INTRODUCTION
Elucidating the factors contributing to population divergence and speciation is the core question in evolutionary biology and has attracted interest from biologists for a long time (Darwin, 1859; Dobzhansky, 1982; Mayr, 1947; Turelli, Barton, & Coyne, 2001). Speciation is a gradual process occurring through the cumulative action of natural selection and genetic drift in which populations diverge into groups that cease or limit allele exchange (Coyne & Orr, 2004; Futuyma, 1998; Raman, 1998). For example, some models of speciation with gene flow suggest that reproductive isolation (RI) begins at a single locus but will progressively expand and facilitate divergence of other linked genes throughout speciation (Rieseberg & Blackman, 2010; Wu, 2001).
The narrowly differentiated peak in the genome can arise at multiple prezygotic and postzygotic life-history stages (Coyne & Orr, 2004; Haerty & Singh, 2006; Presgraves, 2010). During population divergence, prezygotic isolation mechanisms are more important than postzygotic barriers because they act earlier before hybrid zygotes are formed. Additionally, morphological divergence is expected to be associated with prezygotic reproductive barriers. When taxa begin to evolve morphological differences, they can directly or indirectly reduce mattings, sperm transfer, or fertilization (Coyne, 2016; Feder et al., 1994). For example, Turbek et al. found that assortative mating based on both plumage coloration and song can maintain premating isolation between Sporophila iberaensis and S. hypoxantha (Turbek et al., 2021). This view is also applicable to plants. For many taxa, including Aquilegia , flower color differences leading to pollinator shifts likely contribute to RI between populations (Des Marais & Rausher, 2010; Hodges, Whittall, Fulton, & Yang, 2002; Kuriya, Hattori, Nagano, & Itino, 2015; Quattrocchio et al., 1999; Schwinn et al., 2006). Additional differences between species that result in further RI can also accumulate after speciation is already complete. Furthermore, distinguishing between genetic changes that play determinant roles in speciation and those that arise after speciation is complete may be challenging. Thus, organisms in the early stages of speciation provide an opportunity to identify causal differentiation in speciation of the genomic landscape.
With the publication of new species, Aquilegia currently includes approximately 110 species worldwide, with approximately 70 species based on the record of Munz, and is widely distributed in the northern temperate zone (A. S. Erst et al., 2020; A. S. Erst et al., 2017; Luo, Erst, Yang, Deng, & Li, 2018; Munz, 1946). Lu et al. analyzed the genomes and methylomes of 10 Aquilegia plants worldwide and speculated that the specific genetic and epigenetic structure endowsAquilegia with a high degree of adaptability and promotes adaptive radiation speciation of the genus (Lu et al., 2019). However, these species are mature in the late stage of differentiation. In addition to the gene regions that began to differentiate in the early stage of speciation, various factors have led to obvious differentiation of the genome among species. Unlike its congeners, the phenotypes of different populations of A. viridiflora showed high diversity, which occurs in the early stage of speciation. A. viridiflora is an ideal material to study the key genes that lead to RI in the early stage of speciation.
Aquilegia viridiflora shows rich morphological variation in different populations, so Andrey S. Erst et al. identified A. viridiflora with laminae purple or lilac-blue petals as the new speciesA. kamelinii (A. Erst, Shaulo, & Schmakov, 2013) and A. viridiflora with dark purple petals in North China as the new speciesA. hebeica (A. S. Erst et al., 2017). However, the publication of these new species lacks support from molecular data. According to field surveys, the phenotypic variation of A. viridiflora is continuous (see Figure 1). Therefore, A. viridiflora , A. kameliniiand A. hebeica were considered the same species (A. viridiflora ) in our study. A. viridiflora is primarily distributed in northern China, extending between 75.14-132.58° E and 33.24-53.28° N, in which the east-west span is wide, and the environment changes gradually. These factors suggest that environment-mediated divergent selection may be the driving force for the early differentiation of A. viridiflora . Additionally, other species ofAquilegia are produced through ecological speciation, such asA. formosa and A. pubescens (Hodges et al., 2002; Whittall, 2005). Therefore, this species provided a test case to reveal the effects of adapting to different ecological environments in the early stage of the speciation process.
In the present study, we collected population samples covering the main distribution range of A. viridiflora for genomic analyses and morphological characteristic measurements to explore the following: (1) the morphological divergence during the early stages of speciation, (2) the genomic basis of such phenotypic traits, and (3) the role of the environment in maintaining population divergence. Answering these questions will reveal the speciation process of A. viridifloraand the environmental factors that lead to its differentiation.