DOI: 10.5061/dryad.v6wwpzgsp
title: Data from: Using molecular and crowd-sourcing methods to assess breeding ground diet of a migratory brood parasite of conservation concern

description: Breeding ground food availability is critical to the survival and
 productivity of adult birds. The common cuckoo Cuculus canorus is a
 brood-parasitic Afro-Palearctic migrant bird exhibiting long-term
 (breeding) population declines in many European countries. Variation in
 population trend between regions and habitats suggests breeding ground
 drivers such as adult food supply. However, cuckoo diet has not been
 studied in detail since before the most significant population declines in
 Europe began in the mid-1980s. 20th century studies of cuckoo diet largely
 comprised field observations likely to carry bias towards larger prey
 taxa. Here we demonstrate the potential value of 1) using high-throughput
 DNA sequencing of invertebrate prey in faeces to determine cuckoo diet
 with minimal bias towards large prey taxa, and 2) using crowd-sourced
 digital photographs from across Britain to identify lepidopteran cuckoo
 prey taxa during recent years post-decline (2005-2016). DNA analysis found
 a high frequency of Lepidoptera, including moths of family Lasiocampidae,
 prominent within the past literature, but also grasshoppers (Orthoptera)
 and flies (Diptera) that may be overlooked by field observation
 methodologies. The range of larval lepidopteran prey identified from
 photographs largely agreed with those previously documented, with
 potential signs of reduced diversity, and identities of key adult prey
 taxa were supported by molecular results. Notably, many identified cuckoo
 prey taxa have shown severe declines due to agricultural intensification,
 suggesting this has driven spatial patterns of cuckoo loss.
 Landscape-scale, lowland rewilding interventions provide opportunities to
 understand the scale of reversal of previous agricultural intensification
 that may be necessary to restore prey populations sufficiently to permit
 recolonization by cuckoos.
description: <i>Study
 site and faecal sample collection </i> We collected
 faeces from common cuckoo adults in Dartmoor National Park, UK (Fig. 1)
 between April and June 2017 (and one bird ringed in May 2016). 52 samples
 were obtained by collecting fresh faeces dropped by birds using natural
 perches (trees, shrubs and rocks). Seven further samples were collected
 through licensed mist-netting. Within Dartmoor, Warren House (50.61194°N;
 3.87028°W) and Burrator (50.52333°N; 4.01528°W) were selected as valleys
 with scattered trees, with abundant perches and short-grazed grass
 beneath, facilitating detection of cuckoos and their faeces. Sites were
 walked from 06:00 am when weather conditions were not wet and windy. Once
 located, each cuckoo was watched until defecation took place. Faeces were
 searched for within a 3 m radius of the estimated location of defecation,
 and collected within 20 minutes, allowing time to watch for further
 defecation events or to note the bird’s new location if it moved. All
 visibly fresh faeces found were collected in separate 8 ml vials
 containing 90% ethanol and refrigerated at 5°C within 12 h. For data
 analysis, we pooled all faecal samples collected from the same 3 x 3 m
 area at the same time, into groups referred to as ‘sampling
 events’. Mist-netting
 was carried out at Holne Moor, Dartmoor (50.52717°N; 3.86433°W). from
 04:00 am using a triangular array of three 18 m mist-nets (30 x 30 mm
 mesh) (Ecotone, Sopot, Poland) with a decoy female cuckoo at the centre
 and a sound lure of male and female cuckoo calls, at a BTO guideline
 volume (Blackburn <i>et al.</i> 2006). Each trapped bird was
 placed for ten minutes in a veterinary carry case with a clean, laminated
 card floor. All faecal material deposited was collected using the same
 protocols as above. <i>DNA
 extraction and sequencing</i> DNA was
 extracted using a CTAB-based protocol. DNA sequencing of a 290 base-pair
 (bp) region of the mitochondrial COI DNA was used to confirm bird species
 (Hebert <i>et al.</i> 2004, Kerr <i>et al.</i>
 2009) from the faecal samples, unless collected by mist-netting, together
 with a 157 bp region to identify prey taxa using arthropod COI primers
 (Zeale <i>et al.</i> 2011). Libraries for a total of 48
 samples confirmed as originating from cuckoo by sequencing or collection
 during mist-netting, plus controls, were prepared in triplicate and 300
 paired-end sequenced using the Illumina MiSeq v3 SBS reagents (Illumina
 Corp, USA). Details of the methods used to extract the DNA, confirm the
 species, and prepare, sequence and identify taxa are provided in
 supplementary materials. <i>Sequencing data
 analysis</i> DNA sequence
 reads from Illumina MiSeq runs were de-multiplexed, and trimmed to remove
 primer sequence, duplicates and low quality regions (Ewing & Green
 1998, Trevelline <i>et al</i>. 2016). Dada2 (Callahan
 <i>et al.</i> 2016) was used to identify amplicon sequence
 variants (ASVs). The sequence and total number of reads of each ASV (n =
 268) in each replicate PCR from faecal samples were exported into a
 summarising table. The mean number of unique ASVs read per sample was
 14.49 (range 3-38, supplementary materials Table A1). For taxonomic
 identification, ASVs were entered as search queries in the Barcode of Life
 Database (BOLD) version 4 (Ratnasingham & Hebert 2007) Species
 Level Barcode Records. We noted that Lepidoptera and other species
 previously given as cuckoo prey in the literature (e.g. Wyllie 1981) have
 accessioned sequences in BOLD.<i> </i>Sequences from samples
 were considered to match a database sequence if similarity exceeded 98%
 (following Clare <i>et al.</i> (2011) and King <i>et
 al.</i> (2015)). The species with the highest percentage similarity
 was taken to be the prey species of origin (following King <i>et
 al</i>. (2015)). 160 ASVs matched eukaryote taxa, accounting for
 1,864,169 reads on the MiSeq platform. Sequences matching prokaryotic taxa
 were excluded. 108 read sequences of appropriate length (&gt;150 bp)
 for which no match could be found through BOLD, were recorded as the
 aggregate result 'No Match'. UK records for each top-matching
 species were searched online using Google. Species with no previous
 records of UK occurrence were highlighted (following King <i>et
 al.</i> (2015)). Sequences relating to these species were assumed to
 originate from a UK-occurring species within an encompassing higher taxon
 and were included in calculations of frequency of taxa on this
 basis. Crowd-sourced digital photography The search terms “cuckoo” and “<i>Cuculus</i>” were entered in the search tools of Flickr (http://www.flickr.com), Birdforum Gallery (http://www.birdforum.net/gallery/ search.php), Birdguides Gallery (http://www.birdguides.com/ gallery/), Rare Bird Alert gallery (http://www.rarebirdalert.co.uk/RealData/gallery. asp) and Google Images (http://images.google.com). Images had to meet the following criteria: i) contained a full-grown, nutritionally independent common cuckoo handling an identifiable and wild-caught prey item and ii) identified the country and month in which the image was recorded. Plumage was used to classify each bird as juvenile or adult. Prey items in images were identified to the most specific possible taxonomic level, with all identifications made by one person (BH) with relevant taxonomic expertise (Henwood & Sterling 2020). The geographic distribution of the images extended throughout Britain, from 58 locations comprising both inland and coastal sites, though images in Scotland were more clustered to coastal and island locations and few images were available from Wales. The appearance, date, location and accompanying information were used to identify images of the same bird and prey item (referred to in subsequent text as ‘prey handling events’). Photographs of 44 prey handling events in adult cuckoos, and 24 in juveniles, met the criteria for inclusion in analysis.
description: NCBI Sequence Read Archive under BioProject
 number PRJNA606798