Dorota L. Porazinska

Sequencing our way towards understanding global eukaryotic biodiversity

Microscopic eukaryotes are abundant, diverse and fill critical ecological roles across every ecosystem on Earth, yet there is a well-recognized gap in understanding of their global biodiversity. Fundamental advances in DNA sequencing and bioinformatics now allow accurate en masse biodiversity assessments of microscopic eukaryotes from environmental samples. Despite a promising outlook, the field of eukaryotic marker gene surveys faces significant challenges: how to generate data that are most useful to the community, especially in the face of evolving sequencing technologies and bioinformatics pipelines, and how to incorporate an expanding number of target genes.

Ultrasequencing of the meiofaunal biosphere: practice, pitfalls and promises

Biodiversity assessment is the key to understanding the relationship between biodiversity and ecosystem functioning, but there is a well‐acknowledged biodiversity identification gap related to eukaryotic meiofaunal organisms. Meiofaunal identification is confounded by the small size of taxa, morphological convergence and intraspecific variation. However, the most important restricting factor in meiofaunal ecological research is the mismatch between diversity and the number of taxonomists that are able to simultaneously identify and catalogue meiofaunal diversity. Accordingly, a molecular operational taxonomic unit (MOTU)‐based approach has been advocated for en mass meiofaunal biodiversity assessment, but it has been restricted by the lack of throughput afforded by chain termination sequencing. Contemporary pyrosequencing offers a solution to this problem in the form of environmental metagenetic analyses, but this represents a novel field of biodiversity assessment. Here, we provide an overview of meiofaunal metagenetic analyses, ranging from sample preservation and DNA extraction to PCR, sequencing and the bioinformatic interrogation of multiple, independent samples using 454 Roche sequencing platforms. We report two examples of environmental metagenetic nuclear small subunit 18S (nSSU) analyses of marine and tropical rainforest habitats and provide critical appraisals of the level of putative recombinant DNA molecules (chimeras) in metagenetic data sets. Following stringent quality control measures, environmental metagenetic analyses achieve MOTU formation across the eukaryote domain of life at a fraction of the time and cost of traditional approaches. The effectiveness of Roche 454 sequencing brings substantial advantages to studies aiming to elucidate the molecular genetic richness of not only meiofaunal, but also all complex eukaryotic communities.

Relationships at the aboveground-belowground interface: Plants, soil biota, and soil processes

Interactions at the aboveground-below ground interface provide important feedbacks that regulate ecosystem processes. Organisms within soil food webs are involved in processes of decomposition and nutrient mineralization, and their abundance and activity have been linked to plant ecophysiological traits such as species identity and the quality and quantity of plant tissue. We tested aboveground-below ground diversity relationships in a naturally developed plant community of native tallgrass prairie by taking soil samples from beneath naturally established grass tillers of chosen characteristics (e.g., homogeneous vs. heterogeneous plant combinations or C-4 vs. C-3 photosynthetic pathway) without imposing any disturbances to existing plant-soil relationships. The goal of this study was to elucidate the consequences, for soil microbiota (microflora phospholipid fatty acids, protozoa, and nematode functional groups) and for C and N mineralization, of plant community properties such as species richness, resource quality, resource heterogeneity, species identity, and presence of exotics. None of the biotic or abiotic soil variables was related to plant resource heterogeneity. Protozoa were not responsive to any of the plant community traits. Some bacterial and nematode groups were affected by plant characteristics specific to a particular plant species, but no uniform pattern emerged. Invasive and native plants generally were similar with respect to soil variables tested in this study. The lack of clear responses of soil variables to plant community traits indicates that idiosyncratic effects dominate both at the plant and soil biotic level and that generalized plant and soil diversity effects are hard to predict.

Evaluating high‐throughput sequencing as a method for metagenomic analysis of nematode diversity

Nematodes play an important role in ecosystem processes, yet the relevance of nematode species diversity to ecology is unknown. Because nematode identification of all individuals at the species level using standard techniques is difficult and time‐consuming, nematode communities are not resolved down to the species level, leaving ecological analysis ambiguous. We assessed the suitability of massively parallel sequencing for analysis of nematode diversity from metagenomic samples. We set up four artificial metagenomic samples involving 41 diverse reference nematodes in known abundances. Two samples came from pooling polymerase chain reaction products amplified from single nematode species. Two additional metagenomic samples consisted of amplified products of DNA extracted from pooled nematode species. Amplified products involved two rapidly evolving ~400‐bp sections coding for the small and large subunit of rRNA. The total number of reads ranged from 4159 to 14771 per metagenomic sample. Of these, 82% were > 199 bp in length. Among the reads > 199 bp, 86% matched the referenced species with less than three nucleotide differences from a reference sequence. Although neither rDNA section recovered all nematode species, the use of both loci improved the detection level of nematode species from 90 to 97%. Overall, results support the suitability of massively parallel sequencing for identification of nematodes. In contrast, the frequency of reads representing individual species did not correlate with the number of individuals in the metagenomic samples, suggesting that further methodological work is necessary before it will be justified for inferring the relative abundances of species within a nematode community.

The Biodiversity and Biogeochemistry of Cryoconite Holes from McMurdo Dry Valley Glaciers, Antarctica

Abstract Once thought of as inert, ice has been increasingly recognized as a habitat suitable for life. The landscape of the MCMurdo Dry Valleys (MCM) of Antarctica is dominated by glaciers, and glacier melt is the primary water source for life in soils, streams, and lakes. The glaciers, despite their cold and lifeless appearance, offer functioning habitats for life. The major objective of this study was to examine biogeochemical characteristics of miniecosystems present in cryoconite holes and to determine links to other components (soils, streams, and lakes) of the dry valley landscape. We examined cryoconite holes from 5 glaciers spanning the length of Taylor Valley, one of many valleys in the MCM. Cryoconite biotic communities were composed of the same species observed in streams and lakes, namely, cyanobacteria (Chlorococcus, Chroococcus, Crinalium, Oscillatoria, Nostoc, and Sprirulina), rotifers (Philodina gregaria and Cephalodella catellina), tardigrades (Acutuncus antarcticus and Hypsibius spp.), and ciliates. Biotic communities did not reflect the composition of the immediately surrounding environments, suggesting the effects of eolian mixing and transport of sediments and biota across the valley. Gradients of chemistry and biotic abundance in cryoconite holes reflected the position of each glacier in the valley. Nitrogen and organic carbon concentration patterns across glaciers potentially resulted from biological activities in cryoconite holes. Properties of holes were stable from one to the next sampling season, suggesting that changes of cryoconite hole properties develop on longer than yearly time scales.

Metabolic activity and diversity of cryoconites in the Taylor Valley, Antarctica

[1] Metabolic activity and biogeochemical diversity within cryoconites from the Canada, Commonwealth, Howard, and Hughes glaciers in the McMurdo Dry Valleys revealed the presence of a productive microbial refuge in this polar desert ecosystem. Fluorescent in situ hybridization showed a high percentage of Cytophaga-Flavobacteria cells in cryoconite sediments (87.2%), while b-Proteobacterial cells dominated the ice overlying the sediment layer (54.2%). The biomass of bacterial cells in the sediments was also greater (4.82 mgC ml � 1 ) than that in the overlying ice (0.18 mgC ml � 1 ) and was related to bacterial productivity (on the basis of thymidine incorporation), which ranged from 36 ng Cl � 1 d � 1 in the overlying ice to 3329 ng C l � 1 d � 1 in the sediment-containing layers. Bacteria within both the sediments and overlying ice were able to actively incorporate and respire radio-labeled glucose, as well as 17 other dissolved organic carbon compounds. The cryoconites in the Taylor Valley support an active, diverse assemblage of organisms despite the fact that they may remain sealed from the atmosphere for decades. Given the density of the cryoconites in the dry valleys (� 4–6% of ablation zone surfaces), flushing of the cryoconites during warm years could provide a vital nutrient and organic carbon source to the surrounding polar desert.

Snow-Patch Influence on Soil Biogeochemical Processes and Invertebrate Distribution in the McMurdo Dry Valleys, Antarctica

Abstract The McMurdo Dry Valleys is the largest of the ice-free areas in Antarctica. Precipitation events in excess of 1 cm of snow accumulation are rare. During the winter, snow is transported by strong katabatic winds blowing from the polar plateau, and deposited into the lee of topographic features (e.g., stream channels and other topographic depressions). At the start of the austral summer (early October), as much as 10% of the valley soils may be covered by distributed snow patches. Because liquid water is the primary driver of biological, physical, and chemical processes in this polar desert, quantifying fluxes of water from snow patches is important to understanding the influence of hydrology on soil biology and nutrient cycling. During the austral summer of 1999–2000, four snow patches that had developed during the previous winter in Taylor Valley were studied. We measured snow-patch area, depth, and snow water equivalent, as well as subnivian (under snow) and nearby exposed (control) soil temperature, light intensity, soil moisture, invertebrate abundance, soil organic matter content, and 95-d labile pools of C and N. Subnivian soils differed from exposed soils being as much as 26.8°C colder than exposed soils; average soil moisture ranging from 6.9 to 13.6% compared to 0.4% in exposed soils; soil invertebrate populations exceeding 7900 individuals kg−1 dry soil versus less than 1200 individuals kg−1 dry soil in exposed soils; and soil invertebrate species richness values greater than 2 taxa, compared to 1.3 taxa in exposed soils. The results of this study show that these seasonal, sparse snow patches may be an important source of moisture and control habitat of soil ecosystems in this extreme environment.

Reproducibility of read numbers in high-throughput sequencing analysis of nematode community composition and structure

Although nematodes are the most abundant metazoan animals on Earth, their diversity is largely unknown. To overcome limitations of traditional approaches (labour, time, and cost) for assessing biodiversity of nematode species in environmental samples, we have previously examined the suitability of high‐throughput sequencing for assessing species level diversity with a set of control experiments employing a mixture of nematodes of known number and with known sequences for target diagnostic loci. Those initial experiments clearly demonstrated the suitability of the approach for identification of nematode taxa but lacked the replicate experiments necessary to evaluate reproducibility of the approach. Here, we analyze reads generated from three different PCR amplifications and three different sequencing reactions to examine the differential PCR amplification, the possibility of emulsion PCR artefacts, and differences between sequencing reactions. Our results suggest that both qualitative and quantitative data are consistent and highly reproducible. Variation associated with in‐house PCR amplification or emPCR and sequencing are present but the representation of each nematode is very consistent from experiment to experiment and supports the use of read counts to estimate relative abundance of taxa in a metagenetic sample.

Toward Effective Probiotics for Autism and Other Neurodevelopmental Disorders

Hsaio and colleagues link gut microbes to autism spectrum disorders (ASD) in a mouse model. They show that ASD symptoms are triggered by compositional and structural shifts of microbes and associated metabolites, but symptoms are relieved by a Bacteroides fragilis probiotic. Thus probiotics may provide therapeutic strategies for neurodevelopmental disorders.

Ecometagenetics confirm high tropical rainforest nematode diversity.

The general patterns of increasing biodiversity from the poles to the equator have been well documented for large terrestrial organisms such as plants and vertebrates but are largely unknown for microbiota. In contrast to macrobiota, microbiota have long been assumed to exhibit cosmopolitan, random distributions and a lack of spatial patterns. To evaluate the assumption, we conducted a survey of nematode diversity within the soil, litter and canopy habitats of the humid lowland tropical rainforest of Costa Rica using an ultrasequencing ecometagenetic approach at a species‐equivalent taxonomic level. Our data indicate that both richness and diversity of nematode communities in the tropical rainforests of Costa Rica are high and exceed observed values from temperate ecosystems. The majority of nematode species were unknown to science, providing evidence for the presence of highly endemic (not cosmopolitan) species of still completely undiscovered biodiversity. Most importantly, the greater taxonomic resolution used here allowed us to reveal predictable habitat associations for specific taxa and thus gain insights into their nonrandom distribution patterns.