Phylogenetic comparisons of bacterial communities from serpentine and nonserpentine soils

I present the results of a culture-independent survey of soil bacterial communities from serpentine soils and adjacent nonserpentine comparator soils using a variety of newly developed phylogenetically based statistical tools. The study design included site-based replication of the serpentine-to-nonserpentine community comparison over a regional scale ( approximately 100 km) in Northern California and Southern Oregon by producing 16S rRNA clone libraries from pairs of samples taken on either side of the serepentine-nonserpentine edaphic boundary at three geographical sites. At the division level, the serpentine and nonserpentine communities were similar to each other and to previous data from forest soils. Comparisons of both richness and Shannon diversity produced no significant differences between any of the libraries, but the vast majority of phylogenetically based tests were significant, even with only 50 sequences per library. These results suggest that most samples were distinct, consisting of a collection of lineages generally not found in other samples. The pattern of results showed that serpentine communities tended to be more similar to each other than they were to nonserpentine communities, and these differences were at a lower taxonomic scale. Comparisons of two nonserpentine communities generally showed differences, and some results suggest that the geographical site may control community composition as well. These results show the power of phylogenetic tests to discern differences between 16S rRNA libraries compared to tests that discard DNA data to bin sequences into operational taxonomic units, and they stress the importance of replication at larger scales for inferences regarding microbial biogeography.     

Comparison of ectomycorrhizas of Quercus garryana (Fagaceae) on serpentine and non-serpentine soils in southwestern Oregon

The diversity of ectomycorrhizal communities associated with Quercus garryana on and off serpentine soils was compared and related to landscape-level diversity. Serpentine soils are high in magnesium, iron, and heavy metals and low in fertility. In plant communities on serpentine soils, a high proportion of flowering plant species are endemic. At three sites with paired serpentine and nonserpentine soils in southwestern Oregon, we sampled Q. garryana roots and categorized ectomycorrhizas by morphotyping and by restriction fragment length patterns. Ectomycorrhizas were abundant at all sites; no single fungal species dominated in the ectomycorrhizas. Of 74 fungal species characterized by morphotype and pattern of restriction fragment length polymorphisms, 46 occurred on serpentine soils, and 32 were unique to serpentine soil. These species are potentially endemic to serpentine soil. Similarities in species composition between paired serpentine and nonserpentine soils were not significantly lower than among three serpentine sites or among three nonserpentine sites. We conclude that mycorrhizal communities associated with oaks on serpentine soil do not differ in species richness or species evenness from those on neighboring nonserpentine soil.     

Serpentine soils promote ectomycorrhizal fungal diversity

Serpentine soils impose physiological stresses that limit plant establishment and diversity. The degree to which serpentine soils entail constraints on other organisms is, however, poorly understood. Here, I investigate the effect of serpentine soils on ectomycorrhizal (ECM) fungi by conducting a reciprocal transplant experiment, where serpentine and nonserpentine ECM fungal communities were cultured in both their native and non-native soils. Contrary to expectation, serpentine soils hosted higher fungal richness compared to nonserpentine, and most species were recovered from serpentine soil, suggesting ECM fungi are not overall specialized or strongly affected by serpentine edaphic constraints.     

Effect of PCR amplicon size on assessments of clone library microbial diversity and community structure

PCR-based surveys of microbial communities commonly use regions of the small-subunit ribosomal RNA (SSU rRNA) gene to determine taxonomic membership and estimate total diversity. Here we show that the length of the target amplicon has a significant effect on assessments of microbial richness and community membership. Using operational taxonomic unit (OTU)- and taxonomy-based tools, we compared the V6 hypervariable region of the bacterial SSU rRNA gene of three amplicon libraries of c. 100, 400 and 1000 base pairs (bp) from each of two hydrothermal vent fluid samples. We found that the smallest amplicon libraries contained more unique sequences, higher diversity estimates and a different community structure than the other two libraries from each sample. We hypothesize that a combination of polymerase dissociation, cloning bias and mispriming due to secondary structure accounts for the differences. While this relationship is not linear, it is clear that the smallest amplicon libraries contained more different types of sequences, and accordingly, more diverse members of the community. Because divergent and lower abundant taxa can be more readily detected with smaller amplicons, they may provide better assessments of total community diversity and taxonomic membership than longer amplicons in molecular studies of microbial communities.     

Ectomycorrhizal communities of Quercus garryana are similar on serpentine and nonserpentine soils

Serpentine soils, rich in iron, magnesium, and heavy metals, select for unique plant communities and for endemic species. Because mycorrhizal fungi mediate the interaction between plants and soil, we hypothesized that distinct ectomycorrhizal fungi would colonize Quercus garryana roots on serpentine and nonserpentine soils. We sampled roots of Q. garryana on serpentine soils at two locations in the Klamath-Siskiyou Mountains of southwestern Oregon and identified ectomycorrhizas by morphological and molecular methods. The same six most abundant and most frequent mycorrhizal species, Cenococcum geophilum, Tuber candidum, Genea harknessii, Tomentella sp., Sebacina sp., and Inocybe sp., were found on serpentine and nonserpentine soils. Based on similarities calculated using the Sørensen index in Non-metric Multidimensional Scaling, mycorrhizal communities on serpentine and nonserpentine soils were not significantly different. This study showed that ectomycorrhizal species associated with Q. garryana exhibit edaphic tolerance and were neither reduced nor excluded by serpentinite or peridotite parent materials.     

Congeneric Serpentine and Nonserpentine Shrubs Differ More in Leaf Ca:Mg than in Tolerance of Low N, Low P, or Heavy Metals

Serpentine soils limit plant growth by NPK deficiencies, low Ca availability, excess Mg, and high heavy metal levels. In this study, three congeneric serpentine and nonserpentine evergreen shrub species pairs were grown in metalliferous serpentine soil with or without NPKCa fertilizer to test which soil factors most limit biomass production and mineral nutrition responses. Fertilization increased biomass production and allocation to leaves while decreasing allocation to roots in both serpentine and nonserpentine species. Simultaneous increases in biomass and leaf N:P ratios in fertilized plants of all six species suggest that N is more limiting than P in this serpentine soil. Neither N nor P concentrations, however, nor root to shoot translocation of these nutrients, differed significantly between serpentine and nonserpentine congeners. All six species growing in unfertilized serpentine soil translocated proportionately more P to leaves compared to fertilized plants, thus maintaining foliar P. Leaf Ca:Mg molar ratios of the nonserpentine species were generally equal to that of the soil. The serpentine species, however, maintained significantly higher leaf Ca:Mg than both their nonserpentine counterparts and the soil. Elevated leaf Ca:Mg in the serpentine species was achieved by selective Ca transport and/or Mg exclusion operating at the root-to-shoot translocation level, as root Ca and Mg concentrations did not differ between serpentine and nonserpentine congeners. All six species avoided shoot toxicity of heavy metals by root sequestration. The comparative data on nutrient deficiencies, leaf Ca:Mg, and heavy metal sequestration suggest that the ability to maintain high leaf Ca:Mg is a key evolutionary change needed for survival on serpentine soil and represents the physiological feature distinguishing the serpentine shrub species from their nonserpentine congeners. The results also suggest that high leaf Ca:Mg is achieved in these serpentine species by selective translocation of Ca and/or inhibited transport of Mg from roots, rather than by uptake/exclusion at root surfaces.     

Low sequencing efforts bias analyses of shared taxa in microbial communities

The potential for comparing microbial community population structures has been greatly enhanced by developments in next generation sequencing methods that can generate hundreds of thousands to millions of reads in a single run. Conversely, many microbial community comparisons have been published with no more than 1,000 sequences per sample. These studies have presented data on levels of shared operational taxonomic units (OTUs) between communities. Due to lack of coverage, that approach might compromise the conclusions about microbial diversity and the degree of difference between environments. In this study, we present data from recent studies that highlight this problem. Also, we analyzed datasets of 16 rRNA sequences with small and high sequence coverage from different environments to demonstrate that the level of sequencing effort used for analyzing microbial communities biases the results. We randomly sampled pyrosequencing-generated 16S rRNA gene libraries with increasing sequence effort. Sequences were used to calculate Good's coverage, the percentage of shared OTUs, and phylogenetic distance measures. Our data showed that simple counts of presence/absence of taxonomic unities do not reflect the real similarity in membership and structure of the bacterial communities and that community comparisons based on phylogenetic tests provide a way to test statistically significant differences between two or more environments without need an exhaustive sampling effort.     

Composition of freshwater bacterial communities associated with cyanobacterial blooms in four Swedish lakes

The diversity of freshwater bacterioplankton communities has not been extensively studied despite their key role in foodwebs and the cycling of carbon and associated major elements. In order to explore and characterize the composition of bacterioplankton associated with cyanobacterial blooms, large 16S rRNA clone libraries from four lakes experiencing such blooms were analysed. The four libraries contained 1461 clones, of which 559 were prokaryotic sequences of non-cyanobacterial origin. These clones were classified into 158 operational taxonomic units affiliated mainly with bacterial divisions commonly found in freshwater systems, e.g. Proteobacteria, Bacteriodetes, Actinobacteria, Verrucomicrobia and Planctomycetes. Richness and evenness of non-cyanobacterial clones were similar to other clone libraries obtained for freshwater bacterioplankton, suggesting that bacterial communities accompanying cyanobacterial blooms are as diverse as non-bloom communities. Many of the identified operational taxonomic units grouped with known freshwater clusters but the libraries also contained novel clusters of bacterial sequences that may be characteristic for cyanobacterial blooms. About 25% of the operational taxonomic units were detected in more than one lake. Even so, 16S rRNA heterogeneity analysis demonstrated large differences in community composition between lakes regardless of their similar characteristics and close proximity. Hence even the similar environmental conditions created by different cyanobacterial blooms may foster very dissimilar bacterial communities, which could indicate that the genetic diversity in lake bacteria have been underestimated in the past.     

Extensive profiling of a complex microbial community by high-throughput sequencing

Complex microbial communities remain poorly characterized despite their ubiquity and importance to human and animal health, agriculture, and industry. Attempts to describe microbial communities by either traditional microbiological methods or molecular methods have been limited in both scale and precision. The availability of genomics technologies offers an unprecedented opportunity to conduct more comprehensive characterizations of microbial communities. Here we describe the application of an established molecular diagnostic method based on the chaperonin-60 sequence, in combination with high-throughput sequencing, to the profiling of a microbial community: the pig intestinal microbial community. Four libraries of cloned cpn60 sequences were generated by two genomic DNA extraction procedures in combination with two PCR protocols. A total of 1,125 cloned cpn60 sequences from the four libraries were sequenced. Among the 1,125 cloned cpn60 sequences, we identified 398 different nucleotide sequences encoding 280 unique peptide sequences. Pairwise comparisons of the 398 unique nucleotide sequences revealed a high degree of sequence diversity within the library. Identification of the likely taxonomic origins of cloned sequences ranged from imprecise, with clones assigned to a taxonomic subclass, to precise, for cloned sequences with 100% DNA sequence identity with a species in our reference database. The compositions of the four libraries were compared and differences related to library construction parameters were observed. Our results indicate that this method is an alternative to 16S rRNA sequence-based studies which can be scaled up for the purpose of performing a potentially comprehensive assessment of a given microbial community or for comparative studies.     

PCR primers that amplify fungal rRNA genes from environmental samples

Two PCR primer pairs were designed to amplify rRNA genes (rDNA) from all four major phyla of fungi: Ascomycota, Basidiomycota, Chytridomycota, and Zygomycota. PCRs performed with these primers showed that both pairs amplify DNA from organisms representing the major taxonomic groups of fungi but not from nonfungal sources. To test the ability of the primers to amplify fungal rDNA from environment samples, clone libraries from two avocado grove soils were constructed and analyzed. These soils possess different abilities to inhibit avocado root rot caused by Phythophthora cinnamomi. Analysis of the two rDNA clone libraries revealed differences in the two fungal communities. It also revealed a markedly different depiction of the soil fungal community than that generated by a culture-based analysis, confirming the value of rDNA-based approaches for identifying organisms that may not readily grow on agar media. Additional evidence of the usefulness of the primers was obtained by identifying fungi associated with avocado leaves. In both the soil and leaf analyses, no nonfungal rDNA sequences were identified, illustrating the selectivity of these PCR primers. This work demonstrates the ability of two newly developed PCR primer sets to amplify fungal rDNA from soil and plant tissue, thereby providing unique tools to examine this vast and mostly undescribed community of organisms.     

Cultivation-independent analysis reveals a shift in ciliate 18S rRNA gene diversity in a polycyclic aromatic hydrocarbon-polluted soil

Using cultivation-independent methods the ciliate communities of a clay-rich soil with a 90-year record of pollution by polycyclic aromatic hydrocarbons (PAH) (4.5 g kg(-1) PAH) were compared with that of a nonpolluted soil collected in its vicinity and with similar properties. A ciliate-specific set of 18S rRNA gene targeting primers was designed and used to amplify DNA extracted from both soils (surface and 20 cm depth). Four clone libraries were generated with PCR products that covered an 18S rRNA gene fragment of up to 670 bp. Comparative sequence analysis of representative clones proved that the primer set was highly specific for ciliates. Calculation of similarity indices based on operational taxonomic units after amplified ribosomal DNA restriction analysis of the clones showed that the community from the nonpolluted surface soil was highly dissimilar to the other communities. The presence of several taxa, namely sequences affiliated to the orders Phyllopharyngia, Haptoria, Nassophorea, Peniculida and Scuticociliatia in samples from nonpolluted soil, points to the existence of various trophic functional groups. In contrast, the 18S rRNA gene diversity was much lower in the clone libraries from the polluted soil. More than 90% of these sequences belonged to the class Colpodea, a well-known clade of mainly bacterivorous and r-selected species, thus potentially also indicating a lower functional diversity.     

Niche partitioning in arbuscular mycorrhizal communities in temperate grasslands: a lesson from adjacent serpentine and nonserpentine habitats

Arbuscular mycorrhizal fungi (AMF) represent an important soil microbial group playing a fundamental role in many terrestrial ecosystems. We explored the effects of deterministic (soil characteristics, host plant life stage, neighbouring plant communities) and stochastic processes on AMF colonization, richness and community composition in roots of Knautia arvensis (Dipsacaceae) plants from three serpentine grasslands and adjacent nonserpentine sites. Methodically, the study was based on 454‐sequencing of the ITS region of rDNA. In total, we detected 81 molecular taxonomical operational units (MOTUs) belonging to the Glomeromycota. Serpentine character of the site negatively influenced AMF root colonization, similarly as higher Fe concentration. AMF MOTUs richness linearly increased along a pH gradient from 3.5 to 5.8. Contrary, K and Cr soil concentration had a negative influence on AMF MOTUs richness. We also detected a strong relation between neighbouring plant community composition and AMF MOTUs richness. Although spatial distance between the sampled sites (c. 0.3–3 km) contributed to structuring AMF communities in K. arvensis roots, environmental parameters were key factors in this respect. In particular, the composition of AMF communities was shaped by the complex of serpentine conditions, pH and available soil Ni concentration. The composition of AMF communities was also dependent on host plant life stage (vegetative vs. generative). Our study supports the dominance of deterministic factors in structuring AMF communities in heterogeneous environment composed of an edaphic mosaic of serpentine and nonserpentine soils.     

DIFFERENTIAL RESPONSES TO MAGNESIUM AND CALCIUM BY NATIVE POPULATIONS OF AGROPYRON SPICATUM

Plants of Agropyron spicatum (Pursh) Scribn. and Smith. from populations native to serpentine and nonserpentine soils were grown at varying levels of magnesium and calcium in culture solutions. The yields of plants from the two populations were different. At high Mg levels (low Ca) the yield of the serpentine population was significantly higher than that of the nonserpentine population. At low Mg the yield of the serpentine population leveled off at a Mg: Ca ratio of 1:2, while the yield of the nonserpentine population increased up to a Mg:Ca ratio of 1:8 and showed no leveling off. Chemical analyses of tissue showed that the Ca uptake of plants from the serpentine population was significantly higher than that of the nonserpentine population. In addition, the serpentine population maintained a lower Mg concentration in the shoots than the nonserpentine population at comparable Mg substrate levels. The two populations showed differences in Ca and Mg uptake efficiency and Mg/Ca, Ca + Mg/K + Na, and Ca + Mg + K + Na in the shoots. The ecotypic differentiation with respect to Mg and Ca between native populations of serpentine and nonserpentine A. spicatum does not appear to be due to any single mechanism but, rather, a combination of several possible mechanisms, i.e., differences in root morphology, uptake mechanisms, translocation of nutrients, and interactions between cations.     

Investigation of bacterial diversity in Brazilian tropical estuarine sediments reveals high actinobacterial diversity

Phylogenetic and statistical analyses of 16S rRNA gene libraries were used for the investigation of actinobacterial communities present in two tropical estuarine sediments (Santos-São Vicente estuary, Brazil). The libraries were constructed from samples collected at the brackish end of the estuary, highly hydrocarbon-contaminated, and at the marine end, uncontaminated. Clones from the marine end of the estuary were all related to sequences from non-cultured Actinobacteria and unidentified bacteria recovered from a wide range of environmental samples, whereas clones from the brackish end were mainly related to sequences from cultured Actinobacteria. Statistical analyses showed that the community recovered from the hydrocarbon-contaminated sediment sample, at the brackish end, was less diverse than the uncontaminated one, at the marine end, and that the communities from the two libraries were differently structured, suggesting that these may have not originated from the same community. The recognition of the spatial pattern of actinobacterial distribution in a natural environment is a first step towards understanding the way these communities are organized, providing valuable data for further investigations of their taxonomic and functional diversity.     

Hybridization between Arctostaphylos viscida and A. canescens in Oregon

Evidence of natural hybridization betweenArctostaphylos viscida andA. canescens in southwestern Oregon was obtained from morphological studies of field populations. Hybridization occurs where populations ofA. viscida andA. canescens meet in areas where serpentine and nonserpentine soils abut. At these contacts,A. viscida forms large populations only on serpentine andA. canescens only on nonserpentine. Most of the indentifiable hybrids survive on either transitional or nonserpentine soils. The small proportion that are found on serpentine are considered to be backcrosses toA. viscida. The hybrid plants are similar to several putative species maintained in the standard floras, and it is suggested that these no longer be given taxonomic recognition.     

Bacterial and Archaeal 16S rRNA Genes in Late Pleistocene to Holocene Muddy Sediments from the Kanto Plain of Japan

Microbial communities in ancient marine sediments composed of clay and silt obtained from the terrestrial subsurface were phylogenetically analyzed based on their 16S rRNA gene sequences. Chloroflexi and Miscellaneous Crenarchaeotic Group were predominant in bacterial and archaeal clone libraries, respectively. Of 44 operational taxonomic units (OTUs) that had close relatives in the database, 30 were close to sequences obtained from marine environments. Some sequences belonged to the candidate groups JS1, ANME-I, and Marine Benthic Group-C, which are typically found in marine sediments. Low chloride concentrations in the sediments suggest that these marine-affiliated sequences may not reflect currently active microbial communities. Our results indicate the existence of long-term preserved DNA or descendants of ancient oceanic microbial components in subsurface muddy sediments in a temperate region, which may reflect indigenous population of paleoenvironments.     

Molecular diversity of soil and marine 16S rRNA gene sequences related to beta-subgroup ammonia-oxidizing bacteria.

We have conducted a preliminary phylogenetic survey of ammonia-oxidizing beta-proteobacteria, using 16S rRNA gene libraries prepared by selective PCR and DNA from acid and neutral soils and polluted and nonpolluted marine sediments. Enrichment cultures were established from samples and analyzed by PCR. Analysis of 111 partial sequences of c. 300 bases revealed that the environmental sequences formed seven clusters, four of which are novel, within the phylogenetic radiation defined by cultured autotrophic ammonia oxidizers. Longer sequences from 13 cluster representatives support their phylogenetic positions relative to cultured taxa. These data suggest that known taxa may not be representative of the ammonia-oxidizing beta-proteobacteria in our samples. Our data provide further evidence that molecular and culture-based enrichment methods can select for different community members. Most enrichments contained novel Nitrosomonas-like sequences whereas novel Nitrosospira-like sequences were more common from gene libraries of soils and marine sediments. This is the first evidence for the occurrence of Nitrosospira-like strains in marine samples. Clear differences between the sequences of soil and marine sediment libraries were detected. Comparison of 16S rRNA sequences from polluted and nonpolluted sediments provided no strong evidence that the community composition was determined by the degree of pollution. Soil clone sequences fell into four clusters, each containing sequences from acid and neutral soils in varying proportions. Our data suggest that some related strains may be present in both samples, but further work is needed to resolve whether there is selection due to pH for particular sequence types.     

Microbial community composition in soils of Northern Victoria Land, Antarctica

Biotic communities and ecosystem dynamics in terrestrial Antarctica are limited by an array of extreme conditions including low temperatures, moisture and organic matter availability, high salinity, and a paucity of biodiversity to facilitate key ecological processes. Recent studies have discovered that the prokaryotic communities in these extreme systems are highly diverse with patchy distributions. Investigating the physical and biological controls over the distribution and activity of microbial biodiversity in Victoria Land is essential to understanding ecological functioning in this region. Currently, little information on the distribution, structure and activity of soil communities anywhere in Victoria Land are available, and their sensitivity to potential climate change remains largely unknown. We investigated soil microbial communities from low- and high-productivity habitats in an isolated Antarctic location to determine how the soil environment impacts microbial community composition and structure. The microbial communities in Luther Vale, Northern Victoria Land were analysed using bacterial 16S rRNA gene clone libraries and were related to soil geochemical parameters and classical morphological analysis of soil metazoan invertebrate communities. A total of 323 16S rRNA gene sequences analysed from four soils spanning a productivity gradient indicated a high diversity (Shannon-Weaver values > 3) of phylotypes within the clone libraries and distinct differences in community structure between the two soil productivity habitats linked to water and nutrient availability. In particular, members of the Deinococcus/Thermus lineage were found exclusively in the drier, low-productivity soils, while Gammaproteobacteria of the genus Xanthomonas were found exclusively in high-productivity soils. However, rarefaction curves indicated that these microbial habitats remain under-sampled. Our results add to the recent literature suggesting that there is a higher biodiversity within Antarctic soils than previously expected.     

UniFrac: a new phylogenetic method for comparing microbial communities

We introduce here a new method for computing differences between microbial communities based on phylogenetic information. This method, UniFrac, measures the phylogenetic distance between sets of taxa in a phylogenetic tree as the fraction of the branch length of the tree that leads to descendants from either one environment or the other, but not both. UniFrac can be used to determine whether communities are significantly different, to compare many communities simultaneously using clustering and ordination techniques, and to measure the relative contributions of different factors, such as chemistry and geography, to similarities between samples. We demonstrate the utility of UniFrac by applying it to published 16S rRNA gene libraries from cultured isolates and environmental clones of bacteria in marine sediment, water, and ice. Our results reveal that (i) cultured isolates from ice, water, and sediment resemble each other and environmental clone sequences from sea ice, but not environmental clone sequences from sediment and water; (ii) the geographical location does not correlate strongly with bacterial community differences in ice and sediment from the Arctic and Antarctic; and (iii) bacterial communities differ between terrestrially impacted seawater (whether polar or temperate) and warm oligotrophic seawater, whereas those in individual seawater samples are not more similar to each other than to those in sediment or ice samples. These results illustrate that UniFrac provides a new way of characterizing microbial communities, using the wealth of environmental rRNA sequences, and allows quantitative insight into the factors that underlie the distribution of lineages among environments.     

Toward a census of bacteria in soil

For more than a century, microbiologists have sought to determine the species richness of bacteria in soil, but the extreme complexity and unknown structure of soil microbial communities have obscured the answer. We developed a statistical model that makes the problem of estimating richness statistically accessible by evaluating the characteristics of samples drawn from simulated communities with parametric community distributions. We identified simulated communities with rank-abundance distributions that followed a truncated lognormal distribution whose samples resembled the structure of 16S rRNA gene sequence collections made using Alaskan and Minnesotan soils. The simulated communities constructed based on the distribution of 16S rRNA gene sequences sampled from the Alaskan and Minnesotan soils had a richness of 5,000 and 2,000 operational taxonomic units (OTUs), respectively, where an OTU represents a collection of sequences not more than 3% distant from each other. To sample each of these OTUs in the Alaskan 16S rRNA gene library at least twice, 480,000 sequences would be required; however, to estimate the richness of the simulated communities using nonparametric richness estimators would require only 18,000 sequences. Quantifying the richness of complex environments such as soil is an important step in building an ecological framework. We have shown that generating sufficient sequence data to do so requires less sequencing effort than completely sequencing a bacterial genome.