Fungal disease and temperature alter skin microbiome structure in an experimental salamander system

Pathogens compete with host microbiomes for space and resources. Their shared environment impacts pathogen–microbiome–host interactions, which can lead to variation in disease outcome. The skin microbiome of red‐backed salamanders (Plethodon cinereus) can reduce infection by the pathogen Batrachochytrium dendrobatidis (Bd) at moderate infection loads, with high species richness and high abundance of competitors as putative mechanisms. However, it is unclear if the skin microbiome can reduce epizootic Bd loads across temperatures. We conducted a laboratory experiment to quantify skin microbiome and host responses (P. cinereus: n = 87) to Bd at mimicked epizootic loads across temperatures (13, 17 and 21°C). We quantified skin microbiomes using 16S rRNA gene metabarcoding and identified operational taxonomic units (OTUs) taxonomically similar to culturable bacteria known to kill Bd (anti‐Bd OTUs). Prior to pathogen exposure, temperature changed the microbiome (OTU richness decreased by 12% and the abundance of anti‐Bd OTUs increased by 18% per degree increase in temperature), but these changes were not predictive of disease outcome. After exposure, Bd changed the microbiome (OTU richness decreased by 0.1% and the abundance of anti‐Bd OTUs increased by 0.2% per 1% increase in Bd load) and caused high host mortality across temperatures (35/45: 78%). Temperature indirectly impacted microbiome change and mortality through its direct effect on pathogen load. We did not find support for the microbiome impacting Bd load or host survival. Our research reveals complex host, pathogen, microbiome and environmental interactions to demonstrate that during epizootic events the microbiome will be unlikely to reduce pathogen invasion, even for putatively Bd‐resistant species.     

Exploring Symbiodinium diversity and host specificity in Acropora corals from geographical extremes of Western Australia with 454 amplicon pyrosequencing

Scleractinian corals have demonstrated the ability to shuffle their endosymbiotic dinoflagellate communities (genus Symbiodinium) during periods of acute environmental stress. This has been proposed as a mechanism of acclimation, which would be increased by a diverse and flexible association with Symbiodinium. Conventional molecular techniques used to evaluate Symbiodinium diversity are unable to identify genetic lineages present at background levels below 10%. Next generation sequencing (NGS) offers a solution to this problem and can resolve microorganism diversity at much finer scales. Here we apply NGS to evaluate Symbiodinium diversity and host specificity in Acropora corals from contrasting regions of Western Australia. The application of 454 pyrosequencing allowed for detection of Symbiodinium operational taxonomic units (OTUs) occurring at frequencies as low as 0.001%, offering a 10 000‐fold increase in sensitivity compared to traditional methods. All coral species from both regions were overwhelmingly dominated by a single clade C OTU (accounting for 98% of all recovered sequences). Only 8.5% of colonies associated with multiple clades (clades C and D, or C and G), suggesting a high level of symbiont specificity in Acropora assemblages in Western Australia. While only 40% of the OTUs were shared between regions, the dominance of a single OTU resulted in no significant difference in Symbiodinium community structure, demonstrating that the coral‐algal symbiosis can remain stable across more than 15° of latitude and a range of sea surface temperature profiles. This study validates the use of NGS platforms as tools for providing fine‐scale estimates of Symbiodinium diversity and can offer critical insight into the flexibility of the coral‐algal symbiosis.     

Concordance of bacterial communities of two tick species and blood of their shared rodent host

High‐throughput sequencing is revealing that most macro‐organisms house diverse microbial communities. Of particular interest are disease vectors whose microbiome could potentially affect pathogen transmission and vector competence. We investigated bacterial community composition and diversity of the ticks Dermacentor variabilis (n = 68) and Ixodes scapularis (n = 15) and blood of their shared rodent host, Peromyscus leucopus (n = 45) to quantify bacterial diversity and concordance. The 16S rRNA gene was amplified from genomic DNA from field‐collected tick and rodent blood samples, and 454 pyrosequencing was used to elucidate their bacterial communities. After quality control, over 300 000 sequences were obtained and classified into 118 operational taxonomic units (OTUs, clustered at 97% similarity). Analysis of rarefied communities revealed that the most abundant OTUs were tick species‐specific endosymbionts, Francisella and Rickettsia, and the commonly flea‐associated bacterium Bartonella in rodent blood. An Arsenophonus and additional Francisella endosymbiont were also present in D. variabilis samples. Rickettsia was found in both tick species but not in rodent blood, suggesting that it is not transmitted during feeding. Bartonella was present in larvae and nymphs of both tick species, even those scored as unengorged. Relatively, few OTUs (e.g. Bartonella, Lactobacillus) were found in all sample types. Overall, bacterial communities from each sample type were significantly different and highly structured, independent of their dominant OTUs. Our results point to complex microbial assemblages inhabiting ticks and host blood including infectious agents, tick‐specific endosymbionts and environmental bacteria that could potentially affect arthropod‐vectored disease dynamics.     

Lacustrine Nostoc (Nostocales) and associated microbiome generate a new type of modern clotted microbialite

Microbialites are mineral formations formed by microbial communities that are often dominated by cyanobacteria. Carbonate microbialites, known from Proterozoic times through the present, are recognized for sequestering globally significant amounts of inorganic carbon. Recent ecological work has focused on microbial communities dominated by cyanobacteria that produce microbial mats and laminate microbialites (stromatolites). However, the taxonomic composition and functions of microbial communities that generate distinctive clotted microbialites (thrombolites) are less well understood. Here, microscopy and deep shotgun sequencing were used to characterize the microbiome (microbial taxa and their genomes) associated with a single cyanobacterial host linked by 16S sequences to Nostoc commune Vaucher ex Bornet & Flahault, which dominates abundant littoral clotted microbialites in shallow, subpolar, freshwater Laguna Larga in southern Chile. Microscopy and energy‐dispersive X‐ray spectroscopy suggested the hypothesis that adherent hollow carbonate spheres typical of the clotted microbialite begin development on the rigid curved outer surfaces of the Nostoc balls. A surface biofilm included >50 nonoxygenic bacterial genera (taxa other than Nostoc) that indicate diverse ecological functions. The Laguna Larga Nostoc microbiome included the sulfate reducers Desulfomicrobium and Sulfospirillum and genes encoding all known proteins specific to sulfate reduction, a process known to facilitate carbonate deposition by increasing pH. Sequences indicating presence of nostocalean and other types of nifH, nostocalean sulfide:ferredoxin oxidoreductase (indicating anoxygenic photosynthesis), and biosynthetic pathways for the secondary products scytonemin, mycosporine, and microviridin toxin were identified. These results allow comparisons with microbiota and microbiomes of other algae and illuminate biogeochemical roles of ancient microbialites.     

Diazotrophic diversity in the Caribbean coral, Montastraea cavernosa

Previous research on the Caribbean coral Montastraea cavernosa reported the presence of cyanobacterial endosymbionts and nitrogen fixation in orange, but not brown, colonies. We compared the diversity of nifH gene sequences between these two color morphs at three locations in the Caribbean and found that the nifH sequences recovered from M. cavernosa were consistent with previous studies on corals where members of both the α-proteobacteria and cyanobacteria were recovered. A number of nifH operational taxonomic units (OTUs) were significantly more abundant in the orange compared to the brown morphs, and one specific OTU (OTU 17), a cyanobacterial nifH sequence similar to others from corals and sponges and related to the cyanobacterial genus Cyanothece, was found in all orange morphs of M. cavernosa at all locations. The nifH diversity reported here, from a community perspective, was not significantly different between orange and brown morphs of M. cavernosa.     

Diurnal variations in the auto- and heterotrophic activity of cyanobacterial phycospheres (Gloeotrichia echinulata) and the identity of attached bacteria

1. We assessed the role of cyanobacterial–bacterial consortia (Gloeotrichia echinulata phycospheres) for net changes in inorganic carbon, primary production (PP) and secondary production in Lake Erken (Sweden). 2. At the time of sampling, large colonies of G. echinulata formed a massive bloom with abundances ranging from 102 colonies L^?1 in the pelagic zone to 5000 colonies L^?1 in shallow bays. These colonies and their surrounding phycospheres contributed between 17 and 92% of total PP, and phycosphere‐associated bacteria contributed between 8.5 and 82% of total bacterial secondary production. PP followed a diurnal cycle, whereas bacterial production showed no such pattern. Over a 24 h period, carbon dioxide measurements showed that the phycospheres were net autotrophic in the top layer of the water column, whereas they were net heterotrophic below 2 m depth. 3. Sequencing and phylogenetic analysis of 16S rRNA genes of attached bacteria revealed a diverse bacterial community that included populations affiliated with Proteobacteria, Bacteriodetes, Acidobacteria, Fusobacteria, Firmicutes, Verrucomicrobia, and other Cyanobacteria. 4. Compared with their planktonic counterparts, bacteria associated with cyanobacterial phycospheres had lower affinity for arginine, used as a model compound to assess uptake of organic compounds. 5. Extrapolation of our data to the water column of lake Erken suggests that microorganisms that were not associated with cyanobacteria dominated CO₂ production at the ecosystem scale during our experiments, as CO₂ fixation balanced CO₂ production in the cyanobacterial phycospheres.     

Fingerprinting and phylogeny of some heterocystous cyanobacteria using short tandemly repeated repetitive and highly iterated palindrome sequences

The presence of repeated DNA, viz. short tandemly repeated repetitive (STRR) and highly iterated palindrome (HIP) sequences was used as a typing technique for assessing genetic variability and phylogenetic relatedness of heterocystous cyanobacteria. Primers analogous to the STRR and HIP sequences were used to generate specific fingerprints for the twelve heterocystous cyanobacterial strains and a dendrogram was constructed. STRRmod and HIPTG primers revealed 100% polymorphism and yielded almost identical patterns. Anabaena sp. PCC 7120 clustered with Nostoc muscorum with both primers. Primer STRRmod supported the heterogeneity between Nostoc and Anabaena but HIPTG placed these two genera distinctly apart. STRRmod and HIPTG revealed that the members of the two orders were intermixed and thus suggesting a monophyletic origin of heterocystous cyanobacteria.     

Geographic mosaic of symbiont selectivity in a genus of epiphytic cyanolichens

In symbiotic systems, patterns of symbiont diversity and selectivity are crucial for the understanding of fundamental ecological processes such as dispersal and establishment. The lichen genus Nephroma (Peltigerales, Ascomycota) has a nearly cosmopolitan distribution and is thus an attractive model for the study of symbiotic interactions over a wide range of spatial scales. In this study, we analyze the genetic diversity of Nephroma mycobionts and their associated Nostoc photobionts within a global framework. The study is based on Internal Transcribed Spacer (ITS) sequences of fungal symbionts and tRNA^Leu (UAA) intron sequences of cyanobacterial symbionts. The full data set includes 271 Nephroma and 358 Nostoc sequences, with over 150 sequence pairs known to originate from the same lichen thalli. Our results show that all bipartite Nephroma species associate with one group of Nostoc different from Nostoc typically found in tripartite Nephroma species. This conserved association appears to have been inherited from the common ancestor of all extant species. While specific associations between some symbiont genotypes can be observed over vast distances, both symbionts tend to show genetic differentiation over wide geographic scales. Most bipartite Nephroma species share their Nostoc symbionts with one or more other fungal taxa, and no fungal species associates solely with a single Nostoc genotype, supporting the concept of functional lichen guilds. Symbiont selectivity patterns within these lichens are best described as a geographic mosaic, with higher selectivity locally than globally. This may reflect specific habitat preferences of particular symbiont combinations, but also the influence of founder effects.     

MOLECULAR CHARACTERIZATION OF THE DIVERSITY AND POTENTIAL TOXICITY OF CYANOBACTERIAL MATS IN TWO TROPICAL LAGOONS IN THE SOUTH PACIFIC OCEAN1

Marine benthic cyanobacteria in tropical areas have recently been associated with several human poisoning events. To enhance the characterization of these microorganisms and their potential toxicity, benthic cyanobacterial communities were sampled in the lagoons of two islands (Raivavae and Rurutu) located in French Polynesia where human poisoning events by seafood had been reported. The morphological appearance of the mats was used to identify four types of cyanobacterial mat. By a 16S rRNA sequencing approach, it appeared that these mats were usually dominated by a restricted number of operational taxonomic units (OTUs), which were closely related to Leptolyngbya, Oscillatoria, Hydrocoleum, and Anabaena sequences, as previously reported in other tropical lagoons. Interestingly, we determined that these dominant filamentous OTUs were associated in the mats with other cyanobacteria, including unicellular species. By using a population genetic approach based on the sequencing of the internally transcribed spacer (ITS) of the rRNA operon, we found a very restricted genetic diversity in the most common OTU, which displayed a high sequence similarity with Leptolyngbya sp. In addition, there was no geographic differentiation at various spatial scales in the distribution of the different genotypes, suggesting that this species is able to spread over large distances. Finally, PCR screening of genes involved in the biosynthesis of known cyanotoxins revealed the presence of the saxitoxin gene (stxG) in two mats containing a mix of filamentous and unicellular cyanobacterial species.     

Developmental patterns related to nitrogen fixation in theNostoc-Gunnera magellanica Lam. symbiosis

Developmental patterns related to nitrogen fixation in the heterocystous cyanobacteriumNostoc harboured in distinct colonies along the stem ofGunnera magellanica Lam. plantlets were examined using successive plant sections. Pronounced morphological, physiological and biochemical alterations in the cyanobacterium were demonstrated. Close to the growing apex the cyanobacterial biomass, contained in smallGunnera cells, was low and consisted mostly of vegetative cells showing a high density of different storage structures except for cyanophycin granules. In contrast, both the total and specific nitrogenase activity and the relative nitrogenase protein level were at maximum within this part; while the frequency of heterocysts increased from zero to 30% within the same area. The nitrogenase protein was localized only in the heterocysts throughout the plant. Further down theGunnera stem there was a progressive increase in both the cyanobacterial biomass and the heterocyst frequency, which finally constituted about 60% of the cyanobacterial cell population. Throughout this part of the stem, cyanophycin granules were frequent in the vegetativeNostoc cells. At the base of the stem, degeneratedNostoc cells dominated and the nitrogenase activity was close to zero, although the nitrogenase protein remained. Degeneration of theNostoc cells and leaf shedding coincided. Both intact plants (approx. 20 mm in height) and plant stem sections (2 mm in length) showed substantial nitrogenase activity, although sectioning caused a 30% reduction in total nitrogenase activity.     

Biological and chemical factors driving the temporal distribution of cyanobacteria and heterotrophic bacteria in a eutrophic lake (West Lake,China)

Physico-chemical parameters, hydrological conditions, and microbial interactions can affect the growth and persistence of cyanobacteria, but the interacting effects among these bloom-forming factors are still poorly known. This hampers our capacity to predict the occurrence of cyanobacterial bloom accurately. Here, we studied the relationship between temperature, N and P cycles, and the microbial community abundance and diversity at 0.5 m under the surface of West Lake (China) from January 21 to November 20, 2015, in order to better understand the key factors regulating temporal changes in the cyanobacterial community. Using high throughput sequencing of the 16S rRNA gene V3-V4 region, we studied the diversity and abundance of bacteria. In parallel, we measured physico-chemical parameters and followed the abundance of key genes involved in N fixation, denitrification, and nutrient uptake. Multivariate analyses suggest that P concentration and water temperature are the key factors controlling the outbreak of summer cyanobacterial bloom. RT-qPCR analyses of the bacterial community and measurements of the copy number of denitrification-related gene (nirK, nosZ, nirS) show that denitrification potential and denitrifying bacteria relative abundance (Pseudomonas and Bacillus) increased in concert with diazotrophic cyanobacterial genera (Anabaena, Nostoc, Aphanizomenon flos-aquae) and the common bloom-forming non-diazotrophic cyanobacterium genus Microcystis. The present study brings new insights on the complex interplay between physico-chemical parameters, heterotrophic bacterial community composition, nitrogen cycle, and cyanobacteria dominance in a eutrophic lake.

     

The microbiome associated with two Synechococcus ribotypes at different levels of ecological interaction

Planktonic cyanobacteria belonging to the genus Synechococcus are ubiquitously distributed in marine and fresh waters, substantially contributing to total carbon fixation on a global scale. While their ecological relevance is acknowledged, increasing resolution in molecular techniques allows disentangling cyanobacteria's role at the micro‐scale, where complex microbial interactions may drive the overall community assembly. The interplay between phylogenetically different Synechococcus clades and their associated bacterial communities can affect their ecological fate and susceptibility to protistan predation. In this study, we experimentally promoted different levels of ecological interaction by mixing two Synechococcus ribotypes (MW101C3 and LL) and their associated bacteria, with and without a nanoflagellate grazer (Poterioochromonas sp.) in laboratory cultures. The beta‐diversity of the Synechococcus‐associated microbiome in laboratory cultures indicated that the presence of the LL ribotype was the main factor determining community composition changes (41% of total variance), and prevailed over the effect of protistan predation (18% of total variance). Our outcomes also showed that species coexistence and predation may promote microbial diversity, thus highlighting the underrated ecological relevance of such micro‐scale factors.     

Genetic variability,community structure,and horizontal transfer of endosymbionts among three Asia II‐Bemisia tabaci mitotypes in Pakistan

Endosymbionts associated with the whitefly Bemisia tabaci cryptic species are known to contribute to host fitness and environmental adaptation. The genetic diversity and population complexity were investigated for endosymbiont communities of B. tabaci occupying different micro‐environments in Pakistan. Mitotypes of B. tabaci were identified by comparative sequence analysis of the mitochondria cytochrome oxidase I (mtCOI) gene sequence. Whitefly mitotypes belonged to the Asia II‐1, ‐5, and ‐7 mitotypes of the Asia II major clade. The whitefly–endosymbiont communities were characterized based on 16S ribosomal RNA operational taxonomic unit (OTU) assignments, resulting in 43 OTUs. Most of the OTUs occurred in the Asia II‐1 and II‐7 mitotypes (r² = .9, p < .005), while the Asia II‐5 microbiome was less complex. The microbiome OTU groups were mitotype‐specific, clustering with a basis in phylogeographical distribution and the corresponding ecological niche of their whitefly host, suggesting mitotype‐microbiome co‐adaptation. The primary endosymbiont Portiera was represented by a single, highly homologous OTU (0%–0.67% divergence). Two of six Arsenophonus OTUs were uniquely associated with Asia II‐5 and ‐7, and one occurred exclusively in Asia II‐1, two only in Asia II‐5, and one in both Asia II‐1 and ‐7. Four other secondary endosymbionts, Cardinium, Hemipteriphilus, Rickettsia, and Wolbachia OTUs, were found at ≤29% frequencies. The most prevalent Arsenophonus OTU was found in all three Asia II mitotypes (55% frequency), whereas the same strain of Cardinium and Wolbachia was found in both Asia II‐1 and ‐5, and a single Hemipteriphilus OTU occurred in Asia II‐1 and ‐7. This pattern is indicative of horizontal transfer, suggestive of a proximity between mitotypes sufficient for gene flow at overlapping mitotype ecological niches.     

Phylogenetic diversity of bacteria associated with the endemic freshwater sponge Lubomirskia baicalensis

In this study, for the first time the diversity of bacteria associated with the endemic freshwater sponge Lubomirskia baicalensis collected from the Sousern Basin of Lake Baikal was investigated employing cultivation-independent approaches. In total, 102 bacterial 16S rRNA clones were screened using restriction fragment length polymorphism (RFLP) and 30 were selected for sequencing. BLASTN and phylogenetic analysis based on near full length 16S rDNA sequences showed that 22 operational taxonomic units (OTUs) were clustered in six known phyla: Actinobacteria (8 OTUs), alpha-Proteobacteria (4 OTUs), beta-Proteobacteria (4 OTUs), Verrucomicrobia (4 OTUs), Nitrospiracea (1 OTU) and Bacteroidetes (1 OTU). Remarkably all phylotypes were affiliated to uncultured microorganisms, however, all alpha-Proteobacteria sequences were closely related to bacteria derived from the freshwater sponge Spongilla lacustris. Our results reveal a high diversity in the L. baicalensis bacterial community and provide an insight into microbial ecology and diversity within freshwater sponges inhabiting the ancient Lake Baikal ecosystem.     

Abdominal microbial communities in ants depend on colony membership rather than caste and are linked to colony productivity

Gut bacteria aid their host in digestion and pathogen defense, and bacterial communities that differ in diversity or composition may vary in their ability to do so. Typically, the gut microbiomes of animals living in social groups converge as members share a nest environment and frequently interact. Social insect colonies, however, consist of individuals that differ in age, physiology, and behavior, traits that could affect gut communities or that expose the host to different bacteria, potentially leading to variation in the gut microbiome within colonies. Here we asked whether bacterial communities in the abdomen of Temnothorax nylanderi ants, composed largely of the gut microbiome, differ between different reproductive and behavioral castes. We compared microbiomes of queens, newly eclosed workers, brood carers, and foragers by high‐throughput 16S rRNA sequencing. Additionally, we sampled individuals from the same colonies twice, in the field and after 2 months of laboratory housing. To disentangle the effects of laboratory environment and season on microbial communities, additional colonies were collected at the same location after 2 months. There were no large differences between ant castes, although queens harbored more diverse microbial communities than workers. Instead, we found effects of colony, environment, and season on the abdominal microbiome. Interestingly, colonies with more diverse communities had produced more brood. Moreover, the queens' microbiome composition was linked to egg production. Although long‐term coevolution between social insects and gut bacteria has been repeatedly evidenced, our study is the first to find associations between abdominal microbiome characteristics and colony productivity in social insects.     

Coexisting cryptic species of the Litoditis marina complex (Nematoda) show differential resource use and have distinct microbiomes with high intraspecific variability

Differences in resource use or in tolerances to abiotic conditions are often invoked as potential mechanisms underlying the sympatric distribution of cryptic species. Additionally, the microbiome can provide physiological adaptations of the host to environmental conditions. We determined the intra‐ and interspecific variability of the microbiomes of three cryptic nematode species of the Litoditis marina species complex that co‐occur, but show differences in abiotic tolerances. Roche 454 pyrosequencing of the microbial 16S rRNA gene revealed distinct bacterial communities characterized by a substantial diversity (85–513 OTUs) and many rare OTUs. The core microbiome of each species contained only very few OTUs (2–6), and four OTUs were identified as potentially generating tolerance to abiotic conditions. A controlled experiment in which nematodes from two cryptic species (Pm1 and Pm3) were fed with either an E. coli suspension or a bacterial mix was performed, and the 16S rRNA gene was sequenced using the MiSeq technology. OTU richness was 10‐fold higher compared to the 454 data set and ranged between 1118 and 7864. This experiment confirmed the existence of species‐specific microbiomes, a core microbiome with few OTUs, and high interindividual variability. The offered food source affected the bacterial community and illustrated different feeding behaviour between the cryptic species, with Pm3 exhibiting a higher degree of selective feeding than Pm1. Morphologically similar species belonging to the same feeding guild (bacterivores) can thus have substantial differences in their associated microbiomes and feeding strategy, which in turn may have important ramifications for biodiversity–ecosystem functioning relationships.     

Host population genetics and biogeography structure the microbiome of the sponge Cliona delitrix

Sponges occur across diverse marine biomes and host internal microbial communities that can provide critical ecological functions. While strong patterns of host specificity have been observed consistently in sponge microbiomes, the precise ecological relationships between hosts and their symbiotic microbial communities remain to be fully delineated. In the current study, we investigate the relative roles of host population genetics and biogeography in structuring the microbial communities hosted by the excavating sponge Cliona delitrix. A total of 53 samples, previously used to demarcate the population genetic structure of C. delitrix, were selected from two locations in the Caribbean Sea and from eight locations across the reefs of Florida and the Bahamas. Microbial community diversity and composition were measured using Illumina‐based high‐throughput sequencing of the 16S rRNA V4 region and related to host population structure and geographic distribution. Most operational taxonomic units (OTUs) specific to Cliona delitrix microbiomes were rare, while other OTUs were shared with congeneric hosts. Across a large regional scale (>1,000 km), geographic distance was associated with considerable variability of the sponge microbiome, suggesting a distance–decay relationship, but little impact over smaller spatial scales (<300 km) was observed. Host population structure had a moderate effect on the structure of these microbial communities, regardless of geographic distance. These results support the interplay between geographic, environmental, and host factors as forces determining the community structure of microbiomes associated with C. delitrix. Moreover, these data suggest that the mechanisms of host regulation can be observed at the population genetic scale, prior to the onset of speciation.     

BIOLOGICAL NITROGEN INFLUX IN AN OHIO RELICT PRAIRIE

The principal contributors of biologically fixed N in natural grassland ecosystems appear to be asymbiotic bacteria and heterocystous cyanobacteria. The environmental factors of light, moisture, and temperature play important roles in the magnitude of the N₂-fixation activity. Biological N₂-fixation was measured in the Elizabeth's Prairie section of the Lynx Prairie Preserve, Adams County, Ohio, during 15 site visits beginning 29 March through 8 November 1980. In situ N₂-fixation activity was measured using the acetylene-reduction technique. The percentage cover of cyanobacterial colonies (Nostoc sp.) was determined using Point-Frame Analysis. Soil and air temperatures and soil water potentials also were measured. Intact soil cores with a surface cover of Nostoc were collected and returned to the laboratory to quantify the effect of decreasing water potential on the N₂(C₂H₂)ase activity of Nostoc. The N₂(C₂H₂)ase activity of Nostoc on the intact soil cores displayed a linear response of approximately 10% decrease in N₂(C₂H₂)ase activity per one bar decrease in soil water potential. The cyanobacteria contributed almost all of the biologically fixed N at the site until late June. From late June through to mid September, heterotrophic diazotrophs played the major role in the N₂-fixation activity. These changes are attributed to fluctuations in Nostoc sp. colony cover, temperature, and soil water potentials. Extrapolation of the measured rates, and assuming an average of 10 hr per day of activity, Nostoc sp. is shown to have contributed 4.60 ± 1.17 kg N ha⁻¹ yr⁻¹. Heterotrophic diazotrophs contributed an estimated 3.19 ± 1.18 kg N ha⁻¹ yr⁻¹. The total biological N₂-fixation for the site was calculated at 8.2 ± 2.55 kg N ha⁻¹ yr⁻¹, from additional measurements which estimated total diazotrophic activity of the site. These rates of N₂-fixation are among the highest reported for temperate grassland habitats.     

Algal and Fungal Diversity in Antarctic Lichens

The composition of lichen ecosystems except mycobiont and photobiont has not been evaluated intensively. In addition, recent studies to identify algal genotypes have raised questions about the specific relationship between mycobiont and photobiont. In the current study, we analyzed algal and fungal community structures in lichen species from King George Island, Antarctica, by pyrosequencing of eukaryotic large subunit (LSU) and algal internal transcribed spacer (ITS) domains of the nuclear rRNA gene. The sequencing results of LSU and ITS regions indicated that each lichen thallus contained diverse algal species. The major algal operational taxonomic unit (OTU) defined at a 99% similarity cutoff of LSU sequences accounted for 78.7–100% of the total algal community in each sample. In several cases, the major OTUs defined by LSU sequences were represented by two closely related OTUs defined by 98% sequence similarity of ITS domain. The results of LSU sequences indicated that lichen‐associated fungi belonged to the Arthoniomycetes, Eurotiomycetes, Lecanoromycetes, Leotiomycetes, and Sordariomycetes of the Ascomycota, and Tremellomycetes and Cystobasidiomycetes of the Basidiomycota. The composition of major photobiont species and lichen‐associated fungal community were mostly related to the mycobiont species. The contribution of growth forms or substrates on composition of photobiont and lichen‐associated fungi was not evident.