DOI: 10.5061/dryad.k3j9kd5h8
title: DNA methylation associates with sex-specific effects of experimentally increased yolk testosterone in wild nestlings

description: Maternal hormones can profoundly impact offspring physiology and behaviour
 in sex-dependent ways. Yet little is known on the molecular mechanisms
 linking these maternal effects to offspring phenotypes. DNA methylation,
 an epigenetic mechanism, is suggested to facilitate maternal androgens’
 effects. To assess whether phenotypic changes induced by maternal
 androgens associate with DNA methylation changes, we experimentally
 manipulated yolk testosterone levels in wild great tit eggs (Parus major)
 and quantified phenotypic and DNA methylation changes in the hatched
 offspring. While we found no effect on the handing stress response,
 increased yolk testosterone levels decreased the begging probability,
 emphasised sex-differences in fledging mass and affected methylation at
 763 CpG sites, but always in a sex-specific way. These sites associated
 with genes involved in growth, oxidative stress and reproduction,
 suggesting sex-specific trade-offs to balance the costs and benefits of
 exposure to high yolk testosterone levels. Future studies should assess if
 these effects extend beyond the nestling stage and impact fitness.
description: # DNA methylation associates with sex-specific effects of experimentally
 increased yolk testosterone in wild nestlings All data and scripts related
 to the manuscript "DNA methylation associates with sex-specific
 effects of experimentally increased yolk testosterone in wild
 nestlings" by Bernice Sepers, Suvi Ruuskanen, Tjomme van Mastrigt and
 Kees van Oers. --- Explanation of column names that are included in many
 data files: ID = identity of an individual great tit (Parus major);
 brood_origin = brood of origin, the number of the nest box where an
 individual was born; brood_rearing = brood of rearing, the number of the
 nest box where an individual was raised; treatment = experimental
 treatment group, either testosterone (i.e. hatched from egg injected with
 testosterone + sesame oil) or control (i.e. hatched from egg injected with
 sesame oil); sex = sex of the individual. * "HS_data.xlsx"
 includes the raw handling stress data (sheet HS) and the handling stress
 estimates (sheet HS_ESTIMATES) for each nestling on day 14 after hatching.
 Column explanations: april_date = April day on which the breath rate (i.e.
 handling stress test) was recorded; temperature = air temperature in °C at
 the test location; bout = bout number (15 seconds) within one minute;
 breath rate = number of breaths (breast movements) per bout; time minutes
 = time in minutes since midnight; estimate = handling stress estimate. *
 "video_data.xlsx" includes the video data, including nestling
 food solicitation behaviour, on day eight after hatching. Column
 explanations: visit_number = parental visit number; recorded_brood =
 recorded brood, i.e. brood of rearing; fed_ID = ID of the nestling that
 received the prey item; fed_treatment = treatment of the nestling that
 received the prey item; fed_sex = sex of the nestling that received the
 prey item; fed_brood_origin = brood of origin of the nestling that
 received the prey item; ID_all = nestling ID; brood_origin_all = brood of
 origin; begging_all = indicates which nestlings showed a begging response
 right before feeding (1 = begging response shown, 0 = no begging response
 shown); fed_all = indicates which nestling received the prey item (1 =
 received the prey item, 0 = did not receive the prey item); treatment_all
 = experimental treatment group; sex_all = nestling sex. *
 "barcode_files_epiGBS2020_B19-B23.xlsx" includes the unique
 barcode combinations needed to demultiplex the raw DNA methylation
 (epiGBS2) data on NCBI (see below). Column name explanation: Flowcell =
 sequencing flowcell; Lane = sequencing lane; Barcode_R1 = barcode ligated
 to the 5 prime – 3 prime DNA strands (i.e. R1 or BA); Barcode_R2 = barcode
 ligated to the 3 prime – 5 prime DNA strands (i.e. R2 or CO); Sample =
 individual; ENZ_R1 = restriction enzyme; ENZ_R2 = restriction enzyme;
 Wobble_R1 = length (in nucleotides) of the random nucleotide sequence
 (i.e. UMI) in the adapter of the 5 prime – 3 prime DNA strands; Wobble_R2
 = length (in nucleotides) of the random nucleotide sequence (i.e. UMI) in
 the adapter of the 3 prime – 5 prime DNA strands; Species = study species.
 * "info_nestlings_epigbs.txt" includes nestling information to
 link to the DNA methylation data. Column name explanation: File_location =
 location of the DNA methylation call file name, output of the epiGBS2
 pipeline. Any cells that contain "NA" represent missing values
 or the column does not apply to that individual. --- *
 "Rscript_HS.R" includes the script to analyse the nestling
 handling stress data in "HS_data.xlsx" *
 "Rscript_video.R" includes the script to analyse the video data
 (nestling food solicitation behaviour and feeding) in
 "video_data.xlsx". * "Rscript_brood_characteristics.R"
 includes the script to analyse the incubation length data, the clutch size
 data, the hatching success data and the egg weight data. The data are
 archived in the SPI-Birds Database
 ([https://spibirds.org/en](https://spibirds.org/en)) and can be requested.
 * "Rscript_biometry_nestlings.R" includes the script to analyse
 the nestling biometry data (weight on day two, six, eight and 14 and
 tarsus length on day 14). The data are archived in the SPI-Birds Database
 ([https://spibirds.org/en](https://spibirds.org/en)) and can be requested.
 * "Rscript_methylation_filtering.R" includes the script to
 filter the DNA methylation (epiGBS2) data. *
 "Rscript_methylation_analyses.R" includes the script to analyse
 the DNA methylation data. * "Rscript_methylation_annotation.R"
 includes the script to annotate CpG sites. --- * The raw genomic epiGBS2
 reads are available on NCBI under BioProject PRJNA208335 under the SRA
 accessions SRX22027777, SRX22027778, SRX22027779, SRX22027780 and
 SRX22027781. * The epiGBS2 bioinformatics pipeline can be accessed on
 github
 ([https://github.com/nioo-knaw/epiGBS2](https://github.com/nioo-knaw/epiGBS2)). --- For more information on data collection and analysis, please refer to the published manuscript and the Supplementary Material. Also, please feel free to contact Bernice Sepers ([b.sepers@nioo.knaw.nl](mailto:b.sepers@nioo.knaw.nl) or [bernice.sepers@uni-bielefeld.de](mailto:bernice.sepers@uni-bielefeld.de)) or Kees van Oers ([k.vanoers@nioo.knaw.nl](mailto:k.vanoers@nioo.knaw.nl)) if you have any further questions or comments.