Using digital PCR to predict ciliate abundance from ribosomal RNA gene copy numbers.

Ciliates play a key role in most ecosystems. Their abundance in natural samples is crucial for answering many ecological questions. Traditional methods of quantifying individual species, which rely on microscopy, are often labour-intensive, time-consuming and can be highly biassed. As a result, we investigated the potential of digital polymerase chain reaction (dPCR) for quantifying ciliates. A significant challenge in this process is the high variation in the copy number of the taxonomic marker gene (ribosomal RNA [rRNA]). We first quantified the rRNA gene copy numbers (GCN) of the model ciliate, Paramecium tetraurelia, during different stages of the cell cycle and growth phases. The per-cell rRNA GCN varied between approximately 11,000 and 130,000, averaging around 50,000 copies per cell. Despite these variations in per-cell rRNA GCN, we found a highly significant correlation between GCN and cell numbers. This is likely due to the coexistence of different cellular stages in an uncontrolled (environmental) ciliate population. Thanks to the high sensitivity of dPCR, we were able to detect the target gene in a sample that contained only a single cell. The dPCR approach presented here is a valuable addition to the molecular toolbox in protistan ecology. It may guide future studies in quantifying and monitoring the abundance of targeted (even rare) ciliates in natural samples.

Simultaneous analysis of seven 16S rRNA hypervariable gene regions increases efficiency in marine bacterial diversity detection.

Environmental DNA sequencing is the gold standard to reveal microbial community structures. In most applications, a one-fragment PCR approach is applied to amplify a taxonomic marker gene, usually a hypervariable region of the 16S rRNA gene. We used a new reverse complement (RC)-PCR-based assay that amplifies seven out of the nine hypervariable regions of the 16S rRNA gene, to interrogate bacterial communities in sediment samples collected from different coastal marine sites with an impact gradient. In parallel, we employed a traditional one-fragment analysis of the hypervariable V3-V4 region to investigate whether the RC-PCR reveals more of the 'unseen' diversity obtained by the one-fragment approach. As a benchmark for the full deck of diversity, we subjected the samples to PCR-free metagenomic sequencing. None of the two PCR-based approaches recorded the full taxonomic repertoire obtained from the metagenomics datasets. However, the RC-PCR approach detected 2.8 times more bacterial genera compared to the near-saturation sequenced V3-V4 samples. RC-PCR is an ideal compromise between the standard one-fragment approach and metagenomics sequencing and may guide future environmental sequencing studies, in which bacterial diversity is a central subject.

High salinity gradients and intermediate spatial scales shaped similar biogeographical and co-occurrence patterns of microeukaryotes in a tropical freshwater-saltwater ecosystem.

Microeukaryotes play key ecological roles in the microbial web of aquatic ecosystems. However, large knowledge gaps urgently need to be filled regarding the biogeography with associated shaping mechanisms and co-occurrence patterns of microeukaryotes under freshwater-saltwater gradients, especially true in tropical regions. Here, we investigated microeukaryotes of six mixed freshwater-saltwater regions in the Pearl River Estuary and surrounding coasts in southern China, with salinity ranging 0.1-32.0% and distances spanned up to 500 km, using molecular ecological methods. Results indicate that the biogeography of abundant and rare microeukaryotic communities was similar, both their co-occurrence patterns and biogeographical patterns were driven by deterministic and stochastic processes. The environmental factors with higher selective pressure than dispersal limitation meant that the role of deterministic process in structuring communities was more significant than that of stochastic process, and salinity played important role in structuring both microeukaryotic communities and networks. The abundant communities had stronger influence on entire microeukaryotic communities and seemed to be more sensitive to environmental changes than their rare counterparts, while rare ones had stronger interspecific relationships. Finally, the geographic scale and environmental gradients of study regions should firstly be clarified in future research on the ecological processes of microeukaryotes before conclusions are drawn.

Supervised machine learning is superior to indicator value inference in monitoring the environmental impacts of salmon aquaculture using eDNA metabarcodes.

Increasing anthropogenic impact and global change effects on natural ecosystems has prompted the development of less expensive and more efficient bioassessments methodologies. One promising approach is the integration of DNA metabarcoding in environmental monitoring. A critical step in this process is the inference of ecological quality (EQ) status from identified molecular bioindicator signatures that mirror environmental classification based on standard macroinvertebrate surveys. The most promising approaches to infer EQ from biotic indices (BI) are supervised machine learning (SML) and the calculation of indicator values (IndVal). In this study we compared the performance of both approaches using DNA metabarcodes of bacteria and ciliates as bioindicators obtained from 152 samples collected from seven Norwegian salmon farms. Results from standard macroinvertebrate-monitoring of the same samples were used as reference to compare the accuracy of both approaches. First, SML outperformed the IndVal approach to infer EQ from eDNA metabarcodes. The Random Forest (RF) algorithm appeared to be less sensitive to noisy data (a typical feature of massive environmental sequence data sets) and uneven data coverage across EQ classes (a typical feature of environmental compliance monitoring scheme) compared to a widely used method to infer IndVals for the calculation of a BI. Second, bacteria allowed for a more accurate EQ assessment than ciliate eDNA metabarcodes. For the implementation of DNA metabarcoding into routine monitoring programmes to assess EQ around salmon aquaculture cages, we therefore recommend bacterial DNA metabarcodes in combination with SML to classify EQ categories based on molecular signatures.

Aquatic food webs in deep temperate lakes: Key species establish through their autecological versatility.

Microbial planktonic communities are the basis of food webs in aquatic ecosystems since they contribute substantially to primary production and nutrient recycling. Network analyses of DNA metabarcoding data sets emerged as a powerful tool to untangle the complex ecological relationships among the key players in food webs. In this study, we evaluated co-occurrence networks constructed from time-series metabarcoding data sets (12 months, biweekly sampling) of protistan plankton communities in surface layers (epilimnion) and bottom waters (hypolimnion) of two temperate deep lakes, Lake Mondsee (Austria) and Lake Zurich (Switzerland). Lake Zurich plankton communities were less tightly connected, more fragmented and had a higher susceptibility to a species extinction scenario compared to Lake Mondsee communities. We interpret these results as a lower robustness of Lake Zurich protistan plankton to environmental stressors, especially stressors resulting from climate change. In all networks, the phylum Ciliophora contributed the highest number of nodes, among them several in key positions of the networks. Associations in ciliate-specific subnetworks resembled autecological species-specific traits that indicate adaptions to specific environmental conditions. We demonstrate the strength of co-occurrence network analyses to deepen our understanding of plankton community dynamics in lakes and indicate biotic relationships, which resulted in new hypotheses that may guide future research in climate-stressed ecosystems.

Ecosystems monitoring powered by environmental genomics: A review of current strategies with an implementation roadmap.

A decade after environmental scientists integrated high-throughput sequencing technologies in their toolbox, the genomics-based monitoring of anthropogenic impacts on the biodiversity and functioning of ecosystems is yet to be implemented by regulatory frameworks. Despite the broadly acknowledged potential of environmental genomics to this end, technical limitations and conceptual issues still stand in the way of its broad application by end-users. In addition, the multiplicity of potential implementation strategies may contribute to a perception that the routine application of this methodology is premature or "in development", hence restraining regulators from binding these tools into legal frameworks. Here, we review recent implementations of environmental genomics-based methods, applied to the biomonitoring of ecosystems. By taking a general overview, without narrowing our perspective to particular habitats or groups of organisms, this paper aims to compare, review and discuss the strengths and limitations of four general implementation strategies of environmental genomics for monitoring: (a) Taxonomy-based analyses focused on identification of known bioindicators or described taxa; (b) De novo bioindicator analyses; (c) Structural community metrics including inferred ecological networks; and (d) Functional community metrics (metagenomics or metatranscriptomics). We emphasise the utility of the three latter strategies to integrate meiofauna and microorganisms that are not traditionally utilised in biomonitoring because of difficult taxonomic identification. Finally, we propose a roadmap for the implementation of environmental genomics into routine monitoring programmes that leverage recent analytical advancements, while pointing out current limitations and future research needs.

A fundamental difference between macrobiota and microbial eukaryotes: protistan plankton has a species maximum in the freshwater-marine transition zone of the Baltic Sea.

Remane's Artenminimum at the horohalinicum is a fundamental concept in ecology to describe and explain the distribution of organisms along salinity gradients. However, a recent metadata analysis challenged this concept for protists, proposing a species maximum in brackish waters. Due to data bias, this literature-based investigation was highly discussed. Reliable data verifying or rejecting the species minimum for protists in brackish waters were critically lacking. Here, we sampled a pronounced salinity gradient along a west-east transect in the Baltic Sea and analysed protistan plankton communities using high-throughput eDNA metabarcoding. A strong salinity barrier at the upper limit of the horohalinicum and 10 psu appeared to select for significant shifts in protistan community structures, with dinoflagellates being dominant at lower salinities, and dictyochophytes and diatoms being keyplayers at higher salinities. Also in vertical water column gradients in deeper basins (Kiel Bight, Arkona and Bornholm Basin) appeared salinity as significant environmental determinant influencing alpha- and beta-diversity patterns. Importantly, alpha-diversity indices revealed species maxima in brackish waters, that is, indeed contrasting Remane's Artenminimum concept. Statistical analyses confirmed salinity as the major driving force for protistan community structuring with high significance. This suggests that macrobiota and microbial eukaryotes follow fundamentally different rules regarding diversity patterns in the transition zone from freshwater to marine waters.

Improving eDNA-based protist diversity assessments using networks of amplicon sequence variants.

Effective and precise grouping of highly similar sequences remains a major bottleneck in the evaluation of high-throughput sequencing datasets. Amplicon sequence variants (ASVs) offer a promising alternative that may supersede the widely used operational taxonomic units (OTUs) in environmental sequencing studies. We compared the performance of a recently developed pipeline based on the algorithm DADA2 for obtaining ASVs against a pipeline based on the algorithm SWARM for obtaining OTUs. Illumina-sequencing of 29 individual ciliate species resulted in up to 11 ASVs per species, while SWARM produced up to 19 OTUs per species. To improve the congruency between species diversity and molecular diversity, we applied sequence similarity networks (SSNs) for second-level sequence grouping into network sequence clusters (NSCs). At 100% sequence similarity in SWARM-SSNs, NSC numbers decreased from 7.9-fold overestimation without abundance filter, to 4.5-fold overestimation when an abundance filter was applied. For the DADA2-SSN approach, NSC numbers decreased from 3.5-fold to 3-fold overestimation. Rand index cluster analyses predicted best binning results between 97% and 94% sequence similarity for both DADA2-SSNs and SWARM-SSNs. Depending on the ecological questions addressed in an environmental sequencing study with protists we recommend ASVs as replacement for OTUs, best in combination with SSNs.

A Proposed Timescale for the Evolution of Armophorean Ciliates: Clevelandellids Diversify More Rapidly Than Metopids.

Members of the class Armophorea occur in microaerophilic and anaerobic habitats, including the digestive tract of invertebrates and vertebrates. Phylogenetic kinships of metopid and clevelandellid armophoreans conflict with traditional morphology-based classifications. To reconcile their relationships and understand their morphological evolution and diversification, we utilized the molecular clock theory as well as information contained in the estimated time trees and morphology of extant taxa. The radiation of the last common ancestor of metopids and clevelandellids very likely occurred during the Paleozoic and crown diversification of the endosymbiotic clevelandellids dates back to the Mesozoic. According to diversification analyses, endosymbiotic clevelandellids have higher net diversification rates than predominantly free-living metopids. Their cladogenic success was very likely associated with sharply isolated ecological niches constituted by their hosts. Conflicts between traditional classifications and molecular phylogenies of metopids and clevelandellids very likely come from processes, leading to further diversification without extinction of ancestral lineages as well as from morphological plesiomorphies incorrectly classified as apomorphies. Our study thus suggests that diversification processes and reconstruction of ancestral morphologies improve the understanding of paraphyly which occurs in groups of organisms with an apparently long evolutionary history and when speciation prevails over extinction.

A Comparison of Different Ciliate Metabarcode Genes as Bioindicators for Environmental Impact Assessments of Salmon Aquaculture.

Ciliates are powerful indicators for monitoring the impact of aquaculture and other industrial activities in the marine environment. Here, we tested the efficiency of four different genetic markers (V4 and V9 regions of the SSU rRNA gene, D1 and D2 regions of the LSU rRNA gene, obtained from environmental (e)DNA and environmental (e)RNA) of benthic ciliate communities for environmental monitoring. We obtained these genetic metabarcodes from sediment samples collected along a transect extending from below salmon cages toward the open sea. These data were compared to benchmark data from traditional macrofauna surveys of the same samples. In beta diversity analyses of ciliate community structures, the V4 and V9 markers had a higher resolution power for sampling sites with different degrees of organic enrichment compared to the D1 and D2 markers. The eDNA and eRNA V4 markers had a higher discriminatory power than the V9 markers. However, results obtained with the eDNA V9 marker corroborated better with the traditional macrofauna monitoring. This allows for a more direct comparison of ciliate metabarcoding with the traditional monitoring. We conclude that the ciliate eDNA V9 marker is the best choice for implementation in routine monitoring programs in marine aquaculture.

Morphology, Morphogenesis and Molecular Phylogeny of a New Obligate Halophile Ciliate, Schmidtiella ultrahalophila gen. nov., spec. nov. (Ciliophora, Hypotrichia) Isolated from a Volcanic Crater on Sal (Cape Verde Islands).

A new hypotrichous ciliate, Schmidtiella ultrahalophila gen. nov., spec. nov., was isolated from a solar saltern on the island of Sal, Cape Verde. The possession of only one short dorsal kinety clearly distinguishes S. ultrahalophila from other known hypotrichous genera and species. Further diagnostic characters include: a flexible and slender body, an average size of 85 × 15 µm in vivo; a bipartite adoral zone with two hypertrophied frontal adoral membranelles and nine to twelve ventral adoral membranelles; three frontal, one parabuccal, two frontoventral, two or three postoral ventral, and two or three frontoterminal cirri; and marginal cirral rows variable in number, usually one on each side. Ontogenetic data indicate the following: the frontal-ventral cirri originate from six or five anlagen; the proter inherits the parental adoral zone; the frontal and ventral cirri originate from five or six anlagen; and the marginal cirral rows and the dorsal kinety tend to originate intrakinetally. Additional marginal rows are rarely derived from de novo anlagen. Based on its morphology, morphogenesis and its SSU rRNA phylogenetic placement, the new species should be assigned to the order Sporadotrichida Fauré-Fremiet, 1961. Due to low taxon sampling, however, its exact position in this order remains enigmatic.

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.

A Contribution to the Morphology and Phylogeny of Chlamydodon, with Three New Species from China (Ciliophora, Cyrtophoria).

Three new cyrtophorian ciliates isolated from coastal areas of China were described based on morphological and genetic data. The Chlamydodon mnemosyne-like species Chlamydodon similis sp. n. differs from its congeners mainly by its number of somatic kineties. Chlamydodon oligochaetus sp. n. is distinguished from its congeners mainly by having fewer somatic kineties, and/or an elongated body shape. Chlamydodon crassidens sp. n. is characterized mainly by an inverted triangular body shape, a posteriorly interrupted cross-striated band (5-6 µm wide), and a large cytostome. Moreover, we provided small-subunit (SSU) rDNA sequences of C. similis sp. n. and C. oligochaetus sp. n. Maximum likelihood (ML) and Bayesian inference (BI) consistently placed C. similis sp. n. as a sister to C. paramnemosyne, but showed different branching position of C. oligochaetus sp. n., which may be due to a low taxon sampling in the Chlamydodontidae and/or an insufficient resolution of the marker gene at species level.

Transition boundaries for protistan species turnover in hypersaline waters of different biogeographic regions.

The identification of environmental barriers which govern species distribution is a fundamental concern in ecology. Even though salt was previously identified as a major transition boundary for micro- and macroorganisms alike, the salinities causing species turnover in protistan communities are unknown. We investigated 4.5 million high-quality protistan metabarcodes (V4 region of the SSU rDNA) obtained from 24 shallow salt ponds (salinities 4%-44%) from South America and Europe. Statistical analyses of protistan community profiles identified four salinity classes, which strongly selected for different protistan communities: 4-9%, 14-24%, 27-36% and 38-44%. The proportion of organisms unknown to science is highest in the 14-24% salinity class, showing that environments within this salinity range are an unappreciated reservoir of as yet undiscovered organisms. Distinct higher-rank taxon groups dominated in the four salinity classes in terms of diversity. As increasing salinities require different cellular responses to cope with salt, our results suggest that different evolutionary lineages of protists have evolved distinct haloadaptation strategies. Salinity appears to be a stronger selection factor for the structuring of protistan communities than geography. Yet, we find a higher degree of endemism in shallow salt ponds compared with less isolated ecosystems such as the open ocean. Thus, rules for biogeographic structuring of protistan communities are not universal, but depend on the ecosystem under consideration.

Ciliate diversity and distribution patterns in the sediments of a seamount and adjacent abyssal plains in the tropical Western Pacific Ocean.

BACKGROUND: Benthic ciliates and the environmental factors shaping their distribution are far from being completely understood. Likewise, deep-sea systems are amongst the least understood ecosystems on Earth. In this study, using high-throughput DNA sequencing, we investigated the diversity and community composition of benthic ciliates in different sediment layers of a seamount and an adjacent abyssal plain in the tropical Western Pacific Ocean with water depths ranging between 813 m and 4566 m. Statistical analyses were used to assess shifts in ciliate communities across vertical sediment gradients and water depth. RESULTS: Nine out of 12 ciliate classes were detected in the different sediment samples, with Litostomatea accounting for the most diverse group, followed by Plagiopylea and Oligohymenophorea. The novelty of ciliate genetic diversity was extremely high, with a mean similarity of 93.25% to previously described sequences. On a sediment depth gradient, ciliate community structure was more similar within the upper sediment layers (0-1 and 9-10 cm) compared to the lower sediment layers (19-20 and 29-30 cm) at each site. Some unknown ciliate taxa which were absent from the surface sediments were found in deeper sediments layers. On a water depth gradient, the proportion of unique OTUs was between 42.2% and 54.3%, and that of OTUs shared by all sites around 14%. However, alpha diversity of the different ciliate communities was relatively stable in the surface layers along the water depth gradient, and about 78% of the ciliate OTUs retrieved from the surface layer of the shallowest site were shared with the surface layers of sites deeper than 3800 m. Correlation analyses did not reveal any significant effects of measured environmental factors on ciliate community composition and structure. CONCLUSIONS: We revealed an obvious variation in ciliate community along a sediment depth gradient in the seamount and the adjacent abyssal plain and showed that water depth is a less important factor shaping ciliate distribution in deep-sea sediments unlike observed for benthic ciliates in shallow seafloors. Additionally, an extremely high genetic novelty of ciliate diversity was found in these habitats, which points to a hot spot for the discovery of new ciliate species.

Beyond the "Code": A Guide to the Description and Documentation of Biodiversity in Ciliated Protists (Alveolata, Ciliophora).

Recent advances in molecular technology have revolutionized research on all aspects of the biology of organisms, including ciliates, and created unprecedented opportunities for pursuing a more integrative approach to investigations of biodiversity. However, this goal is complicated by large gaps and inconsistencies that still exist in the foundation of basic information about biodiversity of ciliates. The present paper reviews issues relating to the taxonomy of ciliates and presents specific recommendations for best practice in the observation and documentation of their biodiversity. This effort stems from a workshop that explored ways to implement six Grand Challenges proposed by the International Research Coordination Network for Biodiversity of Ciliates (IRCN-BC). As part of its commitment to strengthening the knowledge base that supports research on biodiversity of ciliates, the IRCN-BC proposes to populate The Ciliate Guide, an online database, with biodiversity-related data and metadata to create a resource that will facilitate accurate taxonomic identifications and promote sharing of data.

Microbial eukaryote plankton communities of high-mountain lakes from three continents exhibit strong biogeographic patterns.

Microbial eukaryotes hold a key role in aquatic ecosystem functioning. Yet, their diversity in freshwater lakes, particularly in high-mountain lakes, is relatively unknown compared with the marine environment. Low nutrient availability, low water temperature and high ultraviolet radiation make most high-mountain lakes extremely challenging habitats for life and require specific molecular and physiological adaptations. We therefore expected that these ecosystems support a plankton diversity that differs notably from other freshwater lakes. In addition, we hypothesized that the communities under study exhibit geographic structuring. Our rationale was that geographic dispersal of small-sized eukaryotes in high-mountain lakes over continental distances seems difficult. We analysed hypervariable V4 fragments of the SSU rRNA gene to compare the genetic microbial eukaryote diversity in high-mountain lakes located in the European Alps, the Chilean Altiplano and the Ethiopian Bale Mountains. Microbial eukaryotes were not globally distributed corroborating patterns found for bacteria, multicellular animals and plants. Instead, the plankton community composition emerged as a highly specific fingerprint of a geographic region even on higher taxonomic levels. The intraregional heterogeneity of the investigated lakes was mirrored in shifts in microbial eukaryote community structure, which, however, was much less pronounced compared with interregional beta-diversity. Statistical analyses revealed that on a regional scale, environmental factors are strong predictors for plankton community structures in high-mountain lakes. While on long-distance scales (>10 000 km), isolation by distance is the most plausible scenario, on intermediate scales (up to 6000 km), both contemporary environmental factors and historical contingencies interact to shift plankton community structures.

Description of the Halophile Euplotes qatarensis nov. spec. (Ciliophora, Spirotrichea, Euplotida) Isolated from the Hypersaline Khor Al-Adaid Lagoon in Qatar.

The morphology, ontogenesis, and phylogenetic relationships of a halophile euplotid ciliates, Euplotes qatarensis nov. spec., isolated from the Khor Al-Adaid Lagoon in Qatar were investigated based on live observation as well as protargol- and silver nitrate-impregnated methods. The new species is characterised by a combination of features: the halophile habitat, a cell size of 50-65 × 33-40 µm, seven dorsal ridges, 10 commonly sized frontoventral cirri, two widely spaced marginal cirri, 10 dorsolateral kineties, and a double silverline pattern. The morphogenesis is similar to that of its congeners: (i) the oral primordium develops hypoapokinetally and the parental oral apparatus is retained; (ii) the frontoventral-transverse field of five streaks gives rise to the frontal, ventral, and transverse cirri, but not to the cirri I/1 and the marginal cirri; (iii) the dorsal somatic ciliature develops by intrakinetal proliferation of basal bodies in two anlagen per kinety that are just anterior and posterior to the future division furrow; (iv) the caudal cirri are formed by the two rightmost dorsolateral kineties. The SSU rDNA sequence of E. qatarensis branches with full support in the Euplotopsis elegans-Euplotes nobilii-Euplotopsis raikovi clade. The closest related publicly available SSU rDNA sequence is the one of E. nobilii, with which E. qatarensis has 93.4% sequence similarity. Euplotes parawoodruffi Song & Bradbury, 1997 is transferred to the genus Euplotoides based on the absence of frontoventral cirrus VI/3.

Testing ecological theories with sequence similarity networks: marine ciliates exhibit similar geographic dispersal patterns as multicellular organisms.

BACKGROUND: High-throughput sequencing technologies are lifting major limitations to molecular-based ecological studies of eukaryotic microbial diversity, but analyses of the resulting millions of short sequences remain a major bottleneck for these approaches. Here, we introduce the analytical and statistical framework of sequence similarity networks, increasingly used in evolutionary studies and graph theory, into the field of ecology to analyze novel pyrosequenced V4 small subunit rDNA (SSU-rDNA) sequence data sets in the context of previous studies, including SSU-rDNA Sanger sequence data from cultured ciliates and from previous environmental diversity inventories. RESULTS: Our broadly applicable protocol quantified the progress in the description of genetic diversity of ciliates by environmental SSU-rDNA surveys, detected a fundamental historical bias in the tendency to recover already known groups in these surveys, and revealed substantial amounts of hidden microbial diversity. Moreover, network measures demonstrated that ciliates are not globally dispersed, but are structured by habitat and geographical location at intermediate geographical scale, as observed for bacteria, plants, and animals. CONCLUSIONS: Currently available 'universal' primers used for local in-depth sequencing surveys provide little hope to exhaust the significantly higher ciliate (and most likely microbial) diversity than previously thought. Network analyses such as presented in this study offer a promising way to guide the design of novel primers and to further explore this vast and structured microbial diversity.