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

Abstract

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.

Multiple species‐specific molecular markers using nanofluidic array as a tool to detect prey DNA from carnivore scats

“…Our diagnostic method with species‐specific markers adds to the more frequent studies using DNA from carnivore scats to identify prey (Hacker et al., 2021 ; Quéméré et al., 2021 ; Roffler et al., 2021 ; Shi et al., 2021 ; Smith et al., 2018 ; Xiong et al., 2017 ). These studies primarily used DNA metabarcoding, which produces a vast amount of valuable information but sometimes also needs consideration of potential bias sources in key steps in the data handling process, lack of reference databases of barcodes for many prey species, but also intensive laboratory procedures and considerable bioinformatics training (Hacker et al., 2021 ; Tercel et al., 2021 ; Zinger et al., 2019 ). The relatively simple molecular method developed here, applying the nanofluidic array technology to detect prey DNA, represents a promising and valid alternative to other methods.…”

“…The cost of using this technology in the year 2021 was ca 20 €/sample including DNA extraction. This aspect, together with the high sensitivity of detection when amplifying very short DNA fragment lengths (Broquet et al., 2007 ), potentially makes nanofluidic array technology a good contender to, for example, metabarcoding with NGS and conventional sequencing (Tercel et al., 2021 ) for detecting prey species from large sample sizes for ecological studies.…”

Using multi-marker DNA metabarcoding to reveal the diet of a scarce woodland bird

“…It is also important to acknowledge the possibility of secondary consumption via lepidopteran taxa within the diet (Tercel et al, 2021), which may result in indirect species associations. Secondary consumption may result in falsely inflated detection of plant taxa through co-amplification of plant DNA within the guts of lepidopteran taxa consumed by Hawfinch.…”

“…Furthermore, characterising consumers' resources can provide information about niche specialisation at both inter-and intra-specific scales (Kratina, LeCraw, Ingram, & Anholt, 2012). The accurate identification of components within an omnivorous diet is however, still considered challenging (da Silva et al, 2019;De Barba et al, 2014;Tercel, Symondson, & Cuff, 2021). Due to the costly, laborious and taxonomically demanding nature of exploring omnivorous diet, studies attempting to elucidate all dietary aspects are rare (Pompanon et al, 2012;Tercel et al, 2021).…”

Handling of targeted amplicon sequencing data focusing on index hopping and demultiplexing using a nested metabarcoding approach in ecology

“…There is a large range of possible TAS applications including variant detection and tumour profiling in cancer research, the detection of somatic mutations or those associated with susceptibility to disease, new findings in the field of phylogeny and taxonomy studies or the discovery of useful genes for applications in molecular breeding 2 , 3 , 9 , 10 . In the field of environmental sciences, TAS is becoming increasingly important, as it facilitates the assessment of the taxonomic composition of environmental samples with the help of metabarcoding approaches such as environmental DNA (eDNA) based biomonitoring or food web studies 11 – 13 .…”