Effects of long‐term differential fertilization on eukaryotic microbial communities in an arable soil: a multiple barcoding approach

To understand the fine‐scale effects of changes in nutrient availability on eukaryotic soil microorganisms communities, a multiple barcoding approach was used to analyse soil samples from four different treatments in a long‐term fertilization experiment. We performed PCR amplification on soil DNA with primer pairs specifically targeting the 18S rRNA genes of all eukaryotes and three protist groups (Cercozoa, Chrysophyceae‐Synurophyceae and Kinetoplastida) as well as the ITS gene of fungi and the 23S plastid rRNA gene of photoautotrophic microorganisms. Amplicons were pyrosequenced, and a total of 88 706 quality filtered reads were clustered into 1232 operational taxonomic units (OTU) across the six data sets. Comparisons of the taxonomic coverage achieved based on overlapping assignment of OTUs revealed that half of the eukaryotic taxa identified were missed by the universal eukaryotic barcoding marker. There were only little differences in OTU richness observed between organic‐ (farmyard manure), mineral‐ and nonfertilized soils. However, the community compositions appeared to be strongly structured by organic fertilization in all data sets other than that generated using the universal eukaryotic 18S rRNA gene primers, whereas mineral fertilization had only a minor effect. In addition, a co‐occurrence based network analysis revealed complex potential interaction patterns between OTUs from different trophic levels, for example between fungivorous flagellates and fungi. Our results demonstrate that changes in pH, moisture and organic nutrients availability caused shifts in the composition of eukaryotic microbial communities at multiple trophic levels.     

Mock communities highlight the diversity of host‐associated eukaryotes

Host‐associated microbes are ubiquitous. Every multicellular eukaryote, and even many unicellular eukaryotes (protists), hosts a diverse community of microbes. High‐throughput sequencing (HTS) tools have illuminated the vast diversity of host‐associated microbes and shown that they have widespread influence on host biology, ecology and evolution (McFall‐Ngai et al. 2013 ). Bacteria receive most of the attention, but protists are also important components of microbial communities associated with humans (Parfrey et al. 2011 ) and other hosts. As HTS tools are increasingly used to study eukaryotes, the presence of numerous and diverse host‐associated eukaryotes is emerging as a common theme across ecosystems. Indeed, HTS studies demonstrate that host‐associated lineages account for between 2 and 12% of overall eukaryotic sequences detected in soil, marine and freshwater data sets, with much higher relative abundances observed in some samples (Ramirez et al. 2014 ; Simon et al. 2015 ; de Vargas et al. 2015 ). Previous studies in soil detected large numbers of predominantly parasitic lineages such as Apicomplexa, but did not delve into their origin [e.g. (Ramirez et al. 2014 )]. In this issue of Molecular Ecology, Geisen et al. ( 2015 ) use mock communities to show that many of the eukaryotic organisms detected by environmental sequencing in soils are potentially associated with animal hosts rather than free‐living. By isolating the host‐associated fraction of soil microbial communities, Geisen and colleagues help explain the surprisingly high diversity of parasitic eukaryotic lineages often detected in soil/terrestrial studies using high‐throughput sequencing (HTS) and reinforce the ubiquity of these host‐associated microbes. It is clear that we can no longer assume that organisms detected in bulk environmental sequencing are free‐living, but instead need to design studies that specifically enumerate the diversity and function of host‐associated eukaryotes. Doing so will allow the field to determine the role host‐associated eukaryotes play in soils and other environments and to evaluate hypotheses on assembly of host‐associated communities, disease ecology and more.     

Protists in Arctic drift and land‐fast sea ice

Global climate change is having profound impacts on polar ice with changes in the duration and extent of both land‐fast ice and drift ice, which is part of the polar ice pack. Sea ice is a distinct habitat and the morphologically identifiable sympagic community living within sea ice can be readily distinguished from pelagic species. Sympagic metazoa and diatoms have been studied extensively since they can be identified using microscopy techniques. However, non‐diatom eukaryotic cells living in ice have received much less attention despite taxa such as the dinoflagellate Polarella and the cercozoan Cryothecomonas being isolated from sea ice. Other small flagellates have also been reported, suggesting complex microbial food webs. Since smaller flagellates are fragile, often poorly preserved, and are difficult for non‐experts to identify, we applied high throughput tag sequencing of the V4 region of the 18S rRNA gene to investigate the eukaryotic microbiome within the ice. The sea ice communities were diverse (190 taxa) and included many heterotrophic and mixotrophic species. Dinoflagellates (43 taxa), diatoms (29 taxa) and cercozoans (12 taxa) accounted for ~80% of the sequences. The sympagic communities living within drift ice and land‐fast ice harbored taxonomically distinct communities and we highlight specific taxa of dinoflagellates and diatoms that may be indicators of land‐fast and drift ice.     

Vertical Micro‐Zonation of Testate Amoebae and Ciliates in Peatland Waters in Relation to Potential Food Resources and Grazing Pressure

The aim of this study was to examine the community structure and vertical micro‐distribution of testate amoebae and ciliates in a raised bog in eastern Poland, as well as to assess the influence of potential food resources (Chl‐a, bacteria, heterotrophic flagellates) and predators (rotifers and copepods) on protozoa communities. Samples were taken from surface, bottom and interstitial waters. At each type of micro‐habitat and each sampling date water was sampled using a plexiglass corer or mini‐piezometers. Additionally, in order to evaluate grazing pressure, field enclosures were used in which metazoan abundance and composition was manipulated by size‐fractionation. Over experiments, metazoan populations shifted from dominance of rotifers to copepods. In the first experiment, with rotifers dominating, metazoa had only a modest predatory impact on the protozoa. In contrast, the second experiment, with copepods prevailing, demonstrated a clear top‐down control of the protozoa communities by metazoan. The density and biomass of protozoa significantly differed between the studied stations, with the lowest numbers in the interstitial water and the highest in the surface water. Surface sampling were dominated by mixotrophic taxa, whereas the deepest sampling level was characterized by increase in the proportion of bacterivore species. These differences between micro‐habitats may be due to differences in environmental conditions (food resources and grazing pressure). Ordination analysis revealed that bacteria can strongly regulate the abundance and taxonomic composition of protozoa in the bottom and interstitial waters. Metazoan predators could be the main regulators of protozoa communities in surface water. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

High‐throughput metabarcoding of eukaryotic diversity for environmental monitoring of offshore oil‐drilling activities

As global exploitation of available resources increases, operations extend towards sensitive and previously protected ecosystems. It is important to monitor such areas in order to detect, understand and remediate environmental responses to stressors. The natural heterogeneity and complexity of communities means that accurate monitoring requires high resolution, both temporally and spatially, as well as more complete assessments of taxa. Increased resolution and taxonomic coverage is economically challenging using current microscopy‐based monitoring practices. Alternatively, DNA sequencing‐based methods have been suggested for cost‐efficient monitoring, offering additional insights into ecosystem function and disturbance. Here, we applied DNA metabarcoding of eukaryotic communities in marine sediments, in areas of offshore drilling on the Norwegian continental shelf. Forty‐five samples, collected from seven drilling sites in the Troll/Oseberg region, were assessed, using the small subunit ribosomal RNA gene as a taxonomic marker. In agreement with results based on classical morphology‐based monitoring, we were able to identify changes in sediment communities surrounding oil platforms. In addition to overall changes in community structure, we identified several potential indicator taxa, responding to pollutants associated with drilling fluids. These included the metazoan orders Macrodasyida, Macrostomida and Ceriantharia, as well as several ciliates and other protist taxa, typically not targeted by environmental monitoring programmes. Analysis of a co‐occurrence network to study the distribution of taxa across samples provided a framework for better understanding the impact of anthropogenic activities on the benthic food web, generating novel, testable hypotheses of trophic interactions structuring benthic communities.     

Phagotrophic Protist Diversity in the Groundwater of a Karstified Aquifer – Morphological and Molecular Analysis

To clarify the structure of microbial food webs in groundwater, knowledge about the protist diversity and feeding strategies is essential. We applied cultivation‐dependent approaches and molecular methods for further understanding of protist diversity in groundwater. Groundwater was sampled from a karstified aquifer located in the Thuringian Basin (Thuringia, Germany). Cultivable protist abundance estimated up to 8,000 cells/L. Eleven flagellates, 10 naked amoebae, and one ciliate morpho‐species were detected in groundwater enrichment cultures. Most of the flagellates morpho‐species, typically < 10 μm, were sessile or free swimming suspension feeders, e.g., Spumella spp., Monosiga spp., and mobile, surface‐associated forms that grasp biofilms, e.g., Bodo spp. Naked amoebae, typically < 35 μm, that grasp biofilms were represented by, e.g., Vahlkampfia spp., Vannella spp., and Hartmanella spp. The largest fraction of the 18S rRNA gene sequences was affiliated with Spumella‐like Stramenopiles. Besides, also sequences affiliated with fungi and metazoan grazers were detected in clone libraries of the groundwater. We hypothesize that small sized protist species take refuge in the structured surface of the fractures and fissures of the karstified aquifer and mainly feed on biofilm‐associated or suspended bacteria.     

Dietary‐driven variation effects on the symbiotic flagellate protist communities of the subterranean termite Reticulitermes grassei Clément

The ability of subterranean termites to digest lignocellulose relies not only on their digestive tract physiology, but also on the symbiotic relationships established with flagellate protists and bacteria. The objective of this work was to test the possible effect of different cellulose‐based diets on the community structure (species richness and other diversity metrics) of the flagellate protists of the subterranean termite Reticulitermes grassei. Termites belonging to the same colony were subjected to six different diets (natural diet, maritime pine wood, European beech, thermally modified European beech, cellulose powder and starvation), and their flagellate protist community was evaluated after the trials. All non‐treated sound woods produced similar flagellate protist communities that were more diverse and of high evenness (low dominance). On the contrary, flagellate protist communities from cellulose‐fed termites and starving termites were considered to be significantly different from all non‐treated woods; they were less diverse and some morphotypes became dominant as a consequence of flagellate protist communities having suffered major adaptations to these diets. The flagellate protist communities of untreated beech and thermally modified beech‐fed termites were considered to be significantly different in terms of abundance and morphotype diversity. This may be caused by a decrease in lignocellulose quality available for termites and from an interference of thermally treated wood with the chemical stability of the termite hindgut. Our study suggests that as a consequence of the strong division of labour among these protists to accomplish the intricate process of lignocellulose digestion, termite symbiotic flagellate protist communities are a dynamic assemblage able to adapt to different conditions and diets. This study is important for the community‐level alteration approach, and it is the first study to investigate the effects of thermally modified wood on the flagellate protist communities of subterranean termites.     

An improved method for utilizing high‐throughput amplicon sequencing to determine the diets of insectivorous animals

DNA analysis of predator faeces using high‐throughput amplicon sequencing (HTS) enhances our understanding of predator–prey interactions. However, conclusions drawn from this technique are constrained by biases that occur in multiple steps of the HTS workflow. To better characterize insectivorous animal diets, we used DNA from a diverse set of arthropods to assess PCR biases of commonly used and novel primer pairs for the mitochondrial gene, cytochrome oxidase C subunit 1 (COI). We compared diversity recovered from HTS of bat guano samples using a commonly used primer pair “ZBJ” to results using the novel primer pair “ANML.” To parameterize our bioinformatics pipeline, we created an arthropod mock community consisting of single‐copy (cloned) COI sequences. To examine biases associated with both PCR and HTS, mock community members were combined in equimolar amounts both pre‐ and post‐PCR. We validated our system using guano from bats fed known diets and using composite samples of morphologically identified insects collected in pitfall traps. In PCR tests, the ANML primer pair amplified 58 of 59 arthropod taxa (98%), whereas ZBJ amplified 24–40 of 59 taxa (41%–68%). Furthermore, in an HTS comparison of field‐collected samples, the ANML primers detected nearly fourfold more arthropod taxa than the ZBJ primers. The additional arthropods detected include medically and economically relevant insect groups such as mosquitoes. Results revealed biases at both the PCR and sequencing levels, demonstrating the pitfalls associated with using HTS read numbers as proxies for abundance. The use of an arthropod mock community allowed for improved bioinformatics pipeline parameterization.     

How does litter quality affect the community of soil protists (testate amoebae) of tropical montane rainforests?

Litter quality and diversity are major factors structuring decomposer communities. However, little is known on the relationship between litter quality and the community structure of soil protists in tropical forests. We analyzed the diversity, density, and community structure of a major group of soil protists of tropical montane rainforests, that is, testate amoebae. Litterbags containing pure and mixed litter of two abundant tree species at the study sites (Graffenrieda emarginata and Purdiaea nutans) differing in nitrogen concentrations were exposed in the field for 12?months. The density and diversity of testate amoebae were higher in the nitrogen-rich Graffenrieda litter suggesting that nitrogen functions as an important driving factor for soil protist communities. No additive effects of litter mixing were found, rather density of testate amoebae was reduced in litter mixtures as compared to litterbags with Graffenrieda litter only. However, adding of high-quality litter to low-quality litter markedly improved habitat quality, as evaluated by the increase in diversity and density of testate amoebae. The results suggest that local factors, such as litter quality, function as major forces shaping the structure and density of decomposer microfauna that likely feed back to decomposition processes.     

Consistent patterns of high alpha and low beta diversity in tropical parasitic and free‐living protists

Tropical animals and plants are known to have high alpha diversity within forests, but low beta diversity between forests. By contrast, it is unknown whether microbes inhabiting the same ecosystems exhibit similar biogeographic patterns. To evaluate the biogeographies of tropical protists, we used metabarcoding data of species sampled in the soils of three lowland Neotropical rainforests. Taxa–area and distance–decay relationships for three of the dominant protist taxa and their subtaxa were estimated at both the OTU and phylogenetic levels, with presence–absence and abundance‐based measures. These estimates were compared to null models. High local alpha and low regional beta diversity patterns were consistently found for both the parasitic Apicomplexa and the largely free‐living Cercozoa and Ciliophora. Similar to animals and plants, the protists showed spatial structures between forests at the OTU and phylogenetic levels, and only at the phylogenetic level within forests. These results suggest that the biogeographies of macro‐ and micro‐organismal eukaryotes in lowland Neotropical rainforests are partially structured by the same general processes. However, and unlike the animals and plants, the protist OTUs did not exhibit spatial structures within forests, which hinders our ability to estimate the local and regional diversity of protists in tropical forests.     

High‐throughput environmental sequencing reveals high diversity of litter and moss associated protist communities along a gradient of drainage and tree productivity

Although previous studies, mostly based on microscopy analyses of a few groups of protists, have suggested that protists are abundant and diverse in litter and moss habitats, the overall diversity of moss and litter associated protists remains elusive. Here, high‐throughput environmental sequencing was used to characterize the diversity and community structure of litter‐ and moss‐associated protists along a gradient of soil drainage and forest primary productivity in a temperate rainforest in British Columbia. We identified 3262 distinct protist OTUs from 36 sites. Protists were strongly structured along the landscape gradient, with a significant increase in alpha diversity from the blanket bog ecosystem to the zonal forest ecosystem. Among all investigated environmental variables, calcium content was the most strongly associated with the community composition of protists, but substrate composition, plant cover and other edaphic factors were also significantly correlated with these communities. Furthermore, a detailed phylogenetic analysis of unicellular opisthokonts identified OTUs covering most lineages, including novel OTUs branching with Discicristoidea, the sister group of Fungi, and with Filasterea, one of the closest unicellular relatives to animals. Altogether, this study provides unprecedented insight into the community composition of moss‐ and litter‐associated protists.     

Links between soil microbial communities and plant traits in a species‐rich grassland under long‐term climate change

Climate change can influence soil microorganisms directly by altering their growth and activity but also indirectly via effects on the vegetation, which modifies the availability of resources. Direct impacts of climate change on soil microorganisms can occur rapidly, whereas indirect effects mediated by shifts in plant community composition are not immediately apparent and likely to increase over time. We used molecular fingerprinting of bacterial and fungal communities in the soil to investigate the effects of 17 years of temperature and rainfall manipulations in a species‐rich grassland near Buxton, UK. We compared shifts in microbial community structure to changes in plant species composition and key plant traits across 78 microsites within plots subjected to winter heating, rainfall supplementation, or summer drought. We observed marked shifts in soil fungal and bacterial community structure in response to chronic summer drought. Importantly, although dominant microbial taxa were largely unaffected by drought, there were substantial changes in the abundances of subordinate fungal and bacterial taxa. In contrast to short‐term studies that report high resistance of soil fungi to drought, we observed substantial losses of fungal taxa in the summer drought treatments. There was moderate concordance between soil microbial communities and plant species composition within microsites. Vector fitting of community‐weighted mean plant traits to ordinations of soil bacterial and fungal communities showed that shifts in soil microbial community structure were related to plant traits representing the quality of resources available to soil microorganisms: the construction cost of leaf material, foliar carbon‐to‐nitrogen ratios, and leaf dry matter content. Thus, our study provides evidence that climate change could affect soil microbial communities indirectly via changes in plant inputs and highlights the importance of considering long‐term climate change effects, especially in nutrient‐poor systems with slow‐growing vegetation.     

The 12th International Workshops on Opportunistic Protists (IWOP‐12)

The 12th International Workshops on Opportunistic Protists (IWOP‐12) was held in August 2012 in Tarrytown, New York. The objectives of the IWOP meetings are to: (1) serve as a forum for exchange of new information among active researchers concerning the basic biology, molecular genetics, immunology, biochemistry, pathogenesis, drug development, therapy, and epidemiology of these immunodeficiency‐associated pathogenic eukaryotic microorganisms that are seen in patients with AIDS and (2) foster the entry of new and young investigators into these underserved research areas. The IWOP meeting focuses on opportunistic protists, e.g. the free‐living amoebae, Pneumocystis, Cryptosporidium, Toxoplasma, the Microsporidia, and kinetoplastid flagellates. This conference represents the major conference that brings together research groups working on these opportunistic pathogens. Slow but steady progress is being achieved on understanding the biology of these pathogenic organisms, their involvement in disease causation in both immune‐deficient and immune‐competent hosts, and is providing critical insights into these emerging and reemerging pathogens. This IWOP meeting demonstrated the importance of newly developed genomic level information for many of these pathogens and how analysis of such large data sets is providing key insights into the basic biology of these organisms. A great concern is the loss of scientific expertise and diversity in the research community due to the ongoing decline in research funding. This loss of researchers is due to the small size of many of these research communities and a lack of appreciation by the larger scientific community concerning the state of art and challenges faced by researchers working on these organisms.     

PCR Amplification-Independent Methods for Detection of Microbial Communities by the High-Density Microarray PhyloChip

Environmental microbial community analysis typically involves amplification by PCR, despite well-documented biases. We have developed two methods of PCR-independent microbial community analysis using the high-density microarray PhyloChip: direct hybridization of 16S rRNA (dirRNA) or rRNA converted to double-stranded cDNA (dscDNA). We compared dirRNA and dscDNA communities to PCR-amplified DNA communities using a mock community of eight taxa, as well as experiments derived from three environmental sample types: chromium-contaminated aquifer groundwater, tropical forest soil, and secondary sewage in seawater. Community profiles by both direct hybridization methods showed differences that were expected based on accompanying data but that were missing in PCR-amplified communities. Taxon richness decreased in RNA compared to that in DNA communities, suggesting a subset of 20% in soil and 60% in groundwater that is active; secondary sewage showed no difference between active and inactive populations. Direct hybridization of dscDNA and RNA is thus a viable alternative to PCR-amplified microbial community analysis, providing identification of the active populations within microbial communities that attenuate pollutants, drive global biogeochemical cycles, or proliferate disease states.     

Efficient but occasionally imperfect vertical transmission of gut mutualistic protists in a wood‐feeding termite

Although mutualistic associations between animals and microbial symbionts are widespread in nature, the mechanisms that have promoted their evolutionary persistence remain poorly understood. A vertical mode of symbiont transmission (from parents to offspring) is thought to ensure partner fidelity and stabilization, although the efficiency of vertical transmission has rarely been investigated, especially in cases where hosts harbour a diverse microbial community. Here we evaluated vertical transmission rates of cellulolytic gut oxymonad and parabasalid protists in the wood‐feeding termite Reticulitermes grassei. We sequenced amplicons of the 18S rRNA gene of protists from 24 colonies of R. grassei collected in two populations. For each colony, the protist community was characterized from the gut of 14 swarming reproductives and from a pool of 10 worker guts. A total of 98 operational taxonomic units belonging to 13 species‐level taxa were found. The vertical transmission rate was estimated for each protist present in a colony based on its frequency among the reproductives. The results revealed that transmission rates were high, with an average of 0.897 (±0.164) per protist species. Overall, the protist community did not differ between reproductive sexes, suggesting that both the queen and the king could contribute to the gut microbiota of the offspring. A positive relationship between the transmission rate of protists and their prevalence within populations was also detected. However, transmission rates alone do not explain the prevalence of protists. In conclusion, these findings reveal key forces behind a conserved, multispecies mutualism, raising further questions on the roles of horizontal transfer and negative selection in shaping symbiont prevalence.     

Soil pathogen communities associated with native and non‐native Phragmites australis populations in freshwater wetlands

Soil pathogens are believed to be major contributors to negative plant–soil feedbacks that regulate plant community dynamics and plant invasions. While the theoretical basis for pathogen regulation of plant communities is well established within the plant–soil feedback framework, direct experimental evidence for pathogen community responses to plants has been limited, often relying largely on indirect evidence based on above‐ground plant responses. As a result, specific soil pathogen responses accompanying above‐ground plant community dynamics are largely unknown. Here, we examine the oomycete pathogens in soils conditioned by established populations of native noninvasive and non‐native invasive haplotypes of Phragmites australis (European common reed). Our aim was to assess whether populations of invasive plants harbor unique communities of pathogens that differ from those associated with noninvasive populations and whether the distribution of taxa within these communities may help to explain invasive success. We compared the composition and abundance of pathogenic and saprobic oomycete species over a 2‐year period. Despite a diversity of oomycete taxa detected in soils from both native and non‐native populations, pathogen communities from both invaded and noninvaded soils were dominated by species of Pythium. Pathogen species that contributed the most to the differences observed between invaded and noninvaded soils were distributed between invaded and noninvaded soils. However, the specific taxa in invaded soils responsible for community differences were distinct from those in noninvaded soils that contributed to community differences. Our results indicate that, despite the phylogenetic relatedness of native and non‐native P. australis haplotypes, pathogen communities associated with the dominant non‐native haplotype are distinct from those of the rare native haplotype. Pathogen taxa that dominate either noninvaded or invaded soils suggest different potential mechanisms of invasion facilitation. These findings are consistent with the hypothesis that non‐native plant species that dominate landscapes may “cultivate” a different soil pathogen community to their rhizosphere than those of rarer native species.     

The 13th International Workshops on Opportunistic Protists (IWOP13)

The 13th International Workshops on Opportunistic Protists (IWOP‐13) was held November 13–15, 2014 in Seville, Spain. The objectives of the IWOP meetings are to: (1) serve as a forum for exchange of new information among active researchers concerning the basic biology, molecular genetics, immunology, biochemistry, pathogenesis, drug development, therapy, and epidemiology of these immunodeficiency‐associated pathogenic eukaryotic microorganisms that are seen in patients with AIDS and; (2) to foster the entry of new and young investigators into these underserved research areas. The IWOP meeting focuses on opportunistic protists; e.g. the free‐living amoebae, Pneumocystis, Cryptosporidium, Toxoplasma, the Microsporidia, and kinetoplastid flagellates. This conference represents the major conference which brings together research groups working on these opportunistic pathogens. Progress has been achieved on understanding the biology of these pathogenic organisms, their involvement in disease causation in both immune deficient and immune competent hosts and is providing important insights into these emerging and reemerging pathogens. A continuing concern of the participants is the ongoing loss of scientific expertise and diversity in this research community. This decline is due to the small size of these research communities and an ongoing lack of understanding by the broader scientific community of the challenges and limitations faced by researchers working on these organisms, which makes these research communities very sensitive to declines in research funding.     

On the use of high‐throughput sequencing for the study of cyanobacterial diversity in Antarctic aquatic mats

The study of Antarctic cyanobacterial diversity has been mostly limited to morphological identification and traditional molecular techniques. High‐throughput sequencing (HTS) allows a much better understanding of microbial distribution in the environment, but its application is hampered by several methodological and analytical challenges. In this work, we explored the use of HTS as a tool for the study of cyanobacterial diversity in Antarctic aquatic mats. Our results highlight the importance of using artificial communities to validate the parameters of the bioinformatics procedure used to analyze natural communities, since pipeline‐dependent biases had a strong effect on the observed community structures. Analysis of microbial mats from five Antarctic lakes and an aquatic biofilm from the Sub‐Antarctic showed that HTS is a valuable tool for the assessment of cyanobacterial diversity. The majority of the operational taxonomic units retrieved were related to filamentous taxa such as Leptolyngbya and Phormidium, which are common genera in Antarctic lacustrine microbial mats. However, other phylotypes related to different taxa such as Geitlerinema, Pseudanabaena, Synechococcus, Chamaesiphon, Calothrix, and Coleodesmium were also found. Results revealed a much higher diversity than what had been reported using traditional methods and also highlighted remarkable differences between the cyanobacterial communities of the studied lakes. The aquatic biofilm from the Sub‐Antarctic had a distinct cyanobacterial community from the Antarctic lakes, which in turn displayed a salinity‐dependent community structure at the phylotype level.     

Don't put all your eggs in one basket: a cost‐effective and powerful method to optimize primer choice for rRNA environmental community analyses using the Fluidigm Access Array

With the increasing democratization of high‐throughput sequencing (HTS) technologies, along with the concomitant increase in sequence yield per dollar, many researchers are exploring HTS for microbial community ecology. Many elements of experimental design can drastically affect the final observed community structure, notably the choice of primers for amplification prior to sequencing. Some targeted microbes can fail to amplify due to primer‐targeted sequence divergence and be omitted from obtained sequences, leading to differences among primer pairs in the sequenced organisms even when targeting the same community. This potential source of taxonomic bias in HTS makes it prudent to investigate how primer choice will affect the sequenced community prior to investing in a costly community‐wide sequencing effort. Here, we use Fluidigm's microfluidic Access Arrays (IFC) followed by Illumina^® MiSeq Nano sequencing on a culture‐derived local mock community to demonstrate how this approach allows for a low‐cost combinatorial investigation of primer pairs and experimental samples (up to 48 primer pairs and 48 samples) to determine the most effective primers that maximize obtained communities whilst minimizing taxonomic biases.