Introduction

Polyphenism is a unique type of phenotypic plasticity, in which the outputs are not continuous but relatively discrete, arising from the same genotype (Mayr, 1963; Moran, 1992; C.-H. Yang & Andrew Pospisilik, 2019). Its diverse traits and importance in conferring ecological fitness have been widely acknowledged (Darwin, 1897; Simpson, Sword, & Lo, 2011) in both animals and plants (Abouheif & Wray, 2002; Fawcett et al., 2018; Yiyang Liu et al., 2021; Zhang et al., 2021). As one of the most widespread polyphenisms (Joly & Schoen, 2021), dimorphic flowers evolved independently in roughly 700 species from 50 families of plants (Culley & Klooster, 2007). Such flowers are usually chasmogamous (CH) and cleistogamous (CL) growing on a single plant and having contrasting shapes, colors, and smells (Campbell, Quinn, Cheplick, & Bell, 1983; Lord, 1981). CH flowers (hereafter CHs) have bright and colorful petals and nectaries and remain open for cross-pollination, while CL flowers (hereafter CLs) have green petals and remain closed for self-fertilization (Darwin, 1897; Lord, 1981). In addition, CLs are always smaller and have a simpler structure than CHs, leading to lower costs and an automatic transmission advantage (Wang, Du, & Wang, 2017). Therefore, as a ’pessimistic strategy’, CLs can ensure reproductive success under harsh or uncertain conditions (Schnee & Waller, 1986; Waller, 1980). However, selfing through CLs rapidly leads to inbreeding depression and adversely affects genetic load (building up harmful mutations) (Ansaldi, Weber, & Franks, 2018; Charlesworth & Charlesworth, 1987). Thus, outcrossing CHs can effectively increase recombination and overcome these weakness (Culley & Klooster, 2007; Culley & Wolfe, 2001). Such a trade-off with a mixed mating system provides high reproductive assurance, allowing plants to survive unpredictable and extreme environments (Ansaldi et al., 2018; Koontz, Weekley, Haller Crate, & Menges, 2017).
In addition to ecological significance, it would be interesting to know what genes and their expressions have led to the differentiation and maintenance of such dimorphic flowers with the same genotype (Ansaldi et al., 2018; Morinaga et al., 2008). Two recent studies have investigated this genetic differentiation through sequencing the genome and examining gene expression of the dimorphic flowers (Yiyang Liu et al., 2021; Zhang et al., 2021). For Amphicarpaea edgeworthii (Fabaceae) with dimorphic flowers, the identified genes with contrasting expressions between aerial CH and subterranean CL flowers were mainly related to MADS-box genes (Yiyang Liu et al., 2021). Research on Cleistogenes songorica (Gramineae) with dimorphic flowers suggests miRNA, MYBtranscription factors, and targeted genes are involved in the differential development of the highly reduced CH and CL flowers in this grass (Zhang et al., 2021). However, the typical structures of the dimorphic flowers of these two species differ from most species with aerial dimorphic flowers (Campbell et al. , 1983; Culley and Klooster, 2007). In this study, we used multi-omics data to examine differentiation and maintenance of dimorphic flowers in a more typical species, Sinoswertia tetraptera (Gentianaceae), an endangered, alpine biennial restricted to the Qinghai–Tibet Plateau (L. Yang, Zhou, & Chen, 2011). In the entire family Gentianaceae only this monotypic genus has a mixed mating system with both CH and CL flowers on the top or basal stem of a single plant (Figure 1a) and CH flowers may disappear in some plants in the high-altitude extremes (T. He, Liu, & Liu, 2013). The open CH flowers are pale-blue and large with distinct nectaries, while the closed CLs are green without nectaries. Such contrasting shapes and colors are similar to most dimorphic flowers of other species (Culley & Klooster, 2007). In addition, this species has been used as a traditional Tibetan medicine since the 6th century BCE (Rao et al. , 2010) and is rich in iridoid compounds (Brahmachari et al., 2004; Yue Liu et al., 2017). It remains unknown whether the two types of flowers contain the same or different concentrations of iridoid compounds and related gene expressions.
We assembled a chromosome-level de novo genome of S. tetraptera using long Nanopore reads, Illumina short reads, and Hi-C data. By comparative genomic analyses, we first explored the genome evolution of S. tetraptera , the first representative of the family Gentianaceae. Then, based on transcriptome and metabolome analyses, we further examined the omics differentiation and maintenance of dimorphic flowers in S. tetraptera . Finally, we identified candidate genes involved in iridoid biosynthesis according to the weighted gene co-expression network analysis (WGCNA) and gene expressions. Our data provide essential insights into how CH and CL flowers are differentially maintained and iridoids are synthesized in this alpine plant.