bioRxiv preprint doi: https://doi.org/10.1101/782821; this version posted September 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Running title: Adaptive evolution of Aquilegia species 2 3 Genetic and epigenetic mechanisms underpinning the 4 adaptive radiation of Aquilegia species 5 6 Tianyuan Lu1,2,3, Ming-Rui Li1, Ning Ding1, Zhen-Hui Wang4, Li-Zhen Lan1, Xiang Gao5, and Lin-Feng Li1,* 7 8 1Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, School of Life 9 Sciences, Fudan University, Shanghai 200438, China; 10 2McGill University and Genome Quebec Innovation Center, Montreal H3A 0G1, Quebec, Canada; 11 3Lady Davis Institute, SMBD JGH, Montreal H3A 1A3, Quebec, Canada; 12 4Faculty of Agronomy, Jilin Agricultural University, Changchun 130118, China; 13 5Key Laboratory of Molecular Epigenetics of the Ministry of Education, Northeast Normal University, 14 Changchun 130024, China. 15 16 17 *Correspondence author: 18 Lin-Feng Li ([email protected]) 19 bioRxiv preprint doi: https://doi.org/10.1101/782821; this version posted September 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 20 Summary 21 • Elucidating how the intrinsic factors interact with extrinsic triggers to determine species diversification 22 is crucial to understanding the evolution and persistence of biodiversity. The genus Aquilegia is a model 23 system to address the evolutionary mechanisms underpinning rapid adaptive radiation. 24 • We surveyed the genomes and methylomes of ten worldwide Aquilegia species to investigate whether 25 specific genetic and epigenetic architectures are involved in the diversification of Asian, European and 26 North American columbine species. 27 • The resulting phylogenies and population structure inferences revealed high divergence among the 28 Asian, European and North American species. Candidate genes identified at the genetic and epigenetic 29 levels are functionally correlated with diverse adaptation-related traits such as cell reproduction, flower 30 development, and stress tolerance. In particular, a considerable proportion of the selection genes and 31 their associated pathways show overlaps among the intra- and inter-lineage comparisons. Moreover, 32 while CG-loss variations can lead to depletion of cytosine methylation level, epigenetic modification is a 33 complementary intrinsic factor that intertwines with genetic mechanism to facilitate the diversification 34 of Aquilegia species. 35 • Our findings suggest that specific genetic and epigenetic architectures have conferred high 36 adaptability to the columbine species to cope with diverse external conditions, which eventually led to 37 the rapid radiation of Aquilegia species. 38 39 Key words: Adaptive radiation; Aquilegia; Methylome; Speciation; Whole genome resequencing 40 bioRxiv preprint doi: https://doi.org/10.1101/782821; this version posted September 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 41 Introduction 42 Adaptive radiation is the rapid diversification of a single ancestral species into a vast array of common 43 descendants that inhabit different ecological niches or use a variety of resources and differ in 44 phenotypic traits required to exploit diverse environments (Schluter, 1996; Givnish, 1997; Schluter, 45 2000; Givnish, 2015). Disentangling the evolutionary mechanisms underpinning adaptive radiation is 46 fundamental to understanding the evolution and persistence of biodiversity (Simpson & Olson, 1953; 47 Losos, 2010). Well-known animal and plant lineages that diversified through adaptive radiation include 48 Hawaiian silversword, Caribbean anoles, Darwin’s finches, and African cichlids (Carlquist & Motley, 2003; 49 Losos & Ricklefs, 2009; Lamichhaney et al., 2015; Irisarri et al., 2018). However, it still remains under- 50 investigated as to why some lineages can diversify rapidly but their close relatives or other sympatrically 51 distributed lineages do not. In the past decades, accumulating evidence from diverse radiation lineages 52 suggests that extrinsic environmental variables (e.g., resource availability) and intrinsic factors (e.g., 53 lineage-specific genetic variations) can interact to determine the rate and volume of species 54 diversification (Wagner et al., 2012). Among the extrinsic triggers, ecological opportunity is considered 55 as the primary mechanism that causes rapid adaptive radiation through the acquisition of key 56 innovations, invasion of new environments and extinction of competitors (Simpson, 1949; Schluter, 57 2000). On the other hand, new species can also arise as a result of natural selection acting on intrinsic 58 factors (e.g., advantage alleles) that ultimately generate both the phenotypic disparity and similarity 59 among closely related species (Berner & Salzburger, 2015). An illustration of rapid adaptive radiation is 60 the African cichlid fishes, on which the extrinsic environmental factors (e.g., ecological specialization) 61 and extrinsic traits (e.g., adaptive introgression) have acted together to provoke the repeated adaptive 62 radiation in geographically isolated lakes (Wagner et al., 2012; Brawand et al., 2014; Ford et al., 2016; 63 Irisarri et al., 2018). 64 The genus Aquilegia L. (columbine) is a well-recognized model system to address the evolutionary 65 mechanisms underlying rapid adaptive radiation (Fior et al., 2013; Filiault et al., 2018). It includes 66 approximately 70 recently diversified species that are widely distributed in the temperate areas of North 67 America and Eurasia (Munz, 1946). Phylogenetic and geographic inferences have illustrated two 68 independent adaptive radiations of North American and European lineages from the distinct ancestral 69 Asian species (Bastida & Herrera, 2010; Fior et al., 2013). Floral diversification of the North American 70 species is highly correlated with the pollinator specialization (Whittall & Hodges, 2007; Alcántara et al., 71 2010; Kramer & Hodges, 2010; Sharma et al., 2014). In contrast, ecological adaptation and geographic 72 isolation are thought as the major driving forces that promoted the rapid radiation of European species bioRxiv preprint doi: https://doi.org/10.1101/782821; this version posted September 26, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 73 (Fior et al., 2013; Garrido et al., 2017). In Asia, shifts in pollinator and ecological habitat are both 74 proposed to be the underpinning mechanisms that resulted in the diversification of more than 20 75 morphologically distinct species (Tang et al., 2007; Li et al., 2014). Unlike the North American and 76 European lineages, the Asian columbines consist of four highly divergent lineages corresponding to 77 respective geographic origins and evolved independently under distinct external triggers (Tang et al., 78 2007; Fior et al., 2013; Li et al., 2014). The independent adaptive radiations of North American and 79 European lineages from the more ancestral but independently diversified Asian columbine species 80 provide an ideal system to examine how the extrinsic and intrinsic triggers interact to promote the 81 diversification of Aquilegia species. 82 In this study, we surveyed the genomes and DNA methylomes of 36 accessions from ten worldwide 83 columbine species to address the underlying evolutionary mechanisms that caused the diversification of 84 Asian, European and North American columbine species. Of the Asian lineage, four phylogenetically 85 distinct species (A. yabeana, A. viridiflora, A. oxysepala and A. japonica) were selected according to their 86 geographic distributions and ecological habitats. Aquilegia japonica and A. oxysepala are sister species 87 inhabiting alpine tundra and low altitude forest niches in northeastern China, respectively (Li et al., 2011; 88 Li et al., 2014). Our previous studies have documented that natural selection during ecological 89 specialization together with genetic drift under geographic isolation caused the rapid evolution of 90 reproductive isolation between the two species (Li et al., 2014; Li et al., 2019). Here we further 91 investigate how the genetic and epigenetic mechanisms conferred adaptability to the two species to 92 cope with contrasting ecological conditions. In addition, patterns of nucleotide variation and cytosine 93 methylation were also evaluated for the A. yabeana and A. viridiflora. The former species shares highly 94 similar morphological traits and ecological niches with the A. oxysepala but is allopatrically distributed in 95 the northern China. In contrast, while the A. viridiflora is sympatrically distributed with A. yabeana and A. 96 oxysepala in the northern and northeastern China, it often occupies rocky and sandy ecological niches. 97 These attributes allow