Chemical Constituents of Species in the Genus Pleione (Orchidaceae) and the Implications from Molecular Phylogeny.

Pleiones are popular ornamental orchids and different species of Pleione are long being used as traditional medicine in many Asian countries. However, previous chemical investigations of the genus Pleione are restricted to only a few species. In the present study, high performance liquid chromatography (HPLC) fingerprint of Pleione plants was established, which in particular, eight common peaks were confirmed in 16 species/hybrids. Three of the compounds corresponding to the chromatographic peaks were identified by electrospray ionization tandem mass spectrometry (ESI-tandem-MS). HPLC analysis confirmed the studied taxa shared most of chemical compounds but the content of chemical compounds was significantly different between species. Comparison of hierarchical clustering result with phylogenetic tree revealed that closely related species have higher similarities in chemical constituents. In consideration of low chemical similarity between spring-flowering and autumn-flowering species, we suggest a discrimination of these two groups during medicinal use of the genus Pleione. Species with a large pseudobulb and with high content of a certain compound should be given priority in future artificial cultivation and medicinal cultivar breeding. We hope our findings will contribute to the quality control and promote conservation of such endangered plant group.

FTIR-based comparative analysis of glucomannan contents in some tuberous orchids, and effects of pre-processing on glucomannan measurement.

BACKGROUND: Glucomannan (GM) is a polysaccharide of the mannan family of compounds found in some plant species. The dried and powdered tubers of some orchid species, collectively known as 'salep powder,' are a commercially important crop for human consumption and are one of the primary sources of GM. GM content is the primary indicator for the yield and quality of salep powder. We hypothesized that it would be more practical and accurate to measure GM content within tuber powder directly, prior to any purification or pre-processing. The GM content of tubers of 14 different orchid species was evaluated and compared using Fourier transform infrared (FTIR) spectroscopy and an enzymatic colorimetric method. RESULTS: Among the analyzed modes, the sum of the peak areas at 873 and 812 cm-1 , which represent the CH bending attributed to the ß-pyranose form of d-glucose and d-mannose, respectively, gave the only confirmation using colorimetric methods. It was found that the tubers of Himantoglossum caprinum and Serapias vomeracea had the highest GM concentrations among the analyzed species. After conducting different pre-processing steps on Serapias vomeracea tubers, it was found that treating the tubers with milk, or high temperature resulted in an apparent increase in GM concentrations. CONCLUSION: Himantoglossum caprinum and Serapias vomeracea give the highest yields of GM and should be used for commercial horticulture. GM estimation should be made prior to any pre-processing. FTIR spectroscopy is effective and reliable for directly comparing GM content of different orchid species, without the need for any purification or pre-processing. © 2019 Society of Chemical Industry.

[DNA barcoding identification of endangered medicinal plants of Orchidaceae].

In this study, DNA barcoding was used to validate the traditional morphological classification of medicinal plants of Orchidaceae. The 163 samples of 135 species belong to 49 genera which have been confirmed by morphological identification were collected. Candidate sequences, including matK, psbA-trnH and ITS2 sequences, were amplified, bidirectionally sequenced, and assembled. All the sequences were blasted to GenBank database at NCBI, then analyzed using Neighbor-joining tree method by MEGA 7.0. The results showed that the DNAs of 163 samples were successfully extracted. The amplification efficiency of matK, psbA-trnH and ITS2 sequences were 100%, 100% and 98.77%, respectively. The 487 sequences were obtained, 345 sequences of which have matched corresponding sequences in the GenBank database and 142 sequences were new sequences. The topology of NJ tree which were constructed with the matK sequences was better than the trees of psbA-trnH and ITS2 sequences. In conclusion, the matK, psbA-trnH and ITS2 sequences were complementary and suitable for identification of medicinal plants of Orchidaceae. DNA barcoding can be used as an auxiliary means for identification of medicinal plants of Orchidaceae.

Evaluating five different loci (rbcL, rpoB, rpoC1, matK, and ITS) for DNA barcoding of Indian orchids.

Orchidaceae, one of the largest families of angiosperms, is represented in India by 1600 species distributed in diverse habitats. Orchids are in high demand owing to their beautiful flowers and therapeutic properties. Overexploitation and habitat destruction have made many orchid species endangered. In the absence of effective identification methods, illicit trade of orchids continues unabated. Considering DNA barcoding as a potential identification tool, species discrimination capability of five loci, ITS, matK, rbcL, rpoB, and rpoC1, was tested in 393 accessions of 94 Indian orchid species belonging to 47 genera, including one listed in Appendix I of CITES and 26 medicinal species. ITS provided the highest species discrimination rate of 94.9%. While, among the chloroplast loci, matK provided the highest species discrimination rate of 85.7%. None of the tested loci individually discriminated 100% of the species. Therefore, multi-locus combinations of up to five loci were tested for their species resolution capability. Among two-locus combinations, the maximum species resolution (86.7%) was provided by ITS+matK. ITS and matK sequences of the medicinal orchids were species specific, thus providing unique molecular identification tags for their identification and detection. These observations emphasize the need for the inclusion of ITS in the core barcode for plants, whenever required and available.

Sexual Deception in the Eucera-Pollinated Ophrys leochroma: A Chemical Intermediate between Wasp- and Andrena-Pollinated Species.

Ophrys flowers mimic sex pheromones of attractive females of their pollinators and attract males, which attempt to copulate with the flower and thereby pollinate it. Virgin females and orchid flowers are known to use the same chemical compounds in order to attract males. The composition of the sex pheromone and its floral analogue, however, vary between pollinator genera. Wasp-pollinated Ophrys species attract their pollinators by using polar hydroxy acids, whereas Andrena-pollinated species use a mixture of non-polar hydrocarbons. The phylogeny of Ophrys shows that its evolution was marked by episodes of rapid diversification coinciding with shifts to different pollinator groups: from wasps to Eucera and consequently to Andrena and other bees. To gain further insights, we studied pollinator attraction in O. leochroma in the context of intra- and inter-generic pollinator shifts, radiation, and diversification in the genus Ophrys. Our model species, O. leochroma, is pollinated by Eucera kullenbergi males and lies in the phylogeny between the wasp and Andrena-pollinated species; therefore, it is a remarkable point to understand pollinator shifts. We collected surface extracts of attractive E. kullenbergi females and labellum extracts of O. leochroma and analyzed them by using gas chromatography with electroantennographic detection (GC-EAD) and gas chromatography coupled with mass spectrometry (GC-MS). We also performed field bioassays. Our results show that O. leochroma mimics the sex pheromone of its pollinator's female by using aldehydes, alcohols, fatty acids, and non-polar compounds (hydrocarbons). Therefore, in terms of the chemistry of pollinator attraction, Eucera-pollinated Ophrys species might represent an intermediate stage between wasp- and Andrena-pollinated orchid species.

[Newly records of medicinal species in Jiangxi].

Seven species were reported as new records of Jiangxi province, which collected from Wugongshan region, including Huperzia kunmingensis(Lycopodiaceae), Hydrangea mangshanensis(Saxifragaceae), Itea glutinosa(Saxifragaceae), Stellaria monosperma var. japonica(Lycopodiaceae), Youngia pratti(Compositae), and Calanthe henryi(Orchidaceae), Collabium formosanum(Orchidaceae). Among these species, H. kunmingensis, H. mangshanensis, I. glutinosa, Y. pratti and C. henryi are endemic in China. C. henryi is stenotopic distribution in China and regarded as vulnerable endangered plant by IUCN. The report is of great significance to the plant diversity and floristic composition in Jiangxi. All the specimens examined are preserved in Jishou University (JIU) and Sun yat-sen University (SYS).

[New records of medicinal plants in Hubei].

In this paper, we make a report on new records of medicinal plants in Hubei, which include one newly recorded genera and seven newly recorded species and a newly recorded variety. The newly recorded genera is Anoectochilus and its corresponding species is Anoectochilus roxburghii; These newly recorded species are Euphorbia micractina, Astragalus wulingensis, Blumea megacephala, Potentilla saundersiana, Blumea formosana, Lycoris houdyshelii and Colocasia gigantea ; The newly recorded variety is Neottia puberula var. maculata. Among these species, Anoectochilus roxburghii and N. puberula var. maculata are considered as the second-class protection in our country, A. roxburghii is regarded as Endangered（EN）and Astragalus wulingensis is regarded as Critically Endangered (CN) by IUCN. The report of these newly recorded plants borden the distribution and enrich the plant diversity of Hubei.

The complete chloroplast genome sequence of Anoectochilus roxburghii.

The complete chloroplast sequence of the Anoectochilus roxburghii, a popular traditional Chinese medicine for the treatment of cancer, was determined in this study. The chloroplast genome (cpDNA)^ was 152,802 bp in length, containing a pair of inverted repeats of 52,728 bp separated by a large single-copy region and a small single-copy region of 82,641 bp and 17,433 bp, respectively. The chloroplast genome encodes 116 predicted functional genes, including 81 protein-coding genes, four ribosomal RNA genes, and 31 transfer RNA genes, 25 of which are duplicated in the inverted repeat regions. The cpDNA is GC-rich (36.9%).

[Identification of Bletillae Rhizoma and its adulterants by SNPs in ITS2].

To establish a molecular identification method for Bletillae Rhizoma, this paper extracted genome DNA from Bletillae Rhizoma and its adulterants. The sequences of rDNA ITS2 were sequenced after amplifying. Then multiple alignments of ITS2 were constructed phylogenetic tree with Neighbor Joining by MEGA 5. 1 and found out SNPs loci. The result showed that rDNA ITS2 region could identify Bletillae Rhizoma and its adulterants. There existed the SNPs loci, which could identify Bletilla striata and B. ochracea. Furthermore, we designed specific primers against the SNPs loci of B. striata and B. ochracea, then screened primers and optimized the PCR amplification conditions. Finally, the DNA of B. striata and B. ochracea were specifically amplified by BJ59-412F, BJ59-412R and HHBJ-225R. The length of amplification products were respectively about 350 bp and 520 bp that were effectively identified of B. striata and B. ochracea. While, the adulterants of Bletillae Rhizoma were no-reaction occurring. To sum up, the amplification conditions of the primers can identify B. striata, B. ochracea and their adulterants successfully at the same time. This method was easy, time-saving, and reliable, which can be used as a rapid method for molecular identification of Bletillae Rhizoma.

[Multiple analysis of relationship between morphologic traits and yield formation of Anoectochilus roxburghii].

Nine morphologic traits, plant height, ground diameter, leaf length, leaf width, leaf area of plant, leaf fresh weight, blades, length/width ratio, plant fresh weight of Anoectochilus roxburghii from 13 different areas were determined for correlation analysis, path analysis and principal components analysis. Different source of morphological trait variation coefficient of A. roxburghii was 2.96% -12.59%, plant fresh weight was significant positively correlated with ground diameter, plant height and leaf number, and positively correlated with leaf fresh weight. Path analysis showed that plant height had the largest positive direct effect on plant fresh weight, the leaf fresh weight and blades number had indirect effects on the plant fresh weight. Through principal component analysis, morphological traits of A. roxburghii can be divided into "Determinants of high-yielding morphology" and "Determinants of leaf production". In the actual process of production and breeding of A. roxburghii, we should pay attention to plant height, leaf fresh weight, blades numbers and other traits.

[Influence of seedling grade on plant growth, yield and quality of Anoectochilus roxburghii].

The morphological index of the seedlings including the plants height, the ground diameter, the leaf amounts, the fresh weight of the whole plant and the ratio of height to diameter was measured and the principal components were analyzed so as to determine the grading index, and stepwise cluster analysis was applied for clustering analysis. Pot experiments were used to measure the indicators of plant growth and development, the yield and the quality. The results showed that the height and ground diameter were determined as the quality indicators of the seedlings grading and the standard quality grading of seedlings of Anoectochilus roxburghii was initially set up, different seeding plants influenced the plants growth and the yield. The ground diameter of the class I was larger than that of the class II and III, so as the yield. The seedling grading had no obvious effect on the internal quality of medicinal materials. The results of the study provide the basis for standard cultivation of A. roxburghii.

Volatile fingerprint of italian populations of orchids using solid phase microextraction and gas chromatography coupled with mass spectrometry.

The volatile fraction of Ophrys sphegodes Mill. subsp. sphegodes, Ophrys bertolonii subsp. benacensis (Reisigl) O. Danesch, E. Danasch & Ehrend. and Neotinea tridentata (Scop.) R.M. Bateman, Pridgeon & M.W. Case, three orchid species with different pollinator attraction strategies, sampled in vivo and in situ, were evaluated by headspace solid phase microextraction coupled with gas-chromatography and mass spectrometry. The results were compared with the volatile compounds emitted by flowering plant samples picked from the same populations of orchid species. Hydrocarbons, aldehydes, alcohols and terpenes were the major constituents of "in vivo" orchid scents and some distinctive differences in volatile metabolite composition were observed between Ophrys and Neotinea species. Moreover, the odour bouquets of the picked flowering plant samples were different from the in vivo ones and in particular different proportions of the various terpenes and an increase of α-pinene were observed. In conclusion HS/SPME GCMS proved to be a suitable technique for analyzing and distinguishing the volatile fingerprint of different orchid species, sampled in vivo and in situ in a non-disruptive way, with potentially great advantages for ecophysiological studies of rare and endangered species.

[DNA barcoding research and its application on medicinal plants of Bletilla H. G. Reichenbach].

To identify adulterants from medicinal plants of Bletilla H. G. Reichenbach, the suitable candidate DNA barcoding of Bletilla was evaluated. In this study, the internal transcribed spacer (ITS) of nuclear ribosomal DNA, the LFY homologous gene intron 2 and chloroplast ycfl gene were amplified and sequenced from forty-one samples. The intra-specific and inter-specific divergences of Bletilla were calculated, and the identification efficiency was assessed using Barcoding Gap, NJ tree by K2P distance and BLAST1 method. The result showed the intra-specific divergence of nrDNA ITS and ycJfl (0.022-0.106 and 0.017-0.106) were obviously higher than the inter-specific divergence (0-0.012 and 0-0.015), and four species of Bletilla were also accurately distinguished in NJ trees. Whereas, there was no Barcoding Gap on LFY homologous gene intron 2, thus it cannot effectively identify species of Bletilla. Using NJ tree of nrDNA ITS and ycfl gene, powdery medicine and the adulterants of Bletilla were successfully unidentified. In conclusion, nrDNA ITS and ycfl can be used as a potential DNA barcoding to identify the medicinal plants in Bletilla and its adulterants. There were only three basic differences on nrDNA ITS between "Jujing baiji" and Bletilla striata of Lu'an in Anhui province, and two basic differences in ycfl. Based on morphological and molecular data, "Jujing baiji" could be recognized as the species of Bletilla striata.

Testes fitoquímicos em extratos foliares de Oeceoclades maculata Lindl. (Orchidaceae) / Phytochemical tests in Oeceoclades maculata Lindl. (Orchidaceae) leaf extracts

O presente trabalho teve por objetivo determinar a capacidade antioxidante de Oeceoclades maculata. A atividade foi determinada de acordo com a capacidade do extrato hidroalcoólico das folhas reduzir o radical DPPH. Os resultados obtidos permitem concluir que os conteúdos de polifenóis e flavonas/flavonóis são maiores no extrato preparado a frio (EBHF) do que no extrato preparado a quente (EBHQ), sugerindo que o aquecimento usado na preparação do extrato altera esses componentes ativos. Portanto, para esta espécie deve ser realizado o método de extração a frio para conservar as substâncias que possuem atividade antioxidante.

This work aimed to determine the antioxidant capacity of Oeceoclades maculata. The antioxidant activity was determined in accordance with the ability of the hydroalcoholic extract of the leaves to reduce the DPPH radical. The results showed that the polyphenol and flavonoid contents are greater in the cold extract (EBHF) than in the hot extract (EBHQ), which suggests that the heating effect in the preparation of the extract modifies these active compounds. Therefore, for this species, the cold extraction method must be carried in order to conserve the substances with antioxidant activity.

Floral isolation is the main reproductive barrier among closely related sexually deceptive orchids.

Floral isolation is an important component of pollinator-driven speciation. However, up to now, only a few studies have quantified its strength and relative contribution to total reproductive isolation. In this study, we quantified floral isolation among three closely related, sympatric orchid species of the genus Ophrys by directly tracking pollen flow. Ophrys orchids mimic their pollinators' mating signals, and are pollinated by male insects during mating attempts. This pollination system, called sexual deception, is usually highly specific. However, whether pollinator specialization also conveys floral isolation is currently under debate. In this study, we found strong floral isolation: among 46 tracked pollen transfers in two flowering seasons, all occurred within species. Accounting for observation error rate, we estimated a floral isolation index ≥0.98 among each pair of species. Hand pollination experiments suggested that postpollination barriers were effectively absent among our study species. Genetic analysis based on AFLP markers showed a clear species clustering and very few F(1) hybrids in natural populations, providing independent evidence that strong floral isolation prevents significant interspecies gene flow. Our results provide the first direct evidence that floral isolation acts as the main reproductive barrier among closely related plant species with specialized pollination.

Ménage à trois-two endemic species of deceptive orchids and one pollinator species.

In the sexually deceptive orchid genus Ophrys, reproductive isolation is based on the specific attraction of males of a single pollinator species by mimicking the female species-specific sex pheromone. Changes in the odor composition can lead to hybridization and speciation by the attraction of a new pollinator that acts as an isolation barrier toward other sympatrically occurring Ophrys species. On Sardinia, we investigated the evolutionary origin of two sympatrically occurring endemic species, Ophrys chestermanii and O. normanii, which are both pollinated by males of the cuckoo bumblebee Bombus vestalis. Chemical and electrophysiological analyses of floral scent and genetic analyses with amplified fragment length polymorphisms and plastid-markers clearly showed that O. normanii is neither a hybrid nor a hybrid species. The two species evolved from different ancestors, viz. O. normanii from O. tenthredinifera and O. chestermanii from O. annae, and converged to the same pollinator attracted by the same bouquet of polar compounds. In spite of sympatry, pollinator sharing and overlapping blooming periods, no evidence has been obtained for gene flow between O. chestermanii and O. normanii indicating an unusual case among sexually deceptive orchids in which postmating rather than premating reproductive isolation mechanisms strongly prevent interspecific gene flow.

Dating the origin of the Orchidaceae from a fossil orchid with its pollinator.

Since the time of Darwin, evolutionary biologists have been fascinated by the spectacular adaptations to insect pollination exhibited by orchids. However, despite being the most diverse plant family on Earth, the Orchidaceae lack a definitive fossil record and thus many aspects of their evolutionary history remain obscure. Here we report an exquisitely preserved orchid pollinarium (of Meliorchis caribea gen. et sp. nov.) attached to the mesoscutellum of an extinct stingless bee, Proplebeia dominicana, recovered from Miocene amber in the Dominican Republic, that is 15-20 million years (Myr) old. This discovery constitutes both the first unambiguous fossil of Orchidaceae and an unprecedented direct fossil observation of a plant-pollinator interaction. By applying cladistic methods to a morphological character matrix, we resolve the phylogenetic position of M. caribea within the extant subtribe Goodyerinae (subfamily Orchidoideae). We use the ages of other fossil monocots and M. caribea to calibrate a molecular phylogenetic tree of the Orchidaceae. Our results indicate that the most recent common ancestor of extant orchids lived in the Late Cretaceous (76-84 Myr ago), and also suggest that the dramatic radiation of orchids began shortly after the mass extinctions at the K/T boundary. These results further support the hypothesis of an ancient origin for Orchidaceae.

Exine micromorphology of Orchidinae (Orchidoideae, Orchidaceae): phylogenetic constraints or ecological influences?

BACKGROUND AND AIMS: Pollen characters have been widely used in defining evolutionary trends in orchids. In recent years, information on pollination biology and phylogenetic patterns within Orchidinae has become available. Hence, the aim of the presented work is to re-evaluate exine micromorphology of Orchidinae in light of recent phylogenetic studies and to test whether pollen micromorphology strictly depends on phylogenetic relationships among species or whether it is influenced by the marked differences in pollination ecology also reported among closely related species. METHODS: Pollen sculpturing of 45 species of Orchidinae and related taxa was investigated using scanning electron microscopy. To cover potential intraspecific variation, several accessions of the same species were examined. KEY RESULTS: Orchidinae show remarkable variation in exine sculpturing, with a different level of variation within species groups. In some genera, such as Serapias (rugulate) and Ophrys (psilate to verrucate), intrageneric uniformity corresponds well to a common pollination strategy and close relationships among species. However, little exine variability (psilate-scabrate and scabrate-rugulate) was also found in the genus Anacamptis in spite of striking differences in floral architecture and pollination strategies. A larger variety of exine conditions was found in genera Dactylorhiza (psilate, psilate-scabrate and reticulate) and Orchis s.s. (psilate, reticulate, perforate-rugulate and baculate) where no unequivocal correspondence can be found to either phylogenetic patterns or pollination strategies. CONCLUSIONS: Changes in pollen characteristics do not consistently reflect shifts in pollination strategy. A unique trend of exine evolution within Orchidinae is difficult to trace. However, the clades comprising Anacamptis, Neotinea, Ophrys and Serapias show psilate to rugulate or scabrate pollen, while that of the clade comprising Chamorchis, Dactylorhiza, Gymnadenia, Orchis s.s., Platanthera, Pseudorchis and Traunsteinera ranges from psilate to reticulate. Comparison of the data with exine micromorphology from members of the tribe Orchidieae and related tribes suggests a possible general trend from reticulate to psilate.

[Study on resource and varieties of Guoshangye].

OBJECTIVE: To investigate the varieties, resoures and identification authentication of Guoshangye used in Tuja and Miao's folk. METHOD: Through field investigations and comparing the collected specimens and literatures, the classification and identification of the species were studied. RESULT AND CONCLUSION: Origin of Guoshangye plant was from Pholidota yunnanensis, Bulbophyllum andersonii, B. odoratissimum, B. kamgtimgemse, ect. their morphological characters were identified. Due to limited resource of Guoshangye, the development and protection should be paid more attention.

[Advances in studies on chemical components and pharmacology of epiphytic type medicinal plants in the Orohid family].

Many of the epiphytic Orchids are used as traditional Chinese medicine. The chemical components and pharmacology have been studied in recent 15 years. This article reviewed the studies which will be beneficial to reveal the relatives among these medicinal plants in the Orchid Family and be helpful to develop new drugs.