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Phylogenetic relationships of dennstaedtioid ferns: evidence from rbcL sequences.

The ferns are an ancient group of vascular plants that have yielded a staggering array of systematic problems. Among fern classifications, the number of genera in some families has ranged over 10-fold, and some genera have been treated in up to five different families. Three main groups of leptosporangiate ferns have been recognized; the adiantoid, polypodioid, and dennstaedtioid lines. To clarify relationships among genera and families of dennstaedtioid ferns, we sequenced 1320 bp of the chloroplast gene rbcL from 45 species representing 13 families. Sequence divergence for rbcL averaged 0.9% among species within genera, 10.3% among genera within families, and 14.8% among families, suggesting that the data are appropriate for phylogenetic analysis at the generic and familial levels in ferns. Maximum parsimony analysis resulted in four shortest trees of equal length. The strict consensus tree supported many aspects of previously published hypotheses of relationship based on morphological and cytological variations. For example, the tree ferns (which form a single clade) and Hymenophyllaceae appear as sister groups to the dennstaedtioid ferns on all shortest trees. However, Polypodiaceae and adiantoid ferns, groups traditionally considered separate from the dennstaedtioid families, emerged within the dennstaedtioid clade. This analysis also suggests relationships of some problematical genera, such as Monachosorum, Calochlaena, and Lonchitis. Examination of additional DNA sequences of nuclear genes and developmental studies are needed to evaluate further the relationships suggested by phylogenetic analysis of rbcL sequence data.

DNA, Chloroplast↗

X-ray and scanning electron microscopic analysis of the structural composition of tear ferns.

Dried tears from keratoconjunctivitis sicca eyes fail to exhibit the fern-like crystallization patterns observed with tears from eyes with normal tear function. To test our hypothesis that the extent of ferning depends on the ratio of salts to protein and mucin in the tear sample, dried tears from six normal subjects were subjected to scanning electron microscopy (SEM) and X-ray analyses. X-ray diffraction identified sodium chloride and potassium chloride as the major components of tear fern crystals. X-ray fluorescence detected the elements potassium, chlorine, calcium, and sulfur in the dried tear samples, with sulfur indicating the presence of protein and/or mucin. As well as confirming the presence of cubic fern nuclei, SEM revealed two kinds of material, having crystalline and globular appearances, that are hypothesized to be composed of salts and protein/mucin, respectively. Globular material appeared to block extension of crystal fern arms or to coat crystalline material, but did not crystallize. These findings suggest that tear fern crystals are composed of sodium and potassium chloride, with proteinaceous material controlling crystallization indirectly by coating crystal faces and blocking fern extension. This structural composition is consistent with the hypothesis that the ratio of salt to macromolecular species is an important determinant of tear ferning.

Adolescent↗

Characterization of arsenate reductase in the extract of roots and fronds of Chinese brake fern, an arsenic hyperaccumulator.

Root extracts from the arsenic (As) hyperaccumulating Chinese brake fern (Pteris vittata) were shown to be able to reduce arsenate to arsenite. An arsenate reductase (AR) in the fern showed a reaction mechanism similar to the previously reported Acr2p, an AR from yeast (Saccharomyces cerevisiae), using glutathione as the electron donor. Substrate specificity as well as sensitivity toward inhibitors for the fern AR (phosphate as a competitive inhibitor, arsenite as a noncompetitive inhibitor) was also similar to Acr2p. Kinetic analysis showed that the fern AR had a Michaelis constant value of 2.33 mM for arsenate, 15-fold lower than the purified Acr2p. The AR-specific activity of the fern roots treated with 2 mM arsenate for 9 d was at least 7 times higher than those of roots and shoots of plant species that are known not to tolerate arsenate. A T-DNA knockout mutant of Arabidopsis (Arabidopsis thaliana) with disruption in the putative Acr2 gene had no AR activity. We could not detect AR activity in shoots of the fern. These results indicate that (1) arsenite, the previously reported main storage form of As in the fern fronds, may come mainly from the reduction of arsenate in roots; and (2) AR plays an important role in the detoxification of As in the As hyperaccumulating fern.

Arsenate Reductases↗

Incongruence between primary sequence data and the distribution of a mitochondrial atp1 group II intron among ferns and horsetails.

Using DNA sequence data from multiple genes (often from more than one genome compartment) to reconstruct phylogenetic relationships has become routine. Augmenting this approach with genomic structural characters (e.g., intron gain and loss, changes in gene order) as these data become available from comparative studies already has provided critical insight into some long-standing questions about the evolution of land plants. Here we report on the presence of a group II intron located in the mitochondrial atp1 gene of leptosporangiate and marattioid ferns. Primary sequence data for the atp1 gene are newly reported for 27 taxa, and results are presented from maximum likelihood-based phylogenetic analyses using Bayesian inference for 34 land plants in three data sets: (1) single-gene mitochondrial atp1 (exon+intron sequences); (2) five combined genes (mitochondrial atp1 [exon only]; plastid rbcL, atpB, rps4; nuclear SSU rDNA); and (3) same five combined genes plus morphology. All our phylogenetic analyses corroborate results from previous fern studies that used plastid and nuclear sequence data: the monophyly of euphyllophytes, as well as of monilophytes; whisk ferns (Psilotidae) sister to ophioglossoid ferns (Ophioglossidae); horsetails (Equisetopsida) sister to marattioid ferns (Marattiidae), which together are sister to the monophyletic leptosporangiate ferns. In contrast to the results from the primary sequence data, the genomic structural data (atp1 intron distribution pattern) would seem to suggest that leptosporangiate and marattioid ferns are monophyletic, and together they are the sister group to horsetails--a topology that is rarely reconstructed using primary sequence data.

Amino Acid Sequence↗

Arsenic species in an arsenic hyperaccumulating fern, Pityrogramma calomelanos: a potential phytoremediator of arsenic-contaminated soils.

The fern Pityrogramma calomelanos is a hyperaccumulator of arsenic that grows readily on arsenic-contaminated soils in the Ron Phibun district of southern Thailand. P. calomelanos accumulates arsenic mostly in the fronds (up to 8350 microg As g(-1) dry mass) while the rhizoids contain the lowest concentrations of arsenic (88-310 microg As g(-1) dry mass). The arsenic species in aqueous extracts of the fern and soil were determined by high performance liquid chromatography coupled to an inductively coupled plasma mass spectrometer (HPLC-ICPMS) which served as an arsenic specific detector. Only a small part of the arsenic (6.1-12%) in soil was extracted into water, and most of this arsenic (> 97%) was present as arsenate. The arsenic in the fern rhizoids was approximately 60% water-extractable, 95% of which was present as arsenate. In contrast, arsenic in the fern fronds was readily extracted into water (86-93%) and was present mainly as arsenite (60-72%) with the remainder being arsenate. Methylarsonate and dimethylarsinate were detected as trace constituents in only two fern samples. Preliminary estimates of phytoremediation potential suggest that P. calomelanos might remove approximately 2% of the soil arsenic load per year. With due consideration to the type of arsenic compounds present in the fern, and their water-solubility, the option of disposing high arsenic ferns at sea is raised for discussion.

Arsenic↗

Antioxidant responses of hyper-accumulator and sensitive fern species to arsenic.

Plant species capable of hyper-accumulating heavy metals are of considerable interest for phytoremediation, and differ in their ability to accumulate metals from the environment. This work aims to examine (i) arsenic accumulation in three fern species [Chinese brake fern (Pteris vittata L.), slender brake fern (Pteris ensiformis Burm. f.), and Boston fern (Nephrolepis exaltata L.)], which were exposed to 0, 150, or 300 muM of arsenic (Na(2)HAsO(4).7H(2)O), and (ii) the role of anti-oxidative metabolism in arsenic tolerance in these fern species. Arsenic accumulation increased with an increase in arsenic concentration in the growth medium, the most being found in P. vittata fronds showing no toxicity symptoms. In addition, accumulation was highest in the fronds, followed by the rhizome, and finally the roots, in all three fern species. Thiobarbituric acid-reacting substances, indicators of stress in plants, were found to be lowest in P. vittata, which corresponds with its observed tolerance to arsenic. All three ferns responded differentially to arsenic exposure in terms of anti-oxidative defence. Higher levels of superoxide dismutase, catalase, and ascorbate peroxidase were observed in P. vittata than in P. ensiformis and N. exaltata, showing their active involvement in the arsenic detoxification mechanism. However, no significant increase was observed in either guaiacol peroxides or glutathione reductase in arsenic-treated P. vittata. Higher activity of anti-oxidative enzymes and lower thiobarbituric acid-reacting substances in arsenic-treated P. vittata correspond with its arsenic hyper-accumulation and no symptoms of toxicity.

Antioxidants↗

Divergent trajectories of genome architecture and chromosome evolution in ferns and angiosperms.

Ferns and angiosperms represent the two largest vascular plant lineages but exhibit striking genomic and ecological contrasts. We investigated whether differences in genome size, chromosome architecture, GC content, and stomatal traits reveal divergent evolutionary trajectories between these lineages. We assembled the most comprehensive dataset to date, integrating genome size, chromosome number and size, GC content, and stomatal traits for over 1100 fern species and compared it with an extensive angiosperm dataset. Ferns exhibited markedly lower variability and c. 16-fold slower rates of chromosome size evolution than angiosperms. A persistent positive relationship between genome size and chromosome number in ferns suggests limited cytological post-polyploid diploidization. While ferns generally possess larger stomata, this difference disappears after accounting for genome size, indicating that nucleotypic constraints, rather than lineage-specific physiology, dictate stomatal dimensions. Both groups share a unimodal GC-genome size relationship peaking at c. 14 Gbp. Larger fern chromosomes imply lower genome-wide recombination rates, potentially limiting genetic reshuffling and adaptive potential. Our results highlight fundamentally divergent evolutionary trajectories, likely shaped by meiotic symmetry in ferns and meiotic asymmetry, possibly centromere drive, and post-polyploid diploidization in angiosperms, defining the functional and genomic landscapes of these lineages across deep evolutionary timescales.

Genome, Plant↗

Phytotoxicity and phytoaccumulation of trivalent and hexavalent chromium in brake fern.

A recently recognized hyperaccumulator plant, Chinese brake fern (Pteris vittata), has been found to extract very high concentration of arsenic from arsenic-contaminated soil. Chromium usually is a coexisting contaminant with arsenic in most contaminated soils. The potential application of ferns for phytoremediation of chromium(III)- and chromium(VI)-contaminated soils and their phytotoxicity to ferns has not been studied before. In this study, chromium distribution and phytotoxicity at the plant and cellular levels of brake ferns were studied using chemical analyses and scanning electron microscopy. The results show a higher phytotoxicity of Cr from Cr(VI)-contaminated soil to Chinese brake fern than from Cr(III)-contaminated soil. Phytotoxicity symptoms included significant decreases both in fresh biomass weight and relative water content (RWC), and also in leaf chlorosis during the late stage of growing. At higher concentrations (500 mg/kg Cr[VI] and 1,000 mg/kg Cr[III] addition), plants showed reduction in the number of palisade and spongy parenchyma cells in leaves. Compared with other plant species reported for phytoremediation of Cr(VI)-contaminated soil, brake fern took up and accumulated significant amounts of Cr (up to 1,145 mg/kg in shoots and 5,717 mg/kg in roots) and did not die immediately from phytotoxicity. Our study suggests that Chinese brake fern is a potential candidate for phytoremediation of Cr(VI)-contaminated soils, even though plants showed severe phytotoxic symptoms at higher soil Cr concentrations.

Chromium↗

THE FERN REACTION OF CERVICAL MUCUS.

The fern reaction of endocervical mucus was tested in 100 non-pregnant and 200 pregnant women with a view to establishing the relationship of this fern pattern to normal ovarian cyclical changes and normal pregnancy. The fern appeared at day 7-9 of the cycle and disappeared at day 18-20. Twenty-eight of 100 pregnancies before 10 weeks yielded positive fern reactions; two of 100 pregnancies after 10 weeks yielded positive fern reactions. In the presence of a positive fern reaction, the administration of 10 mg. of norethynodrel (Enovid) daily for seven days led to prompt withdrawal bleeding in the absence of pregnancy. With a negative fern reaction, a spontaneous period ensues within 12 days in the absence of pregnancy. Two hundred women were tested for early pregnancy on this premise; in 198, a correct diagnosis was obtained.

Cervix Mucus↗

Exposure to the fern Onychium contiguum causes increase in lipid peroxidation and alters antioxidant status in urinary bladder.

The status of lipid peroxidation, glutathione, glutathione peroxidase, glutathione reductase, glutathione-S-transferase, superoxide dismutase, catalase, ascorbic acid, and alpha-tocopherol was studied in the urinary bladder of guinea pigs exposed to the carcinogenic fern Onychium contiguum. There was significant increase in the preformed lipid peroxides in the urinary bladders from fern exposed animals. The amount of lipid peroxides produced on incubation of urinary bladder homogenates with or without catalyst was significantly higher in the fern exposed animals. The concentrations of glutathione and alpha-tocopherol and the activities of glutathione reductase and catalase were elevated in the urinary bladders of the animals exposed to the fern. No effect was observed on the concentration of ascorbic acid and the activities of glutathione peroxidase, glutathione-S-transferase, and superoxide dismutase. It is summarized that the fern toxins increased oxidative stress in the urinary bladder and antioxidant status was altered. However, the altered antioxidant status did not provide protection from the toxin induced injury. Histopathology of the urinary bladder in the fern exposed animals revealed oedema, haemorrhages, and congestion. This is the first study to show increase in lipid peroxidation along with altered antioxidant status in the urinary bladder of fern exposed animals.

Animals↗

The ferning and nitrazine tests of amniotic fluid between 12 and 41 weeks gestation.

Most studies investigating the usefulness of ferning and nitrazine methods for detecting the presence of amniotic fluid following ruptured amniotic membranes have employed samples from vaginal pooling. Reported accuracies are 84 to 100% for the ferning test and 87 to 97% for the nitrazine test. Our study was designed to determine if these tests are applicable throughout gestation. Samples were obtained from amniocenteses between 14 and 42 weeks. Part I: Of 112 samples allowed to dry on a slide for 3 minutes only, 86.6% were ferning positive and 100% were nitrazine positive. Flame-drying increased the presence of ferning to 96.4%. Part II: 363 samples were allowed to dry completely for up to 10 minutes. All samples were ferning and nitrazine positive. Part III: 17 samples were tested for ferning and nitrazine at 1 hour, and again at 1 week. Sixty-six samples were tested within 1 week and again at 2 weeks. All samples remained ferning and nitrazine positive. Therefore optimal analysis of suspected amniotic fluid occurs when specimens are air-dried for 10 minutes. These tests may be reliably performed at gestations of 12 to 41 weeks.

Amniotic Fluid↗

Carcinogenicity of bracken fern (Pteridium esculentum) in New Zealand.

Bracken fern (Pteridium esculentum) was harvested from two sites LB and TB one of which (TB) was on a central North Island New Zealand farm where bovine enzootic haematuria (BEH) was known to occur. The fern was dried, ground and incorporated (25% w/w) into a pelleted diet and fed to female rats for a total of 162 days. Fifteen weeks later when the rats were autopsied it was found that numerous tumours, mainly of the ileum and urinary bladder were present in the animals fed the bracken fern from the TB site. Neoplasms were found in 85% of rats from the TB group compared with 11% in the LB group while only a single tumour (a haemangioma of the uterus) was observed in the controls. In all, there were neoplasms in 42, 5 and 1 organ/s from the TB, LB and control rat groups respectively (p<0.001). Analysis of the fern and pellets for ptaquiloside, the carcinogen in bracken fern, showed much higher levels present in the material from the TB site. There was 26 and 2270 microg ptaquiloside/g of dried fern and, for pellets from the same fern, 6.5 and 355 microg ptaquiloside/g of pellets, for one collection from the LB and TB sources respectively.

Journal Article↗

Ocular ferning during the menstrual cycle in healthy women.

PURPOSE: The aim of the study is to investigate whether tear ferning patterns change during different phases of the menstrual cycle. METHODS: The tear ferning test was performed on twelve normal women of childbearing age at three day intervals throughout one complete menstrual cycle. Serum hormone levels (progesterone, estrogen, testosterone) were measured. RESULTS: Eight women showed type I ferning, and the other four had type II ferning initially. These patterns did not change during the menstrual cycle. Serum hormone levels were all in the normal range. Since no change in ferning pattern was detected during the menstrual cycle, the ferning test can be done at any time in women. CONCLUSIONS: This study showed no effect of different menstrual cycle phases on tear ferning patterns.

Adult↗

Spatial location studies on the chemical composition of human tear ferns.

Tear ferns are the patterns formed when tears are allowed to dry on a clean smooth surface. The patterns have been categorised according to confluence of pattern, with less confluent ferns indicative of tear film abnormalities. Little is known about the mechanism that gives rise to this phenomenon. Previous work has concentrated on correlation of alterations in tear film composition on the ferning phenomenon. The present study takes a new approach by analysing the locations of chemical elements in the fern to understand how the ferns form. By means of SEM coupled with EDXA, Na, K and Cl were detected within the fern. Sulphur, indicative of the presence of macromolecules such as mucins and proteins, was only found at the very periphery of the dry tear drop. The results suggest that macromolecules do not play a direct role in the formation of the ferns. A model is put forward to explain the observation whereby proteins and mucins are precipitated selectively by increasing salt concentration at the drop margin.

Adult↗

Phytofiltration of arsenic from drinking water using arsenic-hyperaccumulating ferns.

Arsenic contamination of drinking water poses serious health risks to millions of people worldwide. Current technologies used to clean arsenic-contaminated water have significant drawbacks, such as high cost and generation of large volumes of toxic waste. In this study, we investigated the potential of using recently identified arsenic-hyperaccumulating ferns to remove arsenic from drinking water. Hydroponically cultivated, two arsenic-hyperaccumulating fern species (Pteris vittata and Pteris cretica cv. Mayii) and a nonaccumulating fern species (Nephrolepis exaltata) were suspended in water containing 73As-labeled arsenic with initial arsenic concentrations ranging from 20 to 500 microg L(-1). The efficiency of arsenic phytofiltration by these fern species was determined by continuously monitoring the depletion of 73As-labeled arsenic concentration in the water. With an initial water arsenic concentration of 200 microg L(-1), P. vittata reduced the arsenic concentration by 98.6% to 2.8 microg L(-1) in 24 h. When the initial water arsenic was 20 microg L(-1), P. vittata reduced the arsenic concentration to 7.2 microg L(-1) in 6 h and to 0.4 microg L(-1) in 24 h. At similar plant ages, both P. vittata and P. cretica had similar arsenic phytofiltration efficiency and were able to rapidly remove arsenic from water to achieve arsenic levels below the new drinking water limit of 10 microg L(-1). However, N. exaltata failed to reduce water arsenic to achieve the limit under the same experimental conditions. The significantly higher efficiency of arsenic phytofiltration by arsenic-hyperaccumulating fern species is associated with their ability to rapidly translocate absorbed arsenic from roots to shoots. The nonaccumulating fern N. exaltata was unable to translocate the absorbed arsenic to the shoots. Our results demonstrate that the arsenic-phytofiltration technique may provide the basis for a solar-powered hydroponic technique that enables small-scale cleanup of arsenic-contaminated drinking water.

Absorption↗

Tear ferning and other lacrimal tests in normal persons of different ages.

We investigated in normal persons of different ages ranging from 4 months to 85 years the frequency of different ferning types as well as the results of the Schirmer I test, break-up time and tear protein profile. With increasing age, ferning types of higher degree were significantly more frequent. Furthermore, a correlation was found between the ferning test and low Schirmer test values (p less than 0.05) as well as break-up time (p less than 0.1). No correlation exists between ferning the test and tear protein pattern. These results indicate that tear ferning is independent from single tear proteins but in correlation with secreted watery volume and plays some role in tear film stability. Furthermore, these normal values of ferning and protein pattern can be used as references for different ages.

Adolescent↗

Phylogenetic relationships of ferns deduced from rbcL gene sequence.

Part of the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) gene (rbcL) was sequenced from three fern species: Adiantum capillus-veneris, Botrypus strictus, and Osmunda cinnamomea var. fokiensis. This region included 1,333 base pairs, about 90% of the gene. Maximum likelihood analysis of the deduced amino acid sequences indicated that (1) Botrypus (Ophioglossaceae) clustered monophyletically with other ferns (Adiantum, Angiopteris, Osmunda); the closest relative to Botrypus among the three species was Osmunda, which did not support the hypothesis that the Ophioglossaceae are linked to the progymnosperm-seed plant lineage. (2) Eusporangiate ferns containing Botrypus (Ophioglossaceae) and Angiopteris (Marattiaceae) were a paraphyletic group. (3) Seed plants and the four fern species examined formed a monophyletic group, but ferns and bryophytes (liverwort) did not. Variations in rates of substitution for synonymous and nonsynonymous codons were found in fern lineages.

Amino Acid Sequence↗

Arsenic accumulation by two brake ferns growing on an arsenic mine and their potential in phytoremediation.

In an area near an arsenic mine in Hunan Province of south China, soils were often found with elevated arsenic levels. A field survey was conducted to determine arsenic accumulation in 8 Cretan brake ferns (Pteris cretica) and 16 Chinese brake ferns (Pteris vittata) growing on these soils. Three factors were evaluated: arsenic concentration in above ground parts (fronds), arsenic bioaccumulation factor (BF; ratio of arsenic in fronds to soil) and arsenic translocation factor (TF; ratio of arsenic in fronds to roots). Arsenic concentrations in the fronds of Chinese brake fern were 3-704 mg kg-1, the BFs were 0.06-7.43 and the TFs were 0.17-3.98, while those in Cretan brake fern were 149-694 mg kg-1, 1.34-6.62 and 1.00-2.61, respectively. Our survey showed that both ferns were capable of arsenic accumulation under field conditions. With most of the arsenic being accumulated in the fronds, these ferns have potential for use in phytoremediation of arsenic contaminated soils.

Arsenic↗