High‐throughput sequencing has the potential to describe biological communities with high efficiency yet comprehensive assessment of diversity with species‐level resolution remains one of the most challenging aspects of metabarcoding studies. We investigated the utility of curated ribosomal and mitochondrial nematode reference sequence databases for determining phylum‐specific species‐level clustering thresholds. We compiled 438 ribosomal and 290 mitochondrial sequences which identified 99% and 94% as the species delineation clustering threshold, respectively. These thresholds were evaluated in HTS data from mock communities containing 39 nematode species as well as environmental samples from Vietnam. We compared the taxonomic description of the mocks generated by two read‐merging and two clustering algorithms and the cluster‐free Dada2 pipeline. Taxonomic assignment with the RDP classifier was assessed under different training sets. Our results showed that 36/39 mock nematode species were identified across the molecular markers (18S: 32, JB2: 19, JB3: 21) in UClust_ref OTUs at their respective clustering thresholds, outperforming UParse_denovo and the commonly used 97% similarity. Dada2 generated the most realistic number of ASVs (18S: 83, JB2: 75, JB3: 82), collectively identifying 30/39 mock species. The ribosomal marker outperformed the mitochondrial markers in terms of species and genus‐level detections for both OTUs and ASVs. The number of taxonomic assignments of OTUs/ASVs was highest when the smallest reference database containing only nematode sequences was used and when sequences were truncated to the respective amplicon length. Overall, OTUs generated more species‐level detections, which were, however, associated with higher error rates compared to ASVs. Genus‐level assignments using ASVs exhibited higher accuracy and lower error rates compared to species‐level assignments, suggesting that this is the most reliable pipeline for rapid assessment of alpha diversity from environmental samples.

Terrestrial biota in the Antarctic are more globally distinct and highly structured biogeographically than previously believed, but information on biogeographic patterns and endemism in freshwater communities is largely lacking. We studied biogeographic patterns of Antarctic freshwater diatoms based on the analysis of species occurrences in a dataset of 439 lakes spread across the Antarctic realm. Highly distinct diatom floras, both in terms of composition and richness, characterize Continental Antarctica, Maritime Antarctica and the sub-Antarctic islands, with marked biogeographic provincialism in each region. A total of 44% of all species is estimated to be endemic to the Antarctic, and most of them are confined to a single biogeographic region. The level of endemism significantly increases with increasing latitude and geographic isolation. Our results have implications for conservation planning, and suggest that successful dispersal of freshwater diatoms to and within the Antarctic is limited, fostering the evolution of highly endemic diatom floras.

Antarctic soils are known to be oligotrophic and of having low buffering capacities. It is expected that this is particularly the case for inland high-altitude regions. We hypothesized that the bedrock type and the presence of macrobiota in these soils enforce a high selective pressure on their bacterial communities. To test this, we analyzed the bacterial community structure in 52 soil samples from the western Sør Rondane Mountains (Dronning Maud Land, East Antarctica), using the Illumina MiSeq platform in combination with ARISA fingerprinting. The samples were taken along broad environmental gradients in an area covering nearly 1000 km(2) Ordination and variation partitioning analyses revealed that the total organic carbon content was the most significant variable in structuring the bacterial communities, followed by pH, electric conductivity, bedrock type and the moisture content, while spatial distance was of relatively minor importance. Acidobacteria (Chloracidobacteria) and Actinobacteria (Actinomycetales) dominated gneiss derived mineral soil samples, while Proteobacteria (Sphingomonadaceae), Cyanobacteria, Armatimonadetes and candidate division FBP-dominated soil samples with a high total organic carbon content that were mainly situated on granite derived bedrock.

BackgroundCopper dependent nitrite reductase, NirK, catalyses the key step in denitrification, i.e. nitrite reduction to nitric oxide. Distinct structural NirK classes and phylogenetic clades of NirK-type denitrifiers have previously been observed based on a limited set of NirK sequences, however, their environmental distribution or ecological strategies are currently unknown. In addition, environmental nirK-type denitrifiers are currently underestimated in PCR-dependent surveys due to primer coverage limitations that can be attributed to their broad taxonomic diversity and enormous nirK sequence divergence. Therefore, we revisited reported analyses on partial NirK sequences using a taxonomically diverse, full-length NirK sequence dataset.ResultsDivision of NirK sequences into two phylogenetically distinct clades was confirmed, with Clade I mainly comprising Alphaproteobacteria (plus some Gamma- and Betaproteobacteria) and Clade II harbouring more diverse taxonomic groups like Archaea, Bacteroidetes, Chloroflexi, Gemmatimonadetes, Nitrospirae, Firmicutes, Actinobacteria, Planctomycetes and Proteobacteria (mainly Beta and Gamma). Failure of currently available primer sets to target diverse NirK-type denitrifiers in environmental surveys could be attributed to mismatches over the whole length of the primer binding regions including the 3′ site, with Clade II sequences containing higher sequence divergence than Clade I sequences. Simultaneous presence of both the denitrification and DNRA pathway could be observed in 67 % of all NirK-type denitrifiers.ConclusionThe previously reported division of NirK into two distinct phylogenetic clades was confirmed using a taxonomically diverse set of full-length NirK sequences. Enormous sequence divergence of nirK gene sequences, probably due to variable nirK evolutionary trajectories, will remain an issue for covering diverse NirK-type denitrifiers in amplicon-based environmental surveys. The potential of a single organism to partition nitrate to either denitrification or dissimilatory nitrate reduction to ammonium appeared to be more widespread than originally anticipated as more than half of all NirK-type denitrifiers were shown to contain both pathways in their genome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2465-0) contains supplementary material, which is available to authorized users.

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The application of high-throughput sequencing of the 16S rRNA gene has increased the size of microbial diversity datasets by several orders of magnitude, providing improved access to the rare biosphere compared with cultivation-based approaches and more established cultivation-independent techniques. By contrast, cultivation-based approaches allow the retrieval of both common and uncommon bacteria that can grow in the conditions used and provide access to strains for biotechnological applications. We performed bidirectional pyrosequencing of the bacterial 16S rRNA gene diversity in two terrestrial and seven aquatic Antarctic microbial mat samples previously studied by heterotrophic cultivation. While, not unexpectedly, 77.5% of genera recovered by pyrosequencing were not among the isolates, 25.6% of the genera picked up by cultivation were not detected by pyrosequencing. To allow comparison between both techniques, we focused on the five phyla (Proteobacteria, Actinobacteria, Bacteroidetes, Firmicutes and Deinococcus-Thermus) recovered by heterotrophic cultivation. Four of these phyla were among the most abundantly recovered by pyrosequencing. Strikingly, there was relatively little overlap between cultivation and the forward and reverse pyrosequencing-based datasets at the genus (17.1–22.2%) and OTU (3.5–3.6%) level (defined on a 97% similarity cut-off level). Comparison of the V1–V2 and V3–V2 datasets of the 16S rRNA gene revealed remarkable differences in number of OTUs and genera recovered. The forward dataset missed 33% of the genera from the reverse dataset despite comprising 50% more OTUs, while the reverse dataset did not contain 40% of the genera of the forward dataset. Similar observations were evident when comparing the forward and reverse cultivation datasets. Our results indicate that the region under consideration can have a large impact on perceived diversity, and should be considered when comparing different datasets. Finally, a high number of OTUs could not be classified using the RDP reference database, suggesting the presence of a large amount of novel diversity.

The bacterial and microeukaryotic biodiversity were studied using pyrosequencing analysis on a 454 GS FLX+ platform of partial SSU rRNA genes in terrestrial and aquatic habitats of the Sør Rondane Mountains, including soils, on mosses, endolithic communities, cryoconite holes and supraglacial and subglacial meltwater lenses. This inventory was complemented with Denaturing Gradient Gel Electrophoresis targeting Chlorophyta and Cyanobacteria. OTUs belonging to the Rotifera, Chlorophyta, Tardigrada, Ciliophora, Cercozoa, Fungi, Bryophyta, Bacillariophyta, Collembola and Nematoda were present with a relative abundance of at least 0.1% in the eukaryotic communities. Cyanobacteria, Proteobacteria, Bacteroidetes, Acidobacteria, FBP and Actinobacteria were the most abundant bacterial phyla. Multivariate analyses of the pyrosequencing data revealed a general lack of differentiation of both eukaryotes and prokaryotes according to habitat type. However, the bacterial community structure in the aquatic habitats was dominated by the filamentous cyanobacteria Leptolyngbya and appeared to be significantly different compared with those in dry soils, on mosses, and in endolithic habitats. A striking feature in all datasets was the detection of a relatively large amount of sequences new to science, which underscores the need for additional biodiversity assessments in Antarctic inland locations.

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