Molecular scatology and high-throughput sequencing reveal predominately herbivorous insects in the diets of adult and nestling Western Bluebirds (<i>Sialia mexicana</i>) in California vineyards

Jedlicka, Julie A.; Vo, Anh-Thu E.; Almeida, Rodrigo P. P.

doi:10.1642/auk-16-103.1

Cited by 67 publications

(59 citation statements)

References 41 publications

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“…From our repeat samples we were able to estimate technical repeatability and several measures of biological repeatability (Appendix ). Repeatability of MOTU presence/absence was rather low, consistent with low repeatability estimates found by another study that subsampled avian faecal samples (Jedlicka et al, 2016). An implication is that if the focus of an avian faecal metabarcoding study is on the detection of the presence/absence of a specific taxon, then multiple repeat DNA extractions, amplifications and metabarcoding runs are advisable.…”

Section: Discussionsupporting

confidence: 86%

“…In this study we have demonstrated that faecal metabarcoding can provide a robust and powerful method for assessing passerine diet, allowing greater sample sizes and taxonomic resolution than direct assessment (Betts, 1955). Inclusion of positive and negative controls and repeat samples are part of the standard laboratory practice (Alberdi et al, 2018), although few previous metabarcoding studies have included any of these (but see De Barba et al, 2014; Jedlicka et al, 2016), and have proven invaluable in informing this work. Our protocol yielded fourteen MOTUs for the positive control taxon, suggesting that the 2% divergence rule of thumb used in early barcoding studies to group conspecific COI barcode sequences in Metazoa (Hebert, Cywinska, & Ball, 2003 and http://www.barcodinglife.com) is likely to produce spurious taxa, potentially misleading naïve analyses and underlining the necessity for subsequent quality control steps.…”

Section: Discussionmentioning

confidence: 99%

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Gradients in richness and turnover of a forest passerine's diet prior to breeding: A mixed model approach applied to faecal metabarcoding data

et al. 2020

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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.

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Section: Discussionsupporting

confidence: 86%

Section: Discussionmentioning

confidence: 99%

Gradients in richness and turnover of a forest passerine's diet prior to breeding: A mixed model approach applied to faecal metabarcoding data

et al. 2020

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“…Pooling of samples before DNA extraction has been used to reduce processing time and costs by integrating variability among multiple samples or when individual samples were difficult to separate (Burgar et al., ; Clare et al., 2014a, 2014b; Jedlicka, Vo, & Almeida, ), but our results suggest that this strategy may lead to substantial errors in the estimation of dietary descriptors. We found that pools strongly underestimated diet diversity and the frequency of occurrence of prey items, irrespective of sequencing depth, yielding results comparable to those obtained by analysing a single pellet.…”

Section: Discussionmentioning

confidence: 75%

How much is enough? Effects of technical and biological replication on metabarcoding dietary analysis

et al. 2018

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DNA metabarcoding is increasingly used in dietary studies to estimate diversity, composition and frequency of occurrence of prey items. However, few studies have assessed how technical and biological replication affect the accuracy of diet estimates. This study addresses these issues using the European free-tailed bat Tadarida teniotis, involving high-throughput sequencing of a small fragment of the COI gene in 15 separate faecal pellets and a 15-pellet pool per each of 20 bats. We investigated how diet descriptors were affected by variability among (a) individuals, (b) pellets of each individual and (c) PCRs of each pellet. In addition, we investigated the impact of (d) analysing separate pellets vs. pellet pools. We found that diet diversity estimates increased steadily with the number of pellets analysed per individual, with seven pellets required to detect ~80% of prey species. Most variation in diet composition was associated with differences among individual bats, followed by pellets per individual and PCRs per pellet. The accuracy of frequency of occurrence estimates increased with the number of pellets analysed per bat, with the highest error rates recorded for prey consumed infrequently by many individuals. Pools provided poor estimates of diet diversity and frequency of occurrence, which were comparable to analysing a single pellet per individual, and consistently missed the less common prey items. Overall, our results stress that maximizing biological replication is critical in dietary metabarcoding studies and emphasize that analysing several samples per individual rather than pooled samples produce more accurate results.

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“…This single marker approach has been widely used in many studies (Gordon et al, ; McClenaghan, Nol, & Kerr, ; Moran, Prosser, & Moran, ), but it may produce significant biases due to differential primer affinity for different taxa. For instance, although ZBJ is often used as a ‘universal’ marker for arthropods (Crisol‐Martínez, Moreno‐Moyano, Wormington, Brown, & Stanley, ; Jedlicka, Vo, & Almeida, ; Trevelline, Latta, Marshall, Nuttle, & Porter, ; Trevelline et al, ), it may have strong positive or negative bias depending on the taxa (Clarke, Soubrier, Weyrich, & Cooper, ; Piñol, Mir, Gomez‐Polo, & Agustí, ). The challenge is even worse in the case of omnivorous diets, because the variety of taxonomic clades consumed cannot be analysed using a single marker (De Barba et al, ; Taberlet et al, ).…”

Section: Introductionmentioning

confidence: 99%

Advancing the integration of multi‐marker metabarcoding data in dietary analysis of trophic generalists

Silva

Mata

Lopes

et al. 2019

Molecular Ecology Resources

119

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The application of DNA metabarcoding to dietary analysis of trophic generalists requires using multiple markers in order to overcome problems of primer specificity and bias. However, limited attention has been given to the integration of information from multiple markers, particularly when they partly overlap in the taxa amplified, and vary in taxonomic resolution and biases. Here, we test the use of a mix of universal and specific markers, provide criteria to integrate multi‐marker metabarcoding data and a python script to implement such criteria and produce a single list of taxa ingested per sample. We then compare the results of dietary analysis based on morphological methods, single markers, and the proposed combination of multiple markers. The study was based on the analysis of 115 faeces from a small passerine, the Black Wheatears (Oenanthe leucura). Morphological analysis detected far fewer plant taxa (12) than either a universal 18S marker (57) or the plant trnL marker (124). This may partly reflect the detection of secondary ingestion by molecular methods. Morphological identification also detected far fewer taxa (23) than when using 18S (91) or the arthropod markers IN16STK (244) and ZBJ (231), though each method missed or underestimated some prey items. Integration of multi‐marker data provided far more detailed dietary information than any single marker and estimated higher frequencies of occurrence of all taxa. Overall, our results show the value of integrating data from multiple, taxonomically overlapping markers in an example dietary data set.

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Molecular scatology and high-throughput sequencing reveal predominately herbivorous insects in the diets of adult and nestling Western Bluebirds (Sialia mexicana) in California vineyards

Cited by 67 publications

References 41 publications

Gradients in richness and turnover of a forest passerine's diet prior to breeding: A mixed model approach applied to faecal metabarcoding data

Gradients in richness and turnover of a forest passerine's diet prior to breeding: A mixed model approach applied to faecal metabarcoding data

How much is enough? Effects of technical and biological replication on metabarcoding dietary analysis

Advancing the integration of multi‐marker metabarcoding data in dietary analysis of trophic generalists

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