Determining diet is a key prerequisite for understanding species interactions, food web structure and ecological dynamics. In recent years, there has been considerable development in both the methodology and application of novel and more traditional dietary tracing methods, yet there is no comprehensive synthesis that systematically and quantitatively compares the different approaches. Here we conceptualise diet tracing in ecology, provide recommendations for method selection, and illustrate the advantages of method integration. We summarise empirical evidence on how different methods quantify diet mixtures, by contrasting estimates of dietary proportions from multiple methods applied to the same consumer‐resource datasets, or from experimental studies with known diet compositions. Our data synthesis revealed an urgent need for more experiential comparisons among the dietary methods. The comparison of diet quantifications from field observations showed that different techniques aligned well in cases with less than six diet items, but diverged considerably when applied to more complex diet mixtures. Efforts are ongoing to further advance dietary estimation, including how reliably compound specific stable isotope analyses and fatty acid profiles can quantify more prey items than bulk stable isotope analyses. Similarly, DNA analyses, which can depict trophic interactions at a higher resolution than any other method, are generating new ways to better quantify diets and differentiate among life‐stages of prey. Such efforts, combined with more empirical testing of each dietary method and establishment of open data repositories for dietary data, promise to greatly advance community and ecosystem ecology.

One of the most difficult interactions to observe in nature is the relationship between a predator and its prey. When direct observations are impossible, we rely on morphological classification of prey remains, although this is particularly challenging among generalist predators whose faeces contain mixed and degraded prey fragments. In this investigation, we used a polymerase chain reaction and sequence-based technique to identify prey fragments in the guano of the generalist insectivore, the eastern red bat (Lasiurus borealis), and evaluate several hypotheses about prey selection and prey defences. The interaction between bats and insects is of significant evolutionary interest because of the adaptive nature of insect hearing against echolocation. However, measuring the successes of predator tactics or particular prey defences is limited because we cannot normally identify these digested prey fragments beyond order or family. Using a molecular approach, we recovered sequences from 89% of the fragments tested, and through comparison to a reference database of sequences, we were able to identify 127 different species of prey. Our results indicate that despite the robust jaws of L. borealis, most prey taxa were softer-bodied Lepidoptera. Surprisingly, more than 60% of the prey species were tympanate, with ears thought to afford protection against these echolocating bats. Moths of the family Arctiidae, which employ multiple defensive strategies, were not detected as a significant dietary component. Our results provide an unprecedented level of detail for the study of predator-prey relationships in bats and demonstrate the advantages which molecular tools can provide in investigations of complex ecological systems and food-web relationships.

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

Predicting whether a predator is capable of affecting the dynamics of a prey species in the field implies the analysis of the complete diet of the predator, not simply rates of predation on a target taxon. Here, we employed the Ion Torrent next-generation sequencing technology to investigate the diet of a generalist arthropod predator. A complete dietary analysis requires the use of general primers, but these will also amplify the predator unless suppressed using a blocking probe. However, blocking probes can potentially block other species, particularly if they are phylogenetically close. Here, we aimed to demonstrate that enough prey sequence could be obtained without blocking probes. In communities with many predators, this approach obviates the need to design and test numerous blocking primers, thus making analysis of complex community food webs a viable proposition. We applied this approach to the analysis of predation by the linyphiid spider Oedothorax fuscus in an arable field. We obtained over two million raw reads. After discarding the low-quality and predator reads, the libraries still contained over 61 000 prey reads (3% of the raw reads; 6% of reads passing quality control). The libraries were rich in Collembola, Lepidoptera, Diptera and Nematoda. They also contained sequences derived from several spider species and from horticultural pests (aphids). Oedothorax fuscus is common in UK cereal fields, and the results showed that it is exploiting a wide range of prey. Next-generation sequencing using general primers but without blocking probes provided ample sequences for analysis of the prey range of this spider and proved to be a simple and inexpensive approach.

We employ molecular methods to profile the diet of the little brown bat, Myotis lucifugus, and describe spatial and temporal changes in diet over their maternity season. We identified 61 prey species of insects and 5 species of arachnid. The largest proportion of prey (∼32%) were identified as species of the mass-emerging Ephemeroptera (mayfly) genus Caenis. Bats roosting in agricultural settings had lower dietary richness than those occupying a roost located on a forest fragment in a conservation area. We detected temporal fluctuations in diet over the maternity season. Dipteran (fly) species dominated the diet early in the season, replaced later by species of mayfly. Because our methodology provides species-level identification of prey, we were able to isolate environmental indicator species in the diet and draw conclusions about the location and type of their foraging habitat and the health of these aquatic systems. The species detected suggested that the bats use variable habitats; members of one agricultural roost foraged on insects originating in rivers or streams while those in another agricultural roost and the forest roost fed on insects from pond or lake environments. All source water for prey was of fair to good quality, though no species detected are intolerant of pollution thus the habitat cannot be classified as pristine. Our study outlines a model system to investigate the abiotic and biotic interactions between habitat factors through this simple food chain to the top predator.

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

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