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.