Loricate choanoflagellates (Acanthoecida) from warm water seas. VIII. Crinolina Thomsen and Diaphanoeca Ellis.

The loricate choanoflagellate genera Diaphanoeca Ellis and Crinolina Thomsen encompass a total of ten species. The majority of these are recorded from the warm water regions reported on here. A distinct morphological dichotomy characterizes the genus Diaphanoeca as currently circumscribed. The species distribute themselves within a 'D. grandis subgroup' and a 'D. pedicellata subgroup' distinguished on e.g., the position of the protoplast inside the lorica chamber and the elaboration of the anterior projections. We are, while awaiting in particular further molecular evidence, taking a conservative approach and abstain from dealing with the subgroup issue at the generic level. The examination of material from the warm water regions of the world's oceans has resulted in the description of D. sargassoensis sp.n., D. pseudoundulata sp.n., and D. throndsenii sp.n., and a thorough re-examination of D. undulata. Species of Crinolina share multiple features with in particular the D. grandis species subgroup. It is yet relevant, both in a morphological and molecular perspective, to retain the genus Crinolina which remains unambiguously defined based on the posteriorly open lorica. A high level of agreement is found when contrasting morphological and molecular based phylogenetic schemes.

Extended divergence estimates and species descriptions of new craspedid choanoflagellates from the Atacama Desert, Northern Chile.

In contrast to previous perspectives, hypersaline environments have been proven to harbour a variety of potentially highly adapted microorganisms, in particular unicellular eukaryotes. The isolated, hypersaline waterbodies in the Atacama Desert, Northern Chile are exposed to high UV radiation and deposition of toxic heavy metals, making them of great interest regarding studies on speciation and evolutionary processes. In the past two years, among a variety of other protist species, five new species of heterotrophic choanoflagellates were described and analysed from this area, showing an adaptation to a broad range of salinities. Morphological data alone does not allow for species delineation within craspedid species, additional molecular data is essential for modern taxonomy. In addition, molecular clock analyses pointed towards a strong selection force of the extreme environmental conditions. Within this study, we describe three additional craspedid choanoflagellate species, isolated from different aquatic environments. Phylogenetic analyses show two distinct clades of choanoflagellates from the Atacama, suggesting two independent invasions of at least two ancestral marine species, and, as indicated by our new data, a possible dispersal by Andean aquifers. The extended molecular clock analysis based on transcriptomic data of choanoflagellate strains from the Salar de Llamará, a hypersaline basin within the Central Depression of the Atacama Desert, reflects colonisation and divergence events which correspond to geological data of the paleohydrology.

Detection of horizontal gene transfer in the genome of the choanoflagellate Salpingoeca rosetta.

Horizontal gene transfer (HGT), the movement of heritable materials between distantly related organisms, is crucial in eukaryotic evolution. However, the scale of HGT in choanoflagellates, the closest unicellular relatives of metazoans, and its possible roles in the evolution of animal multicellularity remains unexplored. We identified at least 175 candidate HGTs in the genome of the colonial choanoflagellate Salpingoeca rosetta using sequence-based tests. The majority of these were orthologous to genes in bacterial and microalgal lineages, yet displayed genomic features consistent with the rest of the S. rosetta genome-evidence of ancient acquisition events. Putative functions include enzymes involved in amino acid and carbohydrate metabolism, cell signaling, and the synthesis of extracellular matrix components. Functions of candidate HGTs may have contributed to the ability of choanoflagellates to assimilate novel metabolites, thereby supporting adaptation, survival in diverse ecological niches, and response to external cues that are possibly critical in the evolution of multicellularity in choanoflagellates.

Evolution and Diversity of Semaphorins and Plexins in Choanoflagellates.

Semaphorins and plexins are cell surface ligand/receptor proteins that affect cytoskeletal dynamics in metazoan cells. Interestingly, they are also present in Choanoflagellata, a class of unicellular heterotrophic flagellates that forms the phylogenetic sister group to Metazoa. Several members of choanoflagellates are capable of forming transient colonies, whereas others reside solitary inside exoskeletons; their molecular diversity is only beginning to emerge. Here, we surveyed genomics data from 22 choanoflagellate species and detected semaphorin/plexin pairs in 16 species. Choanoflagellate semaphorins (Sema-FN1) contain several domain features distinct from metazoan semaphorins, including an N-terminal Reeler domain that may facilitate dimer stabilization, an array of fibronectin type III domains, a variable serine/threonine-rich domain that is a potential site for O-linked glycosylation, and a SEA domain that can undergo autoproteolysis. In contrast, choanoflagellate plexins (Plexin-1) harbor a domain arrangement that is largely identical to metazoan plexins. Both Sema-FN1 and Plexin-1 also contain a short homologous motif near the C-terminus, likely associated with a shared function. Three-dimensional molecular models revealed a highly conserved structural architecture of choanoflagellate Plexin-1 as compared to metazoan plexins, including similar predicted conformational changes in a segment that is involved in the activation of the intracellular Ras-GAP domain. The absence of semaphorins and plexins in several choanoflagellate species did not appear to correlate with unicellular versus colonial lifestyle or ecological factors such as fresh versus salt water environment. Together, our findings support a conserved mechanism of semaphorin/plexin proteins in regulating cytoskeletal dynamics in unicellular and multicellular organisms.

A Needle in the Haystack - Mapping Sequences to Morphology Exemplified by the Loricate Choanoflagellate Enibas thessalia sp. nov. (Acanthoecida, Acanthoecidae).

Environmental sequencing surveys unveil an unexpected magnitude of protist biodiversity and help to understand environmental community structure as well as biogeographical patterns. The interpretation of these data is still hindered by the lack of a verified and reliable reference database, which is the important basis for all analyses. References should rely on detailed and valid taxonomical descriptions including both morphology and autecological properties. In fact, obtaining such data is still a major challenge as cultivation-based approaches are very selective. In the present study, we highlight the potential to resample habitats which showed phylogenetically interesting sequences from environmental molecular surveys. We have been able to reveal a choanoflagellate species with the use of a single cell isolation approach in order to achieve a morphological description to the target sequence. This new species, Enibas thessalia sp. nov. now extends a recently described monospecific genus. In addition, we illustrate a nudiform lorica reproduction of the genus Enibas by observation of living cells. The genus belongs to the family of Acanthoecidae, which comprises five genera. The morphology of the genus Enibas shows a striking resemblance to the genus Stephanoeca, which belongs to the other family of loricate choanoflagellates, the Stephanoecidae, indicating that morphology alone might not reflect phylogenetic relations. We demonstrate that mapping sequences to a taxonomical description of species is a valuable tool to verify the organism behind an environmental amplicon. We emphasize the urgent need of integrative taxonomy matching molecular data with morphological features to verify the outcome of phylogenetic analyses.

Loricate choanoflagellates (Acanthoecida) from warm water seas. VII. Calotheca Thomsen and Moestrup, Stephanacantha Thomsen and Syndetophyllum Thomsen and Moestrup.

The tectiform loricate choanoflagellate genera Calotheca, Stephanacantha and Syndetophyllum have all been first described from warm water habitats and share the presence of flattened and often elaborate costal strips in the lorica. The current reinvestigation does confirm both the widespread occurrence of these taxa within the global warm water belt, and largely corroborates the established genus and species matrix. We describe here Stephanacantha oceanica sp. nov. which closely resembles S. campaniformis, and transfer Parvicorbicula zigzag to the genus Stephanacantha, despite differences in costal strip morphology, but based on a complete agreement in lorica constructional details.

Loricate choanoflagellates (Acanthoecida) from warm water seas. VI. Pleurasiga Schiller and Parvicorbicula Deflandre.

The loricate choanoflagellate genera Pleurasiga and Parvicorbicula are taxonomically ambiguous. Pleurasiga because of the uncertainty that relates to the true identity of the type species, and Parvicorbicula because too many newly described species over time have been dumped here in lack of better options. While all species currently allocated to the genus Pleurasiga (with the exception of the type species) are observed in our samples from the global warm water belt, the genus Parvicorbicula is represented by just a few and mostly infrequently recorded taxa. Two new species, viz. Pl. quadrangiella sp. nov. and Pl. minutissima sp. nov., are described here. While the former is closely related to Pl. echinocostata, the latter is reminiscent of Pl. minima. Core species of Pleurasiga and Parvicorbicula deviate from the vast majority of loricate choanoflagellates in having both the anterior and the mid-lorica transverse costae located exterior to the longitudinal costae. In Pl. quadrangiella there is no mid-lorica transverse costa but rather a small posterior transverse costa located inside the longitudinal costae. In Pl. minutissima the mid-lorica transverse costa has extensive costal strip overlaps which reveal patterns of costal strip junctions that deviate from the norm.

Morphological and molecular investigation on freshwater choanoflagellates (Craspedida, Salpingoecidae) from the River Rhine at Cologne (Germany).

Paramonosiga coloniensis sp. nov., Salpingoeca amphoriscia sp. nov., S. fluviatilis sp. nov. and S. loutrophoria sp. nov. are frequently found craspedid species in the River Rhine which have not yet been described, despite their high abundance. All new species are characterized based on a distinct morphology which is different from all up to now described species and on a molecular level based on transcriptome data. In addition, we give extended redescriptions of S. amphoridium and S. angulosa, based on SSU and LSU rDNA data and morphology. The six-gene phylogenetic analyses place all species into freshwater clades of the craspedids. The separation of the freshwater and marine clades of this group is becoming more distinct with every craspedid sequence added. The River Rhine is one of the largest rivers in Europe but its protist biodiversity is fairly undescribed, especially regarding choanoflagellates. We conclude that the biodiversity of craspedid choanoflagellates is broadly underestimated.

Loricate choanoflagellates (Acanthoecida) from warm water seas. V. Thomsenella Özdikmen (= Platypleura Thomsen).

Loricate choanoflagellate genera that incorporate flattened costal strips in the lorica (i.e. Calotheca, Stephanacantha, Thomsenella (= Platypleura) and Syndetophyllum) are prevalent in warm water habitats. The genus Thomsenella (=Platypleura) thus comprises four species and three of these have an Andaman Sea type locality. Our ongoing examination of loricate choanoflagellate material from all major warm water oceans has provided us with the opportunity of revisiting species of Thomsenella in order to test and fortunately verify, in a morpho-specific context, the robustness of the species matrix initially defined.

Loricate choanoflagellates (Acanthoecida) from warm water seas. IV. Cosmoeca Thomsen.

Aided by an extensive collection of specimens from warm water seas, it has been possible to revisit the loricate choanoflagellate genus Cosmoeca. While the type species C. norvegica and also C. ventricosa sensu stricto have been described from temperate North Atlantic realms and share a cosmopolitan distribution, the remaining species and morphotypes of Cosmoeca are largely confined to warmer waters. The new data broadly validates the initial circumscription of species of Cosmoeca. The persisting taxonomic puzzle with respect to C. ventricosa, which in addition to the core type accommodates no less than five different morphotypes (form A-E), has been further elucidated. The Cosmoeca paper is part of a 'monographic' series of warm water loricate choanoflagellate contributions in progress, where the aim is to provide the best possible account of warm water species diversity, based on traditional light and electron microscopical techniques, as a tool for future identification work based on microscopy, and in support of the work in progress with establishing a quality assured molecular tool for future recognition of diversity.

Loricate choanoflagellates (Acanthoecida) from warm water seas. III. Acanthocorbis Hara and Takahashi and Stephanoeca Ellis.

A large-scale investigation of warm water loricate choanoflagellate communities has revealed the presence of close to 80 species, which is approximately half of all loricate choanoflagellate taxa described when including also hitherto undescribed forms known to us. We are in the process of stepwise providing a monographic treatment of these communities. The overall aim is to contribute the best possible account of species diversity, based on traditional light and electron microscopical techniques, as a tool for future identification work based on microscopy, and in support of the work in progress with establishing a quality assured molecular tool for future recognition of diversity. In this paper we summarize our findings of species of Acanthocorbis and Stephanoeca, which include the description of several new taxa: A. conicella sp. nov., A. gladiella sp. nov., S. broomia sp. nov., S. naja sp. nov., and S. andemanica sp. nov.

Loricate choanoflagellates (Acanthoecida) from warm water seas. II. Bicosta, Apheloecion, Campyloacantha, Crucispina, Calliacantha and Saroeca.

The main outcome of this and subsequent papers is to provide a baseline survey of heterotrophic protist diversity from warm water marine ecosystems, exemplified by loricate choanoflagellates (Acanthoecida). Genera in focus here (i.e. Bicosta, Apheloecion, Campyloacantha, Crucispina, Calliacantha and Saroeca) possess anterior spines or projections and a posterior pedicel, and have 0, 1 or 2 transverse costae. Longitudinal costae are, with the exception of Campyloacantha, external to transverse costal elements across all genera examined here. We describe here Apheloecion eqpacia sp. nov. and Calliacantha magna sp. nov., both of which are so far distributionally confined to warm water habitats. A 'form A' of Bicosta minor is introduced to facilitate the distinction between B. minor sensu stricto and a presumed warm water adapted variant with a posterior lorica chamber twist of the longitudinal costae.

First description of an euryoecious acanthoecid choanoflagellate species, Enibas tolerabilis gen. et sp. nov. from a salar in the Chilean Andes based on morphological and transcriptomic data.

In general, acanthoecid choanoflagellates have been described to occur exclusively in brackish water to marine habitats. Only recently, two studies have proven their existence in inland waters. One of them has shown, that an acanthoecid species from a small lake (near Apia on the island of Upolu, Samoa) is strictly freshwater adapted, not able to tolerate even brackish water. In this study, we present the first euryoecious acanthoecid species, able to live and reproduce in freshwater as well as under hypersaline conditions. The new species, Enibas tolerabilis gen. et sp. nov. was isolated in 2017 from the Salar de Ascotán in the Altiplano at 3750 m a. s. l., Northern Chile. The salinity at the time of sampling was 6 PSU. A series of autecological experiments have revealed a salinity tolerance from freshwater up to 70 PSU. In our phylogenetic analysis, E. tolerabilis gen. et sp. nov. clustered within the family of Acanthoecidae, forming a well-supported sister clade together with two other, environmental choanoflagellate sequences. We erected a new genus, Enibas gen. nov. and described the morphology, molecular biology and autecology for E. tolerabilis gen. et sp. nov. which has a stephanoecid-like lorica morphology. We emphasize that the definition of the genus Stephanoeca, being polyphyletic, is in urgent need of revision as we showed that this morphology is present in both acanthoecid families.

Loricate choanoflagellates (Acanthoecida) from warm water seas. I. Conioeca gen. nov. and Nannoeca Thomsen.

The main outcome of this and subsequent papers is to provide a baseline survey of heterotrophic protist diversity from warm water marine ecosystems, exemplified by loricate choanoflagellates (Acanthoecida). Loricate choanoflagellates are heterotrophic, nano-sized protists that are ubiquitous in marine and brackish water habitats. They dwell in a lorica formed by silicified costal strips organized in species specific patterns. The single anteriorly directed flagellum is surrounded by a collar formed by microvilli which together constitute the feeding apparatus. Keystone benefits from this warm water survey, which covers all three major oceans, is an improved understanding of global biogeographical patterns, and a further consolidation of the morphospecies matrix, that constitutes a highly essential reference framework for the current efforts to provide barcodes for as many species of loricate choanoflagellates as possible, based on e.g. single cell pipetting techniques. We describe here Conioeca gen. et sp. nov., which is so far distributionally confined to warm water habitats, and elaborate on the morphological variability encountered within the N. minuta complex. This leads to both the circumscription of a new N. minuta form A as well as the description of N. mexicana sp. nov.

Four new choanoflagellate species from extreme saline environments: Indication for isolation-driven speciation exemplified by highly adapted Craspedida from salt flats in the Atacama Desert (Northern Chile).

With this study we aim to extend the knowledge on the biogeography of craspedid choanoflagellates with additional data from extreme environments. Up to now, very little is known about choanoflagellates from extreme saline environments, as most studies have focused on marine and freshwater habitats. Though previously investigated high saline ice biota communities have indicated a possible adaptation to environments with high salt concentrations. Hypersaline endorheic basins, so-called salt flats or salares from the Atacama Desert in Northern Chile provide an intense environment regarding fluctuating and extreme salinities, which allow for studies on evolutionary adaptations of protists to hypersaline conditions. This study focused on choanoflagellate species isolated from different salt flats, their morphological characteristics using light and electron microscopy, molecular marker genes (SSU and LSU rDNA) and their salinity tolerance. Here, we described four new craspedid choanoflagellate species, highly adapted to the hypersaline environment of the Atacama Desert. This study extends our knowledge on choanoflagellate phylogeny and ecology and can become the basis for further molecular studies to understand the mechanisms of adaptations. Additionally, we emphasize the need of adding additional data such as autecological characteristics to amend species definitions, which is only possible from cultivated strains. This data would support the use of molecular data originating from metagenomic analyses also in an ecological context.

Gene family innovation, conservation and loss on the animal stem lineage.

Choanoflagellates, the closest living relatives of animals, can provide unique insights into the changes in gene content that preceded the origin of animals. However, only two choanoflagellate genomes are currently available, providing poor coverage of their diversity. We sequenced transcriptomes of 19 additional choanoflagellate species to produce a comprehensive reconstruction of the gains and losses that shaped the ancestral animal gene repertoire. We identified ~1944 gene families that originated on the animal stem lineage, of which only 39 are conserved across all animals in our study. In addition, ~372 gene families previously thought to be animal-specific, including Notch, Delta, and homologs of the animal Toll-like receptor genes, instead evolved prior to the animal-choanoflagellate divergence. Our findings contribute to an increasingly detailed portrait of the gene families that defined the biology of the Urmetazoan and that may underpin core features of extant animals.

Evaluation of single-cell genomics to address evolutionary questions using three SAGs of the choanoflagellate Monosiga brevicollis.

Single-cell genomics (SCG) appeared as a powerful technique to get genomic information from uncultured organisms. However, SCG techniques suffer from biases at the whole genome amplification step that can lead to extremely variable numbers of genome recovery (5-100%). Thus, it is unclear how useful can SCG be to address evolutionary questions on uncultured microbial eukaryotes. To provide some insights into this, we here analysed 3 single-cell amplified genomes (SAGs) of the choanoflagellate Monosiga brevicollis, whose genome is known. Our results show that each SAG has a different, independent bias, yielding different levels of genome recovery for each cell (6-36%). Genes often appear fragmented and are split into more genes during annotation. Thus, analyses of gene gain and losses, gene architectures, synteny and other genomic features can not be addressed with a single SAG. However, the recovery of phylogenetically-informative protein domains can be up to 55%. This means SAG data can be used to perform accurate phylogenomic analyses. Finally, we also confirm that the co-assembly of several SAGs improves the general genomic recovery. Overall, our data show that, besides important current limitations, SAGs can still provide interesting and novel insights from poorly-known, uncultured organisms.

A six-gene phylogeny provides new insights into choanoflagellate evolution.

Recent studies have shown that molecular phylogenies of the choanoflagellates (Class Choanoflagellatea) are in disagreement with their traditional taxonomy, based on morphology, and that Choanoflagellatea requires considerable taxonomic revision. Furthermore, phylogenies suggest that the morphological and ecological evolution of the group is more complex than has previously been recognized. Here we address the taxonomy of the major choanoflagellate order Craspedida, by erecting four new genera. The new genera are shown to be morphologically, ecologically and phylogenetically distinct from other choanoflagellate taxa. Furthermore, we name five novel craspedid species, as well as formally describe ten species that have been shown to be either misidentified or require taxonomic revision. Our revised phylogeny, including 18 new species and sequence data for two additional genes, provides insights into the morphological and ecological evolution of the choanoflagellates. We examine the distribution within choanoflagellates of these two additional genes, EF-1A and EFL, closely related translation GTPases which are required for protein synthesis. Mapping the presence and absence of these genes onto the phylogeny highlights multiple events of gene loss within the choanoflagellates.

Bridging the gap between morphological species and molecular barcodes - Exemplified by loricate choanoflagellates.

Translating the vast amounts of molecular barcodes from global surveys of microbial eukaryotes into ecological insight depends critically on a well-curated reference database with adequate taxonomic coverage. In this respect, the choanoflagellates resemble other eukaryotic lineages: reasonable coverage at higher taxonomic levels, but missing diversity at the species level. The acanthoecid (loricate) choanoflagellates are well-characterized morphologically, with over 115 species described, but less than 10% with any sequence data. Because lorica shape is species-specific, the acanthoecids represent an opportunity to link morphological with molecular data within a lineage of eukaryotes. To match morphospecies to sequences, we sampled the Kattegat and the Isefjord in Denmark in September 2014 and February 2015. We identified 45 morphospecies and sequenced ribosomal DNA of nine previously unsequenced species, roughly doubling the number of acanthoecid species with sequence data, including the first data representing five genera: Bicosta, Calliacantha, Cosmoeca, Crinolina and Pleurasiga. Our phylogenetic analysis is mainly congruent with morphology-based systematics. Five of the newly sequenced species match a previously unidentified barcode from Tara Oceans, providing access to the global distribution of species isolated from Danish waters. One species, Calliacantha natans, is the second most globally abundant choanoflagellate present in Tara Oceans. Our project translating new ribosomal DNA sequences to distributions of described species on a global scale supports the approach linking morphology to molecular barcodes for microbial eukaryote ecology.

A phylogenetic and morphological re-investigation of Diaphanoeca spiralifurca, Didymoeca elongata and Polyoeca dichotoma (Acanthoecida/Choanomonadida) from the Caribbean Sea.

Acanthoecid choanoflagellates are described and classified according to their distinct siliceous lorica, a unique morphological characteristic within protistan species. Only recently it has become clear that beside morphological and developmental data on the lorica molecular data, mainly based on SSU rDNA, it is necessary to resolve phylogenetic relationships within this group. Previously Didymoeca elongata was thought to be an endemic species from Taiwanese waters. However, this species has now been found in the Caribbean Sea at several sampling sites along with two other species, Diaphanoeca spiralifurca and Polyoeca dichotoma. By sequencing the SSU rDNA and other genes and by adding light and electron microscopical data to the description of these species, the taxonomy and phylogeny of Acanthoecida is being better understood.