Deep‐branching Novel Lineages and High Diversity of Haptophytes in the Skagerrak (Norway) Uncovered by 454 Pyrosequencing

Microalgae in the division Haptophyta may be difficult to identify to species by microscopy because they are small and fragile. Here, we used high‐throughput sequencing to explore the diversity of haptophytes in outer Oslofjorden, Skagerrak, and supplemented this with electron microscopy. Nano‐ and picoplanktonic subsurface samples were collected monthly for 2 yr, and the haptophytes were targeted by amplification of RNA/cDNA with Haptophyta‐specific 18S ribosomal DNA V4 primers. Pyrosequencing revealed higher species richness of haptophytes than previously observed in the Skagerrak by microscopy. From ca. 400,000 reads we obtained 156 haptophyte operational taxonomic units (OTUs) after rigorous filtering and 99.5% clustering. The majority (84%) of the OTUs matched environmental sequences not linked to a morphological species, most of which were affiliated with the order Prymnesiales. Phylogenetic analyses including Oslofjorden OTUs and available cultured and environmental haptophyte sequences showed that several of the OTUs matched sequences forming deep‐branching lineages, potentially representing novel haptophyte classes. Pyrosequencing also retrieved cultured species not previously reported by microscopy in the Skagerrak. Electron microscopy revealed species not yet genetically characterised and some potentially novel taxa. This study contributes to linking genotype to phenotype within this ubiquitous and ecologically important protist group, and reveals great, unknown diversity.     

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.     

Patterns of Symbiodinium (Dinophyceae) diversity and assemblages among diverse hosts and the coral reef environment of Lizard Island,Australia

Large‐scale environmental disturbances may impact both partners in coral host–Symbiodinium systems. Elucidation of the assembly patterns in such complex and interdependent communities may enable better prediction of environmental impacts across coral reef ecosystems. In this study, we investigated how the community composition and diversity of dinoflagellate symbionts in the genus Symbiodinium were distributed among 12 host species from six taxonomic orders (Actinaria, Alcyonacea, Miliolida, Porifera, Rhizostoma, Scleractinia) and in the reef water and sediments at Lizard Island, Great Barrier Reef before the 3rd Global Coral Bleaching Event. 454 pyrosequencing of the ITS2 region of Symbiodinium yielded 83 operational taxonomic units (OTUs) at a 97% similarity cut‐off. Approximately half of the Symbiodinium OTUs from reef water or sediments were also present in symbio. OTUs belonged to six clades (A‐D, F‐G), but community structure was uneven. The two most abundant OTUs (100% matches to types C1 and A3) comprised 91% of reads and OTU C1 was shared by all species. However, sequence‐based analysis of these dominant OTUs revealed host species specificity, suggesting that genetic similarity cut‐offs of Symbiodinium ITS2 data sets need careful evaluation. Of the less abundant OTUs, roughly half occurred at only one site or in one species and the background Symbiodinium communities were distinct between individual samples. We conclude that sampling multiple host taxa with differing life history traits will be critical to fully understand the symbiont diversity of a given system and to predict coral ecosystem responses to environmental change and disturbance considering the differential stress response of the taxa within.     

Geomicrobiology of deep-sea deposits: estimating community diversity from low-temperature seafloor rocks and minerals

The role of deep‐sea microbial communities in the weathering of hydrothermal vent deposits is assessed using mineralogical and molecular biological techniques. The phylogenetic diversity of varied deep‐sea bare rock habitats associated with the oceanic spreading centre at the Juan de Fuca Ridge was accessed using restriction fragment length polymorphism (RFLP) and rDNA sequencing. The mineralogical composition of the deposits used for phylogenetic analysis was determined by X‐ray diffraction in order to determine the proportion and composition of sulphide minerals, and to determine degree of alteration associated with each sample. RFLP analyses resulted in 15 unique patterns, or Operational Taxonomic Units (OTUs). Most environments examined were dominated by only one or two OTUs, which often comprised approximately 60% of the rDNA clones generated from that environment. Only one environment, the Mound, had a representative rDNA clone from every OTU identified in this study. For one other environment, ODP sediments, rDNA clones were all contained in a single OTU. The diversity of the microbial community is found to decrease with decreasing reactivity of the sulphide component in the samples and with increasing presence of alteration products. Phylogenetic analyses reveal that OTUs contain representatives of the epsilon‐, beta‐ and gamma‐subdivisions of the Proteobacteria. OTU1, which dominates clone libraries from every environment and is increasingly dominant with increasing rock alteration, is closely related to a group of chemolithoautotrophic iron‐oxidizing bacteria that have been recently isolated from the deep sea. The apparent abundance and widespread distribution within the samples examined of the putative iron‐oxidizing bacteria that may be represented by OTU1 suggests that this physiological group could play an important role in rock‐weathering and carbon fixation at the seafloor.     

Methanogen diversity and community composition in peatlands of the central to northern Appalachian Mountain region, North America

Methanogenic archaea are ubiquitous in peat soils; however, their diversity and distributions within and among peatland ecosystems are not well known. We used comprehensive clone libraries of 16S rRNA gene sequences to investigate spatial patterns in diversity (richness, evenness of taxa) and composition (taxonomic, phylogenetic) of the methanogenic community in six peatlands arrayed 775?km from eastern Ontario, Canada to West Virginia, USA. Five sites were Sphagnum (moss) and shrub dominated; one site was sedge dominated; and, potential rates of methane (CH₄) production ranged from 15 to 450?nmol/g?day. The gradient allowed us to examine influences of site conditions, site history, and climate on community composition. The region had representatives of methanogens from four taxonomic orders. We observed 29 operationally defined units (OTUs) based on >97% sequence identity. One OTU accounted for 43% of all clones, whereas 15 OTUs were rare with <1% of the total number of clones. The number of OTUs per site ranged from 4 to 21, and statistical analysis suggested diversity of 4–43 per site. Eighteen of the OTUs were endemic to one site; albeit, most endemics occurred in the sedge dominated site. One OTU was cosmopolitan, occurring in all six sites. We found a positive relationship between methanogen diversity and rates of CH₄ production per site (Pearson r?=?0.93). Turnover in community composition between sites was weakly related to geographic distance between sites, whereas variation in soil pH and annual temperature played larger roles. About 50% of the variation in community composition was unexplained by distance, pH, mean climate, and site age. We conclude that methanogen diversity in peatlands of the central Appalachian region is shaped by present-day environmental conditions, suggesting an influence of impending climatic and environmental changes.     

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.     

Comparison of morphological,DNA barcoding,and metabarcoding characterizations of freshwater nematode communities

Biomonitoring approaches and investigations of many ecological questions require assessments of the biodiversity of a given habitat. Small organisms, ranging from protozoans to metazoans, are of great ecological importance and comprise a major share of the planet's biodiversity but they are extremely difficult to identify, due to their minute body sizes and indistinct structures. Thus, most biodiversity studies that include small organisms draw on several methods for species delimitation, ranging from traditional microscopy to molecular techniques. In this study, we compared the efficiency of these methods by analyzing a community of nematodes. Specifically, we evaluated the performances of traditional morphological identification, single‐specimen barcoding (Sanger sequencing), and metabarcoding in the identification of 1500 nematodes from sediment samples. The molecular approaches were based on the analysis of the 28S ribosomal large and 18S small subunits (LSU and SSU). The morphological analysis resulted in the determination of 22 nematode species. Barcoding identified a comparable number of operational taxonomic units (OTUs) based on 28S rDNA (n = 20) and fewer OTUs based on 18S rDNA (n = 12). Metabarcoding identified a higher OTU number but fewer amplicon sequence variants (AVSs) (n = 48 OTUs, n = 17 ASVs for 28S rDNA, and n = 31 OTUs, n = 6 ASVs for 18S rDNA). Between the three approaches (morphology, barcoding, and metabarcoding), only three species (13.6%) were shared. This lack of taxonomic resolution hinders reliable community identifications to the species level. Further database curation will ensure the effective use of molecular species identification.     

Haptophyte Diversity and Vertical Distribution Explored by 18S and 28S Ribosomal RNA Gene Metabarcoding and Scanning Electron Microscopy

Haptophyta encompasses more than 300 species of mostly marine pico‐ and nanoplanktonic flagellates. Our aims were to investigate the Oslofjorden haptophyte diversity and vertical distribution by metabarcoding, and to improve the approach to study haptophyte community composition, richness and proportional abundance by comparing two rRNA markers and scanning electron microscopy (SEM). Samples were collected in August 2013 at the Outer Oslofjorden, Norway. Total RNA/cDNA was amplified by haptophyte‐specific primers targeting the V4 region of the 18S, and the D1‐D2 region of the 28S rRNA. Taxonomy was assigned using curated haptophyte reference databases and phylogenetic analyses. Both marker genes showed Chrysochromulinaceae and Prymnesiaceae to be the families with highest number of Operational Taxonomic Units (OTUs), as well as proportional abundance. The 18S rRNA data set also contained OTUs assigned to eight supported and defined clades consisting of environmental sequences only, possibly representing novel lineages from family to class. We also recorded new species for the area. Comparing coccolithophores by SEM with metabarcoding shows a good correspondence with the 18S rRNA gene proportional abundances. Our results contribute to link morphological and molecular data and 28S to 18S rRNA gene sequences of haptophytes without cultured representatives, and to improve metabarcoding methodology.     

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.     

Statistical approaches for estimating actinobacterial diversity in marine sediments

Bacterial diversity in a deep-sea sediment was investigated by constructing actinobacterium-specific 16S ribosomal DNA (rDNA) clone libraries from sediment sections taken 5 to 12, 15 to 18, and 43 to 46 cm below the sea floor at a depth of 3,814 m. Clones were placed into operational taxonomic unit (OTU) groups with >/= 99% 16S rDNA sequence similarity; the cutoff value for an OTU was derived by comparing 16S rRNA homology with DNA-DNA reassociation values for members of the class Actinobacteria. Diversity statistics were used to determine how the level of dominance, species richness, and genetic diversity varied with sediment depth. The reciprocal of Simpson's index (1/D) indicated that the pattern of diversity shifted toward dominance from uniformity with increasing sediment depth. Nonparametric estimation of the species richness in the 5- to 12-, 15- to 18-, and 43- to 46-cm sediment sections revealed a trend of decreasing species number with depth, 1,406, 308, and 212 OTUs, respectively. Application of the LIBSHUFF program indicated that the 5- to 12-cm clone library was composed of OTUs significantly (P = 0.001) different from those of the 15- to 18- and 43- to 46-cm libraries. F(ST) and phylogenetic grouping of taxa (P tests) were both significant (P < 0.00001 and P < 0.001, respectively), indicating that genetic diversity decreased with sediment depth and that each sediment community harbored unique phylogenetic lineages. It was also shown that even nonconservative OTU definitions result in severe underestimation of species richness; unique phylogenetic clades detected in one OTU group suggest that OTUs do not correspond to real ecological groups sensu Palys (T. Palys, L. K. Nakamura, and F. M. Cohan, Int. J. Syst. Bacteriol. 47:1145-1156, 1997). Mechanisms responsible for diversity and their implications are discussed.     

Comparison of the diversity,composition, and host recurrence of xylariaceous endophytes in subtropical,cool temperate,and subboreal regions in Japan

The diversity, composition, and host recurrence of endophytic fungi in the Xylariaceae were compared in subtropical (ST), cool temperate (CT), and subboreal forests (SB) in Japan based on the 28S ribosomal DNA sequences from fungal isolates. A total of 610 isolates were obtained from the leaves of 167 tree species in three sites, which were classified into 42 operational taxonomic units (OTUs) at the 99 % similarity level of the 28S rDNA sequence. ST, CT, and SB yielded 31, 13, and three OTUs, respectively. The OTU richness, diversity, and evenness of fungal communities were in the order: ST > CT > SB. The 42 OTUs were assigned to nine genera in the Xylariaceae: Xylaria, Annulohypoxylon, Anthostomella, Biscogniauxia, Nemania, Hypoxylon, Muscodor, Daldinia, and Rosellinia. Xylarioid isolates in the subfamily Xylarioideae outnumbered Hypoxyloid isolates in the subfamily Hypoxyloideae in ST and CT, whereas the opposite was found in SB. Sørensen’s quotient of similarity was generally low between the three sites. Host recurrence of fungal OTUs was evaluated with the degree of specialization of interaction network between xylariaceous endophytes and plant species and compared between the three sites. We found that the networks in the three sites showed a significantly higher degree of specialization than simulated networks, where partners were associated randomly. Permutational multivariate analyses of variance indicated that plant family and leaf trait significantly affected the OTU composition in ST, which can account for the specialization of interaction network and host recurrence of xylariaceous endophytes.     

Simulated nitrogen deposition affects community structure of arbuscular mycorrhizal fungi in northern hardwood forests

Our previous investigation found elevated nitrogen deposition caused declines in abundance of arbuscular mycorrhizal fungi (AMF) associated with forest trees, but little is known about how nitrogen affects the AMF community composition and structure within forest ecosystems. We hypothesized that N deposition would lead to significant changes in the AMF community structure. We studied the diversity and community structure of AMF in northern hardwood forests after more than 12 years of simulated nitrogen deposition. We performed molecular analyses on maple (Acer spp.) roots targeting the 18S rDNA region using the fungal‐specific primers AM1 and NS31. PCR products were cloned and identified using restriction fragment length polymorphism (RFLP) and sequencing. N addition significantly altered the AMF community structure, and Glomus group A dominated the AMF community. Some Glomus operational taxonomic units (OTUs) responded negatively to N inputs, whereas other Glomus OTUs and an Acaulospora OTU responded positively to N inputs. The observed effect on community structure implies that AMF species associated with maples differ in their response to elevated nitrogen. Given that functional diversity exists among AMF species and that N deposition has been shown to select less beneficial fungi in some ecosystems, this change in community structure could have implications for the functioning of this type of ecosystem.     

Bacterial diversity of the digestive gland of Sydney rock oysters,Saccostrea glomerata infected with the paramyxean parasite,Marteilia sydneyi

Aims: To determine whether the infestation by the protozoan paramyxean parasite, Marteilia sydneyi, changes the bacterial community of the digestive gland of Sydney rock oysters, Saccostrea glomerata. Methods and Results: Six 16S rDNA clone libraries were established from three M. sydneyi‐infected and three un‐infected oysters. Restriction enzyme analysis followed by sequencing representative clones revealed a total of 23 different operational taxonomic units (OTUs) in un‐infected oysters, comprising the major phyla: Firmicutes, Proteobacteria, Cyanobacteria and Spirocheates, where the clone distribution was 44, 36, 7 and 5%, respectively. Close to half of the OTUs are not closely related to any other hitherto determined sequence. In contrast, S. glomerata infected by M. sydneyi had only one OTU present in the digestive gland. Phylogenetic analysis of the 16S rDNA sequence reveals that this dominant OTU, belonging to the α‐Proteobacteria, is closely related to a Rickettsiales‐like prokaryote (RLP). Conclusions: The microbiota of the digestive gland of Sydney rock oysters is changed by infection by M. sydneyi, becoming dominated by a RLP, and generally less diverse. The bacterial community of un‐infected S. glomerata differs from previous studies in that we identified the dominant taxa as Firmicutes and α‐Proteobacteria, rather than heterotrophic γ‐Proteobacteria. Significance and Impact of the Study: This is the first culture‐independent study of the microbiota of the digestive glands of edible oysters to the species level. The commercial viability of the Sydney rock oyster industry in Australia is currently threatened by Queensland Unknown disease and the changes in the bacterial community of S. glomerata corresponding with infection by M. sydneyi sheds further light on the link between parasite infection and mortality in this economically damaging disease.     

Divergence thresholds and divergent biodiversity estimates: can metabarcoding reliably describe zooplankton communities?

DNA metabarcoding is a promising method for describing communities and estimating biodiversity. This approach uses high‐throughput sequencing of targeted markers to identify species in a complex sample. By convention, sequences are clustered at a predefined sequence divergence threshold (often 3%) into operational taxonomic units (OTUs) that serve as a proxy for species. However, variable levels of interspecific marker variation across taxonomic groups make clustering sequences from a phylogenetically diverse dataset into OTUs at a uniform threshold problematic. In this study, we use mock zooplankton communities to evaluate the accuracy of species richness estimates when following conventional protocols to cluster hypervariable sequences of the V4 region of the small subunit ribosomal RNA gene (18S) into OTUs. By including individually tagged single specimens and “populations” of various species in our communities, we examine the impact of intra‐ and interspecific diversity on OTU clustering. Communities consisting of single individuals per species generated a correspondence of 59–84% between OTU number and species richness at a 3% divergence threshold. However, when multiple individuals per species were included, the correspondence between OTU number and species richness dropped to 31–63%. Our results suggest that intraspecific variation in this marker can often exceed 3%, such that a single species does not always correspond to one OTU. We advocate the need to apply group‐specific divergence thresholds when analyzing complex and taxonomically diverse communities, but also encourage the development of additional filtering steps that allow identification of artifactual rRNA gene sequences or pseudogenes that may generate spurious OTUs.     

Bacterial diversity in the rumen of Indian Surti buffalo (Bubalus bubalis), assessed by 16S rDNA analysis

Bacterial communities in buffalo rumen were characterized using a culture-independent approach for a pooled sample of rumen fluid from 3 adult Surti buffaloes. Buffalo rumen is likely to include species of various bacterial phyla, so 16S rDNA sequences were amplified and cloned from the sample. A total of 191 clones were sequenced and similarities to known 16S rDNA sequences were examined. About 62.82% sequences (120 clones) had >90% similarity to the 16S rDNA database sequences. Furthermore, about 34.03% of the sequences (65 clones) were 85–89% similar to 16S rDNA database sequences. For the remaining 3.14%, the similarity was lower than 85%. Phylogenetic analyses were also used to infer the makeup of bacterial communities in the rumen of Surti buffalo. As a result, we distinguished 42 operational taxonomic units (OTUs) based on unique 16S r DNA sequences: 19 OTUs affiliated to an unidentified group (45.23% of total OTUs), 11 OTUs of the phylum Firmicutes, also known as the low G+C group (26.19%), 7 OTUs of theCytophaga-Flexibacter-Bacteroides phylum (16.66%), 4 OTUs of Spirochaetes (9.52%), and 1 OTU of Actinobacteria (2.38%). These include 10 single-clone OTUs, so Good’s coverage (94.76%) of 16S rRNA libraries indicated that sequences identified in the libraries represent the majority of bacterial diversity present in rumen.     

Organic farming increases richness of fungal taxa in the wheat phyllosphere

Organic farming is often advocated as an approach to mitigate biodiversity loss on agricultural land. The phyllosphere provides a habitat for diverse fungal communities that are important for plant health and productivity. However, it is still unknown how organic farming affects the diversity of phyllosphere fungi in major crops. We sampled wheat leaves from 22 organically and conventionally cultivated fields in Sweden, paired based on their geographical location and wheat cultivar. Fungal communities were described using amplicon sequencing and real‐time PCR. Species richness was higher on wheat leaves from organically managed fields, with a mean of 54 operational taxonomic units (OTUs) compared with 40 OTUs for conventionally managed fields. The main components of the fungal community were similar throughout the 350‐km‐long sampling area, and seven OTUs were present in all fields: Zymoseptoria, Dioszegia fristingensis, Cladosporium, Dioszegia hungarica, Cryptococcus, Ascochyta and Dioszegia. Fungal abundance was highly variable between fields, 10³–10⁵ internal transcribed spacer copies per ng wheat DNA, but did not differ between cropping systems. Further analyses showed that weed biomass was the strongest explanatory variable for fungal community composition and OTU richness. These findings help provide a more comprehensive understanding of the effect of organic farming on the diversity of organism groups in different habitats within the agroecosystem.     

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.     

Spatial Variation in <Emphasis Type="Italic">Streptomyces</Emphasis> Genetic Composition and Diversity in a Prairie Soil

Understanding how microbial genotypes are arrayed in space is crucial for identifying local factors that may influence the spatial distribution of genetic diversity. In this study we investigated variation in 16S rDNA sequences and rep-PCR fingerprints of Streptomyces stains isolated from prairie soil among three locations and four soil depths. Substantial variation in Streptomyces OTU (operational taxonomic unit) and BOX-PCR fingerprint diversity was found among locations within a limited spatial area (1 m²). Further, phylogenetic lineages at each location were distinct. However, there was little variation in genetic diversity among isolates from different soil depths and similar phylogenetic lineages were found at each depth. Some clones were found at a localized scale while other clones had a relatively widespread distribution. There was poor correspondence between 16S rDNA groupings and rep-PCR fingerprint groupings. The finding of distinct phylogenetic lineages and the variation in spatial distribution of clones suggests that selection pressures may vary over the soil landscape.     

Bacterial diversity in the bulk soil and rhizosphere fractions of Lolium perenne and Trifolium repens as revealed by PCR restriction analysis of 16S rDNA

The rhizosphere of Trifolium repens and Lolium perenne was divided into three fractions: the bulk soil, the soil adhering to the roots and the washed roots (rhizoplane and endorhizosphere). After isolation and purification of DNA from these fractions, 16S rDNA was amplified by PCR and cloned to obtain a collection of 16S rRNA genes representative of the bacterial communities of these three fractions. The genes were then characterized by PCR restriction analysis. Each different profile was used to define an operational taxonomic unit (OTU). The numbers of OTUs and the numbers of clones among these OTUs allowed to calculate a diversity index. The number of OTUs decreased as root proximity increased and a few OTUs became dominant, resulting in a lower diversity index. In the root fraction of T. repens, the restriction profile of the dominant OTU matched the theoretical profile of the 16S rRNA gene of Rhizobium leguminosarum. This study showed that plant roots create a selective environment for microbial populations.     

Karst pools in subsurface environments: collectors of microbial diversity or temporary residence between habitat types

We studied bacterial diversity and community composition in three shallow pools of a Swiss karst cave system with contrasting hydrological and hydrochemical properties. The microbial assemblages in the pools were remarkably different, and only one operational taxonomic unit of 16S rRNA genes (OTU, 97% similarity) was shared between the three of them (total OTU number in all pools: 150). Unexpectedly high microbial phylotype richness was found even in the two pools without groundwater contact and with low concentrations of organic carbon and total cell numbers (< 10⁴ ml^?1). One of these seepage water fed systems harboured 15 distinct OTUs from several deeply branching lineages of the candidate phylum OP3, whereas representatives of this group were not detected in the other two pools. A tentative phylogeographic analysis of available OP3‐related sequences in the context of our data set revealed that there was generally little agreement between the habitats of origin of closely related sequence types. Two bacterial clades affiliated with the obligate methylamine utilizer Methylotenera mobilis were only found in the pool that was exposed to repeated flooding events. These bacteria formed relatively stable populations of up to 6% of total cell counts over periods of several months irrespective of inundation by groundwater. This suggests that karst water may provide a means of transport for these bacteria from terrestrial to freshwater habitats.