Introduction
Whole-genome duplication (WGD) or polyploidization has been suggested to be prevalent throughout the evolutionary history of plants, acting as an important evolutionary force promoting diversification, speciation, and adaptation to new environments (Jiao et al., 2011; Levin, 1983; Soltis & Soltis, 2016; Wood et al., 2009). Neofunctionalization or subfunctionalization of duplicated genes post-WGD, as well as species-specific gene retention and loss, lead to expansion and contraction of gene families, resulting in the generation of morphological and adaptive diversity (Force et al., 1999; Ren et al., 2022; Van de Peer et al., 2017). However, the process of species differentiation after ancient WGD is often accompanied by the occurrence of genome rearrangement events, resulting in the generation of new genes or changes in the expression and regulation mode of existing genes, which may result in the emergence of new traits and promote speciation (Estep et al., 2014; Hu et al., 2021). Based on the origin and level of ploidy, polyploidy can be divided into two types, autopolyploidy and allopolyploidy. The former involves duplication of the diploid genome in a single species; whereas the latter is formed by a genomic combination between two or more distant species (Soltis & Soltis, 2016; Stebbins, 1947). Although the relative proportion of the two types are comparable, allopolyploids are generally expected to have higher evolutionary advantages due to the pairing of chromosomes from each parent, which often results in more chromosomal structural variations, novel gene interactions, and morphological innovations (Soltis & Soltis, 2016; Taylor & Larson, 2019). In general, polyploid plants exhibit greater vigor, stronger resistance, wider environmental adaptability, and higher biomass and economic yield than their diploid relatives (Barker et al., 2016; Ding et al., 2018; Estep et al., 2014; Taylor & Larson, 2019). However, the underlying genomic basis of ecological adaptation and subsequent diversification remain poorly understood in most polyploid species.
Chloridoideae, containing approximately 124 genera and 1,602 species, is the fourth largest subfamily of the grass family (Gramineae; Soreng et al., 2017). Most species of this subfamily use the C4 photosynthetic pathway, and the first evolutionary transition from C3 to C4 photosynthesis was found in this subfamily about 32–25 million years ago (Ma; Christin et al. 2008). Species of this subfamily possess strong tolerance to arid environments (Soreng et al. 2017), such asOropetium thomaeum and Cleistogenes songorica (VanBuren et al., 2015; Zhang et al., 2021a). With a base chromosome number of x = 10, it is suggested that more than 90% of species within this subfamily are polyploids, including the staple grain crop teff in Ethiopia and many turfgrass and forage species (Roodt & Spies, 2003). Ancient WGD events should have contributed to the stress tolerance, emergence of valuable traits and diversification of these grasses (Roodt and Spies, 2003; Zhang et al., 2021a). Thus, this subfamily may serve as a valuable system for investigating potential impacts of WGD, subsequent subgenome divergence, functional innovations, genome rearrangement, and diversification in polyploid plants. Recent advances in sequencing technologies have facilitated genome assemblies for polyploid species. To date, 16 reference genomes of this subfamily have been reported, 13 of which are polyploid. Five genomes were used for comparative examination of desiccation tolerance and sensitivity (Chávez Montes et al., 2021; Pardo et al., 2020). Whereas the rest mainly focused on the genomes themselves and investigated the divergence between subgenomes or the genomic basis of adaptation to extreme environments (e.g.,VanBuren et al., 2015; VanBuren et al., 2021; Wang et al., 2022; Zhang et al., 2021a). Among these studies, however, few have focused specifically on evolutionary divergence after polyploidization. Thus, a comprehensive understanding of how subsequent speciation and diversification occurs in these polyploid genomes remains unknown (Hu et al., 2021).
We focus on a newly named subtribe, Orininae, belonging to the tribe Cynodonteae, Chloridoideae (Peterson et al., 2016; Soreng et al., 2017). This subtribe includes two genera, Orinus (3 species) andCleistogenes (17 species), which are often difficult to separate morphologically. For example, Hao (1938) has treated one Orinusspecies, Orinus kokonorica (K.S. Hao) Tzvelev, as aCleistogenes . However, there are two distinct morphological differences between the two genera. Cleistogenes has hidden cleistogamous spikelets concealed within the upper sheaths, which is not found in Orinus ; and Orinus has elongated rhizomes covered with leathery and glossy scales, while Cleistogenes has a cespitose habit or very short rhizomes (Chen et al., 2006). Cleistogenes songorica is an allotetraploid species (2n = 4x = 40) and possesses a genomic basis for its dimorphic flower differentiation and drought adaptation (Zhang et al., 2021a). Our karyotyping also revealed a chromosome number of 40 for O. kokonorica (Figure 1A), indicating that this species is a tetraploid plant. However, there is no reference genome for Orinus , which significantly limits our understanding of its evolutionary history and genomic divergence between these two genera. Here, we selected O. kokonorica as a case study and performed comparative genomics between O. kokonorica and C. songorica to investigate genomic basis of adaptive evolution and diversification after polyploidization.
Orinus kokonorica is mainly distributed in the northeastern Qinghai-Tibet Plateau (QTP) at elevations between 2400 and 4200 m (Su et al., 2015), whereas C. songorica widely occurs in semi-arid and desert areas in central Asia at relatively low altitude (Chen et al., 2006). Both species are important forage resources with good palatability and high nutritious value for local livestock in arid regions. The two species display differences mainly in flower and rhizome morphology and in altitudinal distributions. In addition, with elongated rhizomes, O. kokonorica has developed a strong underground root system (Figure 1A), which helps to improve sand fixation and water conservation in arid areas. In this study, we first performed genome assembly and RNA-seq-based transcriptomics under different stress treatments for O. kokonorica . Then, using publicly available genomic and transcriptomic data for C. songorica , we conducted comparative genomic and transcriptomic analyses to elucidate the evolutionary history of the two species and the genomic basis of their subsequent divergence.