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.