Spatial genetic patterns and distribution dynamics of Begonia grandis (Begoniaceae), a widespread herbaceous species in China.

Introduction: Begonia L., one of the 10 largest plant genera, contains over 2,100 species, most of which have a very limited distribution range. Understanding the spatial genetic structure and distribution dynamics of a widespread species in this genus will contribute to clarifying the mechanism responsible for Begonia speciation. Methods: In this study, we used three chloroplast DNA markers (ndhF-rpl32, atpI-atpH, and ndhA intron), coupled with species distribution modeling (SDM), to investigate the population genetic structure and distribution dynamics of Begonia grandis Dryand., the species of Begonia with the widest distribution in China. Results: Thirty-five haplotypes from 44 populations clustered into two groups, and haplotype divergence began in the Pleistocene (1.75 Mya). High genetic diversity (H d = 0.894, H T = 0.910), strong genetic differentiation (F ST = 0.835), and significant phylogeographical structure (G ST/N ST = 0.848/0.917, P < 0.05) were observed. The distribution range of B. grandis migrated northwards after the last glacial maximum, but its core distribution area remained stable. Discussion: Combined, the observed spatial genetic patterns and SDM results identified the Yunnan-Guizhou Plateau, the Three Gorges region, and the Daba Mountains as potential refugia of B. grandis. BEAST-derived chronogram and haplotype network analysis do not support the Flora Reipublicae Popularis Sinicae and Flora of China for subspecies classification based on morphological characteristics. Our results support the hypothesis that population-level allopatric differentiation may be an important speciation process for the Begonia genus and a key contributor to its rich diversity.

Genetic diversity and inferred ancestry of Asian lotus (Nelumbo nucifera) germplasms in Thailand and Vietnam.

Tropical lotus (Nelumbo) is an important and unique ecological type of lotus germplasm. Understanding the genetic relationship and diversity of the tropical lotus is necessary for its sustainable conservation and utilization. Using 42 EST-SSR (expressed sequence tag-simple sequence repeats) and 30 SRAP (sequence-related amplified polymorphism) markers, we assessed the genetic diversity and inferred the ancestry of representative tropical lotus from Thailand and Vietnam. In total, 164 and 41 polymorphic bands were detected in 69 accessions by 36 EST-SSR and seven SRAP makers, respectively. Higher genetic diversity was revealed in Thai lotus than in Vietnamese lotus. A Neighbor-Joining tree of five main clusters was constructed using combined EST-SSR and SRAP markers. Cluster I included 17 accessions of Thai lotus; cluster II contained three Thai accessions and 11 accessions from southern Vietnam; and cluster III was constituted by 13 accessions of seed lotus. Consistent with the results from the Neighbor-Joining tree, the genetic structure analysis showed that the genetic background of most Thai and Vietnamese lotus was pure, as artificial breeding has been rare in both countries. Furthermore, these analyses indicate that Thai and Vietnamese lotus germplasms belong to two different gene pools or populations. Most lotus accessions are genetically related to geographical distribution patterns in Thailand or Vietnam. Our findings showed that the origin or genetic relationships of some unidentified lotus sources can be evaluated by comparing morphological characteristics and the data of molecular markers. In addition, these findings provide reliable information for the targeted conservation of tropical lotus and parent selection in breeding novel cultivars of lotus.

Jasmonate-Responsive Transcription Factors NnWRKY70a and NnWRKY70b Positively Regulate Benzylisoquinoline Alkaloid Biosynthesis in Lotus (Nelumbo nucifera).

Lotus (Nelumbo nucifera) is a large aquatic plant that accumulates pharmacologically significant benzylisoquinoline alkaloids (BIAs). However, little is known about their biosynthesis and regulation. Here, we show that the two group III WRKY transcription factors (TFs), NnWRKY70a and NnWRKY70b, positively regulate the BIA biosynthesis in lotus. Both NnWRKY70s are jasmonic acid (JA) responsive, with their expression profiles highly correlated to the BIA concentration and BIA pathway gene expression. A dual-luciferase assay showed that NnWRKY70a could transactivate the NnTYDC promoter, whereas NnWRKY70b could activate promoters of the three BIA structural genes, including NnTYDC, NnCYP80G, and Nn7OMT. In addition, the transient overexpression of NnWRKY70a and NnWRKY70b in lotus petals significantly elevated the BIA alkaloid concentrations. Notably, NnWRKY70b seems to be a stronger BIA biosynthesis regulator, because it dramatically induced more BIA structural gene expressions and BIA accumulation than NnWRKY70a. A yeast two-hybrid assay further revealed that NnWRKY70b physically interacted with NnJAZ1 and two other group III WRKY TFs (NnWRKY53b and NnWRKY70a), suggesting that it may cooperate with the other group III WRKYs to adjust the lotus BIA biosynthesis via the JA-signaling pathway. To illustrate the mechanism underlying NnWRKY70b-mediated BIA regulation in the lotus, a simplified model is proposed. Our study provides useful insights into the regulatory roles of WRKY TFs in the biosynthesis of secondary metabolites.

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.

Begoniaparvibracteata, a new species in Begonia sect. Platycentrum (Begoniaceae) from Guangxi of China, based on morphological and molecular evidence.

The previously reported begonias in a limestone forest of Guangxi mainly belong to Begoniasect.Coelocentrum Irmscher. In this article, we described and illustrated a new species in sect. Platycentrum (Klotzsch) A.DC., Begoniaparvibracteata X.X.Feng, R.K.Li & Z.X.Liu, which was discovered in a karst forest of south-western Guangxi. The begonia shows high morphological similarity to B.subhowii S.H. Huang and B.psilophylla Irmscher, but differs from the latter two in its narrower oblique-ovate asymmetric leaf blade, 4 (occasionally 6) tepals of pistillate flower and smaller membranous inflorescence bracts. Molecular phylogenetic analysis, based on ITS sequence data, supports the new species as monophyletic and distinct from B.subhowii and B.psilophylla. Considering its narrow distribution and the disturbance of human activities, the conservation status of new taxon is evaluated as "Vulnerable" (VU B1, B2 ab (i, iv, v), D2) according to the IUCN Red List Categories and Criteria.

Begoniapseudoedulis, a new species in Begoniasect.Platycentrum (Begoniaceae) from southern Guangxi of China.

Begoniapseudoedulis, a new species in Begoniasect.Platycentrum (Klotzsch) A.DC. (Begoniaceae) from southern Guangxi of China, is here described and illustrated. It morphologically resembles B.edulis H.Lév. and B.dielsiana E.Pritz. ex Diels but differs easily by its hairy petioles and inflorescences, and red hispidulous flower tepals, ovary and capsules. The molecular phylogenetic analysis based on ITS supported that the new species was a monophyletic lineage, separating from both B.dielsiana and B.edulis. Due to its isolated distribution with several small populations, which are possibly disturbed by human activities, the species is considered as "Near Threatened" (NT) according to the IUCN Red List Categories and Criteria.

Metabolite analysis in Nymphaea 'Blue Bird' petals reveal the roles of flavonoids in color formation, stress amelioration, and bee orientation.

In this study, metabolome of open petals (OP) and closed petals (CP) from Nymphaea 'Blue Bird' was firstly investigated. A total of 455 metabolites was identified in Nymphaea 'Blue Bird' petals, which was mainly composed of 100 flavonoids, 83 phenolic acids, 64 amino acids and derivatives, 60 lipids, 32 alkaloids, 32 organic acids, 24 nucleotides and derivatives, and 12 lignans and coumarins. By differential analysis, 192 metabolites were identified with variable importance in project ≥ 1, among which 83 and 109 metabolites were up- and down-regulated in OP group, respectively. Further analysis (Log2 fold change ≥ 1) identified 26 and 7 metabolites exhibited significantly lower and higher contents in CP group, relative to OP group. Importantly, KEGG analysis indicated that flavonoid biosynthesis exhibited the most significant enrichment. qRT-PCR analysis indicated that the PAL, CHS, and HCDBR genes showed a significantly higher expression in OP group than in CP group. These data explain the increase of naringenin chalcone and phloretin in OP. However, there was no significant difference of total flavonoids between OP and CP groups. Considering the increase of H2O2 content and ultraviolet (UV) absorption peak in OP, our results implied that diurnal stressful conditions induced the degradation of flavonoids, which contributed to environmental stress amelioration. Moreover, a higher absorption peak of 360-380 nm UV was observed in the extract liquor of OP. The sensitivity maximum of the UV-photoreceptor of bees is situated around 340-380 nm UV. This suggested, as noted for the maximum absorption of dihydrokaempferol in 340-370 nm, rhythmic accumulation and loss of these differential flavonoids in Nymphaea 'Blue Bird' petals might enhance UV pattern to some degree, influencing pollinator attraction.

Transcriptome Analysis Reveals the Senescence Process Controlling the Flower Opening and Closure Rhythm in the Waterlilies (Nymphaea L.).

Most waterlily flowers open at dawn and close after noon usually for three to four days, and thereafter wilt. The short lifespan of flowers restricts the development of the flower postharvest industry. The termination of flower movements is a key event during flower aging process. However, it is still unclear when the senescence process initiates and how it terminates the movement rhythm. In this study, we observed that the opening diameter of flowers was the smallest on the fourth (last) flowering day. Subsequent transcriptome profiles generated from petals at different flowering stages showed that the multiple signaling pathways were activated at the last closure stage (Time 3, T3) of the flowers, including Ca2+, reactive oxygen species and far red light signaling pathways, as well as auxin, ethylene and jasmonic acid signaling pathways. Moreover, In terms of cell metabolism regulation, the genes related to hydrolase (protease, phospholipase, nuclease) were upregulated at T3 stage, indicating that petals entered the senescence stage at that time; and the genes related to water transport and cell wall modification were also differentially regulated at T3 stage, which would affect the ability of cell expand and contract, and eventually lead to petal not open after the fourth day. Collectively, our data provided a new insight into the termination of flower opening in the waterlilies, and a global understanding of the senescence process of those opening-closure rhythm flowers.

Molecular Cloning and Expression Profile of Class E Genes Related to Sepal Development in Nelumbo nucifera.

The lotus (Nelumbo Adans.) is an important aquatic plant with ornamental, medicinal and edible values and cultural connotations. It has single-, semi-double-, double- and thousand-petalled types of flower shape and is an ideal material for developmental research of flower doubling. The lotus is a basal eudicot species without a morphological difference between the sepals and petals and occupies a critical phylogenetic position in flowering plants. In order to investigate the genetic relationship between the sepals and petals in the lotus, the class E genes which affect sepal formation were focused on and analyzed. Here, SEPALLATA 1(NnSEP1) and its homologous genes AGAMOUS-LIKE MADS-BOXAGL9 (NnAGL9) and MADS-BOX TRANSCRIPTION FACTOR 6-like (NnMADS6-like) of the class E gene family were isolated from the flower buds of the Asian lotus (Nelumbo nucifera Gaertn.). The protein structure, subcellular localization and expression patterns of these three genes were investigated. All three genes were verified to locate in the nucleus and had typical MADS-box characteristics. NnSEP1 and NnMADS6-like were specifically expressed in the sepals, while NnAGL9 was highly expressed in the petals, suggesting that different developmental mechanisms exist in the formation of the sepals and petals in the lotus. The significant functional differences between NnSEP1, NnMADS6-like and NnAGL9 were also confirmed by a yeast two-hybrid assay. These results expand our knowledge on the class E gene family in sepal formation and will benefit fundamental research on the development of floral organs in Nelumbo.

Begonia shenzhenensis, a new species of Begoniaceae from Guangdong, China.

Begonia shenzhenensis D.K.Tian & X.Yun Wang, sp. nov., a new species in Begonia sect. Platycentrum of Begoniaceae from Shenzhen of Guangdong province, China, is described and illustrated. Morphologically, it is primarily similar to B. coelocentroides in the same section but differs by its denser hairs on leaf, petiole, and pedicel, abtuse anther apex, hairy ovary, and narrower adaxial fruit wing. Based on only one small population found to date, its conservation status is assigned to Critical Endangered according to the IUCN Red List Categories and Criteria.

A new species (Begoniagiganticaulis) of Begoniaceae from southern Xizang (Tibet) of China.

Begoniagiganticaulis, a huge new species in Begoniasect.Platycentrum of Begoniaceae from southern Xizang (Tibet) of China, is described. Morphologically, it is mostly similar to B.longifolia and B.acetosella, but clearly differs from the former mainly by its dioecious and taller plants, sparse hairs on abaxial veins, longer inflorescence, unique shape of fruits, and differs from the latter mainly by its late and longer flowering time, 6-tepals of female flower and 3-loculed ovary. The phylogenetic analyses also support the separation of the new species from other taxa. Based on the current data, its conservation status is assigned to Endangered (B2a) according to the IUCN Red List Categories and Criteria.

Several new records, synonyms, and hybrid-origin of Chinese begonias.

Begonia is a mega-genus with about 2500 species by most estimates, with China having over 210 accepted species. After field surveys, literature review and examination of herbarium specimens, some new taxa, new records, synonyms and the hybrid-origin of some taxa have been confirmed. Here, we report that Begonia dioica Buch.-Ham. ex D.Don and B. flagellaris Hara, both from Xizang (Tibet) are new to China; Begonia lipingensis Hance, B. muliensis T.T.Yu and B. sizemoreae Kiew are synonyms of B. circumlobata Hance, B. taliensis Gagnepain and B. longiciliata C.Y.Wu, respectively; and Begonia × lancangensis S.H.Huang and B. × malipoensis S.H.Huang & Y.M.Shui are natural hybrids.

Histological and Cytological Characterization of Anther and Appendage Development in Asian Lotus (Nelumbo nucifera Gaertn.).

The lotus (Nelumbo Adans.) is a perennial aquatic plant with important value in horticulture, medicine, food, religion, and culture. It is rich in germplasm and more than 2000 cultivars have been cultivated through hybridization and natural selection. Microsporogenesis and male gametogenesis in the anther are important for hybridization in flowering plants. However, little is known about the cytological events, especially related to the stamen, during the reproduction of the lotus. To better understand the mechanism controlling the male reproductive development of the lotus, we investigated the flower structure of the Asian lotus (N. nucifera). The cytological analysis of anther morphogenesis showed both the common and specialized cytological events as well as the formation of mature pollen grains via meiosis and mitosis during lotus anther development. Intriguingly, an anatomical difference in anther appendage structures was observed between the Asian lotus and the American lotus (N. lutea). To facilitate future study on lotus male reproduction, we categorized pollen development into 11 stages according to the characterized cytological events. This discovery expands our knowledge on the pollen and appendage development of the lotus as well as improving the understanding of the species differentiation of N. nucifera and N. lutea.

Diversity and conservation of Chinese wild begonias.

Begonia, one of the most diverse plant taxa and the fifth or sixth largest angiosperm genus, consists of over 1800 accepted species. The number of species recognized within this genus has greatly increased over the past 20 years, rising from 80 to 200 species in China alone. Based on recent field surveys, the number of begonia species in China is predicted to be between 250 and 300. Given the large number of begonia species that still remain to be described, further taxonomical work is urgently required. This is especially true for Chinese Begonia, in which there is a huge diversity of habitat, habit, plant size, leaf type, flower and fruit morphology, and most species are narrowly distributed in isolated habitats that are subject to negative disturbances from climate change, as well as agricultural and industrial activities. Although the conservation status for the majority of species has been evaluated using the standards of the International Union for Conservation of Nature, the results don't represent the truth in many species, and also about 11.5% of which are data-absent. In addition, illegal collection and over-harvesting of wild begonias for ornamental or medicinal use has increased due to the rapid development of internet commerce. Far more often than predicted, these species should be categorized as rare and endangered and require immediate protection. Ex situ conservation of Chinese begonias started in 1995 and over 60% of the total species have been so far introduced into cultivation by several major botanical gardens in China. However, only few research institutions, limited funds and human resources have been involved in Begonia conservation; moreover, no project has conducted reintroduction. Therefore, more conservation-based work remains to be done. Improved conservation of Chinese begonias in the future depends on further field survey, an improved understanding of population diversity, and integrative approaches, including in situ and ex situ conservation, seed banking, and plant reintroduction. Species-targeted conservation zones should be established for endangered species excluded from the existing nature reserves. Additionally, laws pertaining to plant protection should be extended to prevent the illegal collection and transaction of wild plants, particularly for those species with unique habitats and small populations.

Characterization of flower-bud transcriptome and development of genic SSR markers in Asian lotus (Nelumbo nucifera Gaertn.).

BACKGROUND: Asian lotus (Nelumbo nucifera Gaertn.) is the national flower of India, Vietnam, and one of the top ten traditional Chinese flowers. Although lotus is highly valued for its ornamental, economic and cultural uses, genomic information, particularly the expressed sequence based (genic) markers is limited. High-throughput transcriptome sequencing provides large amounts of transcriptome data for promoting gene discovery and development of molecular markers. RESULTS: In this study, 68,593 unigenes were assembled from 1.34 million 454 GS-FLX sequence reads of a mixed flower-bud cDNA pool derived from three accessions of N. nucifera. A total of 5,226 SSR loci were identified, and 3,059 primer pairs were designed for marker development. Di-nucleotide repeat motifs were the most abundant type identified with a frequency of 65.2%, followed by tri- (31.7%), tetra- (2.1%), penta- (0.5%) and hexa-nucleotide repeats (0.5%). A total of 575 primer pairs were synthesized, of which 514 (89.4%) yielded PCR amplification products. In eight Nelumbo accessions, 109 markers were polymorphic. They were used to genotype a sample of 44 accessions representing diverse wild and cultivated genotypes of Nelumbo. The number of alleles per locus varied from 2 to 9 alleles and the polymorphism information content values ranged from 0.6 to 0.9. We performed genetic diversity analysis using 109 polymorphic markers. A UPGMA dendrogram was constructed based on Jaccard's similarity coefficients revealing distinct clusters among the 44 accessions. CONCLUSIONS: Deep transcriptome sequencing of lotus flower buds developed 3,059 genic SSRs, making a significant addition to the existing SSR markers in lotus. Among them, 109 polymorphic markers were successfully validated in 44 accessions of Nelumbo. This comprehensive set of genic SSR markers developed in our study will facilitate analyses of genetic diversity, construction of linkage maps, gene mapping, and marker-assisted selection breeding for lotus.