Unravelling the palaeobiogeographical history of the living fossil genus Rehderodendron (Styracaceae) with fossil and extant pollen and fruit data.

BACKGROUND: The relict genus Rehderodendron (Styracaceae), the species of which are restricted to mostly warm temperate to tropical climate in East Asia today, is known from fossil fruits and pollen in Europe during warmer periods from the lower Eocene to Pliocene. To infer which extant species are most closely related to the fossils, new data of pollen and fruit morphologiesy of six extant species, and additional new data of fossil pollen and previously described fossil fruits of Rehderodendron, are compared. RESULTS: Both fossil pollen and fruits resemble a morphological mixture of the extant species R. indochinense, R. kwantungense, R. macrocarpum, and R. microcarpum, thus implying that these extant taxa and the fossil European taxa represent an old Eurasian lineage, whereas the pollen and fruit morphology of the extant R. kweichowense and R. truongsonense differ considerably from the fossils and other extant species investigated, and are considered to have evolved independently. CONCLUSIONS: The palaeobiogeographical history of Rehderodendron reveals that its fossil members of the European lineage were most prominent during climatic optima such as the Palaeocene-Eocene Thermal Maximum (PETM), Early Eocene Climate Optimum (EECO) and Middle Miocene Thermal Maximum (MMTM). However, when during the Pliocene the climate changed to colder and less humid conditions, the genus went extinct in Europe but migrated eastwards, most likely in two dispersal events along the Tethys Sea prior to extinction. One of the former most westerly stepping stones is suggested by the refugial occurrence of R. microcarpum in the southeastern Himalaya, whereas R. macrocarpum and R. kwangtungense, the taxa distributed more to the east, might have migrated eastwards already before the Miocene.

Chloroplast Genome Evolution and Species Identification of Styrax (Styracaceae).

The genus Styrax L. consists of approximately 130 species distributed in the Americas, eastern Asia, and the Mediterranean region. The phylogeny and evolutionary history of this genus are not clear. Knowledge of the phylogenetic relationships and the method for species identification will be critical for the evolution of this genus. In this study, we sequenced the chloroplast genome of 17 Styrax samples and added 17 additional chloroplast genome sequences from GenBank. The data were used to investigate chloroplast genome evolution, infer phylogenetic relationships, and access the species identification rate within Styrax. The Styrax chloroplast genome contains typical quadripartite structures, ranging from 157,641 bp to 159,333 bp. The chloroplast genome contains 114 unique genes. The P distance among the Styrax species ranged from 0.0003 to 0.00611. Seventeen small inversions and SSR sites were discovered in the Styrax chloroplast genome. By comparing with the chloroplast genome sequences, six mutation hotspots were identified, and the markers ycf1b and trnT-trnL were identified as the best Styrax-specific DNA barcodes. The specific barcodes and superbarcode exhibited higher discriminatory power than universal barcodes. Chloroplast phylogenomic results improved the resolution of the phylogenetic relationships of Styrax compared to previous analyses.

Plastome structure and phylogenetic relationships of Styracaceae (Ericales).

BACKGROUND: The Styracaceae are a woody, dicotyledonous family containing 12 genera and an estimated 160 species. Recent studies have shown that Styrax and Sinojackia are monophyletic, Alniphyllum and Bruinsmia cluster into a clade with an approximately 20-kb inversion in the Large Single-Copy (LSC) region. Halesia and Pterostyrax are not supported as monophyletic, while Melliodendron and Changiostyrax always form sister clades. Perkinsiodendron and Changiostyrax are newly established genera of Styracaceae. However, the phylogenetic relationship of Styracaceae at the generic level needs further research. RESULTS: We collected 28 complete plastomes of Styracaceae, including 12 sequences newly reported here and 16 publicly available sequences, comprising 11 of the 12 genera of Styracaceae. All species possessed the typical quadripartite structure of angiosperm plastomes, with sequence differences being minor, except for a large 20-kb (14 genes) inversion found in Alniphyllum and Bruinsmia. Seven coding sequences (rps4, rpl23, accD, rpoC1, psaA, rpoA and ndhH) were identified to possess positively selected sites. Phylogenetic reconstructions based on seven data sets (i.e., LSC, SSC, IR, Coding, Non-coding, combination of LSC + SSC and concatenation of LSC + SSC + one IR) produced similar topologies. In our analyses, all genera were strongly supported as monophyletic. Styrax was sister to the remaining genera. Alniphyllum and Bruinsmia form a clade. Halesia diptera does not cluster with Perkinsiodendron, while Perkinsiodendron and Rehderodendron form a clade. Changiostyrax is sister to a clade of Pterostyrax and Sinojackia. CONCLUSION: Overall, our results demonstrate the power of plastid phylogenomics in improving estimates of phylogenetic relationships among genera. This study also provides insight into plastome evolution across Styracaceae.

Plastid phylogenomics resolves infrafamilial relationships of the Styracaceae and sheds light on the backbone relationships of the Ericales.

Relationships among the genera of the small, woody family Styracaceae and among families of the large, diverse order Ericales have resisted complete resolution with sequences from one or a few genes. We used plastome sequencing to attempt to resolve the backbone relationships of Styracaceae and Ericales and to explore plastome structural evolution. Complete plastomes for 23 species are newly reported here, including 18 taxa of Styracaceae and five of Ericales (including species of Sapotaceae, Clethraceae, Symplocaceae, and Diapensiaceae). Combined with publicly available complete plastome data, this resulted in a data set of 60 plastomes, including 11 of the 12 genera of Styracaceae and 12 of 22 families of Ericales. Styracaceae plastomes were found to possess the quadripartite structure typical of angiosperms, with sizes ranging from 155 to 159 kb. Most of the plastomes were found to possess the full complement of typical angiosperm plastome genes. Unusual structural features were detected in plastomes of Alniphyllum and Bruinsmia, including the presence of a large 20-kb inversion (14 genes) in the Large Single-Copy region, the loss or pseudogenization of the clpP and accD genes in Bruinsmia, and the loss of the first exon of rps16 in B. styracoides. Likewise, the second intron from clpP was found to be lost in Alniphyllum and Huodendron. Phylogenomic analyses including all 79 plastid protein-coding genes provided improved resolution for relationships among the genera of Styracaceae and families of Ericales. Styracaceae was strongly supported as monophyletic, with Styrax, Huodendron, and a clade of Alniphyllum + Bruinsmia successively sister to the remainder of the family, all with strong support. All genera of Styracaceae were recovered as monophyletic, except for Halesia and Pterostyrax, which were each recovered as polyphyletic with strong support. Within Ericales, all families were recovered as monophyletic with strong support, with Balsaminaceae sister to remaining Ericales. Most relationships recovered in plastome analyses are congruent with previous analyses based on smaller data sets. Our results demonstrate the power of plastid phylogenomics to improve phylogenetic hypotheses among genera and families, and provide new insight into plastome evolution across Ericales.

Deep phylogeographical structure and parallel host range evolution in the leaf beetle Agelasa nigriceps.

To understand the mechanisms behind the diversification of herbivorous insects through insect-plant interactions, it is important to know how the insects change their diet breadth in response to environmental changes. In this study, we investigated the phylogeographical pattern of the leaf beetle Agelasa nigriceps to infer the evolutionary history of its host range. While this beetle commonly uses Actinidia arguta (Actinidiaceae) as a host plant, it has been recorded recently on Pterostyrax hispidus (Styracaceae), which is now increasing in abundance at some localities in Japan due to the indirect effects of high population size of a mammalian herbivore. Considerable variation among populations in the ability of Ag. nigriceps to use P. hispidus suggests that P. hispidus is a newly acquired host plant for this beetle. Phylogenetic analyses using mitochondrial DNA sequences and amplified fragment length polymorphism (AFLP) revealed a high degree of phylogeographical structure in Ag. nigriceps throughout Japan, which is consistent with the hypothesis that several glacial refugia existed in the Japanese archipelago. In contrast, no genetic structure associated with the host plants was detected. Both the mitochondrial DNA and AFLP analyses showed that populations that can use P. hispidus are polyphyletic. These results and geographical variation in host use suggest that the host range expansion to a novel host, P. hispidus, is a very recent and possibly ongoing phenomenon and has occurred independently in several regions. Our study illustrates that the host range of herbivorous insects can evolve repeatedly in response to similar environmental changes.

Antibacterial evaluation of Styrax pohlii and isolated compounds

The antibacterial activity of the compounds egonol (1) and homoegonol (2), of the crude ethanolic extract of Styrax pohlii (Styracaceae) aerial parts (EE), and of its n-hexane (HF), EtOAc (EF), n-BuOH (BF), and hydromethanolic (HMF) fractions was evaluated against the following microorganisms: Streptococcus pneumoniae (ATCC 6305), S. pyogenes (ATCC 19615), Haemophilus influenzae (ATCC 10211), Pseudomonas aeruginosa (ATCC 27853), and Klebsiella pneumoniae (ATCC 10031). The broth microdilution method was used for determination of the minimum inhibitory concentration (MIC) during preliminary evaluation of antibacterial activity. The EE yielded MIC values of 400 µg/mL for S. pneumoniae and P. aeruginosa and 300 µg/mL for H. influenzae. The HF and EF fractions exhibited enhanced antibacterial activity, with MIC values of 200 µg/mL against S. pneumoniae, but only EF displayed activity against H. influenzae (MIC 200 µg/mL). The best MIC value with compounds 1 and 2 (400 µg/mL) was obtained for (1) against S. pneumoniae and P. aeruginosa. Therefore, 1 exhibited weak antibacterial activity against these standard strains.

As atividades antimicrobianas das substâncias egonol (1) e homoegonol (2), do extrato etanólico das partes aéreas de Styrax pohlii (Styracaceae) (EE), bem como das frações n-hexano (HF), AcOEt (EF), n-BuOH (BF) e hidrometanólica (HMF) foram avaliadas frente aos seguintes microorganismos: Streptococcus pneumoniae (ATCC 6305), S. pyogenes (ATCC 19615), Haemophilus influenzae (ATCC 10211), Pseudomonas aeruginosa (ATCC 27853) e Klebsiella pneumoniae (ATCC 10031). O método de microdiluição em caldo foi utilizado para a determinação da concentração inibitória mínima (CIM) na avaliação preliminar da atividade antimicrobiana. EE mostrou valores de CIM de 400 µg/mL para S. pneumoniae e P. aeruginosa, e 300 µg/mL para H. influenzae. As frações HF e EF apresentaram melhora na atividade antimicrobiana, com valores de CIM de 200 µg/mL frente S. pneumoniae, mas apenas EF apresentou ação contra H. influenzae (200 µg/mL). Em relação às substâncias 1 e 2, o melhor valor de CIM (400 µg/mL) foi obtido por 1 frente a S. pneumoniae e P. aeruginosa, que exibiu fraca atividade antimicrobiana contra estas cepas padrões.

Anticancer activity of 2α, 3α, 19ß, 23ß-Tetrahydroxyurs-12-en-28-oic acid (THA), a novel triterpenoid isolated from Sinojackia sarcocarpa.

BACKGROUND: Natural products represent an important source for agents of cancer prevention and cancer treatment. More than 60% of conventional anticancer drugs are derived from natural sources, particularly from plant-derived materials. In this study, 2α, 3α, 19ß, 23ß-tetrahydroxyurs-12-en-28-oic acid (THA), a novel triterpenoid from the leaves of Sinojackia sarcocarpa, was isolated, and its anticancer activity was investigated both in vitro and in vivo. PRINCIPAL FINDINGS: THA possessed potent tumor selected toxicity in vitro. It exhibited significantly higher cytotoxicity to the cancer cell lines A2780 and HepG2 than to IOSE144 and QSG7701, two noncancerous cell lines derived from ovary epithelium and liver, respectively. Moreover, THA showed a dose-dependent inhibitory effect on A2780 ovary tumor growth in vivo in nude mice. THA induced a dose-dependent apoptosis and G2/M cell cycle arrest in A2780 and HepG2 cells. The THA-induced cell cycle arrest was accompanied by a downregulation of Cdc2. The apoptosis induced by THA was evident by induction of DNA fragmentation, release of cytoplasmic Cytochrome c from mitochondria, activation of caspases, downregulation of Bcl-2 and upregulation of Bax. CONCLUSION: The primary data indicated that THA exhibit a high toxicity toward two cancer cells than their respective non-cancerous counterparts and has a significant anticancer activity both in vitro and in vivo. Thus, THA and/or its derivatives may have great potential in the prevention and treatment of human ovary tumors and other malignancies.

Liquid chromatography-electrospray ionization tandem mass spectrometry for the quantification of styraxlignolide A in rat plasma.

Styraxlignolide A is a pharmacologically active ingredient isolated from Styrax japonica Sieb. et Zucc. A rapid, selective, and sensitive liquid chromatographic method with electrospray ionization tandem mass spectrometry was developed for use in the quantification of styraxlignolide A in rat plasma. Styraxlignolide A was extracted from rat plasma using ethyl acetate at neutral pH. The analytes were separated on an Atlantis dC18 column using a mixture of methanol and ammonium formate (10 mM, pH 3.0) (70:30, v/v) and detected by tandem mass spectrometry in multiple reaction monitoring mode. The standard curve was linear (r(2) =0.9978) over the concentration range of 100-10000 ng/mL. The lower limit of quantification was 100 ng/mL using 50 µL of plasma sample. The coefficient of variation and relative error for intra- and inter-assays at four QC levels were 1.6-8.3% and from -12.0 to -1.7%, respectively. The present method was applied successfully to the pharmacokinetic study of styraxlignolide A after intravenous administration of styraxlignolide A at a dose of 10 mg/kg in male Sprague-Dawley rats.

Purification and anticancer activity investigation of pentacyclic triterpenoids from the leaves of Sinojackia sarcocarpa L.Q. Luo by high-speed counter-current chromatography.

Sinojackia sarcocarpa L.Q. Luo has high aesthetic value, which is clinically used to treat diseases such as the variations of arthritis, thromboangiitis obliterans, angina pectoris as well as many other diseases. Pentacyclic triterpenoids are the main pharmacological effective compounds. High-speed counter-current chromatography method was performed on a Midi-DE centrifuge at 25°C. The solvent system was n-hexane-ethylacetate-methanol-water (10:5:3:1, v/v). Peak fractions were collected according to the elution profile for subsequent high-performance liquid chromatography analysis. The structure identification was performed by ultraviolet, infrared, mass spectrometry, (1)H-NMR and (13)C-NMR. Compound 2α,3α,19ß,23ß-tetrahydroxyurs-12-en-28-oic acid and 2α,3α,23ß-trihydroxyurs-12-en-28-oic acid were purified from the plant of Styracaceaen genius for the first time, whose purities were 96.57% and 97.33%, respectively. Compared with the same dose of oral 5-fluorouracil with 57.6% inhibition rate, the S(180) tumour inhibition rates of 20 mg kg(-1)d(-1) two compounds were 59.5% and 48.9%, respectively.

Fine-scale spatial genetic structure and gene flow in a small, fragmented population of Sinojackia rehderiana (Styracaceae), an endangered tree species endemic to China.

Populations of Sinojackia rehderiana are highly threatened and have small and scattered distribution due to habitat fragmentation and human activities. Understanding changes in genetic diversity, the fine-scale spatial genetic structure (SGS) at different life stages and gene flow of S. rehderiana is critical for developing successful conservation strategies for fragmented populations of this endangered species. In this study, 208 adults, 114 juveniles and 136 seedlings in a 50 × 100-m transect within an old-growth forest were mapped and genotyped using eight microsatellite makers to investigate the genetic diversity and SGS of this species. No significant differences in genetic diversity among different life-history stages were found. However, a significant heterozygote deficiency in adults and seedlings may result from substantial biparental inbreeding. Significant fine-scale spatial structure was found in different life-history stages within 19 m, suggesting that seed dispersal mainly occurred near a mother tree. Both historical and contemporary estimates of gene flow (13.06 and 16.77 m) indicated short-distance gene dispersal in isolated populations of S. rehderiana. The consistent spatial structure revealed in different life stages is most likely the result of limited gene flow. Our results have important implications for conservation of extant populations of S. rehderiana. Measures for promoting pollen flow should be taken for in situ conservation. The presence of a SGS in fragmented populations implies that seeds for ex situ conservation should be collected from trees at least 19-m apart to reduce genetic similarity between neighbouring individuals.

Complexidade estrutural e diversidade de insetos galhadores em Styrax pohlii Fritsch (Styracaceae) / Structural complexity and diversity of insects gall makers in Styrax pohlii Fritsch (Styracaceae)

Esse trabalho teve por objetivo testar o efeito da complexidade estrutural da planta hospedeira Styrax pohlii Fritsch (Styracaceae) na riqueza e abundância de insetos galhadores. No estudo 30 indivíduos da planta hospedeira foram amostrados em um fragmento de mata semidecídua no Campus da Universidade Federal de Goiás em Goiânia, Goiás, Brasil. De cada indivíduo foram medidos a altura, circunferência do caule ao nível do solo, diâmetro da copa e número de folhas por ramo. Para estimativa da riqueza e abundância de galhas foram coletados 10 ramos apicais de cada indivíduo. Segundo as análises realizadas, a altura da planta hospedeira, o diâmetro da copa e a circunferência do caule não influenciaram no número de folhas com galhas, galhas por folha, galhas por indivíduo e riqueza de morfotipos. O número de folhas por ramo, por outro lado, esteve positivamente e significativamente relacionado com a riqueza e abundância de galhas por indivíduo em S. pohlli. Como estratégia de reprodução, muitos insetos galhadores realizam a oviposição em plantas com grande abundância de folhas, uma vez que, oferecem uma maior disponibilidade de sítios de oviposição.

This work was aimed to test whether the structural complexity of the host plant Styrax pohlii Fritsch (Styracaceae) in the diversity of insects gall makers. In the study, 30 individuals of the host plant were sampled in a fragment of semideciduous forest Campus of the Universidade Federal de Goiás, Goiânia, Goiás, Brazil. From each individual, height, circumference of the stem at ground level, crown diameter and number of leaves per branch were measured, In addition, 10 branches apical for each individual were collected to estimate the richness and abundance of galls. According to our analyses, canopy diametes and trunk circumference of the host plant did not effect the number of leaf with galls, galls per leaf, galls for individual and richness of morphotypes. The number of leaf per stem, on the other hand, was the parameter positively and significantly correlated to the richness and abundance of galls in S. pohlii. As a strategy of reproduction, many gall-inducing insects perform the oviposition in plants with great abundance of leaves, since they offer a greater availability of sites for oviposition.

Phylogeny of Sinojackia (Styracaceae) based on DNA sequence and microsatellite data: implications for taxonomy and conservation.

BACKGROUND AND AIMS: The genus Sinojackia consists of eight species, all endemic to China. All species of Sinojackia are endangered or threatened owing to poor recruitment within populations. Information on molecular phylogenetics is critical for developing successful conservation strategies for this genus. METHODS: Combined DNA sequence data from the nuclear ribosomal internal transcribed spacer regions and plastid psbA-trnH intergenic spacer and microsatellite data were used to infer a phylogeny of the genus. KEY RESULTS: Parsimony analysis of the combined sequence data and multivariate analysis based on fruit characters indicated that Sinojackia dolichocarpa is monophyletic and genetically well separated from the other Sinojackia species, thus supporting its rank at the generic level as Changiostyrax. Phylogenetic relationships within Sinojackia sensu stricto are unresolved from the combined sequence data. A UPGMA dendrogram based on seven microsatellite loci of 96 individual plants yielded a first-diverging cluster of all individuals of S. microcarpa. The remaining species form another cluster without any definitive patterns corresponding to current species circumscriptions, suggesting either extensive hybridization or incipient speciation. CONCLUSIONS: The results suggest that there are too many species recognized within Sinojackia sensu stricto, but this must be further assessed with comprehensive morphological and taxonomic revisionary work. The implications of the phylogenetic data for conservation are discussed.

Microsatellite analysis reveals interpopulation differentiation and gene flow in the endangered tree Changiostyrax dolichocarpa (Styracaceae) with fragmented distribution in central China.

Polymorphic simple sequence repeat (SSR) markers were used to investigate the impact of habitat fragmentation on the population structure and gene flow of Changiostyrax dolichocarpa, a critically endangered tree in central China. Intrapopulation genetic diversity, population structure and gene flow in the five extant populations of this species were analysed by eight SSR markers. Intrapopulation genetic diversity results suggest that C. dolichocarpa remnants maintained a relatively high degree of genetic diversity despite severe fragmentation. Low genetic differentiation among populations was found based on Wright's F(ST) and amova analysis. Both the F(ST)-based estimate and private allele method revealed high historical gene flow among the remnant populations. Recent immigrants, detected by assignment tests, tend to decrease from the grandparent generation to the current generation. The potentially highly restricted current gene flow among fragments may render the fragmented populations of C. dolichocarpa at a higher risk of local extinction several generations after fragmentation. Both in situ and ex situ conservation management for the remnant populations of C. dolichocarpa are therefore urgently needed to rescue remaining genetic diversity.

Saponins from Styrax officinalis.

Three triterpene saponins named Styrax-saponin A-C (1-3) were found in pericarps of Styrax officinalis together with the deacylsaponin (4). Structural determinations were achieved using 1D-, 2D-NMR and mass spectrometry.

Allocation of resources to reproduction in Styrax obassia in a masting year.

An analysis is presented of three possible pathways of reproductive allocation, namely, allocation of resources to reproductive organs from reproductive shoots, from non-reproductive shoots and from the main trunk. These pathways were examined by comparing the amount of storage starch in reproductive shoots, non-reproductive shoots and the main trunk in Styrax obassia, a typical masting tree species, during a year of little flowering (1999) and in a mass-flowering year (2000). In addition, we measured rates of light-saturated photosynthesis in leaves of reproductive and non-reproductive shoots to examine the contribution of photosynthetic production to reproductive costs. In both the main trunk and non-reproductive shoots the pattern of seasonal variation in the amount of starch did not differ between 1999 and 2000. However, in the mass-flowering year, the amount of starch in the reproductive shoots was less than that in non-reproductive shoots during the growing season. Thus, reproductive shoots bore most of the cost of reproduction, although non-reproductive shoots and the main trunk also bore some of the cost. Mass-based rates of light-saturated photosynthesis of the leaves of reproductive shoots were significantly higher than those of non-reproductive shoots during both the flowering and the fruiting period. However, leaves of reproductive shoots had a significantly smaller area, a lower mass per area, and lower concentrations of nitrogen than leaves of non-reproductive shoots, although the number of leaves did not differ between the two types of shoots. Therefore, the amount of photosynthate per shoot was significantly lower in reproductive shoots than in non-reproductive shoots. These results suggest that the cost of reproduction depends predominantly on storage starch in reproductive shoots, although it is still unclear how much photosynthate is allocated to reproductive organs from non-reproductive shoots.