DOI: 10.5061/dryad.zkh1893k8
title: Data from: Geographic differences in the phenology of gonadal development and moult, but not of egg laying, are genetically based in a small songbird

description: To forecast how fast populations can adapt to climate change, it is
 essential to determine the evolutionary potential of different life-cycle
 stages under selection. In birds, timing of gonadal development and moult
 are primarily regulated by photoperiod, while laying date is highly
 phenotypically plastic to temperature. We tested whether geographic
 variation in phenology of these life-cycle events between populations of
 great tits (Parus major) has a genetic basis, indicating that contemporary
 genetic adaptation is possible. We carried out a common garden experiment
 in which we bred first- and second-generation pairs in captivity
 originating from eggs from Gotland (Sweden) and Hoge Veluwe (Netherlands),
 two populations that showed different temperature sensitivity of laying
 date in a recent meta-analysis. We recorded the phenology of egg-laying,
 moult and gonadal size in early spring. We found no significant
 differences in laying date between the populations, but they did differ in
 moult timing and testis size. This implies that under climate change the
 timing of gonadal development and moult, which are mainly regulated by
 photoperiod, will not respond to increased temperature but can respond by
 genetic adaptation in response to selection, while the opposite holds for
 laying date, perhaps indicating that plasticity is constraining genetic
 adaptation.
description: <em>Setting up common garden
 experiment</em> Birds: In the
 spring of 2021, freshly laid, unincubated eggs were transported from eight
 populations to the NIOO: La Rouvière (France), Boshoek (Belgium), Wytham
 Woods (England), Gotland (Sweden) and four Dutch populations (Hoge Veluwe,
 Vlieland, Oosterhout and Liesbos). The eggs were then taken to a Dutch
 population (Bennekomse Bos, lat: 52,003; long: 5,708) where they were
 placed in foster nests for incubating and early parental care.  At 10 days
 post-hatching, these chicks were transported to the Netherlands Institute
 of Ecology and hand raised, following procedures as outlined in [1]. They
 were blood sampled and based on their genotypes (using five microsatellite
 regions Pma-TGAn33, PmaC25, PmaTAGAn71, PmaGAn27 and PmaD10 [2]) and that
 of the potential parents, they were assigned to a family following a
 standard protocol [3]. For a number of these
 populations there were severe problems leading to very low numbers of
 chicks (see Appendix 1 for more info on all eight populations). Hence, we
 only include offspring from Gotland (Sweden, lat: 57,063, long: 18,278)
 and Hoge Veluwe (Netherlands, lat: 52,041, long: 5,856) in our analysis.
 Note that due to the different timing of eggs laying the two populations
 the Gotland eggs were placed seven days later at the foster parent nests
 than the Hoge Veluwe eggs (7 May vs 30 April). The
 following year (2022) we formed first generation (F1) breeding pairs
 (within populations) from these birds and kept them from January onwards
 in pairs in open aviaries at the Netherlands Institute of Ecology with
 <em>ad libitum</em> food (constant daily amount of food
 consisting of a mixture of minced beef, proteins and vitamins, sunflower
 seeds, fat, a mix of dried insects, a mixture of proteins, vitamins,
 minerals and trace elements (Ce´De´-mix), a surplus of calcium, water for
 drinking and bathing, nesting material and four nestboxes as nesting
 opportunities. The eggs produced where collected every morning and put in
 an egg turner (i.e. a device that gently rocks eggs during storage – a
 CocinaCo 154 Eggs Quail Turner Tray Container). Eggs
 were, within five days of laying, taken to the Bennekomse Bos to foster
 parents. We put Gotland and Hoge Veluwe eggs together in foster broods
 (with a total clutch between 5 and 11 eggs, 9 on average) to ensure common
 conditions during incubation and early chick rearing. Note that as there
 was no difference in laying date between the populations for the F1 pairs,
 eggs were taken to their foster nests during the same period. At day 10
 post-hatching the chicks were taken to the Netherlands Institute of
 Ecology, hand raised, blood sampled and assigned to a family.
 In the following year (2023) 20 Gotland and 20 Hoge Veluwe second
 generation (F2) breeding pairs were set-up. As these F2 birds originate
 from eggs produced in a common garden setting, and thus any carry over
 effects of the location the eggs were produced are excluded, any
 differences between them will be genetic. In total 7 Hoge Veluwe and 8
 Gotland F1 pairs and 20 Hoge Veluwe and 20 Gotland F2 pairs were used
 (number of pairs that produced a clutch was 7, 7, 20 and 17
 respectively). Aviaries: Breeding
 pairs were set up in 40 outdoor aviaries of 4 m x 2 m x 2 m (l x w x h)
 with on one side a mesh, allowing natural light and ambient temperatures.
 Despite being exposed to natural light, all aviaries are still darker than
 natural conditions. This causes the birds in aviaries to consistently lay
 later than wild birds if left without additional light. Thus, a
 fluorescent light tube provided additional light in the morning for all
 breeding pairs. In January and February lights were on from sunrise until
 midday (<em>i.e.</em> same as normal housing conditions) and
 from March onwards lights were on 2h and 15 min before sunrise until
 midday. This additional light was crucial for eggs to be laid while foster
 nests were still available to produce the F2 birds. The aviary building
 consisted of two rows of aviaries (20 West and 20 East facing) and to
 minimize the impact of any systematic variation in conditions between
 aviaries we kept the Gotland and Hoge Veluwe pairs in alternating
 aviaries. <em>Phenotyping</em>
 Laying date: Laying dates were recorded for
 both the Hoge Veluwe and Gotland F1 and F2 generations (see Appendix 1 for
 laying dates of F1 birds from other populations). Nest boxes were checked
 daily for nest building progress and new eggs, and the laying date was the
 day the first egg was laid. Eggs were replaced by plastic dummy eggs, and
 upon clutch completion females were allowed to incubate for four complete
 days after which nests and dummy eggs were removed on the fifth day.
 Frequently, pairs would initiate replacement clutches. Here, we only
 analyse the laying date of the first broods, i.e. the first clutch of the
 season. Moult: Moult was scored once
 in both years at the end of the breeding season (F1 =
 17<sup>th</sup> of June in 2022 and F2 =
 16<sup>th</sup> of June in 2023). We inspected the right wing
 of each bird and gave 10 scores per bird, one for each primary feather
 (P1-P10), from 0 to 5 (0 old feather, 5 fully grown), reflecting how much
 it had grown [4]. Then, we obtained a moult score per individual by
 converting each of the 0-5 moult scores into an approximate proportion of
 feather grown [following 5], multiplied by the respective mass of that
 particular feather, and finally summed the values of all feathers. This
 resulted in a single value ranging between 0 and 1. The mass-corrected
 moult score serves as a proxy for moult timing because all birds were
 scored on the same day and feather mass increases fairly linearly
 throughout the season [5–7]. Thus, the larger the score, the furthest the
 bird is in its moult progress and consequently the earlier it started
 moulting. Gonadal size: We measured
 the gonadal sizes of F2 birds on 22, 23, 26 & 27 Feb 2024. Birds,
 19 Hoge Veluwe and 19 Gotland pairs, were kept in the same outdoor
 aviaries as in the F2 breeding season of 2023. The birds, alternating
 between pairs from Gotland and Hoge Veluwe, were put under isoflurane
 after which they were decapitated and the length (mm), width (mm) and
 fresh mass (mg) of their gonads measured. We calculated testis and ovary
 volume as V = 4/3.π.a<sup>2</sup>.b, where a is width (mm)/2
 and b is length (mm)/2.
 <em>Statistics</em> Laying date,
 moult score and gonad volume were analysed using generalized linear models
 using the lm function of lme4 [8] in R version 4.3.2 [9], with population
 (Gotland or Hoge Veluwe), generation (F1 or F2) and (for moult and gonads)
 sex (male or female), and their interactions, as explanatory variables.
 For testis the right testis was analysed (as one left testis was missing,
 reducing the sample size and as left and right testis volume were highly
 correlated (Pearson correlation: 0.96)). Following Schaper et al. [10], we
 use the <sup>10</sup>log gonadal volume as the distribution of
 the logged values better follow a normal distribution.
 References: 1.           Drent PJ, van Oers K,
 van Noordwijk AJ. 2003 Realized heritability of personalities in the great
 tit (Parus major). <em>Proc. R. Soc. Lond. Ser. B-Biol.
 Sci.</em> <strong>270</strong>, 45–51.
 2.           Saladin V, Bonfils D, Binz T, Richner H. 2003
 Isolation and characterization of 16 microsatellite loci in the Great Tit
 <em>Parus major</em>. <em>Mol. Ecol. Notes</em>
 <strong>3</strong>, 520–522. 3.          
 Greives TJ <em>et al.</em> 2015 Costs of sleeping in:
 circadian rhythms influence cuckoldry risk in a songbird. <em>Funct.
 Ecol.</em> <strong>29</strong>, 1300–1307.
 (doi:10.1111/1365-2435.12440) 4.           Ginn HB,
 Melville DS. 1983 <em>Moult in Birds</em>. British Trust for
 Ornithology. See https://books.google.nl/books?id=Sd9FAAAAYAAJ.
 5.           Underhill LG, Zucchini W. 1988 A model for avian
 primary moult. <em>Ibis</em> <strong>130</strong>,
 358–372. (doi:10.1111/j.1474-919X.1988.tb00993.x)
 6.           Dawson A, Newton I. 2004 Use and validation of a
 molt score index corrected for primary feather mass.
 <em>Auk</em> <strong>121</strong>,
 372–379. 7.           Dawson A. 2003 A detailed
 analysis of primary feather moult in the Common Starling Sturnus vulgaris–
 new feather mass increases at a constant rate. <em>Ibis</em>
 <strong>145</strong>, E69–E76.
 (doi:10.1046/j.1474-919X.2003.00161.x) 8.          
 Bates D, Machler M, Bolker BM, Walker SC. 2015 Fitting Linear
 Mixed-Effects Models Using lme4. <em>J. Stat. Softw.</em>
 <strong>67</strong>, 1–48. 9.          
 R_Core_Team. 2021 <em>R: A Language and Environment for Statistical
 Computing</em>. Vienna, Austria: R Foundation for Statistical
 Computing. 10.        Schaper SV, Gienapp P, Dawson A,
 Visser ME. 2013 Heritability of gonad size varies across season in a wild
 songbird. <em>J. Evol. Biol.</em>
 <strong>26</strong>, 2739–2745.
 (doi:10.1111/jeb.12249)
description: # Geographic differences in the phenology of gonadal development and
 moult, but not of egg laying, are genetically based in a small songbird
 [https://doi.org/10.5061/dryad.zkh1893k8](https://doi.org/10.5061/dryad.zkh1893k8) ## Description of the data and file structure To study to what extent geographic variation in laying date in great tits has a genetic basis, we carried out a two-generation common garden experiment in which we bred great tits that originated from eggs retrieved from two populations that in the Bailey et al. (2002, Nature Communications) analysis showed different temperature sensitivity. Such a common garden approach can show that there are genetic differences underlying this difference in sensitivity, as the confounding effects of different environments that shape the phenotype, as it occurs in the wild, are lifted. Such local adaptation would demonstrate that evolution has occurred over time, providing insights into the potential for genetic adaptation in current populations under selection. We measured three life cycle events: laying date of the breeding pairs (primarily sensitive to temperature) as well as seasonal timing of moult and gonadal development (primarily sensitive to photoperiod). ### Files and variables #### File: Common\_garden\_gonads\_follicle.csv **Description:** We measured the gonadal sizes of F2 birds on 22, 23, 26 & 27 Feb 2024. Birds, 19 Hoge Veluwe and 19 Gotland pairs, were kept in the same outdoor aviaries as in the F2 breeding season of 2023. The birds, alternating between pairs from Gotland and Hoge Veluwe, were put under isoflurane after which they were decapitated and the length (mm), width (mm) and fresh mass (mg) of their gonads measured. We calculated testis and ovary volume as V = 4/3.π.a2.b, where a is width (mm)/2 and b is length (mm)/2. ##### Variables * RingNumber: Unique number per individual * Population: Population (Hoge Veluwe or Gotland) * FollicleVolume: Volume of female follicle (mm3) * LogFollicleVolume: 10log of FollicleVolume  * FemaleMother: ring number of female's mother #### File: Common\_garden\_gonads\_testis.csv **Description:** We measured the gonadal sizes of F2 birds on 22, 23, 26 & 27 Feb 2024. Birds, 19 Hoge Veluwe and 19 Gotland pairs, were kept in the same outdoor aviaries as in the F2 breeding season of 2023. The birds, alternating between pairs from Gotland and Hoge Veluwe, were put under isoflurane after which they were decapitated and the length (mm), width (mm) and fresh mass (mg) of their gonads measured. We calculated testis and ovary volume as V = 4/3.π.a2.b, where a is width (mm)/2 and b is length (mm)/2. ##### Variables * RingNumber: Unique number per individual * Population: Population (Hoge Veluwe or Gotland) * RightTestisVolume: Volume of male right testis (mm3) * LogRightTestisVolume: 10log of RightTestisVolume * MaleMother: ringnumber of male's mother #### File: Common\_garden\_gonads.R **Description:** R script to analyse the gonadal data #### File: Common\_garden\_gonads.xlsx **Description:** Excel version of Common_garden_gonads_follicle.csv & Common_garden_gonads_testis.csv #### File: Common\_garden\_laying\_date.csv **Description:** Nest boxes were checked daily for nest building progress and new eggs, and the laying date was the day the first egg was laid. We only analyse the laying date of the first broods, i.e. the first clutch of the season. ##### Variables * Year:   Year * Aviary:   Aviary number in building 9 of the NIOO (1-40, missing numbers are aviaries in which no first brood was produced * Female:   Unique number per individual female * Female: Generation F1=first generation, F2=second generation female * FemaleMother: Ring number of female's mother * FemaleArea:  Population from which the female originates (Hoge Veluwe or Gotland) * Male:   Unique number per individual male * MaleGeneration:  F1=first generation, F2=second generation male * MaleMother: Ring number of male's mother * MaleArea:  Population from which the male originates (Hoge Veluwe or Gotland) * BroodType:  0 = first brood (see above) * LayDate:   Laying date in DD/MM/YYYY * LayAprilDate:  Laying date in April days (i.e. 1 April = day 1) #### File: Common\_garden\_laying\_date.R **Description:** R script to analyse the laying date data #### File: Common\_garden\_laying\_date.xlsx **Description:** Excel version of Common_garden_laying date.csv #### File: Common\_garden\_moult.csv **Description:** Moult was scored once in both years at the end of the breeding season (F1 = 17th of June in 2022 and F2 = 16th of June in 2023). We inspected the right wing of each bird and gave 10 scores per bird, one for each primary feather (P1-P10), from 0 to 5 (0 old feather, 5 fully grown), reflecting how much it had grown [1]. Then, we obtained a moult score per individual by converting each of the 0-5 moult scores into an approximate proportion of feather grown [following 2], multiplied by the respective mass of that particular feather, and finally summed the values of all feathers. This resulted in a single value ranging between 0 and 1. The mass-corrected moult score serves as a proxy for moult timing because all birds were scored on the same day and feather mass increases fairly linearly throughout the season [2–4]. Thus, the larger the score, the furthest the bird is in its moult progress and consequently the earlier it started moulting. 1\. Ginn HB, Melville DS. 1983 Moult in Birds. British Trust for Ornithology. See [https://books.google.nl/books?id=Sd9FAAAAYAAJ](https://books.google.nl/books?id=Sd9FAAAAYAAJ). 2\. Underhill LG, Zucchini W. 1988 A model for avian primary moult. Ibis 130, 358–372. (doi:10.1111/j.1474-919X.1988.tb00993.x) 3\. Dawson A, Newton I. 2004 Use and validation of a molt score index corrected for primary feather mass. Auk 121, 372–379. 4\. Dawson A. 2003 A detailed analysis of primary feather moult in the Common Starling Sturnus vulgaris– new feather mass increases at a constant rate. Ibis 145, E69–E76. (doi:10.1046/j.1474-919X.2003.00161.x) ##### Variables * RingNumber: Unique number per individual * Sexe:  1=female, 2=male * Generation: F1=first generation, F2=second generation * Area:  Population (Hoge Veluwe or Gotland) * MoultWeight: Moult score - see above * Mother: Ring number of individual's mother #### File: Common\_garden\_moult.R **Description:** R script to analyse the moult data #### File: Common\_garden\_moult.xlsx **Description:** Excel version of Common_garden_moult.csv