Phenotype- and gene-driven Speciation under Environment-mediated Natural Selection
The differentiation of floral characteristics is considered the core of prezygotic isolation in plants and plays an important role in speciation (Celep et al., 2020; Xu et al., 2011). In the present study, the four identified groups of A. viridiflora were located in different climate types and had continuous phenotypic variation. The areas where NE and NW were located showed large mean diurnal temperature ranges, while the areas where EL and CN were located showed small mean diurnal temperature ranges. Populations located in environments with a wide mean diurnal temperature range exhibit longer spurs and pistils than those located in environments with a narrow mean diurnal temperature range. The differences in the spur and pistil lengths may promote the formation of reproductive isolation (Brothers & Delph, 2017; Kramer, 2009; Sun, Schlüter, Gross, & Schiestl, 2015). Additionally, plant growth is greatly affected by the environment; therefore, adaptive adjustments to different environments are necessary (Anderson & Song, 2020). Among the adaptable traits, flower size (corolla diameter, petal length, and spur length) was significantly negatively correlated with the inflorescence number and leaf area (Table 1 and Figure S5). NE and NW had larger and fewer flowers and smaller leaves, while CN and EL had smaller flowers, a greater number of flowers and larger leaves. This phenomenon has also been reported in Pedicularis siphonantha populations (Dai, Amboka, Kadiori, Wang, & Yang, 2017). The wide mean diurnal temperature range limits the activities of pollinators. To ensure the production of offspring, A. viridiflora uses more energy for reproductive growth and less energy for vegetative growth. Additionally, the populations in this area (NW and NE) used the limited energy available for reproductive growth to form a small number of flowers, thus enhancing their ability to attract pollinators. By contrast, abundant pollinators were found in areas (EL and CN) with a narrow mean diurnal temperature range, and A. viridiflora populations in such areas use more energy for vegetative growth to ensure their long-term survival. The groups in these areas use their reproductive growth energy to form more flowers and produce more seeds.
According to Wu’s “Genic view of the process of speciation”, the key genes or sites of speciation determine the adaptive differentiation of the population to the environment in which it is located (Wang, He, Shi, & Wu, 2020; Wu, 2001). Among selected and differentially expressed genes related to the environment, Aqcoe5G182600 (Cytokinin oxidase 5) might have played an important role in the early speciation of A. viridiflora . Cytokinin oxidase 5 and its orthologous genes play an important role in plant grain yield.Os CKX2knockout results in increases in the numbers of both panicle branches and grains per plant under stress conditions in Oryza sativa(Ashikari et al., 2005). Accordingly, Aqcoe5G182600 may regulate the activity of flower primordia and control the number of inflorescences by controlling the levels of cytokinin. Furthermore, five candidate genes were related to resistance to environmental stress,Aqcoe1G340100 (Seong et al., 2007), Aqcoe1G488100(Koussevitzky et al., 2008), Aqcoe3G242600 (Magadlela et al., 2019), Aqcoe5G182600 (S. Li et al., 2019) andAqcoe7G135600 (He et al., 2019), indicating that the differences in the growth environment drive the genetic differentiation of different groups of A. viridiflora .
The appearance of nectar spurs is a critical morphological characteristic of highly diversified Aquilegia . Nosil et al. proposed that a ‘speciation gene’ is defined as any gene contributing to the evolution of reproductive isolation (Nosil & Schluter, 2011). Because Aqcoe5G459400(OBP3-responsive gene 1) was shown to be related to nectar spurs by integrating the results of WGCNA, and its expression level was negatively correlated with the spur length, as well as the selected SNP in Aqcoe5G459400 was differentiated and almost fixed in NE and EL, Aqcoe5G459400 could be considered a key gene in the early stage of A. viridiflora speciation. Previous studies have shown that OBP3-responsive gene 3 (ORG3 ) controls the size of petals by controlling the number of cells (Omidbakhshfard et al., 2018). Additionally, A. ecalcarata has short or no spurs because cell division ceased and cell differentiation began earlier (Ballerini, Kramer, & Hodges, 2019). Therefore, Aqcoe5G459400 may control the spur size by controlling the number of cells in A. viridiflora . Additionally, the ABI3/VP1 transcription factor located upstream of Aqcoe5G459400 and its orthologs are key genes involved in regulating abscisic acid (ABA), which not only plays a role in the dormancy of seeds and buds but also affects the flowering time of plants (Riboni, Robustelli Test, Galbiati, Tonelli, & Conti, 2016; Shu et al., 2018; Shu et al., 2016; Shu et al., 2013). In summary, a cascade reaction between ABI3/VP1 and ORG3 likely exists, controlling the spur size in A. viridiflora . The expression level ofAqcoe5G459400 was lower in the lineages from regions with a wide mean diurnal temperature range, causing A. viridiflora to produce larger flowers to attract more pollinators and increasing the chance of successful reproduction. By contrast, its expression level is higher in the lineages from regions with a narrow mean diurnal temperature range, and A. viridiflora produces smaller flowers in such areas to increase the number of seeds produced. Thus, the differentiation ofAqcoe5G49500 enables A. viridiflora to develop different adaptive evolution mechanisms in response to different environments and promotes speciation. During speciation of A. viridiflora , except for the differentiation of Aqcoe5G49500 , the gene flow between populations may increase at other sites because of the genetic variation mediated by natural selection, which accelerates the divergence ofA. viridiflora into different species (X. Wang et al., 2020). Additionally, Aqcoe2G056600 , Aqcoe2G172400 ,Aqcoe3G439700 , Aqcoe7G244300 and Aqcoe7G395200interacting with Aqcoe5G459400 in the module are related to histone methylation and ion transport across membranes and therefore may improve the ability of plants to resist insect pests and adverse stresses (Fedoreyeva, Vanyushin, & Baranova, 2020; Liao et al., 2018; Philippe, Ralph, Külheim, Jancsik, & Bohlmann, 2010; Rodríguez-Celma, Chou, Kobayashi, Long, & Balk, 2019; Surya, 2020).