Analysis of the Fungal Community Composition in Endemic Orchids with Terrestrial Habitat in Subtropical Regions.

Habenaria and Liparis are well-known orchid genera that grow in terrestrial habitats in the tropics, subtropics or temperate zones. Three species have been found in subtropical regions of China, inhabiting terrestrial to epiphytic habitats. This study focuses on three species, H. dentata (distributed in Asia), H. yachangensis, and L. gigantea. For H. yachangensis and L. gigantea, there is no information about the mycorrhizal community in these species. This study aims to conduct the fungal community screening of Chinese ground orchids from subtropical regions. We performed a comparative analysis of the fungal community among H. dentata, H. yachangensis, and L. gigantea, determining their ITS regions using NGS paired-end sequences. The results clarified the diversity and the predominance of fungal genera. Ascomycota was abundant compared to Basidiomycota or other fungi groups in all communities, with a high dominance in all populations, especially for L. gigantea. At different root spatial locations, the fungal community diversity and richness were higher in the soil than in the rhizosphere or inner root. However, the results suggest that L. gigantea has a different fungal community compared to Habenaria species. In this order, the subtropical terrestrial orchids have a different fungal network compared to the northern terrestrial orchids. Also, there is a high probability of co-existence and co-evolution of endophytic fungi in these terrestrial orchids, indicating the potential role of host plants in selecting an endophytic fungal community. Furthermore, our results highlight the need to elucidate the microbe interactions of these unique orchids for long-term purposes, such as isolating indigenous fungi for suitable inoculants for further orchid propagation, restoration, and conservation.

Evaluation of Habenaria aitchisonii Reichb. for antioxidant, anti-inflammatory, and antinociceptive effects with in vivo and in silico approaches.

Habenaria aitchisonii Reichb was analyzed in this research, including its chemical composition and its in vitro antioxidant, anti-inflammatory, acute oral toxicity, and antinociceptive activity. The chloroform and ethyl acetate fractions were found to be the most powerful based on in vitro antioxidant, anti-inflammatory, and analgesic assays. The acute oral toxicity of the crude methanolic extract was determined before in vivo studies. The acetic acid and formalin tests were used to measure the antinociceptive effect, and the potential mechanisms involved in antinociception were explored. The carrageenan-induced paw edema test was used to examine the immediate anti-inflammatory effect, and many phlogistic agents were used to determine the specific mechanism. Furthermore, for ex vivo activities, the mice were sacrificed, the forebrain was isolated, and the antioxidant levels of glutathione (GSH), superoxide dismutase (SOD), thiobarbituric acid reactive substances (TBARS) and catalase (CAT) were estimated using a UV spectrophotometer. No toxicity was seen at oral dosages up to 3,000 mg/kg. The antinociceptive impact was much higher than the standard drug. Both the inflammatory and neurogenic phases of the formalin experiment revealed an analgesic effect in the chloroform and ethyl acetate fractions. In carrageenan anti-inflammatory assays, the chloroform fraction (Ha.Chf) was the most potent fraction. We further studied the GC-MS of crude plant extract and found a total of 18 compounds. In the anti-inflammatory mechanism, it was observed that the Ha.Chf inhibits the COX-2 as well as 5-LOX pathways. The results exhibited that this species is a good source of phytocomponents like germacrone, which can be employed as a sustainable and natural therapeutic agent, supporting its traditional use in folk medicine for inflammatory conditions and pain.

Phenolic phytochemistry, in vitro, in silico, in vivo, and mechanistic anti-inflammatory and antioxidant evaluations of Habenaria digitata.

Excessive and imbalance of free radicals within the body lead to inflammation. The objective of the current research work was to explore the anti-inflammatory and antioxidant potential of the isolated compounds from Habenaria digitata. In this study, the isolated phenolic compounds were investigated for in vitro and in vivo anti-inflammatory potential along with the antioxidant enzyme. The anti-inflammatory and antioxidant potential of the phenolic compounds was assayed via various enzymes like COX-1/2, 5-LOX and ABTS, DPPH, and H2O2 free radical enzyme inhibitory assay. These compounds were also explored for their in vivo antioxidant activity like examining SOD, CAT, GSH-Px, and MDA levels in the brain, heart, and liver. The anti-inflammatory potential was evaluated using the carrageenan-induced pleurisy model in mice. On the basis of initial screening of isolated compounds, the most potent compound was further evaluated for the anti-inflammatory mechanism. Furthermore, the molecular docking study was also performed for the potent compound. The phenolic compounds were isolated and identified by GC-MS/NMR analysis by comparing its spectra to the library spectra. The isolated phenolic compounds from H. digitata were 5-methylpyrimidine-24,4-diol (1), 3,5-dihydroxy-6-methyl-2,3-dihydropyran-4-one (2), 2-isopropyl-5-methylphenol (3), 3-methoxy-4-vinylphenol (4), and 2,6-dimethoxy-4-vinylphenol (5). In in vitro antioxidant assay, the most potent compound was compound 1 having IC50 values of 0.98, 0.90, and 5 µg/mL against ABTS, DPPH, and H2O2, respectively. Similarly, against COX1/2 and 5-LOX ,compound 1 was again the potent compound with IC50 values of 42.76, 10.70, and 7.40 µg/mL. Based on the in vitro results, compound 1 was further evaluated for in vivo antioxidant and anti-inflammatory potential. Findings of the study suggest that H. digitata contains active compounds with potential anti-inflammatory and antioxidant effects. These compounds could be screened as drug candidates for pharmaceutical research, targeting conditions associated with oxidative stress and inflammatory conditions in medicinal chemistry and support their ethnomedicinal use for inflammation and oxidative stress.

The Genetic Diversity and Genetic Structure of the Germplasm Resources of the Medicinal Orchid Plant Habenaria dentata.

Habenaria dentata has medicinal and ornamental value, but the number of wild populations is decreasing dramatically. Thus, conducting research on its genetic diversity and structure is necessary to provide a basis for its conservation. This study aimed to explore the genetic diversity of the wild plant H. dentata and protect and optimize its wild resources. The genetic diversity of 133 samples from six wild populations of H. dentata was analyzed using Inter Simple Sequence Repeat molecular markers to provide a scientific basis for the screening of improved germplasm resources. The results showed that the average number of alleles was 1.765, the average number of effective alleles was 1.424, the average Nei's gene diversity index was 0.252, the average Shannon diversity index was 0.381, and the average percentage of polymorphic loci was 76.499%. The variation within the populations was 77.34%, and the variation between the populations was 22.66%. The gene flow was 1.705, which was greater than 1. The results of the cluster analysis showed that the six populations were mainly divided into four clusters and were not classified according to their geographical location. There was no significant correlation between the geographical location and genetic distance between the populations (r = 0.557, p > 0.05). The genetic diversity of H. dentata is high. Among the six wild populations, the genetic diversity of the Mulun population was the highest and this population can be used as a key protection unit. The study on the genetic diversity of H. dentata can not only reveal the reasons for the decrease in the number of individuals in the population to a certain extent, and put forward the protection strategy, but also provide a scientific basis for the breeding of excellent seed resources.

Contrasting range changes of terrestrial orchids under future climate change in China.

Climate change has impacted the distribution and abundance of numerous plant and animal species during the last century. Orchidaceae is one of the largest yet most threatened families of flowering plants. However, how the geographical distribution of orchids will respond to climate change is largely unknown. Habenaria and Calanthe are among the largest terrestrial orchid genera in China and around the world. In this paper, we modeled the potential distribution of eight Habenaria species and ten Calanthe species in China under the near-current period (1970-2000) and the future period (2081-2100) to test the following two hypotheses: 1) narrow-ranged species are more vulnerable to climate change than wide-ranged species; 2) niche overlap between species is positively correlated with their phylogenetic relatedness. Our results showed that most Habenaria species will expand their ranges, although the climatic space at the southern edge will be lost for most Habenaria species. In contrast, most Calanthe species will shrink their ranges dramatically. Contrasting range changes between Habenaria and Calanthe species may be explained by their differences in climate-adaptive traits such as underground storage organs and evergreen/deciduous habits. Habenaria species are predicted to generally shift northwards and to higher elevations in the future, while Calanthe species are predicted to shift westwards and to higher elevations. The mean niche overlap among Calanthe species was higher than that of Habenaria species. No significant relationship between niche overlap and phylogenetic distance was detected for both Habenaria and Calanthe species. Species range changes in the future was also not correlated with their near current range sizes for both Habenaria and Calanthe. The results of this study suggest that the current conservation status of both Habenaria and Calanthe species should be adjusted. Our study highlights the importance of considering climate-adaptive traits in understanding the responses of orchid taxa to future climate change.

The complete chloroplast genome sequence of Habenaria dentata (Orchidaceae).

Habenaria dentata is a rare species with high ornamental value in China. In this study, we report the complete chloroplast (cp) genome of H. dentata using the Illumina sequencing data. The total genome of H. dentata is 153,682 bp in length and the GC content is 36.62%, with a pair of inverted repeats (IRs) regions of 26,339 bp each, a large single-copy (LSC) region of 83,963 bp and a small single-copy (SSC) region of 17,041 bp. The cp genome encoded 133 genes, including 87 protein-coding genes (PCG), eight rRNA genes, and 38 tRNA genes. The maximum-likelihood phylogenetic analysis based on 12 cp genomes showed that H. dentata was sister to Habenaria chejuensis and Habenaria ciliolaris. This work will be valuable for genetic and phylogenetic studies on H. dentata.

Genetic Diversity on a Rare Terrestrial Orchid, Habenaria linearifolia in South Korea: Implications for Conservation Offered by Genome-Wide Single Nucleotide Polymorphisms.

Monitoring intraspecific diversity offers invaluable insights on conservation practices as the variation is the product of species evolution. Accordingly, the role of population genetic diversity has drawn great attention over the last century responding to the biodiversity loss induced by a series of anthropogenic changes. Orchids are one of the most diverse, yet ironically most rapidly disappearing plant groups due to the specialized habitat preferences. Thus, population-level genetic diversity studies may offer a powerful tool for orchid conservation programs. Using the 3 restriction site-associated DNA (3RAD) approach, 2,734 genome-wide single nucleotide polymorphisms (SNPs) were isolated. With the 2,734 SNPs, we investigated genetic diversity and population structure on 72 individuals of Habenaria linearifolia and Habenaria cruciformis in South Korea. Overall, the genetic diversity was well maintained in South Korean Habenaria, but high F ST values were estimated suggesting large population diversification with limited gene flow. Bayesian assignment analysis revealed a morphologically cryptic diversity pattern in Jeju Island populations, which might serve as an evolutionarily significant unit.

Low Specificity but Dissimilar Mycorrhizal Communities Associating with Roots May Contribute to the Spatial Pattern of Four Co-Occurring Habenaria (Orchidaceae) Species.

Fungi with orchid roots have been increasingly proven to play important roles in orchid growth, spatial distribution, and coexistence of natural communities. Here, we used 454 amplicon pyrosequencing with two different primer combinations to investigate the spatial variations in the community of OMF and endophytic fungi associates within the roots of four co-occurring Habenaria species. The results showed that all investigated Habenaria species were generalists and the different fungi communities may contribute to the spatial separation of the four Habenaria species. Firstly, the fungal OTUs identified in the roots of the four species overlapped but their presence differed amongst species and numerous distinct OMF families were unique to each species. Second, NMDS clustering showed samples clustered together based on associated species and PERMANOVA analyses indicated that fungi communities in the roots differed significantly between the Habenaria species, both for all endophytic fungi communities and for OMF communities. Third, the network structure of epiphytic fungi was highly specialized and modular but demonstrated lowly connected and anti-nested properties. However, it calls for more soil nutrition and soil fungal communities' studies to elucidate the contribution of habitat-specific adaptations in general and mycorrhizal divergence.

A comparative study on the reproductive success of two rewarding Habenaria species (Orchidaceae) occurring in roadside verge habitats.

BACKGROUND: Most orchid species have been shown to be severely pollination limited, and the factors affecting reproductive success have been widely studied. However, the factors determining the reproductive success vary from species to species. Habenaria species typically produce nectar but exhibit variable fruit set and reproductive success among species. Here, we investigated the influence of the flowering plant density, inflorescence size, breeding system, and pollinator behaviour on the reproductive success of two rewarding Habenaria species. RESULTS: Our observations indicated that Habenaria limprichtii and H. petelotii co-occur in roadside verge habitats and present overlapping flowering periods. Both species were pollination limited, although H. limprichtii produced more fruits than H. petelotii under natural conditions during the 3-year investigation. H. petelotii individuals formed distinct patches along roadsides, while nearly all H. limprichtii individuals clustered together. The bigger floral display and higher nectar sugar concentration in H. limprichtii resulted in increased attraction and visits from pollinators. Three species of effective moths pollinated for H. limprichtii, while Thinopteryx delectans (Geometridae) was the exclusive pollinator of H. petelotii. The percentage of viable seeds was significantly lower for hand geitonogamy than for hand cross-pollination in both species. However, H. limprichtii may often be geitonogamously pollinated based on the behaviours of the pollinators and viable embryo assessment. CONCLUSIONS: In anthropogenic interference habitats, the behaviours and abundance of pollinators influence the fruit set of the two studied species. The different pollinator assemblages in H. limprichtii can alleviate pollinator specificity and ensure reproductive success, whereas the more viable embryos of natural fruit seeds in H. petelotii suggested reducing geitonogamy by pollinators in the field. Our results indicate that a quantity-quality trade-off must occur between species with different breeding strategies so that they can fully exploit the existing given resources.

The pollination of Habenaria rhodocheila (Orchidaceae) in South China: When butterflies take sides.

Habenaria is one of the largest terrestrial genera in the family Orchidaceae. Most field studies on Habenaria species with greenish-white and nocturnal scented flowers are pollinated by nocturnal hawkmoths and settling moths. However, H. rhodocheila presents reddish flowers lacking a detectable scent and fails to fit the moth pollination syndrome. We investigated the pollinators, breeding system, and functional traits of H. rhodocheila in South China and found that two diurnal swallowtail butterflies Papilio helenus and Papilio nephelus (Papilionidae) were the effective pollinators. When butterflies foraged for nectar in the spur, the pollinia became attached between the palpi. A triangular projected median rostellar lobe was found at the entrance (sinus) of the spur of H. rhodocheila. This lobe divided the spur opening into two entrances forcing butterflies to enter their proboscides through the left or right side. When the projection of median rostellar lobe was removed, the site of pollinium attachment changed to the eyes of the butterflies, leading to a higher rate of pollinium removal but lower rate of pollinium deposition. Our quartz glass cylinder choice experiment suggested that visual rather than olfactory cues provided the major stimuli for butterflies to locate these flowers. Hand pollination experiments suggested this species was self-compatible but pollinator-dependent. However, the proportion of seeds with large embryos produced in self-pollinated fruits was significantly lower than in cross-pollinated fruits, indicating a significant inbreeding depression. Unlike many other orchid species, fruit set was higher than rates of pollinium removal, indicating a high level of pollination efficiency in a species with friable pollinia. Shifts from moth to butterfly pollination in the genus Habenaria parallel other orchid lineages providing insights into the potential for pollinator-mediated floral trait selection.

Phytochemical profiling of bioactive compounds, anti-inflammatory and analgesic potentials of Habenaria digitata Lindl.: Molecular docking based synergistic effect of the identified compounds.

ETHNOPHARMACOLOGICAL RELEVANCE: Members of Orchidaceae family has a long history in herbal and Chinese medicines. Members of this family are most commonly famous in the management of inflammation and analgesia in folk medicine. Habenaria digitata, an unexplored specie of Orchidaceae is found in North areas of Pakistan and is used by the local population for the management of analgesia and inflammation. AIM OF THE STUDY: Based on the effective outcomes of the natural products as alternative therapies, we have evaluated Habenaria digitata for the management of analgesia and inflammation. The aim of the designed project is to provide a scientific basis of using this plant for the management of analgesia and inflammation. MATERIALS AND METHODS: The H. digitata crude extract (Hd.Cr) and subfractions, i.e. n-hexane (Hd.Hex), chloroform (Hd.Chf), ethyl acetate (Hd.EtAc), n-butanol (Hd.Bt) and aqueous (Hd.Aq) were used. The GC-MS analysis was used for the identification of phytochemicals. The plants samples were subjected to cyclooxygenase (COX 2) and lipoxygenase (5-LOX) enzymes assays. The hot plate model, acetic acid induced writhing and formalin induced paw licking models were used for in-vivo analgesic studies. The in-vivo anti-inflammatory potential was determined with carrageenan induced paw edema test. Molecular docking studies of the identified compounds were carried out by using Molecular Operating Environment (MOE, 2016.08). RESULTS: The GC-MS analysis confirmed sixty-five compounds in Hd.Cr. Among the fractions, Hd.Chf and Hd.EtAc displayed highest activities. The observed IC50 values were 21.30 and 32.39 µg/ml against COX 2 while 14.42and 16.40 µg/ml for 5-LOX respectively. The in-vivo inflammatory and analgesic studies were pre-requisited with acute toxicity tests. In carrageenan induced inflammation, Hd.Chf excelled the standard drug aspirin by giving 62.92% inhibition of paw edema at 4th h. Similarly, at highest concentration (75 mg/kg) of acetic acid induced analgesia, Hd.Chf was more potent than the standard drug. In formalin method, Hd.Chf exhibited 85.81% inhibition at phase-I and 74.15% at Phase-II. In hot plate model, Hd.Chf exhibited average reaction time of 10.90 at 15, 30, 45 and 60 min intervals. Docking studies supported our results and confirm the synergistic effects of phytochemicals. CONCLUSIONS: Our experimental results concluded that H. digitata contains several bioactive compounds. These bioactive compounds synergistically have therapeutic efficacy for the management of inflammation and analgesia. We have confirmed both of these potentials from the in-vitro and in-vivo experiments. Moreover, it is also obvious that the chloroform and ethyl acetate fractions are rich in these bioactive compounds. Specifically, the Hd.Chf is observed to be more practical in all the tested models of analgesia and inflammation. Computed binding energies of the compounds revealed that all the compounds have synergistic effect to prevent analgesia and inflammation.

Diversification in Qinghai-Tibet Plateau: Orchidinae (Orchidaceae) clades exhibiting pre-adaptations play critical role.

We explore the origins of the extraordinary plant diversity in the Qinghai-Tibetan Plateau (QTP) using Orchidinae (Orchidaceae) as a model. Our results indicate that six major clades in Orchidinae exhibited substantial variation in the temporal and spatial sequence of diversification. Our time-calibrated phylogenetic model suggests that the species-richness of Orchidinae arose through a combination of in situ diversification, colonisation, and local recruitment. There are multiple origins of species-richness of Orchidinae in the QTP, and pre-adaptations in clades from North Temperate and alpine regions were crucial for in situ diversification. The geographic analysis identified 29 dispersals from Asia, Africa and Europe into the QTP and 15 dispersals out. Most endemic species of Orchidinae evolved within the past six million years.

Phylogenetic relationships amongst the African genera of subtribe Orchidinae s.l. (Orchidaceae; Orchideae): Implications for subtribal and generic delimitations.

Phylogenetic relationships within the Orchideae sensu Pridgeon et al, remain one of the biggest unresolved issues in our understanding of the taxonomy of the orchids. Members of the Orchideae are numerous and widespread in Africa but remain poorly represented in phylogenetic research. In this study we included a broad sampling of African taxa for which we sequenced three plastid (rbcl, matK and trnL + trnL-F) and two nuclear regions (ITS and 18S). We used 368 sequences representing 278 species and 49 genera to infer relationships using the Bayesian Inference and Maximum Likelihood method. Our results show strong support for three clades, two of which almost entirely match the historical circumscription of Orchidinae and Habenariinae, and the third, Bartholininae, sister to the former two, includes the genera Holothrix and Bartholina. Stenoglottis should be assigned to Orchidinae and not to Habenariinae. Several genera such as Habenaria, Cynorkis and Benthamia are shown to be para- or polyphyletic: Bonatea, Centrostigma, Platycoryne and Roeperocharis are all embedded in Habenaria; Physoceras, Arnottia and part of Benthamia are embedded in Cynorkis. We propose a subdivision of Orchideae sensu lato into nine subtribes, but refrain from making generic re-arrangements until more extensive or more in-depth studies have been done.

Morphological and Genetic Diversities of Habenaria radiata (Orchidaceae) in the Kinki Area, Japan.

Floral organs have evolved from leaves for reproduction, and the morphological analyses help to understand the plant diversity and evolution. Habenaria radiata (syn. Pecteilis radiata) is a terrestrial orchid living in wetlands in Japan, Russia, South Korea, and China. The habitats of this plant in Japan have been reduced because of environmental destruction and overexploitation, and thus it is on the Red List of Japan as a Near Threatened species. One of the three petals of the H. radiata flower is called a lip or labellum, which resembles a flying white bird, egret, or white heron, with its proposed function being to attract pollinators. To understand the diversity of H. radiata plants in different areas, we examined the lip morphology and phylogeny of populations from eight habitats in the Kinki area, Japan. The complex shapes of the lips were quantified and presented as a radar chart, enabling characterization of the morphological difference among populations. Phylogenetic analysis with microsatellite markers that we generated showed the variation of genetic diversity among populations, suggesting the different degrees of inbreeding, outbreeding, and vegetative propagation. Our approach offers a basic method to characterize the morphological and genetic diversity in natural populations.

The complete chloroplast genome sequence of Habenaria ciliolaris (Orchidaceae).

Habenaria ciliolaris is a kind of orchid with ornamental value. In this study, we reported the complete chloroplast genome of H. ciliolaris. The complete chloroplast genome is 154,544 bp in length, consists of a pair of inverted repeat (IR, 25,455 bp) regions, a large single-copy region (LSC, 84,032 bp) and a small single-copy region (SSC, 19,602 bp). It contains 179 genes, including 133 protein-coding genes, 38 tRNAs, and 8 rRNAs. A maximum-likelihood phylogenetic analysis demonstrated a close relationship between H. ciliolaris and Habenaria radiata. This work will be valuable for genetic and phylogenetic studies on H. ciliolaris.

Phylogenetics of subtribe Orchidinae s.l. (Orchidaceae; Orchidoideae) based on seven markers (plastid matK, psaB, rbcL, trnL-F, trnH-psba, and nuclear nrITS, Xdh): implications for generic delimitation.

BACKGROUND: Subtribe Orchidinae (Orchidaceae, Orchidoideae) are a nearly cosmopolitan taxon of terrestrial orchids, comprising about 1800 species in 47 to 60 genera. Although much progress has been made in recent years of phylogenetics of Orchidinae, considerable problems remain to be addressed. Based on molecular phylogenetics, we attempt to illustrate the phylogenetic relationships and discuss generic delimitation within Orchidinae. Seven DNA markers (five plastid and two nuclear), a broad sampling of Orchidinae (400 species in 52 genera) and three methods of phylogenetic analysis (maximum likelihood, maximum parsimony and Bayesian inference) were used. RESULTS: Orchidinae s.l. are monophyletic. Satyrium is sister to the rest of Orchidinae s.l. Brachycorythis and Schizochilus are successive sister to Asian-European Orchidinae s.s. Sirindhornia and Shizhenia are successive sister to clade formed by Tsaiorchis-Hemipilia-Ponerorchis alliance. Stenoglottis is sister to the Habenaria-Herminium-Peristylus alliance. Habenaria, currently the largest genus in Orchidinae, is polyphyletic and split into two distant clades: one Asian-Australian and the other African-American-Asian. Diplomeris is sister to Herminium s.l. plus Asian-Australian Habenaria. CONCLUSIONS: We propose to recognize five genera in the Ponerorchis alliance: Hemipilia, Ponerorchis s.l., Sirindhornia, Shizhenia and Tsaiorchis. Splitting Habenaria into two genera based on morphological characters and geographical distribution may be the least disruptive approach, and it is reasonable to keep Satyrium in Orchidinae.

The complete plastome sequence of the endangered orchid Habenaria radiata (Orchidaceae).

In this study, we determined the complete chloroplast sequence of Habenaria radiata (Thunb.) Spreng. (Orchidaceae) (NCBI acc. no. KX871237), an endangered plant species protected by the national law of Korea. The gene order and gene content of the H. radiata plastome are similar to those of typical angiosperm plastomes. The 11 ndh genes, which are usually lost in orchid plastomes, are intact in the H. radiata plastome. The complete plastome is 155,353 bp in length and consists of a large single copy of 84,833 bp and a small single copy of 17,718 bp, separated by two inverted repeats of 26,401 bp. The plastome contains 113 genes, of which 79 are protein-coding genes, 30 are tRNA genes, and four are rRNA genes. Sixteen genes contain one intron and two genes (clpP, ycf3) have two introns. A total of 76 simple sequence repeat (SSR) loci, which consist of 58 mono-SSR, 17 di-SSR, and 1 tri-SSR, are scattered along the H. radiata plastome. Some of these plastome SSR and high sequence divergent regions may be useful for development of genetic markers for the populations of H. radiata and other congeneric species. Phylogenetic analysis identified the sister relationship between H. radiata and H. pantlingiana within the tribe Orchideae.

Ultramicroscopic examination of mature massulae of Habenaria arinaria (Orchidaceae).

The mature massula of H. arinaria was examined by means of transmission electron microscopy, with the aim to understand the nature of cohesion between grains, the accumulation of pollen storage reserves, and the behavior of the nucleus of the vegetative cell in this composite type of pollen. The massula was a union of a large number of polygonal pollen grains that were tightly linked together. The exine within the massula were highly simplified, consisting of a single layer of nexine-2, lacking tectum, bacula, and nexine-1, while all the four layers comprised the exine on the massula surface. The two layers of nexine-2 of adjacent grains fused into a seamless whole. Undoubtedly the fusion of the nexine-2 was the mechanism by which the grains of the massula were linked together. No starch grains, lipid bodies, or storage proteins were present in the mature massula, and so the composite pollen of this species belonged to a novel type with regard to storage reserves. The vegetative nucleus was not lobed and revealed a huge amount of highly condensed chromatin, indicating a quiescent status. The condensed status of the vegetative nuclei in this composite type of pollen system is in striking contrast to the highly decondensed status reported in the free type of pollen grains.

Protective effect of Habenaria intermedia tubers against acute and chronic physical and psychological stress paradigms in rats

The present study was undertaken to evaluate the adaptogenic activity of ethanol (EtHI), ethyl acetate (EAHI) fractions of Habenaria intermedia D. Don, Orchidaceae (HI), tubers using immobilization induced acute stress (AS), chronic stress (CS) and swimming induced stress in experimental animals. The tested doses of EtHI (100 and 200 mg/kg, p.o.) and higher dose of EAHI (200 mg/kg, p.o.) normalized altered serum biochemical parameters and the severity of ulcers in both AS and CS. EAHI and EtHI restored the hyperthrophy of adrenal gland and atrophy of spleen and thymus gland in AS and CS. Greater swimming time was noted in the mice pretreated with EtHI and EAHI. Levels of adrenal ascorbic acid and cortisol were restored significantly. EAHI exhibited prominent scavenging effect of DPPH, hydroxyl radical and lipid peroxidation in vitro. Phytochemical studies resulted in the isolation of scopoletin and gallic acid as marker compounds. Our results proved the traditional claim of HI as anti-stress/adaptogen in Ayurvdea.