Field relationships between scytonemin density,growth, and irradiance in cyanobacteria occurring in low illumination regimes

In situ measurements of ultraviolet (UV) irradiance, carbon fixation, and scytonemin pigmentation were made on Scytonema populations from contrasting localities in England. Significant negative correlations were obtained between the following variate pairs: pigmentation and UV irradiance; pigmentation and carbon fixation rate. A significant positive correlation was found between pigmentation and sheath thickness.The negative correlation between pigmentation and UV irradiance was unexpected and appeared contrary to the results of previous studies, which indicated a positive correlation between the variates and the recognition of scytonemin as a radiation shield. However, by considering how radiation damage is related to cell division and the water relations of the sites investigated, it was shown that scytonemin is still functioning as a radiation shield, even in shaded sites. Rivularia colonies produced scytonemin only upon their upper, sun-exposed surfaces and were positively correlated with UV irradiance. This paper also describes the successful use of some new and inexpensive techniques to measure pigments in cyanobacterium sheaths, and integrated in situ UV-irradiance.     

NUCLEAR RIBOSOMAL DNA PHYLOGENY OF HALIMEDALES REVEALS SEVERAL POLYPHYLETIC GENERA

Halimedales (Chlorophyta) are widely distributed in reefs and lagoons throughout the tropics and subtropics. One genus, Chlorodesmis, consists of simple, branching siphons that persist into the mature sexual phase. In most other genera, only the juvenile stages exhibit such simple anatomy. Their mature, often‐calcified thalli possess complex siphonal anatomy and differentiation depending on the location in the thallus. We obtained a nuclear ribosomal DNA phylogeny encompassing many genera in order to unravel the evolution of this complexity. Results pinpoint Caulerpa as outgroup to a clade with all other genera included in this study. The first dichotomy separates Halimeda from the remaining genera. Distinct gametangial anatomy defines these two principal clades. Within the remainder, Tydemania and its sister Flabellia are nearest neighbors to a clade containing Rhipocephalus, Udotea, Penicillus and Chlorodesmis. The latter three genera are polyphyletic. Therefore, character states of thallus superstructure defining these genera appear to be homoplasies. Chlorodesmis represents a case of neoteny where the juvenile stage persists into reproductive maturity. We will evaluate which morphological characters do change state in agreement with the molecular phylogeny and can, therefore, serve as diagnostic characters for a more natural classification.     

Bioinformatic,phylogenetic and chemical analysis of the UV-absorbing compounds scytonemin and mycosporine-like amino acids from the microbial mat communities of Shark Bay,Australia

Shark Bay, Western Australia is a World Heritage area with extensive microbial mats and stromatolites. Microbial communities that comprise these mats have developed a range of mitigation strategies against changing levels of photosynthetically active and ultraviolet radiation, including the ability to biosynthesise the UV-absorbing natural products scytonemin and mycosporine-like amino acids (MAAs). To this end, the distribution of photoprotective pigments within Shark Bay microbial mats was delineated in the present study. This involved amplicon sequencing of bacterial 16S rDNA from communities at the surface and subsurface in three distinct mat types (smooth, pustular and tufted), and correlating this data with the chemical and molecular distribution of scytonemin and MAAs. Employing UV spectroscopy and MS/MS fragmentation, mycosporine-glycine, asterina and an unknown MAA were identified based on typical fragmentation patterns. Marker genes for scytonemin and MAA production (scyC and mysC) were amplified from microbial mat DNA and placed into phylogenetic context against a broad screen throughout 363 cyanobacterial genomes. Results indicate that occurrence of UV screening compounds is associated with the upper layer of Shark Bay microbial mats, and the occurrence of scytonemin is closely dependent on the abundance of cyanobacteria.     

MORPHOLOGY,LIFE HISTORY,AND MOLECULAR PHYLOGENY OF STSCHAPOVIA FLAGELLARIS (TILOPTERIDALES,PHAEOPHYCEAE) AND THE ERECTION OF THE STSCHAPOVIACEAE FAM. NOV.1

The phenology, life history, ultrastructure of reproductive structures, and molecular phylogeny using rbcL and rDNA (5.8S, internal transcribed spacer 2, and partial 26S) gene sequences of Stschapovia flagellaris, endemic to the northwestern Pacific Ocean, were studied. This species was first classified in the order Delamareales together with Delamarea, Coelocladia, and Cladothele. Those three genera, however, were later transferred to Dictyosiphonales, whereas the systematic position of Stschapovia remained unclear. At Abashiri, Hokkaido, Japan, the species regenerated by forming a new erect thallus from a perennial crustose holdfast or by presumably parthenogenetic development of eggs released from the erect thallus. There was no alternation of generations. In winter, the monoecious erect thallus formed reproductive structures (i.e. plurilocular antheridia and oogonia) in the thickened part of the thallus. Sperm had a chloroplast with an eyespot and a long anterior and short posterior flagellum. Eggs contained numerous disc‐shaped chloroplasts, physodes, and vacuoles. Neither sexual attraction of the presumptive sperm by eggs nor their sexual fusion was observed. Molecular phylogenetic analyses revealed the closest phylogenetic relationship between Stschapovia and Halosiphonaceae, and they grouped with Phyllariaceae and Tilopteridaceae (Tilopteridales s. s.). Stschapovia and Tilopteridaceae have several important morphological similarities: chloroplasts lacking pyrenoids, lack of sexual reproduction despite the release of obvious sperm, occurrence of monoecious gametophytes, and similarity in the early developmental pattern of the erect thallus. In conclusion, we propose the establishment of the new family Stschapoviaceae to accommodate Stschapovia and the placement of the family in the order Tilopteridales together with Tilopteridaceae, Halosiphonaceae, and Phyllariaceae.     

Screening of different algae for green synthesis of gold nanoparticles

The cyanobacteria Phormidium valderianum, P. tenue and Microcoleus chthonoplastes and the green algae Rhizoclonium fontinale, Ulva intestinalis, Chara zeylanica and Pithophora oedogoniana were exposed to hydrogen tetrachloroaurate solution and were screened for their suitability for producing nano‐gold. All three cyanobacteria genera and two of the green algae (Rhizoclonium fontinale and Ulva intestinalis) produced gold nanoparticles intracellularly, confirmed by purple colouration of the thallus within 72?h of treatment at 20°C. Extracted nanoparticle solutions were examined by UV‐vis spectroscopy, transmission electron microscopy (TEM) and X‐ray diffractometry (XRD). XRD confirmed the reduction of Au (III) to Au (0). UV‐vis spectroscopy and TEM studies indicated the production of nanoparticles having different shapes and sizes. Phormidium valderianum synthesized mostly spherical nanoparticles, along with hexagonal and triangular nanoparticles, at basic and neutral pHs (pH 9 and pH 7, respectively). Medicinally important gold nanorods were synthesized (together with gold nanospheres) only by P. valderianum at acidic pH (pH 5); this was initially determined by two surface plasmon bands in UV‐vis spectroscopy and later confirmed by TEM. Spherical to somewhat irregular particles were produced by P. tenue and Ulva intestinalis (TEM studies). The UV‐vis spectroscopy of the supernatant of other algal extracts indicated the formation of mostly spherical particles. Production of gold nanoparticles by algae is more ecofriendly than purely chemical synthesis. However, the choice of algae is important: Chara zeylanica and Pithophora oedogoniana were found to be unable to produce nanoparticles.     

Ulva and Enteromorpha as indicators of heavy metal pollution

We studied the use of two genera of green macroalgae, Ulva and Enteromorpha, as indicators of heavy metal contamination on the northwest coast of Spain. We evaluated the extent of external contamination by fine particles adhering to the algal thallus and found that although not a problem in Ulva, it may be in Enteromorpha samples. The mean levels of metals in both seaweeds were in accordance with previously reported levels in different species of the genera in clean areas. A large number of significant correlations between levels of metals in macroalgae and in sediment (for both the total and labile fractions) were found. However, almost all of the coefficients of correlation decreased after geochemical normalization of sediment metal concentrations.     

A re-examination of the morphology, ultrastructure and classification of genera in the Lemaneaceae (Batrachospermales, Rhodophyta)

Vegetative and reproductive morphology and ultrastructure were examined for the three genera of the freshwater red algal family Lemaneaceae: Lemanea (two species, seven populations), Paralemanea (two species, three populations) and Psilosiphon (one species, one population). Psilosiphon is readily distinguished from the other two genera in having an outer cortex composed of well-defined filaments interconnected with a dense medulla (both cell types being little vacuolated), spores cleaving off obliquely, putative spermatangia scattered on the thallus surface and reproduction by adventitious filaments. Based on the distinctness of this genus (and corroborated by molecular phylogenies in preparation), a new family is described, the Psilosiphonaceae. Lemanea and Paralemanea appear to be closely related, with an outer cortex that has cells of increasing size and vacuolation from the periphery to the interior and not in obvious rows, a central lumen that contains few ray cells but no medullary filaments, sexual reproduction with spermatangia in distinct clusters, and carpospores in chains, some of which can germinate in the thallus lumen. Lemanea and Paralemanea have distinguishing characteristics that the other genus does not: for Lemanea, hair cells, ray cells abutting the outer cortex and spermatangia in patches; for Paralemanea, no hair cells or ray cells appressed to the outer cortex, inner cortical filaments surrounding the central axis and spermatangia in rings.     

Do secondary substances in the thallus of a lichen promote CO2 diffusion and prevent depression of net photosynthesis at high water content?

Many lichens show seriously depressed net photosynthesis (NP) at high thallus water contents due to increased carbon dioxide diffusion resistance through blockage of diffusion pathways by water. The soil lichen Diploschistes muscorum, however, shows no depression and NP is close to maximal even at the highest thallus water content. We investigated whether lichen substances (lecanoric and diploschistesic acids) in the cortex and medulla contributed to this ability to maintain high NP. Dry thalli were extracted with water-free acetone and, after this treatment, were found to be fully viable to the extent of continued growth after replanting in the field. No differences were found in the response of NP to thallus water content between the normal and extracted thalli, in fact the response curves were often nearly identical. Thus, in this species it seems that lichen substances did not maintain the water-free diffusion pathways and some other explanation, possibly structural, needs to be sought. Received: 5 April 1997 / Accepted: 26 April 1997     

DIATOMS LIVING INSIDE THE THALLUS OF THE GREEN ALGAL LICHEN COENOGONIUM LINKII IN NEOTROPICAL LOWLAND RAIN FORESTS1

Coenogonium linkii Ehrenb. is a very common filamentous lichen, growing on stems, hanging roots, and lianas in the understory of neotropical lowland rain forests. We investigated several thalli of this species from locations in Panama and French Guiana. All thalli were inhabited by various species of terrestrial diatoms, which were found between the thallus filaments on extracellular material of the mycobiont. We identified 18 species of diatoms belonging to nine genera: Diadesmis, Eunotia, Hantzschia, Luticola, Melosira, Nitzschia, Orthoseira, Pinnularia, and Stauroneis. The potential benefits both diatoms and lichens could derive from symbiosis in relation to water, irradiance, and nitrogen availability are discussed.     

Occurrence of UV-Absorbing, Mycosporine-Like Compounds among Cyanobacterial Isolates and an Estimate of Their Screening Capacity

A survey of 20 strains of cyanobacteria (belonging to 13 genera) isolated from habitats exposed to strong insolation revealed that 13 strains contained one or more water-soluble, UV-absorbing, mycosporine amino acid (MAA)-like compounds. Some of the compounds were identical in several strains. In all, 13 distinct compounds were found. The UV absorption spectra of MAAs complemented well that of the extracellular sunscreen pigment scytonemin, which many of the strains also produced. Even though the specific MAA contents were variable among strains, they were invariably higher when the cultures were grown with UV radiation than when it was absent. In five strains tested, the MAA complement accumulated as a solute in the cytoplasmic cell fraction. The sunscreen capacities of MAA and scytonemin and their combined capacity were estimated for each strain and condition on the basis of the specific contents, cell size, and cellular location of the compounds. The estimates suggested that significant, albeit not complete, protection from UV photodamage could be gained from the possession of either MAA or scytonemin but especially from simultaneous screening by both types of compounds.     

Dasycladales from the Upper Maastrichtian of Salento Peninsula (Puglia, southern Italy)

Summary A rich dasycladalean assemblage, mainly consisting of new taxa, has been discovered in upper Maastrichtian coarse bioclastic limestones of shelf margin facies cropping out along the southeastern coast of Salento peninsula (Puglia, Southern Italy). It consists of 8 species grouped into 6 genera:Cymopolia decastroi Parente 1994;C. barattoloi Parente, 1994;Zittelina fluegeli n.sp;Jodotella koradae (Dieni, Massari & Radoicic, 1983) nov. comb.;Barattoloporella salentina n. gen. n. sp.;Morelletpora dienii n. sp.;Neomeris spp. (two different species). Zittelina fluegeli n. sp. is characterized by an ovoid thallus with calcification made by a calcareous wall enveloping only the proximal part of the branches (except in basal whorls) and by closely packed, and partly coalescent, calcified ampullae arranged all around the median and distal portion of branches. The transferral ofNeomeris (Larvaria) koradae Dieni, Massari & Radoicic, 1983 into the genusJodotella, with the new combinationJodotella koradae, is proposed on the basis of new observations on the number and arrangement of fertile ampullae. The new genusBarattoloporella, type-speciesBarattoloporella salentina n. gen. n. sp., is erected for dasycladalean algae characterized by a segmented thallus. Each segment consists of: a) basal and apical sterile whorls made by primary branches only, b) central fertile whorls made by primary branches bearing in terminal position one fertile ampulla and one or two secondary branches. morelletpora dienii n. sp. is characterized by a segmented thallus with barrel shaped to pear shaped repetitive elements, consisting of simple whorls of first order branches only. Shape of the branches varies from regularly phloiophorous to more or less differentiated in a stalk and a swollen portion, with or without a subterminal constriction. This is by far the most diverse dasycladalean assemblage ever found in the Maastrichtian. Its diversity supports the conclusion that, within the Late Cretaceous, the Maastrichtian represents a maximum in dasycladalean diversity. Its taxonomic composition strenghtens the hypothesis that dasycladaleans were hardly affected by K/T mass extinction.     

Characterization of Armillaria spp. from peach orchards in the southeastern United States using fatty acid methyl ester profiling

Limited information is available regarding the composition of cellular fatty acids in Armillaria and the extent to which fatty acid profiles can be used to characterize species in this genus. Fatty acid methyl ester (FAME) profiles generated from cultures of A. tabescens, A. mellea, and A. gallica consisted of 16–18 fatty acids ranging from 12–24 carbons in length, although some of these were present only in trace amounts. Across the three species, 9-cis,12-cis-octadecadienoic acid (9,12-C18:2), hexadecanoic acid (16:0), heneicosanoic acid (21:0), 9-cis-octadecenoic acid (9-C18:1), and 2-hydroxy-docosanoic acid (OH-22:0) were the most abundant fatty acids. FAME profiles from different thallus morphologies (mycelium, sclerotial crust, or rhizomorphs) displayed by cultures of A. gallica showed that thallus type had no significant effect on cellular fatty acid composition (P > 0.05), suggesting that FAME profiling is sufficiently robust for species differentiation despite potential differences in thallus morphology within and among species. The three Armillaria species included in this study could be distinguished from other lignicolous basidiomycete species commonly occurring on peach (Schizophyllum commune, Ganoderma lucidum, Stereum hirsutum, and Trametes versicolor) on the basis of FAME profiles using stepwise discriminant analysis (average squared canonical correlation = 0.953), whereby 9-C18:1, 9,12-C18:2, and 10-cis-hexadecenoic acid (10-C16:1) were the three strongest contributors. In a separate stepwise discriminant analysis, A. tabescens, A. mellea, and A. gallica were separated from one another based on their fatty acid profiles (average squared canonical correlation = 0.924), with 11-cis-octadecenoic acid (11-C18:1), 9-C18:1, and 2-hydroxy-hexadecanoic acid (OH-16:0) being most important for species separation. When fatty acids were extracted directly from mycelium dissected from naturally infected host tissue, the FAME-based discriminant functions developed in the preceding experiments classified all samples (n = 16) as A. tabescens; when applied to cultures derived from the same naturally infected samples, all unknowns were similarly classified as A. tabescens. Thus, FAME species classification of Armillaria unknowns directly from infected tissues may be feasible. Species designation of unknown Armillaria cultures by FAME analysis was identical to that indicated by IGS-RFLP classification with AluI.     

Euopsis and Harpidium,Genera of the Lichinaceae (Lichenes) with Rostrate Asci*

Based on corresponding ascocarp ontogeny and thallus structure, the genera Euopsis and Harpidium are included in the family Lichinaceae. In the two species of Euopsis, E. granatina and E. pulvinata, the apothecia develop from ascogonia in generative tissue, while in H. rutilans they are pycnoascocarps. In thallus anatomy, the species of Euopsis resemble Pyrenopsis haematopsis and allied species, while H. rutilans corresponds in structure and development of the thallus and apothecia to Pyrenopsis haemaleella (syn. P. sphinctotricha). H. rutilans is the first member of Lichinaceae known to have only a green algal symbiont. In E. granatina, two phycobionts are always present, a species of Gloeocapsa and a chlorococcalean alga. In Euopsis and Harpidium, the ascus wall is composed of an outer, non-expansible and an inner, expansible layer; the latter surrounds the protoplast as an amyloid collar, which expands during spore release into a long, tapering rostrum. In Euopsis, the outer wall layer is strongly amyloid and the upper part separated from the expanded amyloid rostrum by a non-amyloid zone, appearing like a slit in LM studies. The ultrastructure and function of the ascus in E. granatina has been studied in TEM and is interpreted as functionally unitunicate-rostrate. Unitunicate asci with short rostrum are described for P. haemaleella and P. haematopsis.     

THALLUS DIFFERENTIATION OF PHOTOSYNTHESIS,GROWTH, REPRODUCTION,AND UV‐B SENSITIVITY IN THE GREEN ALGA ULVA PERTUSA (CHLOROPHYCEAE)1

The chlorophyte Ulva is perceived as a simple and uniform algal form, with little functional differentiation within a thallus. We compared morphology, pigmentation, photosynthesis, growth, reproduction, and UV‐B sensitivity between different thallus regions of Ulva pertusa Kjellman. Thallus thickness and cell size were significantly greater, whereas cell number was less in the basal region than in other regions. Photosynthetic pigment contents were lowest in the basal region and increased toward the marginal region. Photosynthetic capacity and photosynthetic efficiency normalized to fresh weight, area, volume, and cell number showed a progressive increase from the basal to marginal parts; however, on a chl basis those values were equal regardless of thallus part. Values of light saturation point were not statistically different between regions. Growth rates increased from marginal to basal and to middle parts of the thallus, whereas sporulation was highest in marginal (100%) followed by middle (30%) and basal parts (0%). Daily observation over 9 days showed that 56% of the basal cells divided once and did not produce spores, whereas every marginal cell went through its first division and 89% of the primary daughter cells also divided, resulting in 100% sporulation. A 7‐day treatment with PAR and PAR + UV‐A caused a significant decrease in the effective quantum yield of all thallus regions, followed by a recovery toward the initial values, whereas PAR + UV‐A + UV‐B irradiation led to greater photoinhibition and less recovery. Marked differences in the UV‐B sensitivity were observed, with marginal parts being more sensitive and basal parts most resistant.     

The response regulator Npun_F1278 is essential for scytonemin biosynthesis in the cyanobacterium Nostoc punctiforme ATCC 29133

Following exposure to long‐wavelength ultraviolet radiation (UVA), some cyanobacteria produce the indole‐alkaloid sunscreen scytonemin. The genomic region associated with scytonemin biosynthesis in the cyanobacterium Nostoc punctiforme includes 18 cotranscribed genes. A two‐component regulatory system (Npun_F1277/Npun_F1278) directly upstream from the biosynthetic genes was identified through comparative genomics and is likely involved in scytonemin regulation. In this study, the response regulator (RR), Npun_F1278, was evaluated for its ability to regulate scytonemin biosynthesis using a mutant strain of N. punctiforme deficient in this gene, hereafter strain Δ1278. Following UVA radiation, the typical stimulus to initiate scytonemin biosynthesis, Δ1278 was incapable of producing scytonemin. A phenotypic characterization of Δ1278 suggests that aside from the ability to produce scytonemin, the deletion of the Npun_F1278 gene does not affect the cellular morphology, cellular differentiation capability, or lipid‐soluble pigment complement of Δ1278 compared to the wildtype. The mutant, however, had a slower specific growth rate under white light and produced ~2.5‐fold more phycocyanin per cell under UVA than the wildtype. Since Δ1278 does not produce scytonemin, this study demonstrates that the RR gene, Npun_F1278, is essential for scytonemin biosynthesis in N. punctiforme. While most of the evaluated effects of this gene appear to be specific for scytonemin, this regulator may also influence the overall health of the cell and phycobiliprotein synthesis, directly or indirectly. This is the first study to identify a regulatory gene involved in the biosynthesis of the sunscreen scytonemin and posits a link between cell growth, pigment synthesis, and sunscreen production.     

CHARACTERIZATION AND BIOLOGICAL IMPLICATIONS OF SCYTONEMIN,A CYANOBACTERIAL SHEATH PIGMENT1

Scytonemin, the yellow-brown pigment of cyanobacterial (blue-green algal) extracellular sheaths, was found in species thriving in habitats exposed to intense solar radiation. Scytonemin occurred predominantly in sheaths of the outermost parts or top layers of cyanobacterial mats, crusts, or colonies. Scytonemin appears to be a single compound identified in more than 30 species of cyanobacteria from cultures and natural populations. It is lipid soluble and has a prominent absorption maximum in the near-ultraviolet region of the spectrum (384 nm in acetone; ca. 370 nm in vivo) with a long tail extending to the infrared region. Microspectrophotometric measurements of the transmittance of pigmented sheaths and the quenching of ultraviolet excitation of phycocyanin fluorescence demonstrate that the pigment was effective in shielding the cells from incoming near-ultraviolet-blue radiation, but not from green or red light. High light intensity (between 99 and 250 μmol photon · m^?2· S^?1, depending on species) promoted the synthesis of scytonemin in cultures of cyanobacteria. In cultures, high light intensity caused reduction in the specific content of Chl a and phycobilins, increase in the ratio of total carotenoids to Chl a, and scytonemin increase. UV-A (320–400 nm) radiation was very effective in eliciting scytonemin synthesis. Scytonemin production was physiological and not due to a mere photochemical conversion. These results strongly suggest that scytonemin production constitutes an adaptive strategy of photoprotection against short-wavelength solar irradiance.     

Laying the foundations for a new classification of Agaonidae (Hymenoptera: Chalcidoidea), a multilocus phylogenetic approach

A phylogeny of the Agaonidae (Chalcidoidea) in their restricted sense, pollinators of Ficus species (Moraceae), is estimated using 4182 nucleotides from six genes, obtained from 101 species representing 19 of the 20 recognized genera, and four outgroups. Data analysed by parsimony and Bayesian inference methods demonstrate that Agaonidae are monophyletic and that the previous classification is not supported. Agaonidae are partitioned into four groups: (i) Tetrapus, (ii) Ceratosolen + Kradibia, (iii) some Blastophaga + Wiebesia species, and (iv) all genera associated with monoecious figs and a few Blastophaga and Wiebesia. The latter group is subdivided into subgroups: (i) Pleistodontes, (ii) Blastophaga psenes and neocaledonian Dolichoris, (iii) some Blastophaga and Wiebesia species, and (iv) Platyscapa, all afrotropical genera and all genera associated with section Conosycea. Eleven genera were recovered as monophyletic, six were para‐ or polyphyletic, and two cannot be tested with our data set. Based on our phylogeny we propose a new classification for the Agaonidae. Two new subfamilies are proposed: Tetrapusiinae for the genus Tetrapus, and Kradibiinae for Ceratosolen + Kradibia. Liporrhopalum is synonymized with Kradibia and the subgenus Valisia of Blastophaga is elevated to generic rank. These changes resulted in 36 new combinations. Finally, we discuss the hypothesis of co‐speciation between the pollinators and their host species by comparing the two phylogenies. © The Willi Hennig Society 2009.