Analysis of rbcL sequences reveals the global biodiversity,community structure,and biogeographical pattern of thermoacidophilic red algae (Cyanidiales)

Thermoacidophilic cyanidia (Cyanidiales) are the primary photosynthetic eukaryotes in volcanic areas. These red algae also serve as important model organisms for studying life in extreme habitats. The global biodiversity and community structure of Cyanidiales remain unclear despite previous sampling efforts. Here, we surveyed the Cyanidiales biodiversity in the Tatun Volcano Group (TVG) area in Taiwan using environmental DNA sequencing. We generated 174 rbcL sequences from eight samples from four regions in the TVG area, and combined them with 239 publicly available rbcL sequences collected worldwide. Species delimita‐tion using this large rbcL data set suggested at least 20 Cyanidiales OTUs (operational taxono‐mic units) worldwide, almost three times the presently recognized seven species. Results from environmental DNA showed that OTUs in the TVG area were divided into three groups: (i) dominant in hot springs with 92%–99% sequence identity to Galdieria maxima; (ii) largely distributed in drier and more acidic microhabitats with 99% identity to G. partita; and (iii) primarily distributed in cooler microhabitats and lacking identity to known cyanidia species (a novel Cyanidiales lineage). In both global and individual area analyses, we observed greater species diversity in non‐aquatic than aquatic habitats. Community structure analysis showed high similarity between the TVG community and West Pacific‐Iceland communities, reflecting their geographic proximity to each other. Our study is the first examination of the global species diversity and biogeographic affinity of cyanidia. Additionally, our data illuminate the influence of microhabitat type on Cyanidiales diversity and highlight intriguing questions for future ecological research.     

An unusual cyanobacterium from saline thermal waters with relatives from unexpected habitats

Cyanobacteria that grow above seawater salinity at temperatures above 45°C have rarely been studied. Cyanobacteria of this type of thermo-halophilic extremophile were isolated from siliceous crusts at 40–45°C in a geothermal seawater lagoon in southwest Iceland. Iceland Clone 2e, a Leptolyngbya morphotype, was selected for further study. This culture grew only at 45–50°C, in medium ranging from 28 to 94 g L⁻¹ TDS, It showed 3 doublings 24 h⁻¹ under continuous illumination. This rate at 54°C was somewhat reduced, and death occurred at 58°C. A comparison of the 16S rDNA sequence with all others in the NCBI database revealed 2 related Leptolyngbya isolates from a Greenland hot spring (13–16 g L⁻¹ TDS). Three other similar sequences were from Leptolyngbya isolates from dry, endolithic habitats in Yellowstone National Park. All 6 formed a phylogenetic clade, suggesting common ancestry. These strains shared many similarities to Iceland Clone 2e with respect to temperature and salinity ranges and optima. Two endolithic Leptolyngbya isolates, grown previously at 23°C in freshwater medium, grew well at 50°C but only in saline medium. This study shows that limited genotypic similarity may reveal some salient phenotypic similarities, even when the related cyanobacteria are from vastly different and remote habitats.     

An <Emphasis Type="Italic">in silico</Emphasis> analysis of the mitochondrial protein import apparatus of plants

Background  

An in silico analysis of the mitochondrial protein import apparatus from a variety of species; including Chlamydomonas reinhardtii, Chlorella variabilis, Ectocarpus siliculosus, Cyanidioschyzon merolae, Physcomitrella patens, Selaginella moellendorffii, Picea glauca, Oryza sativa and Arabidopsis thaliana was undertaken to determine if components differed within and between plant and non-plant species.     

Cyanidium chilense (Cyanidiophyceae,Rhodophyta) from tuff rocks of the archeological site of Cuma,Italy

Phlegrean Fields is a large volcanic area situated southwest of Naples (Italy), including both cave and thermoacidic habitats. These extreme environments host the genus Cyanidium; the species C. chilense represents a common phototrophic microorganism living in anthropogenic caves. With a view to provide a comprehensive characterization for a correct taxonomic classification, morpho‐ultrastructural investigations of C. chilense from Sybil's cave (Phlegren Fields) was herein carried out and compared with the thermoacidophilic C. caldarium. The biofilm was also analyzed to define the role of C. chilense in the establishment of a biofilm within cave environments. Despite the peculiar ecological and molecular divergences, C. chilense and C. caldarium shared all the main diacritic features, suggesting morphological convergence within the genus; cytological identity was found among C. chilense strains geographically distant and adapted to different substrates, such as the porous yellow tuff of Sybil cave, and calcyte, magnesite and basaltic rocks from other caves. C. chilense is generally dominant in all biofilms, developing monospecific islets, developing both superficially or between fungal hyphae and coccoid cyanobacteria. Extracellular polymeric substances (EPS) were recorded in C. chilense biofilms from Sybil cave, confirming the role of EPS in facilitating cells adhesion to the surface, creating a cohesive network of interconnecting biofilm cells.     

Comparative genomic analyses of transport proteins encoded within the red algae Chondrus crispus,Galdieria sulphuraria,and Cyanidioschyzon merolae11

Galdieria sulphuraria and Cyanidioschyzon merolae are thermo‐acidophilic unicellular red algal cousins capable of living in volcanic environments, although the former can additionally thrive in the presence of toxic heavy metals. Bioinformatic analyses of transport systems were carried out on their genomes, as well as that of the mesophilic multicellular red alga Chondrus crispus (Irish moss). We identified transport proteins related to the metabolic capabilities, physiological properties, and environmental adaptations of these organisms. Of note is the vast array of transporters encoded in G. sulphuraria capable of importing a variety of carbon sources, particularly sugars and amino acids, while C. merolae and C. crispus have relatively few such proteins. Chondrus crispus may prefer short chain acids to sugars and amino acids. In addition, the number of encoded proteins pertaining to heavy metal ion transport is highest in G. sulphuraria and lowest in C. crispus. All three organisms preferentially utilize secondary carriers over primary active transporters, suggesting that their primary source of energy derives from electron flow rather than substrate‐level phosphorylation. Surprisingly, the percentage of inorganic ion transporters encoded in C. merolae more closely resembles that of C. crispus than G. sulphuraria, but only C. crispus appears to signal via voltage‐gated cation channels and possess a Na⁺/K⁺‐ATPase and a Na⁺ exporting pyrophosphatase. The results presented in this report further our understanding of the metabolic potential and toxic compound resistances of these three organisms.     

CYANIDIA (CYANIDIALES) POPULATION DIVERSITY AND DYNAMICS IN AN ACID‐SULFATE‐CHLORIDE SPRING IN YELLOWSTONE NATIONAL PARK1

The unicellular eukaryotic algae Cyanidium, Galdieria, and Cyanidioschyzon (herein referred to as “cyanidia”) are the only photoautotrophs occurring in acidic (pH<4.0) geothermal environments at temperatures above 40°C. In Yellowstone National Park (YNP), we examined an annual event we refer to as “mat decline,” where cyanidial mats undergo a seasonably defined color fading. Monthly sampling of chemical, physical, and biological features revealed that spring aqueous chemistry was essentially invariant over the 1‐year sampling period. However, multiple regression analysis suggested that a significant proportion of algal most probable number (MPN) count variation could be explained by water temperature and UV–visible (VIS) light exposure. Irradiance manipulations (filtering) were then coupled with ¹⁴CO₂ incorporation experiments to directly demonstrate UV inhibition of photosynthesis. Population dynamics were also evident in 18S rDNA PCR clone libraries, which were different in composition at MPN maxima and minima, and again evident in PCR‐amplified chloroplast genomic short sequence repeat (SSR) analysis. PCR‐cloned SSRs of the YNP isolates and mats were very similar to Cyanidioschyzon merolae Luca, Taddei et Varano, although distance analysis could distinguish the YNP cyanidia from the genome sequenced C. merolae that was isolated in Italy. Unexpectedly, while phylogenetic analysis of 18S rDNA sequences and SSR sequences derived from YNP cyanidial mats and pure cultures suggested these algae are most closely related to C. merolae (99.7% identity), cell morphology was consistent with the genera Galdieria and Cyanidium.     

Contribution to the taxonomy and distribution of Cyclops abyssorum Sars (Crustacea,Copepoda) in several lakes and ponds of central Italy

A preliminary study has been carried out on the taxonomy and distribution of Cyclops abyssorum Sars in several deep and shallow lakes and in long lasting ponds of central Italy.A morphological and morphometric analysis of material from Lake Albano has shown that its cyclopoids belong to the praealpinus-group of C. abyssorum. This group has also been found in lakes Vico, Martignano, and Bracciano. All these populations have continuous reproduction and do not show any seasonal variation in size.In some of the long lasting Latial ponds forms of the mixed group divulsus have been found. They are characterized by a marked seasonal polymorphism and their life-cycles vary according to whether the basin is filled with water continuously or is subject to desiccation from time to time. The analysis of a population recently collected in Lake Montepulciano (Tuscany) resulted in the identification of an ecotype similar to that described as C. a. apenninae in other eutrophic lakes of the Apennine region.Causal environmental factors responsible for the distribution of these different types of C. abyssorum are discussed.     

A combined molecular and morphological approach to species delimitation in black-fruited, endolithic Caloplaca: high genetic and low morphological diversity

A revision based on the morphological and genetic analyses of 133 specimens of black-fruited, endolithic Caloplaca belonging to subgenus Pyrenodesmia is presented. The material was collected in 16 sites distributed along a transept from Gargano (Central Italy) to the southeastern Alps, from sea level to ca 1500 m. The nuclear ITS was sequenced for all the mycobionts and selected representatives of photobionts. Except for the sorediate C. erodens, all species share the same algal lineage of Trebouxia as photobiont. The haplotype analysis of the mycobionts revealed an unexpected, high genetic heterogeneity. Three main morphotypic clusters were recognized among five species: C. albopruinosa (syn. C. agardhiana auct.), C. alociza, C. badioreagens, C. erodens, and C. variabilis. A phylogenetic analysis, including already available Caloplaca sequence data, revealed that these lichens form a monophyletic group within the genus. For each species, notes on ecology, distribution in Italy, and nomenclature are given.     

Isolation of the cell-nuclear,mitochondrial, and chloroplast DNA from the ultra-small eukaryoteCyanidioschyzon merolae

Summary The amounts of cell-nuclear DNA (cl-DNA), mitochondrial DNA (mt-DNA) and chloroplast DNA (cp-DNA) inCyanidioschyzon merolae were estimated by using a video-intensified microscope (VIM) system.C. merolae had the smallest amount of cell-nuclear DNA among eukaryotes. The results show that a cell-nucleus, a mitochondrion and a chloroplast contain an average 8.0×10³kbp, 1.6×10³kbp, and 5.0×10³kbp, respectively. To confirm these results, cl-DNA, mt-DNA, and cp-DNA were isolated from cells by density centrifugation on Hoechst 33258/CsCl after density centrifugation on ethidium bromide/CsCl. The amounts of cl-DNA, mt-DNA, and cp-DNA obtained from the bands supported the data shown by the VIM-system. The cytochemical and biochemical characteristics were compared with those ofCyanidium caldarium RK-1 andC. caldarium Forma A. The values of cl-DNA and cp-DNA ofC. merolae were about 1.716 and 1.709, respectively. The order in density was different from that ofC. caldarium Forma A but very similar to that ofC. caldarium RK-1. However, the restriction patterns of cp-DNA inC. merolae differed from those ofC. caldarium RK-1.     

Identification of novel proteins in isolated polyphosphate vacuoles in the primitive red alga Cyanidioschyzon merolae

Plant vacuoles are organelles bound by a single membrane, and involved in various functions such as intracellular digestion, metabolite storage, and secretion. To understand their evolution and fundamental mechanisms, characterization of vacuoles in primitive plants would be invaluable. Algal cells often contain polyphosphate‐rich compartments, which are thought to be the counterparts of seed plant vacuoles. Here, we developed a method for isolating these vacuoles from Cyanidioschyzon merolae, and identified their proteins by MALDI TOF‐MS. The vacuoles were of unexpectedly high density, and were highly enriched at the boundary between 62 and 80% w/v iodixanol by density‐gradient ultracentrifugation. The vacuole‐containing fraction was subjected to SDS–PAGE, and a total of 46 proteins were identified, including six lytic enzymes, 13 transporters, six proteins for membrane fusion or vesicle trafficking, five non‐lytic enzymes, 13 proteins of unknown function, and three miscellaneous proteins. Fourteen proteins were homologous to known vacuolar or lysosomal proteins from seed plants, yeasts or mammals, suggesting functional and evolutionary relationships between C. merolae vacuoles and these compartments. The vacuolar localization of four novel proteins, namely CMP249C (metallopeptidase), CMJ260C (prenylated Rab receptor), CMS401C (ABC transporter) and CMT369C (o‐methyltransferase), was confirmed by labeling with specific antibodies or transient expression of hemagglutinin‐tagged proteins. The results presented here provide insights into the proteome of C. merolae vacuoles and shed light on their functions, as well as indicating new features.     

Influence of weather and phenotypic characteristics on pregnancy rates of female roe deer in central Italy

In many ungulates, female fecundity is influenced by weather, population density and body condition. Based on a six-year survey of the reproductive tracts of adult female roe deer (Capreolus capreolus) harvested in the province of Pisa (Tuscany, central Italy), we evaluated the influence of weather, population density and individual characteristics on pregnancy rates. Eighty-five percent of females were pregnant, with a median litter size of two (range one—three). We found that pregnancy rate was positively correlated with summer rainfall and body mass of females, whereas early-winter conditions, spring rainfall, the age of females and density-dependent factors did not appear to influence pregnancy rate. These results reflect the particular seasonal variation in the abundance and quality of resources in Mediterranean habitats and show that heavier females (high-quality mothers) are more productive than lighter females.     

Infestation of the invasive mollusc <Emphasis Type="Italic">Crepidula fornicata</Emphasis> by the native shell borer <Emphasis Type="Italic">Cliona celata</Emphasis>: a case of high parasite load without detrimental effects

The temporal prevalence of the widespread boring sponge Cliona celata and its effects were analysed in a population of the invasive mollusc Crepidula fornicata. This mollusc produces extra shell material when infested with the endolithic sponge, suggesting that infestation may be detrimental for C. fornicata growth and/or reproduction. For 37 months, size, sex, female reproductive status and sponge-infestation stage were recorded for 300 individuals sampled every month in the Bay of Morlaix (France). In the 12,049 individuals examined, the prevalence of C. celata was high with a monthly average of 43.1% of the individuals hosting the sponge. The relative proportion of heavily infested individuals generally increased over time. Nevertheless, a cyclic decrease occurred every 10 months, suggesting putative episodes of mortality of heavily infested individuals. The gregarious behaviour of the mollusc seemed to promote the high prevalence of the sponge, which may propagate through contact between neighbouring C. fornicata individuals. Due to the sex-size relationship in protandrous C. fornicata, females were far more infested than males. We did not find evidence for a cost of producing extra shell material on somatic growth or on female fertility, and the boring sponge is unlikely to substantially affect the sex-change patterns in C. fornicata. The limited effects of the endolithic sponge on C. fornicata contrasts with the documented damage to some local species, including commercially exploited shellfish, suggesting that C. fornicata may alter the infestation dynamics in the surrounding native community. Dedicated studies are now needed to investigate the extent and mechanisms of these species interactions.     

Evolutionary diversification of cryophilic <Emphasis Type="Italic">Grylloblatta</Emphasis>species (Grylloblattodea: Grylloblattidae) in alpine habitats of California

Background  

Climate in alpine habitats has undergone extreme variation during Pliocene and Pleistocene epochs, resulting in repeated expansion and contraction of alpine glaciers. Many cold-adapted alpine species have responded to these climatic changes with long-distance range shifts. These species typically exhibit shallow genetic differentiation over a large geographical area. In contrast, poorly dispersing organisms often form species complexes within mountain ranges, such as the California endemic ice-crawlers (Grylloblattodea: Grylloblattidae: Grylloblatta). The diversification pattern of poorly dispersing species might provide more information on the localized effects of historical climate change, the importance of particular climatic events, as well as the history of dispersal. Here we use multi-locus genetic data to examine the phylogenetic relationships and geographic pattern of diversification in California Grylloblatta.     

Diversity and stability of coral endolithic microbial communities at a naturally high pCO2 reef

The health and functioning of reef‐building corals is dependent on a balanced association with prokaryotic and eukaryotic microbes. The coral skeleton harbours numerous endolithic microbes, but their diversity, ecological roles and responses to environmental stress, including ocean acidification (OA), are not well characterized. This study tests whether pH affects the diversity and structure of prokaryotic and eukaryotic algal communities associated with skeletons of Porites spp. using targeted amplicon (16S rRNA gene, UPA and tufA) sequencing. We found that the composition of endolithic communities in the massive coral Porites spp. inhabiting a naturally high pCO₂ reef (avg. pCO₂ 811 μatm) is not significantly different from corals inhabiting reference sites (avg. pCO₂ 357 μatm), suggesting that these microbiomes are less disturbed by OA than previously thought. Possible explanations may be that the endolithic microhabitat is highly homeostatic or that the endolithic micro‐organisms are well adapted to a wide pH range. Some of the microbial taxa identified include nitrogen‐fixing bacteria (Rhizobiales and cyanobacteria), algicidal bacteria in the phylum Bacteroidetes, symbiotic bacteria in the family Endozoicomoniaceae, and endolithic green algae, considered the major microbial agent of reef bioerosion. Additionally, we test whether host species has an effect on the endolithic community structure. We show that the endolithic community of massive Porites spp. is substantially different and more diverse than that found in skeletons of the branching species Seriatopora hystrix and Pocillopora damicornis. This study reveals highly diverse and structured microbial communities in Porites spp. skeletons that are possibly resilient to OA.     

Subcellular distribution of central carbohydrate metabolism pathways in the red alga Cyanidioschyzon merolae

Main conclusion

Comprehensive subcellular localization analysis revealed that the subcellular distribution of carbohydrate metabolic pathways in the red alga Cyanidioschyzon is essentially identical with that in Arabidopsis , except the lack of transaldolase. In plants, the glycolysis and oxidative pentose phosphate pathways (oxPPP) are located in both cytosol and plastids. However, in algae, particularly red algae, the subcellular localization of enzymes involved in carbon metabolism is unclear. Here, we identified and examined the localization of enzymes related to glycolysis, oxPPP, and tricarboxylic acid (TCA) and Calvin–Benson cycles in the red alga Cyanidioschyzon merolae. A gene encoding transaldolase of the oxPPP was not found in the C. merolae genome, and no transaldolase activity was detected in cellular extracts. The subcellular localization of 65 carbon metabolic enzymes tagged with green fluorescent protein or hemagglutinin was examined in C. merolae cells. As expected, TCA and Calvin–Benson cycle enzymes were localized to mitochondria and plastids, respectively. The analyses also revealed that the cytosol contains the entire glycolytic pathway and partial oxPPP, whereas the plastid contains a partial glycolytic pathway and complete oxPPP, with the exception of transaldolase. Together, these results suggest that the subcellular distribution of carbohydrate metabolic pathways in C. merolae is essentially identical with that reported in the photosynthetic tissue of Arabidopsis thaliana; however, it appears that substrates typically utilized by transaldolase are consumed by glycolytic enzymes in the plastidic oxPPP of C. merolae.     

Molecular variation in Galdieria sulphuraria (Galdieri) Merola and its bearing on taxonomy

Cyanidium caldarium, Cyanidioschyzon merolae and Galdieria sulphuraria are three unicellular algae characteristic, of acid thermal environments. Recently, on the basis of morphological characters, three new species of Galdieria (G. partita, G. daedala, G. maxima ) isolated from acid-thermal springs in Russia have been instituted. A selected region of rbcL and the sequence of the intergenic spacer between the rbcL and rbcS have been amplified and sequenced from different Galdieria species and strains, in order to define molecular relationship among these interesting algae. The obtained cladogram shows that Cyanidium caldarium and Cyanidioschyzon merolae form a sister group which, in turn, is in a sister group relationship with Galdieria. This last genus is divided in two clades, one of which includes G. sulphuraria accessions from Naples (Italy), California, and Yellowstone and the other one includes G. sulphuraria accessions from Java (Indonesia) and from the Russian species. These results support the status of the genus Galdieria and suggest that G. daedala, G. maxima and G. partita are three very similar strains of G. sulphuraria; the rbcL variation within Galdieria accessions has a pattern which is broadly connected to the geographial distribution. The data obtained from the intergenic rbcL-rbcS spacer partly confirm those from the rbcL analysis.     

An anatomical investigation of calcicolous endolithic lichens from the Trieste karst (NE Italy)

ABSTRACT

The anatomy of five endolithic lichens (Acrocordia conoidea, Petractis clausa, Rinodina immersa, Verrucaria baldensis, and V. marmorea) from the Trieste Karst (north-eastern Italy) was thoroughly investigated. Samples already used in previous ecophysiological studies were examined by histological and mineralogical techniques, and by SEM. Biomineralisation products were searched for by X-ray diffractometry, X-ray microdiffractometry, and Fourier Transformed Infrared spectrophotometry. The results confirm that the photobiont layer is located approximately at the same depth in the substratum, although the species occur in habitats with strongly different light regimes; the thallus development is relatively constant within populations of a single species, but differs considerably among species. Several peculiarities of each species were revealed, such as the presence of large clews of hyphae in the inner layer of P. clausa, forming large voids in the substratum, or the development of morphologically different oil-hyphae. Calcium oxalate crystals were not detected. Some terms currently used to describe the anatomy of endolithic lichens are critically discussed, and the new term “lithocortex” and “pseudo-medulla” are introduced.     

Phylogeography of the Italian vairone (<Emphasis Type="Italic">Telestes muticellus</Emphasis>, Bonaparte 1837) inferred by microsatellite markers: evolutionary history of a freshwater fish species with a restricted and fragmented distribution

Background  

Owing to its independence from the main Central European drainage systems, the Italian freshwater fauna is characterized by a high degree of endemicity. Three main ichthyogeographic districts have been proposed in Italy. Yet, the validity of these regions has not been confirmed by phylogenetic and population genetic analyses and a phylogeographic scenario for Italy's primary freshwater fish fauna is still lacking. Here, we investigate the phylogeography of the Italian vairone (Telestes muticellus).