DOI: 10.5061/dryad.98sf7m0k7
title: Endogenous biomarkers reveal diet partitioning among three sympatric species of swallows

description: Since the early 1990s, aerial insectivorous birds have shown serious
 population declines in North America, but it is not clear if
 factors common to all species within this guild account for these
 declines. Among sympatric swallows, population trends differ, and
 this may be due to differences in ecology operating throughout the annual
 cycle. Although these species all feed on aerial insects, prey
 taxa can differ tremendously in their “aeroecology” and use by swallows.
 We examined the potential for dietary differences among three
 species of swallows, Barn Swallow (Hirundo rustica),
 Cliff Swallow (Petrochelidon pyrrhonota), and Tree Swallow
 (Tachycineta bicolor), breeding sympatrically in southern
 Ontario, Canada. Potential interspecific differences in nestling
 diet were examined using two endogenous biomarkers, DNA barcoding
 of nestling feces and stable isotope analysis (δ 2H, δ 13C, δ 15N) of
 nestling feathers. We found evidence for differences in dietary
 sources of provisioned young where Barn Swallows provisioned more
 terrestrial-based prey, Cliff Swallows provisioned an
 intermediate diet, and Tree Swallows the most aquatic-emergent insect
 diet. We suggest this information may help to identify potential
 factors contributing to differential declines of aerial insectivores
 operating on the breeding grounds, including diet quality.
description: Fieldwork was conducted at
 nine farms from May-July 2018 within Wellington County, near Guelph,
 Ontario, Canada (43.55° N, 80.25° W). This area is a rural landscape
 characterized by a mixture of row crops, pasture, small woodlots, and a
 few large conservation areas (Kusack et al. 2020). The region includes one
 large lake (Guelph Lake) and numerous small wetlands and ponds (Kusack et
 al., unpublished data). These locations were grouped into seven sites
 because two sets of two farms were adjacent to each other such that we
 considered them the same site. Six of these sites had all three species of
 swallow breeding sympatrically (average 2018 clutch initiation was 25 May
 for Barn Swallows, 29 May for Cliff Swallows, and 24 May for Tree
 Swallows). However, Cliff Swallow nests failed at one of the sites, so
 only Barn and Tree Swallow samples were taken there. At another site
 (Guelph Lake Conservation Area), only Tree Swallows were present. All Barn
 Swallow and Cliff Swallow colonies were located inside barns, whereas Tree
 Swallows were in nest boxes. <strong>Nest Monitoring and
 Sampling</strong> Nest monitoring began in early
 May and involved visiting sites one to two times weekly to establish the
 approximate onset of laying. If eggs were present upon first inspection,
 and the clutch was not complete, it was assumed that one egg had been laid
 per day for all three species (Winkler et al. 2011; Brown et al. 2017;
 Brown and Brown 2019). Once the clutch initiation date was estimated,
 nests were monitored at least twice per week to determine final clutch
 size and approximate hatch date. Timing of hatching was predicted based on
 a 14-day incubation period after the penultimate egg was laid for Barn
 Swallows (Brown and Brown 2019), Cliff Swallows (Brown et al. 2017), and
 Tree Swallows (Winkler et al. 2011). To determine hatch day as accurately
 as possible, nests were visited before predicted hatch day and every few
 days thereafter. Hatch Day was assigned based on evidence of hatching;
 this included eggshells present, unhatched eggs remaining, and nestlings
 still being wet. Hatch Day was considered Day Zero. For nests where hatch
 day could not be assigned, nestlings were aged by feather tract
 development (Stoner 1935; Stoner 1945; Marsh 1980). We only focused on
 sampling first clutches. All nests were visited between
 Day Six and Ten after hatching, during which Tree Swallow and Barn Swallow
 nestlings were banded with a uniquely numbered United States Geological
 Survey (USGS) aluminum leg band. For each nestling, mass, age and relaxed
 wing chord were documented. Fecal samples were collected by holding a
 clean piece of paper under the young as they were removed from their nest,
 these samples were grouped by nest. Cliff Swallow nestlings were not
 accessible for banding and therefore cardboard was placed beneath the nest
 for roughly 30 minutes to collect fecal samples while we processed other
 nestlings from the same site. Barn Swallow and Tree Swallow
 nests were visited and morphometric measurements taken when young were 15
 days old. It was not possible to visit all nests at Day 15 and so some
 Tree Swallows were measured at Day 16 or 17. At that time, feathers were
 taken from all but one nestling per nest for stable isotope analyses.
 Hatch-year Cliff Swallows were visited close to fledging (~Day 20) and
 when they could be coaxed from the nest structure or accessed by
 shortening the entrance tube. At that stage, they were also weighed,
 measured, and had a central tail feather taken for stable isotope
 analyses, then returned to their nests. After sampling, young were
 expected to fledge within 1 - 7 days. Average fledging age is 19-20 days,
 20-21 days, and 18-22 days for Barn Swallows (Brown and Brown 2019), Cliff
 Swallows (Brown et al. 2017), and Tree Swallows (Winkler et al. 2011),
 respectively. All young were banded with a USGS numbered band during
 feather sampling. <strong>Stable Isotope
 Analyses</strong> Tail feathers were soaked with
 2:1 chloroform:methanol overnight, rinsed and dried in a fume hood at
 ambient temperature for 24 h. Samples analyzed for stable hydrogen (δ2H)
 isotopes were weighed (0.35 mg) into silver capsules using the feather
 barbs only. Capsules were compressed and analyzed using the LSIS-AFAR
 stable isotope facility at the University of Western Ontario. Samples were
 loaded into a Uni-prep carousel (Eurovector®, Milan, ITA) held at 60ºC,
 evacuated and maintained under pressure with dry helium and then combusted
 in a Eurovector 3000 elemental analyzer (Eurovector, Milan) pyrolytically
 on glassy carbon at 1350ºC. Separated H2 was analyzed using a Thermo Delta
 V Plus (Thermo scientific®, Bremen, DEU) continuous-flow isotope ratio
 mass spectrometer via a Conflo device (Thermo Scientific, Bremen, DEU).
 Sample results were expressed in the standard delta (δ) notation in parts
 per thousand (‰) deviation from the Vienna Standard Mean Ocean Water
 (VSMOW) standard. In-house keratin standards (CBS: -197‰; KHS: -54.1‰)
 were used to derive the δ2H value of the non-exchangeable H fraction
 according to the comparative equilibration approach (Wassenaar and Hobson
 2003). Based on within-run (n = 5 each) keratin standards, measurement
 error was estimated to be ±2‰. Feather samples analyzed for
 stable carbon (δ13C) and nitrogen (δ15N) isotope values were weighed (1.0
 mg) in tin capsules, compressed and analyzed at the Environment and
 Climate Change Canada stable isotope laboratory in Saskatoon,
 Saskatchewan, Canada. Samples were combusted at 1030°C in a Carlo Erba
 NA1500 (Thermo Scientific; Waltham, United States) or Eurovector 3000
 (Eurovector, Milan) elemental analyzer. The resulting N2 and CO2 were
 separated chromatographically and introduced into an Elementar Isoprime
 (Elementar; Langenselbold, Germany) or a Nu Instruments Horizon (Nu
 Instruments Ltd.; Wrexham, United Kingdom) isotope ratio mass
 spectrometer. Sample results were expressed in the standard delta (δ)
 notation in parts per thousand (‰) deviation from international standards
 (Vienna Pee Dee Belemnite [VPDB] and AIR for δ13C and δ15N, respectively).
 Internal laboratory calibration standards were BWBIII keratin (δ13C =
 -20.18‰, δ15N = 14.31 ‰) and Pugel (δ13C = -13.64‰, δ15N = 5.07‰).
 Measurement precision was based on replicate (n = 5) within-run
 measurements of internal reference material and estimated to be ±0.1‰ for
 both δ13C and δ15N. <strong>Fecal DNA
 Analyses</strong> Fecal samples were transferred in
 the field into scintillation vials containing 95% ethanol and kept cool
 with ice packs. Upon returning from the field, samples were immediately
 frozen at -20<sup>o</sup>C and later processed at the Canadian
 Centre for DNA Barcoding (Guelph, Canada). Samples were amplified
 separately, using arthropod-specific primers targeting a 157 base-pair
 region of the mitochondrial cytochrome c oxidase subunit 1 (COI) gene
 (Hebert et al. 2003). Amplified samples were pooled and sequenced using an
 Ion Torrent PGM high-throughput sequencer (Thermo Fisher Scientific),
 trimmed to remove the primer, and filtered for a minimum size of 100 base
 pairs. Filtered reads were queried against the Barcode of Life Database
 (BOLD; www.boldsystems.org) reference using a basal local alignment search
 tool (BLAST) algorithm to assign taxonomic identity. Results were accepted
 if they had a minimum of 50 reads that matched reference sequence with at
 least 95% identity across at least 100 base pairs.
description: Bumelis_Fecal-DNA:
 This file contains
 information for prey species found in nestling fecal samples. Column B
 shows which species was sampled (BARS = Barn Swallow, CLSW = Cliff
 Swallow, TRES = Tree Swallow). Columns C-E show the age (days since
 hatching), site code, and collection date. Column F is the number of reads
 for each species, and Columns G-L identify the prey to the lowest
 taxonomic level possible. Bumelis_Feather-Isotopes This file contains information on nestling feather isotope values. Column B shows which species was sampled (BARS = Barn Swallow, CLSW = Cliff Swallow, TRES = Tree Swallow). Column C&D are site and date information. Columns E-G are stable isotope values (‰)