Phylogeny, character evolution, and biogeography of the fern genus Bolbitis (Dryopteridaceae).

Bolbitis is a pantropical fern genus of Dryopteridaceae with ca. 80 species mainly in tropical Asia. Earlier studies confirmed the monophyly of Bolbitis when Mickelia is excluded and identified three major clades in Bolbitis. However, earlier studies are based on relatively small sampling and the majority of Asian species are not sampled. In this study, DNA sequences of three plastid markers of 169 accessions representing ca. 68 (85 % of total) species of Bolbitis in nine out of the 10 series recognized by Hennipman (1977), and 54 accessions representing the five remaining bolbitidoid genera are used to infer a global phylogeny with a focus on Asian species. The major results include: (1) Bolbitis is strongly supported as monophyletic; (2) species of Bolbitis are resolved into four major clades and their relationships are: the Malagasy/Mascarene clade is sister to the rest, followed by the African clade which is sister to the American clade + the Asian clade; (3) six well-supported subclades are identified in the most speciose Asian clade; (4) the free-veined Egenolfia is embedded in Bolbitis and is paraphyletic in relation to species with anastomosing venation; (5) three series sensu Hennipman (1977), B. ser. Alienae, B. ser. Egenolfianae, and B. ser. Heteroclitae, are paraphyletic or polyphyletic; (6) evolution of six morphological characters is analyzed and free venation is found to have evolved from anastomosing venation and reversed to free venation in Bolbitis; and (7) biogeographical implications are drawn and it is shown that a single recent dispersal from Asia resulted in continental disjunction of closely related ferns of Bolbitis between Africa and America.

Palynological analysis of the genus Ctenitis (C. Chr.) C. Chr. (Dryopteridaceae) in the Southern Cone of America.

The spore morphology and wall ultrastructure of 12 species of Ctenitis from Southern Cone of America were studied using light microscope, scanning and transmission electron microscope. The study was carried out with herbarium material from Argentine and Brazilian institutions. Equatorial diameters, polar diameters and laesura length were measured. The spores are monolete with echinate or folded ornamentation. In the echinate type, the spines are conical, with broad base and attenuate apex. In the rugate type, the folds are inflated, linear, sinuous, subglobose or handle-shape. The perispore surface is scabrate, rugulate, microverrucose or psilate. Stratification and ultrastructure in the species analyzed are very similar. The exospore is smooth and two-layered in section. Simple and branched channels are observed mainly in the outer exospore. The perispore is composed of two layers, the inner one forms the ornamentation and the outer covers all the outer and inner surfaces. Immature spores were found in all samples of C. fenestralis. The characteristics of the studied spores like macro-ornamentation, color and fold length provide relevant information to differentiate some species or groups of species within the genus.

Phylogeny, biogeography, and character evolution in the fern family Hypodematiaceae.

The Old World fern genera Hypodematium and Leucostegia had long been placed in the families Dryopteridaceae and Davalliaceae, respectively, before the advent of molecular phylogenetics. Recent molecular studies confirmed the recognition of the family Hypodematiaceae composed of these two genera, but the relationships within each of these two genera have been unclear. In the present study we performed phylogenetic analyses (MP, ML, BI) based on DNA data from six plastid markers (atpB, atpB-rbcL, matK, rbcL, rps4 & rps4-trnS, and trnL & trnL-F) of 165 accessions representing 31 species in two genera of Hypodematiaceae as the ingroup and 26 accessions representing Cystopteridaceae, Didymochlaenaceae, Dryopteridaceae, Davalliaceae, Oleandraceae, and Woodsiaceae as the outgroups. Our analyses supported the monophyly of the currently defined Hypodematiaceae only including Hypodematium and Leucostegia and found that the family to be sister to the remaining eupolypods I. Our data resolved three taxa of Leucostegia into two clades. In Hypodematium, 28 taxa are resolved into seven strongly supported clades or single-accession clades. The evolution of important morphological characters are inferred in the phylogenetic context. Our dated phylogeny suggested a latest Jurassic-earliest Cretaceous origin of the family and Upper Cretaceous origin of two genera, with Hypodematiaceae originated from East Asia; extant lineages of Hypodematium originated from East Asia and subsequently into Africa, the Indian region, the Madagascar region, and Southeast Asia; and Leucostegia originated from East Asia and/or Southeast Asia.

Application of spore morphology to solve identification problems in certain species of family Dryopteridaceae from Malakand Division, Pakistan.

Here we investigate the morphology of the spores of 12 native taxa of Dryopteridaceae that grow in Malakand division, Pakistan; most of these species not fully described before. This study intends to survey the taxonomic significance of spore morphological features and their variation useful for species identification in 7 species of Polystichum and 5 taxa of Dryopteris. The spores examinations were accomplished utilizing a light microscope (LM), and scanning electron microscope (SEM). The spores are monolete, medium-sized, with variable shapes in both equatorial and polar views. The mean estimation of equatorial diameter ranging from 28.3 to 58.3 µm and the polar diameter varied from 27.6 to 45.8 µm. The exospore thickness ranges between 1.6 to 3.8 µm, and the thickness of perispore is 0.8-6.7 µm. The perispore is perforated, ornamentation elements occur in different ways in the studied taxa of Polystichum. In investigated Dryopteris taxa, rugate perispore type is found. Results of multivariate statistical analysis (UPGMA, and PCA) established on quantitative and qualitative morphological traits of spores demonstrate that the species form distinct groups, not reflecting any phylogenetic relationships. A key to the spores types is provided for species identification, which dependent on the diagnostic characters of spores. The results of this study indicate that variation of spore morphological traits; in particular, the ornamentation and perispore characteristics, which is surface texture, have diagnostic value at the species level. Our data will help in the identification of spores of the family Dryopteridaceae in palynological and taxonomic studies.

Simple and Divided Leaves in Ferns: Exploring the Genetic Basis for Leaf Morphology Differences in the Genus Elaphoglossum (Dryopteridaceae).

Despite the implications leaves have for life, their origin and development remain debated. Analyses across ferns and seed plants are fundamental to address the conservation or independent origins of megaphyllous leaf developmental mechanisms. Class I KNOX expression studies have been used to understand leaf development and, in ferns, have only been conducted in species with divided leaves. We performed expression analyses of the Class I KNOX and Histone H4 genes throughout the development of leaf primordia in two simple-leaved and one divided-leaved fern taxa. We found Class I KNOX are expressed (1) throughout young and early developing leaves of simple and divided-leaved ferns, (2) later into leaf development of divided-leaved species compared to simple-leaved species, and (3) at the leaf primordium apex and margins. H4 expression is similar in young leaf primordia of simple and divided leaves. Persistent Class I KNOX expression at the margins of divided leaf primordia compared with simple leaf primordia indicates that temporal and spatial patterns of Class I KNOX expression correlate with different fern leaf morphologies. However, our results also indicate that Class I KNOX expression alone is not sufficient to promote divided leaf development in ferns. Class I KNOX patterns of expression in fern leaves support the conservation of an independently recruited developmental mechanism for leaf dissection in megaphylls, the shoot-like nature of fern leaves compared with seed plant leaves, and the critical role marginal meristems play in fern leaf development.

Class I KNOX Is Related to Determinacy during the Leaf Development of the Fern Mickelia scandens (Dryopteridaceae).

Unlike seed plants, ferns leaves are considered to be structures with delayed determinacy, with a leaf apical meristem similar to the shoot apical meristems. To better understand the meristematic organization during leaf development and determinacy control, we analyzed the cell divisions and expression of Class I KNOX genes in Mickelia scandens, a fern that produces larger leaves with more pinnae in its climbing form than in its terrestrial form. We performed anatomical, in situ hybridization, and qRT-PCR experiments with histone H4 (cell division marker) and Class I KNOX genes. We found that Class I KNOX genes are expressed in shoot apical meristems, leaf apical meristems, and pinnae primordia. During early development, cell divisions occur in the most distal regions of the analyzed structures, including pinnae, and are not restricted to apical cells. Fern leaves and pinnae bear apical meristems that may partially act as indeterminate shoots, supporting the hypothesis of homology between shoots and leaves. Class I KNOX expression is correlated with indeterminacy in the apex and leaf of ferns, suggesting a conserved function for these genes in euphyllophytes with compound leaves.

Assessing effects of legacy nuclear waste on plants: Sensitive fern (Onoclea sensibilis) gametophyte viability at the Chalk River site.

We evaluated the effects of chronic exposure to environmental radiological contamination on the reproductive fitness of sensitive fern (Onoclea sensibilis) by quantifying viability in haploid gametophytes of spores collected from ferns from background and contaminated areas of the Chalk River site. Dose rates measured in situ at field sites ranged from 60 to 849 µGy h-1, with effects possible at the more contaminated sites (greater than 400 µGy h-1). Fern spores were also irradiated from 1 to 1000 Gy to develop dose-response curves. We found no effects on gametophyte viability at the most contaminated areas of the Chalk River site, where we estimated growing season doses of 0.3-3.7 Gy. Dose-response curves show evidence of hormesis, with an increase in gametophyte viability up to 10 Gy, followed by a rapid decline to no viable gametophytes at doses of 1000 Gy. The sensitive fern is not a radiosensitive plant species, but effects do occur within the normal range (10-1000 Gy) of most plant species, making it useful as a sentinel species from a community perspective. Sensitive fern spore germination is high and stable over field dose ranges, with effects primarily on gametophyte viability. This method shows promise as an effects monitoring tool for sites with radiological contamination.

The role of water-water cycle in regulating the redox state of photosystem I under fluctuating light.

The regulation of photosystem I (PSI) redox state under fluctuating light was investigated for four species using P700 measurement and electrochromic shift analysis. Species included the angiosperms Camellia japonica, Bletilla striata and Arabidopsis thaliana and the fern Cyrtomium fortunei. For the first seconds after transition from low to high light, all species showed relatively low levels of the proton gradient (ΔpH) across the thylakoid membranes. At this moment, PSI was highly oxidized in C. japonica and C. fortunei but was over-reduced in B. striata and A. thaliana. In B. striata and A. thaliana, the redox state of PSI was largely dependent on ΔpH. In contrast, the rapid oxidation of P700 in C. japonica was relatively independent of ΔpH, but was mainly dependent on the outflow of electrons to O2 via the water-water cycle. In the fern C. fortunei, PSI redox state was rapidly regulated by the fast photo-reduction of O2 rather than the ΔpH. These results indicate that mechanisms regulating PSI redox state under fluctuating light differ greatly between species. We propose that the water-water cycle is an important mechanism regulating the PSI redox state in angiosperms.

A plastid phylogeny of the fern genus Arachniodes (Dryopteridaceae).

Arachniodes (Dryopteridaceae) is one of the most confusing and controversial fern genera in terms of its circumscription, nomenclature, and taxonomy. Estimates of species number range from 40 to 200. Previous molecular works included only 2-17 accessions representing 2-12 species of Arachniodes and allied genera, leaving most of the Asian species remain unsampled and the infragneric relationships unclear. In this study DNA sequences of seven plastid markers of 343 accessions representing ca. 68 species of Arachniodes (275 accessions), and 64 outgroup accessions from subfam. Dryopteridoideae and subfam. Polybotryoideae were used to infer a phylogeny with maximum likelihood, Bayesian inference, and maximum parsimony approaches. Our major results include: (1) Two species currently assigned in Arachniodes (A. macrostegia and A. ochropteroides are resolved outside of the core Arachniodes making the currently defined Arachniodes polyphyletic, confirming earlier findings; (2) Lithostegia, Leptorumohra, and Phanerophlebiopsis are indeed synonyms of Arachniodes; (3) Leptorumohra is confirmed to be monophyletic, but Phanerophlebiopsis is polyphyletic; (4) The New World species of Arachniodes are confirmed to be not monophyletic with A. denticulata being nested within the Old World species, suggesting that this species is dispersed from the Old World; (5) Arachniodes s.s is resolved into 12 major clades, some of which are further divisable into recognizable subclades and groups, with A. mutica from Japan being resolved as the sister to the rest of the genus; (6) A number of systematic implications of the phylogeny have been suggested; and (7) the genus is estimated to contain ca. 83 species.

Global phylogeny and biogeography of the fern genus Ctenitis (Dryopteridaceae), with a focus on the Indian Ocean region.

The diverse and pantropical genus Ctenitis, in the Dryopteridaceae, has been largely ignored in phylogenetic studies until now. In this study, we fill in this gap by reconstructing the first comprehensive phylogeny of the genus including 53 species currently recognized in the genus Ctenitis, among which seven species formerly were assigned to the genus Pseudotectaria and one to Heterogonium. Special emphasis was given to the sampling of species occurring in the African-Indian Ocean region. The presented results include reconstruction of a biogeographic scenario based on estimated divergence times and ancestral area reconstruction. Our findings confirm the inclusion, within Ctenitis, of the Indian Ocean species formerly placed in Pseudotectaria and Heterogonium. The crown group divergence was estimated to date back to the Oligocene or Early Miocene. The biogeographical scenario indicates an initial divergence of the Asian-Pacific ranges and the neotropical ranges, and a subsequent colonization of the Afro-Madagascan region by a lineage with neotropical ancestors. The Afro-Madagascan lineage splits into a lineage endemic to the Mascarene islands and a lineage occurring in Madagascar, the Comoros and Africa. The range expansion towards Africa and Madagascar was estimated to date back to the late Miocene, whereas the estimated ages for the onset of the diversification of the Mascarene diversity is consistent with the ages of these young, volcanic islands. The absence of any extant species of Ctenitis with a multi-continental distribution range and the rarity of inter-island dispersal and speciation in the Indian Ocean region suggest a limited contribution of long distance dispersal to the biogeographical history of this fern genus, versus a high contribution of local speciation.

The Relationship between Mating System and Genetic Diversity in Diploid Sexual Populations of Cyrtomium falcatum in Japan.

The impact of variation in mating system on genetic diversity is a well-debated topic in evolutionary biology. The diploid sexual race of Cyrtomium falcatum (Japanese holly fern) shows mating system variation, i.e., it displays two different types of sexual expression (gametangia formation) in gametophytes: mixed (M) type and separate (S) type. We examined whether there is variation in the selfing rate among populations of this species, and evaluated the relationship between mating system, genetic diversity and effective population size using microsatellites. In this study, we developed eight new microsatellite markers and evaluated genetic diversity and structure of seven populations (four M-type and three S-type). Past effective population sizes (Ne) were inferred using Approximate Bayesian computation (ABC). The values of fixation index (FIS), allelic richness (AR) and gene diversity (h) differed significantly between the M-type (FIS: 0.626, AR: 1.999, h: 0.152) and the S-type (FIS: 0.208, AR: 2.718, h: 0.367) populations (when admixed individuals were removed from two populations). Although evidence of past bottleneck events was detected in all populations by ABC, the current Ne of the M-type populations was about a third of that of the S-type populations. These results suggest that the M-type populations have experienced more frequent bottlenecks, which could be related to their higher colonization ability via gametophytic selfing. Although high population differentiation among populations was detected (FST = 0.581, F'ST = 0.739), there was no clear genetic differentiation between the M- and S-types. Instead, significant isolation by distance was detected among all populations. These results suggest that mating system variation in this species is generated by the selection for single spore colonization during local extinction and recolonization events and there is no genetic structure due to mating system.

A well-sampled phylogenetic analysis of the polystichoid ferns (Dryopteridaceae) suggests a complex biogeographical history involving both boreotropical migrations and recent transoceanic dispersals.

Intercontinental disjunctions in ferns have often been considered as the result of long-distance dispersal (LDD) events rather than of vicariance. However, in many leptosporangiate groups, both processes appear to have played a major role in shaping current geographical distribution. In this study, we reconstructed the phylogenetic relationships and inferred the ancestral distribution areas of the polystichoid ferns (Cyrtomium, Phanerophlebia, and Polystichum), to evaluate the relative impact of vicariance and LDD on the biogeography of this group. We used a molecular dataset including 3346 characters from five plastid loci. With 190 accessions our taxon coverage was about three times as large as any previous worldwide sampling. Biogeographical analyses were performed using S-DIVA and S-DEC and divergence times were estimated by integrating fossil and secondary calibrations. The polystichoid ferns are a monophyletic clade that may have originated in East Asia during the Eocene, an age much younger than previously estimated. Three transoceanic disjunctions between East Asia and New World were identified in the Paleogene: one for Phanerophlebia during late Eocene (34Ma, 19-51Ma), and two in Polystichum at the Eocene-Oligocene boundary (30Ma, 18-43Ma; 28Ma, 19-39Ma respectively). During the Neogene, further range expansions took place from Asia to Africa, Hawaii, and the Southwestern Indian Ocean region. Our results indicate that early transfers between the Old and the New World are compatible with a boreotropical migration scenario. After evolving in Asia during the Eocene, the polystichoid ferns reached the New World in independent migrations at the Eocene-Oligocene boundary through the boreotropical belt. However, although less likely, the alternative hypothesis of independent transoceanic dispersals from the Old to the New World cannot be ruled out. Further range expansion during the Neogene was most likely the result of long-distance dispersal (LDD).

Using a multilocus phylogeny to test morphology-based classifications of Polystichum (Dryopteridaceae), one of the largest fern genera.

BACKGROUND: Polystichum (Dryopteridaceae) is probably the third largest fern genus in the world and contains ca. 500 species. Species of Polystichum occur on all continents except Antarctica, but its highest diversity is found in East Asia, especially Southwest China and adjacent regions. Previous studies typically had sparse taxon sampling and used limited DNA sequence data. Consequently, the majority of morphological hypotheses/classifications have never been tested using molecular data. RESULTS: In this study, DNA sequences of five plastid loci of 177 accessions representing ca. 140 species of Polystichum and 13 species of the closely related genera were used to infer a phylogeny using maximum likelihood, Bayesian inference, and maximum parsimony. Our analyses show that (1) Polystichum is monophyletic, this being supported by not only molecular data but also morphological features and distribution information; (2) Polystichum is resolved into two strongly supported monophyletic clades, corresponding to the two subgenera, P. subg. Polystichum and P. subg. Haplopolystichum; (3) Accessions of P. subg. Polystichum are resolved into three major clades: clade K (P. sect. Xiphophyllum), clade L (P. sect. Polystichum), and the HYMASO superclade dominated by accessions of P. sect. Hypopeltis, P. sect. Macropolystichum, and P. sect. Sorolepidium, while those of P. subg. Haplopolystichum are resolved into eight major clades; and (4) The monophyly of the Afra clade (weakly supported), the Australasian clade (weakly supported), and the North American clade (strongly supported) is confirmed. CONCLUSIONS: Of the 23 sections of Polystichum recognized in a recent classification of the genus, four (P. sect. Hypopeltis, P. sect. Neopolystichum, P. sect. Sorolepidium, P. sect. Sphaenopolystichum) are resolved as non-monophyletic, 16 are recovered as monophyletic, and three are monospecific. Of the 16 monophyletic sections, two (P. sect. Adenolepia, P. sect. Cyrtogonellum) are weakly supported and 14 are strongly supported as monophyletic. The relationships of 11 sections (five in P. subg. Haplopolystichum; six in P. subg. Polystichum) are well resolved.

Immuno-enhancement effects of ethanol extract from Cyrtomium macrophyllum (Makino) Tagawa on cyclophosphamide-induced immunosuppression in BALB/c mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Cyrtomium macrophyllum (Makino) Tagawa has been traditionally used as a herbal medicine for the treatment of various infectious diseases such as tapeworm infestation, colds, and viral diseases. However, no systematic study of the immunity of Cyrtomium macrophyllum ethanol extracts (CM) has yet been reported. The present work evaluates these traits. MATERIALS AND METHODS: 120 male BALB/c mice were divided into 6 groups of 20 mice each: (1) normal group (sterile physiological saline), which served as a blank control; (2) model group (Cyclophosphamide, CY) group (sterile physiological saline), which served as a negative control; (3) low-dose CM (50 mg/kg BW); (4) intermediate-dose CM (100 mg/kg BW); (5) high-dose CM (200 mg/kg BW); (6) CM group (200 mg/kg BW). CY (0.2 ml) was administered via intraperitoneal injection. The other regimens were administered via gavage in 0.2 ml solution. Phytochemical of CM was characterized by HPLC-LTQ-Orbitrap. The acute toxicity effect of the ethanol extract of Cyrtomium macrophyllum was also investigated. RESULTS: The spleen and thymus indices of mice receiving low, intermediate, and high doses of CM recovered more quickly than those of CY mice, and they did so in a dose-dependent manner. These mice also showed higher T cell and B cell proliferation responses and macrophage function than those of CY mice, and their serum levels of interleukin-6 and interferon-γ had become normal. In acute toxicity test, CM exhibited no mortality and behavioral changes in mice. Quantitative phytochemical analysis showed flavonoids, polyphenols, and tannins to be the major compounds present in the extract, at 27.64%, 30.87%, and 11.22%, respectively. We found that 16 compounds were characterized by the interpretation of their mass spectra obtained by the MS/MS. CONCLUSION: The current study demonstrates that Cyrtomium macrophyllum ethanol extract improved immune function in CY-treated mice.

Two new C-methyl flavanones from the rhizomes and frond bases of Matteuccia struthiopteris.

Two new C-methyl flavanones, (2S)-5,7-dihydroxy-6,8-dimethyl-4'-methoxydihydroflavone-7-O-(6″-O-acetyl)-ß-d-glucopyranoside (1) and (2S)-5,7-dihydroxy-6,8-dimethyldihydroflavone-7-O-(6″-O-acetyl)-ß-d-glucopyranoside (2), together with five known compounds, demethoxymatteucinol-7-O-ß-d-glucopyranoside (3), matteucinol-7-O-ß-d-glucopyranoside (4), 5,7-dihydroxy-6-methyl-4'-methoxydihydroflavone (5), methoxymatteucin (6), and thunberginol C (7), were first isolated from the EtOH extract of the rhizomes and frond bases of Matteuccia struthiopteris. The structures were established by spectral analyses, mainly HR-ESI-MS and 1D and 2D NMR experiments (COSY, HSQC, and HMBC).

Significant spatial aggregation and fine-scale genetic structure in the homosporous fern Cyrtomium falcatum (Dryopteridaceae).

Spores of homosporous ferns are small, wind-borne and thus have the potential for long-distance dispersal. This common perception has led to a prediction of near-random spatial genetic structure within fern populations. Spore dispersal and spore bank studies, however, indicate that most spores fall close to the maternal plant (< 5 m), supporting a prediction of significant fine-scale genetic structure (FSGS) within populations. To determine which of these two hypotheses is more likely to occur in nature, we measured inbreeding and quantified the spatial distribution of individuals and allozyme-based genotypes using spatial autocorrelation methods within four populations of the fern Cyrtomium falcatum in southern South Korea. Inbreeding levels were low, and all populations exhibited significant aggregation of individuals and strong FSGS. The present results support the second hypothesis, and the substantial FSGS in C. falcatum could reflect the unique features of most homosporous ferns (outcrossing mating systems that lead a majority of spores to occur at short distances and a very limited dispersal distance of male gametes). Although fern spores are physically analogous to orchid seeds, the intensity of FSGS exhibited in C. falcatum is four times stronger than that in 16 terrestrial orchid species.

Symptoms of Fern Distortion Syndrome resulting from inoculation with opportunistic endophytic fluorescent Pseudomonas spp.

BACKGROUND: Fern Distortion Syndrome (FDS) is a serious disease of Leatherleaf fern (Rumohra adiantiformis). The main symptom of FDS is distortion of fronds, making them unmarketable. Additional symptoms include stunting, irregular sporulation, decreased rhizome diameter, and internal discoloration of rhizomes. We previously reported an association of symptoms with increased endophytic rhizome populations of fluorescent pseudomonads (FPs). The aim of the current study was to determine if FPs from ferns in Costa Rica with typical FDS symptoms would recreate symptoms of FDS. METHODOLOGY AND FINDINGS: Greenhouse tests were conducted over a 29-month period. Micro-propagated ferns derived from tissue culture were first grown one year to produce rhizomes. Then, using an 8×9 randomized complete block experimental design, 8 replicate rhizomes were inoculated by dipping into 9 different treatments before planting. Treatments included water without bacteria (control), and four different groups of FPs, each at a two concentrations. The four groups of FPs included one group from healthy ferns without symptoms (another control treatment), two groups isolated from inside rhizomes of symptomatic ferns, and one group isolated from inside roots of symptomatic ferns. Symptoms were assessed 12 and 17 months later, and populations of FPs inside newly formed rhizomes were determined after 17 months. Results showed that inoculation with mixtures of FPs from ferns with FDS symptoms, but not from healthy ferns, recreated the primary symptom of frond deformities and also the secondary symptoms of irregular sporulation, decreased rhizome diameter, and internal discoloration of rhizomes. CONCLUSIONS: These results suggest a model of causation of FDS in which symptoms result from latent infections by multiple species of opportunistic endophytic bacteria containing virulence genes that are expressed when populations inside the plant reach a minimum level.

Assessing the resistance and bioremediation ability of selected bacterial and protozoan species to heavy metals in metal-rich industrial wastewater.

BACKGROUND: Heavy-metals exert considerable stress on the environment worldwide. This study assessed the resistance to and bioremediation of heavy-metals by selected protozoan and bacterial species in highly polluted industrial-wastewater. Specific variables (i.e. chemical oxygen demand, pH, dissolved oxygen) and the growth/die-off-rates of test organisms were measured using standard methods. Heavy-metal removals were determined in biomass and supernatant by the Inductively Couple Plasma Optical Emission Spectrometer. A parallel experiment was performed with dead microbial cells to assess the biosorption ability of test isolates. RESULTS: The results revealed that the industrial-wastewater samples were highly polluted with heavy-metal concentrations exceeding by far the maximum limits (in mg/l) of 0.05-Co, 0.2-Ni, 0.1-Mn, 0.1-V, 0.01-Pb, 0.01-Cu, 0.1-Zn and 0.005-Cd, prescribed by the UN-FAO. Industrial-wastewater had no major effects on Pseudomonas putida, Bacillus licheniformis and Peranema sp. (growth rates up to 1.81, 1.45 and 1.43 d-1, respectively) compared to other test isolates. This was also revealed with significant COD increases (p < 0.05) in culture media inoculated with living bacterial isolates (over 100%) compared to protozoan isolates (up to 24% increase). Living Pseudomonas putida demonstrated the highest removal rates of heavy metals (Co-71%, Ni-51%, Mn-45%, V-83%, Pb-96%, Ti-100% and Cu-49%) followed by Bacillus licheniformis (Al-23% and Zn-53%) and Peranema sp. (Cd-42%). None of the dead cells were able to remove more than 25% of the heavy metals. Bacterial isolates contained the genes copC, chrB, cnrA3 and nccA encoding the resistance to Cu, Cr, Co-Ni and Cd-Ni-Co, respectively. Protozoan isolates contained only the genes encoding Cu and Cr resistance (copC and chrB genes). Peranema sp. was the only protozoan isolate which had an additional resistant gene cnrA3 encoding Co-Ni resistance. CONCLUSION: Significant differences (p < 0.05) observed between dead and living microbial cells for metal-removal and the presence of certain metal-resistant genes indicated that the selected microbial isolates used both passive (biosorptive) and active (bioaccumulation) mechanisms to remove heavy metals from industrial wastewater. This study advocates the use of Peranema sp. as a potential candidate for the bioremediation of heavy-metals in wastewater treatment, in addition to Pseudomonas putida and Bacillus licheniformis.

[Textual research on original plant of Chinese herbal medicine Cyrtomium Rhizome].

Through studies on morphological characteristics, distribution and ecological habitat described in ancient literatures, Osmunda japonica is believed to the original plant of Chinese herbal medicine Cyrtomium Rhizome. Meanwhile, analysis is also made on causes for descriptions that do not comply with characteristics of O. japonica such as toxicity, flowers and fruits, illustrations, indumentums and flakes as well as appearance of other original plants. It is suggested to list O. japonica as the only original plant of Cyrtomium Rhizome and distinguish it from other frequently seen medical plants. Separated studies are also conducted for pesticide effect and active ingredients of Dryoteris Crassrhizomae Rhizome and Woodwardia japonica that show better effects.

Acylphloroglucinols from Elaphoglossum crassipes: antidepressant-like activity of crassipin A.

Nine new terpenylated acylphloroglucinols, crassipins A-I, were isolated from the rhizomes and roots of the fern Elaphoglossum crassipes, and their structures were elucidated by analysis of spectroscopic data and chemical derivatization. The absolute configurations of some of the compounds were established by CD and VCD in combination with a quantum mechanical method. Crassipin A (1), the major acylphloroglucinol of the Et(2)O extract of E. crassipes, as well as its peracetylated derivative (8), displayed antidepressant-like activity in a mouse forced-swimming test when administered orally at a dose of 15 mg/kg.