Bacterial diversity assessment in Antarctic terrestrial and aquatic microbial mats : a comparison between bidirectional pyrosequencing and cultivation

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

Biodiversity studies in Open-Top Chambers in continental Antarctica

Open Top Chambers are passive warming systems used to experimentally assess the effect of climate change on terrestrial ecosystems, and they were used in several Antarctic regions to study the response of biotic communities. In the BELSPO BELDIVA project, OTCs were used in continental Eastern Antarctica, where environmental conditions are very extreme. In January 2010, 8 Open-Top Chambers (OTC) were installed in four ice-free regions of the Sör Rondane Mountains, namely on the Utsteinen ridge, the Tanngarden granite outcrop, the Teltet nunatak and the fourth nunatak of the Pingvinane range.ANTAR-IMPACT, BELDIV

Analyse bioinformatique des résultats de séquençages d'amplicons

Amplicon sequencing can be a very powerful approach for detecting toxic cyanobacteria or any other kind of microorganism during monitoring programs. However, owing to the huge size of next-generation sequencing (NGS) datasets (up to several Gb), there is an obvious need for semi-automatic data processing and statistical analysis, as well as visualization of the patterns found. Importantly, raw NGS data contain errors, some of which are easily detected (e.g. too short or low-quality reads), while others remain hidden even after the most stringent quality controls (e.g. chimeras, contaminations, reads with large insertions or deletions, referred to as “indels”). As a consequence, NGS data need to be interpreted with caution, and bioinformatics analysis implementing poor error identification can easily lead to erroneous conclusions. Hence, a crucial step in the analysis of NGS data is the detection and removal of as many erroneous reads as possible. Moreover, bioinformatics involve additional preprocessing steps, including demultiplexing (i.e. grouping reads to samples according to the barcode sequence), deleting non-biological tags together with the adaptors and primer sequences, and removing chimeric sequences. In addition, the bioinformatics pipelines enable the quality-filtered sequences to be clustered into biologically relevant operational taxonomic units (OTUs), which form the basis of the statistical analysis, including the calculation of alpha- and beta-diversity.B-BLOOMS, CCAMBIO, CYANOCOST, PYROCYAN

BIOGEOGRAPHIC PATTERNS IN ANTARCTIC LACUSTRINE PROKARYOTES

Amplified climate change, increased human activity and the introduction of alien species likely form the biggest threat to Antarctic terrestrial ecosystems through range size expansions and contractions, regional extirpation and impacts on ecosystem functions. Despite their crucial role in the functioning of Antarctic terrestrial ecosystems , little is known about the present -day diversity and biogeography of microorganisms such as prokaryotes and microeukaryotes in the Antarctic Biogeographic Realm. Furthermore, identification of the key processes underlying microbial biodiversity dynamics is essential to understand and predict the consequences of global change on Antarctic lacustrine ecosystems. We analysed bacterial biodiversity in a total of 152 lacustrine microbial mat samples, distributed over the three main Biogeographic regions in the Antarctic Realm, including continental Antarctica, Maritime Antarctica and the Sub-Antarctic Islands comprising the southern Indian Ocean Province (SIOP) and the southern Pacific Ocean Province (SPOP). We targeted the V1-V3 variable regions of the 16S rRNA gene. Amplicon sequencing was done on an Illumina PE300 MiSeq. Sequences were processed using Usearch and Uparse, Mothur and custom scripts for basic parsing. An OTU cut-off was defined at 97 % sequence similarity, and sequences were mapped against a local GreenGenes database. Downstream analyses were performed using several R packages. We obtained about three million high quality sequences, with an average length of 500 bp. Sequences belonged to 8237 OTUs, and were distributed over 51 phyla and 366 genera. In addition, 649 OTUs remained unclassified at the phylum level and 6263 at the genus level. Mean OTU richness differed strongly between the four biogeographic regions. The lakes from Maritime Antarctica had a higher richness than those from Continental Antarctica. Interestingly, in sub-Antarctica OTU richness was strongly variable, with Marion Island (SIOP) having the lowest and Macquarie Island (SPOP) having on average the highest diversity of all studied regions. Multivariate Analyses showed that microbial community composition varied between biogeographic regions, with Macquarie Island being most different from the other regions. Continental Antarctica, Maritime Antarctica and the lakes from the SIOP share many OTUs, both in the case of Cyanobacteria and other bacteria, but are also characterised by a considerable number of unique OTUs. Within Antarctica, some regions harbour distinct bacterial communities such as the lakes in Schirmacher Oasis, Dronning Maud Land, and those from the eastern and western part of the Antarctic Peninsula.CCAMBI

The diversity and tolerance to osmotic stress of East Antarctic filamentous Cyanobacteria

Filamentous cyanobacteria are keystone species in Antarctic lake ecosystems; they are the basis of the simple foodwebs, play a crucial role in biogeochemical cycling and form the structure of benthic microbial mats which act as habitats for other prokaryotic and (micro-eukaryotic biota. Despite this, little is known about their diversity, adaptation and survival strategies in the extreme Antarctic conditions. We studied the uncultivated prokaryotic diversity using a 454 metagenomic analysis at the 16S rRNA level (V1-V3 region) in Continental Antarctic lakes situated along a conductivity gradient (0.014-142.02 mS/cm). The quality and length of the amplicons was analyzed with a custom-made Mothur pipeline and the resulting sequences were mapped against the Greengenes database, which includes CyanoDB. Almost 27% of the sequences could be assigned to the phylum of the cyanobacteria. The most abundant cyanobacteria in the dataset belonged to the genera Microcoleus, Leptolyngbya, Pseudanabaena, Nodularia and Phormidum. Some 16S rRNA types (at the 97% similarity level), such as sequences related to Leptolynbya antarctica, were present in both freshwater and hypersaline lakes. In order to further investigate this distribution, we isolated filaments of Leptolyngbya from seven lakes with conductivities ranging between 26.8 mS/cm and 0.038 mS/cm. The complete 16S rRNA and ITS genes of the isolates were subsequently sequenced. We found several 16S types related to different lineages of filamentous cyanobacteria in the seven lakes that were supported by ITS data. Two 16S types, belonging to a Leptolyngbya antarctica and Leptolyngbya sp., were each present in two different freshwater lakes. Two different 16S types, both belonging to Leptolynbya antarctica were present in a freshwater and hypersaline lake, which indicates a high ‘intraspecific’ molecular diversity. In order to better understand the adaptation and/or wide tolerance to osmotic stress, we are currently performing ecophysiological experiments with these isolates aimed at assessing the potential local adaptation of these strains to conductivity and desiccation.CC

Human impacts on Antarctic ecosystems: do not forget the microorganisms!

The tiny and microscopic creatures that are the permanent inhabitants of the Antarctic continent are often overlooked in environmental impact assessments and when new management and protection strategies are designed. This lack of consideration is probably due to their small size and the need of sophisticated molecular methods to study their diversity, evolution and geographic distribution. However, considerable progress has been made in the field of molecular diversity in the last two decennia, and is still ongoing for Antarctic bacteria, cyanobacteria, protists, fungi, etc. Recent studies have shown the presence of highly diverse microbial communities and the existence of species endemic to Antarctic in some taxonomic groups. With the emergence of High Throughput Sequencing methodologies that are able to detect ‘rare’ taxa, it becomes crucial to find Antarctic locations that have not yet been impacted by human presence. These ‘pristine’ areas are essential to serve as reference sites and allow to distinguish the true Antarctic organisms from the imported ones. Indeed, recent studies have shown that humans unintentionally disperse their own microbial flora but may also spread organisms from other locations. In the extreme biotopes with a reduced diversity that are currently found in Antarctica, such contaminations might have a profound impact. It is important to raise the awareness of scientists, environmental managers and policy makers about the necessity to single out some areas that are kept untouched, or where stringent biosecurity measures are taken. The purpose is not to hinder scientific research, but to weigh carefully, when exploring a new area, the importance of the acquired piece of knowledge in relation to the possibility of hindering future microbiological research. Some parallels with other fields of research are interesting to consider. Archeologists are used to keeping some parts of the explored caves untouched because they foresee that technological progress will allow better analyses in future. The COSPAR Panel on Planetary Protection makes recommendations to avoid the contamination of other planets with microbes from Earth, which would obscure any discovery of extraterrestrial indigenous life forms. These examples illustrate the essential need to integrate the delineation of reference areas for future analyses in the design and execution of scientific research. In fact, the Madrid Protocol foresees the possibility to designate ‘inviolate areas’ (Annex V, Article 3), though this tool has rarely been used. It would be useful if scientists of all disciplines would reflect how to use this management option.CCAMBI

La diversité microbienne antarctique: importance des facteurs géographiques et écologiques

Antarctica is a prime region to test whether microbes have a biogeography and to study their metacommunity dynamics, because (i) it is isolated from the other continents, (ii) its extreme environmental conditions allow microorganisms to dominate its ecosystems, and (iii) lacustrine and terrestrial habitats occur isolated in a matrix of ice and ocean. We compiled a large set of samples from benthic microbial mats from Antarctic lakes in different ice-free regions and used a polyphasic approach to study their microbial biodiversity by combining morphological characterization of diatoms with molecular techniques such as Denaturing Gradient Gel Electrophoresis (green algae and cyanobacteria), 454 pyrosequencing and cultivation (prokaryotes).AMBI

Antarctic microbial biodiversity: the importance of geographical and ecological factors

Two contrasting views dominate the vivid debate on microbial biogeography. Proponents of the ubiquity or Baas-Becking hypothesis, which states that ‘everything is everywhere, but the environment selects’, argue that unlimited dispersal prevents isolation and thus allopatric speciation. Opponents of this ‘ubiquity’ concept argue that, similar to larger organisms, dispersal limitation matters in microorganisms and that restricted patterns in geographical distribution and endemism do exist. Antarctic continental ecosystems (including coastal and inland lakes, meltwater streams, cryoconites) are dominated by microbial organisms, which play a crucial role in their functioning. However, little is known about Antarctic microbial biodiversity in comparison with more temperate and/or accessible regions of the world. The BelSPO funded project AMBIO aims to test whether (i) microbial communities are structured by the same factors as those shaping communities of macroorganisms, and (ii) endemism among microbes does exist. To this end, we analysed the microbial biodiversity (cyanobacteria, bacteria, and green algae) in a variety of lacustrine and terrestrial habitats and determined the ‘baseline’ data needed to understand the contribution of various processes that are responsible for the distribution patterns in Antarctic microbial diversity. In all groups studied, we observed a relatively large amount of potentially endemic taxa. A study of the uncultivated diversity of cyanobacteria using Denaturing Gradient Gel Electrophoresis (DGGE) analysis of the SSU rRNA gene revealed that ca 25% of the Operational Taxonomic Units (OTU) is potentially endemic. In bacteria, the cultivated diversity was large and distributed over the major phylogenetic groups with Actinobacteria and Alphaproteobacteria well represented in all samples and with many species and genera new to science. In green algae, microchlorophyte strains isolated from lacustrine habitats were analysed on the basis of SSU rRNA sequences, which revealed a wide phylogenetic diversity of apparently endemic Antarctic lineages of which the majority have estimated ages between 17 and 84 Ma. This supports the hypothesis that long-term survival took place in glacial refugia, resulting in a specific Antarctic flora. A preliminary multivariate analysis revealed that in East Antarctica the microbial mat community structure (cyanoabcteria and eukaryotes s.l.) is regulated by both geographical and local environmental factors. Our study is now enlarged with samples from the Antarctic Peninsula. We also used primers to target more specific groups (i) to test the pertinence of the conclusions obtained in East Antarctica and (ii) to assess whether differences in life cycle characteristics (e.g. formation of resting stages) influence metacommunity dynamics in microorganisms