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.

Identification of osmoadaptive strategies in the halophile, heterotrophic ciliate Schmidingerothrix salinarum.

Hypersaline environments pose major challenges to their microbial residents. Microorganisms have to cope with increased osmotic pressure and low water activity and therefore require specific adaptation mechanisms. Although mechanisms have already been thoroughly investigated in the green alga Dunaliella salina and some halophilic yeasts, strategies for osmoadaptation in other protistan groups (especially heterotrophs) are neither as well known nor as deeply investigated as for their prokaryotic counterpart. This is not only due to the recent awareness of the high protistan diversity and ecological relevance in hypersaline systems, but also due to methodological shortcomings. We provide the first experimental study on haloadaptation in heterotrophic microeukaryotes, using the halophilic ciliate Schmidingerothrix salinarum as a model organism. We established three approaches to investigate fundamental adaptation strategies known from prokaryotes. First, proton nuclear magnetic resonance (1H-NMR) spectroscopy was used for the detection, identification, and quantification of intracellular compatible solutes. Second, ion-imaging with cation-specific fluorescent dyes was employed to analyze changes in the relative ion concentrations in intact cells. Third, the effect of salt concentrations on the catalytic performance of S. salinarum malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICDH) was determined. 1H-NMR spectroscopy identified glycine betaine (GB) and ectoine (Ect) as the main compatible solutes in S. salinarum. Moreover, a significant positive correlation of intracellular GB and Ect concentrations and external salinity was observed. The addition of exogenous GB, Ect, and choline (Ch) stimulated the cell growth notably, indicating that S. salinarum accumulates the solutes from the external medium. Addition of external 13C2-Ch resulted in conversion to 13C2-GB, indicating biosynthesis of GB from Ch. An increase of external salinity up to 21% did not result in an increase in cytoplasmic sodium concentration in S. salinarum. This, together with the decrease in the catalytic activities of MDH and ICDH at high salt concentration, demonstrates that S. salinarum employs the salt-out strategy for haloadaptation.

Perspectives from Ten Years of Protist Studies by High-Throughput Metabarcoding.

During the last decade, high-throughput metabarcoding became routine for analyzing protistan diversity and distributions in nature. Amid a multitude of exciting findings, scientists have also identified and addressed technical and biological limitations, although problems still exist for inference of meaningful taxonomic and ecological knowledge based on short DNA sequences. Given the extensive use of this approach, it is critical to settle our understanding on its strengths and weaknesses and to synthesize up-to-date methodological and conceptual trends. This article summarizes key scientific and technical findings, and identifies current and future directions in protist research that uses metabarcoding.

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 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.

Glycine Betaine and Ectoine Are the Major Compatible Solutes Used by Four Different Halophilic Heterotrophic Ciliates.

One decisive factor controlling the distribution of organisms in their natural habitats is the cellular response to environmental factors. Compared to prokaryotes, our knowledge about salt adaptation strategies of microbial eukaryotes is very limited. We, here, used a recently introduced approach (implementing proton nuclear magnetic resonance spectroscopy) to investigate the presence of compatible solutes in halophilic, heterotrophic ciliates. Therefore, we isolated four ciliates from solar salterns, which were identified as Cyclidium glaucoma, Euplotes sp., Fabrea salina, and Pseudocohnilembus persalinus based on their 18S rRNA gene signatures and electron microscopy. The results of 1H-NMR spectroscopy revealed that all four ciliates employ the "low-salt-in" strategy by accumulating glycine betaine and ectoine as main osmoprotectants. We recorded a linear increase of these compatible solutes with increasing salinity of the external medium. Ectoine in particular stands out as its use as compatible solute was thought to be exclusive to prokaryotes. However, our findings and those recently made on two other heterotroph species call for a re-evaluation of this notion. The observation of varying relative proportions of compatible solutes within the four ciliates points to slight differences in haloadaptive strategies by regulatory action of the ciliates. Based on this finding, we provide an explanatory hypothesis for the distribution of protistan diversity along salinity gradients.

Correction to: Glycine Betaine and Ectoine Are the Major Compatible Solutes Used by Four Different Halophilic Heterotrophic Ciliates.

The original version of this article unfortunately contained mistakes in the author affiliation, the references given in two tables and in a figure legend.

Characterization of protistan plankton diversity in ancient salt evaporation ponds located in a volcanic crater on the island Sal, Cape Verde.

Salinity is an important factor when exploring the limits known for life. Therefore, hypersaline systems have attracted much attention in recent years. In this study, we investigated the protistan diversity and community composition in two natural salt evaporation ponds (27-30% salinity) located in an ancient volcanic crater on the Cape Verde island Sal using high-throughput DNA sequencing. Our study revealed a broad range of protistan taxa and a high taxonomic diversity within the Ciliophora, Dinophyceae, and Chlorophyta. We detected a total of 23 Dinophyceae families, although Dinophyceae were generally considered to be only this diverse in aquatic environments of less than 10% salinity. Moreover, we uncovered a high degree of genetic novelty in this habitat. The mean similarity of all detected OTUs to previously described sequences was only 93.6%. These findings strongly dispute the traditional view that extreme hypersaline environments generally maintain low protistan diversity. A meta-analysis covering our and previously published data from other inland and coastal salt ponds clearly showed that our samples clustered according to salinity and not biogeography. This result further supports the claim that salinity is a major transition boundary for protistan communities, regardless of their biogeographic origin.

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.

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.

Protistan diversity in a permanently stratified meromictic lake (Lake Alatsee, SW Germany).

Protists play a crucial role for ecosystem function(ing) and oxygen is one of the strongest barriers against their local dispersal. However, protistan diversity in freshwater habitats with oxygen gradients received very little attention. We applied high-throughput sequencing of the V9 region (18S rRNA gene) to provide a hitherto unique spatiotemporal analysis of protistan diversity along the oxygen gradient of a freshwater meromictic lake (Lake Alatsee, SW Germany). In the mixolimnion, the communities experienced most seasonal structural changes, with Stramenopiles dominating in autumn and Dinoflagellata in summer. The suboxic interface supported the highest diversity, but only 23 OTUs95% (mainly Euglenozoa, after quality check and removal of operational taxonomic units (OTUs) with less than three sequences) were exclusively associated with this habitat. Eukaryotic communities in the anoxic monimolimnion showed the most stable seasonal pattern, with Chrysophyta and Bicosoecida being the dominant taxa. Our data pinpoint to the ecological role of the interface as a short-term 'meeting point' for protists, contributing to the coupling of the mixolimnion and the monimolimnion. Our analyses of divergent genetic diversity suggest a high degree of previously undescribed OTUs. Future research will have to reveal if this result actually points to a high number of undescribed species in such freshwater habitats.

Deep sequencing uncovers protistan plankton diversity in the Portuguese Ria Formosa solar saltern ponds.

We used high-throughput sequencing to unravel the genetic diversity of protistan (including fungal) plankton in hypersaline ponds of the Ria Formosa solar saltern works in Portugal. From three ponds of different salinity (4, 12 and 38 %), we obtained ca. 105,000 amplicons (V4 region of the SSU rDNA). The genetic diversity we found was higher than what has been described from solar saltern ponds thus far by microscopy or molecular studies. The obtained operational taxonomic units (OTUs) could be assigned to 14 high-rank taxonomic groups and blasted to 120 eukaryotic families. The novelty of this genetic diversity was extremely high, with 27 % of all OTUs having a sequence divergence of more than 10 % to deposited sequences of described taxa. The highest degree of novelty was found at intermediate salinity of 12 % within the ciliates, which traditionally are considered as the best known and described taxon group within the kingdom Protista. Further substantial novelty was detected within the stramenopiles and the chlorophytes. Analyses of community structures suggest a transition boundary for protistan plankton between 4 and 12 % salinity, suggesting different haloadaptation strategies in individual evolutionary lineages as a result of environmental filtering. Our study makes evident the gaps in our knowledge not only of protistan and fungal plankton diversity in hypersaline environments, but also in their ecology and their strategies to cope with these environmental conditions. It substantiates that specific future research needs to fill these gaps.

Comparing High-throughput Platforms for Sequencing the V4 Region of SSU-rDNA in Environmental Microbial Eukaryotic Diversity Surveys.

High-throughput sequencing platforms are continuing to increase resulting read lengths, which is allowing for a deeper and more accurate depiction of environmental microbial diversity. With the nascent Reagent Kit v3, Illumina MiSeq now has the ability to sequence the eukaryotic hyper-variable V4 region of the SSU-rDNA locus with paired-end reads. Using DNA collected from soils with analyses of strictly- and nearly identical amplicons, here we ask how the new Illumina MiSeq data compares with what we can obtain with Roche/454 GS FLX with regard to quantity and quality, presence and absence, and abundance perspectives. We show that there is an easy qualitative transition from the Roche/454 to the Illumina MiSeq platforms. The ease of this transition is more nuanced quantitatively for low-abundant amplicons, although estimates of abundances are known to also vary within platforms.

A morphogenetic survey on ciliate plankton from a mountain lake pinpoints the necessity of lineage-specific barcode markers in microbial ecology.

Analyses of high-throughput environmental sequencing data have become the 'gold-standard' to address fundamental questions of microbial diversity, ecology and biogeography. Findings that emerged from sequencing are, e.g. the discovery of the extensive 'rare microbial biosphere' and its potential function as a seed-bank. Even though applied since several years, results from high-throughput environmental sequencing have hardly been validated. We assessed how well pyrosequenced amplicons [the hypervariable eukaryotic V4 region of the small subunit ribosomal RNA (SSU rRNA) gene] reflected morphotype ciliate plankton. Moreover, we assessed if amplicon sequencing had the potential to detect the annual ciliate plankton stock. In both cases, we identified significant quantitative and qualitative differences. Our study makes evident that taxon abundance distributions inferred from amplicon data are highly biased and do not mirror actual morphotype abundances at all. Potential reasons included cell losses after fixation, cryptic morphotypes, resting stages, insufficient sequence data availability of morphologically described species and the unsatisfying resolution of the V4 SSU rRNA fragment for accurate taxonomic assignments. The latter two underline the necessity of barcoding initiatives for eukaryotic microbes to better and fully exploit environmental amplicon data sets, which then will also allow studying the potential of seed-bank taxa as a buffer for environmental changes.

Schmidingerothrix salinarum nov. spec. is the molecular sister of the large oxytrichid clade (ciliophora, hypotricha).

In 2012, Foissner described a curious hypotrich: Schmidingerothrix extraordinaria. This ciliate, which he discovered in hypersaline soils (~100‰) from Namibia, had a frayed buccal lip, three-rowed adoral membranelles, only one frontal cirrus, and a miniaturized first frontal membranelle, while a paroral membrane, dorsal bristle rows and buccal, transverse and caudal cirri were absent. All opisthe structures developed de novo, while parental structures were involved in the proter. When Foissner's study became available, we discovered a similar species in a Portuguese solar saltern, differing from S. extraordinaria mainly by the number of frontoventral cirral rows (3 vs. 1). Furthermore, parental structures were involved in the ontogenesis of both proter and opisthe. The small subunit (SSU) rDNA shows Schmidingerothrix as sister of a large clade containing most classical oxytrichids (e.g. Sterkiella, Oxytricha, Steinia) and many related taxa (e.g. Pattersoniella, Bistichella, Uroleptus). This clade shows a bifurcation named "Oxytricha subclade" and "Uroleptus subclade". Foissner () interpreted the peculiarities of Schmidingerothrix as a reduction caused by the extreme habitat. However, the molecular data do not exclude that Schmidingerothrix presents an ancient state. A morphology-based scheme is presented, showing how the subclades might have evolved from a Schmidingerothrix-like ancestor.