The mechanisms underlying seasonal timing of breeding a multi-level approach using bi-directional genomic selection on timing of egg-laying Irene C. Verhagen Thesis committee Promotor Prof. Dr Marcel E. Visser Special Professor Ecological Genomics Wageningen University & Research Head of department, Department of Animal Ecology Netherlands Institute of Ecology (NIOO-KNAW), Wageningen Co-promotor Dr Veronika N. Laine Postdoctoral researcher, Department of Animal Ecology Netherlands Institute of Ecology (NIOO-KNAW), Wageningen Other members Prof. Dr Barbara Helm, Rijksuniversiteit Groningen Prof. Dr Bas J. Zwaan, Wageningen University & Research Dr Tyler J. Stevenson, University of Glasgow, UK Dr Koen J. F. Verhoeven, Netherlands Institute of Ecology (NIOO-KNAW) This research is conducted under the auspices of the Wageningen Institute of Animal Sciences (WIAS) graduate school. The mechanisms underlying seasonal timing of breeding a multi-level approach using bi-directional genomic selection on timing of egg-laying Irene Charlotte Verhagen Thesis submitted in fulfilment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. Dr A.J.P. Mol, in the presence of the Thesis Committee appointed by the Academic Board, to be defended in public on Friday 8 November 2019 at 11:00 a.m. in the Aula Irene C. Verhagen The mechanisms underlying seasonal timing of breeding: a multi-level approach using bi-directional genomic selection on timing of egg-laying 222 pages PhD thesis, Wageningen University, Wageningen, the Netherlands (2019) With references, with summaries in English and in Dutch ISBN 9778-94-6395-039-8 DOI 10.18174/496393 “We snatch in vain at Nature's veil, She is mysterious in broad daylight, No screws or levers can compel her to reveal, The secrets she has hidden from our sight.” (Johann Wolfgang von Goethe, 1808) Table of contents Chapter 1 General introduction 9 Chapter 2 Genetic and phenotypic responses to genomic selection for 23 timing of breeding in a wild songbird Chapter 3 Temperature has a causal and plastic effect on timing of 49 breeding in a small songbird Chapter 4 Exploration of tissue-specific gene expression patterns 61 underlying timing of breeding in contrasting temperature environments in a songbird Chapter 5 Fine-tuning of seasonal timing of breeding is regulated 97 downstream in the underlying neuro-endocrine system in a small songbird Chapter 6 Exploring temporal changes in DNA methylation and 135 RNA expression in a small songbird – correlations within and between tissues Chapter 7 General discussion 169 References 182 210 Summary Samenvatting 212 215 Curriculum vitae List of publications 216 219 Acknowledgements WIAS Training and Education Statement 221 CHAPTER 1 General introduction Chapter 1 Evolution and natural selection Evolution, the process that leads to the formation of and change in biological systems in response to their environment, has shaped life since its beginning and continues to do so. All organisms, simple and complex, evolve over different scales in both time and space. In general, evolution is described and studied as two distinct hierarchical processes; macro- evolution and micro-evolution. Macro-evolution refers to the origin of new morphological forms, species and divisions of the taxonomic hierarchy above the species level, together with the origin of complex adaptations, such as the eye (Reznick & Ricklefs 2009), but on which I will not dwell further. In contrast, micro-evolution refers to the processes of adaptive modifications (i.e. through alteration in allele frequencies in gene pools) within and among populations, propelled by mutation, migration, genetic drift and natural selection. The latter, put first into words by, independently, both Charles Darwin (1859) and Alfred Russel Wallace (1858), is usually defined as the consequence of three organismal properties: (1) variation among organisms of a population, (2) differential reproduction, and (3) traits that are important for survival or reproduction show heritability (Darwin 1859; Wallace 1858). In general, natural selection is the tendency of beneficial traits to increase in frequency in populations to make them better able to survive and reproduce in its (changing) environment or habitat (Maynard Smith 1989). Basically, when variation in a heritable trait is caused by differential reproduction, the change in the average phenotype of the population is the result from the greater contribution to each generation by the fittest individuals (i.e. individuals with traits best adapted to its environment to survive) (Lewontin 1974; Roughgarden 1979), or as Darwin (1859) stated “One general law, leading to the advancement of all organic beings, namely, multiply, vary, let the strongest live and the weakest die.” Therefore, natural selection ‘prefers’ the fittest organism (or population) with respect to a given trait, leading to adaptation of that trait. A textbook example is the peppered moth (Biston betularia), where natural selection acted in favour of dark-coloured moths as a consequence of air pollution during the Industrial Revolution in Great Britain and a subsequent increase in predation of white-coloured moths by birds (Kettlewell 1958). As such, the population adapted by increasing the occurrence of dark-coloured moths and persisted. Adapting to a warming world: novel and intensified selection pressures However, due to twentieth-century anthropogenic disturbances, most particularly the continuous increase in ambient temperatures (IPCC 2014), many species experience novel and intensified selection pressures (Merilä & Hoffmann 2016). As such, global warming has important ecological consequences and evolutionary impacts on wild populations (Peñuelas & Filella 2001; Walther et al. 2002). Shifts in phenology, i.e. recurring seasonal events, by organisms are among the most observed and studied in relation to climate change (e.g. Parmesan & Yohe 2003; Root et al. 2003; Walther et al. 2002, but see Cohen et al. 2018). Species across taxa have shown mostly advancements in seasonal timing of, among others, 10 General introduction migration (Hüppop & Hüppop 2003), breaking hibernation (Inouye et al. 2000), flowering time (Cayan et al. 2001; Menzel & Dose 2005), spawning date (Beebee 1995) and emergence (Forister & Shapiro 2003; Roy & Sparks 2000). However, these shifts differ in rate between trophic levels, most notably in higher trophic levels (Thackeray et al. 2010, 2016). Therefore, species-specific variation in phenological responses to the environment can disrupt the synchrony of ecological interactions (Harrington et al. 1999; Peñuelas & Filella 2001; Visser & Both 2005). As such, the impacts of climate change may be mediated through these so-called ‘mismatches’, as they result in major consequences for individual fitness and population persistence (Both et al. 2006; Møller et al. 2008; Platt et al. 2003; Visser & Gienapp 2019; Winder & Schindler 2004; but see Reed et al. 2013). Before predictions can be made about how future climate change will affect populations, we however need to obtain a far better understanding of the underlying physiological mechanisms that link environmental cues to life-history decisions, like timing of breeding (Pörtner & Farrell 2008) and if so, how environmental cues affects these. It proves difficult, at least in wild populations, to determine the actual environmental factor that changes phenotypes and drives selection, which could be numerous (Merilä & Hendry 2014). Avian seasonal timing of breeding In birds from temperate zones, continuously rising temperatures have the most predominant effect on the timing of their breeding, i.e. the date at which females initiate egg-laying (e.g. Both et al. 2004; Brown et al. 1999; Crick et al. 1997; Dunn & Winkler 1999; Love et al. 2010; Matthysen et al. 2011). Temperate zone birds rely on food resources to raise their offspring, which usually become available or increases in abundance for a brief period each year (Lack 1968; Perrins 1970; Verhulst & Tinbergen 1991), making them seasonal breeders. This period varies every year due to yearly varying environmental conditions. As such, the onset of breeding needs to be optimally timed, or synchronized, to this increased availability of food resources in order to support successful rearing of offspring (Charmantier et al. 2008; van Noordwijk et al. 1995; Perrins 1965; Sheldon, Kruuk & Merilä 2003). This synchronization is a delicate matter, because the recrudescence of gonads and early offspring development occurs well before offspring care, which is due to the length of the egg incubation period. As such, synchronization of the breeding season to environmental conditions that favour offspring needs, requires extensive physiological and behavioural ‘preparations’. Growth and maturation of the reproductive system can take up 6-8 weeks and is accompanied and followed by finding a mate and nest site, nest building, and ultimately egg-laying and incubation. It follows from this temporal separation of reproductive events that birds need to be able to anticipate or predict the onset of the breeding season, for which they use information from environmental cues. These cues are predominantly photoperiod (Dawson et al. 2001; Farner 1985; Follett 1984; Gwinner 1986; Sharp 1996; Silverin et al. 1993; Wingfield 1993) and temperature (Caro & Visser 2009; Lambrechts & Visser 1999; Williams 2012), which provide information about the time of 11 Chapter 1 the