Morphological and molecular characterization of cyanobacteria from a Brazilian facultative wastewater stabilization pond and evaluation of microcystin production

The cyanobacterial population in the Cajati waste stabilization pond system (WSP) from São Paulo State, Brazil was assessed by cell isolation and direct microscope counting techniques. Ten strains, belonging to five genera (Synechococcus, Merismopedia, Leptolyngbya, Limnothrix, and Nostoc), were isolated and identified by morphological and molecular analyses. Morphological identification of the isolated strains was congruent with their phylogenetic analyses based on 16S rDNA gene sequences. Six cyanobacterial genera (Synechocystis, Aphanocapsa, Merismopedia, Lyngbya, Phormidium, and Pseudanabaena) were identified by direct microscope inspection. Both techniques were complementary, since, of the six genera identified by direct microscopic inspection, only Merismopedia was isolated, and the four other isolated genera were not detected by direct inspection. Direct microscope counting of preserved cells showed that cyanobacteria were the dominant members (>90%) of the phytoplankton community during both periods evaluated (summer and autumn). ELISA tests specific for hepatotoxic microcystins gave positive results for six strains (Synechococcus CENA108, Merismopedia CENA106, Leptolyngbya CENA103, Leptolyngbya CENA112, Limnothrix CENA109, and Limnothrix CENA110), and for wastewater samples collected from raw influent (3.70 μg microcystins/l) and treated effluent (3.74 μg microcystins/l) in summer. Our findings indicate that toxic cyanobacteria in WSP systems are of concern, since the treated effluent containing cyanotoxins will be discharged into rivers, irrigation channels, estuaries, or reservoirs, and can affect human and animal health.     

Clinical Characteristics and Outcomes in Patients with Pulmonary Blastomycosis

Background  Blastomycosis is an uncommon granulomatous infection caused by the thermally dimorphic fungus Blastomyces dermatitidis. The most frequent clinical infections involve the lung, skin, and bone. Pulmonary manifestations range from asymptomatic self-limited infection to severe diffuse pneumonia causing respiratory failure. Objectives  To establish the clinical characteristics and outcomes of patients with pulmonary blastomycosis diagnosed at hospitals in Manitoba and northwestern Ontario, Canada. Methods  A retrospective review of medical records was done for 318 patients with blastomycosis in these regions. Results  The majority of patients were Caucasian (198 (62.5%) patients), male (193 (61%) patients), and residents of Ontario (209 (65.7%) patients). Most patients were treated in an inpatient hospital ward (266 (84%) patients) and survived (294 (92%) patients). Pulmonary involvement, either alone or associated with other sites, was present in 296 (93%) of the 318 patients; 22 (7%) patients had no evidence of pulmonary blastomycosis. The majority of patients had localized lung disease (1–3 quadrants on chest radiograph involved; 225 (82%) patients). Of 294 (92%) patients requiring hospitalization, 266 (90%) patients received all inpatient care on a general medical ward; 28 (10%) patients received some care in the intensive care unit (ICU). Factors associated with ICU admission included diffuse pulmonary disease (four quadrants involved on chest radiograph), diabetes, and prior use of antimicrobial therapy. Twenty-four (8%) patients died, and multivariate analysis showed that older age and Aboriginal ethnicity were the significant risk factors for death from blastomycosis. Conclusion  Blastomycosis is a cause of serious, potentially life-threatening pulmonary infection in this geographic region. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Identification and characterization of <Emphasis Type="Italic">Gluconacetobacter diazotrophicus</Emphasis> mutants defective in the solubilization of phosphorus and zinc

Gluconacetobacter diazotrophicus is a plant-growth-promoting bacterium, which is able to colonize sugarcane and other plant species of economic importance. The potentially beneficial effects promoted by this bacterium on plants are nitrogen-fixation, production of phythormones, action against pathogens and mineral nutrient solubilization. In this study, the molecular mechanisms associated with phosphorus and zinc solubilization were analyzed. A transposon mutant library was constructed and screened to select for mutants defective for phosphorous [Ca₅(PO₄)₃OH] and zinc (ZnO) solubilization. A total of five mutants were identified in each screen. Both screenings, performed independently, allowed to select the same mutants. The interrupted gene in each mutant was identified by sequencing and the results demonstrate that the production of gluconic acid is a required pathway for solubilization of such nutrients in G. diazotrophicus. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. A. C. Intorne and M. V. V. de Oliveira contributed equally to this work.     

Developmental plasticity and biomechanics of treelets and lianas in Manihot aff. quinquepartita (Euphorbiaceae): a branch-angle climber of French Guiana

Background and Aims

Most tropical lianas have specialized organs of attachment such as twining stems, hooks or tendrils but some do not. Many climbers also have an early self-supporting phase of growth and in some species this can produce treelet-sized individuals. This study focuses on how a liana can climb without specialized attachment organs and how biomechanical properties of the stem are modulated between self-supporting treelets and canopy-climbing lianas.

Methods

Biomechanics and stem development were investigated in self-supporting to climbing individuals of Manihot aff. quinquepartita (Euphorbiaceae) from tropical rain forest at Saül, central French Guiana. Bending tests were carried out close to the site of growth. Mechanical properties, including Young''s elastic modulus, were observed with reference to habit type and changes in stem anatomy during development.

Key Results

This liana species can show a remarkably long phase of self-supporting growth as treelets with stiff, juvenile wood characterizing the branches and main stem. During the early phase of climbing, stiff but unstable stem segments are loosely held in a vertical position to host plants via petiole bases. The stiffest stems – those having the highest values of Young''s modulus measured in bending – belonged to young, leaning and climbing stems. Only when climbing stems are securely anchored into the surrounding vegetation by a system of wide-angled branches, does the plant develop highly flexible stem properties. As in many specialized lianas, the change in stiffness is linked to the development of wood with numerous large vessels and thin-walled fibres.

Conclusions

Some angiosperms can develop highly effective climbing behaviour and specialized flexible stems without highly specialized organs of attachment. This is linked to a high degree of developmental plasticity in early stages of growth. Young individuals in either open or closed marginal forest conditions can grow as substantial treelets or as leaning/climbing plants, depending on the availability of host supports. The species of liana studied differs both in terms of development and biomechanics from many other lianas that climb via twining, tendrils or other specialized attachment organs.Key words: Biomechanics, bending, developmental plasticity, French Guiana, liana, Manihot aff. quinquepartita (Euphorbiaceae), treelet, branch angle climber, Young''s modulus     

Flowering phenology of invasive alien plant species compared with native species in three Mediterranean-type ecosystems

Background and Aims

Flowering phenology is a potentially important component of success of alien species, since elevated fecundity may enhance invasiveness. The flowering patterns of invasive alien plant species and related natives were studied in three regions with Mediterranean-type climate: California, Spain and South Africa''s Cape region.

Methods

A total of 227 invasive–native pairs were compared for seven character types across the regions, with each pair selected on the basis that they shared the same habitat type within a region, had a common growth form and pollination type, and belonged to the same family or genus.

Key Results

Invasive alien plant species have different patterns of flowering phenology from native species in the three regions. Whether the alien species flower earlier, later or at the same time as natives depends on the climatic regime in the native range of the aliens and the proportion of species in the invasive floras originating from different regions. Species invading at least two of the regions displayed the same flowering pattern, showing that flowering phenology is a conservative trait. Invasive species with native ranges in temperate climates flower earlier than natives, those from Mediterranean-type climates at the same time, and species from tropical climates flower later. In California, where the proportion of invaders from the Mediterranean Basin is high, the flowering pattern did not differ between invasive and native species, whereas in Spain the high proportion of tropical species results in a later flowering than natives, and in the Cape region early flowering than natives was the result of a high proportion of temperate invaders.

Conclusions

Observed patterns are due to the human-induced sympatry of species with different evolutionary histories whose flowering phenology evolved under different climatic regimes. The severity of the main abiotic filters imposed by the invaded regions (e.g. summer drought) has not been strong enough (yet) to shift the flowering pattern of invasive species to correspond with that of native relatives. It does, however, determine the length of the flowering season and the type of habitat invaded by summer-flowering aliens. Results suggest different implications for impacts at evolutionary time scales among the three regions.Key words: Biological invasions, flowering phenology, genetic inertia, Cape Floristic Region, California, Spain, Mediterranean-type ecosystems, water availability, climatic origin     

Calibrating the Tree of Life: fossils, molecules and evolutionary timescales

Background

Molecular dating has gained ever-increasing interest since the molecular clock hypothesis was proposed in the 1960s. Molecular dating provides detailed temporal frameworks for divergence events in phylogenetic trees, allowing diverse evolutionary questions to be addressed. The key aspect of the molecular clock hypothesis, namely that differences in DNA or protein sequence between two species are proportional to the time elapsed since they diverged, was soon shown to be untenable. Other approaches were proposed to take into account rate heterogeneity among lineages, but the calibration process, by which relative times are transformed into absolute ages, has received little attention until recently. New methods have now been proposed to resolve potential sources of error associated with the calibration of phylogenetic trees, particularly those involving use of the fossil record.

Scope and Conclusions

The use of the fossil record as a source of independent information in the calibration process is the main focus of this paper; other sources of calibration information are also discussed. Particularly error-prone aspects of fossil calibration are identified, such as fossil dating, the phylogenetic placement of the fossil and the incompleteness of the fossil record. Methods proposed to tackle one or more of these potential error sources are discussed (e.g. fossil cross-validation, prior distribution of calibration points and confidence intervals on the fossil record). In conclusion, the fossil record remains the most reliable source of information for the calibration of phylogenetic trees, although associated assumptions and potential bias must be taken into account.     

Molecular markers reveal no genetic differentiation between Myrica rivas-martinezii and M. faya (Myricaceae)

Background and Aims

Myrica rivas-martinezii is a critically endangered endemic of the laurel forest of the Canary Islands and co-occurs very close to M. faya. Some authors suggest that M. rivas-martinezii and M. faya are two morphs of the same species, so molecular markers were used to estimate the levels and structuring of genetic variation within and among natural populations in order to evaluate genetic relationships between these two congeners.

Methods

Six polymorphic microsatellite (simple sequence repeat, SSR) markers were used to determine the genetic diversity and the genetic relationship between both Myrica species.

Key Results

Most of the natural populations analysed were in Hardy–Weinberg equilibrium for both taxa. Analysis of molecular variance (AMOVA) for both species revealed that most of the genetic variability detected was contained within populations (92·48 and 85·91 % for M. faya and M. rivas-martinezii, respectively), which it is consistent with outcrossing and dioecious plants. Estimates of interpopulation genetic variation, calculated from F_ST and G′_ST, were quite low in the two taxa, and these values did not increase substantially when M. rivas-martinezii and M. faya populations were compared. The UPGMA dendrogram based on Nei''s genetic distance clustered the populations by their island origin, independently of taxon. In fact, the mixture of individuals of both taxa did not appreciably disrupt the intrapopulational genetic cohesion, and only 3·76 % variation existed between species.

Conclusions

All the results obtained using molecular markers indicate clearly that both taxa share the same genetic pool, and they are probably the same taxa. Considering that M. rivas-martinezii is classified as at risk of extinction, there should be a change of focus of the current management actions for the conservation of this putatively endangered Canarian endemic.Key words: Canary Islands, conservation genetics, microsatellites, Myrica rivas-martinezii, Myrica faya, plant conservation     

Mycorrhization and phosphorus nutrition affect water relations and CAM induction by drought in seedlings of Clusia minor

Background and Aims

Crassulacean acid metabolism (CAM) is currently viewed as an adaptation to water deficit. In plants of Clusia minor, which grow mostly on acidic, P-deficient soils, CAM is induced by water deficit. The symbiosis between plants and mycorrhizal fungi alleviates the symptoms of P deficiency and may influence drought resistance. Therefore, the effect of P supply, modified by three different experimental treatments, on the induction of CAM by drought in C. minor was investigated to test the hypothesis that P deficiency will produce greater CAM activity and, in addition, that treatment will modify drought tolerance.

Methods

Seedlings were grown in forest soil sterilized and inoculated with Scutellospora fulgida (SF treatment), sterilized and supplemented with P (Ph treatment) or non-sterilized and containing native mycorrhizae (Nat treatment). Leaf turgor potential (ψ_T) was determined psychrometrically, and CAM activity as nocturnal acid accumulation (ΔH⁺) by titration of dawn and dusk leaf sap.

Key Results

Plant mass and P content were higher in SF and Ph than in Nat seedlings. After 21 d of water deficit, ψ_T increased in SF, decreased in Ph and remained unchanged in Nat, and, after 7 and 14 d of water deficit, ΔH⁺ in Nat was three times higher than at the beginning of drought, whereas in SF and Ph ΔH⁺ was lower than on day 0.

Conclusions

P deficiency in Nat seedlings was ameliorated by inoculation or P addition. The SF and Nat seedlings showed greater tolerance of drought than Ph. P deficiency promoted the induction of CAM by drought in Nat seedlings, whereas P fertilization and mycorrhization did not. Nocturnal acid accumulation was highly and negatively correlated with plant P and N contents, indicating that P and N deficiencies are promoters of CAM in droughted plants of C. minor.Key words: Clusia minor, crassulacean acid metabolism, CAM, mycorrhiza, drought, phosphorus deficiency, nitrogen–water relations     

Inherited variability in multiple traits determines fitness in populations of an annual legume from contrasting latitudinal origins

Background and Aims

Variation in fitness depends on corresponding variation in multiple traits which have both genetically controlled and plastic components. These traits are subjected to varying degrees of local adaptation in specific populations and, consequently, are genetically controlled to different extents. In this study it is hypothesized that modulation of different traits would have contrasting relevance for the fitness of populations of diverse origins. Specifically, assuming that environmental pressures vary across a latitudinal gradient, it is suggested that inherited variation in traits differentially determines fitness in annual Lupinus angustifolius populations from contrasting latitudinal origins in western Spain.

Methods

Seeds of L. angustifolius from three contrasting origins were grown in a common garden. Traits related to more plastic vegetative growth and more genetically conserved phenology were measured, together with estimates of reproductive success. Fitness was estimated by the number of viable seeds per plant. Structural Equation Models were used to infer causal relationships among multiple traits and fitness, separating the direct and indirect effects of morphological, phenological and reproductive traits.

Key Results

Phenological, vegetative and reproductive traits accounted for most of the fitness variation. Fitness was highest in plants of southernmost origin, mainly due to earlier flowering. Fitness within each seed origin was controlled by variation in different traits. Southern origin plants that grew to a larger size achieved higher fitness. However, plant size in plants of northernmost origin was irrelevant, but early flowering promoted higher fitness. Variation in fruit and seed set had a greater effect on the fitness of plants of central origin than phenological and size variation.

Conclusions

It is concluded that modulation of a functional trait can be relevant to fitness in a given population (i.e. affecting intensity and direction), but irrelevant in other populations. This points to the need to consider integrated phenotypes when trying to unravel local adaptation effects over single traits.Key words: Lupinus, Structural Equation Models, fitness, phenology, functional traits, reproductive success, SLA, seed size     

Evolution and biogeography of Centaurea section Acrocentron inferred from nuclear and plastid DNA sequence analyses

Background and Aims

Section Acrocentron of the genus Centaurea is one of the largest sections of Centaurea with approx. 100 species. The geographic distribution, centred in the Mediterranean, makes it an excellent example for studies of the biogeographic history of this biodiversity-rich region.

Methods

Plastid (trnH-psbA) and nuclear (ITS and ETS) DNA sequence analysis was used for phylogenetic reconstruction. Ancestral biogeographic patterns were inferred by dispersal-vicariance analysis (DIVA).

Key Results

The resulting phylogeny has implications for the sectional classification of Acrocentron and confirms merging sect. Chamaecyanus into Acrocentron as a subsection. Previous suggestions of an eastern Mediterranean origin of the group are confirmed. The main centres of diversification established in previous studies are now strongly supported. Expansion of the group in two different radiations that followed patently diverse paths is inferred.

Conclusions

Radiation followed two waves, widely separated in time scale. The oldest one, from Turkey to Greece and the northern Balkans and then to North Africa and Iberia, should be dated at the end of the Miocene in the Messinian period. It reached the Iberian Peninsula from the south, following a route that is landmarked by several relictic taxa in Sicily and North Africa. A later radiation during the Holocene interglacial periods followed, involving species from the north of the Balkan Peninsula, along a Eurasian pathway running from Central Iberia to the steppes of Kazakhstan. A generalized pattern of reticulation is also evident from the results, indicating past contacts between presently separated species. Molecular data also confirmed the extent of hybridization within Acrocentron and were successful in reconstructing the paleogeography of the section.Key words: Centaurea sect. Acrocentron, Cardueae, dispersal-vicariance, ETS, ITS, Mediterranean, phylogeny, psbA-trnH, radiation, reticulation