Hidden biodiversity of the extremophilic Cyanidiales red algae

The Cyanidiales is a group of asexual, unicellular red algae, which thrive in acidic and high temperature conditions around hot springs. These unicellular taxa have a relatively simple morphology and are currently classified into three genera, Cyanidium, Cyanidioschyzon and Galdieria. Little is known, however, about the biodiversity of Cyanidiales, their population structure and their phylogenetic relationships. Here we used a taxonomically broadly sampled three-gene data set of plastid sequences to infer a robust phylogenetic framework for the Cyanidiales. The phylogenetic analyses support the existence of at least four distinct Cyanidiales lineages: the Galdieria spp. lineage (excluding Galdieria maxima), the Cyanidium caldarium lineage, a novel monophyletic lineage of mesophilic Cyanidium spp. and the Cyanidioschyzon merolae plus Galdieria maxima lineage. Our analyses do not support the notion of a mesophilic ancestry of the Cyanidiales and suggest that these algae were ancestrally thermo-acidotolerant. We also used environmental polymerase chain reaction (PCR) for the rbcL gene to sample Cyanidiales biodiversity at five ecologically distinct sites at Pisciarelli in the Phlegrean Fields in Italy. This analysis showed a high level of sequence divergence among Cyanidiales species and the partitioning of taxa based on environmental conditions. Our research revealed an unexpected level of genetic diversity among Cyanidiales that revises current thinking about the phylogeny and biodiversity of this group. We predict that future environmental PCR studies will significantly augment known biodiversity that we have discovered and demonstrate the Cyanidiales to be a species-rich branch of red algal evolution.     

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.     

Algal species and light microenvironment in a low-pH, geothermal microbial mat community

Unicellular algae are the predominant microbial mat-forming phototrophs in the extreme environments of acidic geothermal springs. The ecology of these algae is not well known because concepts of species composition are inferred from cultivated isolates and microscopic observations, methods known to provide incomplete and inaccurate assessments of species in situ. We used sequence analysis of 18S rRNA genes PCR amplified from mat samples from different seasons and different temperatures along a thermal gradient to identify algae in an often-studied acidic (pH 2.7) geothermal creek in Yellowstone National Park. Fiber-optic microprobes were used to show that light for algal photosynthesis is attenuated to < 1% over the 1-mm surface interval of the mat. Three algal sequences were detected, and each was present year-round. A Cyanidioschyzon merolae sequence was predominant at temperatures of > or = 49 degrees C. A Chlorella protothecoides var. acidicola sequence and a Paradoxia multisita-like sequence were predominant at temperatures of < or = 39 degrees C.     

Characterization of rbcL group IA introns from two colonial volvocalean species (Chlorophyceae)

Group I introns were reported for the first time in the large subunit of Rubisco (rbcL) genes, using two colonial green algae, Pleodorina californica and Gonium multicoccum (Volvocales). The rbcL gene of P. californica contained an intron (PlC intron) of 1320 bp harboring an open reading frame (ORF). The G. multicoccum rbcL gene had two ORF-lacking introns of 549 (GM1 intron) and 295 (GM2 intron) base pairs. Based on the conserved nucleotide sequences of the secondary structure, the PlC and GM1 introns were assigned to group IA2 whereas the GM2 intron belonged to group IA1. Southern hybridization analyses of nuclear and chloroplast DNAs indicated that such intron-containing rbcL genes are located in the chloroplast genome. Sequencing RNAs from the two algae revealed that these introns are spliced out during mRNA maturation. In addition, the PlC and GM1 introns were inserted in the same position of the rbcL exons, and phylogenetic analysis of group IA introns indicated a close phylogenetic relationship between the PlC and GM1 introns within the lineage of bacteriophage group IA2 introns. However, P. californica and G. multicoccum occupy distinct clades in the phylogenetic trees of the colonial Volvocales, and the majority of other colonial volvocalean species do not have such introns in the rbcL genes. Therefore, these introns might have been recently inserted in the rbcL genes independently by horizontal transmission by viruses or bacteriophage.     

FLORIDOSIDES IN CYANIDIUM CALDARIUM,CYANIDIOSCHYZON MEROLAE AND GALDIERIA SULPHURARIA(RHODOPHYTA,CYANIDIOPHYCEAE)1

Cyanidium caldarium Geitler, Cyanidioschyzon merolae De Luca, Taddei & Varano and Galdieria sulphuraria (Galdieri) Merola are the three thermoacidophilic algae characterized by a chloroplast which is bounded by a single membrane. The presence of this atypical chloroplast made the inclusion of these algae in the Rhodophyta difficult. The discovery in the three algae of floridoside and isofloridoside, typical storage products of red algae, in compatible with their inclusion in the Rhodophyta     

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.     

Establishment of endolithic populations of extremophilic Cyanidiales (Rhodophyta)

Background  

Cyanidiales are unicellular extremophilic red algae that inhabit acidic and high temperature sites around hot springs and have also adapted to life in endolithic and interlithic habitats. Comparative genomic analysis of Cyanidioschyzon merolae and Galdieria sulphuraria predicts that the latter may be more broadly distributed in extreme environments because its genome contains membrane transporters involved in the uptake of reduced carbon compounds that are absent from C. merolae. Analysis of an endolithic site in the Phlegrean Fields near Naples, Italy is consistent with this prediction showing this population to be comprised solely of the newly described lineage Galdieria -B and C. merolae to be limited to humid habitats. Here, we conducted an environmental PCR survey of another extreme environment in Tuscany, Italy and contrasted Cyanidiales population structure at endolithic and interlithic habitats in Naples and Tuscany.     

Molecular characterisation of an Australian isolate of Bonamia exitiosa

An Australian (New South Wales) isolate of Bonamia was characterised at the molecular level by sequencing the 18S-ITS-1 region of the small subunit rRNA operon obtained from flat oysters Ostrea angasi shown to be infected by histological examination. Sequence data alignment with homologous genes from 2 other isolates of Bonamia (New Zealand and France) revealed high levels of nucleotide identity with both isolates. However, the Australian Bonamia is shown to be more closely related to the New Zealand isolate, suggesting the existence of an oceanic subgroup of Bonamia.     

In search of monophyletic taxa in the family Desmidiaceae (Zygnematophyceae, Viridiplantae): the genus Cosmarium

Nuclear-encoded small subunit (SSU) rDNA, 1506 group I introns, and chloroplast rbcL genes were sequenced from 97 strains representing the largest desmid genus Cosmarium (45 spp.), its putative relatives Actinotaenium (5 spp.), Xanthidium (4 spp.), Euastrum (9 spp.), Staurodesmus (13 spp.), and other Desmidiaceae (Zygnematophyceae, Streptophyta) and used to assess phylogenetic relationships in the family. Analyses of single genes and of a concatenated data set (3260 nt) established 10 well-supported clades in the family with Cosmarium species distributed in six clades and one nonsupported assemblage. Most of the clades contained representatives of at least two genera highlighting the polyphyletic nature of the genera Cosmarium, Euastrum, Staurodesmus, and Actinotaenium. To enhance resolution between clades, we extended the data set by sequencing the slowly evolving chloroplast-encoded large subunit (LSU) rRNA gene from 40 taxa. Phylogenetic analyses of a concatenated data set (5509 nt) suggested a sister relationship between two clades that consisted mainly of Cosmarium species and included C. undulatum, the type species of the genus. We describe molecular signatures in the SSU rRNA for two clades and conclude that more studies involving new isolates, additional molecular markers, and reanalyses of morphological traits are necessary before the taxonomic revision of the genus Cosmarium can be attempted.     

Korarchaeota diversity, biogeography, and abundance in Yellowstone and Great Basin hot springs and ecological niche modeling based on machine learning

Over 100 hot spring sediment samples were collected from 28 sites in 12 areas/regions, while recording as many coincident geochemical properties as feasible (>60 analytes). PCR was used to screen samples for Korarchaeota 16S rRNA genes. Over 500 Korarchaeota 16S rRNA genes were screened by RFLP analysis and 90 were sequenced, resulting in identification of novel Korarchaeota phylotypes and exclusive geographical variants. Korarchaeota diversity was low, as in other terrestrial geothermal systems, suggesting a marine origin for Korarchaeota with subsequent niche-invasion into terrestrial systems. Korarchaeota endemism is consistent with endemism of other terrestrial thermophiles and supports the existence of dispersal barriers. Korarchaeota were found predominantly in >55°C springs at pH 4.7-8.5 at concentrations up to 6.6×10(6) 16S rRNA gene copies g(-1) wet sediment. In Yellowstone National Park (YNP), Korarchaeota were most abundant in springs with a pH range of 5.7 to 7.0. High sulfate concentrations suggest these fluids are influenced by contributions from hydrothermal vapors that may be neutralized to some extent by mixing with water from deep geothermal sources or meteoric water. In the Great Basin (GB), Korarchaeota were most abundant at spring sources of pH<7.2 with high particulate C content and high alkalinity, which are likely to be buffered by the carbonic acid system. It is therefore likely that at least two different geological mechanisms in YNP and GB springs create the neutral to mildly acidic pH that is optimal for Korarchaeota. A classification support vector machine (C-SVM) trained on single analytes, two analyte combinations, or vectors from non-metric multidimensional scaling models was able to predict springs as Korarchaeota-optimal or sub-optimal habitats with accuracies up to 95%. To our knowledge, this is the most extensive analysis of the geochemical habitat of any high-level microbial taxon and the first application of a C-SVM to microbial ecology.     

Comparative genomics of two closely related unicellular thermo-acidophilic red algae, Galdieria sulphuraria and Cyanidioschyzon merolae, reveals the molecular basis of the metabolic flexibility of Galdieria sulphuraria and significant differences in carbohydrate metabolism of both algae

Unicellular algae serve as models for the study and discovery of metabolic pathways, for the functional dissection of cell biological processes such as organellar division and cell motility, and for the identification of novel genes and gene functions. The recent completion of several algal genome sequences and expressed sequence tag collections and the establishment of nuclear and organellar transformation methods has opened the way for functional genomics approaches using algal model systems. The thermo-acidophilic unicellular red alga Galdieria sulphuraria represents a particularly interesting species for a genomics approach owing to its extraordinary metabolic versatility such as heterotrophic and mixotrophic growth on more than 50 different carbon sources and its adaptation to hot acidic environments. However, the ab initio prediction of genes required for unknown metabolic pathways from genome sequences is not trivial. A compelling strategy for gene identification is the comparison of similarly sized genomes of related organisms with different physiologies. Using this approach, candidate genes were identified that are critical to the metabolic versatility of Galdieria. Expressed sequence tags and high-throughput genomic sequence reads covering >70% of the G. sulphuraria genome were compared to the genome of the unicellular, obligate photoautotrophic red alga Cyanidioschyzon merolae. More than 30% of the Galdieria sequences did not relate to any of the Cyanidioschyzon genes. A closer inspection of these sequences revealed a large number of membrane transporters and enzymes of carbohydrate metabolism that are unique to Galdieria. Based on these data, it is proposed that genes involved in the uptake of reduced carbon compounds and enzymes involved in their metabolism are crucial to the metabolic flexibility of G. sulphuraria.     

Algal Species and Light Microenvironment in a Low-pH, Geothermal Microbial Mat Community

Unicellular algae are the predominant microbial mat-forming phototrophs in the extreme environments of acidic geothermal springs. The ecology of these algae is not well known because concepts of species composition are inferred from cultivated isolates and microscopic observations, methods known to provide incomplete and inaccurate assessments of species in situ. We used sequence analysis of 18S rRNA genes PCR amplified from mat samples from different seasons and different temperatures along a thermal gradient to identify algae in an often-studied acidic (pH 2.7) geothermal creek in Yellowstone National Park. Fiber-optic microprobes were used to show that light for algal photosynthesis is attenuated to <1% over the 1-mm surface interval of the mat. Three algal sequences were detected, and each was present year-round. A Cyanidioschyzon merolae sequence was predominant at temperatures of ≥49°C. A Chlorella protothecoides var. acidicola sequence and a Paradoxia multisita-like sequence were predominant at temperatures of ≤39°C.     

An Expanded Multilocus Sequence Typing Scheme for Propionibacterium acnes: Investigation of 'Pathogenic', 'Commensal' and Antibiotic Resistant Strains

The Gram-positive bacterium Propionibacterium acnes is a member of the normal human skin microbiota and is associated with various infections and clinical conditions. There is tentative evidence to suggest that certain lineages may be associated with disease and others with health. We recently described a multilocus sequence typing scheme (MLST) for P. acnes based on seven housekeeping genes (http://pubmlst.org/pacnes). We now describe an expanded eight gene version based on six housekeeping genes and two 'putative virulence' genes (eMLST) that provides improved high resolution typing (91eSTs from 285 isolates), and generates phylogenies congruent with those based on whole genome analysis. When compared with the nine gene MLST scheme developed at the University of Bath, UK, and utilised by researchers at Aarhus University, Denmark, the eMLST method offers greater resolution. Using the scheme, we examined 208 isolates from disparate clinical sources, and 77 isolates from healthy skin. Acne was predominately associated with type IA(1) clonal complexes CC1, CC3 and CC4; with eST1 and eST3 lineages being highly represented. In contrast, type IA(2) strains were recovered at a rate similar to type IB and II organisms. Ophthalmic infections were predominately associated with type IA(1) and IA(2) strains, while type IB and II were more frequently recovered from soft tissue and retrieved medical devices. Strains with rRNA mutations conferring resistance to antibiotics used in acne treatment were dominated by eST3, with some evidence for intercontinental spread. In contrast, despite its high association with acne, only a small number of resistant CC1 eSTs were identified. A number of eSTs were only recovered from healthy skin, particularly eSTs representing CC72 (type II) and CC77 (type III). Collectively our data lends support to the view that pathogenic versus truly commensal lineages of P. acnes may exist. This is likely to have important therapeutic and diagnostic implications.     

Microbial community analysis of pH 4 thermal springs in Yellowstone National Park

Extremophiles - The pH of the majority of thermal springs in Yellowstone National Park (YNP) is from 1 to 3 and 6 to 10; relatively few springs (~5%) have a pH range of 4–5. We used 16S rRNA...     

Genetic diversity of root-nodulating bacteria isolated from pea (Pisum sativum) in subtropical regions of China

Diversity of 42 isolates from effective nodules of Pisum sativum in different geographical regions of China were studied using 16S rRNA gene RFLP patterns, 16S rRNA sequencing, 16S–23S rRNA inter-genic spacer (IGS) region RFLP patterns and G-C rich random amplified polymorphic DNA (RAPD). The isolates were distributed in two groups on the basis of their 16S rRNA gene RFLP patterns. The 16S rRNA gene sequences of strains from 16S rRNA gene RFLP patterns group I were very closely related (identities higher than 99.5%) to Rhizobium leguminosarum USDA 2370. Group II consisting of WzP3 and WzP15 was closely related to Rhizobium etli CFN42. The analysis of the 16S–23S IGS RFLP pat-terns divided the isolates into 18 genotypes and four groups. Group I was clustered with R. legumino-sarum USDA2370. Group II consisted of YcP2, YcP3 and CqP7. The strains of group III were distributed abroad. Group IV consisted of WzP3, WzP15 and R. etli CFN42. RAPD divided the isolates into nine clusters in which group IV only consisted of YcP2 and the strains of group V and IX were from Wenzhou and Xiantao, respectively. This assay demonstrated the geographical effect on genetic diversity of pea rhizobia.     

The asteralean affinity of the Mauritian Roussea (Rousseaceae)

Roussea , a monotypic genus endemic to Mauritius, has for a long time been associated with Brexia (Celastraceae). Recently, it has been shown that Roussea is placed correctly in the mainly Australasian Asterales, but the sister group to Roussea has not been unequivocally identified. Cladistic analysis of the chloroplast genes ndhF and rbcL identifies the sister group to.Roussea as Carpodetaceae. Recognizing this relationship, the monotypic Rousseaceae is merged with Carpodetaceae into Rousseaceae s.l. comprising two subfamilies. This group is characterized by many-locular ovaries and similarities in the appearance of the petals. Rousseaceae s.l. exhibit a disjunct distribution in Mauritius, East Australia, New Zealand and New Guinea     

Responses of benthic macroinvertebrate communities to natural geothermal discharges in Yellowstone National Park,USA

We examined responses of benthic macroinvertebrate communities to natural geothermal discharges in 32 streams in Yellowstone National Park (YNP), USA. Geothermal discharges played a major role in structuring benthic communities in YNP, as downstream communities were characterized by low species richness, reduced abundance of EPT taxa and increased abundance of tolerant caddisflies (Trichoptera), chironomids and non-insects. While some taxa were a subset of tolerant organisms that were also common at references sites, others (the damselfly Argia sp., the caddisfly Oxyethira sp. and the exotic New Zealand mudsnail Potamopyrgus antipodarum Gray 1843) were found almost exclusively in geothermal streams. Because geothermal waters are a common feature of YNP, monitoring programs designed to assess long-term status and trends of Yellowstone’s aquatic ecosystems must account for the influence of these discharges. To separate geothermal effects from other potential anthropogenic disturbances in YNP (e.g., atmospheric deposition, road construction, wastewater, global change), we developed a multimetric index based on responses of benthic communities to geothermal discharges. Streams were placed into one of four geothermal categories based on conductivity (reference = < 150 μS/cm; low, moderate and high = 151–300, 301–600 and >600 μS/cm, respectively). The index clearly distinguished among these categories and showed a well-defined threshold response to geothermal effects at very low levels of conductivity. Although the index was specific to geothermal effects, the approach used to develop the index has broad applicability for other systems where impacts of stressors must be assessed within the context of natural environmental gradients. Our findings may provide important insights into how benthic macroinvertebrate communities respond to global change. Reduced discharge and warmer temperatures predicted for Rocky Mountain streams may favor the establishment and expansion of exotic species such as New Zealand mudsnails (P. antipodarum), which are highly tolerant of geothermal influences.     

Geoarchaeota: a new candidate phylum in the Archaea from high-temperature acidic iron mats in Yellowstone National Park

Geothermal systems in Yellowstone National Park (YNP) provide an outstanding opportunity to understand the origin and evolution of metabolic processes necessary for life in extreme environments including low pH, high temperature, low oxygen and elevated concentrations of reduced iron. Previous phylogenetic studies of acidic ferric iron mats from YNP have revealed considerable diversity of uncultivated and undescribed archaea. The goal of this study was to obtain replicate de novo genome assemblies for a dominant archaeal population inhabiting acidic iron-oxide mats in YNP. Detailed analysis of conserved ribosomal and informational processing genes indicates that the replicate assemblies represent a new candidate phylum within the domain Archaea referred to here as ‘Geoarchaeota'' or ‘novel archaeal group 1 (NAG1)''. The NAG1 organisms contain pathways necessary for the catabolism of peptides and complex carbohydrates as well as a bacterial-like Form I carbon monoxide dehydrogenase complex likely used for energy conservation. Moreover, this novel population contains genes involved in the metabolism of oxygen including a Type A heme copper oxidase, a bd-type terminal oxidase and a putative oxygen-sensing protoglobin. NAG1 has a variety of unique bacterial-like cofactor biosynthesis and transport genes and a Type3-like CRISPR system. Discovery of NAG1 is critical to our understanding of microbial community structure and function in extant thermophilic iron-oxide mats of YNP, and will provide insight regarding the evolution of Archaea in early Earth environments that may have important analogs active in YNP today.