4. Discussion
This study demonstrates that faecal metabarcoding can provide detailed
insights into the omnivorous diet of a scarce woodland bird. Previously
unrecorded dietary items were found, despite sampling completeness
recorded at 78.7% and 62.4% for plant and invertebrate genera,
respectively. This highlights the power of DNA metabarcoding to reveal
fine-scale taxonomic detail within dietary research. Our results suggest
that during the breeding season Hawfinch have a wide dietary niche
breadth for both plant and invertebrate species, supporting the
hypotheses that Hawfinch diet differs between populations geographically
and between sexes. As found in other recent studies of bird diet (da
Silva et al., 2020; Jedlicka, Vo, & Almeida, 2017; Mitchell, Horsburgh,
Dawson, Maher, & Arnold, 2021; Shutt et al., 2020; Sullins et al.,
2018), the use of molecular techniques has revealed an unsuspected wide
range of dietary items and provides the first comprehensive analysis of
omnivory within Hawfinch diet. Although this study documents over 100
taxa consumed, previously recorded common food resources such as beech,
cherry and Lepidoptera (Mountford, 1957; Newton, 1967) clearly still
play a dominant role in diet.
Diet is likely to reflect a mixture of prey availability, abundance and
preference, with Hawfinch consumed a broader range of invertebrate taxa
in comparison to plant taxa, reflecting what may be naturally available
within the environment. Food preference, rather than availability or
abundance has been found to contribute towards dietary shifting from
invertebrates to fruit, potentially enabling birds to seasonally balance
nutrient and energy intake (Marshall, Dick, & Guglielmo, 2016).
Invertebrates are typically a high protein to calorie ratio food
resource, with certain species providing specific nutritional value, for
example spiders provide high levels of the amino acid cysteine (Marshall
et al., 2016; Ramsay & Houston, 2003). Hawfinch egg laying begins
around mid April (Kirby, Stanbury, Bellamy, & Lewis, 2019), and the
presence of invertebrates within the diet during the breeding season has
been recorded in other passerine dietary studies conducted over similar
temporal periods (Newton, 1967; Shutt et al., 2020). This may help to
provide specific nutrients beneficial to breeding physiology, such as
egg production in females, as well as providing high protein food for
chicks (Marshall et al., 2016). These dietary patterns are commonly
observed in other passerine species such as chaffinch (Fringilla
coelebs , Linnaeus) (Holland, Hutchison, Smith, & Aebischer, 2006).
Many taxa present in the diet were rare, as has been documented in
previous faecal metabarcoding studies on generalist passerines (Shutt et
al., 2020; Sottas et al., 2020). However, our findings on the more
frequent components of Hawfinch diet agree closely with previous
observations of this species (Bijlsma, 1998; Mountford, 1957). Previous
studies found seeds of hornbeam, cherry and maple were important
throughout the year (Bijlsma, 1998; Mountford, 1957), with buds of ash,
maple and beech as well as Lepidoptera becoming important food resources
during spring and summer (Bijlsma, 1998). The importance of beech as a
food resource was confirmed in this study, being the most prevalent
plant taxon (detected in 38.5% of samples). It is well understood that
birds must balance food handling times with net energy intake, and a
resource is deemed more profitable if it has a higher energy reward per
unit handling time (Molokwu, Nilsson, & Olsson, 2011). It is known that
Hawfinch commonly feed on beech nuts during autumn and winter months
(Mountford, 1957) due to the moderately high fat and carbohydrate levels
of the beechnuts compensating for energy losses due to cold weather
during winter (Renner et al., 2013). The onset of the breeding season
can drive changes in feeding preferences as nutritional needs become
higher (Lima, 2009). As the sampling in this study began during the
pre-breeding season and continued to the end of summer, Hawfinch may
have been gaining a high energy reward from feeding on any remaining
available beech nuts, but also obtaining similar nutritional benefits
from the increased availability of beech buds in the spring. Beech buds
have been shown to contain >15% fat (Lebl, Kürbisch,
Bieber, & Ruf, 2010), and this may be an important energetic
requirement for Hawfinch to boost condition before and during the
breeding season.
Lepidoptera, Coleoptera, Hemiptera, Annelida, Gastropoda and Araneae
have all been observed as prey at the order level (Mountford, 1957) and
all (excluding Annelida) were detected within this study. The high
prevalence of winter moth within Hawfinch diet is not unexpected, as
this larva is an important food resource for other woodland passerine
species, such as nestling tits (Perrins, 1991). The earliest date winter
moth was detected within the diet was mid-April, with prevalence
increasing throughout April and May. Kirby et al. (2019) found
Hawfinch egg laying commonly started during the third week of April and
peaked in mid May. This temporal increase in the number of nests
coincides with the increased incidence of winter moth within the diet,
and most likely corresponds to a change in the availability of winter
moth larvae. This finding raises the possibility that Hawfinch may be
using availability of winter moth as a breeding cue, as has been
suggested in other passerine species (Shutt et al., 2020). In contrast,
the high prevalence of tree slug within the diet was unexpected, as it
was previously thought only snails were consumed (Mountford, 1957). This
may be explained by the availability of algae and lichens within
woodland, which are the main components of tree slug diet (Kappes,
2006). During wet weather, tree slugs feed on algae growing on tree
trunks, but remain under the bark of dead timber during unsuitable
weather (Kappes, 2006). Thus, tree slugs may be taken during periods of
high rainfall when foraging efficiency for defoliating Lepidoptera is
reduced (Morganti, Rubolini, Caprioli, Saino, & Ambrosini, 2017;
Ortega-Jimenez & Dudley, 2012).
The metabarcoding results revealed oak to be widely prevalent within the
diet, something not reported in Mountford’s species monograph (1957).
Past research undertaken on Hawfinch diet during winter (months
unspecified) and during the breeding season (April to August), broadly
fitted with the sampling period of this study, and it is surprising
therefore, that oak was not observed as a food resource. Hawfinch
dietary studies have focused on direct observations of feeding, and
while this method was widely used at the time of Mountford (1957),
direct observation has known limitations, such as observer bias and
error, as well as results being influenced by data recorded from
habitats in which a species is most observable (Matthews, Ridley,
Kaplin, & Grueter, 2020). Furthermore, whether oak species have
specific dietary importance is dependent, in part, on the plant tissue
type consumed and the nutritional value. While this was not investigated
within this study, it is encouraged for further research. Having this
nutritional information may inform how Hawfinch food preferences change
throughout the breeding season and whether there is specific temporal
value available across taxa.
Plant taxa detected within Hawfinch diet varied between geographical
regions. This spatial variation is consistent with similar metabarcoding
studies of birds and insectivorous bats (Clare, Symondson, Broders, et
al., 2014; McClenaghan, Nol, & Kerr, 2019; Shutt et al., 2020). This
could indicate local dietary specialisation, however it is more probable
that Hawfinch are flexible in dietary choice and these patterns arise
from changing availability of food resources.
This may be a result of variation
in tree species abundances within each study region. This variation in
resource use can be seen in the nMDS plot (Figure 3). While there is a
degree of overlap between all regions, the Wye Valley and north Cardiff
sampling regions are situated closer together, indicating dietary taxa
detected from Hawfinch sampled within these regions show higher levels
of similarity than dietary taxa from Hawfinch sampled in north Wales.
The sexual differences in invertebrate dietary composition is likely due
to behavioural, rather than morphometrical differences between males and
females. Hawfinch are judged to have minimal sexual dimorphism, however
biometric measurements such as bill length/depth were not recorded for
this study and therefore future work should incorporate this in order to
improve understanding of possible intra-specific variation. This is one
of only two studies which have used DNA metabarcoding to detect
monomorphic passerine species exhibiting sexual dietary differences (see
da Silva et al., 2020). It has been suggested in some bird species that
females have reduced foraging ranges in order to be closer to offspring,
and as a result, may feed on more abundant or predictable items, even if
these items are less nutritious (da Silva et al., 2020; Sunde, Bølstad,
& Møller, 2003). Freeman (2014) found vertical segregation between the
sexes of two New Guinean whistlers (Pachycephala sp., Schlegel),
with little sexual dimorphism, attributed to territory defence and
intersexual food resource differentiation.
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. Ecologically, it is known that Hawfinch feed
primarily within the canopy (Mountford, 1957), and will only come to the
ground to feed on fallen seed in late winter. This suggests that most
invertebrate taxa were obtained from the vegetation or bark within the
tree canopy, resulting in possible accidental ingestion of plant taxa
when gleaning prey items from trees. Due to metabarcoding methods being
unable to determine which plant tissue is being consumed, in conjunction
with Hawfinch also feeding on the same plant taxa as their prey at
similar times of the year, differentiating what is “true” secondary
predation is extremely challenging.
In conclusion, this study has provided the first molecular insight into
the generalist diet of Hawfinch, at a finer resolution than previous
work. We demonstrate that the diet of Hawfinch, as predicted, varies
both spatially and between sexes. This dietary variation suggests
Hawfinch can respond to changing resource availability by showing
dietary plasticity. In order to maximise the power of dietary analysis,
increasing the temporal scale of sampling would be beneficial for future
work, as would measuring invertebrate abundance to compare with diet
samples. The diet of nestling Hawfinch could be described and geographic
variability of diet assessed in order to quantify the apparent
significance of Lepidoptera for Hawfinch and other woodland birds.
Furthermore, future research could involve faecal metabarcoding of
multiple species from Hawfinch study sites. A large number of
co-existing predator species utilise Lepidoptera and other invertebrates
during the breeding season including great tit (Parus major ,
Linnaeus) (Ramakers, Gienapp, & Visser, 2019), marsh tit (Poecile
palustris , Linnaeus) (Wesołowski & Neubauer, 2017), blue tit
(Cyanistes caeruleus , Linnaeus) (Shutt, Burgess, & Phillimore,
2019) and both great spotted (Dendrocopos major , Linnaeus) and
lesser spotted (Dendrocopos minor , Linnaeus) (Charman et al.,
2012; Smith & Smith, 2013) woodpecker species. Faecal metabarcoding of
adults and nestlings from a range of representative woodland bird
species would help quantify the most important prey species, which could
in turn inform conservation management to maximise their abundance.
The results of this study were only possible due to the high taxonomic
resolution available through metabarcoding methods. As metabarcoding is
becoming more prevalent within ecological research, it becomes
increasingly important to understand how taxonomic resolution can impact
ecological studies, although species-level identification may not always
be necessary, depending on hypotheses studied (Brown et al., 2014;
Renaud, Baudry, & Bessa-Gomes, 2020). The study presented is an example
of how the utilisation of DNA metabarcoding can increase ecological
understanding and improve insights into fine scale ecological patterns.
As this study was focused primarily on elucidating the diet of Hawfinch,
priority was given to maximise the number of faecal samples within the
HTS methodology. This however, limited the number of technical
replicates such as subsampling individual faecal samples throughout the
extraction, amplification and sequencing process (Alberdi, Aizpurua,
Gilbert, & Bohmann, 2018). This resulted in the inability to evaluate
and amend the stochasticity of the results (Alberdi et al., 2018; Zinger
et al., 2019). Artificial communities of known concentrations, or “mock
communities” (Forin-Wiart et al., 2018) were also not included for the
aforementioned reasons. This resulted in the inability to analyse the
sensitivity of the sequencing pipeline among dietary taxa. Compiling a
barcode library specific to the study would also be advantageous. While
work by (Jones et al., 2021) has resulted in increasing the number of
ITS2 sequences within the DNA barcode database by 1105 species, the
utilisation of a custom reference database may allow all future research
to be conducted at the species level.