1. Introduction
To gain a greater understanding of trophic interactions within the
environment it is critical to have an in-depth understanding of species’
diet (Rytkönen et al., 2019). Our understanding of biodiversity and
ecosystem functions is significantly increased by investigating spatial
and temporal variation in trophic interactions within food webs
(Rytkönen et al., 2019; Yu et al., 2012). Estimating diet composition
can provide knowledge crucial for understanding the structure of
ecological communities and the flow of energy and nutrients (Kartzinel
et al., 2015; Nielsen, Clare, Hayden, Brett, & Kratina, 2017).
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).
Morphology-based methods of diet analysis can be time consuming and
biased towards identification of distinguishable and intact undigested
or semi-digested dietary items (Pompanon et al., 2012; Symondson, 2002).
Additionally, morphology-based methodologies often record ingested taxa
at a coarse taxonomic resolution, missing subtle differences in the taxa
consumed and resulting in a lack of ability to make fine scale
inferences relating to species’ ecology (Mata et al., 2016). The use of
molecular techniques such as high-throughput sequencing (HTS) in
conjunction with DNA barcoding, referred to as “metabarcoding” is
being frequently utilised to assess the diet of a range of organisms
(Cuff et al., 2021; Evens et al., 2020; Kartzinel & Pringle, 2020;
Thompson & Newmaster, 2014; Zalewski, Szymura, Kowalczyk, &
Brzeziński, 2021). This technique requires minimal a prioriknowledge on the dietary composition of the target organism (Alberdi et
al., 2017; Valentini et al., 2009) and a wide range of ingested taxa can
be identified to fine taxonomic levels (King, Read, Traugott, &
Symondson, 2008). For the application of metabarcoding to study bird
diet, faecal samples are highly suitable, as they contain residual
dietary DNA and can be collected easily to study species which may
otherwise have been difficult to locate or directly observe (Pompanon et
al., 2012; Taberlet, Bonin, Zinger, & Coissac, 2018).
Within metabarcoding studies, detection of plant species have
traditionally used sections of plant genes rbcL and matK ,
which, when used in conjunction, have the power to provide up to 75%
species-level discrimination (de Vere et al., 2012). Limitations on
amplicon length in HTS (maximum of 2 x 300 base pair reads on an
Illumina Miseq), as well as primers designed to amplify short barcodes
in order to detect DNA in degraded samples (Ando et al., 2013; Dunn et
al., 2018; Pompanon et al., 2012) has resulted in these gene regions
providing reduced taxonomic resolution in analysis of faecal samples
(Pompanon et al., 2012). The Internal Transcribed Spacer 2 (ITS2)
nuclear gene has been proposed as a suitable barcode for herbivorous
dietary analysis (Moorhouse-Gann et al., 2018). Universal primers
targeting the ITS2 region have been developed, producing amplicons
between 187-380 base pairs (Dunn et al., 2018; Moorhouse-Gann et al.,
2018). This has enabled the most variable region within the gene to be
targeted, with the amplicon length suitable for use within DNA
metabarcoding studies of the Turtle dove (Streptopelia turtur ,
Linnaeus), Pink pigeon (Nesoenas mayeri , Prévost) and Telfair’s
skink (Leiolopisma telfairii , Desjardin) (Dunn et al., 2018;
Moorhouse-Gann, 2017; Moorhouse-Gann et al., 2018). In a diet which
contains a wide range of invertebrate taxa, DNA barcodes from the COI
mitochondrial gene region have become the standard and are used in many
species-level identification studies (Kress, García-Robledo, Uriarte, &
Erickson, 2015). This is due to the extensive taxonomic coverage and
depth within the Canadian, European, UK and USA taxonomic COI reference
sequence databases (Porter & Hajibabaei, 2018). Such large databases
reduce the possibility of false taxonomic assignment and improve higher
taxonomic resolution (Andújar, Arribas, Yu, Vogler, & Emerson, 2018;
Porter & Hajibabaei, 2018; Somervuo et al., 2017).
Despite birds being one of the most studied animal classes, few studies
have used molecular techniques to improve understanding of their trophic
ecology (Alonso et al., 2014). In comparison with studies on mammals, in
particular bats, the application of faecal metabarcoding within
passerines studies is limited. However, this is an evolving field, with
research being undertaken on an expanding number of passerine species
(da Silva, Mata, Lopes, Lopes, & Beja, 2020; Shutt et al., 2020). For
woodland birds that forage high in the canopy and are often hidden from
view, obtaining accurate identification of dietary items through
observation alone is particularly challenging, and many studies simply
use foraging location to infer food availability and individual fitness
(Charman, Smith, Dodd, Gruar, & Dillon, 2012; Mackenzie, Hinsley, &
Harrison, 2014). Within temperate environments, passerine birds often
demonstrate a broad dietary range, incorporating a wide range of taxa
(Cholewa & Wesołowski, 2011; Shutt et al., 2020), however, dietary
variability within generalist woodland species is relatively poorly
understood. It has been proposed that the diet of generalists could vary
spatiotemporally, based upon resource availability and preferences of
the consumer. This variation within the diet may result in geographical
patterns in population density, breeding productivity, and local
adaptation to resource use (Shutt et al., 2020).
The Hawfinch (Coccothraustes coccothraustes , Linnaeus) is one of
many bird species closely associated with woodland habitats to have
shown major declines in the UK over a period of a few decades (Kirby et
al., 2018). Hawfinch breed across the Palearctic, with Britain its
western range limit (Kirby et al., 2015). While little is known about
Hawfinch ecology, studies show Hawfinches have declined across Britain,
with the species now too rare to have regular status assessments by
national annual monitoring schemes (Kirby et al., 2015). Instead,
population change is inferred from distribution data compiled from bird
atlas surveys (Balmer et al., 2013). These atlas data indicate a 76%
reduction in the number of 10km squares occupied between 1968 and 2011
(Kirby et al., 2015, 2018) and are further evidenced by Langstonet al. (2002), who estimated a 40% population decline between
the mid 1980’s to the late 1990’s. Hawfinch dietary studies are limited,
with all previous information obtained through personal observations.
Hawfinch are thought to be dietary specialists due to morphological
adaptations (a large, powerful bill) allowing them to feed on large tree
seeds such as cherry (Prunus sp., Linnaeus), hornbeam
(Carpinus betulus , Linnaeus), beech (Fagus sylvatica ,
Linnaeus) and Wych elm (Ulmus glabra , Hudson) (Mountford, 1957).
During the breeding season (typically from April to June), Hawfinch diet
has been observed to include sycamore (Acer pseudoplatanus ,
Linnaeus), hawthorn (Crataegus monogyna , Jacquin), blackthorn
(Prunus spinosa , Linnaeus), wild service tree (Sorbus
torminalis , Crantz), dogwood (Cornus alba , L’Héritier), larch
(Larix decidua , Miller) and oak (Quercus sp., Linnaeus)
(Bijlsma, 1998; Bryant, 2011; Mountford, 1957; Tomiałojć, 2012; von
Haartman, 1978). Hawfinch incorporate invertebrates into their diet,
particularly during the breeding season to provide sufficient nutrition
for egg production, consuming Lepidoptera, Coleoptera, Hemiptera,
Annelida, Gastropoda and Araneae (Mountford 1957).
In this study we used multi-marker DNA metabarcoding to construct a
high-resolution diet profile of a woodland passerine, aiming to
elucidate the taxonomic composition and richness, as well as showing
that HTS is a powerful approach for analysing species interactions. We
hypothesised that Hawfinch diet would show variation across: (a) study
sites and (b) sexes. This study also discusses the suitability of
multi-marker metabarcoding approaches for exploring omnivory.