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Integrating Molecular Phylogenies and Developmental Genetics: a Gesneriaceae Case Study
Abstract
We have analysed and characterised the phylogenetic potential of a nuclear developmental gene, cycloidea (originally isolated from Antirrhinum), involved in the development of floral zygomorphy. We have compared the evolution of part of this putative single copy gene in Old World Gesneriaceae with two contrasting DNA sequence regions, using two sets of data (a 'genus' data set and a 'species' data set); the chloroplast trnL(UAA) intron and the spacer between the trnL (UAA) 3' exon and trnF (GAA) were relatively conserved and suitable for phylogenetic reconstruction at genus level. The multicopy internal transcribed spacers (ITS1 and ITS2) of nuclear ribosomal DNA in contrast appear to be evolving about five times faster and are suitable for resolution at the species level. The putative homologue of cycloidea (Gcyc) has an intermediate substitution rate about three times faster than the chloroplast intron/spacer region. However, the level of pairwise sequence divergence of Gcyc is higher than that of ITS at very low levels of divergence. This difference in apparent rate of molecular evolution between ITS and Gcyc at different levels of the taxonomic hierarchy we attribute to the process of molecular drive in the multicopy ITS. At lower levels of divergence (e.g. between closely related species) fixation of genetic changes in the multicopy ribosomal DNA acts as a restraint on evolutionary rate, whereas third codon position changes in coding single copy nuclear (scnDNA) genes are unconstrained. However, at high levels of divergence (e.g. between general, scnDNA evolution is more functionally constrained than that of ITS and Gcyc therefore Varies less. The small restraining effect of concerted evolution is not noticeable at these levels of sequence divergence. All three regions appear to evolve in a clock-like manner and are found to be suitable for phylogenetic reconstruction by parsimony, resulting in the same or similar topologies. We have examined the Gcyc sequences of three species that have reverted to actinomorphy from a zygomorphic condition. The gene appears to be intact and therefore, by implication, functional in these species. Furthermore, in one of these clades there has been a reversion back to zygomorphy which also implies that the gene is intact. We therefore suggest that in naturally occurring actinomorphic Gesneriaceae Gcyc continues to have a functional role, but zygomorphy is reduced by modifying genes. There is no convincing evidence that Gcyc evolves faster in actinomorphic lineages.
... In total, 57 Gesneriaceae species representing all major lineages, including 54 zygomorphic and 3 actinomorphic species (Conandron ramondioides, Oreocharis leiophylla, Ramonda myconi) covering two subfamilies Didymocarpoideae (Old World) and Gesnerioideae (New World) were selected to investigate the genealogy of CYC-like genes across Gesneriaceae (S1 Table). Forty-four GCYC copies from 27 species were isolated in this study by PCR with Gesneriaceae specific GCYC primers [46] (S1 Table). An additional 49 GCYC copies from 30 species were downloaded from GenBank, representing all publicly available GCYC sequences. ...
... Total genomic DNA was extracted from fresh leaves or leaf material dried by silica gel following a CTAB protocol [47]. Partial sequences of GCYC of the 27 target species were amplified using a degenerate primer pair previously designed for CYC2-like genes in Gesneriaceae: GCYCFs and GCYCR [46]. Amplifications were performed on a DNA Programmable Thermal Cycler (ABI2700, USA) using the following conditions: initial denaturation at 94˚C for 3 min; followed by 35 cycles at 94˚C for 30 s, annealing at 55˚C for 30 s, 72˚C for 50 s; and a subsequent final extension at 72˚C for 10 min. ...
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 (ωpre-duplication = 0.2819, ωpost-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.
... If this is the situation in African violet, we would expect an entire loss of CYC expression in flower buds. Möller et al. (1999) first isolated partial CYC homologs from several Gesneriaceae species. In Saintpaulia, the CYC homologs exist as a pair of recently duplicated CYC paralogs (SiCYC1A, SiCYC1B) and both are direct orthologs FIGURE 1 | The photos, floral diagram, and SEM photos of flowers between zygomorphic wild type (WT) and its two actinomorphic mutants, dorsalized (DA) and ventralized peloria (VA). ...
... Parts of the Saintpaulia SiCYC 1A and 1B sequences were first amplified with DNA and cDNA using primer pairs FS (5 ′ -ATG CTA GGT TTC GAC AAG CC-3 ′ ) and R (5 ′ -ATG AAT TTG TGC TGA TCC AAA ATG-3 ′ ) designed from highly conserved TCP and R domain as in Möller et al. (1999). This primer pair has been demonstrated to efficiently amplify all CYC copies from all major Gesneriaceae lineages (Wang et al., 2004a). ...
With the growing demand for its ornamental uses, the African violet (Saintpaulia ionantha) has been popular owing to its variations in color, shape and its rapid responses to artificial selection. Wild type African violet (WT) is characterized by flowers with bilateral symmetry yet reversals showing radially symmetrical flowers such as dorsalized actinomorphic (DA) and ventralized actinomorphic (VA) peloria are common. Genetic crosses among WT, DA, and VA revealed that these floral symmetry transitions are likely to be controlled by three alleles at a single locus in which the levels of dominance are in a hierarchical fashion. To investigate whether the floral symmetry gene was responsible for these reversals, orthologs of CYCLOIDEA (CYC) were isolated and their expressions correlated to floral symmetry transitions. Quantitative RT-PCR and in situ results indicated that dorsal-specific CYCs expression in WT S. ionantha (SiCYC and SiCYC1B) shifted in DA with a heterotopically extended expression to all petals, but in VA, SiCYC1s' dorsally specific expressions were greatly reduced. Selection signature analysis revealed that the major high-expressed copy of SiCYC had been constrained under purifying selection, whereas the low-expressed helper SiCYC1B appeared to be relaxed under purifying selection after the duplication into SiCYC and SiCYC1B. Heterologous expression of SiCYC in Arabdiopsis showed petal growth retardation which was attributed to limited cell proliferation. While expression shifts of SiCYC and SiCYC1B correlate perfectly to the resulting symmetry phenotype transitions in F1s of WT and DA, there is no certain allelic combination of inherited SiCYC1s associated with specific symmetry phenotypes. This floral transition indicates that although the expression shifts of SiCYC/1B are responsible for the two contrasting actinomorphic reversals in African violet, they are likely to be controlled by upstream trans-acting factors or epigenetic regulations.
... Moreover, Burtt also deduced that the corolla actinomorphy of Tengia should be an evolutionarily secondary condition, derived from corolla zygomorphy and tetrandry or diandry. This has recently been confirmed by molecular studies (Möller et al. 1999, 2009, Wang et al. 2010, Weber et al. 2011. Comparatively, the different numbers and shapes of the stamens and staminodes suggest that they might have retained different characters from their ancestors in the evolutionary process. ...
Petrocodon urceolatus F. Wen, H.F. Cen & L.F. Fu, a new species of Gesneriaceae, is described and illustrated from Zhangjiajie city, Hunan Province, China. Morphologically it resembles P. scopulorum (Chun) Y.Z. Wang in the corolla, but differs in several characters including the shape, length and indumentum of the leaf blade, and the length and indumentum of the stamens and staminode. In addition to morphological description, we provide ecological information, geographic distribution and conservation status for this species.
... Moreover, Burtt concluded that the corolla actinomorphy and pentandry of Tengia is an evolutionarily secondary condition, derived from corolla zygomorphy and tetrandry or diandry (here we confirm its descendance from diandry). Indeed, it has been recently confirmed by molecular studies that genera with actinomorphic flowers do not represent a monophyletic group and that floral actinomorphy has secondarily and independently evolved in several alliances of the didymocarpoid Gesneriaceae (Möller et al. 1999, 2009, Wang et al. 2010. ...
Based on molecular studies, the small Chinese genus Petrocodon (two species and one variety) has been recently enlarged to include the monotypic genera Calcareoboea, Paralagarosolen and Tengia. It is shown here that the (6-7) species of Lagarosolen, the monotypic Dolicholoma, a few species of Didymocarpus, and a number of new species that have recently been published (but not formally described) under Petrocodon and Lagarosolen should be included in this genus. This raises the size of the genus from five to around 20 species. With respect to the floral diversity (corolla form, size, and coloration; with the exception of Tengia, the androecium is always diandrous) and inferred pollination syndromes (different forms of melittophily, ornithophily, psycho- and/or sphingophily), Petrocodon represents one of the most varied genera of Old World Gesneriaceae, comparable to some New World genera.
To produce new ornamental plants by combining Saintpaulia ionantha and Streptocarpus, two closely related genera of the family Gesneriaceae, protoplast fusions were accomplished. Various tissues were evaluated as potential starting materials for protoplast isolation from Streptocarpus saxorum × S. stomandrus and S. caulescens. Sufficient protoplast yields between 2 and 3.6×10⁵ g⁻¹ fresh mass with high viabilities from 68-96% were obtained from the leaves of in vitro shoot cultures, which were grown in liquid medium in the dark. After 14 days of culture, cell divisions reached frequencies between 2.1 and 5.5%, depending on the species, and vigorously growing calluses were obtained. However, only root formation was induced on these calluses; no shoot induction was achieved. For the protoplast fusion, polyethylene glycol was applied and an appropriate fusion protocol was developed. The fusion rate, which was determined by cell nucleus staining with ethidium bromide, reached up to 35% binuclear protoplasts. Cell divisions were observed in four fusion experiments between two Streptocarpus species and two Saintpaulia ionantha varieties, and in two of these experiments, callus formation was also observed. To identify possible hybrid calluses, calluses were preselected by flow cytometry and analysed with RAPD markers. In 27 out of 30 tetraploid calluses the DNA bands from the Streptocarpus parent were dominant or were the only ones detected, in one the bands from the Saintpaulia parent, and only two calluses with bands of both parents were observed. This evidence supports the notion that successful fusions between Saintpaulia and Streptocarpus are possible, but no plants could be regenerated from the hybrid calluses.
The phylogenetic placement of the Old World Gesneriaceae genera Ramonda, Conandron, Bournea, Thamnocharis, and Tengia, all characterized by actinomorphic flowers, has been the subject of much debate. Actinomorphy in Gesneriaceae is rare, with most species exhibiting zygomorphic flowers. The actinomorphic genera have historically been considered “primitive” and lumped in the tribe Ramondeae separate from the remaining Old World Gesneriaceae. In this study, we used nuclear (ITS) and plastid (trnL‐F) DNA for molecular phylogenetic analysis of these five genera along with representative species across the Cyrtandroideae. Our results show that the actinomorphic genera are scattered over several otherwise zygomorphic clades within Cyrtandroideae, and along with previous data, indicate that Ramondeae is an unnatural group. Floral actinomorphy has evolved convergently in different alliances of Old World Gesneriaceae. Ramonda is sister to Haberlea, Bournea is apparently paraphyletic, Conandron seems rather isolated, and Tengia is close to Petrocodon and sister to a group of Chirita sect. Gibbosaccus together with Calcareoboea. We hypothesize that the evolution from zygomorphy to actinomorphy with novel combinations of characters is possibly due to shifts in pollination strategies, such as a switch from nectar‐ to pollen‐rewards.
Background:
Bilateral symmetry flower (zygomorphy) is the ancestral state for Gesneriaceae species. Yet independent reversions to actinomorphy have been parallelly evolved in several lineages. Conandron ramondioides is a natural radially symmetrical species survived in dense shade mountainous habitats where specialist pollinators are scarce. Whether the mutations in floral symmetry genes such as CYC, RAD and DIV genes, or their expression pattern shifts contribute to the reversion to actinomorphy in C. ramondioides thus facilitating shifts to generalist pollinators remain to be investigated. To address this, we isolated putative orthologues of these genes and relate their expressions to developmental stages of flower actinomorphy.
Results:
Tissue specific RT-PCR found no dorsal identity genes CrCYCs and CrRADs expression in petal and stamen whorls, while the ventral identity gene CrDIV was expressed in all petals. Thus, ventralized actinomorphy is evolved in C. ramondioides. However, CrCYCs still persists their expression in sepal whorl. This is congruent with previous findings that CYC expression in sepals is an ancestral state common to both actinomorphic and zygomorphic core Eudicot species.
Conclusions:
The loss of dorsal identity genes CrCYCs and CrRADs expression in petal and stamen whorl without mutating these genes specifies that a novel regulation change, possibly on cis-elements of these genes, has evolved to switch zygomorphy to actinomorphy.
Several ways in which morphology is used in systematic and evolutionary research in angiosperms are shown and illustrated with examples: 1) searches for special structural similarities, which can be used to find hints for hitherto unrecognized relationships in groups with unresolved phylogenetic position; 2) cladistic studies based on morphology and combined morphological and molecular analyses; 3) comparative morphological studies in new, morphologically puzzling clades derived from molecular studies; 4) studies of morphological character evolution, unusual evolutionary directions, and evolutionary lability based on molecular studies; and 5) studies of organ evolution. Conclusions: Goals of comparative morphology have shifted in the present molecular era. Morphology no longer plays the primary role in phylogenetic studies. However, new opportunities for morphology are opening up that were not present in the premolecular era: 1) phylogenetic studies with combined molecular and morphological analyses; 2) reconstruction of the evolution of morphological features based on molecularly derived cladograms; 3) refined analysis of morphological features induced by inconsistencies of previous molecular and molecular phylogenetic analyses; 4) better understanding of morphological features by judgment in a wider biological context; 5) increased potential for including fossils in morphological analyses; and 6) exploration of the evolution of morphological traits by integration of comparative structural and molecular developmental genetic aspects (Evo-Devo); this field is still in its infancy in botany; its advancement is one of the major goals of evolutionary botany.
The taxonomy of the African, Madagascan and Comoro Island (Afro-Malagasy) Gesneriaceae attracts a large amount of interest given the horticultural importance of Cape Primroses (Streptocarpus) and African Violets (Saintpaulia). Earlier studies indicated that the Afro-Malagasy genera form a strongly supported clade, and recent classifications have included some of the genera within an expanded Streptocarpus. Given the global importance of this group, we carried out a comprehensive molecular phylogenetic analysis of all Afro-Malagasy genera in subfamily Didymocarpoideae, tribe Trichosporeae, subtribe Streptocarpinae, to investigate species relationships in these genera as the basis for a new classification. Phylogenetic analyses of the nuclear ribosomal spacer (ITS, 5S NTS) and chloroplast intron and spacer regions (rpl20-rps12 spacer, trnL intron, trnLF spacer) of 226 samples were performed, including all Streptocarpinae genera, except the monotypic Nodonema. The molecular phylogenies demonstrate that the genera with non-twisted fruits are nested within Streptocarpus which has twisted fruits. Two main clades were found, one comprising herbaceous caulescent Streptocarpus that also included Saintpaulia, the caulescents Hovanella and Schizoboea, and the unifoliates Acanthonema and Trachystigma. The second clade comprises the woody caulescents and acaulescent Streptocarpus, Colpogyne and Linnaeopsis. Altogether, twelve well-supported subclades can be recognized, each with a combination of distinct morphological characteristics. A new classification of tribe Streptocarpinae, de facto Streptocarpus, is presented, retaining the two subgenera, Streptocarpus and Streptocarpella, and dividing them into five and seven sections respectively. Nodonema is attributed to subg. Streptocarpus for morphological reasons. The former genus Saintpaulia is classified as Streptocarpus subg. Streptocarpella sect. Saintpaulia with ten species recognized.
To produce new ornamental plants by combining Saintpaulia ionantha and Streptocarpus, two closely related genera of the family Gesneriaceae, protoplast fusions were accomplished. Various tissues were evaluated as potential starting materials for protoplast isolation from Streptocarpus saxorum × S. stomandrus and S. caulescens. Sufficient protoplast yields between 2 and 3.6×10⁵ g⁻¹ fresh mass with high viabilities from 68-96% were obtained from the leaves of in vitro shoot cultures, which were grown in liquid medium in the dark. After 14 days of culture, cell divisions reached frequencies between 2.1 and 5.5%, depending on the species, and vigorously growing calluses were obtained. However, only root formation was induced on these calluses; no shoot induction was achieved. For the protoplast fusion, polyethylene glycol was applied and an appropriate fusion protocol was developed. The fusion rate, which was determined by cell nucleus staining with ethidium bromide, reached up to 35% binuclear protoplasts. Cell divisions were observed in four fusion experiments between two Streptocarpus species and two Saintpaulia ionantha varieties, and in two of these experiments, callus formation was also observed. To identify possible hybrid calluses, calluses were preselected by flow cytometry and analysed with RAPD markers. In 27 out of 30 tetraploid calluses the DNA bands from the Streptocarpus parent were dominant or were the only ones detected, in one the bands from the Saintpaulia parent, and only two calluses with bands of both parents were observed. This evidence supports the notion that successful fusions between Saintpaulia and Streptocarpus are possible, but no plants could be regenerated from the hybrid calluses.
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