DOI: 10.5061/dryad.3ffbg79r1
title: Data from: ultimate drivers of forced extra-pair copulations in birds lacking penises: jackdaws as a case-study

description: Forced copulation is common, presumably because it can increase male
 reproductive success. Forced extra-pair copulation (FEPC) occurs in birds,
 even though most species lack a penis and are widely thought to require
 female cooperation for fertilisation. How FEPC persists, despite a
 presumed lack of siring success and likely non-negligible costs to the
 male, is unknown. Using the jackdaw (Corvus monedula) as a case study, we
 use SNPs to quantify extra-pair paternity rate through FEPC and evaluate
 explanations for the persistence of FEPC in species without a penis. We
 then collate evidence for FEPC across penis-lacking birds. Combining
 genetic and behavioural analyses, our study suggests that the most likely
 explanations for the maintenance of FEPC in jackdaws are that it provides
 a selective advantage to males, or that it is a relic. Our literature
 review shows that across birds lacking a penis, FEPC is taxonomically
 widespread, yet little is known about its evolution. Broader
 implementation of the approach used here, combining both genetic and
 behavioural data, may shed light on why this widespread sexual behaviour
 persists. Additional work is necessary to understand whether a penis is
 needed for paternity through forced copulation, and to quantify the costs
 of FEPC.
description: <em><strong>Behavioural
 data</strong></em> We fitted cameras with
 microphones (380TVL CMOS camera, Handykam, Redruth, U.K.) inside jackdaw
 nest-boxes during the 2014, 2015, 2018 and 2019 breeding seasons. Cameras
 were set to record starting from between 6AM and 11AM BST (n = 575
 recordings). This captures the period when our previous observations, and
 other studies (Gill et al. 2020), indicate the majority of sexual
 behaviour occurs. In addition, a small subset set to record in the
 afternoon (n = 8 recordings; 13:30PM - 14:30PM BST). Videos were recorded
 at three different stages of the breeding season: the nest building stage,
 the incubation stage, and the nestling stage, with one to three videos per
 pair per stage (total number of pairs = 130). The nest building stage was
 filmed from when building had begun and a nest ‘cup’ was becoming apparent
 (Hahn et al., 2021) until the nest was complete; the incubation stage was
 filmed 2 – 10 days after the female’s fertility window until hatching, and
 the nestling stage was filmed at 4 – 10 (nestling 1) and 17 – 23 (nestling
 2) days following the first egg hatching. Note that videos were filmed for
 various research purposes, and in-line with these protocols, no videos
 were filmed during the female’s fertile period (5 days pre-clutch
 initiation to the penultimate lay date; (Gill et al., 2020; Henderson et
 al., 2000)). <strong><em>Sampling, DNA
 Extraction and Sequencing</em></strong> For
 jackdaw chicks that survived until 25 days old, blood samples for DNA
 extraction were collected at ringing. In 2018 and 2019, nest-boxes with
 chicks &lt;25 days old were monitored closely so that deceased chicks
 could be collected before removal from the nest by parents. Deceased
 chicks were frozen at -20℃ as soon after death as possible, and tissue
 samples from the liver were collected for DNA extraction. DNA was
 extracted from blood using Thermo Scientific GeneJET Whole Blood Genomic
 DNA Purification, and from liver using QIAGEN DNeasy Blood &
 Tissue Kit, both following manufacturer’s protocols. See Supplementary
 Materials for further details of DNA extraction and quality
 control. Following DNA extraction, samples were
 selected based on fully sampled family units (i.e. broods where both
 social parents had been sampled), site (sample number was proportional to
 site size), and quality of DNA extraction. Five duplicate samples (from
 the same DNA extraction) were included in the sequencing pool in order to
 estimate approximate sequencing error rate. We also included known social
 half-siblings (known through re-pairing of the social parent) so we could
 examine whether our final analysis had the power to detect half-sib
 relationships, which is key to estimating extra-pair paternity rates. A
 female with no known offspring or parents was also included, because she
 had been observed for several years to associate with a pair who were
 included (along with their offspring) in the sample selection. We also had
 video evidence of this female laying an egg in that pair’s nest-box (see
 Methods: behavioural data). Our final sample for
 sequencing consisted of 188 individuals (plus five duplicates) across two
 sites. 149 individuals were included from Sites Y and Z (treated as one
 site due to gene flow; see Methods: Bioinformatic Pipeline), comprising
 113 (plus two duplicate) offspring from 47 broods and 22 sibships. Two
 parent samples were also duplicates. From Site X, 43 individuals were
 included, which comprised of 33 (plus one duplicate) offspring from 13
 broods and six sibships. Quality control of DNA extracted from liver
 samples revealed a moderate level of degradation in many samples.
 Therefore, only six samples from deceased chicks were included in the
 analysis; all other samples were extracted from blood.
 Samples were sent to Exeter Sequencing Services for library
 preparation and ddRAD sequencing. See Supplementary Materials for detailed
 library prep and sequencing methods.
 <strong><em>Bioinformatic
 pipeline</em></strong> Sequence data
 underwent strict quality filtering, presented in detail in the
 Supplementary Materials. Following quality filtering, reads were run
 through process radtags in STACKS v2.54 (Rochette et al., 2019). Reads
 were then aligned to the jackdaw reference genome (GenBank accession
 JABDSK000000000; Weissensteiner et al., 2020) using GSNAP/GMAP v2020-10-27
 (Wu & Nacu, 2010) specifying a maximum of ten mismatches (-m 10),
 an indel penalty (-i 2) and turning off terminal alignments (–min-coverage
 = 0.95). Only reads that aligned uniquely were retained (-n 1,
 --quiet-if-excessive) (Paris et al., 2017). Following
 alignment, we retained samples with &gt;1 million aligned reads. These
 were run through the ref_map module of STACKS, specifying a minor allele
 frequency of 0.15 (--min-maf 0.15), retaining one random SNP per read
 (--write-random-snp) and retaining only SNPs present in &gt;50% of
 samples (-r 0.5). Finally, we used PLINK v1.9 (Purcell et al., 2007) to
 discard loci in linkage disequilibrium. To do this, we considered all
 individuals without sampled parents as ‘founders’ for Site Y + Site Z, two
 sites with substantial overlap and assumed gene flow (now referred to as
 Site YZ), and Site X, a separate site with little to no observed overlap
 with Site YZ. We tested for linkage disequilibrium using --indep,
 evaluating 50 SNP windows, five SNPs at a time with a variance inflation
 factor cut-off of 2 (Levine et al., 2019), and filtered out SNPs in
 linkage disequilbrium from our final datasets.
description: # Data from: ultimate drivers of forced extra-pair copulations in birds
 lacking penises: jackdaws as a case-study Author: "Beki Hooper"
 Date: '2024-01-07' GENERAL INFORMATION 1. Title of Dataset:
 Ultimate drivers of forced extra-pair copulations in birds lacking
 penises: jackdaws as a case-study 2. Author Information A. Principal
 Investigator Contact Information Name: Alex Thornton Institution:
 University of Exeter Email: B.
 Associate or Co-investigator Contact Information Name: Beki Hooper
 Institution: NA Email: 3. Date of data
 collection (single date, range, approximate date): 2014 – 2019 4.
 Geographic location of data collection: Cornwall, UK 5. Information about
 funding sources that supported the collection of the data: Natural
 Environment Research Council GW4 Studentship (NERC 107672G), NERC
 biomolecular analysis facility (NBAF1152), British Ornithologists’ Union
 (BOU), BBSRC David Phillips Fellowship (BB/H021817/2), Leverhulme Grant
 (RGP-2020-170), Wellcome Trust (Multi-User Equipment Grant award number
 218247/Z/19/Z). SHARING/ACCESS INFORMATION 1. Licenses/restrictions placed
 on the data: CC0 1.0 Universal (CC0 1.0) Public Domain 2. Links to
 publications that cite or use the data: NA 3. Links to other publicly
 accessible locations of the data: None 4. Links/relationships to ancillary
 data sets: None 5. Was data derived from another source? No, except in
 "Supplementary Data.xlsx" in which all sources are reported A.
 If yes, list source(s): NA 6. Recommended citation for this dataset:
 Hooper, R.C., Maher, K., Moore, K., McIvor, G.M., Hosken, D. &
 Thornton, A. (2023). Data from: Ultimate drivers of forced extra-pair
 copulations in birds lacking penises: jackdaws as a case-study. Dryad
 Digital Repository: DATA
 & FILE OVERVIEW 1. File List: A) fine_scale-behav.csv B)
 time_tog_total.csv C) birthyear_sequoia.csv D) site_sequoia.csv E)
 ped_sequoia.csv F) SNPs_site_X.PED G) SNPs_site_YZ.PED H)
 Forced-Extrapair-Copulations-in-Jackdaws.rmd I) sequoia_script.R J)
 Supplementary Video 1 (Y09-220414-0827-E1) K) Supplementary Video 2
 (Y17-100418-0701-B) L) Supplementary Video 3 (Y20-160414-1054-E1) M)
 Supplementary Data.xlsx N) .fastq files 2. Relationship between files, if
 important: Forced-Extrapair-Copulations-in-Jackdaws.rmd, an R script)
 input files: time together (time_tog_total.csv), fine scale behaviour
 (fine_scale_behav.csv) Pedigree reconstruction script (sequoia_script.R,
 an R script) input files: SNP data for site X (SNPs_site_X.ped), SNP data
 for site YZ (SNPs_site_YZ.ped), Site data for each individual
 (site_sequoia.csv), Birth/year data for each individual
 (birthyear_sequoia.csv), Social pedigree for each individual
 (ped_sequoia.csv) 3. Additional related data collected that was not
 included in the current data package: None 4. Are there multiple versions
 of the dataset? A. If yes, name of file(s) that was updated: i. Why was
 the file updated? ii. When was the file updated?
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: fine_scale-behav.csv This file contains the
 fine-scale behavioural data for each pair. It is the concatenated video
 code data, where each new row is a new behaviour that has been recorded
 within a video. 1. Number of variables: 44 2. Number of rows: 11241 3.
 Variable List: male.ID ID of the male fem.ID ID of the female BOX_YEAR
 Nestbox in which the video was filmed and the year it was collected (e.g.
 X2514 = nestbox X25, 2014) METHOD Method of video coding (BORIS or manual,
 where manual was coding via Microsoft Excel) CODER Initials of the person
 who coded the video UNIQUE_ID Concatenation of method, date of video
 recording and nestbox, to give each video a unique identifier YEAR Year of
 video recording STAGE Stage of the breeding season the video was filmed. E
 = incubation. INDIVIDUAL The individual engaging in the recorded behaviour
 (M = male, F = female, Unknown 1 = unknown if male or female, No focal
 subject = extrapair individual) BEHAVIOUR The behaviour that the
 ‘individual’ is engaged in. See the Ethogram in the Supplementary
 Material. TIME_START The time into the video that the behaviour starts
 (seconds) TIME_END The time into the video that the behaviour ends
 (seconds) TREATMENT If the video was collected during the consolation
 experiment (see manuscript) then this column says which treatment the
 video was from (B = before stressor, A = after stressor). If the video
 collected captured a natural intrusion event, it says “INTRUSION”. In two
 videos, there were two intrusions. The first it INTRUSION 1, the second is
 INTRUSION 2, and the time between in BETWEEN. For all videos without an
 intrusion or simulated intrusion event, NA. FEPC_IN_VIDEO Is there an FEPC
 in this video: Yes or No UNIQUE_TR_ID Same as UNIQUE_ID but with Treatment
 included as an identifier. VIDEO_STARTS_DEC The time of day the video
 started but converted to decimal. BEHAVIOUR_DURATION The duration of the
 behaviour (n seconds). OBSERVATION_MINUS_LATENCY The length of the video
 in seconds MALE_VISIT_N The number of the resident male’s visit to the
 nestbox. E.g. on his first visit of the video it is 1; on the second visit
 of the video it is 2. UNIQUE_TR_ID_M_VISIT Same as UNIQUE_TR_ID but with
 MALE_VISIT_N concatenated COPULATION_VISIT Whether the male copulated with
 the female during the visit (Y = Yes, N = No) COPULATION_VIDEO Whether the
 male copulated with the female during the video (Y = Yes, N = No)
 FEPC_in_pairyear Whether there was an FEPC event for the female during the
 breeding season (Y = Yes, N = No) Site The site in which the pair were
 filmed lay.date The date of that the female laid her first egg cl.size The
 number of eggs in the clutch hatch.date The date of the first egg hatching
 n.hatch The number of eggs that hatched fledge.date The date of fledging
 n.fledge The number of chicks that fledge mass.fledge The cumulative mass
 of the fledged chicks in grams pair.ID The male ID and the female ID,
 concatenated FEM_FERTILE Whether the female was in her fertile period (N =
 No, Y = Yes) DAYS_SINCE_FERTILE Number of days since the females fertile
 period ended incubation_duration The duration (in days) of the female’s
 incubation period fem_wing Length of the female’s wing in cm fem_tarsus
 Length of the female’s tarsus in cm fem_exact_age 0 = not exact, 1 = exact
 male_wing Length of the male’s wing in cm male_tarsus Length of the male’s
 tarsus in cm male_exact_age 0 = not exact, 1 = exact min_age_male Minimum
 age of the male min_age_female Minium age of the female years_together_min
 Minimum number of years the pair have been together 4. Missing data codes:
 NA 5. Specialized formats or other abbreviations used: None
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: time_tog_total.csv This file contains the
 amount of time (in seconds) that a pair spend together in a single video
 1. Number of variables: 2 2. Number of rows: 133 3. Variable List:
 UNIQUE_TR_ID As above – unique identifier per video. TIME_TOG_TOTAL Time
 spent together in the nestbox in seconds. 4. Missing data codes: NA 5.
 Specialized formats or other abbreviations used: None
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: birthyear_sequoia.csv This file contains
 birth year and sex data on each individual used in the genomic analysis.
 1. Number of variables: 5 2. Number of rows: 193 3. Variable List: ID ID
 of the jackdaw Sex Sex of the individual (1 is female, 2 is male)
 Birthyear Year of birth (NA = unknown) Minbirthyear If year of birth
 unknown, the minimum possible birth year Maxbirthyear If year of birth
 unknown, the maximum possible birth year 4. Missing data codes: NA 5.
 Specialized formats or other abbreviations used: None
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: site_sequoia.csv This file contains the
 site in which each individual was born. 1. Number of variables: 2 2.
 Number of rows: 4153 3. Variable List: ID ID of the jackdaw LOCATION Site
 where the jackdaw was born 4. Missing data codes: NA 5. Specialized
 formats or other abbreviations used: None
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: ped_sequoia.csv This file contains the dam
 and sire of each sampled individual 1. Number of variables: 3 2. Number of
 rows: 193 3. Variable List: id ID of the jackdaw dam mother of the
 individual. NA if unknown. sire father of the individual. NA if unknown.
 4. Missing data codes: NA 5. Specialized formats or other abbreviations
 used: None
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: SNPS_site_X.ped This file contains the SNP
 data of each sequenced individual. Each individual is a row and columns G
 – ACV are sites. Please read .PED file documentation for more information
 on how to read this file. This file is for individuals from site X only.
 \#########################################################################
 DATA-SPECIFIC INFORMATION FOR: SNPS_site_YZ.ped This file contains the SNP
 data of each sequenced individual. Each individual is a row and columns G
 – AHW are sites. Please read .PED file documentation for more information
 on how to read this file. This file is for individuals from site Y and
 site Z only (merged due to gene flow between sites).
 \#########################################################################
 Forced-Extrapair-Copulations-in-Jackdaws.rmd Running this script, with the
 input data as stated above, will give you all the results for our
 behavioural analysis.
 \#########################################################################
 sequoia_script.R Running this script, with the input data, will give you
 all the results for our pedigree analysis. Sequoia help files clearly
 explain the data structure of each input file. Note you will need to
 download the version of Sequoia used in the script (described within).
 \#########################################################################
 Supplementary Videos 1 – 3 Example footage of internal nestbox footage. 1
 and 2 are examples of attempted FEPC. 3 is an example of a within-pair
 copulation. Note the copulation call made by the male in each. In Video
 S2, the second individual to enter the nestbox is the resident male.
 \#########################################################################
 Supplementary Data.xlsx This file contains information on bird species
 without penises in which FEPCs have been observed. 1. Number of variables:
 13 2. Number of rows: 49 3. Variable List: species (common) The common
 name of the species species (latin) The latin name of the species family
 The Family order The Order mating system* The mating system (U = unknown)
 colonial breeder* Whether the species is a colonial breeder or not (Y =
 yes, Y (sometimes) = sometimes, N = No) intruding male identityᶧ
 (PN:paired neighbouring male, N:neighbouring male, P:paired male,
 UP:unpaired male, U:no info; WPG:within-group male; EPG:extra-group male)
 Self-explanatory unforced extrapair copulations reportedᶧᶧ (Y: reported,
 N: not reported) in previous review whether the fact that this species
 engages in FEPC is in a previous review paper, and the review paper is
 reported. NA = Not reported in a previous review. ᶧprimary source of FEPC
 observation(s) Self-explanatory *Birds of the World citation
 Self-explanatory ᶧᶧsource of unforced extra-pair copulation observation(s)
 Self-explanatory Notes Any associated notes 4\. Missing data codes: NA/U
 5\. Specialized formats or other abbreviations used: None
 \#########################################################################
 .Fastq files All raw samples that were successfully sequenced are shared
 (n = 190 individuals, n = 380 samples consisting of two reads per
 individual – R1 and R2). Please see the manuscript methods and
 Supplementary methods for full details on how these files were generated *
 Files are zipped in DRYAD (fq.gz) * All filtering steps are described in
 our methods (manuscript and supplementary materials)