Trichosporeae
(This chapter has been published in PLOS ONE 14(1): e0210054. 2019)
Kuan-ting Hsin, Jing-Yi Lu, Michael Möller, Chun-neng Wang
Authors’s contribution
KTH, JYL, MM and CNW conceived the context of the manuscript. KTH, JYL and CNW designed the experiment and analysis. KTH and JYL performed experiments and analysis. All authors read and approved the final manuscript.
Abstract
Floral bilateral symmetry is one of the most important acquisitions in flower shape evolution in angiosperms. Members of Gesneriaceae possess predominantly zygomorphic flowers yet natural reversal to actinomorphy have independently evolved multiple times. The development of floral bilateral symmetry relies greatly on the gene CYCLOIDEA (CYC). Our reconstructed GCYC
phylogeny indicated at least five GCYC duplication events occurred over the evolutionary history of Gesneriaceae. However, the patterns of GCYC expression following the duplications and the role of natural selection on GCYC copies in relation to floral symmetry remained largely unstudied. The Asiatic tribe Trichosporeae contains most reversals to actinomorphy. We thus investigated shifts in GCYC gene expression among selected zygomorphic species (Hemiboea bicornuta and Lysionotus pauciflorus) and species with reversals to actinomorphy (Conandron ramondioides) by RT-PCR. In the actinomorphic C. ramondioides, none of the three copies of GCYC was found expressed in petals implying that the reversal was a loss-of-function event. On the other hand, both zygomorphic species retained one GCYC1 copy that was expressed in the dorsal petals but each species utilized a different copy (GCYC1C for H. bicornuta and GCYC1D for L. pauciflorus). Together with previously
published data, it appeared that GCYC1C and GCYC1D copies diversified their expression in a distinct species-specific pattern. To detect whether the selection signal (ω) changed before and after the duplication of GCYC1 in Asiatic Trichosporeae, we reconstructed a GCYC phylogeny using maximum likelihood and Bayesian inference algorithms and examined selection signals using PAML. The PAML analysis detected relaxation from selection right after the GCYC1 duplication (ω pre-duplication = 0.2819, ω post-duplication = 0.3985) among Asiatic Trichosporeae species. We propose that the selection relaxation after the GCYC1 duplication created an "evolutionary window of flexibility" in which multiple copies were retained with randomly diverged roles for dorsal-specific expressions in association with floral symmetry changes.
Key words: reversal, GCYC, duplication, relaxation, flexibility, Trichosporeae, Gesneriaceae
Introduction
Floral symmetry has long been thought to be one of the most extensively researched characters of trait evolution since the studies of Darwin in 1868 (Darwin, 1968; Endress, 1999; Endress, 2001).
Among the various types of floral symmetry identified, zygomorphy and actinomorphy are the two major types across angiosperms. Zygomorphy, or bilateral symmetry, is characterized by a single plane of symmetry that separates flowers into two mirror images. By contrast, actinomorphy, or radial symmetry, is characterized by multiple planes of symmetry. Evolutionary reconstruction of floral symmetry states reveals that actinomorphy is the ancestral state yet multiple transitions to zygomorphy occurred across the angiosperm phylogeny (Citerne et al., 2010; Reyes et al., 2016).
Zygomorphy is considered to be a key innovation, promoting specific ecological and evolutionary interactions between pollinators and floral shape (Endress, 1999; Endress, 2001; Ree & Donoghue, 1999; Preston & Hileman, 2009). However, events of reversals from zygomorphy back to
actinomorphy are also observed in several angiosperm lineages. Some of these reversions have been regarded as adaptations to rare pollinator and/or low pollination efficiency conditions such as high mountains or deep forests (Cronk & Möller, 1997; Hsin & Wang, 2018).
Gesneriaceae is one of the families showing repeated shifts in floral symmetry in their evolutionary history. It is a family with over 3,500 species (Weber et al., 2013) in the order Lamiales with predominantly zygomorphic flowers, with more than half a dozen species showing actinomorphic flowers (Weber et al., 2013; Smith et al., 2004; Weber, 2004; Möller et al., 2009; Möller et al., 2011a). In an early classification Wang & al. (1992) assigned the actinomorphic taxa Ramonda Rich., Conandron Siebold & Zucc, Tengia Chun, Bournea Oliv., Thamnocharis W.T. Wang, to a single tribe, Ramondeae, and considered this tribe also to be the most primitive in subfamily Cyrtandroideae (now Didymocarpoideae, see (Weber et al., 2013)), therefore, regarding
actinomorphic taxa to represent a single ancestral lineage in Gesneriaceae. Burtt (1970), based on the predominance of zygomorphic species and the scattered distribution of actinomorphic species in the
taxonomic system, stated that taxa with actinomorphic flowers were probably reversal from the zygomorphic state and could not be regarded as ancestral. This was supported by molecular phylogenetic studies that indicated the family has a high number of species with reversals to actinomorphy (Smith et al., 2004; Möller et al., 2009; Möller et al, 2011a; Wang et al., 2010). The most recent classification placed the five Old World actinomorphic genera, Bournea, Conandron, Ramonda, Tengia, Thamnocharis in the tribe Trichosporeae of subfamily Didymocarpoideae, and here in different subtribes: R. myconii (subtribe Ramondinae) and the other four in subtribe Didymocarpinae (Weber et al., 2013; Möller et al., 2009; Möller et al., 2011a). The subtribe Didymocarpinae includes the highest number of reversals to actinomorphy, with one event in Conandron (Hsin & Wang, 2018), one in Tengia (now Petrocodon, see (Weber et al., 2011)), one in Thamnocharis (now Oreocharis, see (Möller et al., 2011b)) and two in Bournea (now Oreocharis, see (Möller et al., 2011b)) (Möller et al., 2009; Möller et al., 2011a; Wang et al., 2010; Möller et al., 2011b). The multiple reversals to actinomorphy in Trichosporeae make this tribe suitable for
studying shifts of floral symmetry correlating to gene expression pattern between closely related zygomorphic and actinomorphic species. In addition, the actinomorphic flowers of species of two New World genera also evolved independently in different tribes within subfamily Gesnerioideae, Bellonia in tribe Gesnerieae and Napeanthus in tribe Napeantheae.
The genetics underlying floral symmetry was revealed by studies conducted on CYCLOIDEA (CYC) in Antirrhinum majus (Luo et al., 1996) in Plantaginaceae, a family in Lamiales closely related to Gesneriaceae (Schäferhoff et al., 2010). In core Eudicots, CYC and all its homologues belong to the ECE-CYC2 clade of the CYC/TB1 subfamily in which the dorsal-specific expression evolved in their ancestor (Howarth & Donoghue, 2006). Extensive studies regarding the phylogeny and expression of ECE-CYC2 genes among angiosperm lineages have demonstrated that multiple copies of CYC2-like genes from putative duplication events tend to be retained in both zygomorphic and actinomorphic lineages (Howarth & Donoghue, 2006; Citerne et al., 2000; Wang et al., 2004; Hileman & Baum,
2003; Hileman, 2014). However, it is unclear why CYC2-like genes tend to retain multiple copies and whether these multiple CYC2-like copies exhibit diverse expression patterns in association with floral morphology and floral symmetry transitions.
Within Gesneriaceae, previous studies suggested at least four GCYC duplication events (Citerne et al., 2000; Wang et al., 2004) and these GCYC duplicates all belonged to ECE-CYC2 clade (Song et al., 2009; Gao et al., 2008). These duplications occurred at the subfamily to genus level. The first event occurred during the early diversification of the family, generating GCYC1 and GCYC2.
GCYC2 is apparently lost in subfamily Gesnerioideae and perhaps Sanangoideae. Within tribe Trichosporeae, perhaps two additional independent GCYC1 duplication events were identified. One duplication was detected across the tribe except for European species, forming two subclades, namely GCYC1C and GCYC1D, the other occurred solely for the African genus Streptocarpus (Nishii et al., 2015) generating GCYC1A and GCYC1B (Citerne et al., 2000; Song et al., 2009; Gao et al., 2008; Du & Wang, 2008; Zhou et al., 2008; Hsu et al., 2018]. Together with the fact that
Trichosporeae species have the highest number of reversals to actinomorphy, the high number of GCYC1 duplications may imply that GCYC duplication correlates with the frequent floral symmetry transitions.
Gene duplication is generally required as precursor of functional divergence (Ohno, 1970; Ohta, 1988a; Ohta, 1988b; Force et al., 1999; Wagner et al., 2003). Model prediction and experimental data suggested that following gene duplication, the duplicated copies may have undergone functional divergence through neo-functionalization (shift to new gene expression domain) or
sub-functionalization (modified expression domain partially overlapping with ancestral positions) or may have accumulated deleterious mutations and became non-functionalization (cease to express) (Ohno, 1970; Force et al., 1999). It has been proposed for CYC and other positively selected genes that after gene duplication, natural selection favors the fixation of mutations in one or more copies that adapt
to divergent functions, and subsequently, sequential purifying selection acts to maintain new functions (Ohta, 1988a; Ohta, 1988b; Force et al., 1999; Jabbour et al., 2014; Bello et al., 2017). In other words, genetic constraints are temporarily lifted after duplication events (i.e. dN/dS deviated from 0), and this relaxation provides the opportunity for duplicated genes to adopt divergent functions (Wagner et al., 2003; Bielawski & Yang, 2003). For instance, in Helianthus, shifting strengths of positive selection (dN/dS >1) have acted on three CYC2-like gene copies(CYC2a, b, and c) and functional divergence among them was inferred (Chapman et al., 2008). This was supported by their divergent expression patterns across the copies in which those of one clade were expressed in all floral tissues, another restricted to rays and disk florets, and the other limited to ray florets only, suggesting a case of sub- or neo-functionalization. In addition to functional divergence, loss of function in one copy, non-functionalization, is another possible fate for gene copies (Ohno, 1970;
Ohta, 1988b; Force et al., 1999). In wind-pollinated radially symmetrical flowers among six Plantago species, the loss of showy petals was correlated with a loss or non-expression of A-clade CYC2-like copies, a signature of non-functionalization (Preston et al., 2011). Together, it is likely that natural selection can act on duplicated CYC2-like genes and subsequently their functions diverge, as inferred from shifts of their expression pattern and associated changes in flower morphology.
Only a few studies have examined the correlation between floral symmetry transition in Gesneriaceae and shifts in GCYC expression. Two studies were conducted on actinomorphic reversals in subtribe Didymocarpinae of tribe Trichosporeae, namely Bournea leiophylla (now Oreocharis leiophylla, see (Möller et al., 2011b)) and Tengia scopulorum (now Petrocodon scopulorum, see (Weber et al., 2011)). Distinctive expression patterns between GCYC copies were detected in association with a transition to actinomorphy (Zhou et al., 2008; Pang et al., 2010). In O.
leiophylla, BlCYC1 was found transiently expressed at the floral meristem initiation stage and then quickly vanished at later developmental stages. Thus, the loss of the ECE-CYC2-like gene expression
in later flowering stage seems to correlate with the actinomorphy resulting in mature flowers (Zhou et al., 2008). In P. scopulorum and DA cultivar of African Violets, on the contrary, GCYC duplicated copies were detected across all petals and stamens, correlating with the development of dorsalized actinomorphy (Hsu et al., 2018; Pang et al., 2010). These expression shifts of GCYC copies emphasize the possible associations of expression pattern change and resulting floral symmetry transition. Among zygomorphic Gesneriaceae species, such as Chirita heterotricha (now Primulina heterotricha, see (Wang et al., 2011)) and Opithandra dinghushanensis (now Oreocharis
dinghushanensis, see (Möller et al., 2011b)), it was found that the expression of duplicated GCYC1 copies was mainly confined to the floral dorsal region, even though the expression pattern between copies usually differed and had expanded (Song et al., 2009; Gao et al., 2008; Liu et al., 2014).
Overall, the expression pattern of GCYC duplications seemed to be correlated with their diversified expression patterns not only in actinomophic but also in zygomorphic species. This raises the possibility that potential expression pattern changes, thus functional divergence, has evolved frequently among GCYC duplicates. However, thus far, no integrated study has combined the evolutionary history, patterns of selective pressure, and expression patterns of GCYC copies to provide an overall interpretation to explain the reason why retention of multiple copies seemed to be preferred. Moreover, their association to floral symmetry remains to be studied.
The aim of this study was to investigate the patterns of gene expression and selective pressure of GCYC copies among species of tribe Trichosporeae in Gesneriaceae that contain species with zygomorphic and with actinomorphic flowers. In specific, we addressed (1) whether the expression patterns of these copies vary among zygomorphic and actinomorphy species, by studying two zygomorphic species H. bicornuta and Lysionotus pauciflorus, and one actinomorphic species C.
ramondioides; and (2) whether the rate between nonsynonymous and synonymous substitutions (ω = dN/dS) between different copies changes after GCYC duplication events. Our results will reveal whether the expression pattern of GCYC varies from species to species in general, or whether
copy-specific relaxation signals can be detected after gene duplication. The latter might suggest that evolutionary flexibility plays an important role in maintaining the diversity of flower morphology and floral symmetry transitions, underpinned by the retention of multiple GCYC duplicates in angiosperms.