Assembly and comparative analysis of the complete multichromosomal mitochondrial genome of Cymbidium ensifolium, an orchid of high economic and ornamental value.

BACKGROUND: Orchidaceae is one of the largest groups of angiosperms, and most species have high economic value and scientific research value due to their ornamental and medicinal properties. In China, Chinese Cymbidium is a popular ornamental orchid with high economic value and a long history. However, to date, no detailed information on the mitochondrial genome of any species of Chinese Cymbidium has been published. RESULTS: Here, we present the complete assembly and annotation of the mitochondrial genome of Cymbidium ensifolium (L.) Sw. The mitogenome of C. ensifolium was 560,647 bp in length and consisted of 19 circular subgenomes ranging in size from 21,995 bp to 48,212 bp. The genome encoded 35 protein-coding genes, 36 tRNAs, 3 rRNAs, and 3405 ORFs. Repeat sequence analysis and prediction of RNA editing sites revealed a total of 915 dispersed repeats, 162 simple repeats, 45 tandem repeats, and 530 RNA editing sites. Analysis of codon usage showed a preference for codons ending in A/T. Interorganellar DNA transfer was identified in 13 of the 19 chromosomes, with plastid-derived DNA fragments representing 6.81% of the C. ensifolium mitochondrial genome. The homologous fragments of the mitochondrial genome and nuclear genome were also analysed. Comparative analysis showed that the GC content was conserved, but the size, structure, and gene content of the mitogenomes varied greatly among plants with multichromosomal mitogenome structure. Phylogenetic analysis based on the mitogenomes reflected the evolutionary and taxonomic statuses of C. ensifolium. Interestingly, compared with the mitogenomes of Cymbidium lancifolium Hook. and Cymbidium macrorhizon Lindl., the mitogenome of C. ensifolium lost 8 ribosomal protein-coding genes. CONCLUSION: In this study, we assembled and annotated the mitogenome of C. ensifolium and compared it with the mitogenomes of other Liliidae and plants with multichromosomal mitogenome structures. Our findings enrich the mitochondrial genome database of orchid plants and reveal the rapid structural evolution of Cymbidium mitochondrial genomes, highlighting the potential for mitochondrial genes to help decipher plant evolutionary history.

Species richness disparity in tropical terrestrial herbaceous floras: Evolutionary insight from Collabieae (Orchidaceae).

Species richness is spatially heterogeneous even in the hyperdiverse tropical floras. The main cause of uneven species richness among the four tropical regions are hot debated. To date, higher net diversification rates and/or longer colonization time have been usually proposed to contribute to this pattern. However, there are few studies to clarify the species richness patterns in tropical terrestrial floras. The terrestrial tribe Collabieae (Orchidaceae) unevenly distributes in the tropical regions with a diverse and endemic center in Asia. Twenty-one genera 127 species of Collabieae and 26 DNA regions were used to reconstruct the phylogeny and infer the biogeographical processes. We compared the topologies, diversification rates and niche evolutionary rates of Collabieae and regional lineages on empirical samplings and different simulated samplings fractions respectively. Our results suggested that the Collabieae originated in Asia at the earliest Oligocene, and then independently spread to Africa, Central America, and Oceania since the Miocene via long-distance dispersal. These results based on empirical data and simulated data were similar. BAMM, GeoSSE and niche analyses inferred that the Asian lineages had higher net diversification and niche evolutionary rates than those of Oceanian and African lineages on the empirical and simulated analyses. Precipitation is the most important factor for Collabieae, and the Asian lineage has experienced more stable and humid climate, which may promote the higher net diversification rate. Besides, the longer colonization time may also be associated with the Asian lineages' diversity. These findings provided a better understanding of the regional diversity heterogeneity in tropical terrestrial herbaceous floras.

LC-HRMS/MS-Based Metabolomics Approaches Applied to the Detection of Antifungal Compounds and a Metabolic Dynamic Assessment of Orchidaceae.

The liquid chromatography-mass spectrometry (LC-MS)-based metabolomics approach is a powerful technology for discovering novel biologically active molecules. In this study, we investigated the metabolic profiling of Orchidaceae species using LC-HRMS/MS data combined with chemometric methods and dereplication tools to discover antifungal compounds. We analyze twenty ethanolic plant extracts from Vanda and Cattleya (Orchidaceae) genera. Molecular networking and chemometric methods were used to discriminate ions that differentiate healthy and fungal-infected plant samples. Fifty-three metabolites were rapidly annotated through spectral library matching and in silico fragmentation tools. The metabolomic profiling showed a large production of polyphenols, including flavonoids, phenolic acids, chromones, stilbenoids, and tannins, which varied in relative abundance across species. Considering the presence and abundance of metabolites in both groups of samples, we can infer that these constituents are associated with biochemical responses to microbial attacks. In addition, we evaluated the metabolic dynamic through the synthesis of stilbenoids in fungal-infected plants. The tricin derivative flavonoid- and the loliolide terpenoidfound only in healthy plant samples, are promising antifungal metabolites. LC-HRMS/MS, combined with state-of-the-art tools, proved to be a rapid and reliable technique for fingerprinting medicinal plants and discovering new hits and leads.

Stealing sugar from the honey fungus.

This article comments on: GeSUT4 mediates sucrose import at the symbiotic interface for carbon allocation of heterotrophic Gastrodia elata (Orchidaceae).

Automated conservation assessment of the orchid family with deep learning.

International Union for Conservation of Nature (IUCN) Red List assessments are essential for prioritizing conservation needs but are resource intensive and therefore available only for a fraction of global species richness. Automated conservation assessments based on digitally available geographic occurrence records can be a rapid alternative, but it is unclear how reliable these assessments are. We conducted automated conservation assessments for 13,910 species (47.3% of the known species in the family) of the diverse and globally distributed orchid family (Orchidaceae), for which most species (13,049) were previously unassessed by IUCN. We used a novel method based on a deep neural network (IUC-NN). We identified 4,342 orchid species (31.2% of the evaluated species) as possibly threatened with extinction (equivalent to IUCN categories critically endangered [CR], endangered [EN], or vulnerable [VU]) and Madagascar, East Africa, Southeast Asia, and several oceanic islands as priority areas for orchid conservation. Orchidaceae provided a model with which to test the sensitivity of automated assessment methods to problems with data availability, data quality, and geographic sampling bias. The IUC-NN identified possibly threatened species with an accuracy of 84.3%, with significantly lower geographic evaluation bias relative to the IUCN Red List and was robust even when data availability was low and there were geographic errors in the input data. Overall, our results demonstrate that automated assessments have an important role to play in identifying species at the greatest risk of extinction.

Evaluación Automatizada de la Conservación de la Familia Orchidaceae mediante Aprendizaje Profundo Resumen Los análisis de la Lista Roja de la Unión Internacional para la Conservación de la Naturaleza (UICN) son esenciales para la priorización de las necesidades de conservación, pero requieren de muchos recursos y por lo tanto están disponibles sólo para una fracción de la riqueza mundial de especies. Las evaluaciones automatizadas de la conservación basadas en los registros disponibles de presencia geográfica pueden ser una alternativa rápida pero no está claro cuán confiables son estas evaluaciones. Realizamos evaluaciones automatizadas de la conservación para 13,910 especies (47.3% de las especies conocidas de la familia) de la diversa y mundialmente distribuida familia de las orquídeas (Orchidaceae), en la cual la mayoría de las especies (13,049) no tenían una valoración previa por parte de la UICN. Usamos un método novedoso basado en una red neural profunda (IUC-NN). Identificamos 4,342 especies de orquídeas (31.2% de las especies evaluadas) como posiblemente amenazadas por la extinción (equivalente a las categorías de la UICN en peligro crítico [CR], en peligro [EN] o vulnerable [VU]) y a Madagascar, África Occidental, el sudeste de Asia y varias islas oceánicas como áreas prioritarias para la conservación de orquídeas. La familia Orchidaceae proporcionó un modelo con el cual probar la sensibilidad de los métodos de evaluación automatizada ante problemas con la disponibilidad de datos, la calidad de los datos y los sesgos de muestreo geográfico. La IUC-NN identificó posibles especies amenazadas con una certeza de 84.3% con un sesgo de evaluación geográfica significativamente más bajo en relación con la Lista Roja de la UICN y mostró solidez incluso cuando la disponibilidad de datos fue baja y hubo errores geográficos en los datos de entrada. En general, nuestros resultados demostraron que las evaluaciones automatizadas tienen un papel importante que desempeñar en la identificación de especies con mayor riesgo de extinción.

Assessment of violet-blue color formation in Phalaenopsis orchids.

BACKGROUND: Phalaenopsis represents an important cash crop worldwide. Abundant flower colors observed in Phalaenopsis orchids range from red-purple, purple, purple-violet, violet, and violet-blue. However, violet-blue orchids are less bred than are those of other colors. Anthocyanin, vacuolar pH and metal ions are three major factors influencing flower color. This study aimed to identify the factors causing the violet-blue color in Phalaenopsis flowers and to analyze whether delphinidin accumulation and blue pigmentation formation can be achieved by transient overexpression of heterologous F3'5'H in Phalaenopsis. RESULTS: Cyanidin-based anthocyanin was highly accumulated in Phalaenopsis flowers with red-purple, purple, purple-violet, and violet to violet-blue color, but no true-blue color and no delphinidin was detected. Concomitantly, the expression of PeF3'H (Phalaenopsis equestrsis) was high, but that of PhF3'5'H (Phalaenopsis hybrid) was low or absent in various-colored Phalaenopsis flowers. Transient overexpression of DgF3'5'H (Delphinium grandiflorum) and PeMYB2 in a white Phalaenopsis cultivar resulted a 53.6% delphinidin accumulation and a novel blue color formation. In contrast, transient overexpression of both PhF3'5'H and PeMYB2 did not lead to delphinidin accumulation. Sequence analysis showed that the substrate recognition site 6 (SRS6) of PhF3'5'H was consistently different from DgF3'5'Hs at positions 5, 8 and 10. Prediction of molecular docking of the substrates showed a contrary binding direction of aromatic rings (B-ring) with the SRS6 domain of DgF3'5'H and PhF3'5'H. In addition, the pH values of violet-blue and purple Phalaenopsis flowers ranged from 5.33 to 5.54 and 4.77 to 5.04, respectively. Furthermore, the molar ratio of metal ions (including Al3+, Ca2+ and Fe3+) to anthocyanin in violet-blue color Phalaenopsis was 190-, 49-, and 51-fold higher, respectively, than those in purple-color Phalaenopsis. CONCLUSION: Cyanidin-based anthocyanin was detected in violet-blue color Phalaenopsis and was concomitant with a high pH value and high molar ratio of Al3+, Ca2+ and Fe3+ to anthocyanin content. Enhanced expression of delphinidin is needed to produce true-blue Phalaenopsis.

Metabolomic analysis of Cyrtopodium glutiniferum extract by UHPLC-MS/MS and in vitro antiproliferative and genotoxicity assessment.

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts of orchids have been traditionally used as human phytotherapeutics. Cyrtopodium flavum, for example, due to the analgesic and anti-inflammatory properties, beside the capacity of heal skin lesions has been focus of research. Also Cyrtopodium glutiniferum, an orchid found in the Brazilian southeastern rainforest, is known to synthesize anti-inflammatory glucomannans in the pseudobulbs, as other potentially therapeutic compounds. AIM OF THE STUDY: We have reported the first metabolomic analysis focused on the phenols expression of the neotropical orchid Cyrtopodium glutiniferum Raddi, besides free radical scavenging, anti-inflammatory and antiproliferative activities, and the genotoxicity properties of the aqueous extract. MATERIAL AND METHODS: The metabolomics of C. glutiniferum aqueous extract was performed through UHPLC-MSn acquisition. We have detected the scavenging potential of the extract using DPPH assay. The genotoxic potential was performed by Ames Test (0-5000 µg mL-1) and micronucleous assay (0-5000 µg mL-1) in RAW264.7 cells. The cytotoxic potential of the extract against RAW264.7 was tested by WST-1 assay (0-500 µg mL-1). And after all, the RAW264.7 cells were treated with non-cytotoxic concentrations of C. glutiniferum (0-50 µg mL-1) to evaluate the antiproliferative and anti-inflammatory potential, besides the mitochondrial activity. RESULTS: From the 55 molecules identified, 45.5% belonged to the phenolic compounds database from Phenol Explorer, 29% to an in-house Orchidaceae molecules database, and 25.5% to both. Among the identified phenolic compounds, 18 subclasses were discriminated, being phenanthrenes the most abundant. Doses-dependent of C. glutiniferum extracts were able to induce DPPH free radicals scavenging and also to increase TA100 His+ revertants, in metabolic environment, showing mutagenicity just in the highest concentration, of 5 mg/plate. On Eukaryotic cell models, the extract also has induced dose-response and time-response cytotoxicity against RAW264.7 macrophages, mainly after 48 h and 72 h, even though the extract has not been able to induce the increase of micronucleated cells and mitotic index alteration on Micronucleus assay. The activation and proliferation of macrophages cultures were downregulated after 24 h and 48 h by the non-cytotoxic concentrations of the extract in a dose-dependent manner. CONCLUSIONS: The Cyrtopodium glutiniferum metabolomics, anti-inflammatory and anti-proliferative properties observed in this study suggest a therapeutic efficacy of the orchid extract applied in folk medicine.

Clade-age-dependent diversification under high species turnover shapes species richness disparities among tropical rainforest lineages of Bulbophyllum (Orchidaceae).

BACKGROUND: Tropical rainforests (TRFs) harbour almost half of the world's vascular plant species diversity while covering only about 6-7% of land. However, why species richness varies amongst the Earth's major TRF regions remains poorly understood. Here we investigate the evolutionary processes shaping continental species richness disparities of the pantropical, epiphytic and mostly TRF-dwelling orchid mega-genus Bulbophyllum (c. 1948 spp. in total) using diversification analyses based on a time-calibrated molecular phylogeny (including c. 45-50% spp. each from Madagascar, Africa, Neotropics, and 8.4% from the Asia-Pacific region), coupled with ecological niche modelling (ENM) of geographic distributions under present and past (Last Glacial Maximum; LGM) conditions. RESULTS: Our results suggest an early-to-late Miocene scenario of 'out-of-Asia-Pacific' origin and progressive, dispersal-mediated diversification in Madagascar, Africa and the Neotropics, respectively. Species richness disparities amongst these four TRF lineages are best explained by a time-for-speciation (i.e. clade age) effect rather than differences in net diversification or diversity-dependent diversification due to present or past spatial-bioclimatic limits. For each well-sampled lineage (Madagascar, Africa, Neotropics), we inferred high rates of speciation and extinction over time (i.e. high species turnover), yet with the origin of most extant species falling into the Quaternary. In contrast to predictions of classical 'glacial refuge' theories, all four lineages experienced dramatic range expansions during the LGM. CONCLUSIONS: As the Madagascan, African and Neotropical lineages display constant-rate evolution since their origin (early-to-mid-Miocene), Quaternary environmental change might be a less important cause of their high species turnover than intrinsic features generally conferring rapid population turnover in tropical orchids (e.g., epiphytism, specialization on pollinators and mycorrhizal fungi, wind dispersal). Nonetheless, climate-induced range fluctuations during the Quaternary could still have played an influential role in the origination and extinction of Bulbophyllum species in those three, if not in all four TRF regions.

[Current situation and prospect of resource evaluation and sustainable utilization on Bletilla].

The plants of Bletilla are one of the groups in Orchidaceae with the highest economic value. As the traditional Chinese medicinal material, Bletillae Rhizoma exhibits excellent efficacy in hemostatic, antibiosis, detumescent, anticancer activities and regenerating tissue to heal wound, which has great development potential. However, Bletillae Rhizoma is mainly collected from wild resources. At present, the quantity of wild resources of Bletilla plants has sharply decreased and is far from meeting the needs. Resource appraisal and breeding and cultivation of excellent germplasms of Bletilla plants are important for scientific utilization of the resources of the genus. This paper reviewed the following researches of Chinese Bletilla resources: species and distribution, genetic diversity, active ingredient evaluation, breeding, as well as seeding production and cultivation techniques. Suggestions were also provided in further researches on the resources evaluation, sustainable development and efficient utilization of Chinese Bletilla plants.

Chromosome-level assembly, genetic and physical mapping of Phalaenopsis aphrodite genome provides new insights into species adaptation and resources for orchid breeding.

The Orchidaceae is a diverse and ecologically important plant family. Approximately 69% of all orchid species are epiphytes, which provide diverse microhabitats for many small animals and fungi in the canopy of tropical rainforests. Moreover, many orchids are of economic importance as food flavourings or ornamental plants. Phalaenopsis aphrodite, an epiphytic orchid, is a major breeding parent of many commercial orchid hybrids. We provide a high-quality chromosome-scale assembly of the P. aphrodite genome. The total length of all scaffolds is 1025.1 Mb, with N50 scaffold size of 19.7 Mb. A total of 28 902 protein-coding genes were identified. We constructed an orchid genetic linkage map, and then anchored and ordered the genomic scaffolds along the linkage groups. We also established a high-resolution pachytene karyotype of P. aphrodite and completed the assignment of linkage groups to the 19 chromosomes using fluorescence in situ hybridization. We identified an expansion in the epiphytic orchid lineage of FRS5-like subclade associated with adaptations to the life in the canopy. Phylogenetic analysis further provides new insights into the orchid lineage-specific duplications of MADS-box genes, which might have contributed to the variation in labellum and pollinium morphology and its accessory structure. To our knowledge, this is the first orchid genome to be integrated with a SNP-based genetic linkage map and validated by physical mapping. The genome and genetic map not only offer unprecedented resources for increasing breeding efficiency in horticultural orchids but also provide an important foundation for future studies in adaptation genomics of epiphytes.

Biomimetic Gastrointestinal Tract Functions for Metal Absorption Assessment in Edible Plants: Comparison to In Vivo Absorption.

A biomimetic gastrointestinal tract, including in vitro digestion and biomimetic biomembrane extraction, has been proposed for absorption assessment of metals from edible plants. However, its validity is still unknown. Herein, two species of edible plants, Anoectochilus roxburghii and Radix astragali, were selected and digested in a bionic mouth, stomach, and intestine, and then trace metals (Cr, Mn, Fe, Ni, Cu, Zn, Se, Sr, As, and Pb) were transformed to their final metal species. To check model predictability, in vitro and in vivo metal absorption were imitated and tested by monolayer liposome extraction and rat stomach or single-pass duodenal intestine, respectively. A strong correlation was established between in vivo and in vitro metal absorption ratios, with 0.89 > R2 > 0.66, and a significant relationship (p < 0.05) was exhibited for stomach, intestine, two plant species, and 10 metal species. Our biomimetic system could be used as low-cost alternatives to animal and clinical studies for multi-metal absorption.

A δ(15)N assessment of nitrogen deposition for the endangered epiphytic orchid Laelia speciosa from a city and an oak forest in Mexico.

Atmospheric nitrogen deposition poses a major threat to global biodiversity. Tropical epiphytic plants are especially at risk given their reliance on atmospheric sources of nutrients. The leaf, pseudobulb, and root carbon and nitrogen content, C:N ratio, as well as the nitrogen isotopic composition were studied for individuals of Laelia speciosa from a city and from an oak forest in Mexico. The nitrogen content of leaves was similar between the city and the oak forest, reaching 1.3 ± 0.2 % (dry mass). The δ(15)N of leaves, pseudobulbs, and roots reached 5.6 ± 0.2 ‰ in the city, values found in sites exposed to industrial and vehicular activities. The δ(15)N for plant from the oak forest amounted to -3.1 ± 0.3 ‰, which is similar to values measured from sites with low industrial activities. Some orchids such as Laelia speciosa produce a single pseudobulb per year, i.e., a water and nutrient storage organ, so the interannual nitrogen deposition was studied by considering the ten most recent pseudobulbs for plants from either site formed between 2003 and 2012. The C:N ratio of the ten most recent pseudobulbs from the oak forest, as well as that of the pseudobulbs formed before 2010 for plants in the city were indistinguishable from each other, averaging 132.4 ± 6.5, while it was lower for the two most recent pseudobulbs in the city. The δ(15)N values of pseudobulbs from the oak forest averaged ‒4.4 ± 0.1 ‰ for the entire series. The δ(15)N ranged from 0.1 ± 1.6 ‰ for the oldest pseudobulb to 4.7 ± 0.2 ‰ for the pseudobulb formed in the city from 2008 onwards. Isotopic analysis and the C:N ratio for L. speciosa revealed that rates of nitrogen deposition were higher in the city than in the forest. The δ(15)N values of series of pseudobulbs showed that it is possible to track nitrogen deposition over multiple years.

A New Species of Cleisostoma (Orchidaceae) from the Hon Ba Nature Reserve in Vietnam: A Multidisciplinary Assessment.

A new species, Cleisostoma yersinii J. Ponert & Vuong, is described and illustrated based on the material collected in the Hon Ba Nature Reserve in southern Vietnam. In addition to conventional (macro)morphological examination we comparatively investigated root and leaf anatomy (using light and fluorescent microscopy), assessed nectar characteristics (using HPLC analysis), determined nuclear genome size (using DNA flow cytometry) and reconstructed phylogenetic relationships (using nrITS sequences). Cleisostoma yersinii differs from its putative closest relative C. birmanicum in wider and shorter leaves, larger flowers, distinct lip with S-shaped tip of the mid-lobe, and a shallow spur with two large nectar sacks separated by prominent calli and septum. Nectar is sucrose-dominant and very rich in sugars. Stomata are developed on both sides of the leaf and have prominent hyperstomatal chambers and substomatal cavities. Roots with well-developed exodermis and tracheoidal idioblasts are covered by a two-layer Vanda-type velamen. Chloroplasts occur not only in the cortex but are also abundant in the stele. Mean 1C-value was estimated to 2.57 pg DNA. An updated identification key is provided for SE Asian sections and all Vietnamese species of Cleisostoma.

A direct assessment of realized seed and pollen flow within and between two isolated populations of the food-deceptive orchid Orchis mascula.

Gene flow can counteract the loss of genetic diversity caused by genetic drift in small populations. For this reason, clearly understanding gene flow patterns is of the highest importance across fragmented landscapes. However, gene flow patterns are not only dependent upon the degree of spatial isolation of fragmented populations, but are also dependent upon the life-history traits of the species. Indeed, habitat fragmentation effects appear especially unpredictable for food-deceptive orchid species, because of their highly specialised seed and pollen dispersal mechanisms. In this study we used amplified fragment length polymorphism markers and subsequent parentage and spatial autocorrelation analysis to quantify the extent and the patterns of realized gene flow within and between two adjacent fragmented populations of the food-deceptive Orchis mascula. We observed considerable gene flow between both populations, occurring mainly through pollen dispersal. Seed dispersal, on the other hand, was mainly limited to the first few meters from the mother plant in both populations, although at least one among-population seed dispersal event was observed. This, in turn, resulted in a significant spatial genetic structure for both populations. Although genetic diversity was high in both populations and mainly outcrossing occurred, reproductive output was strongly skewed toward a limited number of successful adult plants. These observed patterns are likely due to the different pollinator behaviour associated with food-deceptive plants. We conclude that these populations can be considered viable under their current fragmented state.

Ex situ germination as a method for seed viability assessment in a peatland orchid, Platanthera blephariglottis.

UNLABELLED: • PREMISE OF THE STUDY: Assessing seed quality in orchids has been hindered by stringent germination requirements. Seed quality has traditionally been assessed in orchids using in vitro or in situ germination protocols or viability staining. However, these methods are not always well suited for rapid assessment of viability in the context of ecological studies.• METHODS: The potential of an ex situ protocol for seed viability assessment of orchids in ecological studies was investigated by sowing seeds of Platanthera blephariglottis on Sphagnum moss collected in the orchid's natural habitat. Ex situ germination results were compared with those obtained by viability staining using triphenyltetrazolium chloride (TTC), and the effect of seed testa color on staining and germination results was investigated.• KEY RESULTS: The ex situ protocol yielded high germination rates, with 66% of the seeds germinating after 9 wk. Depending on the seed testa color class, ex situ germination rates were about 1.4 to 2.5 times higher than viability rates determined using TTC, indicating that the TTC technique underestimated viability compared with the method using ex situ germination. The TTC estimates of viability rates were higher for seeds with dark-colored testae than for pale ones, whereas seed testa color had no effect on germination.• CONCLUSIONS: Our study showed promising results for the use of ex situ germination as an alternative to previously developed protocols for seed viability assessment of orchids in ecological studies. Staining using TTC might not be well suited for this purpose, since it introduced a bias with respect to seed testa color.

Climate-dependent costs of reproduction: survival and fecundity costs decline with length of the growing season and summer temperature.

Costs of reproduction are expected to vary with environmental conditions thus influencing selection on life-history traits. Yet, the effects of habitat conditions and climate on trade-offs among fitness components remain poorly understood. For 2-5 years, we quantified costs of experimentally increased reproduction in two populations (coastal long-season vs. inland short-season) of two long-lived orchids that differ in natural reproductive effort (RE; 30 vs. 75% fruit set). In both species, survival costs were found only at the short-season site, whereas growth and fecundity costs were evident at both sites, and both survival and fecundity costs declined with increasing growing season length and/or summer temperature. The results suggest that the expression of costs of reproduction depend on the local climate, and that climate warming could result in selection favouring increased RE in both study species.

A framework for assessing supply-side wildlife conservation.

Market-based, supply-side interventions such as domestication, cultivation, and wildlife farming have been proposed as legal substitutes for wild-collected plants and animals in the marketplace. Based on the literature, we devised a list of the conditions under which supply-side interventions may yield positive conservation outcomes. We applied it to the trade of the orchid Rhynchostylis gigantea, a protected ornamental plant. We conducted a survey of R. gigantea at Jatujak Market in Bangkok, Thailand. Farmed (legal) and wild (illegal, protected) specimens of R. gigantea were sold side-by-side at market. These results suggest farmed specimens are not being substituted for wild plants in the marketplace. For any given set of physical plant characteristics (size, condition, flowers), the origin of the plants (wild vs. farmed) did not affect price. For all price classes, farmed plants were of superior quality to wild-collected plants on the basis of most physical variables. These results suggest wild and farmed specimens represent parallel markets and may not be substitutable goods. Our results with R. gigantea highlight a range of explanations for why supply-side interventions may lack effectiveness, for example, consumer preferences for wild-collected products and low financial incentives for farming. Our results suggest that market-based conservation strategies may not be effective by themselves and may be best utilized as supplements to regulation and education. This approach represents a broad, multidisciplinary evaluation of supply-side interventions that can be applied to other plant and animal species.

Antimicrobial activity of cold and hot successive pseudobulb extracts of Flickingeria nodosa (Dalz.) Seidenf.

Flickingeria nodosa (Dalz.) Seidenf is a medicinally important orchid plant. It is used for the treatment of asthma, bronchitis, throat infections, dermatological infections and also used as blood purifier. Based on its importance the present study was designed to evaluate its antibacterial and antifungal activity against human pathogens with cold and hot successive extracts. The antimicrobial activities of the plant extracts were evaluated against 7 bacterial and 6 fungal strains using well diffusion method on Mueller Hinton agar medium. The cold water extract has antibacterial activity against S. aureus and S. citreus with maximum zone of inhibition. The cold chloroform extract has good antifungal activity against T. mentagrophytes. The plant can be a source material to herbal drug industry since it has some important antimicrobial components in the extracts that can be used for the development of therapeutic phytomedicine.

Evolutionary demography of iteroparous plants: incorporating non-lethal costs of reproduction into integral projection models.

Understanding the selective forces that shape reproductive strategies is a central goal of evolutionary ecology. Selection on the timing of reproduction is well studied in semelparous organisms because the cost of reproduction (death) can be easily incorporated into demographic models. Iteroparous organisms also exhibit delayed reproduction and experience reproductive costs, although these are not necessarily lethal. How non-lethal costs shape iteroparous life histories remains unresolved. We analysed long-term demographic data for the iteroparous orchid Orchis purpurea from two habitat types (light and shade). In both the habitats, flowering plants had lower growth rates and this cost was greater for smaller plants. We detected an additional growth cost of fruit production in the light habitat. We incorporated these non-lethal costs into integral projection models to identify the flowering size that maximizes fitness. In both habitats, observed flowering sizes were well predicted by the models. We also estimated optimal parameters for size-dependent flowering effort, but found a strong mismatch with the observed flower production. Our study highlights the role of context-dependent non-lethal reproductive costs as selective forces in the evolution of iteroparous life histories, and provides a novel and broadly applicable approach to studying the evolutionary demography of iteroparous organisms.

Development of cDNA-derived ssr markers and their efficiency in diversity assessment of cymbidium accessions

Cymbidium spp. are popular flowering plants. Assessment of the genetic diversity in cultivated Cymbidium facilitates conservation of germplasm and subsequent cultivar improvement. Thus, it is important to develop more efficient polymorphic DNA markers. Although more motifs (403) were identified and more primers (206) were designed in the genomic library compared to the cDNA library, a larger number of successful primers were obtained from the cDNA library (59.9 percent) than from genomic DNA library (51.1 percent). However, higher PIC and gene diversity were identified in genomic SSRs. The average allele number per locus was also higher in genomic SSRs (7.3) than EST-SSRs (5.2), among the 24 evaluated Cymbidium accessions. AT/TA was comparatively high in EST-SSRs, while this motif was not as common in genomic SSRs. The CTT/AAG/TCT/AGA/TTC/GAA and TGC/GCA/GCT/AGC/CTG/CAG motifs were the most abundant tri-nucleotide sequences in EST-SSRs, while GTT/AAC/TGT/ACA/TTG/CAA was the most frequent in genomic SSRs. The number of repeats ranged from 3 to 12 in EST-SSRs. Currently, 52 novel polymorphic SSR markers have been evaluated, which will be useful for germplasm assessments, core set construction, evaluation of genetic diversity, and marker assisted selection (MAS) based Cymbidium breeding.