A longitudinal analysis of telomeres in an insular house sparrow population Aaron Sibma A thesis submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy The University of Sheffield Faculty of Science Department of Animal & Plant Sciences Date of submission: 8th February 2021 Abstract Telomeres show variation in length, both between and within species, and have been proposed as a biomarker of biological ageing, providing information on the current physiological state of individuals, and reflecting (accumulated) somatic damage over time. The underlying causes of variation in telomere length remain unclear. In this thesis, I studied telomere dynamics in a wild passerine population. First, I assessed the methodological consequences of storage of blood in ethanol for the subsequent measurement of telomere length using qPCR. I found that telomere length estimates shorten in a quadratic relationship when blood samples are stored for relatively long periods. I then show that telomeres shorten with age within individuals, and that individuals with shorter telomeres disappear more quickly from the population. Moreover, individuals showed low repeatability of telomere length over time, with some individuals showing significant elongation of telomeres. Using pedigree information, I then estimated the heritability of telomere length, and concluded that this is low in house sparrows, and the variation in telomere length of offspring was explained more by the year of birth. In addition, the age of the biological father had a negative effect on offspring telomere length. I also found that the age of the rearing male had a positive effect on offspring telomere length, but only at the population level. This, again, indicates the selective disappearance of individual adults and/or offspring with shorter telomeres. In conclusion, my results show that telomeres may not reflect accumulated damage but may instead be more plastic than previously thought. Telomeres may instead reflect more immediate circumstances of the individual. Regardless, individuals overall still showed significant telomere attrition with age, and individuals with relatively short telomeres disappeared from the population, supporting the use of telomeres as a biomarker of ageing in wild passerines. 1 2 Acknowledgements First, I would like to thank my supervisor, Professor Terry Burke, who has provided me with guidance throughout my PhD, even after I decided to work part-time and live back in the Netherlands. Our almost weekly meetings were of great help. Moreover, it was a privilege to be able to work on telomeres and have a large multi-year dataset available that was ready to be explored. I would also like to thank Mirre Simons for providing the statistical knowledge that was needed to complete my PhD. I may not always have been able to immediately grasp the statistics you explained in so much detail, but your explanations were immensely helpful, so thank you for having patience with me. And then I would like to thank Julia Schroeder for providing valuable insights in heritability, and for reading many of my chapters. Our fieldwork wintertrips to Lundy, organised in detail by you to accommodate a great number of master students, were a real treat. You provided valuable feedback that I used to improve my work. Many thanks to Nathalie Dos Remedios and Marie-Elena Mannarelli for measuring all the telomeres lengths in thousands of samples and providing me with knowledge on how to measure telomere length on the qPCR. Then I would like to thank all the people on Lundy for keeping me sane during the many months I spent on Lundy, running from nest to nest and trying to catch all sparrows. Without the great people on Lundy providing good company and friendship, I would not have been able to keep going for so long. And of course, a great thanks to my family, who were supportive right from the start, even when they realized I would be living abroad on an island, and then stay 3 to 4 months each year on an even smaller and completely remote island. 3 4 List of contents Abstract ................................................................................................................................................... 1 Acknowledgements ................................................................................................................................. 3 List of contents ........................................................................................................................................ 5 1. General introduction ......................................................................................................................... 10 1.1 Senescence .................................................................................................................................. 10 1.2 Telomeres and senescence ......................................................................................................... 11 1.3 House sparrows on Lundy ........................................................................................................... 12 1.4 Measuring telomeres .................................................................................................................. 13 1.5 Telomeres and sample storage ................................................................................................... 16 1.6 Telomere attrition and elongation .............................................................................................. 16 1.7 Telomeres and survival ............................................................................................................... 21 1.8 Heritability and parental effects ................................................................................................. 22 1.9 Questioning causal involvement ................................................................................................. 23 1.10 Thesis outline ............................................................................................................................ 25 1.11 Contributions made to this thesis ............................................................................................. 25 2. Blood sample storage in ethanol affects telomere length estimation by qPCR ............................... 27 2.1 Summary ..................................................................................................................................... 27 2.2 Introduction ................................................................................................................................ 27 2.3 Methods ...................................................................................................................................... 30 2.3.1 Sample collection ................................................................................................................. 30 2.3.2 DNA extraction ..................................................................................................................... 30 2.3.3 Telomere qPCR measurement ............................................................................................. 31 2.3.4 Experiment ........................................................................................................................... 33 2.3.5 Statistical analysis ................................................................................................................ 35 2.3.6 Repeatability of telomere lengths ....................................................................................... 36 2.4 Results ......................................................................................................................................... 36 2.5 Discussion .................................................................................................................................... 39 Supporting information S2. ............................................................................................................... 43 3. Longitudinal data reveal selective disappearance of individuals with short telomeres ................... 44 3.1 Summary ..................................................................................................................................... 44 3.2 Introduction ................................................................................................................................ 44 5 3.3 Methods ...................................................................................................................................... 48 3.3.1 Study system ........................................................................................................................ 48 3.3.2 Telomere length ................................................................................................................... 48 3.3.3 Storage time effects ............................................................................................................. 49 3.3.4 Age of birds, lifespan, and survival ...................................................................................... 50 3.3.5 Statistical analysis ................................................................................................................ 50 3.3.6 Age effects ........................................................................................................................... 50 3.3.7 Survival and lifespan effects ...............................................................................................