoding analyses of feces using the nucleotide sequences of the chloroplast trnL P6 loop intron region as a molecular marker. The 2 species showed a relatively large degree of niche overlap in plant dietary profiles, feeding mostly on acorn-producing Fagaceae species (assumed to be Quercus crispula). However, A. argenteus was less dependent on Fagaceae species than A. speciosus. Instead, A. argenteus had a wider niche breadth, feeding upon a wider range of plant families such as Betulaceae, Fabaceae, Oleaceae, Pinaceae, Rutaceae, Sapindaceae, Tiliaceae, and Ulmaceae, which were consumed only infrequently by A. speciosus. There was also evidence for species differences in diet across seasons. Oleaceae species (assumed to be Fraxinus mandshurica) were consumed by A. argenteus from June to August, and by A. speciosus from August to October. The results suggested that A. argenteus is a generalist feeder, and A. speciosus a specialist, and the 2 Apodemus species separate their dietary niches not only by the component plant species overall, but also by differences within seasons.

Goldberg A. R., Conway C. J., Tank D. C., Andrews K. R., Gour D. S., Waits L. P. (2020): Diet of a rare herbivore based on DNA metabarcoding of feces: selection, seasonality, and survival. Ecology and Evolution 10: 7627-7643.
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In herbivores, survival and reproduction are influenced by quality and quantity of forage, and hence, diet and foraging behavior are the foundation of an herbivore’s life history strategy. Given the importance of diet to most herbivores, it is imperative that we know the species of plants they prefer, especially for herbivorous species that are at risk for extinction. However, it is often difficult to identify the diet of small herbivores because: (a) They are difficult to observe, (b) collecting stomach contents requires sacrificing animals, and (c) microhistology requires accurately identifying taxa from partially digested plant fragments and likely overemphasizes less‐digestible taxa. The northern Idaho ground squirrel (Urocitellus brunneus) is federally threatened in the United States under the Endangered Species Act. We used DNA metabarcoding techniques to identify the diet of 188 squirrels at 11 study sites from fecal samples. We identified 42 families, 126 genera, and 120 species of plants in the squirrel’s diet. Our use of three gene regions was beneficial because reliance on only one gene region (e.g., only trnL) would have caused us to miss >30% of the taxa in their diet. Northern Idaho ground squirrel diet differed between spring and summer, frequency of many plants in the diet differed from their frequency within their foraging areas (evidence of selective foraging), and several plant genera in their diet were associated with survival. Our results suggest that while these squirrels are generalists (they consume a wide variety of plant species), they are also selective and do not eat plants relative to availability. Consumption of particular genera such as Perideridia may be associated with higher overwinter survival.

Lopes C. M., De Barba M., Boyer F., Mercier C., Galiano D., Kubiak B. B., Maestri R., da Silva Filho P. J. S., Gielly L., Coissac E., de Freitas T. R. O. (2020): Ecological specialization and niche overlap of subterranean rodents inferred from DNA metabarcoding diet analysis. Molecular Ecology 29: 3143-3153.
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Knowledge of how animal species use food resources available in the environment can increase our understanding of many ecological processes. However, obtaining this information using traditional methods is difficult for species feeding on a large variety of food items in highly diverse environments. We amplified the DNA of plants for 306 scat and 40 soil samples, and applied an environmental DNA metabarcoding approach to investigate food preferences, degree of diet specialization and diet overlap of seven herbivore rodent species of the genus Ctenomys distributed in southern and midwestern Brazil. The metabarcoding approach revealed that these species consume more than 60% of the plant families recovered in soil samples, indicating generalist feeding habits of ctenomyids. The family Poaceae was the most common food resource retrieved in scats of all species as well in soil samples. Niche overlap analysis indicated high overlap in the plant families and molecular operational taxonomic units consumed, mainly among the southern species. Interspecific differences in diet composition were influenced, among other factors, by the availability of resources in the environment. In addition, our results provide support for the hypothesis that the allopatric distributions of ctenomyids allow them to exploit the same range of resources when available, possibly because of the absence of interspecific competition.

Aylward C. M., Statham M. J., Barthman‐Thompson L., Kelt D. A., Sacks B. N. (2022): Dietary characterization of the endangered salt marsh harvest mouse and sympatric rodents using DNA metabarcoding. Ecology and Evolution 12: e9121.
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The salt marsh harvest mouse (Reithrodontomys raviventris; RERA) is an endangered species endemic to the coastal wetlands of the San Francisco Estuary, California. RERA are specialized to saline coastal wetlands, and their historical range has been severely impacted by landscape conversion and the introduction of non-native plant and rodent species. A better understanding of their diet is needed to assess habitat quality, particularly in relation to potential competitors. We investigated three questions using DNA metabarcoding with ITS2 and trnL markers: (1) Do RERA specialize on the native plant, pickleweed (Salicornia pacifica), (2) Do RERA consume non-native plants, and (3) What is the dietary niche breadth and overlap with three sympatric native and non-native rodents? RERA diet was dominated by two plants, native Salicornia and non-native salt bush (Atriplex spp.), but included 48 plant genera. RERA diet breadth was narrowest in fall, when they consumed the highest frequencies of Salicornia and Atriplex, and broadest in spring, when the frequencies of these two plants were lowest. Diet breadth was slightly lower for RERA than for co-occurring species in pairwise comparisons. All four species consumed similarly high frequencies of wetland plants, but RERA consumed fewer grasses and upland plants, suggesting that it may be less suited to fragmented habitat than sympatric rodents. Diet overlap was lowest between RERA and the native California vole (Microtis californicus). In contrast, RERA diet overlapped substantially with the native western harvest mouse (R. megalotis) and non-native house mouse (Mus musculus), suggesting potential for competition if these species become sufficiently abundant.

Groen K., Trimbos K. B., Hein S., Blaauw A. I., van Bodegom P. M., Hahne J., Jacob J. (2022): Establishment of a faecal DNA quantification technique for rare and cryptic diet constituents in small mammals. Molecular Ecology Resources 22: 2220-2231.
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DNA-based approaches have greatly improved the applicability of dietary studies aimed at investigating ecological processes. These studies have provided direct insights into, otherwise difficult to measure, interactions between species and trophic levels, food web structure and ecosystem functioning. However, despite these advances, DNA-based methods have struggled to accurately quantify the whole breadth of diet constituents because of methodological biases, such as amplification bias and digestive processes. The present study is, to our knowledge, the first diet study to use droplet digital PCR to quantify diet constituents. We manipulated the diet of wild-caught wood mice (Apodemus sylvaticus) by feeding them with a known amount of small vegetable seeds (onion and carrot) and quantified the DNA traces of these diet constituents in faecal samples. The sensitivity of the technique combined with the control on the experimental design allowed mitigation of methodological bias. We were able to accurately determine DNA concentrations of small vegetable seeds in the diet of wood mice. Quantification of target DNA demonstrated significant differences in DNA content when one vs. five seeds were consumed. These differences remained significant when the age, sex and other diet constituents of the mice were altered. Different DNA markers, targeting different parts of the chloroplast, influenced onion DNA detectability. However, all onion and carrot markers showed higher DNA content for higher seed numbers. Overall, the sensitive DNA-based approach developed in this study allows for minimally invasive quantification of small diet constituents in faeces, which would otherwise be undetectable with traditional methods.

Schuette P., Ebbert S., Droghini A., Nawrocki T. (2022): Small mammal diet indicates plant diversity, vegetation structure, and ecological integrity in a remote ecosystem. Biodiversity and Conservation 31: 909-924.
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We evaluated patterns of herbivory and predation/scavenging by a small mammal, the arctic ground squirrel (Urocitellus parryii), as an indicator of biological diversity, vegetation structure, and ecological integrity on two contrasting islands in the vast and remote Alaska Maritime National Wildlife Refuge. Using DNA metabarcoding techniques, we assessed the taxonomic composition of vertebrates, vascular plants, and bryophytes at family and growth form levels in arctic ground squirrel fecal samples collected on the ecologically intact island of Chowiet, which supports diverse seabird and vegetation communities, and the degraded island of Chirikof, which has incurred centuries of disturbance from introduced arctic fox (Vulpes lagopus) and cattle (Bos taurus taurus). We also evaluated whether fecal samples collected across the growing season (May—August) could act as indicators of herbivore-plant phenology. We did not detect any vertebrate taxa, however, observed herbivory patterns closely matched known differences in island plant diversity based on independent surveys. Diets from the ecologically intact island exhibited higher taxonomic richness, greater evenness, and higher shrub content than the ecologically degraded island, which is a grass- and forb-dominated ecosystem. On Chowiet Island, diets changed seasonally, likely in response to changing availability, nutritional quality, and toxin content associated with primary production. Small mammal diet served as an effective indicator of plant diversity, vegetation structure, and ecological integrity when compared against known composition and status of these remote island ecosystems. Non-invasive fecal sampling and dietary analyses using genetic techniques may provide a useful strategy for monitoring biological diversity, particularly in remote areas where widespread intensive sampling is unfeasible.

UNGULATES

Wam H. K., Hjeljord O. (2010): Moose summer diet from feces and field surveys: a comparative study. Rangeland Ecology & Management 63: 387-395.
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Microhistological analysis of feces is the most applied noninvasive method for assessing diets of wild ungulates. However, the method is complicated by differential digestibility of forage species. To evaluate the efficacy of this method in quantifying browse components in summer diets of moose (Alces alces L.) on Norwegian rangelands, we compared it to parallel field surveys of browsed vegetation on the same range. Although the same principal diet components were identified in the feces and in the field, there were consistent discrepancies between the two methods in estimated proportional diet contents. Birch (Betula spp.) showed the highest field:fecal ratio: 3.3 ± compared to 0.9 ± for Salix spp., 0.8 ± for aspen (Populus tremula L.), and 0.6 ± for rowan (Sorbus aucuparia L.). Until in vivo fecal correction factors for differential forage digestibility are available, we caution against broad application of fecal analyses for estimating proportions of browse in moose diet. Although we could not determine the exact amount of discrepancy implicit in each method, previous studies of moose summer diet in the area clearly indicate that fecal analyses gave a less accurate representation of actual moose browse diet than did the field survey. Fecal analyses are nevertheless needed to identify moose diet components other than browse, which are not easily obtained from field surveys.

Kowalczyk R., Taberlet P., Coissac E., Valentini A., Miquel C., Kamiński T., Wójcik J. M. (2011): Influence of management practices on large herbivore diet—Case of European bison in Białowieża Primeval Forest (Poland). Forest Ecology and Management 261: 821-828.
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Large herbivores are keystone species in many forest areas, as they shape the structure, species diversity and functioning of those ecosystems. The European bison Bison bonasus has been successfully restored after extinction in the wild at the beginning of 20th century. As free-ranging populations of the species were re-established mainly in forest habitats, knowledge of the impact by the largest European terrestrial mammal on tree stands is essential. This helps to make management and conservation decisions for viable population maintenance of the species in the wild. Using a novel DNA-based method of herbivore diet analysis, the trnL approach (DNA-barcoding), we investigated the influence of different foraging conditions (access to supplementary fodder) on bison diet in winter and its potential impact on woody species. Faecal samples were collected from different bison treatment groups: (1) intensively fed; (2) less intensively fed; (3) non-fed utilising forest habitats; and (4) non-fed utilising agricultural areas surrounding the Forest. These were analysed to estimate the proportion of different plant groups consumed by bison. Bison groups differed significantly in their diet. The amount of woody materials (trees and shrubs) consumed by bison increased with decreasing access to supplementary fodder, ranging from 16% in intensively fed bison to 65% in non-fed bison utilising forest habitats. Inversely, the amount of herbs, grasses and sedges decreased from 82% in intensively fed bison to 32% in non-fed bison utilising forest habitats. The species of trees mainly browsed by bison, Carpinus/Corylus, Betula sp. and Salix sp., were of lower economic importance for forest management. The impact of bison on tree species needs further investigation, however, we can predict that browsing by bison, mainly on Carpinus/Corylus, makes an insignificant impact on forestry due to the high and increasing representation of this species in the forest understory. Supplementary feeding has several negative effects on bison ecology and health, therefore reduced and distributed supplementary feeding should be applied as the management practice in the Białowieża Forest.

Hibert F., Taberlet P., Chave J., Scotti-Saintagne C., Sabatier D., Richard-Hansen C. (2013): Unveiling the diet of elusive rainforest herbivores in next generation sequencing era? The tapir as a case study. Plos One 8: e60799.
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Characterizing the trophic relationships between large herbivores and the outstanding plant diversity in rainforest is a major challenge because of their elusiveness. This is crucial to understand the role of these herbivores in the functioning of the rainforest ecosystems. We tested a non-invasive approach based on the high-throughput sequencing of environmental samples using small plant plastid sequences (the trnL P6 loop) and ribosomal ITS1 primers, referred to as DNA metabarcoding, to investigate the diet of the largest neotropical herbivore, the lowland tapir. Sequencing was performed on plant DNA extracted from tapir faeces collected at the Nouragues station, a protected area of French Guiana. In spite of a limited sampling, our approach reliably provided information about the lowland tapir’s diet at this site. Indeed, 95.1% and 74.4% of the plant families and genera identified thanks to the trnL P6 loop, respectively, matched with taxa already known to be consumed by tapirs. With this approach we were able to show that two families and eight new genera are also consumed by the lowland tapir. The taxonomic resolution of this method is limited to the plant family and genera. Complementary barcodes, such as a small portion of ITS1, can be used to efficiently narrow identifications down to the species in some problematic families. We will discuss the remaining limitations of this approach and how useful it is at this stage to unravel the diet of elusive rainforest herbivores and better understand their role as engineers of the ecosystem.

Kartzinel T. R., Chen P. A., Coverdale T. C., Erickson D. L., Kress W. J., Kuzmina M. L., Rubenstein D. I., Wang W., Pringle R. M. (2015): DNA metabarcoding illuminates dietary niche partitioning by African large herbivores. Proceedings of the National Academy of Sciences 112: 8019-8024.
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Niche partitioning facilitates species coexistence in a world of limited resources, thereby enriching biodiversity. For decades, biologists have sought to understand how diverse assemblages of large mammalian herbivores (LMH) partition food resources. Several complementary mechanisms have been identified, including differential consumption of grasses versus nongrasses and spatiotemporal stratification in use of different parts of the same plant. However, the extent to which LMH partition food-plant species is largely unknown because comprehensive species-level identification is prohibitively difficult with traditional methods. We used DNA metabarcoding to quantify diet breadth, composition, and overlap for seven abundant LMH species (six wild, one domestic) in semiarid African savanna. These species ranged from almost-exclusive grazers to almost-exclusive browsers: Grass consumption inferred from mean sequence relative read abundance (RRA) ranged from >99% (plains zebra) to <1% (dik-dik). Grass RRA was highly correlated with isotopic estimates of % grass consumption, indicating that RRA conveys reliable quantitative information about consumption. Dietary overlap was greatest between species that were similar in body size and proportional grass consumption. Nonetheless, diet composition differed between all species—even pairs of grazers matched in size, digestive physiology, and location—and dietary similarity was sometimes greater across grazing and browsing guilds than within them. Such taxonomically fine-grained diet partitioning suggests that coarse trophic categorizations may generate misleading conclusions about competition and coexistence in LMH assemblages, and that LMH diversity may be more tightly linked to plant diversity than is currently recognized.

Gebremedhin B., Flagstad Ø., Bekele A., Chala D., Bakkestuen V., Boessenkool S., Popp M., Gussarova G., Schrøder-Nielsen A., Nemomissa S., Brochmann C. (2016): DNA metabarcoding reveals diet overlap between the endangered Walia ibex and domestic goats – implications for conservation. Plos One 11: e0159133.
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Human population expansion and associated degradation of the habitat of many wildlife species cause loss of biodiversity and species extinctions. The small Simen Mountains National Park in Ethiopia is one of the last strongholds for the preservation of a number of afro-alpine mammals, plants and birds, and it is home to the rare endemic Walia ibex, Capra walie. The narrow distribution range of this species as well as potential competition for resources with livestock, especially with domestic goat, Capra hircus, may compromise its future survival. Based on a curated afro-alpine taxonomic reference library constructed for plant taxon identification, we investigated the diet of the Walia ibex and addressed the dietary overlap with domestic goat using DNA metabarcoding of faecal samples. Faeces of both species were collected from different localities in the National Park. We show that both species are browsers, with forbs, shrubs and trees comprising the largest proportion of their diet, supplemented by grasses. There was a considerable overlap in dietary preferences. Several of the preferred diet items of the Walia ibex (Alchemilla sp., Hypericum revolutum, Erica arborea and Rumex sp.) were also among the most preferred diet items of the domestic goat. These results indicate that there is potential for competition between the two species, especially during the dry season, when resources are limited. Our findings, in combination with the expected increase in domestic herbivores, suggest that management plans should consider the potential threat posed by domestic goats to ensure future survival of the endangered Walia ibex.

Leonard J. L., Perkins L. B., Lammers D. J., Jenks J. A. (2017): Are bison intermediate feeders? Unveiling summer diet selection at the northern fringe of historical distribution. Rangeland Ecology & Management 70: 405-410.
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Bison (Bison bison) were historically distributed throughout North America with the northern edge of the distribution occurring in north-central Manitoba and surrounding provinces. Despite bison occupying the boreal zone of North America, little is known of their forage selection patterns of herbaceous plant material when occupying pastures within a densely forested aspen ecosystem. In 2015 we initiated a study to examine forage selection patterns for bison among and within summer months (June–August). We hypothesized that vegetative composition of bison diets would be consistent with availability, would shift with forage availability, and would predominately consist of grass and sedge species. We opportunistically collected adult female bison fecal samples (N = 99) and identified forage composition using the DNA barcoding method. We estimated availability of forage to the lowest taxonomical level possible using a modified Daubenmire frame. Overall, bison diets were composed of 44.3% grass, 37.7% forb, 16.3% browse, and < 2% sedge and rush. Forage availability comprised 51.2% grass, 28.3% forb, 11.0% sedge, and 7.6% rush. All analyses indicated that use and availability for grass, forb, sedge, and rush differed (P ≤ 0.05) throughout the summer. Grass and forbs were important dietary components for bison, comprising > 80% of bison diets. However, bison selected for these two dietary components independently as the summer progressed. Our results indicate that these bison consume a large portion (~ 54.0%) of low-cellulose, high cell-soluble forages to meet their dietary needs. This suggests that bison may be or become intermediate foragers and are more like elk (Cervus elaphus) than domestic cattle or sheep when inhabiting forested systems at the northern edge of their historical distribution. Herd managers and biologists should be cognizant of the importance of eudicots for bison and adopt a management plan that promotes a spatially heterogenous vegetative schematic.

Bao H., Dou H., Ma Y., Liu H., Jiang G. (2017): Moose winter diet components from feces and field feeding signs: consistency and variability related to forage availability and nutritional requirements. Ecological Research 32: 685-692.
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Diet composition detection of herbivores, relating to feeding strategies, might be influenced by forage availability, geographical variation, and the results of diet estimation also varied in some extent by the different research methods. Therefore, it is difficult to assess how effective various methods of identifying herbivores diet components and their nutritional requirements are. In order to assess the consistency and variability of moose (Alces alces) winter diet composition across a large spatial scale, we compared the results of diet analysis from fecal analysis to those of parallel field survey related to forage availability and crude protein requirements during winter from December to March of 2012, 2013, and 2014 in the Greater Khingan Mountains, China. Our results suggested that: (1) the diet components variability between the two methods exist, and the value of species richness index, shannon wiener index and species evenness index in fecal analysis were greater than in field survey; (2) staple foods (birch, willow and aspen) identified from the two methods exhibited the stable consistency; and the percentage of birch was positive relative to forage availability from both methods; (3) quantitative crude protein of staple food items were different between both method, it was much lower than moose nutritional requirement by fecal analysis, and it was match the nutritional requirement of moose during field survey. These findings suggested that the two methods kept variability in detecting quantitative nutritional components, and only diet composition overlap consistency could not be reflected as quantitative nutritional similarity.

Erickson D. L., Reed E., Ramachandran P., Bourg N. A., McShea W. J., Ottesen A. (2017): Reconstructing a herbivore’s diet using a novel rbcL DNA mini-barcode for plants. AoB Plants 9: plx015.
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Next Generation Sequencing and the application of metagenomic analyses can be used to answer questions about animal diet choice and study the consequences of selective foraging by herbivores. The quantification of herbivore diet choice with respect to native versus exotic plant species is particularly relevant given concerns of invasive species establishment and their effects on ecosystems. While increased abundance of white-tailed deer (Odocoileus virginianus) appears to correlate with increased incidence of invasive plant species, data supporting a causal link is scarce. We used a metabarcoding approach (PCR amplicons of the plant rbcL gene) to survey the diet of white-tailed deer (fecal samples), from a forested site in Warren County, Virginia with a comprehensive plant species inventory and corresponding reference collection of plant barcode and chloroplast sequences. We sampled fecal pellet piles and extracted DNA from 12 individual deer in October 2014. These samples were compared to a reference DNA library of plant species collected within the study area. For 72 % of the amplicons, we were able to assign taxonomy at the species level, which provides for the first time—sufficient taxonomic resolution to quantify the relative frequency at which native and exotic plant species are being consumed by white-tailed deer. For each of the 12 individual deer we collected three subsamples from the same fecal sample, resulting in sequencing 36 total samples. Using Qiime, we quantified the plant DNA found in all 36 samples, and found that variance within samples was less than variance between samples (F = 1.73, P = 0.004), indicating additional subsamples may not be necessary. Species level diversity ranged from 60 to 93 OTUs per individual and nearly 70 % of all plant sequences recovered were from native plant species. The number of species detected did reduce significantly (range 4–12) when we excluded species whose OTU composed <1 % of each sample’s total. When compared to the abundance of native and non-natives plants inventoried in the local community, our results support the observation that white-tailed deer have strong foraging preferences, but these preferences were not consistent for species in either class. Deer forage behaviour may favour some exotic species, but not all.

Robeson M. S., Khanipov K., Golovko G., Wisely S. M., White M. D., Bodenchuck M., Smyser T. J., Fofanov Y., Fierer N., Piaggio A. J. (2018): Assessing the utility of metabarcoding for diet analyses of the omnivorous wild pig (Sus scrofa). Ecology and Evolution 8: 185-196.
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Wild pigs (Sus scrofa) are an invasive species descended from both domestic swine and Eurasian wild boar that was introduced to North America during the early 1500s. Wild pigs have since become the most abundant free‐ranging exotic ungulate in the United States. Large and ever‐increasing populations of wild pigs negatively impact agriculture, sport hunting, and native ecosystems with costs estimated to exceed $1.5 billion/year within the United States. Wild pigs are recognized as generalist feeders, able to exploit a broad array of locally available food resources, yet their feeding behaviors remain poorly understood as partially digested material is often unidentifiable through traditional stomach content analyses. To overcome the limitation of stomach content analyses, we developed a DNA sequencing‐based protocol to describe the plant and animal diet composition of wild pigs. Additionally, we developed and evaluated blocking primers to reduce the amplification and sequencing of host DNA, thus providing greater returns of sequences from diet items. We demonstrate that the use of blocking primers produces significantly more sequencing reads per sample from diet items, which increases the robustness of ascertaining animal diet composition with molecular tools. Further, we show that the overall plant and animal diet composition is significantly different between the three areas sampled, demonstrating this approach is suitable for describing differences in diet composition among the locations.

King S. R., Schoenecker K. A. (2019): Comparison of methods to examine diet of feral horses from noninvasively collected fecal samples. Rangeland Ecology and Management 72: 661-666.
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Feral horses (Equus ferus caballus) have become abundant on public lands in the American West, particularly over the past 10 yr. In areas where they are overabundant, there is risk of habitat degradation. Most previous studies on diet and habitat use of feral horses were conducted more than 20 yr ago; rangelands have changed considerably in that time, so it is useful to revisit horse diets. We conducted a study to examine the diet of feral horses using noninvasive methods and subjectively compare diet analysis techniques. We collected feral horse fecal samples from a sagebrush/pinyon-juniper ecosystem in Colorado in May, August, and October 2014. We analyzed 30 fecal samples from each collection session by both microhistology and plant DNA barcoding. Both microhistology and plant DNA barcoding results indicated horse diet consisted primarily of graminoids (78.5% and 68.8%, respectively, both of which are in greater proportion than availability based on ecological site descriptions); however, the two methods differed in species composition of grasses. Similar to other studies, microhistological analyses underestimated the proportion of forbs in the diet compared with plant DNA barcoding analyses, which showed a surprisingly high contribution of forbs to the diet compared with previous studies. Our results suggest plant DNA barcoding analyses have great potential, although both methods have inherent biases.

Kowalczyk R., Wójcik J. M., Taberlet P., Kamiński T., Miquel C., Valentini A., Craine J. M., Coissac E. (2019): Foraging plasticity allows a large herbivore to persist in a sheltering forest habitat: DNA metabarcoding diet analysis of the European bison. Forest Ecology and Management 449: 117474.
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Large herbivores that survived the Pleistocene/Holocene transition are hypothesized to have been forced to take refuge, as a result of environmental changes and human pressure, into forest habitats. Today, there is an open question of the degree to which extant large herbivores are well adapted to the forests that allowed for the herbivores’ persistence. We studied the diet of European bison (Bison bonasus), the largest terrestrial mammal in Europe, to gain insight into the foraging behaviour of a large herbivore, that appears to be primarily adapted to grazing but has been restored to forested habitats. The study population resided in the Białowieża Primeval Forests, Poland. DNA-based analysis of faecal samples revealed strong seasonal and spatial patterns in bison foraging, consistent between sexes. Bison fed on at least 105 different plant taxa. Woody species constituted 59.4% of DNA sequences, and forbs 33.6%. The two most abundant taxa were raspberry (Rubus idaeus) and European hornbeam (Carpinus betulus), which together comprised 31.0% of the dietary sequences and occurred in over 80% of faecal samples. Seasonally, the diversity of plants eaten by bison increased with increasing food availability. During high plant biomass in summer, bison consumed up to 40 different plant taxa. There was little overlap in the composition of the diet from month to month, reflecting the strong seasonality of vegetation abundance and/or its dietary quality. The results indicate high plasticity in bison foraging strategies and response to seasonal changes in biomass and the species composition of plants. Bison are browsers which continuously adjusts their diet with seasonal availability of easily digestible non-grass vegetation. We propose that dietary plasticity and micro-selection for open habitats (gaps and river valleys) within a forested landscape allowed bison to persist in sheltering forest habitats during the Holocene and accommodate to forest environments during species restoration.

McShea W. J., Sukmasuang R., Erickson D. L., Herrmann V., Ngoprasert D., Bhumpakphan N., Davies S. J. (2019): Metabarcoding reveals diet diversity in an ungulate community in Thailand. Biotropica 51: 923-937.
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The diverse large mammal communities found in Asian dry forests and savannas should segregate based on their diet selection. We examined the diet composition of sympatric ungulate species using metabarcoding to determine whether their diet was segregated and whether obvious attributes (i.e., body size, phylogeny, ecology) explained the structure. We collected fecal samples from eight ungulate species in Huai Kha Khaeng Wildlife Sanctuary in the western forest complex of Thailand. The fecal collections occurred around a plot where all woody species were codified within a genetic barcode library, and this library was supplemented with samples from plant species known to be consumed by these species. Of 273 plant species tested, at least 93 were found within the fecal samples. Over half of the identified species were not previously known by experts as forage species. All ungulate species showed a strong consumption of grasses and forbs. For the three species with sufficient sample size (sambar, banteng, and guar), there were seasonal differences in their diet, with each showing increased occurrence of woody plants during the dry season. The pattern of forage consumption did not follow obvious paradigms of body size or taxonomy, with significant diet differences found in two similar‐sized bovids (gaur, banteng), while the diet of sambar was more similar to bovids than to the other deer species. Asian ungulates differ in their forage consumption and metabarcoding should allow for testing of diet shifts in response to seasonal rains and fires which dominate the phenology of Asian dry forests and savannas.

Iacolina L., Lukassen M. B., Fløjgaard C., Buttenschøn R., Nielsen J. L., Pertoldi C. (2020): eDNA and metabarcoding for rewilding projects monitoring, a dietary approach. Mammalian Biology 100: 411-418.
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There is an increased interest in the possibility to use large animals in the restoration of degraded ecosystems and to increase the capacity of natural areas to sustain richer biodiversity. We quantify the dietary differences and similarities between five moose and 22 red deer introduced in a 2100 ha fenced area under restoration (Lille Vildmose, Denmark) and their potential in the restoration process. Moose and red deer were selected based on their biogeographic affinity and existing knowledge of their diet and feeding behaviour, and are expected to counteract encroachment on open habitats. Using eDNA metabarcoding of dung collected in the field and species identification through saliva recovered from browsed twigs, we investigated the species’ diets and diet overlap after the moose were released into the area, and assessed how their diets match the expected ecosystem function. Despite the partial overlap of some dietary items, such as Betulaceae and Salicaceae, we report a differential use in height of tree species and distinctive use of other food resources between the two species, with red deer eating more herbaceous plants and the moose diet also containing aquatic plants. This study exemplifies a monitoring approach to restoration projects where large herbivores are expected to provide a key ecosystem function through their browsing and grazing.

Shin H. M., Kim J., Jin S. D., Won H. Y., Park S. (2020): Diet composition of the Korean wild boar Sus scrofa coreanus (Suidae) at Mt. Jeombongsan, Korea. Journal of Ecology and Environment 44: 17.
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Korean wild boars (Sus scrofa coreanus Heude), because of their adaptability, are a widespread large mammal; however, they sometimes cause problems by invading farms and eating the crops, creating insufficiencies of some foods in South Korea. To understand the diet composition of Korean wild boars according to sex and body size, we collected their feces from Mt. Jeombongsan, Seoraksan National Park, South Korea. The sizes of fecal samples were measured, and genomic DNA was extracted from the samples. We amplified specific loci targeting plants (rbcL and trnL) and animals (COI) to detect the food sources of this omnivore and amplified the ZF and SRY regions to determine the sex. In the wild boar feces, Rosaceae and Bryophyte were the most frequently detected plant food sources at the family level and Diptera and Haplotaxida were the most frequently detected animal food sources at the order level. As a result of sex determination, the sex ratio of wild boars collected in the Mt. Jeombongsan area was approximately 1:1. Our result suggested that there is no significant difference between the diet composition of male and female boars. Based on the average cross-sectional area of the feces, the top 25% were classified into the large body size group and the bottom 25% were classified into the small body size group. The large body size group mainly preferred Actinidiaceae, and the small body size group most frequently consumed Fagaceae. The diet of the large body size group was more diverse than the small body size group. Our results showed that the wild boars preferred Rosaceae, especially Sanguisorba and Filipendula, as plant food sources, and Diptera and Coleoptera of Insecta as animal food sources. Based on the results, the dietary preferences of wild boar appear to be distinguished by not their sex but their body size. Our study could help to elucidate the feeding ecology and population structure of wild boar, as well as address conservation and management issues.

MULTIPLE SPECIES

ter Schure A. T., Pillai A. A., Thorbek L., Bhavani Shankar M., Puri R., Ravikanth G., de Boer H. J., Boessenkool S. (2021): eDNA metabarcoding reveals dietary niche overlap among herbivores in an Indian wildlife sanctuary. Environmental DNA 3: 681-696.
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As many ecosystems are under increasing pressure from invasive species, habitat degradation, overgrazing and overharvesting, pollution, and climate change, dietary niche monitoring is gaining importance. The Malai Mahadeshwara Wildlife Sanctuary (MMH) in southern India is home to several long-standing ethnic and tribal groups and supports a considerable number of domestic herbivores (cattle, goats and water buffalo) as well as a range of wildlife (including several species of deer, bonnet macaque, and Asian elephant). We reconstructed dietary niche partitioning of the herbivores occurring in MMH using eDNA metabarcoding to quantify diet richness, composition, and overlap. In total, we distinguish 134 diet items (molecular operational taxonomic units), covering 31 plant families. Overall, our results indicate 35% overlap in domestic and wild herbivore diet items. The greatest overlap is found for the dietary niches of cattle and sambar deer (Pianka’s niche overlap index: 0.68), and the dietary niche of cattle also overlaps considerably with those of Indian hare (0.65) and Asian elephant (0.46). This suggests that these herbivores may compete for these food plants in the case of limited availability, which could lead to exclusion of some herbivore species. Particular concern should go to bonnet macaque and Asian elephant as their below average dietary richness could make them vulnerable to changes in their environment. With increasing pressures on local wildlife from a range of different factors, DNA metabarcoding of fecal samples is a non-invasive method for monitoring changes in animal diets, providing valuable information for the management of biodiversity in mosaic natural and anthropogenic landscapes.