Occurrence among macrofungi of the bioconversion of glucosone to cortalcerone

Bioconversion of glucosone to cortalcerone, first discovered in Corticium caeruleum, was found to occur in 43 macrofungi out of the 315 which were screened (13.65%). Relations with taxonomy, ecology and biochemistry are discussed.     

Cortalcerone,a new antibiotic induced by external agents in Corticium caeruleum

The antibacterial compound produced by the fungus Corticium caeruleum in response to external agents was identified as 2-hydroxy-6H- 3-pyrone-2-carboxaldehyde hydrate, a new compound for which the trivial name cortalcerone is proposed.     

Genetic marking of glume color in <Emphasis Type="Italic">Triticum spelta</Emphasis> L. var. <Emphasis Type="Italic">caeruleum</Emphasis> using gliadins

Using gliadins as genetic markers, Triticum spelta L. var. caeruleum accessions were analyzed to identify genetic control of the dark color of glumes. The research material was F₂ and BC₁ plants from crosses between spelt accessions and white-glumed common wheat varieties. The segregation for glume color fitted the monogenic control of the trait. The electrophoretic analysis of gliadins in grains from the hybrid plants has shown that the Gli-Alj* allele in the T. spelta var. caeruleum accessions is linked to the allele for the dark (black) color of glumes at the Rg-A1 locus.     

Crystal structure of bifunctional aldos-2-ulose dehydratase/isomerase from Phanerochaete chrysosporium with the reaction intermediate ascopyrone M

The enzyme aldos-2-ulose dehydratase/isomerase (AUDH) participates in carbohydrate secondary metabolism, catalyzing the conversion of glucosone and 1,5-d-anhydrofructose to the secondary metabolites cortalcerone and microthecin, respectively. AUDH is a homo-dimeric enzyme with subunits of 900 amino acids. The subunit consists of a seven-bladed β-propeller domain, two cupin folds and a C-terminal lectin domain. AUDH contains a structural Zn²⁺ and Mg²⁺ located in loop regions and two zinc ions at the bottom of two putative active-site clefts in the propeller and the cupin domain, respectively. Catalysis is dependent on these two zinc ions, as their specific removal led to loss of enzymatic activity. The structure of the Zn²⁺-depleted enzyme is very similar to that of native AUDH, and structural changes upon metal removal as the cause for the catalytic deficiencies can be excluded. The complex with the reaction intermediate ascopyrone M shows binding of this compound at two different sites, with direct coordination to Zn²⁺ in the propeller domain and as second sphere ligand of the metal ion in the cupin domain. These observations suggest that the two reactions of AUDH might be catalyzed in two different active sites, about 60 Å apart. The dehydration reaction most likely follows an elimination mechanism, where Zn²⁺ acts as a Lewis acid polarizing the C2 keto group of 1,5-d-anhydrofructose. Abstraction of the proton at the C3 carbon atom and protonation of the leaving group, the C4 hydroxyl moiety, could potentially be catalyzed by the side chain of the suitably positioned residue His155.     

Identification and quantification of the glucose degradation product glucosone in peritoneal dialysis fluids by HPLC/DAD/MSMS

Glucose degradation products (GDPs) formed during heat sterilization of peritoneal dialysis (PD) fluids exert cytotoxic effects and promote the formation of advanced glycation end-products in the peritoneal cavity. As a result, long-term application of continuous ambulatory peritoneal dialysis is limited. The composition and concentration of GDPs in PD fluids must be known to evaluate their biological effects. The present study describes a targeted screening for novel GDPs in PD fluids. For this purpose, dicarbonyl compounds were converted with o-phenylenediamine to give the respective quinoxaline derivatives, which were selectively monitored by HPLC/diode array detector. Glucosone was thereby identified as a novel major GDP in PD fluids. Product identity was confirmed by LC/MSMS analysis using independently synthesized glucosone as a reference compound. Furthermore, a method was developed to quantify glucosone in PD fluids by HPLC/UV after derivatization with o-phenylenediamine. The method's limit of detection was 0.6 μM and the limit of quantitation 1.1 μM. A linear calibration curve was obtained between 1.1 and 113.9 μM (R² = 0.9999). Analyzed at three different concentration levels, recovery varied between 95.6% and 102.0%. The coefficient of variation ranged between 0.4% and 4.7%. The method was then applied to the measurement of glucosone in typical PD fluids. Glucosone levels in double chamber bag PD fluids varied between not detectable and 6.7 μM. In single chamber bag fluids, glucosone levels ranged between 28.7 and 40.7 μM.     

Predator recognition learning in rainbow darters Etheostoma caeruleum: specific learning and neophobia

This study investigated whether rainbow darters Etheostoma caeruleum can learn to recognize unfamiliar predators through the process of classical conditioning. Etheostoma caeruleum were conditioned by exposing them simultaneously to their chemical alarm cues (a known fright stimulus) and either chemical cues from larval ringed salamanders Ambystoma annulatum (unfamiliar predator) or to a blank water cue (control). Conditioning could result in either specific learning of the A. annulatum cue or increased wariness in response to any novel cue (neophobia). To distinguish between these possibilities, E. caeruleum in both groups were exposed to either A. annulatum cues alone or to chemical cues from western rat snakes Pantherophis obsoletus (novel cue) 2 days after conditioning. Treatment (A. annulatum‐conditioned) E. caeruleum, but not control E. caeruleum, showed a fright response when they were exposed to both the conditioned (A. annulatum) and novel (P. obsoletus) cues, indicating increased sensitivity to new stimuli. When E. caeruleum were retested after an additional 32 days, however, the fright response occurred only following exposure to the conditioned (A. annulatum) stimulus, indicating that specific learning of the A. annulatum cue had been retained whereas the neophobia to novel stimuli was temporary.     

Use of RAPD analysis to assess the threat of interspecific hybridization to the critically endangered <Emphasis Type="Italic">Polemonium kiushianum</Emphasis> in Japan

Polemonium kiushianum is a critically endangered species of which only eight populations exist in semi-natural grasslands of the Mt. Aso area of Kyushu, Japan. Habitat modification and the risk of hybridization with non-indigenous horticultural congeners, such as P. caeruleum subsp. caeruleum and P. caeruleum subsp. yezoense var. yezoense, pose increasing threats to P. kiushianum. To develop a DNA marker that distinguishes P. kiushianum from other Polemonium species, we performed random amplified polymorphic DNA (RAPD) analysis and selected an approximately 500-bp fragment generated by the OPB06 RAPD primer. In addition, we designed a primer pair, H11F/R, based on the nucleotide sequences of the fragments derived from P. caeruleum subsp. caeruleum and P. caeruleum subsp. yezoense var. yezoense. The results with the H11F/R primers indicated that most extant P. kiushianum plants in natural populations are not genetically contaminated by hybridization with non-indigenous horticultural species.     

Structure of a cyanoglucoside of Lithospermum purpureo-caeruleum

A new glucoside has been isolated from roots of Lithospermum officinale and L. caeruleum. It is not cyanogenetic although containing nitrile group in the aglycone moiety. Its structure has been elucidated by ¹H and ¹³C NMR spectroscopy as 6-O-β-d-glucopyranosyl-1-cyanomethylene-4, 5-dihydroxy-2-cyclohexene.     

Enzymatic production of microthecin by aldos-2-ulose dehydratase from 1,5-anhydro-D-fructose and stability studies of microthecin

Microthecin (2-hydroxy-2-(hydroxymethyl)-2H-pyran-3(6H)-one), which has anti-microbial activity, is one of the end products of an alternative glycogen and starch degrading pathway, the anhydrofructose pathway. It is formed from 1,5-anhydro-d-fructose (AF), a product of α-1,4-glucan lyase (EC 4.2.2.13) from glycogen and starch by aldos-2-ulose dehydratase (AUDH; EC 4. 2.1.110). In the current study, the yield and purity of microthecin was examined with respect to pH and buffers using AUDH purified from the fungus Phanerochaete chrysosporium. It was found that AUDH had a K_m of 5.4 and 4.9 mM towards AF and its natural substrate glucosone, respectively, while its V_max with AF was 5.5 times higher than that with glucosone. Higher molar conversion of 90% was obtained in a reactor with pH controlled around 5.0 and 24°C with de-ionized water as the solvent. Microthecin was found to be most stable in de-ionized water at a pH around 7.0 and stable in freeze-dried form. Under acidic conditions microthecin was degraded to 2′-furyl-2-hydroxymethylketone (FHMK). A ¹³C-NMR method was established to simultaneously monitor the reaction components including AF, microthecin and its intermediates. A mechanism of microthecin formation from AF via the intermediate ascopyrone M (APM) and the degradation of microthecin to FHMK are proposed based on the NMR data obtained.     

Egg viability decreases rapidly with time since ovulation in the rainbow darter Etheostoma caeruleum: implications for the costs of choosiness

Egg viability in the rainbow darter Etheostoma caeruleum, a fish apparently lacking female mate choice, was found to decline rapidly after ovulation. It was observed that the majority of a female's clutch may fail to hatch if she is prevented from mating for as little as 6 h. These data suggest that exercising female mate preferences may be selectively disfavoured in E. caeruleum due to the high cost of delaying mating.     

Male and female contributions to behavioral isolation in darters as a function of genetic distance and color distance

Determining which reproductive isolating barriers arise first between geographically isolated lineages is critical to understanding allopatric speciation. We examined behavioral isolation among four recently diverged allopatric species in the orangethroat darter clade (Etheostoma: Ceasia). We also examined behavioral isolation between each Ceasia species and the sympatric rainbow darter Etheostoma caeruleum. We asked (1) is behavioral isolation present between allopatric Ceasia species, and how does this compare to behavioral isolation with E. caeruleum, (2) does male color distance and/or genetic distance predict behavioral isolation between species, and (3) what are the relative contributions of female choice, male choice, and male competition to behavioral isolation? We found that behavioral isolation, genetic differentiation, and male color pattern differentiation were present between allopatric Ceasia species. Males, but not females, discerned between conspecific and heterospecific mates. Males also directed more aggression toward conspecific rival males. The high levels of behavioral isolation among Ceasia species showed no obvious pattern with genetic distance or male color distance. However, when the E. caeruleum was included in the analysis, an association between male aggression and male color distance was apparent. We discuss the possibility that reinforcement between Ceasia and E. caeruleum is driving behavioral isolation among allopatric Ceasia species.     

Analysis of Early Embryogenesis in Rainbow and Banded Darters (Percidae: Etheostoma) Reveals Asymmetric Postmating Barrier

This study examines early embryogenesis in two species of darters, Etheostoma caeruleum and E. zonale (Teleostei: Percidae), and their hybrids. Results document the course of ontogeny from fertilization until the onset of pigmented eyes. Comparing fertilization and developmental success of conspecific versus heterospecific crosses revealed an asymmetric postmating reproductive barrier. E. caeruleum eggs treated with E. zonale sperm exhibited fertilization and developmental success similar to that of conspecific crosses. In contrast, E. zonale eggs treated with E. caeruleum sperm exhibited reduced fertilization relative to conspecific crosses and abnormal development. Development in this latter cross was compromised at all stages, but appeared to be concentrated around epiboly, or cell migration. As epiboly represents the stage of ontogeny when zygotic genes of both species jointly contribute to embryogenesis, results provide insight into the genetic mechanisms underlying postmating barriers in Etheostoma. Finally, the observed asymmetry in fertilization success is consistent with predictions based on the behavioral ecology of these species. Etheostoma zonale spawn in the open water column, whereas E.␣caeruleum bury their eggs under the substrate. The observed fertilization barrier may have therefore resulted from selection favoring increased fertilization specificity in E. zonale.     

Reproductive isolation between two darter species is enhanced and asymmetric in sympatry

Robust reproductive isolation was found between the rainbow darter Etheostoma caeruleum and the orangethroat darter Etheostoma spectabile, as more offspring were produced when conspecific males and females were crossed as compared with heterospecific crosses. Furthermore, fewer eggs resulted from heterospecific crosses involving sympatric E. spectabile females than those using allopatric E. spectabile females, while a similar pattern was not observed in heterospecific crosses using E. caeruleum females. These results suggest that reinforcement, i.e. selection for pre‐zygotic reproductive barriers driven by reduced hybrid fitness, may have contributed to the evolution and maintenance of reproductive barriers between these potentially hybridizing species in sympatry.     

Pollen wall ontogeny in <Emphasis Type="Italic">Polemonium caeruleum</Emphasis> (Polemoniaceae) and suggested underlying mechanisms of development

By a detailed ontogenetic study of Polemonium caeruleum pollen, tracing each stage of development at high TEM resolution, we aim to understand the establishment of the pollen wall and to unravel the mechanisms underlying sporoderm development. The main steps of exine ontogeny in Polemonium caeruleum, observed in the microspore periplasmic space, are spherical units, gradually transforming into columns, then to rod-like units (procolumellae), the appearance of the initial tectum, growth of columellae in height and tectum in thickness and initial sporopollenin accumulation on them, the appearance of the endexine lamellae and of dark-contrasted particles on the tectum, the appearance of a sponge-like layer and of the intine in aperture sites, the appearance of the foot layer on the base of the sponge-like layer and of spinules on the tectum, and massive sporopollenin accumulation. This sequence of developmental events fits well to the sequence of self-assembling micellar mesophases. This gives (together with earlier findings and experimental exine simulations) strong evidence that genome and self-assembly probably share control of exine formation. It is highly probable that self-assembly is an intrinsic instrument of evolution.     

Characterization of a contemporaneous hybrid zone between two darter species (Etheostoma bison and E. caeruleum) in the Buffalo River System

Hybrid zones have long intrigued evolutionary biologists and provide a natural laboratory to explore the evolution of reproductive isolation (speciation). Molecular characterization of hybrid zone dynamics can provide insight into the strength of reproductive isolation as well as the underlying evolutionary processes shaping gene flow. Approximately one-third of darter species naturally hybridize making this species-rich North American freshwater teleost fish clade an ideal system to investigate the extent and direction of hybridization. The objective of this study was to use diagnostic microsatellite markers to calculate genetic hybrid index scores of two syntopic, but distantly related darter species, Etheostoma bison and Etheostoma caeruleum. A combination of hybrid index scores, assignment tests, and mitochondrial haplotype profiles uncovered mixed ancestry in approximately 6 % of sampled adult individuals, supporting contemporaneous hybridization that was previously undocumented in E. bison. Moreover, hybrids were not limited to the F₁ generation, but encompassed the entire suite of hybrid categories (F₁, F₂ and backcross hybrids). The low number of hybrids assigned to each hybrid category represents a bimodal hybrid zone, suggesting reproductive isolation is strong (but incomplete) and also advocates for the ability of hybrids to produce second-generation hybrids and backcross into both parental species, mediating introgression across species boundaries. To this end, cytonuclear profiles of the sampled parental species and hybrids were consistent with bidirectional gene flow, although there was an overall trend of asymmetric hybridization between E. caeruleum females and E. bison males. The spatiotemporal variation in hybridization rates and resulting cytonuclear patterns expanded on in this study provide a comparative genetic framework on which future studies can begin to elucidate the underlying processes that not only generate a mosaic hybrid zone, but maintain the distinctness of species in the face of gene flow.     

d-Glucosone and l-Sorbosone, Putative Intermediates of l-Ascorbic Acid Biosynthesis in Detached Bean and Spinach Leaves

d-[6-¹⁴C]Glucosone that had been prepared enzymically from d-[6-¹⁴C]glucose was used to compare relative efficiencies of these two sugars for l-ascorbic acid (AA) biosynthesis in detached bean (Phaseolus vulgaris L., cv California small white) apices and 4-week-old spinach (Spinacia oleracea L., cv Giant Noble) leaves. At tracer concentration, ¹⁴C from glucosone was utilized by spinach leaves for AA biosynthesis much more effectively than glucose. Carbon-14 from [6-¹⁴C]glucose underwent considerable redistribution during AA formation, whereas ¹⁴C from [6-¹⁴C]glucosone remained almost totally in carbon 6 of AA. In other experiments with spinach leaves, l-[U-¹⁴C]sorbosone was found to be equivalent to [6-¹⁴C]glucose as a source of ¹⁴C for AA. In the presence of 0.1% d-glucosone, conversion of [6-¹⁴C] glucose into labeled AA was greatly repressed. In a comparable experiment with l-sorbosone replacing d-glucosone, the effect was much less. The experiments described here give substance to the proposal that d-glucosone and l-sorbosone are putative intermediates in the conversion of d-glucose to AA in higher plants.     

Phylogeography and post-glacial colonization patterns of the rainbow darter, Etheostoma caeruleum (Teleostei: Percidae)

Aim To examine the effects of historical climate change and drainage isolation on the distribution of mitochondrial DNA cytochrome b genetic variation within the rainbow darter, Etheostoma caeruleum (Percidae: Etheostomatinae). Location Eastern North American streams including tributaries to the Mississippi River, Great Lakes, Potomac River and Hudson Bay drainages. Methods Parsimony analyses, Bayesian analyses and haplotype networks of mitochondrial DNA sequences. Results Four major clades were recovered from sampled populations of E. caeruleum. Three of four clades are distributed in the western portion of the species’ range (primarily west of the Mississippi River). Samples from this region do not form a monophyletic group, and sequences often vary greatly between samples from adjacent stream systems (up to 7.2% divergence). A basal clade includes samples from the White River system in the Ozark Highlands. The northern Ozarks–upper Midwest clade includes samples from Missouri River tributaries and the upper Midwest (Hudson Bay, upper Mississippi River, and western Lake Michigan drainage). The eastern clade is composed of individuals from the Ohio River, Great Lakes and Potomac River. The Mississippi River corridor clade includes samples from middle and lower Mississippi River tributaries. Main conclusions The four major clades of E. caeruleum are deep allopatric lineages with well‐defined boundaries and have additional phylogeographical structure within each clade. The Ozark Highlands have the greatest levels of diversity relative to distributional area, with marked cytochrome b subdivisions between adjacent stream systems. Samples from previously glaciated areas do not have a subset of the cytochrome b diversity found in unglaciated areas, but four separate source areas are identified based on phylogenetic analyses. Dispersal into previously glaciated areas followed several known glacial outlets and, based on sequence divergence between populations, may have occurred during different glacial or interglacial stages. The disjunct distribution and cytochrome b pattern of E. caeruleum in the Mississippi River corridor clade is consistent with late Pleistocene and Recent changes in the course and characteristics of the middle and lower Mississippi River. Phylogeographical boundaries between clades of E. caeruleum correspond to independent sources of biogeographical information and provide insight into historical stream drainage relationships, post‐glacial colonization and drainage isolation patterns.     

Benthic freshwater cyanobacteria as indicators of anthropogenic pressures

Freshwater cyanobacteria are hardly used as biological indicators of anthropogenic pressures, possibly for two main reasons: (a) their response to anthropogenic pressures is often poorly known; (b) reliably identifying cyanobacteria species is a challenge for technicians and researchers. We assessed the usefulness of cyanobacteria species as biological indicators of two human stressors: the high orthophosphate input from urban wastewaters; the high nitrate concentration produced by agricultural land use. We analysed variation in benthic cyanobacterial assemblages at 85 sites in South-Central Spain as a response to eight environmental variables: pH, conductivity, temperature, altitude, nitrate, orthophosphate, irrigation land use and non-irrigation land use.Results revealed that conductivity was the main environmental factor that contributed to differences between assemblages. Orthophosphate was more influential for community composition than nitrate. Changes in species composition related to human pressures suggested that some cyanobacteria species (e.g. Nostoc verrucosum, Phormidium autumnale, Plectonema tomasinianum, Rivularia haematites, Tolypothrix distorta) could be useful tools for the bioindication of anthropogenic pressures; while others species provide more information about natural physicochemical reference conditions (Nostoc caeruleum, Phormidium fonticola). Further research into cyanobacteria and macroalgae assemblages in different impacted scenarios could help improve macrophyte indices.     

Insect pollinators show constancy for different flower traits between the most- and less-preferred plants: a case study of the long-proboscid tangle-veined fly

• The coevolution of insect pollinators and their host plants is a typical example of natural selection; however, how insect pollinators avoid overdependence on one peculiar plant remains unclear. As most insect pollinators exhibit a diet breadth when showing flower constancy, determining the difference and similarity of most and less preferred flowers by insect pollinators may be helpful to understand their trade-off between flower constancy and overdependence.
• This was addressed using the long-proboscid tangle-veined fly (Nemetrinus spp.). Dietary investigation indicates that the flies show constancy for the morphological characteristic of the Delphinium caeruleum, which is the most preferred plant for this Nemestrinidae fly that has blue, long-tubed flowers.
• In a colour selection experiment, focal individuals showed obvious preference for white, which is the colour of less preferred flowers by the fly in the natural environment. In a scent selection experiment, focal individuals showed obvious preference for D. caeruleum and Dracocephalum heterophyllum but avoidance to Dasiphora fruticosa and Dasiphora davurica. This indicates that long-proboscid tangle-veined flies can forage on other flowers, despite the existing constancy for D. caeruleum, as long as they do not hate the scent. It seems that long-proboscid tangle-veined flies can maximise foraging efficiency by showing constancy for the morphological characteristic of the most preferred plant and for the scent and colour of less-preferred plants.
• The trade-off of long-proboscid tangle-veined flies in the selection of nectar sources may be an adaptation to the risk of overdependence on one plant in evolution.

     

Reproductive biology of the Red List species Polemonium caeruleum (Polemoniaceae)

The majority of flowering plants, including many rare and threatened species, are pollinated by animals, but little is known of pollination and breeding systems of many endangered species. Polemonium caeruleum (Polemoniaceae) is a red‐listed species and is regarded as dichogamous, self‐compatible and bee pollinated. However, some studies show that it is visited by a vast assemblage of anthophilous insects from many taxonomic orders and that breeding systems vary greatly between closely related taxa of this genus. Over a period of 3 years we investigated breeding system, dichogamy, nectar secretion and composition, insect visitations and pollen loads in flowers of P. caeruleum in north‐eastern Poland to determine whether the reproductive biology of the plant explains its rarity. Contrary to published data, our study plants were self‐incompatible and showed a high degree of outcrossing. Our experimental work confirmed the occurrence of protandry in this species, revealed that nectar is sucrose‐dominant and proline‐rich and, for the first time for Polemoniaceae, that nectar secretion and nectar sugar concentration in flowers of P. caeruleum is female‐biased. Although flowers were visited by at least 39 species of insects from five taxonomic orders, overall the plant exhibited many characters associated with bee pollination, and analysis of insect performance showed that bumblebees and honeybees are the key pollinators; occasionally hoverflies and butterflies may also be involved. We conclude that, in terms of pollination system, P. caeruleum demonstrates high apparent generalization, but low realized generalization, and is a functional specialist, as most pollinators belong to a single functional group (guild). Its conservation status, at least in our study population, cannot be explained in terms of the biological properties of its breeding or pollination systems; rather, the present decline of the species is caused by habitat loss. However, if this process and bumblebee decline in Europe continue, P. caeruleum populations may diminish in numbers and density and, owing to the self‐incompatibility of the species, quickly become severely pollen‐limited, thereby accelerating further local extinctions. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173 , 92–107.