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

Abstract

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.

High‐resolution multi‐marker DNA metabarcoding reveals sexual dietary differentiation in a bird with minor dimorphism

“…The current study furthers the previous work of Silva et al. (2019a), using their methodology to explore differences in diet between males and females of the same species. This way, we selected 93 droppings collected upon the first capture of 110 adult birds, thereby avoiding biases that might result from including data from birds captured more than once (pseudoreplication), as well as eventual confounding effects of including a small number of 1st calendar year birds.…”

“…From the 93 droppings thus collected, 62 from males and 31 from females, three were obtained from birds that defecated inside the traps but were not captured. Droppings were stored in 2‐ml tubes with 98% ethanol at 4°C until laboratory analysis (da Silva et al., 2019a).…”

“…The current study examines the diet of black wheatears based on a subset of data generated by da Silva et al. (2019a), who evaluated the limitations and biases associated with single marker metabarcoding in the dietary analysis of trophic generalists and proposed a multi‐marker approach to provide a more comprehensive description of species' diets. The current study furthers the previous work of Silva et al.…”

“…However, previous studies have focused on specialists with a relatively narrow feeding niche, while this methodology remains underexplored in testing sexual dietary in more generalist species such as many omnivorous passerines. Dietary generalists are more challenging to study using metabarcoding because they require a combination of markers to fully encompass the full spectrum of food resources used (da Silva et al., 2019a).…”

“…Here, we aim to show the power of multi‐marker metabarcoding to investigate differences in diet between sexes, by focusing on a generalist passerine judged to have minimal sexual dimorphism, the black wheatear ( Oenanthe leucura ). To address this general goal, the study first documents difference in morphology (bill and body features) between sexes and then use a previously developed approach for integrating metabarcoding dietary data across multiple markers (da Silva et al., 2019a) to describe the diets of both sexes. Using this data, we then tested the hypothesis that diet varies between sexes in terms of (a) diet diversity and (b) frequency of occurrence of the main food items and that (c) sexual dietary differentiation can only be detected at the high taxonomic resolution provided by metabarcoding.…”