University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2017 Contemporary ancestor? Variation in marine threespine stickleback (Gasterosteus aculeatus) and its implications for adaptive divergence Morris, Matthew Morris, M. (2017). Contemporary ancestor? Variation in marine threespine stickleback (Gasterosteus aculeatus) and its implications for adaptive divergence (Unpublished doctoral thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/25434 http://hdl.handle.net/11023/3910 doctoral thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Contemporary ancestor? Variation in marine threespine stickleback (Gasterosteus aculeatus) and its implications for adaptive divergence by Matthew Richard John Morris A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY GRADUATE PROGRAM IN BIOLOGICAL SCIENCES CALGARY, ALBERTA JUNE, 2017 © Matthew Richard John Morris 2017 Abstract Standing genetic variation (SGV) can affect the incidence and pace of adaptation and parallel evolution. The role of SGV versus de novo mutation can be tested in ancestral-derived comparisons when the “contemporary ancestor” is extant. Assumptions about SGV in these contemporary ancestors require formal testing. The threespine stickleback is an icon of adaptive divergence, with multiple freshwater forms having evolved in parallel from a presumably panmictic, evolutionarily static marine population – in part from SGV at Ectodysplasin. Variation among marine stickleback would therefore have consequences for understanding adaptive divergence. I collected marine stickleback from eight locations between Alaska and California. Marine populations varied according to ecogeographic rules. Genotype-by- Sequencing of over 380 000 loci and 5700 SNPs revealed five genetic clusters, including one extending north from Washington to Alaska. Pairwise estimates of genetic differentiation (FST) ranged from 0.02 to 0.18. Tests of phenotypic divergence (PST-FST) for plate counts and body shape fell outside neutral evolutionary expectations, suggesting adaptive divergence may be maintaining this quantitative phenotypic variation among marine populations. Since SGV differed between populations, estimates of candidate loci exhibiting potential selection in response to freshwater colonisation varied depending on the marine population chosen as “ancestral”. It has been theorized that genome-wide heterozygosity improves fitness by buffering against asymmetry. If so, SGV could be maintained if it canalizes plate number. Although heterozygosity and asymmetry varied independently, SGV at Ectodysplasin acted as a genetic stressor that increased asymmetry. Critical thermal minima may have evolved from SGV. Contrary to expectations, marine and freshwater stickleback exhibited the same reaction norm for mitochondrial biogenesis, suggesting that biogenesis has not evolved but has retained an ancestrally adaptive phenotype. Collectively, these results reinforce that SGV is a complex and important factor in the evolution of “contemporary ancestors”, and that failure to take these complexities into account can lead to spurious interpretations of adaptation in derived populations. ii Preface Although this thesis represents my work, no research is conducted in a vacuum. Below is a description of my contribution and the contributions of co-authors (for submitted papers) or research assistants for each of Chapters Three through Six. Morris MRJ, Petrovitch E, Bowles E, Jamniczky HA, Sogers SM (second revised version awaiting approval). Exploring Jordan’s Rule in Pacific threespine stickleback Gasterosteus aculeatus. Journal of Fish Biology. MS 16-580R2. This paper presents Chapter Three in slightly modified form and has been accepted for publication pending minor revisions in the Journal of Fish Biology. Permissions from co-authors can be found in Appendix E. At this point no contract has been signed giving copyright ownership to the journal. The authors contributed as follows: I collected all but the Alaskan stickleback. E. Petrovitch was an undergraduate student who X-rayed all of the fish and did initial vertebral counts and measures of standard length. She submitted this work as an ECOL 507 paper entitled “Assessing Jordan’s Rule in coastal Pacific threespine stickleback.” Her work was the foundation for my work in this chapter; I re- scored all vertebral counts, partitioning them into abdominal and caudal vertebrae, genotyped most individuals at the idh locus to determine sex, counted all other meristic traits, did all of the statistics reported in this chapter, and wrote the manuscript. An additional undergraduate, R. Kaufman, did some of the idh genotyping. E. Bowles collected the Alaskan stickleback, which were vital to the success of this paper. H. Jamniczky and S. Rogers co-supervised E. Petrovitch, and funded this project. Morris MRJ, Bowles E, Allen B, Jamniczky HA, Rogers SM (submitted) Contemporary ancestor? Adaptive divergence from standing genetic variation in Pacific marine threespine stickleback. Evolution. 17-0292. This manuscript presents Chapter Four and has been submitted to Evolution. The authors contributed as follows: E. Bowles collected the Alaskan stickleback. E. Bowles, iii B. Allen and I collaborated to develop the Genotype-by-Sequencing protocol used in this thesis, which included modifications to the Stacks pipeline freely provided by Eric Normandeau, and R-code for using hierfstat and Adegenet. In particular, E. Bowles wrote the script used for GSnap and qqman used in this thesis. H. Jamniczky provided access to the μCT scanner for 3D morphometrics and valuable feedback on the morphology section of this paper. S. Rogers provided funding, feedback, and support throughout this project. Three undergraduates (E. Ellefson, V. Heather, R. Kaufman), not credited as co-authors, provided some assistance in PCR and gel electrophoresis for both idh and Eda, although most of this work was mine. One of these undergraduates also did initial plate counts, although for various reasons I did all of the counts again. I did the following: collected stickleback, extracted DNA, ran most of the PCR, scanned and landmarked all fish, did all morphological analyses, did the entire GBS pipeline and data analysis after initial development with E. Bowles and B. Allen, did all statistical analyses, and wrote the document. Chapter Five has not yet been submitted for publication. The GBS and Eda contributions have already been described. I further assessed plate position on both the left and right side of each stickleback twice to calculate measurement error. I did all statistical analyses and wrote the document. S. Rogers provided funding, feedback, and support. R. Kaufman was an undergraduate student who provided initial plate count information and did initial PCR for Eda and idh for half of the marine stickleback. For various reasons, I redid all of her work, but it is important to note her initial contribution. She published her results for ECOL 528 as “Standing genetic variation and latitudinal clines in threespine stickleback.” I have not sought permission to use her data for this thesis, as I redid the data collection. Chapter Six has not yet been submitted for publication. S. Smith, A. Pistore, and T. Barry collected all stickleback used in this project and did initial fish care at the Bamfield Marine Sciences Center. Once fish were shipped to the University of Calgary, I cared for them with the help of an undergraduate student, N. Hehar, and the occasional help of F. Malik. I designed the experiment. N. Hehar assisted with dissections. All cardiac tissue was processed by N. Tahbaz at the University of Alberta; he took images on the transmission electron microscope and I counted mitochondria from those images. iv W. Dong processed pectoral tissue at the University of Calgary and I took all images on the transmission electron microscope. Pectoral mitochondrial counts were divided between me and an undergraduate student, J. Rosebush. I wrote the manuscript and did all statistics. S. Rogers provided funding, feedback, and support. Publications during my PhD tenure not included in this thesis Morris MRJ, Rogers SM (2013) Overcoming maladaptive plasticity through plastic compensation. Current Zoology. 59: 526-536. Originally written for BIOL 607 – Special topics in biology: Plastic compensation. Morris MRJ (2014) We know in part: James McCosh on evolution and Christian faith. Journal of the History of Biology. 47: 363-410. Originally written for BIOL 607 – Special topics in biology: Darwin’s Origin of Species. Morris MRJ, Rogers SM (2014) Integrating phenotypic plasticity within an ecological genomics framework: recent insights from the genomics, evolution, ecology and fitness of plasticity. In: Ecological Genomics. Eds. CR Landry, N Aubin-Horth. Springer: UK. Originally written as the first chapter of this thesis, before this thesis transformed into something different. Morris MRJ, Richard R, Leder EH, Barrett RDH, Aubin-Horth N, Rogers SM (2014) Gene expression