DIETARY ANALYSIS: BIRDS
Deagle B. E., Gales N. J., Evans K., Jarman S. N., Robinson S., Trebilco R., Hindell M. A. (2007): Studying seabird diet through genetic analysis of faeces: a case study on macaroni penguins (Eudyptes chrysolophus). Plos One 2: e831.
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Determination of seabird diet usually relies on the analysis of stomach-content remains obtained through stomach flushing; this technique is both invasive and logistically difficult. We evaluate the usefulness of DNA-based faecal analysis in a dietary study on chick-rearing macaroni penguins (Eudyptes chrysolophus) at Heard Island. Conventional stomach-content data was also collected, allowing comparison of the approaches. Prey-specific PCR tests were used to detect dietary DNA in faecal samples and amplified prey DNA was cloned and sequenced. Of the 88 faecal samples collected, 39 contained detectable DNA from one or more of the prey groups targeted with PCR tests. Euphausiid DNA was most commonly detected in the early (guard) stage of chick-rearing, and detection of DNA from the myctophid fish Krefftichthys anderssoni and amphipods became more common in samples collected in the later (crèche) stage. These trends followed those observed in the penguins’ stomach contents. In euphausiid-specific clone libraries the proportion of sequences from the two dominant euphausiid prey species (Euphausia vallentini and Thysanoessa macrura) changed over the sampling period; again, this reflected the trend in the stomach content data. Analysis of prey sequences in universal clone libraries revealed a higher diversity of fish prey than identified in the stomachs, but non-fish prey were not well represented. The present study is one of the first to examine the full breadth of a predator’s diet using DNA-based faecal analysis. We discuss methodological difficulties encountered and suggest possible refinements. Overall, the ability of the DNA-based approach to detect temporal variation in the diet of macaroni penguins indicates this non-invasive method will be generally useful for monitoring population-level dietary trends in seabirds.
Murray D. C., Bunce M., Cannell B. L., Oliver R., Houston J., White N. E., Barrero R. A., Bellgard M. I., Haile J. (2011): DNA-based faecal dietary analysis: a comparison of qPCR and high throughput sequencing approaches. Plos One 6: e25776.
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The genetic analysis of faecal material represents a relatively non-invasive way to study animal diet and has been widely adopted in ecological research. Due to the heterogeneous nature of faecal material the primary obstacle, common to all genetic approaches, is a means to dissect the constituent DNA sequences. Traditionally, bacterial cloning of PCR amplified products was employed; less common has been the use of species-specific quantitative PCR (qPCR) assays. Currently, with the advent of High-Throughput Sequencing (HTS) technologies and indexed primers it has become possible to conduct genetic audits of faecal material to a much greater depth than previously possible. To date, no studies have systematically compared the estimates obtained by HTS with that of qPCR. What are the relative strengths and weaknesses of each technique and how quantitative are deep-sequencing approaches that employ universal primers? Using the locally threatened Little Penguin (Eudyptula minor) as a model organism, it is shown here that both qPCR and HTS techniques are highly correlated and produce strikingly similar quantitative estimates of fish DNA in faecal material, with no statistical difference. By designing four species-specific fish qPCR assays and comparing the data to the same four fish in the HTS data it was possible to directly compare the strengths and weaknesses of both techniques. To obtain reproducible quantitative data one of the key, and often overlooked, steps common to both approaches is ensuring that efficient DNA isolation methods are employed and that extracts are free of inhibitors. Taken together, the methodology chosen for long-term faecal monitoring programs is largely dependent on the complexity of the prey species present and the level of accuracy that is desired. Importantly, these methods should not be thought of as mutually exclusive, as the use of both HTS and qPCR in tandem will generate datasets with the highest fidelity.
Joo S., Park S. (2012): Identification of bird species and their prey using DNA barcode on feces from Korean traditional village groves and forests (maeulsoop). Animal Cells and Systems 16: 488-497.
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A DNA barcode based on 648 bp of cytochrome c oxidase I (COI) gene aims to build species-specific libraries for animal groups. However, it is hard to recover full-length (648 bp) barcode gene from environmental fecal samples due to DNA degradation. In this study, we designed a new primer set (K_Bird), which amplifies a 226 bp fragment targeted an inner position of full-length COI barcode based on 102 species of Korean birds to improve amplification success, and we attempted to identify bird species from 39 avian fecal samples collected during 4 months from Jinan, South Korea. Simultaneously, we conducted a dietary analysis using a universal DNA mini-barcode (Uni_Minibar) from same fecal samples. In silico analysis on newly designed mini-barcode represented that genetic distances were 0.5% in species and 9.1% in genera. Intraspecific variations of 149 species out of 174 species (86%) between Korea and North America were within the threshold (5.3% threshold in this study). From environmental fecal samples collected in Jinan, we identified seven avian species, which have high similarity (99–100%) with registered COI sequences in GenBank. Eight kinds of prey species, such as moth, spider, fly, and dragonfly, were identified in dietary analysis. We suppose that our strategy applying mini-barcode for environmental fecal samples, might be a useful and convenient tool for species identification and dietary analysis for birds.
Jedlicka J. A., Sharma A. M., Almeida R. P. (2013): Molecular tools reveal diets of insectivorous birds from predator fecal matter. Conservation Genetics Resources 5: 879-885.
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The emerging field of molecular scatology enables critical testing of food web theory. The non-invasive application of molecular tools allows for sequencing of prey DNA from predator fecal matter, evaluating diet breadth and foraging guild. While insectivorous bats are obscure foragers compared to most insectivorous birds, more is known about which arthropod species bats consume because molecular techniques have been optimized for mammalian systems, not avian physiology. Our research objective was to use molecular tools to detect arthropod prey in the fecal matter of an insectivorous avian predator. We used Western Bluebird (Sialia mexicana) as a model predator due to its generalist foraging strategy. We compared two fecal DNA extraction kits: (1) Qiagen’s DNA stool mini kits, used widely in dietary studies on bats and (2) Zymo’s Soil/Fecal DNA MiniPrep kits, not currently cited in the molecular scatology literature. We successfully extracted DNA only with the Zymo kit, amplified mitochondrial cytochrome oxidase c subunit I genes, sequenced, and identified the arthropod prey. A spiked PCR experiment showed evidence of possible inhibitors remaining in the Qiagen kit extractions. Overall, arthropod prey from seven different orders and five different classes were identified. We discuss the ecological implications of these data and suggest areas of future research applying molecular techniques to avian fecal matter. Consistent methodological advancement will enable molecular scatology to identify ecosystem services provided by insectivorous birds, develop ecological theory, and inform predator conservation efforts.
Buś M. M., Żmihorski M., Romanowski J., Balčiauskienė L., Cichocki J., Balčiauskas L. (2014): High efficiency protocol of DNA extraction from Micromys minutus mandibles from owl pellets: a tool for molecular research of cryptic mammal species. Acta Theriologica 59: 99-109.
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Owl pellets have high potential as a source of DNA. However, this noninvasive method of collecting DNA is rarely used, and its methodological aspects are poorly understood. We investigated the methodology for DNA extraction and amplification from owl pellets containing the smallest European rodent—the Harvest mouse Micromys minutus – as an example. We used mandibles identified in owl pellets for mitochondrial and nuclear DNA amplification. For DNA extraction, we tested two commercial protocols and utilized a protocol being a combination of two commercial kits which ensured high efficiency of DNA extraction. Additionally, we recorded that the amount of DNA was five times higher in extracts from teeth as compared to DNA extracts from jawbones derived from the same mandible. The quantity of DNA was significantly positively correlated with biological sample weight; however, the age of the pellet remains had an impact on the level of inhibition. We recorded inhibition in 40 % of mtDNA extracts derived from pellets older than 150 months, whereas in DNA extracts from pellets younger than 80 months, we did not observe a negative impact of inhibition on PCR efficiency. The amplification success rate was 89.9 % for the mitochondrial fragment and 39.4 % in the case of the nuclear fragment. We observed partial degradation of DNA evidenced by the fact that the longest fragments that we were able to amplify in the case of mtDNA were 450 and 200 bp for nuDNA. The study shows that pellets can be considered as a source of DNA and have high potential for molecular research in the case of threatened species and species that are difficult to study using standard field techniques.
González‐Varo J. P., Arroyo J. M., Jordano P. (2014): Who dispersed the seeds? The use of DNA barcoding in frugivory and seed dispersal studies. Methods in Ecology and Evolution 5: 806-814.
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Assessing dispersal events in plants faces important challenges and limitations. A methodological issue that limits advances in our understanding of seed dissemination by frugivorous animals is identifying ‘which species dispersed the seeds’. This is essential for assessing how multiple frugivore species contribute distinctly to critical dispersal events such as seed delivery to safe sites, long‐distance dispersal and the colonization of non‐occupied habitats. Here, we describe DNA‐barcoding protocols successfully applied to bird‐dispersed seeds sampled in the field. Avian DNA was extracted from the surface of defecated or regurgitated seeds, allowing the identification of the frugivore species that contributed each dispersal event. Disperser species identification was based on a 464‐bp mitochondrial DNA region (COI: cytochrome c oxidase subunit I). We illustrate the possible applications of this method with bird‐dispersed seeds sampled in the field. DNA‐barcoding provides a non‐invasive technique that allows quantifying frugivory and seed dispersal interaction networks, assessing the contribution of each frugivore species to seed rain in different microhabitats, and testing whether different frugivore species select different fruit/seed sizes. DNA barcoding of animal‐dispersed seeds can resolve the distribution of dispersal services provided by diverse frugivore assemblages, allowing a robust and precise estimation of the different components of seed dispersal effectiveness, previously unattainable to traditional field studies at individual seed level. Given that seeds are sampled at the end of the dispersal process, this technique enables us to link the identity of the disperser species responsible for each dispersal event to plant traits and environmental features, thereby building a bridge between frugivory and seed deposition patterns.
Novcic I., Mizrahi D. S., Veit R. R., Symondson W. O. (2015): Molecular analysis of the value of horseshoe crab eggs to migrating shorebirds. Avian Biology Research 8: 210-220.
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Detection of prey DNA from faecal samples is a non-invasive method being successfully applied to the dietary analysis of birds as well as other vertebrates and invertebrates. We have employed this technique to examine the significance of horseshoe crab eggs (Limulus polyphemus) for dunlins (Calidris alpina), semipalmated sandpipers (Calidris pusilla), least sandpipers (Calidris minutilla) and short-billed dowitchers (Limnodromus griseus) during northbound migration at their major stopover area in Delaware Bay. This was accomplished through polymerase chain reaction amplification of Limulus DNA from faecal samples using new horseshoe crab-specific primers. We detected the consumption of crab eggs in all study species, although results suggest that eggs may be a less important food source for least sandpipers than for the other species. Consumption of eggs increases as the migration season progresses, implying a strong preference of birds for this resource at the end of May, when eggs are readily accessible. The results confirm the value of horseshoe crab eggs for migrating shorebirds in this crucial stopover area. The Limulus-specific primers we designed can be effectively used for detection of semidigested Limulus DNA from faecal samples and thus they can be employed in the dietary analysis of other predators that seasonally capitalise on horseshoe crabs or their eggs.
Rocha R. G., Justino J., Leite Y. L. R., Costa L. P. (2015): DNA from owl pellet bones uncovers hidden biodiversity. Systematics and Biodiversity 13: 403-412.
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Owl pellets are a useful tool for inventorying small mammals and a robust complement to traditional trapping methods. Here we assessed if bone samples recovered from Barn owl pellets in the Neotropics represent a viable source of modern DNA. We used the upper incisor of rodents and jaws of marsupials of pellet material for DNA extraction and amplification. Extraction and amplification success depended on the type of bone element used, being much higher from rodent incisors. The amplification success of small (429 bp) and large (801 bp) mitochondrial cytochrome b fragments was relatively similar, suggesting that DNA was not severely degraded, probably because our pellet samples were less than 8 years old. Eleven species of small mammals were identified, from which two are new to the study area: Gracilinanus emiliae and Marmosops pinheiroi. These records represent the southeastern boundary of both species, and extended their geographic distribution 370 km (G. emiliae) and 700 km (M. pinheiroi) to the south. Moreover, two of three detected species of Oecomys occurring in sympatry may represent undescribed species. The use of molecular analysis as a complement to morphological analysis of pellet bones is very effective in species identification, especially considering poorly known and elusive species, such as those reported here. The search for owl pellets during fieldwork should be a common practice among mammalogists in the Neotropics because this material provides a valuable non-invasive source of DNA, often revealing previously undetected species and sometimes uncovering new species.
Gaglio D., Cook T. R., Connan M., Ryan P. G., Sherley R. B. (2017): Dietary studies in birds: testing a non‐invasive method using digital photography in seabirds. Methods in Ecology and Evolution 8: 214-222.
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Dietary studies give vital insights into foraging behaviour, with implications for understanding changing environmental conditions and the anthropogenic impacts on natural resources. Traditional diet sampling methods may be invasive or subject to biases, so developing non-invasive and unbiased methods applicable to a diversity of species is essential. We used digital photography to investigate the diet fed to chicks of a prey-carrying seabird and compared our approach (photo-sampling) to a traditional method (regurgitations) for the greater crested tern Thalasseus bergii. Over three breeding seasons, we identified >24 000 prey items of at least 48 different species, more than doubling the known diversity of prey taken by this population of terns. We present a method to estimate the length of the main prey species (anchovy Engraulis encrasicolus) from photographs, with an accuracy <1 mm and precision ~ 0·5 mm. Compared to regurgitations at two colonies, photo-sampling produced similar estimates of prey composition and size, at a faster species accumulation rate. The prey compositions collected by two researchers photo-sampling concurrently were also similar. Photo-sampling offers a non-invasive tool to accurately and efficiently investigate the diet composition and prey size of prey-carrying birds. It reduces biases associated with observer-based studies and is simple to use. This methodology provides a novel tool to aid conservation and management decision-making in the light of the growing need to assess environmental and anthropogenic change in natural ecosystems.
McInnes J. C., Alderman R., Lea M. A., Raymond B., Deagle B. E., Phillips R. A., Stanworth A., Thompson D. R., Catry P., Weimerskirch H., Suazo C. G. (2017): High occurrence of jellyfish predation by black‐browed and Campbell albatross identified by DNA metabarcoding. Molecular Ecology 26: 4831-4845.
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Gelatinous zooplankton are a large component of the animal biomass in all marine environments, but are considered to be uncommon in the diet of most marine top predators. However, the diets of key predator groups like seabirds have conventionally been assessed from stomach content analyses, which cannot detect most gelatinous prey. As marine top predators are used to identify changes in the overall species composition of marine ecosystems, such biases in dietary assessment may impact our detection of important ecosystem regime shifts. We investigated albatross diet using DNA metabarcoding of scats to assess the prevalence of gelatinous zooplankton consumption by two albatross species, one of which is used as an indicator species for ecosystem monitoring. Black‐browed and Campbell albatross scats were collected from eight breeding colonies covering the circumpolar range of these birds over two consecutive breeding seasons. Fish was the main dietary item at most sites; however, cnidarian DNA, primarily from scyphozoan jellyfish, was present in 42% of samples overall and up to 80% of samples at some sites. Jellyfish was detected during all breeding stages and consumed by adults and chicks. Trawl fishery catches of jellyfish near the Falkland Islands indicate a similar frequency of jellyfish occurrence in albatross diets in years of high and low jellyfish availability, suggesting jellyfish consumption may be selective rather than opportunistic. Warmer oceans and overfishing of finfish are predicted to favour jellyfish population increases, and we demonstrate here that dietary DNA metabarcoding enables measurements of the contribution of gelatinous zooplankton to the diet of marine predators.
Gaglio D., Cook T. R., McInnes A., Sherley R. B., Ryan P. G. (2018): Foraging plasticity in seabirds: a non-invasive study of the diet of greater crested terns breeding in the Benguela region. Plos One 13: e0190444.
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Marine predators, such as seabirds, are useful indicators of marine ecosystem functioning. In particular, seabird diet may reflect variability in food-web composition due to natural or human-induced environmental change. Diet monitoring programmes, which sample diet non-invasively, are valuable aids to conservation and management decision-making. We investigated the diet of an increasing population of greater crested terns Thalasseus bergii in the Western Cape, South Africa, during three successive breeding seasons (2013 to 2015), when populations of other seabirds feeding on small pelagic schooling fish in the region were decreasing. Breeding greater crested terns carry prey in their bills, so we used an intensive photo-sampling method to record their diet with little disturbance. We identified 24,607 prey items from at least 47 different families, with 34 new prey species recorded. Fish dominated the diet, constituting 94% of prey by number, followed by cephalopods (3%), crustaceans (2%) and insects (1%). The terns mainly targeted surface-schooling Clupeiformes, with anchovy Engraulis encrasicolus the most abundant prey in all three breeding seasons (65% overall). Prey composition differed significantly between breeding stages and years, with anchovy most abundant at the start of the breeding season, becoming less frequent as the season progressed. The proportion of anchovy in the diet also was influenced by environmental factors; anchovy occurred more frequently with increasing wind speeds and was scarce on foggy days, presumably because terns rely in part on social facilitation to locate anchovy schools. The application of this intensive and non-invasive photo-sampling method revealed an important degree of foraging plasticity for this seabird within a context of locally reduced food availability, suggesting that, unlike species that specialise on a few high-quality prey, opportunistic seabirds may be better able to cope with reductions in the abundance of their preferred prey.
Sullins D. S., Haukos D. A., Craine J. M., Lautenbach J. M., Robinson S. G., Lautenbach J. D., Kraft J. D., Plumb R. T., Reitz J. H., Sandercock B. K., Fierer N. (2018): Identifying the diet of a declining prairie grouse using DNA metabarcoding. The Auk: Ornithological Advances 135: 583-608.
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Diets during critical brooding and winter periods likely influence the growth of Lesser Prairie-Chicken (Tympanuchus pallidicinctus) populations. During the brooding period, rapidly growing Lesser Prairie-Chicken chicks have high calorie demands and are restricted to foods within immediate surroundings. For adults and juveniles during cold winters, meeting thermoregulatory demands with available food items of limited nutrient content may be challenging. Our objective was to determine the primary animal and plant components of Lesser Prairie-Chicken diets among native prairie, cropland, and Conservation Reserve Program (CRP) fields in Kansas and Colorado, USA, during brooding and winter using a DNA metabarcoding approach. Lesser Prairie-Chicken fecal samples (n = 314) were collected during summer 2014 and winter 2014–2015, DNA was extracted, amplified, and sequenced. A region of the cytochrome oxidase I (COI) gene was sequenced to determine the arthropod component of the diet, and a portion of the trnL intron region was used to determine the plant component. Relying on fecal DNA to quantify dietary composition, as opposed to traditional visual identification of gut contents, revealed a greater proportion of soft-bodied arthropods than previously recorded. Among 80 fecal samples for which threshold arthropod DNA reads were obtained, 35% of the sequences were most likely from Lepidoptera, 26% from Orthoptera, 14% from Araneae, 13% from Hemiptera, and 12% from other orders. Plant sequences from 137 fecal samples were composed of species similar to Ambrosia (27%), followed by species similar to Lactuca or Taraxacum (10%), Medicago (6%), and Triticum (5%). Forbs were the predominant (>50% of reads) plant food consumed during both brood rearing and winter. The importance both of native forbs and of a broad array of arthropods that rely on forbs suggests that disturbance regimes that promote forbs may be crucial in providing food for Lesser Prairie-Chickens in the northern portion of their distribution.
Thalinger B., Oehm J., Zeisler C., Vorhauser J., Traugott M. (2018): Sex‐specific prey partitioning in breeding piscivorous birds examined via a novel, noninvasive approach. Ecology and Evolution 8: 8985-8998.
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Piscivorous birds frequently display sex‐specific differences in their hunting and feeding behavior, which lead to diverging impacts on prey populations. Cormorants (Phalacrocoracidae), for example, were previously studied to examine dietary differences between the sexes and males were found to consume larger fish in coastal areas during autumn and winter. However, information on prey partitioning during breeding and generally on sex‐specific foraging in inland waters is missing. Here, we assess sex‐specific prey choice of Great Cormorants (Phalacrocorax carbo) during two subsequent breeding seasons in the Central European Alpine foreland, an area characterized by numerous stagnant and flowing waters in close proximity to each other. We developed a unique, noninvasive approach and applied it to regurgitated pellets: molecular cormorant sexing combined with molecular fish identification and fish‐length regression analysis performed on prey hard parts. Altogether, 364 pellets delivered information on both, bird sex, and consumed prey. The sexes differed significantly in their overall prey composition, even though Perca fluviatilis, Rutilus rutilus, and Coregonus spp. represented the main food source for both. Albeit prey composition did not indicate the use of different water bodies by the sexes, male diet was characterized by higher prey diversity within a pellet and the consumption of larger fish. The current findings show that female and male cormorants to some extent target the available prey spectrum at different levels. Finally, the comprehensive and noninvasive approach has great potential for application in studies of other piscivorous bird species.
Trevelline B. K., Nuttle T., Hoenig B. D., Brouwer N. L., Porter B. A., Latta S. C. (2018): DNA metabarcoding of nestling feces reveals provisioning of aquatic prey and resource partitioning among Neotropical migratory songbirds in a riparian habitat. Oecologia 187: 85-98.
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Riparian habitats are characterized by substantial flows of emergent aquatic insects that cross the stream-forest interface and provide an important source of prey for insectivorous birds. The increased availability of prey arising from aquatic subsidies attracts high densities of Neotropical migratory songbirds that are thought to exploit emergent aquatic insects as a nestling food resource; however, the prey preferences and diets of birds in these communities are only broadly understood. In this study, we utilized DNA metabarcoding to investigate the extent to which three syntopic species of migratory songbirds—Acadian Flycatcher, Louisiana Waterthrush, and Wood Thrush—breeding in Appalachian riparian habitats (Pennsylvania, USA) exploit and partition aquatic prey subsidies as a nestling food resource. Despite substantial differences in adult foraging strategies, nearly every nestling in this study consumed aquatic taxa, suggesting that aquatic subsidies are an important prey resource for Neotropical migrants nesting in riparian habitats. While our results revealed significant interspecific dietary niche divergence, the diets of Acadian Flycatcher and Wood Thrush nestlings were strikingly similar and exhibited significantly more overlap than expected. These results suggest that the dietary niches of Neotropical migrants with divergent foraging strategies may converge due to the opportunistic provisioning of non-limiting prey resources in riparian habitats. In addition to providing the first application of DNA metabarcoding to investigate diet in a community of Neotropical migrants, this study emphasizes the importance of aquatic subsidies in supporting breeding songbirds and improves our understanding of how anthropogenic disturbances to riparian habitats may negatively impact long-term avian conservation.
Xavier J. C., Cherel Y., Medeiros R., Velez N., Dewar M., Ratcliffe N., Carreiro A. R., Trathan P. N. (2018): Conventional and molecular analysis of the diet of gentoo penguins: contributions to assess scats for non-invasive penguin diet monitoring. Polar Biology 41: 2275-2287.
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There is a growing search for less invasive methods while studying the diet of Antarctic animals in the wild. Therefore, we compared the diet of gentoo penguins from stomach contents (i.e. through visual identification of prey remains) and scats (i.e. faeces), and further compared prey DNA assay in fresh and old scats. Prey remains identified visually in stomach contents and scats were broadly comparable: the crustaceans and fish were the most important components, with Themisto gaudichaudii clearly being the most frequent and numerous prey species in both sampling methods. By mass, differences in species frequency were observed in stomach contents (Parachaenichthys georgianus) and scats (Champsocephalus gunnari), with the former fish species absent in scats. Differences were detected in the most frequent prey (T. gaudichaudii and Euphausia superba) and in various fish species, most with bigger sizes in scats. Allometric equations to estimate most crustacean’s sizes (i.e. relationships between carapace and mass/total length) are needed. For DNA studies, when comparing DNA from fresh and old scats, both provided similar results that, in general, were also similar to the visual analysis. In order to use penguin scats (along with the use of DNA analyses) for monitoring purposes, allometric equations to estimate mass and size of prey (most crustaceans) and better designed species-specific primers are needed for targeting key prey species (e.g. Euphausia superba, T. gaudichaudii). These DNA methodologies can complement other methods (i.e. visual analyses and stomach contents analyses) in monitoring programs of penguins.
Gong M., Ning Y., Han M., Zhao C., Tian J., Li L., Xiao H., Liu G. (2019): A comparison of next-generation sequencing with clone sequencing in the diet analysis of Asian great bustard. Conservation Genetics Resources 11: 15-17.
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Comparative studies of next-generation sequencing (NGS) and clone sequencing in diet analysis have not been conducted. This study compares the performance of NGS and clone sequencing to analyze the diet of Asian great bustard (Otis tarda dybowskii). Identification of known taxa using NGS method was significantly greater than by clone method (t = 8.12, df = 14, p < 0.01), which has highlighted the higher sensitivity of the NGS approach. The proportion of occurrence for NGS method and clone method was significantly correlated (r = 0.63, p = 0.01), suggesting the prey taxa present at low frequency could not be identified by clone method. The cost and time of NGS method was also low compared with the clone method. We recommend the NGS opens up for new possibilities in the diet study, and has wide applicability to other species whose faeces can be collected easily and non-invasively in the field.
Naude V. N., Smyth L. K., Weideman E. A., Krochuk B. A., Amar A. (2019): Using web-sourced photography to explore the diet of a declining African raptor, the Martial Eagle (Polemaetus bellicosus). The Condor: Ornithological Applications 121: duy015.
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Understanding a species’ diet can be critical for effective conservation. While several traditional methods for assessing raptor diet exist, many pose inherit biases and often require extensive fieldwork that can limit sample sizes and the geographic scope of studies. This is especially true for species that nest at low densities (e.g., large eagles). Recently, several studies have demonstrated the value of web-sourced photographs in tackling ecological and evolutionary questions. Specialized software (e.g., MORPHIC) has been developed to systematically extract Google Images for this purpose. We used this approach to explore the diet of Martial Eagles (Polemaetus bellicosus). A shortage of prey is one of the proposed hypotheses for recent population declines across their range. Of the 4,872 photographs selected by MORPHIC, 254 were usable (5%). Birds, mammals, and reptiles each contributed similarly to overall identified prey. Helmeted Guineafowl (Numida meleagris) were the most important bird prey identified (12% of overall prey). The 4 most important mammalian prey species were Thompson’s gazelle (Eudorcas thomsonii; 5%), impala (Aepyceros melampus; 4%), common duiker (Sylvicapra grimmia; 4%), and banded mongoose (Mungos mungo; 4%). Reptile prey was dominated by monitor lizards (Varanus spp.; 21%). Prey class proportions differed significantly by regions with mammalian prey being more common in eastern Africa and reptile prey being more common in southern Africa. Within South Africa, reptile prey proportion was greater in the east than in the west. These corroborate existing prey composition studies. Prey composition differed between age classes, with adult eagles preying on more birds than non-adults. There was no significant difference in the average estimated prey weight by eagle age or feeding position (ground, perched, or flying). Our results suggest that this approach may offer a useful method to explore the diet for raptor species that are well photographed across their range, at minimal cost and research effort.
Nota K., Downing S., Iyengar A. (2019): Metabarcoding-based dietary analysis of hen harrier (Circus cyaneus) in Great Britain using buccal swabs from chicks. Conservation Genetics 20: 1389-1404.
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The hen harrier is a heavily persecuted bird of prey in Great Britain since its diet includes Red grouse, a game bird shot in driven and walked-up grouse shooting. Unlike walked-up shooting where shooters walk up moors and flush grouse with dogs, in driven grouse shooting the grouse are driven by beaters towards static shooters. Driven grouse moors (DGMs) are increasingly being managed to sustain high densities of Red grouse intensifying a long-standing conservation conflict between conservationists and grouse moor keepers. A metabarcoding approach was used with degenerate universal cytochrome oxidase I and cytochrome b primers along with hen harrier blocking primers. A novel sampling method was used to detect prey in buccal swab samples from chicks from broods across Great Britain from both managed and unmanaged moorland habitats. This resulted in detection of 62 species of prey across 51 broods with Meadow pipit, Red grouse, Wren, Skylark, and voles being most frequently detected. Frequency of occurrence data and species accumulation curves reveal high incidence of Red grouse and low prey species richness in the diet of hen harriers in DGMs but low incidence of Red grouse and high prey species richness in walked-up and unmanaged moors. Waders were only detected within walked-up and unmanaged moors and not within DGMs where they have been reported to occur at high densities. Regional species detected included endemic species such as the Orkney vole seen only in Orkney. This study represents the first metabarcoding-based dietary analysis in a raptor using buccal swabs.
Rytkönen S., Vesterinen E. J., Westerduin C., Leviäkangas T., Vatka E., Mutanen M., Välimäki P., Hukkanen M., Suokas M., Orell M. (2019): From feces to data: a metabarcoding method for analyzing consumed and available prey in a bird‐insect food web. Ecology and Evolution 9: 631-639.
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Diets play a key role in understanding trophic interactions. Knowing the actual structure of food webs contributes greatly to our understanding of biodiversity and ecosystem functioning. The research of prey preferences of different predators requires knowledge not only of the prey consumed, but also of what is available. In this study, we applied DNA metabarcoding to analyze the diet of 4 bird species (willow tits Poecile montanus, Siberian tits Poecile cinctus, great tits Parus major and blue tits Cyanistes caeruleus) by using the feces of nestlings. The availability of their assumed prey (Lepidoptera) was determined from feces of larvae (frass) collected from the main foraging habitat, birch (Betula spp.) canopy. We identified 53 prey species from the nestling feces, of which 11 (21%) were also detected from the frass samples (eight lepidopterans). Approximately 80% of identified prey species in the nestling feces represented lepidopterans, which is in line with the earlier studies on the parids’ diet. A subsequent laboratory experiment showed a threshold for fecal sample size and the barcoding success, suggesting that the smallest frass samples do not contain enough larval DNA to be detected by high‐throughput sequencing. To summarize, we apply metabarcoding for the first time in a combined approach to identify available prey (through frass) and consumed prey (via nestling feces), expanding the scope and precision for future dietary studies on insectivorous birds.
Shutt J. D., Nicholls J. A., Trivedi U. H., Burgess M. D., Stone G. N., Hadfield J. D., Phillimore A. B. (2020): Gradients in richness and turnover of a forest passerine’s diet prior to breeding: A mixed model approach applied to faecal metabarcoding data. Molecular Ecology 29: 1199-1213.
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Rather little is known about the dietary richness and variation of generalist insectivorous species, including birds, due primarily to difficulties in prey identification. Using faecal metabarcoding, we provide the most comprehensive analysis of a passerine’s diet to date, identifying the relative magnitudes of biogeographic, habitat and temporal trends in the richness and turnover in diet of Cyanistes caeruleus (blue tit) along a 39 site and 2° latitudinal transect in Scotland. Faecal samples were collected in 2014–2015 from adult birds roosting in nestboxes prior to nest building. DNA was extracted from 793 samples and we amplified COI and 16S minibarcodes. We identified 432 molecular operational taxonomic units that correspond to putative dietary items. Most dietary items were rare, with Lepidoptera being the most abundant and taxon-rich prey order. Here, we present a statistical approach for estimation of gradients and intersample variation in taxonomic richness and turnover using a generalised linear mixed model. We discuss the merits of this approach over existing tools and present methods for model-based estimation of repeatability, taxon richness and Jaccard indices. We found that dietary richness increases significantly as spring advances, but changes little with elevation, latitude or local tree composition. In comparison, dietary composition exhibits significant turnover along temporal and spatial gradients and among sites. Our study shows the promise of faecal metabarcoding for inferring the macroecology of food webs, but we also highlight the challenge posed by contamination and make recommendations of laboratory and statistical practices to minimise its impact on inference.
Berryman A. J., Kirwan G. M. (2021): Is the Tiny Hawk (Accipiter superciliosus) really a specialized predator on hummingbirds? Using citizen science data to elucidate dietary preferences of a little-known Neotropical raptor. Journal of Raptor Research 55: 276-280.
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We used photo-documented citizen science data held in publicly accessible databases to better elucidate the diet of one of the smallest Neotropical Accipitridae, the Tiny Hawk (Accipiter superciliosus). Previous data are exclusively anecdotal, but a suggestion that this species is to some extent a specialized predator on hummingbirds (Trochilidae) is widely reported in the literature, despite being based on few observations. Using a total of more than 680 photographs of Tiny Hawks, we identified 33 items of avian prey from 15 families and at least 25 genera, including one hummingbird and some comparatively large non-passerines. Prey also included one bat. Although the Tiny Hawk is perhaps more liable to predate hummingbirds than most other Neotropical raptors, the available data suggest that the Tiny Hawk, like many other Accipiter species, is primarily a bird specialist. As evidenced by other recently published studies, internet-sourced photographs might prove useful for similar dietary analyses for other poorly known raptors.
Bourbour R. P., Aylward C. M., Tyson C. W., Martinico B. L., Goodbla A. M., Ely T. E., Fish A. M., Hull J. M. (2021): Falcon fuel: metabarcoding reveals songbird prey species in the diet of juvenile Merlins (Falco columbarius) migrating along the Pacific Coast of western North America. Ibis 163: 1282-1293.
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During autumn migration, bird-eating raptors are thought to rely on flocks of migrant songbirds (Passeriformes) as a critical resource to fuel the energetic demands of long-distance migration. However, this hypothesis has been challenging to investigate, and the foraging ecology during migration of most migrant raptors remains unexplored. To address these knowledge gaps, our objective was to document the diet of a bird-eating falcon on active migration. We swabbed visible and trace prey residues from the exterior surface of beaks and talons of migrant juvenile Merlins Falco columbarius in the autumn of 2015 and 2016 at a raptor migration monitoring station positioned on the Pacific Coast of western North America. We used a DNA metabarcoding approach and detected the presence of 40 distinct prey species derived from 210 individual prey species detections on 63 of the 72 (87.5%) migrant juvenile Merlins sampled. We detected an average of 3.3 ± 1.6 prey species on individual Merlins. We found that juvenile males selected smaller prey on average compared with juvenile females. Of the prey species detected, over 80% were migratory songbird species within the Pacific Flyway. In 2015, we detected a greater proportion of irruptive migrants in juvenile Merlin diet compared with 2016. In 2016, we found that the proportion of annual migrants consumed by Merlins corresponded to the timing of peak annual songbird migration in the Pacific Flyway. This study represents one of the first detailed descriptions of songbird prey species consumed by a migrating raptor and supports the hypothesis that migrating juvenile Merlins rely on migrant songbirds to support the energetic demands of migration.
Cabodevilla X., Mougeot F., Bota G., Mañosa S., Cuscó F., Martínez-García J., Arroyo B., Madeira M. J. (2021): Metabarcoding insights into the diet and trophic diversity of six declining farmland birds. Scientific Reports 11: 21131.
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Knowledge of feeding ecology of declining species, such as farmland birds, is essential to address their conservation requirements, especially when their habitats are suffering important reductions of trophic resources. In this study, we apply a metabarcoding approach to describe the diet composition of six of the most significant farmland birds inhabiting European cereal pseudo-steppes: little bustard, great bustard, pin-tailed sandgrouse, black-bellied sandgrouse, red-legged partridge, and common quail. We further studied seasonal diet variations (autumn to spring) in all species but the common quail, whose diet was studied during spring and summer. We show that study species´ diets mostly consisted of plants, although in the case of little bustard and great bustard arthropods are also highly relevant. Among arthropods, we found high proportions of thrips, arachnids, and springtails, which were previously unreported in their diet, and some taxa that could be used as antiparasitic food. Moreover, we report that little bustard’s diet is the least rich of that of all studied species, and that diet of all these species is less diverse in winter than in autumn and spring. Diet composition of these declining species supports the importance of natural and semi-natural vegetation and landscape mosaics that can provide a wide variety of arthropods, plants, and seeds all year-round.
Chua P. Y. S., Lammers Y., Menoni E., Ekrem T., Bohmann K., Boessenkool S., Alsos I. G. (2021): Molecular dietary analyses of western capercaillies (Tetrao urogallus) reveal a diverse diet. Environmental DNA 3: 1156-1171.
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Conservation strategies centered around species habitat protection rely on species’ dietary information. One species at the focal point of conservation efforts is the herbivorous grouse, the western capercaillie (Tetrao urogallus), which is an indicator species for forest biodiversity conservation. Non-molecular means used to study their diet are time-consuming and at low taxonomic resolution. This delays the implementation of conservation strategies including resource protection due to uncertainty about its diet. Thus, limited knowledge on diet is hampering conservation efforts. Here, we use non-invasive environmental DNA (eDNA) metabarcoding on DNA extracted from faces to present the first large-scale molecular dietary analysis of capercaillies. Facal samples were collected from seven populations located in Norway (Finnmark, Troms, Trøndelag, Innlandet) and France (Vosges, Jura, Pyrenees) (n = 172). We detected 122 plant taxa belonging to 46 plant families of which 37.7% of the detected taxa could be identified at species level. The average dietary richness of each sample was 7 ± 5 SD taxa. The most frequently occurring plant groups with the highest relative read abundance (RRA) were trees and dwarf shrubs, in particular, Pinus and Vaccinium myrtillus, respectively. There was a difference in dietary composition (RRA) between samples collected from the different locations (adonis pseudo F5,86 = 11.01, r2 = 0.17, p = 0.001) and seasons (adonis pseudo F2,03 = 0.64, r2 = 0.01, p = 0.036). Dietary composition also differed between sexes at each location (adonis pseudo F1,47 = 2.77, r2 = 0.04, p = 0.024), although not significant for all data combined. In total, 35 taxa (36.8% of taxa recorded) were new capercaillie food items compared with existing knowledge from non-molecular means. The non-invasive molecular dietary analysis applied in this study provides new ecological information of capercaillies’ diet, improving our understanding of adequate habitat required for their conservation.
Huang P. Y., Poon E. S. K., Wong A. T. C., So I. W. Y., Sung Y. H., Sin S. Y. W. (2021): DNA metabarcoding reveals the dietary composition in the endangered black-faced spoonbill. Scientific Reports 11: 18773.
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Extensive loss of natural wetlands caused by changes in land use largely diminishes the food resources essential for the survival of migratory waterbirds. Globally, the decline in waterbird populations in East Asia is the most serious, with 64% of these populations showing a decreasing trend. In this study, we applied DNA metabarcoding to examine the spatiotemporal variations and diversities in the dietary compositions of migratory waterbirds in a natural/artificial wetland complex in Asia. By investigating 110 fecal samples from the endangered black-faced spoonbill (Platalea minor) wintering in the wetland, our results show that P. minor had a broad dietary spectrum. The birds fed on at least 26 species in the classes Actinopterygii and Malacostraca, with Mugiliformes, Cichliformes, and Gobiiformes being the main taxa in their diets. Our results also demonstrated clear patterns of the spatiotemporal variations between the roosting groups and intraspecific variations between the individuals, which potentially reflect some of their feeding habits, and the probable usage of different habitat types in the wetland complex. Using high-throughput sequencing, we were able to elucidate the food resources that are critical to P. minor non-invasively, this method can also be used to provide invaluable information for the conservation of many other waterbird species.
Bumelis K. H., Cadman M. D., Hobson K. A. (2022): Endogenous biomarkers reveal diet partitioning among three sympatric species of swallows. The Auk 139: ukab078.
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Since the early 1990s, aerial insectivorous birds have shown serious population declines in North America, but it is not clear if factors common to all species within this guild account for these declines. Among sympatric swallows, population trends differ, and this may be due to differences in ecology operating throughout the annual cycle. Although these species all feed on aerial insects, prey taxa can differ tremendously in their “aeroecology” and use by swallows. We examined the potential for dietary differences among three species of swallows, Barn Swallow (Hirundo rustica), Cliff Swallow (Petrochelidon pyrrhonota), and Tree Swallow (Tachycineta bicolor), breeding sympatrically in southern Ontario, Canada. Potential interspecific differences in nestling diet were examined using two endogenous biomarkers, DNA barcoding of nestling feces and stable isotope analysis (δ 2H, δ 13C, δ 15N) of nestling feathers. We found evidence for differences in dietary sources of provisioned young where Barn Swallows provisioned more terrestrial-based prey, Cliff Swallows provisioned an intermediate diet, and Tree Swallows the most aquatic-emergent insect diet. We suggest this information may help to identify potential factors contributing to differential declines of aerial insectivores operating on the breeding grounds, including diet quality.
Harris M. P., Albon S. D., Newell M. A., Gunn C., Daunt F., Wanless S. (2022). Long‐term within‐season changes in the diet of Common Guillemot (Uria aalge) chicks at a North Sea colony: implications for dietary monitoring. Ibis 164: 1243-1251.
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Over four decades (1982–2019) there were pronounced within-season changes in the proportion of a key prey species (Lesser Sandeel Ammodytes marinus) in Common Guillemot Uria aalge chick diet. As Sandeels became scarcer their occurrence was largely confined to the early part of the chick period. Consequently, the mean annual proportion of Sandeels was poorly estimated if sampling occurred within a short time window, particularly if this was early or late in the season. Within-season variation is rarely considered in diet monitoring but our results highlight the need for further analyses across other species and sites to develop a deeper understanding of how best to optimize sampling protocols.
Nimz I., Renshaw M. A., Baczenas J., Vanderlip C., Hyrenbach K. D., Iacchei M. (2022): MetaBARFcoding: DNA‐barcoding of regurgitated prey yields insights into Christmas shearwater (Puffinus nativitatis) foraging ecology at Hōlanikū (Kure Atoll), Hawaiʻi. Environmental DNA 4: 254-268.
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Morphological identification of digested prey remains from a generalist predator can be challenging, especially when attempting to match degraded remains to taxonomic keys. DNA techniques, whereby prey is sequenced and matched to large public nucleotide sequence databases, are increasingly being used to augment morphological identification. We used “metaBARFcoding” (DNA metabarcoding) to target a region of the cytochrome c oxidase subunit I (COI) mitochondrial gene to identify prey in highly digested regurgitations from Christmas shearwaters Puffinus nativitatis at Hōlanikū (Kure Atoll). Metabarcoding was used to bulk-process 92 water samples from regurgitations collected from 2009 to 2017, providing an overview of the seabird’s diet. We additionally Sanger-sequenced 100 prey items from 50 randomly chosen regurgitations to verify that metabarcoding characterized key components of the diet. The metabarcoding technique identified 87 unique taxa from 29 families of fish and squid, spanning diverse taxa, including reef-associated, pelagic–oceanic, and mesopelagic species. Rare prey (frequency of occurrence ≤5% of samples) constituted 66% of the species richness, demonstrating the highly diverse diet of this generalist predator. Overall, 81% of the families detected in the contemporary diet were previously documented in Christmas shearwater diets from the Northwestern Hawaiian Islands. Our results indicate that metabarcoding the COI region is useful in identifying a wide range of taxa from highly digested regurgitations, thus facilitating this approach to study seabird diets.
Spence A. R., Wilson Rankin E. E., Tingley M. W. (2022): DNA metabarcoding reveals broadly overlapping diets in three sympatric North American hummingbirds. The Auk 139: ukab074.
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Hummingbirds, a highly diverse avian family, are specialized vertebrate pollinators that feed upon carbohydrate-rich nectar to fuel their fast metabolism while consuming invertebrates to obtain protein. Previous work has found that morphologically diverse hummingbird communities exhibit higher diet specialization on floral resources than morphologically similar hummingbird communities. Due to the difficulties of studying avian diets, we have little understanding whether hummingbirds show similar patterns with their invertebrate prey. Here, we use DNA metabarcoding to analyze floral and invertebrate diets of 3 species of sympatric North American hummingbirds. We collected fecal samples from 89 Anna’s (Calypte anna), 39 Black-chinned (Archilochus alexandri), and 29 Calliope (Selasphorus calliope) hummingbirds in urban and rural localities as well as across an elevational gradient from sea level to 2,500 meters above sea level in California, USA. We found hummingbirds showed high dietary overlap in both invertebrate and plant resources, with few invertebrate and plant families common to most individuals and many families found in only a few individuals. Chironomidae was the most common invertebrate family across all species, and Rosaceae and Orobanchaceae were the most common plant families. Anna’s Hummingbirds had significantly higher invertebrate diet diversity than Black-chinned Hummingbirds when found at the same sites, but we found no difference in plant diet diversity among any of the 3 species. Hummingbirds in urban sites had higher plant diet diversity than in rural sites, but we found no effect of elevation on dietary richness. Our study shows how DNA metabarcoding can be used to non-invasively investigate previously unknown life-histories of well-studied birds, lending insight to community structure, function, and evolution.
Stillman A. N., Caiafa M. V., Lorenz T. J., Jusino M. A., Tingley M. W. (2022): DNA metabarcoding reveals broad woodpecker diets in fire-maintained forests. Ornithology 139: ukac009.
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Ecological disturbance is a key agent shaping the spatial and temporal landscape of food availability. In forests of western North America, disturbance from fire can lead to resource pulses of deadwood-associated arthropods that provide important prey for woodpeckers. Although the foraging strategies among woodpecker species often demonstrate pronounced differences, little is known about the ways in which woodpeckers exploit and partition prey in disturbed areas. In this study, we employed DNA metabarcoding to characterize and compare the arthropod diets of 4 woodpecker species in Washington and California, USA—Black-backed Woodpecker (Picoides arcticus), Hairy Woodpecker (Dryobates villosus), Northern Flicker (Colaptes auratus), and White-headed Woodpecker (Dryobates albolarvatus)—primarily using nestling fecal samples from burned forests 1–13 years postfire. Successful sequencing from 78 samples revealed the presence of over 600 operational taxonomic units (OTUs) spanning 32 arthropod orders. The nestling diets of two species in particular—Northern Flicker and Black-backed Woodpecker—proved to be much broader than previous observational studies suggest. Northern Flicker nestlings demonstrated significantly higher diet diversity compared to other focal species, all of which displayed considerable overlap in diversity. Wood-boring beetles, which colonize dead and dying trees after fire, were particularly important diet items for Black-backed, Hairy, and White-headed woodpeckers. Diet composition differed among species, and diets showed limited differences between newer (≤5 yr) and older (>5 yr) postfire forests. Our results show mixed evidence for dietary resource partitioning, with three of the four focal species exhibiting relatively high diet overlap, perhaps due to the pulsed subsidy of deadwood-associated arthropods in burned forests. Woodpeckers are frequently used as management indicator species for forest health, and our study provides one of the first applications of DNA metabarcoding to build a more complete picture of woodpecker diets.
Tang K., Wang Y., Wu M., Wang S., Fu C., Zhang Z., Fu Y. (2022): Metabarcoding of fecal DNA reveals the broad and flexible diet of a globally endangered bird. Current Zoology 2022: zoac071.
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Knowing the diet of endangered wild animals is a prerequisite for species-specific conservation and habitat management. The Sichuan partridge Arborophila rufipectus is a globally endangered Galliformes species endemic to the mountains of southwest China. Existing information on the diet of this species is biased and fragmented owing to traditional observation methods. Little is known about their dietary composition or how they respond to temporal variations in food resources throughout the year. In this study, a dietary analysis was performed on 60 fecal samples using DNA Metabarcoding of invertebrates and plants to determine the primary animal and plant components of the diet across three critical periods of adult life history (breeding, post-breeding wandering, and overwintering). Preys from the dipteran order, followed by the lepidopteran and araneaen spp. were the predominant, animal-derived foods. Symplocos, Rubus, Celastrus, Holboellia, and Actinidia spp. supply a large abundance of fruits and seeds for this omnivorous bird. Substantial temporal dietary changes among the three periods and a general shift toward lower dietary diversity during the breeding season was observed, suggesting that the Sichuan partridge can adjust their diet according to the availability of food resources and their own needs. Characterizing the composition and seasonal changes in Sichuan partridge diets informs the habitat management of native flora (the plant taxa that can generate berries and seeds, such as Symplocos, Rubus, Celastrus and Holboellia, which are likely of conservation interest) to achieve full life-cycle conservation.
Volpe N. L., Thalinger B., Vilacoba E., Braukmann T. W., Di Giacomo A. S., Berkunsky I., Lijtmaer D. A., Steinke D., Kopuchian C. (2022): Diet composition of reintroduced Red-and-Green Macaws reflects gradual adaptation to life in the wild. The Condor 124: duab059.
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Over the last two centuries, the Red-and-Green Macaw (Ara chloropterus) has become locally extinct in Argentina. In an attempt to restore its key ecosystem functions as both disperser and regulator of large-seeded plants, a reintroduction project was initiated at the Iberá National Park in northeastern Argentina. The ability of released individuals to find food is crucial, especially when working with captive-bred animals, as long-term establishment of a self-sustaining population depends on their short-term ability to exploit wild food sources. Monitoring of feeding habits is usually conducted through behavioral observation, but in recent years DNA metabarcoding has emerged as an alternative for obtaining highly resolved data on diet composition. In this study, we use a combination of both techniques to characterize the breadth and composition of the reintroduced macaws’ diet. In addition, we compare the efficiency of both observational and molecular techniques to assess diet composition in a frugivorous bird. Individuals fed on a variety of plant species (n = 49) belonging to a broad phylogenetic spectrum (28 families). Dietary richness estimated by direct observation and DNA metabarcoding was similar, though smaller than the combination of the two datasets as both techniques detected at least 15 species not recorded by the other method. While the total number of detected species was higher for observational data, the rate of species-detection per sampling day was higher for DNA metabarcoding. These results suggest that a combination of both methods is required in order to obtain the most accurate account of the total diversity of the diet of a bird species. The ability of reintroduced macaws to successfully exploit local food resources throughout the year indicates a good level of adjustment to the release site, an important step towards the creation of a stable, self-sustaining population of Red-and-Green Macaws in Northern Argentina.