Fungal microbiomes are determined by host phylogeny and exhibit widespread associations with the bacterial microbiome.

Interactions between hosts and their resident microbial communities are a fundamental component of fitness for both agents. Though recent research has highlighted the importance of interactions between animals and their bacterial communities, comparative evidence for fungi is lacking, especially in natural populations. Using data from 49 species, we present novel evidence of strong covariation between fungal and bacterial communities across the host phylogeny, indicative of recruitment by hosts for specific suites of microbes. Using co-occurrence networks, we demonstrate marked variation across host taxonomy in patterns of covariation between bacterial and fungal abundances. Host phylogeny drives differences in the overall richness of bacterial and fungal communities, but the effect of diet on richness was only evident in the mammalian gut microbiome. Sample type, tissue storage and DNA extraction method also affected bacterial and fungal community composition, and future studies would benefit from standardized approaches to sample processing. Collectively these data indicate fungal microbiomes may play a key role in host fitness and suggest an urgent need to study multiple agents of the animal microbiome to accurately determine the strength and ecological significance of host-microbe interactions.

The problem of omnivory: A synthesis on omnivory and DNA metabarcoding.

Dietary analysis using DNA metabarcoding is a powerful tool that is increasingly being used to further our knowledge of trophic interactions in highly complex food webs but is not without limitations. Omnivores, the most generalist of consumers, pose unique challenges when using such methods. Here, we provide the rationale to understand the problems associated with analysing the complex diets of omnivores. By reviewing existing metabarcoding studies of omnivorous diet, and constructing hypothetical scenarios arising from each, we outline that great caution is required when interpreting sequencing data in such cases. In essence, the problems of accidental consumption and secondary ingestion are significant sources of error when investigating omnivorous diets. The integration of multiple high throughput sequencing markers increases the taxonomic breadth of taxa detected but we reveal how some detections may be misleading. Disentangling which taxa have been deliberately or accidentally consumed by the focal omnivore is challenging and can falsely emphasise those that were not intentionally consumed, obscuring biologically meaningful interactions. Although we suggest ways to disentangle these issues, we urge that the results of such analyses should be interpreted with caution and all possible scenarios for the presence of biota within omnivores given due consideration.

The choice of universal primers and the characteristics of the species mixture determine when DNA metabarcoding can be quantitative.

DNA metabarcoding is a technique used to survey biodiversity in many ecological settings, but there are doubts about whether it can provide quantitative results, that is, the proportions of each species in the mixture as opposed to a species list. While there are several experimental studies that report quantitative metabarcoding results, there are a similar number that fail to do so. Here, we provide the rationale to understand under what circumstances the technique can be quantitative. In essence, we simulate a mixture of DNA of S species with a defined initial abundance distribution. In the simulated PCR, each species increases its concentration following a certain amplification efficiency. The final DNA concentration will reflect the initial one when the efficiency is similar for all species; otherwise, the initial and final DNA concentrations would be poorly related. Although there are many known factors that modulate amplification efficiency, we focused on the number of primer-template mismatches, arguably the most important one. We used 15 common primers pairs targeting the mitochondrial COI region and the mitogenomes of ca. 1,200 insect species. The results showed that some primers pairs produced quantitative results under most circumstances, whereas some other primers failed to do so. In conclusion, depending on the primer pair used in the PCR amplification and on the characteristics of the mixture analysed (i.e., high species richness, low evenness), DNA metabarcoding can provide a quantitative estimate of the relative abundances of different species.

The decline of the Turtle Dove: Dietary associations with body condition and competition with other columbids analysed using high-throughput sequencing.

Dietary changes linked to the availability of anthropogenic food resources can have complex implications for species and ecosystems, especially when species are in decline. Here, we use recently developed primers targeting the ITS2 region of plants to characterize diet from faecal samples of four UK columbids, with particular focus on the European turtle dove (Streptopelia turtur), a rapidly declining obligate granivore. We examine dietary overlap between species (potential competition), associations with body condition in turtle doves and spatiotemporal variation in diet. We identified 143 taxonomic units, of which we classified 55% to species, another 34% to genus and the remaining 11% to family. We found significant dietary overlap between all columbid species, with the highest between turtle doves and stock doves (Columba oenas), then between turtle doves and woodpigeons (Columba palumbus). The lowest overlap was between woodpigeons and collared doves (Streptopelia decaocto). We show considerable change in columbid diets compared to previous studies, probably reflecting opportunistic foraging behaviour by columbids within a highly anthropogenically modified landscape, although our data for nonturtle doves should be considered preliminary. Nestling turtle doves in better condition had a higher dietary proportion of taxonomic units from natural arable plant species and a lower proportion of taxonomic units from anthropogenic food resources such as garden bird seed mixes and brassicas. This suggests that breeding ground conservation strategies for turtle doves should include provision of anthropogenic seeds for adults early in the breeding season, coupled with habitat rich in accessible seeds from arable plants once chicks have hatched.

Sex and age-biased nematode prevalence in reptiles.

Prevalence and intensity of parasitic infections are often higher in male than in female vertebrates. This bias may represent either differences between host sex in exposure or susceptibility to parasites. The former may be due to sex-specific behaviour of the host, including differential habitat use or diet. Differences in susceptibility are often regarded as a negative effect of male sex steroid hormones on the immune system. Host-parasite dynamics are of great interest in terms of reptile survival, ecology and conservation. We used, for the first time, molecular diagnostics to track nematode parasitism in wild populations of reptiles noninvasively. Using slow worms (Anguis fragilis) as a model species, we investigated the interacting effects of time of year, sex, length, weight and climatic variables on the prevalence of the gastroenterological parasitic nematode Neoxysomatium brevicaudatum. Faeces were collected from three sites over 2 years. There was an interaction between sex and time of year, with lower nematode prevalence in males than in females in July or August (different between years) but a high prevalence in males in April. As the latter is during the slow worm breeding season, this may be the result of testosterone-induced immunosuppression. A second-order interaction between slow worm length and weight was found to be significant, with a positive association between prevalence and body condition in young slow worms and a negative association in older slow worms. The convex pattern of nematode prevalence with age that emerged suggests an increase with age-related exposure and a decrease with age-related acquired immunity.

An inordinate fondness for beetles? Variation in seasonal dietary preferences of night-roosting big brown bats (Eptesicus fuscus).

Generalist species with numerous food web interactions are thought to provide stability to ecosystem dynamics; however, it is not always clear whether habitat generality translates into dietary diversity. Big brown bats are common across North America and employ a flexible foraging strategy over water, dense forests, forest edges and rural and urban settings. Despite this generalist use of habitat, they are paradoxically characterized as beetle specialists. However, hard carapaces may preferentially survive digestion leading to over-representation during morphological analysis of diet. This specialization has not been evaluated independently using molecular analysis and species-level identification of prey. We used next-generation sequencing to assess the diet of big brown bats. Beetles were consumed in the highest frequency but Lepidoptera species richness was highest among identified prey. The consumption of species showed strong seasonal and annual variation. While Coleoptera consumption varied, Lepidoptera and Ephemeroptera were relatively constant dietary components. Dietary diversity increased in late summer when insect diversity decreases. Our results indicate that big brown bats are dietary generalists and, while beetles are an important component of the diet, Lepidoptera are equally important, and Lepidoptera and Ephemeroptera are the only stable prey resource exploited. As resources become limited, big brown bats may respond by increasing the species richness of prey and thus their connectedness in the ecosystem. This characterization of diet corresponds well with a generalist approach to foraging, making them an important species in encouraging and maintaining ecosystem stability.

Molecular analysis of the diets of snakes: changes in prey exploitation during development of the rare smooth snake Coronella austriaca.

Reptiles are declining in many parts of the world, mainly due to habitat loss and environmental change. A major factor in this is availability of suitable food. For many animals, dietary requirements shift during developmental stages and a habitat will only be suitable for conserving a species if it supports all stages. Conventional methods for establishing diet often rely on visual recognition of morphologically identifiable features of prey in faeces, regurgitation or stomach contents, which suffer from biases and poor resolution of taxa. DNA-based techniques facilitate noninvasive analysis of diet from faeces without these constraints. We tested the hypothesis that diet changes during growth stages of smooth snakes (Coronella austriaca), which have a highly restricted distribution in the UK but are widespread in continental Europe. Small numbers of the sympatric grass snake (Natrix natrix) were analysed for comparison. Faecal samples were collected from snakes and prey DNA analysed using PCR, targeting amphibians, reptiles, mammals and invertebrates. Over 85% of smooth snakes were found to have eaten reptiles and 28% had eaten mammals. Predation on mammals increased with age and was entirely absent among juveniles and subadults. Predation on reptiles did not change ontogenetically. Smooth snakes may, therefore, be restricted to areas of sufficiently high reptile densities to support young snakes.

Diet of the insectivorous bat Pipistrellus nathusii during autumn migration and summer residence.

Migration is widespread among vertebrates, yet bat migration has received little attention and only in the recent decades has a better understanding of it been gained. Migration can cause significant changes in behaviour and physiology, due to increasing energy demands and aerodynamic constraints. Dietary shifts, for example, have been shown to occur in birds before onset of migration. For bats, it is not known if a change in diet occurs during migration, although breeding season-related dietary preference has been documented. It is known that a diet rich in fats and the accumulation of fat deposits do increase the flight range of migratory bats. Some bat species can be regarded as long-distance migrants, covering up to 2000 km between summer and winter roosting areas. Pipistrellus nathusii (Vespertilionidae), a European long-distant migrant, travels each year along the Baltic Sea from north-eastern Europe to hibernate in central and southern Europe. This study presents data on the dietary habits of migrating Pipistrellus nathusii compared with those during the breeding season. We analysed faecal samples from bats on fall migration caught at the Ornithological Field Station in Pape, Latvia and from samples collected in North-Latvian summer roosts. We applied both morphological identification and molecular methods, as morphological methods also recognize life stages of prey and can contribute frequency data. The diets of bats on migration and breeding bats were similar, with Diptera and Lepidoptera comprising the major prey categories. However, certain prey groups could be explained by the different hunting habitats exploited during migration vs. summer residence.

Resource partitioning by insectivorous bats in Jamaica.

In this investigation, we use variation in wing morphology, echolocation behaviour, patterns of habitat use and molecular diet analysis to demonstrate that six species of sympatric insectivorous bats in Jamaica show significant differences that could explain resource partitioning among the species. High-intensity echolocating species that used shorter, broadband signals and had shorter, broader wings (Pteronotus macleayii, Pteronotus quadridens, Mormoops blainvillii) foraged most in edge habitats, but differed in timing of peak activity. P. macleayii and M. blainvillii differed in diet, but low sample size precluded diet analysis for P. quadridens. High-intensity echolocating species that used longer, more narrowband signals and had longer, narrower wings (Molossus molossus, Tadarida brasiliensis) foraged most in open areas and differed in diet from the other species. Two disparate species were most active in clutter (dense vegetation). Pteronotus parnellii used high-duty-cycle echolocation apparently specialized for detecting fluttering targets in clutter. Macrotus waterhousii used low-intensity, broadband echolocation calls and presumably uses prey-generated sounds when foraging. These two species also differed in diet. Our data show that differences in morphology and echolocation behaviour coincide with differences in habitat use and diet, resulting in minimal overlap in resource use among species.

Molecular field analysis of trophic relationships in soil-dwelling invertebrates to identify mercury, lead and cadmium transmission through forest ecosystems.

Contamination pathways in complex food chains in soil ecosystems can be difficult to elucidate. Molecular analysis of predator gut content can, however, rapidly reveal previously unidentified trophic interactions between invertebrates and thereby uncover pathways of pollutant spread. Here, we measured concentrations of the toxic metals lead, cadmium and mercury in carabid beetle predators and their prey. Invertebrates were sampled at one control and four heavy metal-polluted sites to reveal the impact of diet composition and seasonal variation in prey availability on metal burden in carabids and metal transfer pathways through forest ecosystems. This is the first report, to our knowledge, of carabid diet composition based on PCR analysis of gut contents at the forest community level, rather than in cultivated fields. Extensive screening using group- and species-specific primers revealed that carabids ate primarily earthworms and slugs, as well as smaller numbers of woodlice and springtails. Metal concentrations in carabids correlated with seasonal changes in diet. Mercury accumulated in beetle predators more than in their slug prey. As earthworms, slugs and carabid beetles are the major prey of many birds and mammals, prey-predator transfer and associated toxicity are major risks at mercury-contaminated sites. Carabids may be useful bioindicators for assessing the impact of pollutants on soil ecosystems, as long as species and seasonal factors are taken into account.

An holistic ecological analysis of the diet of Cory's shearwaters using prey morphological characters and DNA barcoding.

Knowledge of the dietary choices and trophic niches of organisms is the key to understanding their roles in ecosystems. In seabird diet studies, prey identification is a difficult challenge, often yielding results with technique-specific biases. Additionally, sampling efforts are often not extensive enough to reveal intrapopulational variation. Immature animals, which may constitute up to 50% of a population, may occupy a significantly different trophic niche to more experienced birds, but this remains largely unexplored. We investigated the diet of Cory's shearwater (Calonectris diomedea) from Selvagem Grande, an island located off the northwest African coast, collecting a total of 698 regurgitate samples over three consecutive breeding seasons. The diet was assessed using two complementary approaches for prey identification: conventional morphological analysis (using fish vertebrae, otoliths and cephalopod beaks) and DNA barcoding of the 16S rRNA mitochondrial gene, in cases where a positive identification could not be retrieved. Species assignments employed BLAST and distance-based methods, as well as direct optimization of the tree length based on unaligned sequences in POY. This method resulted in robust tree estimates and species assignments, showing its potential for DNA barcoding of stomach contents using hypervariable markers such as the 16S. The molecular approach increased taxonomic resolution and revealed an additional 17 taxa. Diet differed significantly according to breeding status, sex, breeding phase (prelaying and chick rearing) and year. Such direct evidence of trophic segregation within the same population has rarely been shown in seabirds and highlights the importance of including such variables in ecosystem-based management approaches.

The diet of Myotis lucifugus across Canada: assessing foraging quality and diet variability.

Variation in prey resources influences the diet and behaviour of predators. When prey become limiting, predators may travel farther to find preferred food or adjust to existing local resources. When predators are habitat limited, local resource abundance impacts foraging success. We analysed the diet of Myotis lucifugus (little brown bats) from Nova Scotia (eastern Canada) to the Northwest Territories (north-western Canada). This distribution includes extremes of season length and temperature and encompasses colonies on rural monoculture farms, and in urban and unmodified areas. We recognized nearly 600 distinct species of prey, of which ≈30% could be identified using reference sequence libraries. We found a higher than expected use of lepidopterans, which comprised a range of dietary richness from ≈35% early in the summer to ≈55% by late summer. Diptera were the second largest prey group consumed, representing ≈45% of dietary diversity early in the summer. We observed extreme local dietary variability and variation among seasons and years. Based on the species of insects that were consumed, we observed that two locations support prey species with extremely low pollution and acidification tolerances, suggesting that these are areas without environmental contamination. We conclude that there is significant local population variability in little brown bat diet that is likely driven by seasonal and geographical changes in insect diversity, and that this prey may be a good indicator of environment quality.

Non-native ants drive dramatic declines in animal community diversity: A meta-analysis.

Non-native ants can cause ecosystem-wide ecological change, and these changes are generally assumed to be negative. Despite this, the evidence base has never been holistically synthesised to quantify whether and to what degree non-native ants impact native species diversity.In this study, we performed a meta-analysis of the effects of ant invasion on animal communities. We extracted data from 46 published articles investigating abundance (156 effect sizes) and richness (53 effect sizes) responses of animal taxa to ant invasion in locations relatively unimpacted by other stressors (e.g. human disturbance, other non-native species) to help isolate the effects of invasion.Overall, local animal diversity declined severely, with species abundance and richness lower by 42.79% and 53.56%, respectively, in areas with non-native ants compared with intact uninvaded sites. We then combined responses of individual animal taxa extracted from an article into a single response to represent the 'community' abundance (40 effect sizes) or richness (28 effect sizes) response to non-native ants represented in each article. Local communities decreased substantially in total abundance (52.67%) and species richness (53.47%) in invaded sites.These results highlight non-native ants as the drivers, rather than passengers, of large net-negative reductions to animal community diversity in relatively undisturbed systems around the world, approximately halving local species abundance and richness in invaded areas. Improved international prevention processes, early detection systems harnessing emerging technologies, and well-designed control measures deployable by conservation practitioners are urgently needed if these effects are to be mitigated, prevented or reversed.

Otterly delicious: Spatiotemporal variation in the diet of a recovering population of Eurasian otters (Lutra lutra) revealed through DNA metabarcoding and morphological analysis of prey remains.

Eurasian otters are apex predators of freshwater ecosystems and a recovering species across much of their European range; investigating the dietary variation of this predator over time and space, therefore, provides opportunities to identify changes in freshwater trophic interactions and factors influencing the conservation of otter populations. Here we sampled feces from 300 dead otters across England and Wales between 2007 and 2016, conducting both morphological analyses of prey remains and dietary DNA metabarcoding. Comparison of these methods showed that greater taxonomic resolution and breadth could be achieved using DNA metabarcoding but combining data from both methodologies gave the most comprehensive dietary description. All otter demographics exploited a broad range of taxa and variation likely reflected changes in prey distributions and availability across the landscape. This study provides novel insights into the trophic generalism and adaptability of otters across Britain, which is likely to have aided their recent population recovery, and may increase their resilience to future environmental changes.

Herbivorous dietary selection shown by hawfinch (Coccothraustes coccothraustes) within mixed woodland habitats.

Knowledge of diet and dietary selectivity is vital, especially for the conservation of declining species. Accurately obtaining this information, however, is difficult, especially if the study species feeds on a wide range of food items within heterogeneous and inaccessible environments, such as the tree canopy. Hawfinches (Coccothraustes coccothraustes), like many woodland birds, are declining for reasons that are unclear. We investigated the possible role that dietary selection may have in these declines in the UK. Here, we used a combination of high-throughput sequencing of 261 hawfinch faecal samples assessed against tree occurrence data from quadrats sampled in three hawfinch population strongholds in the UK to test for evidence of selective foraging. This revealed that hawfinches show selective feeding and consume certain tree genera disproportionally to availability. Positive selection was shown for beech (Fagus), cherry (Prunus), hornbeam (Carpinus), maples (Acer) and oak (Quercus), while Hawfinch avoided ash (Fraxinus), birch (Betula), chestnut (Castanea), fir (Abies), hazel (Corylus), rowan (Sorbus) and lime (Tilia). This approach provided detailed information on hawfinch dietary choice and may be used to predict the effects of changing food resources on other declining passerines populations in the future.

Multi-marker DNA metabarcoding reveals spatial and sexual variation in the diet of a scarce woodland bird.

Avian diet can be affected by site-specific variables, such as habitat, as well as intrinsic factors such as sex. This can lead to dietary niche separation, which reduces competition between individuals, as well as impacting how well avian species can adapt to environmental variation. Estimating dietary niche separation is challenging, due largely to difficulties in accurately identifying food taxa consumed. Consequently, there is limited knowledge of the diets of woodland bird species, many of which are undergoing serious population declines. Here, we show the effectiveness of multi-marker fecal metabarcoding to provide in-depth dietary analysis of a declining passerine in the UK, the Hawfinch (Coccothraustes coccothraustes). We collected fecal samples from (n = 262) UK Hawfinches prior to, and during, the breeding seasons in 2016-2019. We detected 49 and 90 plant and invertebrate taxa, respectively. We found Hawfinch diet varied spatially, as well as between sexes, indicating broad dietary plasticity and the ability of Hawfinches to utilize multiple resources within their foraging environments.

Who is eating what: diet assessment using next generation sequencing.

The analysis of food webs and their dynamics facilitates understanding of the mechanistic processes behind community ecology and ecosystem functions. Having accurate techniques for determining dietary ranges and components is critical for this endeavour. While visual analyses and early molecular approaches are highly labour intensive and often lack resolution, recent DNA-based approaches potentially provide more accurate methods for dietary studies. A suite of approaches have been used based on the identification of consumed species by characterization of DNA present in gut or faecal samples. In one approach, a standardized DNA region (DNA barcode) is PCR amplified, amplicons are sequenced and then compared to a reference database for identification. Initially, this involved sequencing clones from PCR products, and studies were limited in scale because of the costs and effort required. The recent development of next generation sequencing (NGS) has made this approach much more powerful, by allowing the direct characterization of dozens of samples with several thousand sequences per PCR product, and has the potential to reveal many consumed species simultaneously (DNA metabarcoding). Continual improvement of NGS technologies, on-going decreases in costs and current massive expansion of reference databases make this approach promising. Here we review the power and pitfalls of NGS diet methods. We present the critical factors to take into account when choosing or designing a suitable barcode. Then, we consider both technical and analytical aspects of NGS diet studies. Finally, we discuss the validation of data accuracy including the viability of producing quantitative data.

Seasonal and ontological variation in diet and age-related differences in prey choice, by an insectivorous songbird.

The diet of an individual animal is subject to change over time, both in response to short-term food fluctuations and over longer time scales as an individual ages and meets different challenges over its life cycle. A metabarcoding approach was used to elucidate the diet of different life stages of a migratory songbird, the Eurasian reed warbler (Acrocephalus scirpaceus) over the 2017 summer breeding season in Somerset, the United Kingdom. The feces of adult, juvenile, and nestling warblers were screened for invertebrate DNA, enabling the identification of prey species. Dietary analysis was coupled with monitoring of Diptera in the field using yellow sticky traps. Seasonal changes in warbler diet were subtle, whereas age class had a greater influence on overall diet composition. Age classes showed high dietary overlap, but significant dietary differences were mediated through the selection of prey; (i) from different taxonomic groups, (ii) with different habitat origins (aquatic vs. terrestrial), and (iii) of different average approximate sizes. Our results highlight the value of metabarcoding data for enhancing ecological studies of insectivores in dynamic environments.

DNA metabarcoding reveals introduced species predominate in the diet of a threatened endemic omnivore, Telfair's skink (Leiolopisma telfairii).

Introduced species can exert disproportionately negative effects on island ecosystems, but their potential role as food for native consumers is poorly studied. Telfair's skinks are endemic omnivores living on Round Island, Mauritius, a globally significant site of biodiversity conservation. We aimed to determine the dietary diversity and key trophic interactions of Telfair's skinks, whether introduced species are frequently consumed, and if diet composition changes seasonally between male and female skinks. We used DNA metabarcoding of skink fecal samples to identify animals (COI) and plants (ITS2) consumed by skinks. There were 389 dietary presence counts belonging to 77 dietary taxa found across the 73 Telfair's skink fecal samples. Introduced taxa were cumulatively consumed more frequently than other categories, accounting for 49.4% of all detections, compared to cryptogenic (20.6%), native (20.6%), and endemic taxa (9.5%). The most frequently consumed introduced species was the ant, Pheidole megacephala, present in 40% of samples. Blue latan palm, Latania loddigesii, was the most frequently consumed endemic species, present in 33% of samples but was only detected in the dry season, when fruits are produced. We found a strong seasonal difference in diet composition explained by the presence of certain plant species solely or primarily in one season and a marked increase in the consumption of animal prey in the dry season. Male and female skinks consumed several taxa at different frequencies. These results present a valuable perspective on the role of introduced species in the trophic network of their invaded ecosystem. Both native and introduced species provide nutritional resources for skinks, and this may have management implications in the context of species conservation and island restoration.