K. R. Freeman

The effects of chronic nitrogen fertilization on alpine tundra soil microbial communities: implications for carbon and nitrogen cycling

Many studies have shown that changes in nitrogen (N) availability affect primary productivity in a variety of terrestrial systems, but less is known about the effects of the changing N cycle on soil organic matter (SOM) decomposition. We used a variety of techniques to examine the effects of chronic N amendments on SOM chemistry and microbial community structure and function in an alpine tundra soil. We collected surface soil (0-5 cm) samples from five control and five long-term N-amended plots established and maintained at the Niwot Ridge Long-term Ecological Research (LTER) site. Samples were bulked by treatment and all analyses were conducted on composite samples. The fungal community shifted in response to N amendments, with a decrease in the relative abundance of basidiomycetes. Bacterial community composition also shifted in the fertilized soil, with increases in the relative abundance of sequences related to the Bacteroidetes and Gemmatimonadetes, and decreases in the relative abundance of the Verrucomicrobia. We did not uncover any bacterial sequences that were closely related to known nitrifiers in either soil, but sequences related to archaeal nitrifiers were found in control soils. The ratio of fungi to bacteria did not change in the N-amended soils, but the ratio of archaea to bacteria dropped from 20% to less than 1% in the N-amended plots. Comparisons of aliphatic and aromatic carbon compounds, two broad categories of soil carbon compounds, revealed no between treatment differences. However, G-lignins were found in higher relative abundance in the fertilized soils, while proteins were detected in lower relative abundance. Finally, the activities of two soil enzymes involved in N cycling changed in response to chronic N amendments. These results suggest that chronic N fertilization induces significant shifts in soil carbon dynamics that correspond to shifts in microbial community structure and function.

Evidence that chytrids dominate fungal communities in high-elevation soils

Periglacial soils are one of the least studied ecosystems on Earth, yet they are widespread and are increasing in area due to retreat of glaciers worldwide. Soils in these environments are cold and during the brief summer are exposed to high levels of UV radiation and dramatic fluctuations in moisture and temperature. Recent research suggests that these environments harbor immense microbial diversity. Here we use sequencing of environmental DNA, culturing of isolates, and analysis of environmental variables to show that members of the Chytridiomycota (chytrids) dominate fungal biodiversity and perhaps decomposition processes in plant-free, high-elevation soils from the highest mountain ranges on Earth. The zoosporic reproduction of chytrids requires free water, yet we found that chytrids constituted over 70% of the ribosomal gene sequences of clone libraries from barren soils of the Himalayas and Rockies; by contrast, they are rare in other soil environments. Very few chytrids have been cultured, although we were successful at culturing chytrids from high-elevation sites throughout the world. In a more focused study of our sites in Colorado, we show that carbon sources that support chytrid growth (eolian deposited pollen and microbial phototrophs) are abundant and that soils are saturated with water for several months under the snow, thus creating ideal conditions for the development of a chytrid-dominated ecosystem. Our work broadens the known biodiversity of the Chytridomycota, and describes previously unsuspected links between aquatic and terrestrial ecosystems in alpine regions.

Biogeography and habitat modelling of high-alpine bacteria

Soil microorganisms dominate terrestrial biogeochemical cycles; however, we know very little about their spatial distribution and how changes in the distributions of specific groups of microbes translate into landscape and global patterns of biogeochemical processes. In this paper, we use a nested sampling scheme at scales ranging from 2 to 2,000 m to show that bacteria have significant spatial autocorrelation in community composition up to a distance of 240 m, and that this pattern is driven by changes in the relative abundance of specific bacterial clades across the landscape. Analysis of clade habitat distribution models and spatial co-correlation maps identified soil pH, plant abundance and snow depth as major variables structuring bacterial communities across this landscape, and revealed an unexpected and important oligotrophic niche for the Rhodospirillales in soil. Furthermore, our global analysis of high-elevation soils from the Andes, Rockies, Himalayas and Alaskan range shows that habitat distribution models for bacteria have a strong predictive power across the entire globe.

Phylogeography of microbial phototrophs in the dry valleys of the high Himalayas and Antarctica

High-elevation valleys in dry areas of the Himalayas are among the most extreme, yet least explored environments on Earth. These barren, rocky valleys are subjected to year-round temperature fluctuations across the freezing point and very low availability of water and nutrients, causing previous workers to hypothesize that no photoautotrophic life (primary producers) exists in these locations. However, there has been no work using modern biogeochemical or culture-independent molecular methods to test the hypothesis that photoautotrophs are absent from high Himalayan soil systems. Here, we show that although microbial biomass levels are as low as those of the Dry Valleys of Antarctica, there are abundant microbial photoautotrophs, displaying unexpected phylogenetic diversity, in barren soils from just below the permanent ice line of the central Himalayas. Furthermore, we discovered that one of the dominant algal clades from the high Himalayas also contains the dominant algae in culture-independent surveys of both soil and ice samples from the Dry Valleys of Antarctica, revealing an unexpected link between these environmentally similar but geographically very distant systems. Phylogenetic and biogeographic analyses demonstrated that although this algal clade is globally distributed to other high-altitude and high-latitude soils, it shows significant genetic isolation by geographical distance patterns, indicating local adaptation and perhaps speciation in each region. Our results are the first to demonstrate the remarkable similarities of microbial life of arid soils of Antarctica and the high Himalayas. Our findings are a starting point for future comparative studies of the dry valleys of the Himalayas and Antarctica that will yield new insights into the cold and dry limits to life on Earth.

Soil rotifer communities are extremely diverse globally but spatially autocorrelated locally

Bdelloid rotifers are important contributors to biogeochemical cycling and trophic dynamics of both aquatic and terrestrial ecosystems, but little is known about their biogeographic distribution and community structure in terrestrial environments. This lack of knowledge stems from a lack of phylogenetic information and assumptions that microbial eukaryotes are globally distributed and have very limited diversity across vast geographic distances. However, these assumptions have been based more on assessments of their morphology than any measure of their true genetic diversity and biogeographic distribution. We developed specific primers for the cytochrome c oxidase subunit 1 (cox1) gene of bdelloid rotifers and amplified and cloned sequences using a nested sampling scheme that represented local (0–10 m) to global (up to 10,000 km) scales. Using phylogenetic community analyses (UniFrac) and geospatial statistics (semivariograms, mantel tests), we were able to reject the hypothesis that communities of rotifers are the same across even fairly small geographic distances. Bdelloid communities showed highly significant spatial structuring with spatial autocorrelation ranges of 54–133 m, but beyond that distance communities were extremely dissimilar. Furthermore, we show that these spatial patterns are driven not only by changes in relative abundance of phylotypes but also by absolute changes in phylotype occurrence (richness). There is almost no overlap in phylotype [or operational taxonomic unit (OTU)] occurrence between communities at distances beyond the autocorrelation range (~133 m). Such small species ranges, combined with their ubiquity in soils, make it increasingly clear that the biodiversity of bdelloid rotifers (and other less easily dispersed microbes) is much higher than previously thought.

Environmental DNA sequencing primers for eutardigrades and bdelloid rotifers

BackgroundThe time it takes to isolate individuals from environmental samples and then extract DNA from each individual is one of the problems with generating molecular data from meiofauna such as eutardigrades and bdelloid rotifers. The lack of consistent morphological information and the extreme abundance of these classes makes morphological identification of rare, or even common cryptic taxa a large and unwieldy task. This limits the ability to perform large-scale surveys of the diversity of these organisms.Here we demonstrate a culture-independent molecular survey approach that enables the generation of large amounts of eutardigrade and bdelloid rotifer sequence data directly from soil. Our PCR primers, specific to the 18s small-subunit rRNA gene, were developed for both eutardigrades and bdelloid rotifers.ResultsThe developed primers successfully amplified DNA of their target organism from various soil DNA extracts. This was confirmed by both the BLAST similarity searches and phylogenetic analyses. Tardigrades showed much better phylogenetic resolution than bdelloids. Both groups of organisms exhibited varying levels of endemism.ConclusionThe development of clade-specific primers for characterizing eutardigrades and bdelloid rotifers from environmental samples should greatly increase our ability to characterize the composition of these taxa in environmental samples. Environmental sequencing as shown here differs from other molecular survey methods in that there is no need to pre-isolate the organisms of interest from soil in order to amplify their DNA. The DNA sequences obtained from methods that do not require culturing can be identified post-hoc and placed phylogenetically as additional closely related sequences are obtained from morphologically identified conspecifics. Our non-cultured environmental sequence based approach will be able to provide a rapid and large-scale screening of the presence, absence and diversity of Bdelloidea and Eutardigrada in a variety of soils.