Genetic and epigenetic characteristics associated with the rapid radiation of Aquilegia species

Elucidating the genetic and epigenetic bases underlying species diversification is crucial to understanding the evolution and persistence of biodiversity. As a well-known horticultural plant grown worldwide, the genus Aquilegia (columbine) is also a model system in adaptive radiation research. In this study, we surveyed the genomes and DNA methylomes of ten representative Aquilegia species from the Asian, European and North American lineages. Our inferences of the phylogenies and population structure revealed clearly high genetic and DNA methylomic divergence across the three lineages. By multi-levelled genome-wide scanning, we identified candidate genes exhibiting lineage-specific genetic or epigenetic variation patterns that are signatures of inter-specific divergence. We demonstrated that these species diversification-associated genetic variations and epigenetic variabilities were partially independent but were both functionally related to various biological processes vital to adaptation, including stress tolerance, cell reproduction and DNA repair. Our study provides an exploratory overview of how the established genetic and epigenetic signatures are associated with the rapid radiation of Aquilegia species.


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Adaptive radiation is the rapid diversification of a single ancestral species into a vast array of common 42 descendants that inhabit different ecological niches or use a variety of resources, but differ in 43 phenotypic traits required to exploit diverse environments [1][2][3][4] . Disentangling the evolutionary 44 mechanisms underpinning adaptive radiation is fundamental to understanding the evolution and 45 persistence of biodiversity 5,6 . This has been a key focus of many studies which were investigating 46 different animal and plant lineages that diversified through adaptive radiation, including Hawaiian 47 silversword, Caribbean anoles, Darwin's finches, and African cichlids 7-10 . However, it remains under-48 investigated as to why some lineages could diversify rapidly but their close relatives or other 49 sympatrically distributed lineages did not. In the past decades, accumulating evidence from diverse 50 radiation lineages suggest that both the extrinsic environmental factors (e.g., resource availability) and 51 genetic variations can determine the rate and volume of species diversification 11 . Among the 52 environmental triggers, ecological opportunity is considered as the primary mechanism that causes 53 rapid adaptive radiation through acquisition of key innovations, penetration of new environments and

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To further gain an insight into genome-wide nucleotide variation pattern of the ten Aquilegia 122 species, we calculated nucleotide diversity (π) and genetic divergence (F ST ) for each chromosome and for   Candidate genes or genomic regions associated with adaptive divergence were determined from three 152 perspectives. First, we considered genes localized within the regions that showed low intra-specific 153 diversity but high inter-specific divergence to be representative of intra-specific genetic differences. We 154 thus identified 23 genes from the above seven candidate genomic regions that were both HDGRs and 155 LDGRs shared by A. oxysepala and A. japonica (Table S1). Genes within these genomic regions were 156 functionally associated with meiotic nuclear division, adenine methyltransferase and basic cellar 157 activities.

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While the first strategy mainly relied on genome-wide scanning for 100-kb non-overlapping sliding 159 window, we also employed a functional annotation-based approach to identify highly impactful 160 conservative clade-specific variations (CCVs) from both the within and between lineage comparisons.

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Our results revealed that a considerable proportion (17.9-40.5%) of the CCVs were identified in the gene 162 body regions (Table S2). We then examined the potential functional impacts of genes harboring these 163 identified CCVs. Between the A. oxysepala and A. japonica, the CCV-carrying genes were enriched in 164 several vital biological pathways related to cell reproduction, including telomere maintenance, DNA 165 repair, and DNA helicase activity (Figure 2 and Table 1). For example, two candidate genes 166 (Aqcoe6G160300 and Aqcoe7G062500) coding for Xklp2 (TPX2) were functionally correlated with spindle 167 assembly during the mitotic process (27, 28). Among the three phylogenetic lineages, the CCVs-168 harboring genes were also functionally involved in the mitotic chromosome condensation, DNA ligase 169 activity and aminopeptidase activity (Figure 2 and Table 1     photosynthesis-related genes, PsaA/PsaB and CemA, showed significantly differential methylation 235 between the two species in the genic regions ( Figure S7a and b). At the inter-lineage level, apparently more DMGs were identified between the North American and European species (6,087 genes) 237 compared to those of between the two lineages and Asian species (3,308-5,003 genes) ( Table S3 and S4).

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DMGs characterized from the inter-lineage comparisons were mainly involved in the plant growth (e.g.,

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response to auxin) and defense, response to biotic stimulus and wounding (Figure 2).

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We then examined whether the candidate genes (CCV-carrying genes and DMGs) superimposed on 241 the same signature of natural selection. We found while a considerable proportion of the candidate 242 genes were shared for each of the genetics-and epigenetics-based assessments (Figure S8), they 243 showed a segregated distribution pattern across all comparisons ( Figure S9). Likewise, the Gene  Since both genetic variation and differential CG methylation seemed to have crucial and multifaceted 293 influences on the adaptation of the ten Aquilegia species, we wondered whether differential epigenetic 294 modifications were dependent on genetic variations. Among the 588,659 CG loci examined, 224,222

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(38.09%) carried a CG-loss variation. We then illustrated epigenetic variability for the variation-carrying 296 and non-variant CG loci, respectively. As shown in Figure 4, genetic-epigenetic associations of varying 297 magnitude were observed in both types of CG loci. The variation-carrying CG loci conveyed information 298 that highly resembled their genetic background. The overall methylation pattern was highly conserved 299 within the same species but exhibited obvious divergence across the ten Aquilegia species (Figure 4a). In 300 contrast, CG methylation divergence at the non-variant CG loci varied with higher variability at both the 301 intra-and inter-specific levels (Figure 4b). By examining the correlation of genetic variability and 302 cytosine methylation, we found that CG methylation divergence at variation-carrying CG-site was largely 303 attributable to the CG-loss variations (Figure 4c). In particular, 75% of the CG-loss variations occurring at 304 the most highly variable CG-methylated dinucleotides could explain at least 75% of the total epigenetic 305 variability per se. Nevertheless, there was still a considerable proportion of epigenetic variability that 306 could not be sufficiently explained by the variant-CG locus (Figure 4d).

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We also attempted to identify cis-driver mutations for each of the 1,229 DMRs between the A.

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It has been proposed that if a genetic factor is the potential determinant promoting adaptive 387 speciation, one would expect to identify specific genetic architectures in the diversified lineages 8,14,31 . In

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Darwin's finches, for example, polyphyletic topology was observed as a general pattern in 14 389 morphologically distinct species, phenotypic diversity of the beak shape was mainly determined by    regions representing the other driving forces. Therefore, we claim that the candidate genes identified to 451 be associated with adaptive radiation do not necessarily point towards causal evolutionary mechanisms.

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They may also be by-products of the long-term process of adaptive radiation. In addition, we never than 453 less only focused on analysis of CG methylation as puzzles persist regarding the functional roles of CHG 454 and CHH methylation. We also expect that future studies with larger sample sizes will be able to In this study, a total of 36 accessions from ten worldwide Aquilegia species were collected (

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To infer the phylogenetic relationship between the ten Aquilegia species, NJ trees were

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In addition, we identified CG islands from the A. coerulea "Goldsmith" reference genome using EMBOSS

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We are very grateful to Aköz Gökçe for her constructive comments on our manuscript. We appreciate Dr.

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Peng Jiang, Zhang Zhang, and the USDA for kindly providing the seeds of the Aquilegia species.

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Polymorphisms detected on each chromosome were retrieved separately to infer the phylogeny.

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Values on the x-and y-axis are the nucleotide diversity (π) for each sliding window.