Anke Behnke

Depicting more accurate pictures of protistan community complexity using pyrosequencing of hypervariable SSU rRNA gene regions

Initial environmental pyrosequencing studies suggested highly complex protistan communities with phylotype richness decisively higher than previously estimated. However, recent studies on individual bacteria or artificial bacterial communities evidenced that pyrosequencing errors may skew our view of the true complexity of microbial communities. We pyrosequenced two diversity markers (hypervariable regions V4 and V9 of the small-subunit rDNA) of an intertidal protistan model community, using the Roche GS-FLX and the most recent GS-FLX Titanium sequencing systems. After pyrosequencing 24 reference sequences we obtained up to 2039 unique tags (from 3879 V4 GS-FLX Titanium reads), 77% of which were singletons. Even binning sequences that share 97% similarity still emulated a pseudodiversity exceeding the true complexity of the model community up to three times (V9 GS-FLX). Pyrosequencing error rates were higher for V4 fragments compared with the V9 domain and for the GS-FLX Titanium compared with the GS-FLX system. Furthermore, this experiment revealed that error rates are taxon-specific. As an outcome of this study we suggest a fast and efficient strategy to discriminate pyrosequencing signals from noise in order to more realistically depict the structure of protistan communities using simple tools that are implemented in standard tag data-processing pipelines.

Microeukaryote Community Patterns along an O2/H2S Gradient in a Supersulfidic Anoxic Fjord (Framvaren, Norway)

ABSTRACT To resolve the fine-scale architecture of anoxic protistan communities, we conducted a cultivation-independent 18S rRNA survey in the superanoxic Framvaren Fjord in Norway. We generated three clone libraries along the steep O2/H2S gradient, using the multiple-primer approach. Of 1,100 clones analyzed, 753 proved to be high-quality protistan target sequences. These sequences were grouped into 92 phylotypes, which displayed high protistan diversity in the fjord (17 major eukaryotic phyla). Only a few were closely related to known taxa. Several sequences were dissimilar to all previously described sequences and occupied a basal position in the inferred phylogenies, suggesting that the sequences recovered were derived from novel, deeply divergent eukaryotes. We detected sequence clades with evolutionary importance (for example, clades in the euglenozoa) and clades that seem to be specifically adapted to anoxic environments, challenging the hypothesis that the global dispersal of protists is uniform. Moreover, with the detection of clones affiliated with jakobid flagellates, we present evidence that primitive descendants of early eukaryotes are present in this anoxic environment. To estimate sample coverage and phylotype richness, we used parametric and nonparametric statistical methods. The results show that although our data set is one of the largest published inventories, our sample missed a substantial proportion of the protistan diversity. Nevertheless, statistical and phylogenetic analyses of the three libraries revealed the fine-scale architecture of anoxic protistan communities, which may exhibit adaptation to different environmental conditions along the O2/H2S gradient.

A Molecular Approach to Identify Active Microbes in Environmental Eukaryote Clone Libraries

A rapid method for the simultaneous extraction of RNA and DNA from eukaryote plankton samples was developed in order to discriminate between indigenous active cells and signals from inactive or even dead organisms. The method was tested using samples from below the chemocline of an anoxic Danish fjord. The simple protocol yielded RNA and DNA of a purity suitable for amplification by reverse transcription-polymerase chain reaction (RT-PCR) and PCR, respectively. We constructed an rRNA-derived and an rDNA-derived clone library to assess the composition of the microeukaryote assemblage under study and to identify physiologically active constituents of the community. We retrieved nearly 600 protistan target clones, which grouped into 84 different phylotypes (98% sequence similarity). Of these phylotypes, 27% occurred in both libraries, 25% exclusively in the rRNA library, and 48% exclusively in the rDNA library. Both libraries revealed good correspondence of the general community composition in terms of higher taxonomic ranks. They were dominated by anaerobic ciliates and heterotrophic stramenopile flagellates thriving below the fjord’s chemocline. The high abundance of these bacterivore organisms points out their role as a major trophic link in anoxic marine systems. A comparison of the two libraries identified phototrophic dinoflagellates, “uncultured marine alveolates group I,” and different parasites, which were exclusively detected with the rDNA-derived library, as nonindigenous members of the anoxic microeukaryote community under study.

Fungal diversity in oxygen-depleted regions of the Arabian Sea revealed by targeted environmental sequencing combined with cultivation

In order to study fungal diversity in oxygen minimum zones of the Arabian Sea, we analyzed 1440 cloned small subunit rRNA gene (18S rRNA gene) sequences obtained from environmental samples using three different PCR primer sets. Restriction fragment length polymorphism (RFLP) analyses yielded 549 distinct RFLP patterns, 268 of which could be assigned to fungi (Dikarya and zygomycetes) after sequence analyses. The remaining 281 RFLP patterns represented a variety of nonfungal taxa, even when using putatively fungal-specific primers. A substantial number of fungal sequences were closely related to environmental sequences from a range of other anoxic marine habitats, but distantly related to known sequences of described fungi. Community similarity analyses suggested distinctively different structures of fungal communities from normoxic sites, seasonally anoxic sites and permanently anoxic sites, suggesting different adaptation strategies of fungal communities to prevailing oxygen conditions. Additionally, we obtained 26 fungal cultures from the study sites, most of which were closely related (>97% sequence similarity) to well-described Dikarya. This indicates that standard cultivation mainly produces more of what is already known. However, two of these cultures were highly divergent to known sequences and seem to represent novel fungal groups on high taxonomic levels. Interestingly, none of the cultured isolates is identical to any of the environmental sequences obtained. Our study demonstrates the importance of a multiple-primer approach combined with cultivation to obtain deeper insights into the true fungal diversity in environmental samples and to enable adequate intersample comparisons of fungal communities.

Spatio-temporal variations in protistan communities along an O2/H2S gradient in the anoxic Framvaren Fjord (Norway)

Despite its relevance for ecology and biodiversity, the stability of spatial microeukaryote diversity patterns in time has received only little attention using gene-based strategies, and there is little knowledge about the relation of spatial vs. temporal variation. We addressed this subject by investigating seasonal fluctuations in protistan communities in three ecologically distinct marine habitats. We analyzed 3360 eukaryote small subunit rRNA gene sequences collected along an O(2)/H(2)S gradient in a Norwegian fjord in order to reveal shifts in protistan community composition and structure in three different seasons. In all nine clone libraries, ciliates and stramenopiles accounted for the largest proportion. Yet, as expected, at the phylotype level, the protistan communities from distinct habitats differed significantly, with the number of shared phylotypes between two habitats being as low as 18%. This confirmed previous notions that environmental factors along the stratification gradient shape biodiversity patterns. Surprisingly, the intrahabitat community composition and structure varied at a comparable order of magnitude over time, with only 18-28% phylotypes shared within the same habitat. Our study demonstrates that the consideration of local fluctuations in microeukaryote diversity over time offers additional information for diversity surveys and can significantly contribute to the revelation of spatial protistan community patterns.

Meta-analyses of environmental sequence data identify anoxia and salinity as parameters shaping ciliate communities

Nearly 6000 SSU rRNA sequences of ciliated protists, compiled from 50 marine and terrestrial sampling sites worldwide were analysed to identify environmental barriers spatially relating ciliate communities. The hypotheses were: (i) oxygen is a dispersal barrier for strict anaerobes, allowing allopatric speciation in isolated habitats and resulting in high diversity; (ii) as salinity affects metabolism it may lead to distinct clusters with evolutionarily separated lineages; (iii) different oceanic realms support significantly different ciliate communities. Based on the α-diversity of the ciliate sequences in each habitat and the shared diversity among different habitats, ecological and geographic boundaries were revealed. Community similarities between pairs of habitats and all habitats were depicted with a metric distance matrix. Anoxia and salinity emerged as decisive determinants structuring the communities, with anoxia largely overlaying the effect of other environmental parameters. Measurements of α-diversity suggest a tremendous diversity of ciliates in anoxic environments, exceeding the one in normoxic environments. Salinity also exerts a high selection pressure on ciliates, contributing to community structure, composition and distribution patterns. However, marine–freshwater transitions seem to be more recent in ciliates compared with most other protistan taxa. The theory of island biogeography seems to apply to anoxic habitats, making these sites promising targets for the discovery of novel diversity.

Sample pooling obscures diversity patterns in intertidal ciliate community composition and structure.

The aim of this study was to assess the effect of sample pooling on the portrayal of ciliate community structure and composition in intertidal sediment samples. Molecular ciliate community profiles were obtained from nine biological replicates distributed in three discrete sampling plots and from samples that were pooled prior to RNA extraction using terminal restriction fragment polymorphism (T-RFLP) analyses of SSU rRNA. Comparing the individual replicates of one sampling plot with each other, we found a differential variability among the individual biological replicates. T-RFLP profiles of pooled samples displayed a significantly different community composition compared with the cumulative individual biological replicate samples. We conclude that sample pooling obscures diversity patterns in ciliate and possibly also other microbial eukaryote studies. However, differences between pooled samples and replicates were less pronounced when community structure was analyzed. We found that the most abundant T-RFLP peaks were generally shared between biological replicates and pooled samples. Assuming that the most abundant taxa in an ecosystem under study are also the ones driving ecosystem processes, sample pooling may still be effective for the analyses of ecological key players.

The Anoxic Framvaren Fjord as a Model System to Study Protistan Diversity and Evolution

The origin of eukaryotes in an anoxic versus an oxygenated environment is controversially discussed. To date, no conclusive data are available to decide this debate for the one or for the other camp. Yet, a substantial data set, coming from the anaerobic biochemistry of extant eukaryotes, the modeling of Proterozoic ocean chemistry, and earth’s history (specifically when considered in concerto), provides reasonable evidence for the hypothesis of an early eukaryote evolution in an anoxic world, which is summarized in the first part of this chapter. Contemporary anoxic and sulfidic environments are the stage on which to find the key players that can help to strengthen this view. We, in the second part of this chapter, present an ideal natural model system – the supersulfidic, anoxic Framvaren Fjord in south Norway – to study the evolution and diversity of unicellular eukaryotes – the protists – and summarize the knowledge that has been accumulated from the Framvaren Fjord in this field of research, evidencing the importance of such systems in evolution and biodiversity research.