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.

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.

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.

MEDI: Macronutrient Extraction and Determination from invertebrates, a rapid, cheap and streamlined protocol.

Macronutrients, comprising carbohydrates, proteins and lipids, underpin many ecological processes, but their quantification in ecological studies is often inaccurate and laborious, requiring large investments of time and bulk samples, which make individual-level studies impossible. This study presents Macronutrient Extraction and Determination from Invertebrates (MEDI), a protocol for the direct, rapid and relatively low-cost determination of macronutrient content from single small macroinvertebrates.Macronutrients were extracted by a sequential process of soaking in 1:12 chloroform:methanol solution to remove lipid and then solubilising tissue in 0.1 M NaOH. Proteins, carbohydrates and lipids were determined by colorimetric assays from the same individual specimens.The limits of detection of MEDI with the equipment and conditions used were 0.067, 0.065 and 0.006 mg/ml for proteins, carbohydrates and lipids respectively. Adjusting the volume of reagents used for extraction and determination can broaden the range of concentrations that can be detected. MEDI successfully identified taxonomic differences in macronutrient content between five insect species.Macronutrient Extraction and Determination from Invertebrates can directly and rapidly determine macronutrient content in tiny (dry mass ~3 mg) and much larger individual invertebrates. Using MEDI, the total macronutrient content of over 50 macroinvertebrates can be determined within around 3 days of collection at a cost of ~$1.35 per sample.

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.

How animals distribute themselves in space: energy landscapes of Antarctic avian predators.

BACKGROUND: Energy landscapes provide an approach to the mechanistic basis of spatial ecology and decision-making in animals. This is based on the quantification of the variation in the energy costs of movements through a given environment, as well as how these costs vary in time and for different animal populations. Organisms as diverse as fish, mammals, and birds will move in areas of the energy landscape that result in minimised costs and maximised energy gain. Recently, energy landscapes have been used to link energy gain and variable energy costs of foraging to breeding success, revealing their potential use for understanding demographic changes. METHODS: Using GPS-temperature-depth and tri-axial accelerometer loggers, stable isotope and molecular analyses of the diet, and leucocyte counts, we studied the response of gentoo (Pygoscelis papua) and chinstrap (Pygoscelis antarcticus) penguins to different energy landscapes and resources. We compared species and gentoo penguin populations with contrasting population trends. RESULTS: Between populations, gentoo penguins from Livingston Island (Antarctica), a site with positive population trends, foraged in energy landscape sectors that implied lower foraging costs per energy gained compared with those around New Island (Falkland/Malvinas Islands; sub-Antarctic), a breeding site with fluctuating energy costs of foraging, breeding success and populations. Between species, chinstrap penguins foraged in sectors of the energy landscape with lower foraging costs per bottom time, a proxy for energy gain. They also showed lower physiological stress, as revealed by leucocyte counts, and higher breeding success than gentoo penguins. In terms of diet, we found a flexible foraging ecology in gentoo penguins but a narrow foraging niche for chinstraps. CONCLUSIONS: The lower foraging costs incurred by the gentoo penguins from Livingston, may favour a higher breeding success that would explain the species' positive population trend in the Antarctic Peninsula. The lower foraging costs in chinstrap penguins may also explain their higher breeding success, compared to gentoos from Antarctica but not their negative population trend. Altogether, our results suggest a link between energy landscapes and breeding success mediated by the physiological condition.

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.

Species Separation within, and Preliminary Phylogeny for, the Leafhopper Genus Anoscopus with Particular Reference to the Putative British Endemic Anoscopus duffieldi (Hemiptera: Cicadellidae).

The subfamily Aphrodinae (Hemiptera: Cicadellidae) contains ~33 species in Europe within four genera. Species in two genera in particular, Aphrodes and Anoscopus, have proved to be difficult to distinguish morphologically. Our aim was to determine the status of the putative species Anoscopus duffieldi, found only on the RSPB Nature Reserve at Dungeness, Kent, a possible rare UK endemic. DNA from samples of all seven UK Anoscopus species (plus Anoscopusalpinus from the Czech Republic) were sequenced using parts of the mitochondrial cytochrome oxidase I and 16S rRNA genes. Bayesian inference phylogenies were created. Specimens of each species clustered into monophyletic groups, except for Anoscopusalbifrons, A. duffieldi and Anoscopuslimicola. Two A. albifrons specimens grouped with A. duffieldi repeatedly with strong support, and the remaining A. albifrons clustered within A. limicola. Genetic distances suggest that A. albifrons and A. limicola are a single interbreeding population (0% divergence), while A. albifrons and A. duffieldi diverged by only 0.28%. Shared haplotypes between A. albifrons, A. limicola and A. duffieldi strongly suggest interbreeding, although misidentification may also explain these topologies. However, all A. duffieldi clustered together in the trees. A conservative approach might be to treat A. duffieldi, until other evidence is forthcoming, as a possible endemic subspecies.

Insights into aphid prey consumption by ladybirds: Optimising field sampling methods and primer design for high throughput sequencing.

Elucidating the diets of insect predators is important in basic and applied ecology, such as for improving the effectiveness of conservation biological control measures to promote natural enemies of crop pests. Here, we investigated the aphid diet of two common aphid predators in Central European agroecosystems, the native Coccinella septempunctata (Linnaeus) and the invasive Harmonia axyridis (Pallas; Coleoptera: Coccinellidae) by means of high throughput sequencing (HTS). For acquiring insights into diets of mobile flying insects at landscape scale minimizing trapping bias is important, which imposes methodological challenges for HTS. We therefore assessed the suitability of three field sampling methods (sticky traps, pan traps and hand-collection) as well as new aphid primers for identifying aphid prey consumption by coccinellids through HTS. The new aphid primers facilitate identification to species level in 75% of the European aphid genera investigated. Aphid primer specificity was high in silico and in vitro but low in environmental samples with the methods used, although this could be improved in future studies. For insect trapping we conclude that sticky traps are a suitable method in terms of minimizing sampling bias, contamination risk and trapping success, but compromise on DNA-recovery rate. The aphid diets of both field-captured ladybird species were dominated by Microlophium carnosum, the common nettle aphid. Another common prey was Sitobion avenae (cereal aphid), which got more often detected in C. septempunctata compared to H. axyridis. Around one third of the recovered aphid taxa were common crop pests. We conclude that sampling methodologies need constant revision but that our improved aphid primers offer currently one of the best solutions for broad screenings of coccinellid predation on aphids.

Estimation of trophic niches in myrmecophagous spider predators.

Among spiders, taxonomically the most diversified group of terrestrial predators, only a few species are stenophagous and feed on ants. The levels of stenophagy and ant-specialisation vary among such species. To investigate whether stenophagy is only a result of a local specialisation both fundamental and realised trophic niches need to be estimated. Here we investigated trophic niches in three closely-related spider species from the family Gnaphosidae (Callilepis nocturna, C. schuszteri, Nomisia exornata) with different levels of myrmecophagy. Acceptance experiments were used to estimate fundamental trophic niches and molecular methods to estimate realised trophic niches. For the latter two PCR primer sets were used as these can affect the niche breadth estimates. The general invertebrate ZBJ primers were not appropriate for detecting ant DNA as they revealed very few prey types, therefore ant-specific primers were used. The cut-off threshold for erroneous MOTUs was identified as 0.005% of the total number of valid sequences, at individual predator level it was 0.05%. The fundamental trophic niche of Callilepis species included mainly ants, while that of N. exornata included many different prey types. The realised trophic niche in Callilepis species was similar to its fundamental niche but in N. exornata the fundamental niche was wider than realised niche. The results show that Callilepis species are ant-eating (specialised) stenophagous predators, catching mainly Formicinae ants, while N. exornata is an ant-eating euryphagous predator catching mainly Myrmicinae ants.

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.

New universal ITS2 primers for high-resolution herbivory analyses using DNA metabarcoding in both tropical and temperate zones.

DNA metabarcoding is a rapidly growing technique for obtaining detailed dietary information. Current metabarcoding methods for herbivory, using a single locus, can lack taxonomic resolution for some applications. We present novel primers for the second internal transcribed spacer of nuclear ribosomal DNA (ITS2) designed for dietary studies in Mauritius and the UK, which have the potential to give unrivalled taxonomic coverage and resolution from a short-amplicon barcode. In silico testing used three databases of plant ITS2 sequences from UK and Mauritian floras (native and introduced) totalling 6561 sequences from 1790 species across 174 families. Our primers were well-matched in silico to 88% of species, providing taxonomic resolution of 86.1%, 99.4% and 99.9% at the species, genus and family levels, respectively. In vitro, the primers amplified 99% of Mauritian (n = 169) and 100% of UK (n = 33) species, and co-amplified multiple plant species from degraded faecal DNA from reptiles and birds in two case studies. For the ITS2 region, we advocate taxonomic assignment based on best sequence match instead of a clustering approach. With short amplicons of 187-387 bp, these primers are suitable for metabarcoding plant DNA from faecal samples, across a broad geographic range, whilst delivering unparalleled taxonomic resolution.

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.

Resolving large-scale pressures on species and ecosystems: propensity modelling identifies agricultural effects on streams.

Although agriculture is amongst the world's most widespread land uses, studies of its effects on stream ecosystems are often limited in spatial extent. National monitoring data could extend spatial coverage and increase statistical power, but present analytical challenges where covarying environmental variables confound relationships of interest.Propensity modelling is used widely outside ecology to control for confounding variables in observational data. Here, monitoring data from over 3000 English and Welsh river reaches are used to assess the effects of intensive agricultural land cover (arable and pastoral) on stream habitat, water chemistry and invertebrates, using propensity scores to control for potential confounding factors (e.g. climate, geology). Propensity scoring effectively reduced the collinearity between land cover and potential confounding variables, reducing the potential for covariate bias in estimated treatment-response relationships compared to conventional multiple regression.Macroinvertebrate richness was significantly greater at sites with a higher proportion of improved pasture in their catchment or riparian zone, with these effects probably mediated by increased algal production from mild nutrient enrichment. In contrast, macroinvertebrate richness did not change with arable land cover, although sensitive species representation was lower under higher proportions of arable land cover, probably due to greatly elevated nutrient concentrations. Synthesis and applications. Propensity modelling has great potential to address questions about pressures on ecosystems and organisms at the large spatial extents relevant to land-use policy, where experimental approaches are not feasible and broad environmental changes often covary. Applied to the effects of agricultural land cover on stream systems, this approach identified reduced nutrient loading from arable farms as a priority for land management. On this specific issue, our data and analysis support the use of riparian or catchment-scale measures to reduce nutrient delivery to sensitive water bodies.

Discovery of a monophagous true predator, a specialist termite-eating spider (Araneae: Ammoxenidae).

True predators are characterised by capturing a number of prey items during their lifetime and by being generalists. Some true predators are facultative specialists, but very few species are stenophagous specialists that catch only a few closely related prey types. A monophagous true predator that would exploit a single prey species has not been discovered yet. Representatives of the spider family Ammoxenidae have been reported to have evolved to only catch termites. Here we tested the hypothesis that Ammoxenus amphalodes is a monophagous termite-eater capturing only Hodotermes mossambicus. We studied the trophic niche of A. amphalodes by means of molecular analysis of the gut contents using Next Generation Sequencing. We investigated their willingness to accept alternative prey and observed their specific predatory behaviour and prey capture efficiency. We found all of the 1.4 million sequences were H. mossambicus. In the laboratory A. amphalodes did not accept any other prey, including other termite species. The spiders attacked the lateral side of the thorax of termites and immobilised them within 1 min. The paralysis efficiency was independent of predator:prey size ratio. The results strongly indicate that A. amphalodes is a monophagous prey specialist, specifically adapted to feed on H. mossambicus.

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.