The role of natural habitats in agricultural systems for bird conservation: the case of the threatened Lesser Grey Shrike

Birds of agricultural systems are one of the most threatened groups of birds in Europe mainly due to their sharp population decline in recent decades. Habitat intensification resulting from more productive agricultural practices has been proposed as a major cause for these declines. However, especially in some regions such as Eastern European and Mediterranean countries, little is known about the ultimate factors linked to habitat intensification that drive population declines for different species. The Lesser Grey Shrike is a good study species for a better understanding of such processes since it is closely related to agricultural habitats in Europe and has suffered a strong decline in range and population size across the western half of the continent. In this study, we explored variations in breeding parameters of this shrike related to habitat composition and food supply at the territory level. We found that fledgling success of early breeders was related to the presence of natural (shrub lands) and semi-natural (fallows) habitats in the predominantly agricultural matrix that dominated breeding territories. Their influence on fledgling success appeared to be mediated by a higher arthropod availability on these habitats. Indeed, Lesser Grey Shrike showed a strong preference for these habitats as hunting locations. Our results highlight the importance of natural habitats in intensified agricultural land mosaics for the conservation of bird species. We suggest that management plans should pay special attention to the availability of habitats which serve as high quality food reservoirs and can potentially contribute to enhance the species population viability in an area. Finally, we discuss the possible link between agricultural intensification and Lesser Grey Shrike population declines in Western Europe.     

Breeding Barn Swallows Hirundo rustica on organic and conventional arable farms in the Netherlands

Populations of farmland birds are under pressure as a result of agricultural intensification. It has been proposed that less intensive approaches to farming, such as organic farming methods, may halt these population declines. In addition, organic farmers may have a more positive attitude towards nature and the environment which can possibly also have positive effects on the populations of breeding birds. We have compared the attitude of conventional and organic farmers towards the presence of Barn Swallows Hirundo rustica and the abundance of breeding Barn Swallows on organic and conventional arable farms in the Netherlands. We found that the abundance of breeding Barn Swallows did not differ between these two types of farms an that both organic and conventional farmers had a positive attitude towards the presence of Barn Swallows on their farms. Our results show that organic farming does not attract more Barn Swallows. However, agricultural intensification could have resulted in lower breeding success and, consequently, population declines, although there may be other contributory factors as well.     

The significance of seed food in chick development re‐evaluated by tracking day‐to‐day dietary variation in the nestlings of a granivorous passerine

The dietary adjustment of nestlings of granivorous birds to a seed diet and the different morphological characteristics of ingested food have rarely been examined in natural conditions. It has been suggested that the provision of cereal grains to nestlings of some seed‐eating bird species in modern agroecosytems is the result of poor food conditions after agricultural intensification. We analysed the abundance of invertebrate prey in the main foraging habitat of parent birds, daily changes (from hatching to fledging) in the efficiency of cereal seed digestion, and the dietary characteristics, diet composition and prey type delivered to nestlings of the Yellowhammer Emberiza citrinella. Analysis of faecal sacs from nests located in breeding habitat with an abundant invertebrate fauna revealed no relationship between the proportion of cereal seeds in the diet of nestlings and the food supply in the main foraging sites of the parents. Neonate nestlings (1 day old) exclusively received weakly chitinized invertebrate prey (arachnids and flies), but from the second day of life the nestlings were fed a variety of highly chitinized invertebrate prey, the percentage biomass of which did not change for the remainder of the nesting period. Cereal grains started to be delivered to 3‐day‐old nestlings and were already efficiently digested, and the percentage biomass of this food type increased progressively with nestling age. We suggest that the provisioning of cereal grains to nestlings is not forced by external factors, such as modern agricultural intensification; rather, it is an intentional behaviour of parent birds aimed at achieving physiological adjustment to seed food in the early stages of ontogeny.     

Feeding behaviour and diet composition of Marsh Harriers Circus aeruginosus in agricultural landscapes

Capsule Diet composition and feeding behaviour of breeding birds differed between areas of high and low agricultural intensification.

Aims To analyze diet composition and feeding behaviour of Marsh Harriers Circus aeruginosus in agricultural landscapes with different degrees of agricultural intensification.

Methods Diet composition and feeding behaviour was assessed by focal observations at nests during breeding seasons in 2001–03 in two agricultural areas within the eastern Ebro Basin (on the northern Iberian Peninsula) which differed in their degree of agricultural intensification: a traditional non-irrigated and non-intensively managed farmland area; and an irrigated and intensively managed farmland area.

Results Marsh Harriers in the intensively managed area took higher percentages of small mammals than harriers in the traditional area managed at low-intensity, where they also had a more diverse diet. Male delivery rates increased throughout the breeding season in the intensively managed area. On the contrary, no significant trend in delivery rates was observed in the traditional area, where harriers turned to larger prey late in the season.

Conclusion Marsh Harriers probably took advantage of different prey types in intensively managed and traditional farmland according to prey availability. Differences in diet composition seem to be related to different foraging strategies adopted by harriers in traditional and intensively managed farmlands. In the traditionally managed farmlands, harriers responded to increases in the energy requirements of the brood over the course of the breeding season by taking larger prey, whereas in the intensively managed farmlands harriers increased feeding rates.     

Adverse effects of agricultural intensification and climate change on breeding habitat quality of Black‐tailed Godwits Limosa l. limosa in the Netherlands

Agricultural intensification is one of the main drivers of farmland bird declines, but effects on birds may be confounded with those of climate change. Here we examine the effects of intensification and climate change on a grassland breeding wader, the Black‐tailed Godwit Limosa l. limosa, in the Netherlands. Population decline has been linked to poor chick survival which, in turn, has been linked to available foraging habitat. Foraging habitat of the nidifugous chicks consists of uncut grasslands that provide cover and arthropod prey. Conservation measures such as agri‐environment schemes aim to increase the availability of chick foraging habitat but have not yet been successful in halting the decline. Field observations show that since the early 1980s, farmers advanced their first seasonal mowing or grazing date by 15 days, whereas Godwits did not advance their hatching date. Ringing data indicate that between 1945 and 1975 hatching dates advanced by about 2 weeks in parallel with the advancement of median mowing dates. Surprisingly, temperature sums at median mowing and hatching dates suggest that while the agricultural advancement before 1980 was largely due to agricultural intensification, after 1980 it was largely due to climate change. Examining arthropod abundance in a range of differently managed grasslands revealed that chick food abundance was little affected but that food accessibility in intensively used tall swards may be problematic for chicks. Our results suggest that, compared with 25 years ago, nowadays (1) a much higher proportion of clutches and chicks are exposed to agricultural activities, (2) there is little foraging habitat left when chicks hatch and (3) because of climate change, the vegetation in the remaining foraging habitat is taller and denser and therefore of lower quality. This indicates that for agri‐environment schemes to make a difference, they should not only be implemented in a larger percentage of the breeding area than the current maxima of 20–30% but they should also include measures that create more open, accessible swards.     

Nestling diet variation in an insular Mediterranean population of blue tits Parus caeruleus: effects of years,territories and individuals

Mediterranean evergreen forests of Corsica are characterized by relatively high species diversity of arthropods with low population densities. Food is never superabundant for Corsican blue tits Parus caeruleus. This study focused on the composition of the food of blue tit nestlings and especially on two main components, caterpillars and spiders. The nestling diet was studied for two years using 8-mm cameras that automatically took photographs of adult birds with food. The diet was composed of c. 50% caterpillars and c. 30% spiders. There were between-year and between-individual differences in these proportions. In both years of the study the proportion of caterpillars declined during the course of the breeding season. Individual and time effects on prey sizes were also observed. Pairs and individuals were fairly constant in the proportions of prey over the feeding period. Different food items were not brought in runs. These findings suggest that strong food limitation exists on Corsica, which can considerably influence life-history traits of the blue tit.     

The effect of sward height and drainage on Common Starlings Sturnus vulgaris and Northern Lapwings Vanellus vanellus foraging in grassland habitats

Agricultural change is often cited as a causal factor in the decline of the UK's farmland birds because bird declines have mirrored changes in agricultural practices. Although much is known about the mechanisms driving population declines on arable systems, mechanisms in grassland systems are relatively poorly studied, despite receiving a similar degree of intensification. Agricultural intensification may affect bird declines by reducing food abundance or accessibility, forager mobility or predation risk. Here we examine experimentally the effects of sward height on the foraging behaviour of adult Common Starlings Sturnus vulgaris , and the effects of sward height and drainage on the behaviour of Northern Lapwing Vanellus vanellus chicks. Both species are declining across the UK and both forage in farmed grassland habitats, but they differ in their foraging methods because Starlings probe for soil invertebrates whereas Lapwing chicks glean prey from surfaces. Overall, after controlling for prey abundance, short swards were found to be more productive for both species. Prey capture rate within foraging bouts did not differ with sward height for Starlings, but Starlings spent more time foraging on short swards and captured 33.2% more prey. Starlings walked more steps on short swards. Lapwing chick foraging rates declined as sward height increased. Soil moisture was not found to be a predictor of Lapwing chick foraging rates within the observed range. Our results suggest that short swards are a more profitable foraging habitat for soil and surface invertebrate feeders. Short swards may facilitate surface prey detection, improve forager mobility and increase foraging time by altering vigilance patterns. Provision of short swards in areas where these are lacking could be simple method of improving foraging habitats for grassland birds.     

From Puffins to Plankton: A DNA-Based Analysis of a Seabird Food Chain in the Northern Gulf of Maine

The predator-prey interactions within food chains are used to both characterize and understand ecosystems. Conventional methods of constructing food chains from visual identification of prey in predator diet can suffer from poor taxonomic resolution, misidentification, and bias against small or completely digestible prey. Next-generation sequencing (NGS) technology has become a powerful tool for diet reconstruction through barcoding of DNA in stomach content or fecal samples. Here we use multi-locus (16S and CO1) next-generation sequencing of DNA barcodes on the feces of Atlantic puffin (Fratercula arctica) chicks (n=65) and adults (n=64) and the stomach contents of their main prey, Atlantic herring (Clupea harengus, n=44) to investigate a previously studied food chain. We compared conventional and molecular-derived chick diet, tested the similarity between the diets of puffin adults and chicks, and determined whether herring prey can be detected in puffin diet samples. There was high variability in the coverage of prey groups between 16S and CO1 markers. We identified more unique prey with our 16S compared to CO1 barcoding markers (51 and 39 taxa respectively) with only 12 taxa identified by both genes. We found no significant difference between the 16S-identified diets of puffin adults (n=17) and chicks (n=41). Our molecular method is more taxonomically resolved and detected chick prey at higher frequencies than conventional field observations. Many likely planktonic prey of herring were detected in feces from puffin adults and chicks, highlighting the impact secondary consumption may have on the interpretation of molecular dietary analysis. This study represents the first simultaneous molecular investigation into the diet of multiple components of a food chain and highlights the utility of a multi-locus approach to diet reconstruction that is broadly applicable to food web analysis.     

Diet composition and prey preference of tench, Tinca tinca (L.), common bream, Abramis brama (L.), perch, Perca fluviatilis L. and roach, Rutilus rutilus (L.), in two contrasting gravel pit lakes: potential trophic overlap with wildfowl

The abundance of benthic macroinvertebrate taxa and the relative abundance of zooplankton taxa were compared with the diet of bream, tench. perch and roach from two gravel pit lakes during May-July 1986 and 1987. Significant food preferences were demonstrated between species and between lakes. Chironomid pupae dominated the macroinvertebrate diet of perch, roach, ate predominantly either Spirogyra sp. (St Peters Lake) or Daphniu hyalina (Main Lake).
The Main Lake, bream fed largely upon chironomid larvae and the bivalve Sphuerium but in St Peters Lake they positively selected a variety of less abundant benthic invertebrate taxa. Bream switched from benthos to zooplankton in the Main Lake in 1986. Tench ate large numbers of Aselhs and showed positive selection of various macroinvertebrate prey in St Peters but ate D. hjulim in Main Lake. Tropic overlap for chironomids and other macroinvertebrate prey was demonstrated between perch, bream and tench, and potentially with wildfowl which used the gravel pits for breeding and wintering.     

Population decline is accompanied by loss of genetic diversity in the Lesser Grey Shrike Lanius minor

The Lesser Grey Shrike has suffered successive declines in population size and a marked contraction of its breeding range since the early 20th century, largely because of long‐term agricultural intensification. This has resulted in a severely fragmented distribution in Western Europe, with isolated breeding nuclei in Spain, France and Italy and a more continuous distribution in Eastern Europe and Asia. Using a combination of nuclear and mitochondrial markers, we assessed the genetic structure and diversity of Lesser Grey Shrike populations from Western Europe, Central Europe and Asia. There was significant genetic differentiation among three major regional groups, one European and two Asian. Genetic diversity measures were lowest in the smallest and most marginal Spanish population. Limited genetic diversity, combined with rapid population decline, suggests the Spanish population may face extinction in the near future.     

The recent declines of farmland bird populations in Britain: an appraisal of causal factors and conservation actions

In this paper, the main aspects of agricultural intensification that have led to population declines in farmland birds over the past 50 years are reviewed, together with the current state of knowledge, and the effects of recent conservation actions. For each of 30 declining species, attention is focused on: (1) the external causes of population declines, (2) the demographic mechanisms and (3) experimental tests of proposed external causal factors, together with the outcome of (4) specific conservation measures and (5) agri‐environment schemes. Although each species has responded individually to particular aspects of agricultural change, certain groups of species share common causal factors. For example, declines in the population levels of seed‐eating birds have been driven primarily by herbicide use and the switch from spring‐sown to autumn‐sown cereals, both of which have massively reduced the food supplies of these birds. Their population declines have been associated with reduced survival rates and, in some species, also with reduced reproductive rates. In waders of damp grassland, population declines have been driven mainly by land drainage and the associated intensification of grassland management. This has led to reduced reproductive success, as a result of lowered food availability, together with increased disturbance and trampling by farm stock, and in some localities increased nest predation. The external causal factors of population decline are known (with varying degrees of certainty) for all 30 species considered, and the demographic causal factors are known (again with varying degrees of certainty) for 24 such species. In at least 19 species, proposed causal factors have been tested and confirmed by experiment or by local conservation action, and 12 species have been shown to benefit (in terms of locally increased breeding density) from options available in one or more agri‐environment schemes. Four aspects of agricultural change have been the main drivers of bird population declines, each affecting a wide range of species, namely: (1) weed‐control, mainly through herbicide use; (2) the change from spring‐sown to autumn‐sown cereal varieties, and the associated earlier ploughing of stubbles and earlier crop growth; (3) land drainage and associated intensification of grassland management; and (4) increased stocking densities, mainly of cattle in the lowlands and sheep in the uplands. These changes have reduced the amounts of habitat and/or food available to many species. Other changes, such as the removal of hedgerows and ‘rough patches’, have affected smaller numbers of species, as have changes in the timings of cultivations and harvests. Although at least eight species have shown recent increases in their national population levels, many others seem set to continue declining, or to remain at a much reduced level, unless some relevant aspect of agricultural practice is changed.     

Diet and foraging opportunism of the Guiana dolphin (Sotalia guianensis) in the Abrolhos Bank,Brazil

Diet analysis allows exploring how coastal dolphins interact with the environment and their role in the marine food webs. We studied the diet and feeding ecology of the Guiana dolphin, Sotalia guianensis, through analysis of stomach content from 42 animals stranded on the eastern coast of Brazil. A total of 1,336 semidigested prey items (fish, otoliths, cephalopod beaks, and crustaceans) were identified. Teleost fish comprised the most frequent food item (92% of the total), followed by cephalopods, and crustaceans. Prey belonged to 34 taxa and richness in individual stomachs varied from 1 to 15 prey taxa. Prey were generally small, but showed a significant trend to increase in size with dolphin length. The main prey of Guiana dolphins were demersal, estuarine, and sound-making fish such as catfish and sciaenids. No sex-related differences in diet were found. Diet composition varied seasonally and occurrence of prey items was coherent with breeding or high abundance periods of some fish species and squids. Our study shows the importance of demersal prey from estuarine and soft-sediment habitats to Guiana dolphin in the Abrolhos Bank and reveals that feeding habits are generalist and opportunistic, with diet reflecting the seasonal abundance and availability of prey.     

Adélie Penguin Population Diet Monitoring by Analysis of Food DNA in Scats

The Adélie penguin is the most important animal currently used for ecosystem monitoring in the Southern Ocean. The diet of this species is generally studied by visual analysis of stomach contents; or ratios of isotopes of carbon and nitrogen incorporated into the penguin from its food. There are significant limitations to the information that can be gained from these methods. We evaluated population diet assessment by analysis of food DNA in scats as an alternative method for ecosystem monitoring with Adélie penguins as an indicator species. Scats were collected at four locations, three phases of the breeding cycle, and in four different years. A novel molecular diet assay and bioinformatics pipeline based on nuclear small subunit ribosomal RNA gene (SSU rDNA) sequencing was used to identify prey DNA in 389 scats. Analysis of the twelve population sample sets identified spatial and temporal dietary change in Adélie penguin population diet. Prey diversity was found to be greater than previously thought. Krill, fish, copepods and amphipods were the most important food groups, in general agreement with other Adélie penguin dietary studies based on hard part or stable isotope analysis. However, our DNA analysis estimated that a substantial portion of the diet was gelatinous groups such as jellyfish and comb jellies. A range of other prey not previously identified in the diet of this species were also discovered. The diverse prey identified by this DNA-based scat analysis confirms that the generalist feeding of Adélie penguins makes them a useful indicator species for prey community composition in the coastal zone of the Southern Ocean. Scat collection is a simple and non-invasive field sampling method that allows DNA-based estimation of prey community differences at many temporal and spatial scales and provides significant advantages over alternative diet analysis approaches.     

Climate change effects on migration phenology may mismatch brood parasitic cuckoos and their hosts

Phenological responses to climate change vary among taxa and across trophic levels. This can lead to a mismatch between the life cycles of ecologically interrelated populations (e.g. predators and prey), with negative consequences for population dynamics of some of the interacting species. Here we provide, to our knowledge, the first evidence that climate change might disrupt the association between the life cycles of the common cuckoo (Cuculus canorus), a migratory brood parasitic bird, and its hosts. We investigated changes in timing of spring arrival of the cuckoo and its hosts throughout Europe over six decades, and found that short-distance, but not long-distance, migratory hosts have advanced their arrival more than the cuckoo. Hence, cuckoos may keep track of phenological changes of long-distance, but not short-distance migrant hosts, with potential consequences for breeding of both cuckoo and hosts. The mismatch to some of the important hosts may contribute to the decline of cuckoo populations and explain some of the observed local changes in parasitism rates of migratory hosts.     

Breeding ground correlates of the distribution and decline of the Common Cuckoo Cuculus canorus at two spatial scales

Many migratory bird species are undergoing population declines as a result of potentially multiple, interacting mechanisms. Understanding the environmental associations of spatial variation in population change can help tease out the likely mechanisms involved. Common Cuckoo Cuculus canorus populations have declined by 69% in England but increased by 33% in Scotland. The declines have mainly occurred in lowland agricultural landscapes, but their mechanisms are unknown. At both the local scale within the county of Devon (SE England) and at the national (UK) scale, we analysed the breeding season distribution of Cuckoos in relation to habitat variation, the abundance of host species and the abundance of moth species whose caterpillars are a key food of adult Cuckoos. At the local scale, we found that Cuckoos were more likely to be detected in areas with more semi‐natural habitat, more Meadow Pipits Anthus pratensis (but fewer Dunnocks Prunella modularis) and where, later in the summer, higher numbers of moths were captured whose larvae are Cuckoo prey. Nationally, Cuckoos have become more associated with upland heath characterized by the presence of Meadow Pipit hosts, and with wetland habitats occupied by Eurasian Reed Warbler Acrocephalus scirpaceus hosts. The core distribution of Cuckoos has shifted from south to north within the UK. By the end of 2009, the abundance of macro‐moth species identified as prey had also declined four times faster than that of species not known to be taken by Cuckoos. The abundance of these moths has shown the sharpest declines in grassland, arable and woodland habitats and has increased in semi‐natural habitats (heaths and rough grassland). Our study suggests that Cuckoos are likely to remain a very scarce bird in lowland agricultural landscapes without large‐scale changes in agricultural practices.     

Evaluation of the importance of roe deer fawns in the spring–summer diet of red foxes in southeastern Norway

Red fox Vulpes vulpes predation on roe deer Capreolus capreolus fawns has the potential to strongly affect prey population dynamics, but it is unclear whether this relationship is symmetrical or not. We analysed the spring–summer diet of adult foxes and of their cubs in a fragmented agricultural area of southeastern Norway, where a parallel study showed that the predator kills annually 25% of the radio-monitored roe deer fawns. The overall diet was highly varied and was dominated by small mammals (33% volume), especially Microtus agrestis, and medium-large mammals (25%), largely represented by fawns. The frequency of occurrence (FO) of fawns in the diet of adult foxes was highest in early spring, thus, supporting previous studies showing that the predator started actively hunting for fawns from the very beginning of the birth season. During the summer, the FO of both fawns and small mammals markedly declined, while that of berries and invertebrates increased. As expected for central-place foragers, cubs consumed a higher proportion of large prey items compared to adults. In particular, 25% of scats from cubs—versus 9% from adults—contained roe deer remains, suggesting a high profitability of fawns for vixens raising offspring. However, considering the wide food spectrum and the availability of several large prey items in our study area, it seems unlikely that the importance of fawns to the diet and population dynamics of red foxes could be as great as the impact of the predator on roe deer populations. This asymmetrical relationship implies that there are unlikely to be any stabilising feedback mechanisms in the predator–prey relationship.     

Demography of Ferruginous Hawks Breeding in Western Canada

Abstract: We assessed age-dependent survival, site-fidelity, and, together with data on prey and reproduction, trends in the population of ferruginous hawks (Buteo regalis) breeding in western Canada. Our analysis included 7,129 ferruginous hawks banded near Hanna, Alberta, and Kindersley-Alsask, Saskatchewan, from 1972 to 2003. We estimated annual adult survival rate to be 0.708 (SE = 0.024) and first year survival for nestlings was 0.545 (SE = 0.147). Resighting probability was modeled as a constant for nestlings (0.009, SE = 0.010), but it varied among years for adults consistent with our sampling efforts. Band reporting rate was at 0.022 (SE = 0.007) for both nestlings and adults. Fidelity to the study site was 1.00 (SE = 0.000) for adults and 0.035 (SE = 0.014) for nestlings. Nesting density ranged from 3.1 to 14.0 pairs/100 km² and averaged 9.8 pairs/100 km². We observed an average clutch size of 3.2 (SE=0.06) and brood sizes of 2.71 (SE=0.07) near Hanna and 2.79 (SE = 0.99) at Kindersley-Alsask. Richardson's ground squirrels (Spermophilus richardsonii) made up 95% of ferruginous hawk prey. Prey availability was positively correlated with number of offspring near Hanna and Kindersley-Alsask. We believe the lower than expected adult survival did not result in population decrease; rather, declines in reproduction resulting from declines in the abundance of ground squirrels better explain an observed 4.5-fold decline in nesting densities during the study. The results suggest that ferruginous hawk management should address prey in addition to habitat management, and that management needs are regional in scope with particular emphasis on the breeding range within the northern Great Plains.     

Pesticide Acute Toxicity Is a Better Correlate of U.S. Grassland Bird Declines than Agricultural Intensification

Common agricultural birds are in decline, both in Europe and in North America. Evidence from Europe suggests that agricultural intensification and, for some species, the indirect effects of pesticides mediated through a loss of insect food resource is in part responsible. On a state-by-state basis for the conterminous Unites States (U.S.), we looked at several agronomic variables to predict the number of grassland species increasing or declining according to breeding bird surveys conducted between 1980 and 2003. Best predictors of species declines were the lethal risk from insecticide use modeled from pesticide impact studies, followed by the loss of cropped pasture. Loss of permanent pasture or simple measures of agricultural intensification such as the proportion of land under crop or the proportion of farmland treated with herbicides did not explain bird declines as well. Because the proportion of farmland treated with insecticides, and more particularly the lethal risk to birds from the use of current insecticides feature so prominently in the best models, this suggests that, in the U.S. at least, pesticide toxicity to birds should be considered as an important factor in grassland bird declines.     

Pellets of proof: First glimpse of the dietary composition of adult odonates as revealed by metabarcoding of feces

Recent advances in molecular techniques allow us to resolve the diet of unstudied taxa. Odonates are potentially important top‐down regulators of many insects. Yet, to date, our knowledge of odonate prey use is based mainly on limited observations of odonates catching or eating their prey. In this study, we examine the potential use of metabarcoding in establishing the diet of three adult odonate species (Lestes sponsa, Enallagma cyathigerum, and Sympetrum danae) at a site in southwestern Finland. To this purpose, we compared three different methods for extracting DNA from fecal samples: the Macherey‐Nagel Nucleospin XS kit, a traditional salt extraction, and the Zymo Research Fecal Microprep kit. From these extracts, we amplified group‐specific mitochondrial markers (COI and 16S rRNA) from altogether 72 odonate individuals, and compared them to comprehensive reference libraries. The three odonate species show major overlap in diet, with no significant differences between individuals of different size and/or gender, reflecting opportunistic foraging of adult odonates. Of a total of 41 different prey species detected, the most frequently consumed ones were Diptera, with additional records of six other orders. Based on our data, the best DNA extraction method is the traditional salt extraction, as it provides the most information on prey content while also being the most economical. To our knowledge, this is the first study to resolve the species‐level diet of adult odonates. Armed with the appropriate methodological caveats, we are ready to examine the ecological role of odonates in both terrestrial and aquatic food webs, and in transferring subsidies between these two realms.     

When to use next generation sequencing or diagnostic PCR in diet analyses

Next‐generation sequencing (NGS) is increasingly used for diet analyses; however, it may not always describe diet samples well. A reason for this is that diet samples contain mixtures of food DNA in different amounts as well as consumer DNA which can reduce the food DNA characterized. Because of this, detections will depend on the relative amount and identity of each type of DNA. For such samples, diagnostic PCR will most likely give more reliable results, as detection probability is only marginally dependent on other copresent DNA. We investigated the reliability of each method to test (a) whether predatory beetle regurgitates, supposed to be low in consumer DNA, allow to retrieve prey sequences using general barcoding primers that co‐amplify the consumer DNA, and (b) to assess the sequencing depth or replication needed for NGS and diagnostic PCR to give stable results. When consumer DNA is co‐amplified, NGS is better suited to discover the range of possible prey, than for comparing co‐occurrences of diet species between samples, as retested samples were repeatedly different in prey detections with this approach. This shows that samples were incompletely described, as prey detected by diagnostic PCR frequently were missed by NGS. As the sequencing depth needed to reliably describe the diet in such samples becomes very high, the cost‐efficiency and reliability of diagnostic PCR make diagnostic PCR better suited for testing large sample‐sets. Especially if the targeted prey taxa are thought to be of ecological importance, as diagnostic PCR gave more nested and consistent results in repeated testing of the same sample.