Marine DNA Viral Macro- and Microdiversity from Pole to Pole.

Microbes drive most ecosystems and are modulated by viruses that impact their lifespan, gene flow, and metabolic outputs. However, ecosystem-level impacts of viral community diversity remain difficult to assess due to classification issues and few reference genomes. Here, we establish an ∼12-fold expanded global ocean DNA virome dataset of 195,728 viral populations, now including the Arctic Ocean, and validate that these populations form discrete genotypic clusters. Meta-community analyses revealed five ecological zones throughout the global ocean, including two distinct Arctic regions. Across the zones, local and global patterns and drivers in viral community diversity were established for both macrodiversity (inter-population diversity) and microdiversity (intra-population genetic variation). These patterns sometimes, but not always, paralleled those from macro-organisms and revealed temperate and tropical surface waters and the Arctic as biodiversity hotspots and mechanistic hypotheses to explain them. Such further understanding of ocean viruses is critical for broader inclusion in ecosystem models.

Gene Expression Changes and Community Turnover Differentially Shape the Global Ocean Metatranscriptome.

Ocean microbial communities strongly influence the biogeochemistry, food webs, and climate of our planet. Despite recent advances in understanding their taxonomic and genomic compositions, little is known about how their transcriptomes vary globally. Here, we present a dataset of 187 metatranscriptomes and 370 metagenomes from 126 globally distributed sampling stations and establish a resource of 47 million genes to study community-level transcriptomes across depth layers from pole-to-pole. We examine gene expression changes and community turnover as the underlying mechanisms shaping community transcriptomes along these axes of environmental variation and show how their individual contributions differ for multiple biogeochemically relevant processes. Furthermore, we find the relative contribution of gene expression changes to be significantly lower in polar than in non-polar waters and hypothesize that in polar regions, alterations in community activity in response to ocean warming will be driven more strongly by changes in organismal composition than by gene regulatory mechanisms. VIDEO ABSTRACT.

Global Trends in Marine Plankton Diversity across Kingdoms of Life.

The ocean is home to myriad small planktonic organisms that underpin the functioning of marine ecosystems. However, their spatial patterns of diversity and the underlying drivers remain poorly known, precluding projections of their responses to global changes. Here we investigate the latitudinal gradients and global predictors of plankton diversity across archaea, bacteria, eukaryotes, and major virus clades using both molecular and imaging data from Tara Oceans. We show a decline of diversity for most planktonic groups toward the poles, mainly driven by decreasing ocean temperatures. Projections into the future suggest that severe warming of the surface ocean by the end of the 21st century could lead to tropicalization of the diversity of most planktonic groups in temperate and polar regions. These changes may have multiple consequences for marine ecosystem functioning and services and are expected to be particularly significant in key areas for carbon sequestration, fisheries, and marine conservation. VIDEO ABSTRACT.

Exploring the phycosphere of Emiliania huxleyi: From bloom dynamics to microbiome assembly experiments.

Coccolithophores have global ecological and biogeochemical significance as the most important calcifying marine phytoplankton group. The structure and selection of prokaryotic communities associated with the most abundant coccolithophore and bloom-forming species, Emiliania huxleyi, are still poorly known. In this study, we assessed the diversity of bacterial communities associated with an E. huxleyi bloom in the Celtic Sea (Eastern North Atlantic), exposed axenic E. huxleyi cultures to prokaryotic communities derived from bloom and non-bloom conditions, and followed the dynamics of their microbiome composition over one year. Bloom-associated prokaryotic communities were dominated by SAR11, Marine group II Euryarchaeota and Rhodobacterales and contained substantial proportions of known indicators of phytoplankton bloom demises such as Flavobacteriaceae and Pseudoalteromonadaceae. The taxonomic richness of bacteria derived from natural communities associated with axenic E. huxleyi rapidly shifted and then stabilized over time. The succession of microorganisms recruited from the environment was consistently dependent on the composition of the initial bacterioplankton community. Phycosphere-associated communities derived from the E. huxleyi bloom were highly similar to one another, suggesting deterministic processes, whereas cultures from non-bloom conditions show an effect of stochasticity. Overall, this work sheds new light on the importance of the initial inoculum composition in microbiome recruitment and elucidates the temporal dynamics of its composition and long-term stability.

Swarm v3: towards tera-scale amplicon clustering.

MOTIVATION: Previously we presented swarm, an open-source amplicon clustering programme that produces fine-scale molecular operational taxonomic units (OTUs) that are free of arbitrary global clustering thresholds. Here, we present swarm v3 to address issues of contemporary datasets that are growing towards tera-byte sizes. RESULTS: When compared with previous swarm versions, swarm v3 has modernized C++ source code, reduced memory footprint by up to 50%, optimized CPU-usage and multithreading (more than 7 times faster with default parameters), and it has been extensively tested for its robustness and logic. AVAILABILITY AND IMPLEMENTATION: Source code and binaries are available at https://github.com/torognes/swarm. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Seasonal dynamics of marine protist communities in tidally mixed coastal waters.

Major seasonal community reorganizations and associated biomass variations are landmarks of plankton ecology. However, the processes of plankton community turnover rates have not been fully elucidated so far. Here, we analyse patterns of planktonic protist community succession in temperate latitudes, based on quantitative taxonomic data from both microscopy counts (cells >10 µm) and ribosomal DNA metabarcoding (size fraction >3 µm, 18S rRNA gene) from plankton samples collected bimonthly over 8 years (2009-2016) at the SOMLIT-Astan station (Roscoff, Western English Channel). Based on morphology, diatoms were clearly the dominating group all year round and over the study period. Metabarcoding uncovered a wider diversity spectrum and revealed the prevalence of Dinophyceae and diatoms but also of Cryptophyta, Chlorophyta, Cercozoa, Syndiniales and Ciliophora in terms of read counts and or richness. The use of morphological and molecular analyses in combination allowed improving the taxonomic resolution and to identify the sequence of the dominant species and OTUs (18S V4 rDNA-derived taxa) that drive annual plankton successions. We detected that some of these dominant OTUs were benthic as a result of the intense tidal mixing typical of the French coasts in the English Channel. Our analysis of the temporal structure of community changes point to a strong seasonality and resilience. The temporal structure of environmental variables (especially Photosynthetic Active Radiation, temperature and macronutrients) and temporal structures generated by species life cycles and or species interactions, are key drivers of the observed cyclic annual plankton turnover.

The Tara Pacific expedition-A pan-ecosystemic approach of the "-omics" complexity of coral reef holobionts across the Pacific Ocean.

Coral reefs are the most diverse habitats in the marine realm. Their productivity, structural complexity, and biodiversity critically depend on ecosystem services provided by corals that are threatened because of climate change effects-in particular, ocean warming and acidification. The coral holobiont is composed of the coral animal host, endosymbiotic dinoflagellates, associated viruses, bacteria, and other microeukaryotes. In particular, the mandatory photosymbiosis with microalgae of the family Symbiodiniaceae and its consequences on the evolution, physiology, and stress resilience of the coral holobiont have yet to be fully elucidated. The functioning of the holobiont as a whole is largely unknown, although bacteria and viruses are presumed to play roles in metabolic interactions, immunity, and stress tolerance. In the context of climate change and anthropogenic threats on coral reef ecosystems, the Tara Pacific project aims to provide a baseline of the "-omics" complexity of the coral holobiont and its ecosystem across the Pacific Ocean and for various oceanographically distinct defined areas. Inspired by the previous Tara Oceans expeditions, the Tara Pacific expedition (2016-2018) has applied a pan-ecosystemic approach on coral reefs throughout the Pacific Ocean, drawing an east-west transect from Panama to Papua New Guinea and a south-north transect from Australia to Japan, sampling corals throughout 32 island systems with local replicates. Tara Pacific has developed and applied state-of-the-art technologies in very-high-throughput genetic sequencing and molecular analysis to reveal the entire microbial and chemical diversity as well as functional traits associated with coral holobionts, together with various measures on environmental forcing. This ambitious project aims at revealing a massive amount of novel biodiversity, shedding light on the complex links between genomes, transcriptomes, metabolomes, organisms, and ecosystem functions in coral reefs and providing a reference of the biological state of modern coral reefs in the Anthropocene.

Plankton networks driving carbon export in the oligotrophic ocean.

The biological carbon pump is the process by which CO2 is transformed to organic carbon via photosynthesis, exported through sinking particles, and finally sequestered in the deep ocean. While the intensity of the pump correlates with plankton community composition, the underlying ecosystem structure driving the process remains largely uncharacterized. Here we use environmental and metagenomic data gathered during the Tara Oceans expedition to improve our understanding of carbon export in the oligotrophic ocean. We show that specific plankton communities, from the surface and deep chlorophyll maximum, correlate with carbon export at 150 m and highlight unexpected taxa such as Radiolaria and alveolate parasites, as well as Synechococcus and their phages, as lineages most strongly associated with carbon export in the subtropical, nutrient-depleted, oligotrophic ocean. Additionally, we show that the relative abundance of a few bacterial and viral genes can predict a significant fraction of the variability in carbon export in these regions.

EukRef: Phylogenetic curation of ribosomal RNA to enhance understanding of eukaryotic diversity and distribution.

Environmental sequencing has greatly expanded our knowledge of micro-eukaryotic diversity and ecology by revealing previously unknown lineages and their distribution. However, the value of these data is critically dependent on the quality of the reference databases used to assign an identity to environmental sequences. Existing databases contain errors and struggle to keep pace with rapidly changing eukaryotic taxonomy, the influx of novel diversity, and computational challenges related to assembling the high-quality alignments and trees needed for accurate characterization of lineage diversity. EukRef (eukref.org) is an ongoing community-driven initiative that addresses these challenges by bringing together taxonomists with expertise spanning the eukaryotic tree of life and microbial ecologists, who use environmental sequence data to develop reliable reference databases across the diversity of microbial eukaryotes. EukRef organizes and facilitates rigorous mining and annotation of sequence data by providing protocols, guidelines, and tools. The EukRef pipeline and tools allow users interested in a particular group of microbial eukaryotes to retrieve all sequences belonging to that group from International Nucleotide Sequence Database Collaboration (INSDC) (GenBank, the European Nucleotide Archive [ENA], or the DNA DataBank of Japan [DDBJ]), to place those sequences in a phylogenetic tree, and to curate taxonomic and environmental information for the group. We provide guidelines to facilitate the process and to standardize taxonomic annotations. The final outputs of this process are (1) a reference tree and alignment, (2) a reference sequence database, including taxonomic and environmental information, and (3) a list of putative chimeras and other artifactual sequences. These products will be useful for the broad community as they become publicly available (at eukref.org) and are shared with existing reference databases.

Coupling between taxonomic and functional diversity in protistan coastal communities.

The study of protistan functional diversity is crucial to understand the dynamics of oceanic ecological processes. We combined the metabarcoding data of various coastal ecosystems and a newly developed trait-based approach to study the link between taxonomic and functional diversity across marine protistan communities of different size-classes. Environmental DNA was extracted and the V4 18S rDNA genomic region was amplified and sequenced. In parallel, we tried to annotate the operational taxonomic units (OTUs) from our metabarcoding dataset to 30 biological traits using published and accessible information on protists. We then developed a method to study trait correlations across protists (i.e. trade-offs) in order to build the best functional groups. Based on the annotated OTUs and our functional groups, we demonstrated that the functional diversity of marine protist communities varied in parallel with their taxonomic diversity. The coupling between functional and taxonomic diversity was conserved across different protist size classes. However, the smallest size-fraction was characterized by wider taxonomic and functional groups diversity, corroborating the idea that nanoplankton and picoplankton are part of a more stable ecological background on which larger protists and metazoans might develop.

Pan genome of the phytoplankton Emiliania underpins its global distribution.

Coccolithophores have influenced the global climate for over 200 million years. These marine phytoplankton can account for 20 per cent of total carbon fixation in some systems. They form blooms that can occupy hundreds of thousands of square kilometres and are distinguished by their elegantly sculpted calcium carbonate exoskeletons (coccoliths), rendering them visible from space. Although coccolithophores export carbon in the form of organic matter and calcite to the sea floor, they also release CO2 in the calcification process. Hence, they have a complex influence on the carbon cycle, driving either CO2 production or uptake, sequestration and export to the deep ocean. Here we report the first haptophyte reference genome, from the coccolithophore Emiliania huxleyi strain CCMP1516, and sequences from 13 additional isolates. Our analyses reveal a pan genome (core genes plus genes distributed variably between strains) probably supported by an atypical complement of repetitive sequence in the genome. Comparisons across strains demonstrate that E. huxleyi, which has long been considered a single species, harbours extensive genome variability reflected in different metabolic repertoires. Genome variability within this species complex seems to underpin its capacity both to thrive in habitats ranging from the equator to the subarctic and to form large-scale episodic blooms under a wide variety of environmental conditions.

Insights into global diatom distribution and diversity in the world's ocean.

Diatoms (Bacillariophyta) constitute one of the most diverse and ecologically important groups of phytoplankton. They are considered to be particularly important in nutrient-rich coastal ecosystems and at high latitudes, but considerably less so in the oligotrophic open ocean. The Tara Oceans circumnavigation collected samples from a wide range of oceanic regions using a standardized sampling procedure. Here, a total of ∼12 million diatom V9-18S ribosomal DNA (rDNA) ribotypes, derived from 293 size-fractionated plankton communities collected at 46 sampling sites across the global ocean euphotic zone, have been analyzed to explore diatom global diversity and community composition. We provide a new estimate of diversity of marine planktonic diatoms at 4,748 operational taxonomic units (OTUs). Based on the total assigned ribotypes, Chaetoceros was the most abundant and diverse genus, followed by Fragilariopsis, Thalassiosira, and Corethron We found only a few cosmopolitan ribotypes displaying an even distribution across stations and high abundance, many of which could not be assigned with confidence to any known genus. Three distinct communities from South Pacific, Mediterranean, and Southern Ocean waters were identified that share a substantial percentage of ribotypes within them. Sudden drops in diversity were observed at Cape Agulhas, which separates the Indian and Atlantic Oceans, and across the Drake Passage between the Atlantic and Southern Oceans, indicating the importance of these ocean circulation choke points in constraining diatom distribution and diversity. We also observed high diatom diversity in the open ocean, suggesting that diatoms may be more relevant in these oceanic systems than generally considered.

Neobodonids are dominant kinetoplastids in the global ocean.

Kinetoplastid flagellates comprise basal mostly free-living bodonids and derived obligatory parasitic trypanosomatids, which belong to the best-studied protists. Due to their omnipresence in aquatic environments and soil, the bodonids are of ecological significance. Here, we present the first global survey of marine kinetoplastids and compare it with the strikingly different patterns of abundance and diversity in their sister clade, the diplonemids. Based on analysis of 18S rDNA V9 ribotypes obtained from 124 sites sampled during the Tara Oceans expedition, our results show generally low to moderate abundance and diversity of planktonic kinetoplastids. Although we have identified all major kinetoplastid lineages, 98% of kinetoplastid reads are represented by neobodonids, namely specimens of the Neobodo and Rhynchomonas genera, which make up 59% and 18% of all reads, respectively. Most kinetoplastids have small cell size (0.8-5 µm) and tend to be more abundant in the mesopelagic as compared to the euphotic zone. Some of the most abundant operational taxonomic units have distinct geographical distributions, and three novel putatively parasitic neobodonids were identified, along with their potential hosts.

The Evolution of Silicon Transport in Eukaryotes.

Biosilicification (the formation of biological structures from silica) occurs in diverse eukaryotic lineages, plays a major role in global biogeochemical cycles, and has significant biotechnological applications. Silicon (Si) uptake is crucial for biosilicification, yet the evolutionary history of the transporters involved remains poorly known. Recent evidence suggests that the SIT family of Si transporters, initially identified in diatoms, may be widely distributed, with an extended family of related transporters (SIT-Ls) present in some nonsilicified organisms. Here, we identify SITs and SIT-Ls in a range of eukaryotes, including major silicified lineages (radiolarians and chrysophytes) and also bacterial SIT-Ls. Our evidence suggests that the symmetrical 10-transmembrane-domain SIT structure has independently evolved multiple times via duplication and fusion of 5-transmembrane-domain SIT-Ls. We also identify a second gene family, similar to the active Si transporter Lsi2, that is broadly distributed amongst siliceous and nonsiliceous eukaryotes. Our analyses resolve a distinct group of Lsi2-like genes, including plant and diatom Si-responsive genes, and sequences unique to siliceous sponges and choanoflagellates. The SIT/SIT-L and Lsi2 transporter families likely contribute to biosilicification in diverse lineages, indicating an ancient role for Si transport in eukaryotes. We propose that these Si transporters may have arisen initially to prevent Si toxicity in the high Si Precambrian oceans, with subsequent biologically induced reductions in Si concentrations of Phanerozoic seas leading to widespread losses of SIT, SIT-L, and Lsi2-like genes in diverse lineages. Thus, the origin and diversification of two independent Si transporter families both drove and were driven by ancient ocean Si levels.

Nomenclature for the Nameless: A Proposal for an Integrative Molecular Taxonomy of Cryptic Diversity Exemplified by Planktonic Foraminifera.

Investigations of biodiversity, biogeography, and ecological processes rely on the identification of "species" as biologically significant, natural units of evolution. In this context, morphotaxonomy only provides an adequate level of resolution if reproductive isolation matches morphological divergence. In many groups of organisms, morphologically defined species often disguise considerable genetic diversity, which may be indicative of the existence of cryptic species. The diversity hidden by morphological species can be disentangled through genetic surveys, which also provide access to data on the ecological distribution of genetically circumscribed units. These units can be identified by unique DNA sequence motifs and allow studies of evolutionary and ecological processes at different levels of divergence. However, the nomenclature of genetically circumscribed units within morphological species is not regulated and lacks stability. This represents a major obstacle to efforts to synthesize and communicate data on genetic diversity for multiple stakeholders. We have been confronted with such an obstacle in our work on planktonic foraminifera, where the stakeholder community is particularly diverse, involving geochemists, paleoceanographers, paleontologists, and biologists, and the lack of stable nomenclature beyond the level of formal morphospecies prevents effective transfer of knowledge. To circumvent this problem, we have designed a stable, reproducible, and flexible nomenclature system for genetically circumscribed units, analogous to the principles of a formal nomenclature system. Our system is based on the definition of unique DNA sequence motifs collocated within an individual, their typification (in analogy with holotypes), utilization of their hierarchical phylogenetic structure to define levels of divergence below that of the morphospecies, and a set of nomenclature rules assuring stability. The resulting molecular operational taxonomic units remain outside the domain of current nomenclature codes, but are linked to formal morphospecies as regulated by the codes. Subsequently, we show how this system can be applied to classify genetically defined units using the SSU rDNA marker in planktonic foraminifera and we highlight its potential use for other groups of organisms where similarly high levels of connectivity between molecular and formal taxonomies can be achieved.

UniEuk: Time to Speak a Common Language in Protistology!

Universal taxonomic frameworks have been critical tools to structure the fields of botany, zoology, mycology, and bacteriology as well as their large research communities. Animals, plants, and fungi have relatively solid, stable morpho-taxonomies built over the last three centuries, while bacteria have been classified for the last three decades under a coherent molecular taxonomic framework. By contrast, no such common language exists for microbial eukaryotes, even though environmental '-omics' surveys suggest that protists make up most of the organismal and genetic complexity of our planet's ecosystems! With the current deluge of eukaryotic meta-omics data, we urgently need to build up a universal eukaryotic taxonomy bridging the protist -omics age to the fragile, centuries-old body of classical knowledge that has effectively linked protist taxa to morphological, physiological, and ecological information. UniEuk is an open, inclusive, community-based and expert-driven international initiative to build a flexible, adaptive universal taxonomic framework for eukaryotes. It unites three complementary modules, EukRef, EukBank, and EukMap, which use phylogenetic markers, environmental metabarcoding surveys, and expert knowledge to inform the taxonomic framework. The UniEuk taxonomy is directly implemented in the European Nucleotide Archive at EMBL-EBI, ensuring its broad use and long-term preservation as a reference taxonomy for eukaryotes.

Global patterns of pelagic dinoflagellate diversity across protist size classes unveiled by metabarcoding.

Dinoflagellates (Alveolata) are one of the ecologically most important groups of modern phytoplankton. Their biological complexity makes assessment of their global diversity and community structure difficult. We used massive V9 18S rDNA sequencing from 106 size-fractionated plankton communities collected across the world's surface oceans during the Tara Oceans expedition (2009-2012) to assess patterns of pelagic dinoflagellate diversity and community structuring over global taxonomic and ecological scales. Our data and analyses suggest that dinoflagellate diversity has been largely underestimated, representing overall ∼ 1/2 of protistan rDNA metabarcode richness assigned at ≥ 90% to a reference sequence in the world's surface oceans. Dinoflagellate metabarcode diversity and abundance display regular patterns across the global scale, with different order-level taxonomic compositions across organismal size fractions. While the pico to nano-planktonic communities are composed of an extreme diversity of metabarcodes assigned to Gymnodiniales or are simply undetermined, most micro-dinoflagellate metabarcodes relate to the well-referenced Gonyaulacales and Peridiniales orders, and a lower abundance and diversity of essentially symbiotic Peridiniales is unveiled in the meso-plankton. Our analyses could help future development of biogeochemical models of pelagic systems integrating the separation of dinoflagellates into functional groups according to plankton size classes.

Environmental Sequencing Provides Reasonable Estimates of the Relative Abundance of Specific Picoeukaryotes.

UNLABELLED: High-throughput sequencing (HTS) is revolutionizing environmental surveys of microbial diversity in the three domains of life by providing detailed information on which taxa are present in microbial assemblages. However, it is still unclear how the relative abundance of specific taxa gathered by HTS correlates with cell abundances. Here, we quantified the relative cell abundance of 6 picoeukaryotic taxa in 13 planktonic samples from 6 European coastal sites using epifluorescence microscopy on tyramide signal amplification-fluorescence in situ hybridization preparations. These relative abundance values were then compared with HTS data obtained in three separate molecular surveys: 454 sequencing of the V4 region of the 18S ribosomal DNA (rDNA) using DNA and RNA extracts (DNA-V4 and cDNA-V4) and Illumina sequencing of the V9 region (cDNA-V9). The microscopic and molecular signals were generally correlated, indicating that a relative increase in specific 18S rDNA was the result of a large proportion of cells in the given taxa. Despite these positive correlations, the slopes often deviated from 1, precluding a direct translation of sequences to cells. Our data highlighted clear differences depending on the nucleic acid template or the 18S rDNA region targeted. Thus, the molecular signal obtained using cDNA templates was always closer to relative cell abundances, while the V4 and V9 regions gave better results depending on the taxa. Our data support the quantitative use of HTS data but warn about considering it as a direct proxy of cell abundances. IMPORTANCE: Direct studies on marine picoeukaryotes by epifluorescence microscopy are problematic due to the lack of morphological features and due to the limited number and poor resolution of specific phylogenetic probes used in fluorescence in situ hybridization (FISH) routines. As a consequence, there is an increasing use of molecular methods, including high-throughput sequencing (HTS), to study marine microbial diversity. HTS can provide a detailed picture of the taxa present in a community and can reveal diversity not evident using other methods, but it is still unclear what the meaning of the sequence abundance in a given taxon is. Our aim is to investigate the correspondence between the relative HTS signal and relative cell abundances in selected picoeukaryotic taxa. Environmental sequencing provides reasonable estimates of the relative abundance of specific taxa. Better results are obtained when using RNA extracts as the templates, while the region of 18S ribosomal DNA had different influences depending on the taxa assayed.

Hidden diversity of Acoelomorpha revealed through metabarcoding.

Animals with bilateral symmetry comprise the majority of the described species within Metazoa. However, the nature of the first bilaterian animal remains unknown. As most recent molecular phylogenies point to Xenacoelomorpha as the sister group to the rest of Bilateria, understanding their biology, ecology and diversity is key to reconstructing the nature of the last common bilaterian ancestor (Urbilateria). To date, sampling efforts have focused mainly on coastal areas, leaving potential gaps in our understanding of the full diversity of xenacoelomorphs. We therefore analysed 18S rDNA metabarcoding data from three marine projects covering benthic and pelagic habitats worldwide. Our results show that acoels have a greater richness in planktonic environments than previously described. Interestingly, we also identified a putative novel clade of acoels in the deep benthos that branches as sister group to the rest of Acoela, thus representing the earliest-branching acoel clade. Our data highlight deep-sea environments as an ideal habitat to sample acoels with key phylogenetic positions, which might be useful for reconstructing the early evolution of Bilateria.

Placing environmental next-generation sequencing amplicons from microbial eukaryotes into a phylogenetic context.

Nucleotide positions in the hypervariable V4 and V9 regions of the small subunit (SSU)-rDNA locus are normally difficult to align and are usually removed before standard phylogenetic analyses. Yet, with next-generation sequencing data, amplicons of these regions are all that are available to answer ecological and evolutionary questions that rely on phylogenetic inferences. With ciliates, we asked how inclusion of the V4 or V9 regions, regardless of alignment quality, affects tree topologies using distinct phylogenetic methods (including PairDist that is introduced here). Results show that the best approach is to place V4 amplicons into an alignment of full-length Sanger SSU-rDNA sequences and to infer the phylogenetic tree with RAxML. A sliding window algorithm as implemented in RAxML shows, though, that not all nucleotide positions in the V4 region are better than V9 at inferring the ciliate tree. With this approach and an ancestral-state reconstruction, we use V4 amplicons from European nearshore sampling sites to infer that rather than being primarily terrestrial and freshwater, colpodean ciliates may have repeatedly transitioned from terrestrial/freshwater to marine environments.