Comparative chloroplast genomics reveals the phylogeny and the adaptive evolution of Begonia in China.

BACKGROUND: The Begonia species are common shade plants that are mostly found in southwest China. They have not been well studied despite their medicinal and decorative uses because gene penetration, decreased adaptability, and restricted availability are all caused by frequent interspecific hybridization. RESULT: To understand the patterns of mutation in the chloroplast genomes of different species of Begonia, as well as their evolutionary relationships, we collected seven Begonia species in China and sequenced their chloroplast genomes. Begonia species exhibit a quadripartite structure of chloroplast genomes (157,634 - 169,694 bp), consisting of two pairs of inverted repeats (IR: 26,529 - 37,674 bp), a large single copy (LSC: 75,477 - 86,500 bp), and a small single copy (SSC: 17,861 - 18,367 bp). 128-143 genes (comprising 82-93 protein-coding genes, 8 ribosomal RNAs, and 36-43 transfer RNAs) are found in the chloroplast genomes. Based on comparative analyses, this taxon has a relatively similar genome structure. A total of six substantially divergent DNA regions (trnT-UGU-trnL-UAA, atpF-atpH, ycf4-cemA, psbC-trnS-UGA, rpl32-trnL-UAG, and ccsA-ndhD) are found in the seventeen chloroplast genomes. These regions are suitable for species identification and phylogeographic analysis. Phylogenetic analysis shows that Begonia species that were suited to comparable environments grouped in a small clade and that all Begonia species formed one big clade in the phylogenetic tree, supporting the genus' monophyly. In addition, positive selection sites were discovered in eight genes (rpoC1, rpoB, psbE, psbK, petA, rps12, rpl2, and rpl22), the majority of which are involved in protein production and photosynthesis. CONCLUSION: Using these genome resources, we can resolve deep-level phylogenetic relationships between Begonia species and their families, leading to a better understanding of evolutionary processes. In addition to enhancing species identification and phylogenetic resolution, these results demonstrate the utility of complete chloroplast genomes in phylogenetically and taxonomically challenging plant groupings.

Genomes shed light on the evolution of Begonia, a mega-diverse genus.

Clarifying the evolutionary processes underlying species diversification and adaptation is a key focus of evolutionary biology. Begonia (Begoniaceae) is one of the most species-rich angiosperm genera with c. 2000 species, most of which are shade-adapted. Here, we present chromosome-scale genome assemblies for four species of Begonia (B. loranthoides, B. masoniana, B. darthvaderiana and B. peltatifolia), and whole genome shotgun data for an additional 74 Begonia representatives to investigate lineage evolution and shade adaptation of the genus. The four genome assemblies range in size from 331.75 Mb (B. peltatifolia) to 799.83 Mb (B. masoniana), and harbor 22 059-23 444 protein-coding genes. Synteny analysis revealed a lineage-specific whole-genome duplication (WGD) that occurred just before the diversification of Begonia. Functional enrichment of gene families retained after WGD highlights the significance of modified carbohydrate metabolism and photosynthesis possibly linked to shade adaptation in the genus, which is further supported by expansions of gene families involved in light perception and harvesting. Phylogenomic reconstructions and genomics studies indicate that genomic introgression has also played a role in the evolution of Begonia. Overall, this study provides valuable genomic resources for Begonia and suggests potential drivers underlying the diversity and adaptive evolution of this mega-diverse clade.

Multi-tissue transcriptome analysis of two Begonia species reveals dynamic patterns of evolution in the chalcone synthase gene family.

Begonia is an important horticultural plant group, as well as one of the most speciose Angiosperm genera, with over 2000 described species. Genus wide studies of genome size have shown that Begonia has a highly variable genome size, and analysis of paralog pairs has previously suggested that Begonia underwent a whole genome duplication. We address the contribution of gene duplication to the generation of diversity in Begonia using a multi-tissue RNA-seq approach. We chose to focus on chalcone synthase (CHS), a gene family having been shown to be involved in biotic and abiotic stress responses in other plant species, in particular its importance in maximising the use of variable light levels in tropical plants. We used RNA-seq to sample six tissues across two closely related but ecologically and morphologically divergent species, Begonia conchifolia and B. plebeja, yielding 17,012 and 19,969 annotated unigenes respectively. We identified the chalcone synthase gene family members in our Begonia study species, as well as in Hillebrandia sandwicensis, the monotypic sister genus to Begonia, Cucumis sativus, Arabidopsis thaliana, and Zea mays. Phylogenetic analysis suggested the CHS gene family has high duplicate turnover, all members of CHS identified in Begonia arising recently, after the divergence of Begonia and Cucumis. Expression profiles were similar within orthologous pairs, but we saw high inter-ortholog expression variation. Sequence analysis showed relaxed selective constraints on some ortholog pairs, with substitutions at conserved sites. Evidence of pseudogenisation and species specific duplication indicate that lineage specific differences are already beginning to accumulate since the divergence of our study species. We conclude that there is evidence for a role of gene duplication in generating diversity through sequence and expression divergence in Begonia.

Selection of suitable reference genes for qRT-PCR analysis of Begonia semperflorens under stress conditions.

Begonia semperflorens (B. semperflorens), belonging to the family Begoniaceae, has now been widely cultivated worldwide and is famous for its ornamental plants with colourful flowers and distinctive leaves. The selection of appropriate internal reference genes is very important to accurately determine target gene expression via quantitative real-time PCR. However, internal reference gene selection has never been conducted in B. semperflorens. In this study, seven candidate reference genes of B. semperflorens, including 18S ribosomal RNA (Bs18S), pentatricopeptide repeat-containing protein (BsPPR), actin-related protein 5 isoform X2 (BsACT), DNAJ homologue subfamily C member 17 (BsDNAJ), glyceraldehyde-3-phosphate dehydrogenase (BsGAPDH), NAD-dependent malic enzyme 59 kDa isoform, mitochondria (BsNAD-ME), and peptidyl-prolyl cis-trans isomerase CYP26-2, chloroplast (BsCYP), which were obtained from our previous studies, were selected. The stabilities of these genes under stress conditions were analysed using geNorm and NormFinder. Validation of target gene expressions, including phenylalanine ammonia-lyase (BsPAL) and respiratory burst oxidase homologue D (BsRBOHD) under biotic and abiotic conditions, phenylalanine ammonia-lyase (BsPAL), anthocyanidin synthase (BsANS), chalcone synthase (BsCHS), and flavanone-3-hydroxylase (BsF3H) under low temperature, using these seven internal reference genes for normalisation further confirmed the stabilities of the selected genes and indicated the need for reference gene selection for normalising gene expressions in B. semperflorens. Of the seven candidate reference genes, the combination of BsACT, BsDNAJ, and BsNAD-ME was the ideal reference gene set for normalising gene expression in samples under biotic conditions. BsCYP combined with BsACT or BsGAPDH was the best reference gene pair under abiotic conditions. BsACT and BsPPR could be combined to normalise gene expression under low temperature. Our results will benefit future studies on gene expression in plants of Begoniaceae.

Chloroplast and nuclear DNA exchanges among Begonia sect. Baryandra species (Begoniaceae) from Palawan Island, Philippines, and descriptions of five new species.

The Philippine island of Palawan is highly biodiverse. During fieldwork there in 2011 & 2014 we found five unknown species in the large genus Begonia. The species are similar in their rhizomatous stems, four-tepaled flowers, inferior two- or three-locular ovaries with bilamellate placentas, and are assignable to Begonia sect. Baryandra. Our observations support the recognition of these as five new species endemic to Palawan: B. elnidoensis, B. gironellae, B. quinquealata, B. tabonensis and B. tenuibracteata which are described here. The five new species were added to phylogenies based Bayesian analysis of nrDNA (ITS) and chloroplast DNA (ndhA, ndhF-rpl32, rpl32-trnL, trnC-trnD), along with 45 other allied ingroup species. A majority of the species show incongruent positions in the two phylogenies, with evidence of prevalent chloroplast capture. Models show chloroplast capture is more likely in plant populations with high levels of inbreeding following a reduction in selfing rate after hybridisation; we suggest that this is a possible explanation for the massive amount of chloroplast exchange seen in our phylogeny, as Begonia species often exist as small isolated populations and may be prone to inbreeding depression. Our data also indicate a level of nuclear genetic exchange between species. The high prevalence of hybrid events in Begonia is potentially an important factor in driving genomic change and species evolution in this mega-diverse genus.

High-throughput Transcriptome Sequencing Reveals the Role of Anthocyanin Metabolism in Begonia semperflorens Under High Light Stress.

Begonia semperflorens is an ornamental perennial herb. The leaves of B. semperflorens turn red under increased light, which increases the ornamental value of the plant. The color of the leaves is determined by anthocyanin metabolism. In B. semperflorens leaves, anthocyanin metabolism is sensitive to external environmental conditions such as temperature, light and hormone levels. To explore this process in detail and to assess gene expression under high light stress, transcriptome analysis was performed by RNA sequencing using the sequencing-by-synthesis method. A total of 83 699 unigenes were isolated, and 51 754 unigenes were annotated using the NR, Swiss-Prot, KEGG, COG, KOG, GO and Pfam databases. Furthermore, many of the differentially expressed genes were related to factors associated with anthocyanin metabolism, which influences the expression of leaf color.

Comparative Analysis of Begonia Plastid Genomes and Their Utility for Species-Level Phylogenetics.

Recent, rapid radiations make species-level phylogenetics difficult to resolve. We used a multiplexed, high-throughput sequencing approach to identify informative genomic regions to resolve phylogenetic relationships at low taxonomic levels in Begonia from a survey of sixteen species. A long-range PCR method was used to generate draft plastid genomes to provide a strong phylogenetic backbone, identify fast evolving regions and provide informative molecular markers for species-level phylogenetic studies in Begonia.

The Miocene to Pleistocene colonization of the Philippine archipelago by Begonia sect. Baryandra (Begoniaceae).

PREMISE OF THE STUDY: One third of the species-rich Philippine flora is endemic, and most of the islands in the archipelago have never been connected to a continental region. We currently lack any well-sampled angiosperm phylogenies that span the archipelago, prohibiting the formation of informed hypotheses as to the evolution of this rich and highly endemic flora. METHODS: We produced time-calibrated phylogenetic trees from both nuclear (ITS) and chloroplast (ndhA intron, ndhF-rpl32 spacer, rpl32-trnL spacer, trnC-trnD spacer) regions of 41 species of Begonia sect. Baryandra, all except one endemic to the Philippines. Historical biogeography was reconstructed across the chloroplast phylogeny using a Bayesian binary method of character optimization. Comparison of phylogenies from the two genomes permitted insight into the prevalence of hybridization in the group. KEY RESULTS: The Philippine archipelago was colonized by Begonia sect. Baryandra in the late Miocene, via long-distance dispersal from western Malesia and a point of entry likely to be in the northwestern region of the archipelago. Palawan, Luzon, and Panay all bear early-branching lineages from this initial colonization. There have been Plio-Pleistocene dispersals from these islands into Borneo and Mindanao. Hybridization was common between species as evidenced by haplotype sharing and phylogenetic incongruence. CONCLUSIONS: The phylogenies show a high degree of geographic structure, which millions of years of exposure to typhoons have not blurred, showing long-term species and population stability. The recent dispersals to Mindanao are congruent with the geologically recent arrival of the island at its current latitude in the southern Philippines.

[Study on wild germplasm resoures of Begonia fimbristipula in Dinghu District, Zhaoqing City, Guangdong Province].

OBJECTIVE: A systematical investigation on the distribution of wild germplasm of Regonia fimbristipula in Dinghu District, Zhaoqing City, Guangdong Province was conducted at 8 sites. METHODS: Field quadrat survey method was carried out. RESULTS: Begonia fimbristipula had obvious phenotypical plasticity showing three phenotypes with red, green and bicolor leaf, respectively. Its populations lived in the ecological environment of rock. The growth and building of Begonia fimbristipula population were mutually influenced by many ecological factors such as natural habitat, slope-exposure, soil thickness, sunlight, air humidity as well as soil physical and chemical properties. CONCLUSION: Living environment vulnerability and human activities are the main reason causing sharp decrease of wild resources of Begonia fimbristipula. Evaluation on regional distribution of wild Begonia fimbristipula and its protection and use of the rationalization have important value.

The evolution of sex ratio differences and inflorescence architectures in Begonia (Begoniaceae).

PREMISE OF THE STUDY: A major benefit conferred by monoecy is the ability to alter floral sex ratio in response to selection. In monoecious species that produce flowers of a given sex at set positions on the inflorescence, floral sex ratio may be related to inflorescence architecture. We studied the loci underlying differences in inflorescence architecture between two monoecious Begonia species and related this to floral sex ratios. METHODS: We performed trait comparisons and quantitative trait locus (QTL) mapping in a segregating backcross population between Central American Begonia plebeja and B. conchifolia. We focused on traits related to inflorescence architecture, sex ratios, and other reproductive traits. KEY RESULTS: The inflorescence branching pattern of B. conchifolia was more asymmetric than B. plebeja, which in turn affects the floral sex ratio. Colocalizing QTLs of moderate effect influenced both the number of male flowers and the fate decisions of axillary meristems, demonstrating the close link between inflorescence architecture and sex ratio. Additional QTLs were found for stamen number (30% variance explained, VE) and pollen sterility (12.3% VE). CONCLUSIONS: One way in which Begonia species develop different floral sex ratios is through modifications of their inflorescence architecture. The potential pleiotropic action of QTL on inflorescence branching and floral sex ratios may have major implications for trait evolution and responses to selection. The presence of a single QTL of large effect on stamen number may allow rapid divergence for this key floral trait. We propose candidate loci for stamen number and inflorescence branching for future characterization.

Genetic differentiation and species cohesion in two widespread Central American Begonia species.

Begonia is one of the ten largest plant genera, with over 1500 species. This high species richness may in part be explained by weak species cohesion, which has allowed speciation by divergence in allopatry. In this study, we investigate species cohesion in the widespread Central American Begonia heracleifolia and Begonia nelumbiifolia, by genotyping populations at microsatellite loci. We then test for post-zygotic reproductive barriers using experimental crosses, and assess whether sterility barriers are related to intraspecific changes in genome size, indicating major genome restructuring between isolated populations. Strong population substructure was found for B. heracleifolia (FST=0.364, F'ST=0.506) and B. nelumbiifolia (FST=0.277, F'ST=0.439), and Bayesian admixture analysis supports the division of most populations into discrete genetic clusters. Moderate levels of inferred selfing (B. heracleifolia s=0.40, B. nelumbiifolia s=0.62) and dispersal limitation are likely to have contributed to significant genetic differentiation (B. heracleifolia Jost's D=0.274; B. nelumbiifolia D=0.294). Interpopulation crosses involving a divergent B. heracleifolia population with a genome size ∼10% larger than the species mean had a ∼20% reduction in pollen viability compared with other outcrosses, supporting reproductive isolation being polymorphic within the species. The population genetic data suggest that Begonia populations are only weakly connected by gene flow, allowing reproductive barriers to accumulate between the most isolated populations. This supports allopatric divergence in situ being the precursor of speciation in Begonia, and may also be a common speciation mechanism in other tropical herbaceous plant groups.

Natural foliar variegation without costs? The case of Begonia.

BACKGROUND AND AIMS: Foliar variegation is recognized as arising from two major mechanisms: leaf structure and pigment-related variegation. Begonia has species with a variety of natural foliar variegation patterns, providing diverse examples of this phenomenon. The aims of this work are to elucidate the mechanisms underlying different foliar variegation patterns in Begonia and to determine their physiological consequences. METHODS: Six species and one cultivar of Begonia were investigated. Light and electron microscopy revealed the leaf structure and ultrastructure of chloroplasts in green and light areas of variegated leaves. Maximum quantum yields of photosystem II were measured by chlorophyll fluorescence. Comparison with a cultivar of Ficus revealed key features distinguishing variegation mechanisms. KEY RESULTS: Intercellular space above the chlorenchyma is the mechanism of variegation in these Begonia. This intercellular space can be located (a) below the adaxial epidermis or (b) below the adaxial water storage tissue (the first report for any taxa), creating light areas on a leaf. In addition, chlorenchyma cell shape and chloroplast distribution within chlorenchyma cells differ between light and green areas. Chloroplasts from both areas showed dense stacking of grana and stroma thylakoid membranes. The maximum quantum yield did not differ significantly between these areas, suggesting minimal loss of function with variegation. However, the absence of chloroplasts in light areas of leaves in the Ficus cultivar led to an extremely low quantum yield. CONCLUSIONS: Variegation in these Begonia is structural, where light areas are created by internal reflection between air spaces and cells in a leaf. Two forms of air space structural variegation occur, distinguished by the location of the air spaces. Both forms may have a common origin in development where dermal tissue becomes loosely connected to mesophyll. Photosynthetic functioning is retained in light areas, and these areas do not include primary veins, potentially limiting the costs of variegation.

Identification of genome constitutions in Begonia×chungii and its putative parents, B. longifolia and B. palmata, by genomic in situ hybridization (GISH).

Based mainly on morphological features and geographical distribution, Begonia×chungii (2n=22) was recently reported as a natural hybrid between B. longifolia and B. palmata in Taiwan. This study aims to confirm the hybridity of B.×chungii and to sort out the genome constitutions of its putative parents, using genomic in situ hybridization (GISH). Genomic DNAs of both parental species were used as probes for B.×chungii and the experimental F(1) hybrid, B. palmata×B. longifolia, in GISH analyses. Bicolor-GISH analyses in B.×chungii showed that the 22 chromosomes consisted of six chromosomes hybridized with a probe derived from the B. palmata genome, six with another probe from the B. longifolia genome and the remaining ten with both genomes overlapped. Meanwhile, bicolor-GISH in B. palmata×B. longifolia showed a remarkable similarity to that of B.×chungii. The reciprocal GISH results between B. longifolia and B. palmata were comparable. Our GISH analyses confirmed that B.×chungii is a natural F(1) hybrid between B. longifolia and B. palmata. Genomes of the parental species were shown to be partially homologous, suggesting a derived common ancestral genome in them.

Isolation of compound microsatellite markers in Begonia fenicis (Begoniaceae) endemic to East and Southeast Asian islands.

PREMISE OF THE STUDY: We developed compound microsatellite markers for Begonia fenicis, a species endemic to eastern and southeastern Asian islands, to investigate geographical genetic structure. METHODS AND RESULTS: Using the compound microsatellite marker technique, 21 markers were developed and six polymorphic markers were characterized for samples from four islands in Taiwan and southern Japan. The number of alleles per locus ranged from two to six (mean = 4.33). Observed and expected heterozygosities were 0.125-0.725 (0.383) and 0.498-0.719 (0.641), and polymorphic information content was 0.371-0.664 (0.567). The interspecific transferability of the 21 markers was evaluated for eight species of the section Diploclinium from the Philippines; 15 markers were successfully amplified in one to eight species. CONCLUSIONS: These results indicate the utility of the six microsatellite markers in B. fenicis to investigate geographical genetic structure. The transferable markers are potentially useful for other species of the section.

A non-coding plastid DNA phylogeny of Asian Begonia (Begoniaceae): evidence for morphological homoplasy and sectional polyphyly.

Maximum likelihood and Bayesian analyses of non-coding plastid DNA sequence data based on a broad sampling of all major Asian Begonia sections (ndhA intron, ndhF-rpl32 spacer, rpl32-trnL spacer, 3977 aligned characters, 84 species) were used to reconstruct the phylogeny of Asian Begonia and to test the monophyly of major Asian Begonia sections. Ovary and fruit characters which are crucial in current sectional circumscriptions were mapped on the phylogeny to assess their utility in infrageneric classifications. The results indicate that the strong systematic emphasis placed on single, homoplasious characters such as undivided placenta lamellae (section Reichenheimia) and fleshy pericarps (section Sphenanthera), and the recognition of sections primarily based on a suite of plesiomorphic characters including three-locular ovaries with axillary, bilamellate placentae and dry, dehiscent pericarps (section Diploclinium), has resulted in the circumscription of several polyphyletic sections. Moreover, sections Platycentrum and Petermannia were recovered as paraphyletic. Because of the homoplasy of systematically important characters, current classifications have a certain diagnostic, but only poor predictive value. The presented phylogeny provides for the first time a reasonably resolved and supported phylogenetic framework for Asian Begonia which has the power to inform future taxonomic, biogeographic and evolutionary studies.

Meiotic aberrations during 2n pollen formation in Begonia.

Unreduced gametes are the driving force for the polyploidization of plants in nature, and are also an important tool for ploidy breeding. The final heterozygosity of a 2n pollen grain depends on the cytological mechanism behind 2n pollen formation. In this study, chromosome pairing and chromosome segregation during the microsporogenesis of seven Begonia genotypes were analysed using fluorescent chromosome staining on (squashed) pollen mother cells. Among the seven genotypes, five genotypes produce 2n pollen (B. 'Bubbles', B. 'Florence Rita', B. 'Orococo', B. 'Tamo' and B276) and two genotypes produce only normal n pollen (B. fischeri and B243). All 2n pollen producers showed a mechanism equivalent to first division restitution (FDR), in which chromosomes did not segregate during meiosis I but only during meiosis II. This FDR was the result of (a) an irregular chromosome pairing in B. 'Tamo', (b) stickiness of chromosomes associated with numerous chromosome bridges in B. 'Florence Rita' and B276, and (c) a combination of irregular chromosome pairing and stickiness of chromosomes in B. 'Bubbles'. The exact mechanism of the nuclear restitution in B. 'Orococo' could not be determined. Other mechanisms, such as early asymmetric cytokinesis, omission of meiosis II, parallel or tripolar spindle formation, were rather uncommon. Unpaired chromosomes (univalents) were observed in all genotypes, but they had moved to one of the poles by the end of anaphase I or II. Only B. 'Tamo' formed a high number of micronuclei. Consequently, this genotype formed a large number of malformed pollen. Obviously, chromosome behaviour during meiosis in Begonia is very dynamic, which may have important consequences for chromosome evolution and biodiversity within the genus.

Genome size variation in Begonia.

The genome sizes of a Begonia collection comprising 37 species and 23 hybrids of African, Asiatic, Middle American, and South American origin were screened using flow cytometry. Within the collection, 1C values varied between 0.23 and 1.46 pg DNA. Genome sizes were, in most cases, not positively correlated with chromosome number, but with pollen size. A 12-fold difference in mean chromosome size was found between the genotypes with the largest and smallest chromosomes. In general, chromosomes from South American genotypes were smaller than chromosomes of African, Asian, or Middle American genotypes, except for B. boliviensis and B. pearcei. Cytological chromosome studies in different genotypes showed variable chromosome numbers, length, width, and total chromosome volume, which confirmed the diversity in genome size. Large secondary constrictions were present in several investigated genotypes. These data show that chromosome number and structure exhibit a great deal of variation within the genus Begonia, and likely help to explain the large number of taxa found within the genus.

Population genetic divergence corresponds with species-level biodiversity patterns in the large genus Begonia.

Begonia is one of the largest angiosperm genera, containing over 1500 species. Some aspects of the distribution of biodiversity in the genus, such as the geographical restrictions of monophyletic groups, the rarity and morphological variability of widespread species, and a preponderance of narrow endemics, suggest that restricted gene flow may have been a factor in the formation of so many species. In order to investigate whether this inference based on large-scale patterns is supported by data at the population level, we examined the distribution of genetic variation within Begonia sutherlandii in the indigenous forests of Kwazulu-Natal, South Africa, using microsatellite markers. Despite the species being predominantly outbreeding, we found high and significant levels of population structure (standardized =F'ST= 0.896). Even within individual populations, there was evidence for clear differentiation of subpopulations. There is thus congruence in evolutionary patterns ranging from interspecific phylogeny, the distribution of individual species, to the levels of population differentiation. Despite this species-rich genus showing a pan-tropical distribution, these combined observations suggest that differentiation occurs over very local scales. Although strongly selected allelic variants can maintain species cohesion with only low levels of gene flow, we hypothesize that in Begonia, gene flow levels are often so low, that divergence in allopatry is likely to be a frequent occurrence, and the lack of widespread species may in part be attributable to a lack of a mechanism for holding them together.