How good are global DNA-based environmental surveys for detecting all protist diversity? Arcellinida as an example of biased representation.

Metabarcoding approaches targeting microeukaryotes have deeply changed our vision of protist environmental diversity. The public repository EukBank consists of 18S v4 metabarcodes from 12,672 samples worldwide. To estimate how far this database provides a reasonable overview of all eukaryotic diversity, we used Arcellinida (lobose testate amoebae) as a case study. We hypothesised that (1) this approach would allow the discovery of unexpected diversity, but also that (2) some groups would be underrepresented because of primer/sequencing biases. Most of the Arcellinida sequences appeared in freshwater and soil, but their abundance and diversity appeared underrepresented. Moreover, 84% of ASVs belonged to the suborder Phryganellina, a supposedly species-poor clade, whereas the best-documented suborder (Glutinoconcha, 600 described species) was only marginally represented. We explored some possible causes of these biases. Mismatches in the primer-binding site seem to play a minor role. Excessive length of the target region could explain some of these biases, but not all. There must be some other unknown factors involved. Altogether, while metabarcoding based on ribosomal genes remains a good first approach to document microbial eukaryotic clades, alternative approaches based on other genes or sequencing techniques must be considered for an unbiased picture of the diversity of some groups.

Cyphoderia ampulla (Cyphoderiidae: Rhizaria), a tale of freshwater sailors: The causes and consequences of ecological transitions through the salinity barrier in a family of benthic protists.

The salinity barrier that separates marine and freshwater biomes is probably the most important division in biodiversity on Earth. Those organisms that successfully performed this transition had access to new ecosystems while undergoing changes in selective pressure, which often led to major shifts in diversification rates. While these transitions have been extensively investigated in animals, the tempo, mode, and outcome of crossing the salinity barrier have been scarcely studied in other eukaryotes. Here, we reconstructed the evolutionary history of the species complex Cyphoderia ampulla (Euglyphida: Cercozoa: Rhizaria) based on DNA sequences from the nuclear SSU rRNA gene and the mitochondrial cytochrome oxidase subunit I gene, obtained from publicly available environmental DNA data (GeneBank, EukBank) and isolated organisms. A tree calibrated with euglyphid fossils showed that four independent transitions towards freshwater systems occurred from the mid-Miocene onwards, coincident with important fluctuations in sea level. Ancestral trait reconstructions indicated that the whole family Cyphoderiidae had a marine origin and suggest that ancestors of the freshwater forms were euryhaline and lived in environments with fluctuating salinity. Diversification rates did not show any obvious increase concomitant with ecological transitions, but morphometric analyses indicated that species increased in size and homogenized their morphology after colonizing the new environments. This suggests adaptation to changes in selective pressure exerted by life in freshwater sediments.

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.

Revisions to the Classification, Nomenclature, and Diversity of Eukaryotes.

This revision of the classification of eukaryotes follows that of Adl et al., 2012 [J. Euk. Microbiol. 59(5)] and retains an emphasis on protists. Changes since have improved the resolution of many nodes in phylogenetic analyses. For some clades even families are being clearly resolved. As we had predicted, environmental sampling in the intervening years has massively increased the genetic information at hand. Consequently, we have discovered novel clades, exciting new genera and uncovered a massive species level diversity beyond the morphological species descriptions. Several clades known from environmental samples only have now found their home. Sampling soils, deeper marine waters and the deep sea will continue to fill us with surprises. The main changes in this revision are the confirmation that eukaryotes form at least two domains, the loss of monophyly in the Excavata, robust support for the Haptista and Cryptista. We provide suggested primer sets for DNA sequences from environmental samples that are effective for each clade. We have provided a guide to trophic functional guilds in an appendix, to facilitate the interpretation of environmental samples, and a standardized taxonomic guide for East Asian users.

Environmental Sequencing Fills the Gap Between Parasitic Haplosporidians and Free-living Giant Amoebae.

Class Ascetosporea (Rhizaria; Endomyxa) comprises many parasites of invertebrates. Within this group, recent group-specific environmental DNA (eDNA) studies have contributed to the establishment of the new order Mikrocytida, a new phylogeny and characterization of Paramyxida, and illuminated the diversity and distribution of haplosporidians. Here, we use general and lineage-specific PCR primers to investigate the phylogenetic "gap" between haplosporidians and their closest known free-living relatives, the testate amoeba Gromia and reticulate amoeba Filoreta. Within this gap are Paradinium spp. parasites of copepods, which we show to be highly diverse and widely distributed in planktonic and benthic samples. We reveal a robustly supported radiation of parasites, ENDO-3, comprised of Paradinium and three further clades (ENDO-3a, ENDO-3b and SPP). A further environmental group, ENDO-2, perhaps comprising several clades, branches between this radiation and the free-living amoebae. Early diverging haplosporidians were also amplified, often associated with bivalves or deep-sea samples. The general primer approach amplified an overlapping set of novel lineages within ENDO-3 and Haplosporida, whereas the group-specific primer strategy, targeted to amplify from the earliest known divergent haplosporidians to Gromia, generated greater sequence diversity across part of this phylogenetic range.

Clarifying the Relationships between Microsporidia and Cryptomycota.

Some protists with microsporidian-like cell biological characters, including Mitosporidium, Paramicrosporidium, and Nucleophaga, have SSU rRNA gene sequences that are much less divergent than canonical Microsporidia. We analysed the phylogenetic placement and environmental diversity of microsporidian-like lineages that group near the base of the fungal radiation and show that they group in a clade with metchnikovellids and canonical microsporidians, to the exclusion of the clade including Rozella, in line with what is currently known of their morphology and cell biology. These results show that the phylogenetic scope of Microsporidia has been greatly underestimated. We propose that much of the lineage diversity previously thought to be cryptomycotan/rozellid is actually microsporidian, offering new insights into the evolution of the highly specialized parasitism of canonical Microsporidia. This insight has important implications for our understanding of opisthokont evolution and ecology, and is important for accurate interpretation of environmental diversity. Our analyses also demonstrate that many opisthosporidian (aphelid+rozellid+microsporidian) SSU V4 OTUs from Neotropical forest soils group with the short-branching Microsporidia, consistent with the abundance of their protist and arthropod hosts in soils. This novel diversity of Microsporidia provides a unique opportunity to investigate the evolutionary origins of a highly specialized clade of major animal parasites.

Rhizarian 'Novel Clade 10' Revealed as Abundant and Diverse Planktonic and Terrestrial Flagellates, including Aquavolon n. gen.

Rhizarian 'Novel Clade 10' (NC10) is frequently detected by 18S rRNA gene sequencing studies in freshwater planktonic samples. We describe a new genus and two species of eukaryovorous biflagellate protists, Aquavolon hoantrani n. gen. n. sp. and A. dientrani n. gen. n. sp., which represent the first morphologically characterized members of NC10, here named Aquavolonida ord. nov. The slightly metabolic cells possess naked heterodynamic flagella, whose kinetosomes lie at a right angle to each other and are connected by at least one fibril. Unlike their closest known relative Tremula longifila, they rotate around their longitudinal axis when swimming and only very rarely glide on surfaces. Screening of a wide range of environmental DNA extractions with lineage-specific PCR primers reveals that Aquavolonida consists of a large radiation of protists, which are most diversified in freshwater planktonic habitats and as yet undetected in marine environments. Earlier-branching lineages in Aquavolonida include less frequently detected organisms from soils and freshwater sediments. The 18S rRNA gene phylogeny suggests that Aquavolonida forms a common evolutionary lineage with tremulids and uncharacterized 'Novel Clade 12', which likely represents one of the deepest lineages in the Rhizaria, separate from Cercozoa (Filosa), Endomyxa, and Retaria.

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.

Multigene phylogeny resolves deep branching of Amoebozoa.

Amoebozoa is a key phylum for eukaryote phylogeny and evolutionary history, but its phylogenetic validity has been questioned since included species are very diverse: amoebo-flagellate slime-moulds, naked and testate amoebae, and some flagellates. 18S rRNA gene trees have not firmly established its internal topology. To rectify this we sequenced cDNA libraries for seven diverse Amoebozoa and conducted phylogenetic analyses for 109 eukaryotes (17-18 Amoebozoa) using 60-188 genes. We conducted Bayesian inferences with the evolutionarily most realistic site-heterogeneous CAT-GTR-Γ model and maximum likelihood analyses. These unequivocally establish the monophyly of Amoebozoa, showing a primary dichotomy between the previously contested subphyla Lobosa and Conosa. Lobosa, the entirely non-flagellate lobose amoebae, are robustly partitioned into the monophyletic classes Tubulinea, with predominantly tube-shaped pseudopodia, and Discosea with flattened cells and different locomotion. Within Conosa 60/70-gene trees with very little missing data show a primary dichotomy between the aerobic infraphylum Semiconosia (Mycetozoa and Variosea) and secondarily anaerobic Archamoebae. These phylogenetic features are entirely congruent with the most recent major amoebozoan classification emphasising locomotion modes, pseudopodial morphology, and ultrastructure. However, 188-gene trees where proportionally more taxa have sparser gene-representation weakly place Archamoebae as sister to Macromycetozoa instead, possibly a tree reconstruction artefact of differentially missing data.

The Protist Ribosomal Reference database (PR2): a catalog of unicellular eukaryote small sub-unit rRNA sequences with curated taxonomy.

The interrogation of genetic markers in environmental meta-barcoding studies is currently seriously hindered by the lack of taxonomically curated reference data sets for the targeted genes. The Protist Ribosomal Reference database (PR(2), http://ssu-rrna.org/) provides a unique access to eukaryotic small sub-unit (SSU) ribosomal RNA and DNA sequences, with curated taxonomy. The database mainly consists of nuclear-encoded protistan sequences. However, metazoans, land plants, macrosporic fungi and eukaryotic organelles (mitochondrion, plastid and others) are also included because they are useful for the analysis of high-troughput sequencing data sets. Introns and putative chimeric sequences have been also carefully checked. Taxonomic assignation of sequences consists of eight unique taxonomic fields. In total, 136 866 sequences are nuclear encoded, 45 708 (36 501 mitochondrial and 9657 chloroplastic) are from organelles, the remaining being putative chimeric sequences. The website allows the users to download sequences from the entire and partial databases (including representative sequences after clustering at a given level of similarity). Different web tools also allow searches by sequence similarity. The presence of both rRNA and rDNA sequences, taking into account introns (crucial for eukaryotic sequences), a normalized eight terms ranked-taxonomy and updates of new GenBank releases were made possible by a long-term collaboration between experts in taxonomy and computer scientists.

Diverse molecular signatures for ribosomally 'active' Perkinsea in marine sediments.

BACKGROUND: Perkinsea are a parasitic lineage within the eukaryotic superphylum Alveolata. Recent studies making use of environmental small sub-unit ribosomal RNA gene (SSU rDNA) sequencing methodologies have detected a significant diversity and abundance of Perkinsea-like phylotypes in freshwater environments. In contrast only a few Perkinsea environmental sequences have been retrieved from marine samples and only two groups of Perkinsea have been cultured and morphologically described and these are parasites of marine molluscs or marine protists. These two marine groups form separate and distantly related phylogenetic clusters, composed of closely related lineages on SSU rDNA trees. Here, we test the hypothesis that Perkinsea are a hitherto under-sampled group in marine environments. Using 454 diversity 'tag' sequencing we investigate the diversity and distribution of these protists in marine sediments and water column samples taken from the Deep Chlorophyll Maximum (DCM) and sub-surface using both DNA and RNA as the source template and sampling four European offshore locations. RESULTS: We detected the presence of 265 sequences branching with known Perkinsea, the majority of them recovered from marine sediments. Moreover, 27% of these sequences were sampled from RNA derived cDNA libraries. Phylogenetic analyses classify a large proportion of these sequences into 38 cluster groups (including 30 novel marine cluster groups), which share less than 97% sequence similarity suggesting this diversity encompasses a range of biologically and ecologically distinct organisms. CONCLUSIONS: These results demonstrate that the Perkinsea lineage is considerably more diverse than previously detected in marine environments. This wide diversity of Perkinsea-like protists is largely retrieved in marine sediment with a significant proportion detected in RNA derived libraries suggesting this diversity represents ribosomally 'active' and intact cells. Given the phylogenetic range of hosts infected by known Perkinsea parasites, these data suggest that Perkinsea either play a significant but hitherto unrecognized role as parasites in marine sediments and/or members of this group are present in the marine sediment possibly as part of the 'seed bank' microbial community.

Multigene eukaryote phylogeny reveals the likely protozoan ancestors of opisthokonts (animals, fungi, choanozoans) and Amoebozoa.

Animals and fungi independently evolved from the protozoan phylum Choanozoa, these three groups constituting a major branch of the eukaryotic evolutionary tree known as opisthokonts. Opisthokonts and the protozoan phylum Amoebozoa (amoebae plus slime moulds) were previously argued to have evolved independently from the little-studied, largely flagellate, protozoan phylum, Sulcozoa. Sulcozoa are a likely evolutionary link between opisthokonts and the more primitive excavate flagellates that have ventral feeding grooves and the most primitive known mitochondria. To extend earlier sparse evidence for the ancestral (paraphyletic) nature of Sulcozoa, we sequenced transcriptomes from six gliding flagellates (two apusomonads; three planomonads; Mantamonas). Phylogenetic analyses of 173-192 genes and 73-122 eukaryote-wide taxa show Sulcozoa as deeply paraphyletic, confirming that opisthokonts and Amoebozoa independently evolved from sulcozoans by losing their ancestral ventral groove and dorsal pellicle: Apusozoa (apusomonads plus anaerobic breviate amoebae) are robustly sisters to opisthokonts and probably paraphyletic, breviates diverging before apusomonads; Varisulca (planomonads, Mantamonas, and non-gliding flagellate Collodictyon) are sisters to opisthokonts plus Apusozoa and Amoebozoa, and possibly holophyletic; Glissodiscea (planomonads, Mantamonas) may be holophyletic, but Mantamonas sometimes groups with Collodictyon instead. Taxon and gene sampling slightly affects tree topology; for the closest branches in Sulcozoa and opisthokonts, proportionally reducing missing data eliminates conflicts between homogeneous-model maximum-likelihood trees and evolutionarily more realistic site-heterogeneous trees. Sulcozoa, opisthokonts, and Amoebozoa constitute an often-pseudopodial 'podiate' clade, one of only three eukaryotic 'supergroups'. Our trees indicate that evolution of sulcozoan dorsal pellicle, ventral pseudopodia, and ciliary gliding (probably simultaneously) generated podiate eukaryotes from Malawimonas-like excavate flagellates.

Diverse patterns of correspondence between protist metabarcodes and protist metagenome-assembled genomes.

Two common approaches to study the composition of environmental protist communities are metabarcoding and metagenomics. Raw metabarcoding data are usually processed into Operational Taxonomic Units (OTUs) or amplicon sequence variants (ASVs) through clustering or denoising approaches, respectively. Analogous approaches are used to assemble metagenomic reads into metagenome-assembled genomes (MAGs). Understanding the correspondence between the data produced by these two approaches can help to integrate information between the datasets and to explain how metabarcoding OTUs and MAGs are related with the underlying biological entities they are hypothesised to represent. MAGs do not contain the commonly used barcoding loci, therefore sequence homology approaches cannot be used to match OTUs and MAGs. We made an attempt to match V9 metabarcoding OTUs from the 18S rRNA gene (V9 OTUs) and MAGs from the Tara Oceans expedition based on the correspondence of their relative abundances across the same set of samples. We evaluated several metrics for detecting correspondence between features in these two datasets and developed controls to filter artefacts of data structure and processing. After selecting the best-performing metrics, ranking the V9 OTU/MAG matches by their proportionality/correlation coefficients and applying a set of selection criteria, we identified candidate matches between V9 OTUs and MAGs. In some cases, V9 OTUs and MAGs could be matched with a one-to-one correspondence, implying that they likely represent the same underlying biological entity. More generally, matches we observed could be classified into 4 scenarios: one V9 OTU matches many MAGs; many V9 OTUs match many MAGs; many V9 OTUs match one MAG; one V9 OTU matches one MAG. Notably, we found some instances in which different OTU-MAG matches from the same taxonomic group were not classified in the same scenario, with all four scenarios possible even within the same taxonomic group, illustrating that factors beyond taxonomic lineage influence the relationship between OTUs and MAGs. Overall, each scenario produces a different interpretation of V9 OTUs, MAGs and how they compare in terms of the genomic and ecological diversity they represent.

Global perspective of environmental distribution and diversity of Perkinsea (Alveolata) explored by a meta-analysis of eDNA surveys.

Perkinsea constitutes a lineage within the Alveolata eukaryotic superphylum, mainly composed of parasitic organisms. Some described species represent significant ecological and economic threats due to their invasive ability and pathogenicity, which can lead to mortality events. However, the genetic diversity of these described species is just the tip of the iceberg. Environmental surveys targeting this lineage are still scarce and mainly limited to the Northern Hemisphere. Here, we aim to conduct an in depth exploration of the Perkinsea group, uncovering the diversity across a variety of environments, including those beyond freshwater and marine ecosystems. We seek to identify and describe putative novel organisms based on their genetic signatures. In this study, we conducted an extensive analysis of a metabarcoding dataset, focusing on the V4 region of the 18S rRNA gene (the EukBank dataset), to investigate the diversity, distribution and environmental preferences of the Perkinsea. Our results reveal a remarkable diversity within the Perkinsea, with 1568 Amplicon Sequence Variants (ASVs) identified across thousands of environmental samples. Surprisingly, we showed a substantial diversity of Perkinsea within soil samples (269 ASVs), challenging the previous assumption that this group is confined to marine and freshwater environments. In addition, we revealed that a notable proportion of Perkinsea ASVs (428 ASVs) could correspond to putative new organisms, encompassing the well-established taxonomic group Perkinsidae. Finally, our study shed light on previously unveiled taxonomic groups, including the Xcellidae, and revealed their environmental distribution. These findings demonstrate that Perkinsea exhibits far greater diversity than previously detected and surprisingly extends beyond marine and freshwater environments. The meta-analysis conducted in this study has unveiled the existence of previously unknown clusters within the Perkinsea lineage, solely identified based on their genetic signatures. Considering the ecological and economic importance of described Perkinsea species, these results suggest that Perkinsea may play a significant, yet previously unrecognized, role across a wide range of environments, spanning from soil environments to the abyssal zone of the open ocean with important implications for ecosystem functioning.

Predicting global distributions of eukaryotic plankton communities from satellite data.

Satellite remote sensing is a powerful tool to monitor the global dynamics of marine plankton. Previous research has focused on developing models to predict the size or taxonomic groups of phytoplankton. Here, we present an approach to identify community types from a global plankton network that includes phytoplankton and heterotrophic protists and to predict their biogeography using global satellite observations. Six plankton community types were identified from a co-occurrence network inferred using a novel rDNA 18 S V4 planetary-scale eukaryotic metabarcoding dataset. Machine learning techniques were then applied to construct a model that predicted these community types from satellite data. The model showed an overall 67% accuracy in the prediction of the community types. The prediction using 17 satellite-derived parameters showed better performance than that using only temperature and/or the concentration of chlorophyll a. The constructed model predicted the global spatiotemporal distribution of community types over 19 years. The predicted distributions exhibited strong seasonal changes in community types in the subarctic-subtropical boundary regions, which were consistent with previous field observations. The model also identified the long-term trends in the distribution of community types, which suggested responses to ocean warming.

Patterns of eukaryotic diversity from the surface to the deep-ocean sediment.

Remote deep-ocean sediment (DOS) ecosystems are among the least explored biomes on Earth. Genomic assessments of their biodiversity have failed to separate indigenous benthic organisms from sinking plankton. Here, we compare global-scale eukaryotic DNA metabarcoding datasets (18S-V9) from abyssal and lower bathyal surficial sediments and euphotic and aphotic ocean pelagic layers to distinguish plankton from benthic diversity in sediment material. Based on 1685 samples collected throughout the world ocean, we show that DOS diversity is at least threefold that in pelagic realms, with nearly two-thirds represented by abundant yet unknown eukaryotes. These benthic communities are spatially structured by ocean basins and particulate organic carbon (POC) flux from the upper ocean. Plankton DNA reaching the DOS originates from abundant species, with maximal deposition at high latitudes. Its seafloor DNA signature predicts variations in POC export from the surface and reveals previously overlooked taxa that may drive the biological carbon pump.

Genomic evidence for global ocean plankton biogeography shaped by large-scale current systems.

Biogeographical studies have traditionally focused on readily visible organisms, but recent technological advances are enabling analyses of the large-scale distribution of microscopic organisms, whose biogeographical patterns have long been debated. Here we assessed the global structure of plankton geography and its relation to the biological, chemical, and physical context of the ocean (the 'seascape') by analyzing metagenomes of plankton communities sampled across oceans during the Tara Oceans expedition, in light of environmental data and ocean current transport. Using a consistent approach across organismal sizes that provides unprecedented resolution to measure changes in genomic composition between communities, we report a pan-ocean, size-dependent plankton biogeography overlying regional heterogeneity. We found robust evidence for a basin-scale impact of transport by ocean currents on plankton biogeography, and on a characteristic timescale of community dynamics going beyond simple seasonality or life history transitions of plankton.

Oceans are brimming with life invisible to our eyes, a myriad of species of bacteria, viruses and other microscopic organisms essential for the health of the planet. These 'marine plankton' are unable to swim against currents and should therefore be constantly on the move, yet previous studies have suggested that distinct species of plankton may in fact inhabit different oceanic regions. However, proving this theory has been challenging; collecting plankton is logistically difficult, and it is often impossible to distinguish between species simply by examining them under a microscope. However, within the last decade, a research schooner called Tara has travelled the globe to gather thousands of plankton samples. At the same time, advances in genomics have made it possible to identify species based only on fragments of their DNA sequence. To understand the hidden geography of plankton communities in Earth's oceans, Richter et al. pored over DNA from the Tara Oceans expedition. This revealed that, despite being unable to resist the flow of water, various planktonic species which live close to the surface manage to occupy distinct, stable provinces shaped by currents. Different sizes of plankton are distributed in different sized provinces, with the smallest organisms tending to inhabit the smallest areas. Comparing DNA similarities and speeds of currents at the ocean surface revealed how these might stretch and mix plankton communities. Plankton play a critical role in the health of the ocean and the chemical cycles of planet Earth. These results could allow deeper investigation by marine modellers, ecologists, and evolutionary biologists. Meanwhile, work is already underway to investigate how climate change might impact this hidden geography.

Phylogeny of novel naked Filose and Reticulose Cercozoa: Granofilosea cl. n. and Proteomyxidea revised.

Naked filose and reticulose protozoa were long lumped as proteomyxids or left outside higher groups. We cultivated eight naked filose or reticulose strains, did light microscopy, 18S rDNA sequencing and phylogeny (showing all are Cercozoa), and sequenced 80 environmental 18S-types. Filose species belong in subphylum Filosa and reticulose ones in subphylum Endomyxa, making proteomyxids polyphyletic. We therefore transfer the classically mainly reticulose Proteomyxidea to Endomyxa, removing evident filosans as new class Granofilosea (including Desmothoracida, Acinetactis and new heliomonad family Heliomorphidae (new genus Heliomorpha (=Dimorpha)). Five new species of Limnofila gen. n. (L. mylnikovi; L. anglica; L. longa; L. oxoniensis; L. borokensis, previously misidentified as Biomyxa (=Gymnophrys) cometa) form a large freshwater clade (new order Limnofilida). Mesofila limnetica gen., sp. n. and Nanofila marina gen., sp. n. group separately in Granofilosea (Cryptofilida ord. n.). In Endomyxa, a new genus of reticulose proteomyxids (Filoreta marina, F. japonica, F. turcica spp. n., F. (=Corallomyxa) tenera comb. n.) forms a clade (Reticulosida) related to Gromiidea/Ascetosporea. Platyreta germanica gen., sp. n. and Arachnula impatiens are related vampyrellids (Aconchulinida) within a large clade beside Phytomyxea. Biomyxidae and Rhizoplasmidae fam. n. remain incertae sedis within Proteomyxidea. Gymnophrydium and Borkovia are revised. The reticulose Corallomyxa are unlike Filoreta and possibly Amoebozoa, not Cercozoa.

Diversification of unicellular eukaryotes: cryptomonad colonizations of marine and fresh waters inferred from revised 18S rRNA phylogeny.

The cryptomonads is a well-defined lineage of unicellular eukaryotes, composed of several marine and freshwater groups. However, the evolutionary relationships among these groups are unclear due to conflicting inferences between morphological and molecular phylogenies. Here, we have inferred the evolutionary relationships among marine and freshwater species in order to better understand the importance of the marine-freshwater boundary on the historical diversification patterns of cryptomonads. We have constructed improved molecular phylogenies by taking into account rate variation both across sites and across sequences (covarion substitutions), and by analysing the vast majority of publicly available cryptomonad 18S rRNA sequences and related environmental phylotypes. The resulting phylogenies included 55 sequences, and revealed two novel freshwater cryptomonad clades (CRY1 and CRY2) and a large hidden diversity of cryptomonads. CRY1 was placed deeply within the cryptomonad phylogeny together with all the major freshwater lineages (i.e. Goniomonas and Cryptomonas), while CRY2 was placed within a lineage of marine species identified as Plagioselmis-like with the aid of a new sequence generated from a cultured species. The inferred phylogenies suggest only few successful marine-freshwater transitions over the history of cryptomonads. Most of the transitions seem to have occurred from marine to fresh waters, but re-colonizations of marine habitats have also taken place. This implies that the differences in the biogeophysical conditions between marine and fresh waters constitute a substantial barrier for the cross-colonization of these environments by cryptomonads.