Resurrecting an Urban Sunflower Population: Phenotypic and Molecular Changes Over 36 Years A THESIS SUBMITTED TO THE FACULTY OF THE UNIVERSITY OF MINNESOTA BY Marissa Marie Spear IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE Briana L. Gross, Julie R. Etterson December 2019 © 2019 Marissa Marie Spear Acknowledgements I would like to thank my advisor, Dr. Briana Gross, for her counsel and unwavering support throughout the duration of this project. I would also like to thank my co-advisor, Dr. Julie Etterson, for sharing her scientific expertise. From experimental design to data analysis, I appreciate both my advisors’ commitment to high-quality science. I would also like to thank my committee member Dr. Amanda Grusz for her input on this project. Additionally, I would also like to thank members of the Gross Lab, who have helped with data collection and general moral support, as well as the many undergraduate students who assisted with this project. Finally, thank you to my parents, Dwight and Alene, for lending their engineering and crafting skills to this project. Further thanks to the University of Minnesota Duluth Biology Department and Integrated Biosciences Graduate Program for supporting my research. i Abstract Resurrection experiments, in which dormant propagules of antecedent populations are grown alongside modern populations, provide a unique opportunity to directly evaluate phenotypic and molecular evolution in response to environmental challenges. To understand evolution of an urban population of Helianthus annuus (Asteraceae) over 36 years, we resurrected samples obtained from a 1980 USDA National Plant Germplasm System accession alongside contemporary successors gathered in 2016. Molecular changes in transcript expression using RNA-seq data revealed 200 differentially expressed transcripts between antecedent and modern groups. Transcript expression was higher in modern samples as compared to antecedent samples, while expression patterns indicated evolution due to genetic drift, gene introgression, or adaptive evolution. After a refresher generation in greenhouse conditions, we grew the resulting family lines in an outdoor common garden under varied water availability (high-water and low-water) and temperature conditions (ambient and elevated > 3°C) corresponding to cooler 1980 and warmer 2016 conditions to observe phenotypic differences and plastic response. Seventy- seven percent of measured traits differed, with modern individuals displaying traits similar to cultivated varieties and antecedent individuals displaying more customary wild-type traits. For example, modern plants were larger and showed more apical dominance while antecedent plants produced more branches and inflorescences. Modern trait means were often selected for across varied environmental treatments, especially those resembling modern conditions. This indicates that modern plants are well-adapted to their current environment. However, the modern population displayed little genetic variation underlying important reproductive traits which may limit the potential for further evolution of this population in response to changing conditions. The resurrection method allowed us to understand molecular and phenotypic evolution as a response to environmental pressures, gene flow from cultivated H. annuus, or some combination of evolutionary mechanisms resulting in the observed differences between the 1980 and 2016 populations. ii Table of Contents Acknowledgements………………………………………………………………………...i Abstract……………………………………………………………………....……………ii List of Tables………………………………………………………………………….…..iv List of Figures……………………………………………………………………………..v Introduction………………………………………………………………………………..1 Methods……………………………………………………………………………………7 Results…………………………………………………………………………………....14 Discussion……………….……………………………………………………………….21 References………………………………………………………………………………..39 iii List of Tables Table 1: Environmental and genetic influence on traits……..…………..………………..28 Table 2: Selection analysis results………………….……………………………..……...30 iv List of Figures Figure 1: Sample locations ………………………………………………………….…...32 Figure 2: Common garden block design……………………………………….…………33 Figure 3: PCA biplot based on differentially expressed transcripts……….……………..34 Figure 4: Venn diagram of transcript expression………………………………….….…..35 Figure 5: Trait value distribution and direction of selection………………………..……..36 Figure 6: Genetic variation in date of first flower………………………………………..37 Figure 7: Relationships between date of first flower and seed traits………………………38 v Introduction Species living in the Anthropocene face novel combinations of environmental pressures and challenges due to climate change and land use change (Barnosky et al., 2012; Matesanz, Gianoli, & Valladares, 2010). Species’ responses to changing conditions range from adaptation or migration to extinction (Davis et al., 2005). Understanding whether a species has the potential to adapt has major consequences for ecosystems worldwide (Parmesan, 2006; Root et al., 2003; Hoffmann & Sgrò, 2011), and the need for empirical evidence supporting this issue is critical. Resurrection experiments, in which propagules of antecedent populations and modern populations are grown alongside each other, provide a unique opportunity to directly evaluate responses to environmental changes over time and estimate future evolutionary trajectories (Hairston et al., 1999; Franks, Sim, & Weis, 2007; Etterson et al., 2016; Franks, Hamann, & Weis, 2018a). The resurrection approach allows us to observe phenotypic and molecular changes of species that have accrued over time (Etterson et al., 2016). Furthermore, if individuals are grown in contrasting environments, we can examine the extent to which adaptation proceeds through the evolution of fixed genetic changes, plasticity, or both. Treating resurrected individuals to a refresher generation allows the generation of genetic lines with known pedigrees and family structure to tease out the influence of genetic variation while simultaneously minimizing maternal and seed age effects (Franks et al., 2018a). Early applications of this approach revealed genetic differentiation between successional stages of arctic plants (McGraw, 1993) and adaptive evolution of zooplankton in response to environmental disturbances (Hairston et al., 1999). Recent resurrection studies have revealed contemporary evolution of phenology (Franks, Sim, & Weis, 2007; 1 Nevo et al., 2012; Thomann et al., 2015; Dickman et al., 2019), evolution of adaptive plasticity (Sultan et al., 2013), and response to herbivory (Bustos‐Segura, Fornoni, & Núñez‐Farfán, 2014; Franks, et al., 2018b). Comparisons of antecedent and modern populations grown in the same environment allow researchers to quantify the molecular changes underlying trait evolution (Franks et al., 2016) and genetic diversity (Nevo et al., 2012). Direct observation of evolutionary changes can ultimately lead to a better understanding of the interplay of genetic diversity, environmental response, and plasticity as species continue to evolve. Adaptation: Quickly changing environments can elicit rapid adaptive evolutionary responses as populations adjust to new pressures, including those induced by climate change (Shaw and Etterson, 2012). Traits may evolve in response to environmental disturbance if there is genetic variation present in the population (Etterson & Shaw, 2001; Etterson, 2004a; Etterson, 2004b), as observed in Wyeomyia smithii (pitcher plant mosquito) (Bradshaw & Holzapfel, 2001), Brassica rapa (field mustard) (Franks et al., 2007), and Thumus vulgaris (thyme) (Thompson et al., 2013). In the short term, a substantial degree of phenotypic change can be conferred by rapid changes in gene expression rather than the evolution of the genes themselves (King & Wilson, 1975; López-Maury, Marguerat, & Bähler, 2008; Yang & Wang, 2013). Differences in gene expression underlie adaptive trait shifts in response to stress (Swindell et al., 2007) and are important for local adaptation along climatic and latitudinal gradients (Lasky et al., 2014; Slotte et al., 2007). Overall, whether trait means evolve via changes in gene sequence or gene expression, responses must occur rapidly to keep pace with current predictions for climate change. 2 Phenotypic plasticity is a universal trait (Schlichting & Smith, 2002) where in varying environmental conditions a single genotype can result in multiple phenotypes (Bradshaw, 1965; Schlichting, 1986). Plastic phenotypic responses to the environment are commonly maladaptive (Schlichting, 1989; Dorn, Pyle, & Schmitt, 2000; Van Kleunen & Fischer, 2005) but can be adaptive if the reaction norm leads to the maintenance of fitness in for organisms living in a rapidly changing environment (Via & Lande, 1985; Schlichting & Smith, 2002; Ghalambor et al., 2007). This is advantageous because the response is immediate rather than requiring dispersal and range shifts or changes in allele frequencies at structural or regulatory genes. Genotypes with adaptive plastic responses have the potential to persist in a given area even as the climate shifts (Matesanz et al., 2010). However, there are limits on the extent of plastic responses which can lead to the eventual exhaustion of the adaptive capacity of species via phenotypic plasticity (DeWitt, Sih, & Wilson, 1998; Van Kleunen & Fischer, 2005; Schlichting, 1986). In summary, adaptive plastic responses have the potential to buy time by allowing individual organisms to persist in their original