Pond et al., 2009

Pond, K.S., Wadhawan, S., Chiaromonte, F., Ananda, G., Chung, W.Y., Taylor, J., Nekrutenko, A., 2009. Windshield splatter analysis with the Galaxy metagenomic pipeline. Genome Research 19, 2144-2153.

These authors describe a novel software system that integrates several functions relevant for metagenomic analysis, generally defined as examining environmental samples of nucleic acids (typically DNA) without culturing the organisms present, and drawing inferences about the biological community (phylogenetic or functional) from the sequences. This is currently my favourite paper, for the quality of the writing, the density of information, the usefulness of the described methodology, and especially for the dataset they use as their demonstration of their system. I'm going to mostly use direct quotes from this paper, because there's no way I could say any of this better by paraphrasing.

The abstract starts with: 

How many species inhabit our immediate surroundings? A straightforward collection technique suitable for answering this question is known to anyone who has ever driven a car at highway speeds. 

The Introduction describes the existing resources available for metagenomic analyses, and how those resources can be expected to deal with prokaryotic and eukaryotic data. For example, while protein sequences are often employed in studies of prokaryotes (including the use of predicted protein sequences and open reading frames (ORFs) from DNA sequences), the small fraction of eukaryote genomes that codes for proteins makes such strategies less useful for investigating community composition of eukaryotes.

The authors undertook two voyages on sequential days in July of 2007, travelling from Pennsylvania to New Brunswick, in a minivan equiped with sticky tape on its bumper. They frequently refer to "windshield splatter", though this is slightly inaccurate, as the tape was affixed to the bumper of the vehicle, several decimeters closer to ground level than the windshield.

Jumping into the Results:

The most prominent difference between the two trips is in the number of reads identified with green plants (Viridiplantae): 10,242 in trip A versus 612 in trip B. It is unlikely that a two orders of magnitude difference reflects a genuine variation in species abundance of such a ubiquitous taxonomic group between the two trips. Because during each trip we collected two samples (left and right sides of the vehicle; see Methods) we were able to trace the majority (9317) of Viridiplantae reads to the left subsample. The most likely explanation for this overabundance is that a piece of plant material (e.g., a leaf or stem fragment) adhered to the collection surface. 

This illustrates a few of the striking differences between biology at the level of macroscopic organisms (e.g. most of botany, or the animals that a good naturalist would be expected to be familiar with) and microscopic, especially bacterial. A single leaf or stem fragment contains thousands to millions of cells in direct contact with each other in a dense 3-dimensional structure. Bacterial cells in the environment are often found in biofilms, which are typically a single cell layer or only a few cell layers thick, and cover a tiny area. Or they occur as individual cells, separated by multiple cell-length-equivalents from their neighbours. Also, identification to high taxonomic levels such as Order or Phylum is common in environmental microbiology, yet essentially unheard of for multicellular organisms - if it's big enough to see, it can be identified to Family or better by a person equiped with a readily-available guide. Yet they report a "green plant" - anything from roses to ginkos is included in that high-level taxon!

The list included unexpected entries such as the genus Homo even though the two trips were uneventful. Such matches are likely caused by road debris (which often includes roadkill) adhering to the collecting tape. Because few entries in NT and WGS databases are derived from, say, white-tailed deer (Odocoileus virginianus, a prevalent large mammal roadkill in the northeastern United States), reads truly representing this speces are more likely to match abundant human sequences. 

That first sentence, above, is probably my favourite sentence in the entire paper. "the two trips were uneventful." Just savour that, and ponder the meanings...

This is also another striking difference between metagenomics and related microbiological sampling and study strategies and how multicellular eukaryotes are most often studied. No ecologist would normally need to describe the probability of mistaking a sample derived from a white-tailed deer with that from a human, yet here, because of the way the databases used for comparison and identification are structured, consideration of roadkill rates (and roadside clean-up efforts, presumably) are required to refine the raw identifications derived from comparisons of DNA sequences. 

Existing tools for major steps in the environmental-sample-to-phylogeny experimental pipeline are difficult to use and make work together, thus: 

This is why our objective was to build a complete pipeline for homology-based taxonomic labeling of metagenomic reads that was self-contained and guided the user from data acquistion and QC, to database searches, and finally, actual metagenomic analyses. We demonstrate that the classification performance of our solution is on par with currently available applications...
Our second goal was to perform a eukaryotic metagenomic study on the organic matter collected on an automobile's windshield. Specifically, we were interested in addressing two questions: Can one identify eukaryotic taxa from random reads generated by the next-generation sequencing technology from environmental samples? and Is it possible to contrast species abundance between geographic locations? While this pilot analysis provides positive answers to both questions, it also raises important issues and limitations. 

I leave it to you to read this excellent paper and see the "issues and limitations" they describe.

And I *love* their methods: 

The front bumper of a 2006 Dodge Caravan ("The Wanderer") was divided at the license plate into "left" (passenger side) and "right" (driver side), and was taped with a double-sided carpet tape. On top of the carpet tape, a 3M 5414 Water Soluble Wave Solder Tape was affixed, exposing its sticky side. The tapes were applied on June 23, 2007, at 6 am EDT in State College, Pennsylvania, and removed in tubes containing Tris EDTA buffer at 12 pm EDT in Manchester, Connecticut. New tapes were again applied in Portland, Maine, at 5 pm EDT and removed in Moncton, New Brunswick, at 12 pm EDT the following day.

Note that they named the vehicle (with a pretty good name, in my opinion), and they describe "left" and "right" in opposition to the common standard among drivers - their description is based on a person standing in front of the vehicle, facing the windshield; their "left" is the vehicle's starboard side, and "right" is port. It's extremely unlikely "The Wanderer" is a right-hand-drive vehicle.

Their software is web-based and available at:  www.usegalaxy.org

Su et al. 2011

Su, C., Lei, L., Duan, Y., Zhang, K.-Q., Yang, J., 2011. Culture-independent methods for studying environmental microorganisms: methods, application, and perspective. Applied Microbiology and Biotechnology 93, 993-1003.

These authors provide a summary overview of the more recently-developed culture-independent methods and their use in studying microbial communities. Figure 1 shows the basics of each of several methods, that all start with the collection of an environmental sample (e.g. soil, water, mouth swab, etc.), and end with data analysis and evalution dependent on the hypotheses of the study.

(I'm not going to post Figure 1 here, I'm not interested in violating copyright)

In the introductory part of the review, these authors provide a list and a taxonomy of these methods.

PCR-based

·         DGGE/TGGE (denaturing/temperature gradient gel electrophoresis)

·         SSCP (Single-strand-conformation polymorphism)

·         RFLP (restriction fragment length polymorphism)

·         T-RFLP (terminal restriction fragment length polymorphism)

·         qPCR (quantitative PCR)

non-PCR-based

·         FISH (fluorescence in situ hybridization)

·         Microarray

·         Raman microspectroscopy
      NanoSIMS (nano-scale secondary ion mass spectrometry
     
     NGS (Next Generation Sequencing)  
        Pyrosequencing

The field of metagenomics is described apart from these methods, as a broad category of investigations of microorganisms in mixed, uncultured communities.

I found this paper a useful introduction to some of the terminology and methodology of environmental microbiology. At the moment, it seems unlikely I will be citing this paper directly, but its reference list will be useful, and I might want to re-read this in a few months, when I have gained some more familiarity with key concepts.

Palmer et al. 2010

Palmer K, Drake HL, Horn MA. 2010. Association of novel and highly diverse acid-tolerant denitrifiers with N2O fluxes of an acidic fen. Applied and Enironmental Microbiology 76: 1125-1134.

These authors examined soils from an acidic fen in southern Germany, and discovered novel denitrifiers that are apparently adapted to local conditions and contribute to the cycling of nitrogen within the fen. Methods employed included measurement of soil parameters, microcosms to examine denitrification rates (both total denitrification and net production / consumption of N2O), cell counts of cultured organisms, and phylogenetic analysis using narG and nosZ sequences and RFLP.

Palmer et al. 2009

Palmer K, Drake HL, Horn MA. 2009. Genome-derived criteria for assigning environmental narG and nosZ sequences to operational taxonomic units of nitrate reducers. Applied and Environmental Microbiology 75: 5170-5174.

These authors compared the sequences of narG and nosZ genes to corresponding sequences of 16s rRNA genes, using in-silico analysis of sequences downloaded from GenBank. While similarities above 97% are commonly used for species- or genus-level taxonomic delineation for 16s sequences, this analysis found much lower threshold similarities for such delineation using the structural genes.

This paper is confusing to me. One part of the text appears to contradict itself, when the authors state that the Nar operon in Pseudomonas stutzeri A1501 is putatively alien in origin (i.e. recent horizontal transfer), then go on to state in the same paragraph that it appears unlikely that the Nar operon was horizontally transferred in any species. I may just be misunderstanding the meaning of the term “putatively alien” in regards to a bacterial gene sequence.

A greater puzzle is presented by the list of nosZ sequences. These authors downloaded 85 such sequences, where my own attempts to extract data from GenBank resulted in only 42 unique nosZ sequences. The list in a supplementary table includes several cases of multiple accessions of the same species but of different PD. The paper these clusters of PD-sequences are derived from is Dandie et al. (2007); a quick scan of this paper did not reveal what the distinction “PD” indicates.

Schmidt et al. 2008

Schmidt SK, Reed SC, Nemergut DR, Grandy AS, Cleveland CC, Weintraub MN, Hill AW, Costellow EK, Meyer AF, Neff JC, Martin AM. 2008. The earliest stages of ecosystem succession in high-elevation (5000 metres above sea level), recently deglaciated soils. Proceedings of the Royal Society of London, Series B 275: 2793-2802.

These authors describe the microbial community and soil parameters of a chronosequence at the foreground of a receding glacier high in the Peruvian Andes. From a combination of aerial photography and previous work at this site, a series of sites of soils of increasing ages from zero to 79 years old was established. No surface plants, even lichens, are present on any of these new soils, and soil nutrient levels (carbon, nitrogen) are very low; the only organisms present are microorganisms.

Two previous hypotheses had been proposed to explain the dynamics of very early primary succession on new soil. Organic matter has been observed to accumulate slowly in new soils; the source of this material is either aeolian deposits (i.e. wind-borne plant detritus and pollen) or in-situ fixation of CO2 and N2. These are not mutually exclusive hypotheses, but the relative contributions of each are explored in this study.

The methods used here cover an extensive list of soil parameters. Three sets of soil samples were collected: for microbiological analysis, N-fixation measurement, and all other chemical analyses. The other chemical analyses include photosynthetic pigment extraction, soil total and mineral nitrogen, pyrolysis for identifying sources of carbon compounds (i.e. microbial-autotroph, microbial-heterotroph, plant), enzyme assays for common and informative microbial enzymes, and soil stability analysis of the resistance of these new soils to erosion forces such as water runoff.

These authors focused on the cyanobacterial fraction of the microbial community in this study; some details of other components of the biota are described in an earlier paper, Nemergut et al. (2007). Cyanobacteria are autotrophs also capable of fixing atmospheric nitrogen, thus they are ideal primary colonizers of new soil as they require little more than a source of moisture and air. Analysis of the community included the use of the P-test (Martin 2002); note that as in Nemergut et al. (2007), he is one of the authors of this study. The analytical approach is very similar to that employed in the earlier study, with a comparison of discovered sequences to published sequences from around the world. In this study, cyanobacterial sequences from zero and 4-year-old soils were similar to sequences from an extremely broad sample of habitats, including Antarctic lake ice, marine subseafloor sediments, urban aerosols, forest soils, and oil-polluted soils.

The soil chronosequence showed a clear pattern of stages of primary succession at every level of analysis. The soil microbial community became both more abundant and more diverse through time, soil nutrients increased, the chemical environment included increasing amounts and diversity of complex organic molecules, key enzyme pathways became established, and soil stability increased as soils aged. N-fixation showed a peak, with increasing N-fixation activity from the zero to 4-year-old soils (by two orders of magnitude), then declining by about half in the 79-year-old soils. This mirrors and precedes a widely-observed pattern in plant primary succession, in which nitrogen-fixing plants are among the first colonizers, but decline in abundance at later stages of succession. Enzyme and organic molecule patterns were consistent with a total absence of heterotrophs in the extremely young soils, increasing occurrence of organisms capable of decomposing plant matter in the 4-year-old soils, and a molecular ecology qualitatively similar to a mature plant-associated soil in the 79-year-old soil.

The list of procedures and level of detail of analysis in this paper is impressive. Many, though certainly not all, of these techniques will be models for my own work, especially in the summer of 2010. The molecular-diversity techniques pioneered by Martin (Martin 2002, Nemergut et al. 2007, this paper) as well as the techniques of analyzing soil pigments and soil nutrients are all very interesting.

Sørensen et al. 2006

Sørensen LI, Holmstrup M, Maraldo K, Christensen S, Christensen B. 2006. Soil fauna communities and microbial respiration in high Arctic tundra soils at Zackenberg, Northeast Greenland. Polar Biology 29: 189-195.

These authors sampled soil animals from three sites at Zackenberg station, Greenland, over three days in mid-summer. Two of the sites were considered mesic heath, with a mix of Cassiope tetragona and other High Arctic species of plants, while the third site was dominated by Dryas spp. and was considered dry heath; snow melts from the dry heath up to 20 days earlier than from the mesic heaths. Soil samples ranging down to about 6cm depth were collected, stored at 5ºC for up to two weeks, and analyzed by a range of methods in the laboratories in Europe.

Different groups of soil animals were extracted by varying methods. Soil microarthropods, a diverse group dominated by Collebola and Acari, were extracted by modified MacFadyen funnels into Benzoic acid. Enchytraeids and dipteran larvae were extracted in Baermann wet funnels with heating of the samples, into tap water. Protozoa were washed from soils in water and grown on media plates in the dark at 10ºC. Nematodes were collected by the Blender-Cotton wool method of Schouten and Arp (1991). Soil microbial respiration was measured in serum bottles, with the CO2 concentration in the headspace measured at zero, 5 and 25 hours, with a fully factorial design of nutrient amendments of C, N, and P. Soil pH and soil organic matter content, but not moisture content or other nutrient concentrations were determined using methods not clearly described, though presumably these procedures were similar to standard methods.

Once abundance and biomass data was collected, comparisons between plots were made using multivariate analysis and a software package named PRIMER 5.0. My understanding is the species counts were (log+1) transformed to reduce the influence of very abundant species, then analyzed using an approach similar to Principal Components Analysis. The result of this analysis was a clear difference between the dry heath and the two mesic heaths, while the two mesic heaths were not different from each other in parameter-space. A Bray-Curtis similarity matrix was also involved, though I’m not certain I understand how.

Different taxonomic groups were identified to different taxonomic levels; 19 species of Collembola and 7 species of Enchytraeids were found, for example, but Acari were identified to suborder (Cryptostigmata (oribatids), Prostigmata, Mesostigmata) and nematodes and protozoans were counted at those high taxonomic levels. While the two mesic heath sites were only marginally significantly different from each other, there was a clear increase in abundances in the dry heath site. For collembola at least, the dry heath site was also dominated by two highly abundant species, which differed from the majority of species in the mesic sites by being unpigmented and associated with sub-surface, rather than soil-surface, regions in the soil. The higher abundance of probably bacteria-eating nematodes at the dry heath strongly suggests higher turnover of microorganisms as well as generally higher biological activity from the higher populations of most soil animals.

These authors suggest higher organic matter decomposition rates at the dry heath, which seems reasonable given the higher animal populations there. However, their attribution of higher soil pH there to higher respiration levels seems like more of a stretch, absent supporting mineralogical and soil-nutrient data.

This paper provides an excellent example of the data that can be collected and analyzed from a brief but intensive study of soil invertebrates at a High Arctic site. In addition, meaningful information about differences in biodiversity between locations can be derived from studies of organisms not identified to fine taxonomic levels.

O'Neill et al. 2010

O’Neill KP, Godwin HW, Jiménez-Esquilín AE, Battigelli JP. 2010. Reducing the dimensionality of soil microinvertebrate community datasets using Indicator Species Analysis: Implications for ecosystem monitoring and soil management. Soil Biology & Biochemistry 42: 145-154.

These authors used a dataset of soil microarthropods to evaluate a method for identifying indicator species for ecosystem monitoring. The method centres on the Indicator Value (IV) of a species, a number that integrates the degree of uniqueness to a place of a species and the abundance of that species within a given habitat. A high IV value indicates both high information content and a high probability of being sampled. The IV is apparently robust to differences in site number and species absolute abundances, and provides a single value for evaluating observed or expected changes in an ecosystem. Indicator species, furthermore, integrate habitat conditions over their lifespans, in contrast to measures of chemical and physical parameters that capture a snapshot of an ecosystem.

The basic evaluation approach here was to identify indicator species along a clear environmental gradient from meadow to forest in West Virginia. The habitat was divided into three zones, with an edge patch between the open meadows and closed-canopy forest. Near-surface soil cores were collected from each zone every month from April 2004 to April 2005 (n = 180), using the top of the mineral soil as the reference depth. Microarthropods were extracted in a modified Macfadyen funnel with a strong and increasing temperature gradient, into 70% ethanol.

Diversity measures, including Simpson’s and Shannon indices, were based on counts of individuals identified to family level (suborder for Acari). Differences between sites were analyzed by 2-way repeated measures ANOVA and Principle Components Analysis, after rare taxa (those that occurred in less than 10% of samples) were removed; rare taxa are extremely unlikely to be identified as indicator species.

Calculating IV for each taxon, regardless of the taxonomic resolution, provides large advantages in labour time and taxonomic expertise, as many microfauna are very difficult to identify to genus or species. These authors state that enumeration of a single sample required more than 1 hour of a trained taxonomist’s time. In studies such as this one, there are further advantages of IV associated with its robustness in the face of many zero measurements (i.e. taxa absent from samples) and the general messiness of these kinds of datasets. However, the ISA approach is intended for 2-stage studies, where an intensive initial survey identifies indicator species (taxa), and later long-term monitoring ignores other species. For studies specificially designed to address biodiversity, such as my own, excluding rare taxa would not be beneficial, and there may be no easy escape from time-consuming morphotaxa sorting.

I have spoken with Dr. Battigelli, the trained taxonomist in this study. He has indicated that while this IV-based approach may not be appropriate for my own work, it nonetheless demonstrates the types of analyses that can be conducted with soil invertebrates identified to middle taxonomic levels. He has assured me I could probably be trained to identify Collembola to Family and Acari to Suborder in a matter of a few days, and he would be interested in futher studies of collected soil invertebrates based on interesting patterns that emerge at these taxonomic levels.

Philippot et al. 2002

Philippot L, Piutti S, Martin-Laurent F, Hallet S, Germon JC. 2002. Molecular analysis of the nitrate-reducing community from unplanted and maize-planted soils. Applied and Environmental Microbiology 68: 6121-6128.

These authors applied molecular techniques including PCR, RFLP, and sequencing to the study of soil bacteria relevant to crops. Dissimilatory nitrate reduction, the process that converts NO3- to NO2-, is widespread in prokaryotes, with the activity described in alpha, beta, and gamma Proteobacteria, gram-positive bacteria, and some archaea. There are two described enzymes that catalyze the reaction and provide energy to the organism; these authors focused on the membrane-bound protein, specifically one subunit that includes a distinctive set of components. Their approach was to design primers for a well-conserved region of the gene narG that amplify a 650bp region, and then subject the PCR product to cloning, RFLP analysis, and sequencing.

Community structure and diversity was compared between pots planted with maize versus unplanted controls. Maize (Zea mays) is a plant that facilitates gas diffusion in its roots under oxygen-stress soil conditions; this creates an aerobic region in the rhizosphere distinct from anaerobic conditions further from roots. While diversity, as measured by standard indices, did not differ between planted and unplanted soils, the structure of the communities did change, with numerous RFLP phylotypes found in only one or the other treatment. This suggests a role of rhizosphere conditions, likely involving both oxygen and root exudates, in selecting for particular groups of microorganisms.

Nitrate reduction occurs primarily or possibly only under aerobic conditions. The microbial cell gains energy from dissimilatory reduction of nitrate, and if it occurs in the rhizosphere, the plant may gain a readily-accessible form of nitrogen in the form of nitrite. Denitrification, the process that shuttles nitrogen atoms from nitrite to gaseous forms such as N2O or N2, can occur under a range of oxygen conditions, including aerobic, thus denitrifiers in the rhizosphere may compete with plant roots for nitrite. The fate of nitrite produced by dissimilatory nitrate reduction can also be to ammonium, though this appears to be rare in soil and more common in vertebrate guts and digested sludge, two environments typically lacking in oxygen.

This paper provides some molecular tools for my own studies of nitrogen dynamics in soils, especially the sequences of the degenerate primers. In addition, it provides some clarification of parts of the remarkably complex soil-nitrogen cycle.

Li et al. 2009

Li X-R, Du B, Fu H-X, Wang R-F, Shi J-H, Wang Y, Jetten MSM, Quan ZX. 2009. The bacterial diversity in an anaerobic ammonium-oxidizing (anammox) reactor community. Systematic and Applied Microbiology 32: 278-289.

These authors studied the bacterial community that developed inside a bioreactor running on sewage sludge under anaerobic conditions. Like Lim et al. (2008), the main focus of this study was in the applications of ammonia-oxidizing bacteria (AOB) to water treatment facilities. The expected chemistry of anaerobic ammonia oxidation catalyzed by microorganisms (“anammox”) includes the use of nitrite as the electron acceptor in a near-one-to-one ratio with the consumption of ammonia or ammonium. The energy derived from this process is used by the cell to fix CO2, thus making these organisms autotrophs. This alters the underlying stoichiometry slightly, as some nitrite is diverted to CO2 fixation rather than ammonia oxidation.

The study of anaerobic AOB is still quite new, with the five described genera of such organisms all named with “Candidatus” prefixes, indicating recent species descriptions. All are in one group (taxonomic level unknown), the Brocadiales, within the phylum Planctomycetes. Aerobic AOB are in other groups, and include some species within the genus Nitrosomonas in the Beta-Proteobacteria that are capable of limited ammonia-oxidizing activity under anaerobic conditions, and can apparently survive long periods without oxygen.

These authors did not develop novel primers for PCR or qPCR in this study. Instead, they used published primer sets; I gather they did not use the TaqMan double-dye system for qPCR, as no mention of probes is made. The target genomic sequences were portions of the 16s rRNA gene, using E. coli as a standard. Oddly, the overall procedure included normal PCR, followed by cloning and insertion into plasmids, followed by qPCR of plasmid DNA containing the 16s sequences. It is unclear to me exactly why this was done, though later in the paper there are a few sequence-based phylogenetic trees that might have been based on sequences derived from this cloning procedure. In any case, the qPCR did provide informative results regarding the composition of bacterial groups within the reactor.

Of the sequences identified, the great majority were unlike cultivated organisms, highlighting the utility of these techniques in studying environmental samples. AnAOB produced approximately 16% of sequences, with aerobic AOB less than 1%. Non-AOB in three phyla constituted the majority of sequences, including 38% Chlorobi, 21% Chloroflexi, and 7% Bacteriodetes. These are filamentous heterotrophic bacteria, and appear to be closely associated with the granules that formed in the reactor solution after a few months. These authors suggest further research on the ecophysiology of these groups to answer questions regarding energy and material cycles within these systems.

In addition to 16s sequences, the hzo locus was also studied. This is a gene that produces an enzyme that catalyzes the oxidation of hydrazine (rocket fuel; N2H4) to N2 gas. No mention is made of the possibilities for N2O production or consumption in this process. The gene is restricted to AnAOB only, or at least that is the inference based on the observation that hydrazine is a unique intermediate molecule of the anammox process.

This study provides a useful example of the combination of qPCR and molecular-phylogenetic approaches in studying a microbiological system. Applied together, the two approaches allow the extraction of useful information regarding taxonomic diversity, both richness and evenness, among functional groups of organisms.

Kostina et al. 1994

Kostina NV, Stepanov AL, Umarov MM. 1994. Study of the complex of nitrous oxide-reducing microorganisms in the soil. Eurasian Soil Science 26: 81-87.

These authors extracted microorganisms from a range of soils apparently collected from various places in Russia and produced cultures of organisms capable of reducing nitrous oxide. The only nitrogen source in culture vials was N2O, and conditions were rendered anaerobic by flushing with argon gas.

Most cultures gradually lost their ability to reduce N2O, especially mixed-species cultures. A few pure strains were isolated that did not show this loss, and maintained high levels of activity in storage.

Pseudomonas spp. and Bacillus spp. contributed the vast majority of N2O-reducing activity in all soils, with other groups including Aeromonas spp., Micrococcus spp., Flavobacterium spp., Erwinia spp., and an organism identified as “similar to Corynebacterium” also showing some activity. Neither actinomycetes nor eukaryotes were found in any of the cultures capable of N2O-reduction, indicating this is a physiological process not possessed by these organisms.

This is an English translation of a paper that was probably originally in Russian: Pochvovedeniye 1993 25: 72-76.

Wagner et al. 2009

Wagner D, Kobabe S, Liebner S. 2009. Bacterial community structure and carbon turnover in permafrost-affected soils of the Lena Delta, northeastern Siberia. Canadian Journal of Microbiology 55: 73-83.

These authors examined the microbial communities at two depth bands (near-surface and near-permafrost) in low-centred tundra polygons at the vast permafrost wetland of the delta of the Lena River. The delta covers more than 60 000 km^2, and much of it appears to be a reserve or national park of Russia. The CAVM (Walker et al. 2002) describes most of the delta as vegetation type W2, sedge, moss, dwarf-shrub wetland, and satellite images from Google maps shows very extensive lake and pond coverage of the landscape. In short, it’s pretty wet, and generally cold.

The general finding of this paper is that while near-surface communities include a wide diversity of aerobic and facultatively-anaerobic bacteria, the deeper, colder, anaerobic portions of the soil contain almost no aerobes, and are instead dominated by “fermenting” species capable of decomposing recalcitrant organic carbon molecules under negative-redox conditions. There is a sharp temperature gradient, which combined with the poorer quality of carbon, the lack of oxygen and negative redox conditions, and the general water saturation at depth creates conditions near the permafrost suitable only for the slow microbial metabolisms. None of this is particularly surprising, but the observation of decreased biodiversity with water saturation does suggest the worrying possibility that increased water in this system, driven by melting permafrost and climate change (particularly upstream in the long and North-flowing Lena) could drive these microbial communities to lose some “physiological skills” such as the ability to oxidize methane, a metabolic pathway possessed only by some aerobic prokaryotes.

This paper is quite important to my own work, I think. Besides emphasizing the role of water content in structuring soil chemical and especially biological conditions, the description of the methods used to measure microbial biodiversity should be useful. However, while the BIOLOG plates seem interesting, the results of this technique are not at all well explained in this paper. I do not know what is indicated by the relationship shown in Figure 3, for example, of changes in colour development associated with carbon turnover of various categories of organic substrates. Several of the figures are simple plots of principal component analysis (PCA), literally just PC1 vs. PC2 with some outlines drawn around some clusters. I’m sure there is more of interest in this paper besides the coarse outline of biodiversity differences in communities, but without a more thorough explanation of the nearly-raw data I cannot see it.

Nemergut et al. 2007

Nemergut DR, Anderson SP, Cleveland CC, Martin AP, Miller AE, Seimon A, Schmidt SK. 2007. Microbial community succession in an unvegetated, recently deglaciated soil. Microbial Ecology 53: 110-122.

These authors describe the partly-predictable patterns of succession among the soil microbes of a glacial foreland in Peru. Primary succession on new terrain, as found in front of a receding glacier, has been studied to some extent, especially regarding the vegetation. Studies of the microbial communities have been rarer, but the few that have been conducted have suggested that these communities also show predictable patterns of community assembly and turnover associated with soil age. Basic ecological theory has led to the nitrogen paradigm of primary succession in soils: nitrogen is absent from new mineral substrate, thus nitrogen fixing organisms have a competitive advantage and are therefore abundant. The presence of nitrogen fixers is tightly linked to the accumulation of soil nitrogen; hence these organisms may facilitate later successional stages.

The study site in this paper is in Peru, at a glacier that seems to be receding quickly. These authors sampled from 3 transects arranged parallel to the front of the glacier, located adjacent to the glacier on soil less than a year old, 100m away on soil about 4 years old, and 500m away on soil about 20 years old. This area receives very high inputs of pollen, leading to the hypothesis that heterotrophic, nitrogen-fixing organisms may be present, using the pollen as a carbon source but drawing nitrogen from the atmosphere because the C:N ratio of pollen is higher than that of microbial biomass. Surface soil samples were collected, kept at 0C, and analyzed in Colorado. Much of the analyses were detailed phylogenetic examination, including the P-test of Martin (2002); note that he is one of the authors of this paper. OTU and a range of sequence-data fine-tuning techniques were also employed.

Over the study area, autotrophic nitrogen fixers were abundant. The bacteria found were extremely diverse at the highest taxonomic levels, and many sequences identified were not closely related to existing sequences in public databases. Diversity increased rapidly from the youngest soils to the 4-year-old, then plateaued.

One very interesting group of bacteria found are the Comamodaceae; sequences reported in other studies were in many cases derived from glacial or ice-sheet ice. The Comamodaceae found in the youngest soils here may have persisted as viable populations in the glacier; differences between the two closely-examined youngest communities suggest physical and genetic isolation for hundreds to thousands of years, allowing speciation events to accumulate differences.

Other patterns among the sequences identified suggest that the earliest colonizers of new terrain may be cosmopolitan – some of the Comamodaceae sequences, for example, are similar to those derived from a glacier in Nunavut. Later colonizers may be more endemic, and displace the earliest colonizers as soils age. The trophic status of the first colonizers is not clear; these authors did not have a test for definite autotrophs or heterotrophs, as Comamodaceae are known to include both modes. At the macrobiological level, the earliest arrivals on new terrain are typically heterotrophs, insects that feed on deposited organic matter such as wind-blown pollen.

This paper is very useful to me, describing as it does a complete set of analytical procedures for my planned biogeographic / phylogenetic studies, as well as providing data in the form of publicly-accessible sequences and analyzed information on patterns of soil microbial community assembly.

Jones and Henry 2003

Jones GA, Henry GHR. 2003. Primary plant succession on recently deglaciated terrain in the Canadian High Arctic. Journal of Biogeography 30: 277-296.

These authors examined five glacial foregrounds on Ellesmere Island, one intensively and the other 4 “extensively”, to determine patterns of succession among plant communities on sterile ground. The ecological literature recognizes several different modes of succession, including a categorization by Henry and Svoboda (1987) based on the relative strengths of biotic and abiotic factors. This model of succession recognizes 3 modes; directional succession with species replacement, directional succession without species replacement, and non-directional succession without replacement. They are arranged in increasing importance of abiotic factors, referred to here as “environmental resistance”, which operates in opposition to “biological driving forces”.

In temperate regions, where much of the relevant ecological theory has been developed, biotic factors are mainly competition. In the High Arctic, a successional pattern consistent with directional-with-replacement was found, demonstrating that this can occur even in environments with obviously severe abiotic factors. However, these authors argue that the biotic factor driving this succession was probably not competition, because total plant cover remains below 10% by area even at the fourth stage recognized here, and species richness is always very low. The polar oasis landscape with 80-100% plant cover was never reached within the approximately 50-year old glacial forelands examined by these authors, though it is likely that competition is important in that “stage 5” level of High Arctic succession.

Other biotic variables suggested playing a role in successional dynamics in the High Arctic included facilitation and life-history characteristics. These factors are not independent; later successional species such as Salix arctica appear not to be able to establish until soil fertility has been improved by mats of very-early-colonizing mosses, and are long-lived, slow-growing species that contribute little to the early seed bank and seed rain. Thus, multiple plant and environmental characteristics appear to interact when structuring early communities.

I read this paper to try to gain some understanding of ecological succession and the role of time-since-deglaciation among the ecosystems of Alexandra Fjord. Rather than being distinct successional stages in sequence as I had previously supposed, it appears the various lowland ecosystems are all of a similar age, and have different vegetation communities as a result of other factors besides simply relative proximity to the Twin Glacier. Dryas integrefolia and Cassiope tetragona were important parts of this study, and both appear in stage 4, after primary-colonizing mosses, and early-colonizing forbs such as Papaver radicatum and early-colonizing deciduous shrubs like Saxifraga spp. Both Dryas and Cassiope form associations with mycorrhyzal fungi, a requirement that may slow their colonization of novel habitats; earlier-spreading plants do not form these associations, and instead may be limited by seed dispersal.

This was helpful in organizing the structure of the manuscript I am currently working on, which will describe some of the soil biotic communities both in the Alexandra Fjord lowlands and in the adjacent polar desert. It is not a simple story of succession from one ecosystem to the next, but succession does play a role.

Nemergut et al. 2005

Nemergut DR, Costello EK, Meyer AF, Pescador MY, Weintraub MN, Schmidt SK. 2005. Structure and function of alpine and arctic soil microbial communities. Research in Microbiology 156: 775-784.

These authors review the current state of knowledge of microbial communities in cold- and snow-affected soils. Their primary study site is a ridge in Colorado with a range of habitats from sub-alpine forest to glaciated mountain-tops; all areas receive significant snow cover for much of the year. They describe only three studies of microbial communities in the Arctic, stating these are the only such studies to their knowledge at the time of preparation of this paper.

The referenced work in this review clearly demonstrates that microbial communities are active when snow covered, contrary to the previous assumption that low temperatures would effectively prohibit microbial metabolisms during winter. Indeed, microbial biomass is actually highest in winter in the alpine tundra systems studied and lowest in spring after an apparent population crash. A wide diversity of microbes has been found, from Bacteria, Archaea, and Eucaryea, including deeply divergent lineages with no known associations with described groups. The physiologies and ecological functions of many of these microbes are completely unknown.

This paper provides a useful overview of the state of the field of cold-soils microecology, with many interesting references and some surprising synthesized findings. This research group in Colorado appears to be one of the few groups in the world studying cold soil microbial communities and their links to climate change.

Lipson et al. 2009

Lipson DA, Monson RK, Schmidt SK, Weintraub MN. 2009. The trade-off between growth rate and yield in microbial communities and the consequences for under-snow soil respiration in a high elevation coniferous forest. Biogeochemistry 95: 23-35.

These authors conducted a multiply-combined approach study that examined soil microbial communities in the sub-alpine forest of Colorado. They investigated growth and respiration of microbes including both bacteria and fungi, how those processes varied between summer (snow free) and winter (snow covered), and linked these processes to measures of community composition, and built a mathematical model of soil microbial metabolism and temperature. The overall purpose of this study was to thoroughly examine soil microbial processes relating to CO2 emissions and carbon cycling.

The major finding of this study was that there are effectively two distinct microbial communities in this ecosystem. In summer, there is a community of slow-growing, high biomass-yield microbes with a low specific respiration; in other words, the summer microbes grow slowly but efficiently, capturing much of the available carbon as biomass and releasing relatively little CO2 per unit biomass. In winter, the community is composed of fast-growing, low yield microbes that release much more CO2 per unit biomass.

There are effectively two ecological strategies at work, during different seasons. The winter strategy is one of competition. Available nutrients are consumed rapidly, releasing large amounts of CO2 but producing little growth. In summer, the strategy is more cooperative, with slower, less scramble-like growth that more fully uses available nutrients in growing new cells.

In general, the bacteria in the system seem more capable of the high-competition strategy, as these authors found little contribution of fungi to total ecosystem respiration in winter, by using a set of bacterial and fungal inhibitors. The winter community has a much higher response to temperature (Q10) than the summer community. A winter community at intermediate temperatures produces much more CO2 than does a summer community.

In analyzing the composition of the communities, these authors employed the P-test method of Martin (2002), as I intend to as well. I found this paper through a Web of Science search for papers citing Martin (2002); this was one of 153 papers found. The first author of this paper, D.A. Lipson, appears to have a substantial history of publications examining soil microbial communities.

Bohannan and Hughes 2003

Bohannan BJM, Hughes J. 2003. New approaches to analyzing microbial biodiversity data. Current Opinion in Microbiology. 6: 282-287.

These authors review the use of three broad approaches to studying microbial biodiversity in environments. The three are 1) parametric, 2) nonparametric, and 3) community phylogenetics. Each has advantages and disadvantages, and these authors suggest a combined approach may be most beneficial. Both 1) and 2) are based on Operational Taxonomic Units, to avoid the many problems of bacterial species identification, while 3) is based on molecular phylogenies, typically 16s rDNA.

Parametric approaches make simplifying assumptions and are based on some model of species richness in microbial communities; often this model is log-normal, in which some taxa are rare, some are abundant, and most are intermediate. These approaches extrapolate from patterns in a sample to the total environment. The obvious downside to parametric approaches is the vulnerability of the model to incorrect and difficult to test assumptions.

Nonparametric approaches avoid assuming any model, and instead are typically built on an approach analogous to mark-release-recapture. Sequences encountered more than once in a sample are recaptures, and the frequency of these doubletons is assumed to be related to how many unique sequences are present: more doubletons means fewer total sequences. As a downside, these approaches provide only a lower limit to actual richness, thus generally underestimating total diversity.

Community phylogenetics approaches avoid the OTU concept and thereby preserve useful data in the form of genetic information about sequences and sequence relationships. The downside of community phylogenetics approaches is they sample a clone library derived from the environment, not the environment directly, and can therefore not extrapolate from the sample to the environment.
This paper provides several useful examples of each approach, and supports the utility of Martin’s (2002) combined approach, which is what I would like to apply to my data to be collected in 2010. Figure 3 in this paper, for example, provides a useful overview of what Martin (2002) did, and how to make inferences about observed patterns.