Data from: ultimate drivers of forced extra-pair copulations in birds lacking penises: jackdaws as a case-study

Published Jan 08, 2024 on Dryad. https://doi.org/10.5061/dryad.3ffbg79r1

Abstract

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.

Author: “Beki Hooper”
Date: ‘2024-01-07’

GENERAL INFORMATION

Title of Dataset: Ultimate drivers of forced extra-pair copulations in birds lacking penises: jackdaws as a case-study
Author Information
A. Principal Investigator Contact Information
Name: Alex Thornton
Institution: University of Exeter
Email: alex.thornton@exeter.ac.uk
B. Associate or Co-investigator Contact Information
Name: Beki Hooper
Institution: NA
Email: Rebecca.hooper@evobio.eu
Date of data collection (single date, range, approximate date): 2014 – 2019
Geographic location of data collection: Cornwall, UK
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

Licenses/restrictions placed on the data: CC0 1.0 Universal (CC0 1.0) Public Domain
Links to publications that cite or use the data: NA
Links to other publicly accessible locations of the data: None
Links/relationships to ancillary data sets: None
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
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: https://doi.org/10.5061/dryad.3ffbg79r1

DATA & FILE OVERVIEW

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
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)
Additional related data collected that was not included in the current data package: None
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?

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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.

Number of variables: 44
Number of rows: 11241
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
Missing data codes: NA
Specialized formats or other abbreviations used: None

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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

Number of variables: 2
Number of rows: 133
Variable List:
UNIQUE_TR_ID As above – unique identifier per video.
TIME_TOG_TOTAL Time spent together in the nestbox in seconds.
Missing data codes: NA
Specialized formats or other abbreviations used: None

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DATA-SPECIFIC INFORMATION FOR: birthyear_sequoia.csv
This file contains birth year and sex data on each individual used in the genomic analysis.

Number of variables: 5
Number of rows: 193
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
Missing data codes: NA
Specialized formats or other abbreviations used: None

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DATA-SPECIFIC INFORMATION FOR: site_sequoia.csv
This file contains the site in which each individual was born.

Number of variables: 2
Number of rows: 4153
Variable List:
ID ID of the jackdaw
LOCATION Site where the jackdaw was born
Missing data codes: NA
Specialized formats or other abbreviations used: None

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DATA-SPECIFIC INFORMATION FOR: ped_sequoia.csv
This file contains the dam and sire of each sampled individual

Number of variables: 3
Number of rows: 193
Variable List:
id ID of the jackdaw
dam mother of the individual. NA if unknown.
sire father of the individual. NA if unknown.
Missing data codes: NA
Specialized formats or other abbreviations used: None

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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.

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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).

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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.

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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).

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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.

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Supplementary Data.xlsx
This file contains information on bird species without penises in which FEPCs have been observed.

Number of variables: 13
Number of rows: 49
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

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.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)

Behavioural data

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)).

Sampling, DNA Extraction and Sequencing

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 <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.

Bioinformatic pipeline

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 >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 >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.

Data from: ultimate drivers of forced extra-pair copulations in birds lacking penises: jackdaws as a case-study

Data files

Abstract

README: Data from: ultimate drivers of forced extra-pair copulations in birds lacking penises: jackdaws as a case-study

Methods