DNA metabarcoding successfully quantifies relative abundances of arthropod taxa in songbird diets: a validation study using camera-recorded diets

Abstract

Ecological research is often hampered by the inability to quantify animal diets. Large-scale changes in arthropod diversity, abundance and phenologies urge the need to understand the consequences for trophic interactions. Diet composition of insectivorous predators can be tracked through DNA metabarcoding of faecal samples, but to validate the quantitative accuracy of metabarcoding, validation using free-living animals for which their diet can be approximated, is needed.This validation study assesses the use of DNA metabarcoding in quantifying diets of an insectivorous songbird. Using camera footage, we documented food items delivered to nestling Pied Flycatchers Ficedula hypoleuca, and subsequently sequenced the Cytochrome Oxidase subunit I (COI) in their faeces. Our special interest was to retrieve the relative contribution of arthropod taxa with a PCR-based protocol.Assessment of taxonomic coverage of the invertebrate COI primers LCO1490 and HCO1777, previously applied in insectivorous songbirds, demonstrated that COI barcodes were predominantly assigned to arthropod taxa, however, substantial amounts of reads (2–60%) were assigned to flycatchers. Modified primers reduced vertebrate reads to 0.03%, and yielded more spider DNA without significant changing the recovery of other arthropod taxa.To explore digestive biases, contents of stomachs and lower intestines were compared in eight adult male flycatchers, victims of competitors for nest boxes. Similarity in arthropod community composition between stomach and intestines confirmed limited digestive bias.With nest box cameras in 2013, 2015 and 2016, size-adjusted counts of prey items fed to nestlings were recorded, to approximate provided biomass of arthropod orders and families which allowed comparison with abundance of COI barcode reads in nestling faeces. The relative abundances of size-adjusted prey counts and COI reads correlated at R = 0.85 (CI:0.68-0.94) at order level and at R=0.75 (CI:0.67-0.82) at family level. Each common order and all common taxa within Lepidoptera, Diptera and Coleoptera were retrieved in similar proportions, while the taxonomic resolution was higher in barcodes than in camera records.This DNA metabarcoding validation demonstrates that quantitative arthropod diet analyses is possible in songbirds. We discuss the ecological application of our approach in approximating the arthropod diets in insectivorous birds.

Metabarcoding analysis of marine zooplankton confirms the ecological role of a sheltered bight along an exposed continental shelf

“…We estimated species richness and relative abundance, with the latter a key component in diversity estimates and ecosystem health assessments (Nichols et al, 2018). Lower confidence in relative abundance estimates (measured as read counts in metabarcoding) has been expressed by Elbrecht and Leese (2015), Piñol et al (2015) and Jusino et al (2019), although these estimates have been successfully used for nematode communities (Schenk et al, 2019), arthropod taxa in songbird diets (Verkuil et al, 2020) and zooplankton research (Macher et al, 2020;Pitz et al, 2020). In an experimental approach, Govender et al (2022) found a strong relationship between known zooplankton species ratios (including abundant and rare species) and read counts derived from metabarcoding using COI minibarcode primer sets.…”

Extracting abundance information from DNA-based data

“…For instance, Schenk et al (2019) have reported that primers for the 28S D3-D5 and 18S V4 regions return nematode read frequencies that accurately recover relative species abundances. Similarly, Verkuil et al (2020) have reported that modified COI primers can recover the relative biomasses of insect orders from Pied Flycatcher faeces. This method achieves across-species quantification but not within-species quantification.…”

“…In some cases, the target taxon is nearly uniform in body size and DNA-extraction efficiency, and it can be possible to design PCR primers that bind similarly across species. For instance, Schenk et al (2019) have reported that primers for the 28S D3-D5 and 18S V4 regions return nematode read frequencies that accurately recover relative species abundances, Verkuil et al (2020) have reported that modified COI primers can recover the relative biomasses of insect orders from Pied Flycatcher faeces, and Ershova et al (2021) have reported that increasing COI primer degeneracy (Leray-XT) can recover relative biomasses of marine zooplankton. This method achieves across-species quantification, albeit with error, but not within-species quantification.…”

Using eDNA metabarcoding to understand the effect of fire on the diet of small mammals in a woodland ecosystem

“…The presence of misassigned tags to the extraction controls and one of the PCR controls was very low compared with the overall number of sequence reads obtained, and it is not considered to have affected the results of the study. In eDNA metabarcoding results, the number of sequence reads obtained does not represent the abundance of particular ZOTUs present in the sample (Deagle et al, 2018; Verkuil et al, 2020) thus all data were converted to presence/absence before further analysis and ZOTUs that were present in the extraction controls were removed from the dataset.…”