A study on the diversity and ecology of choanoflagellates by integrative taxonomy

As a major component and link between trophic levels, protists fulfil a variety of different ecological functions in microbial food webs and are essential components in all ecosystems. In particular, one group of heterotrophic protists, the choanoflagellates play an important role in aquatic habitats, as they are efficient filter feeders, able to handle several food particles at the same time. As choanoflagellates resemble morphologically the choanocytes of sponges, they got already early attention regarding the evolution of multicellularity. The close relation could be proven by phylogenetic analyses, in which choanoflagellates cluster within the Opisthokonta as a sister group to the metazoans, making them the closest unicellular relatives to the animals. Classical taxonomical studies on this group revealed a high morphological diversity and later on phylogenetic analyses were able to depict the relationship among a variety of species resulting into two major orders, the Craspedida possessing only an organic covering like a theca or a glycocalyx and the Acanthoecida with an inorganic siliceous basket-like covering (lorica). But still, we face a high discrepancy between the number of morphologically described and sequenced species. In addition, investigations on ecological properties proofed to be essential to further extend our knowledge on choanoflagellates concerning evolutionary questions. Moreover, the significance of a comprehensive integrative taxonomy with accurate species descriptions is nowadays undeniable, as modern molecular surveys using high-throughput sequencing methods produce massive amounts of molecular information, which can only be analysed and interpreted with the scaffold of a verified reference database founded on taxonomical data. Within this study, several diverse habitats from freshwater to hypersaline environments were investigated to extend our current knowledge on the diversity and ecology of choanoflagellates. From freshwater and marine habitats, exemplified by studies from the River Rhine at Cologne and a transect of the Atlantic Ocean respectively, new craspedid choanoflagellate species were described based on detailed morphological (light and scanning electron microscopy) and molecular (SSU and LSU rDNA sequencing as well as transcriptomic) data. In addition, by an empirical survey, insights into salinity tolerances of several craspedid choanoflagellates were gained for a better understanding of their ecological dispersal potential, in particular the potential to cross the marine-freshwater boundary. Furthermore, an unexplored and hostile environment, the Atacama Desert in Northern Chile was investigated. This region is characterized by hypersaline water bodies, heavy metal contamination and extreme UV radiation. Studying these extreme environments resulted in the description of seven new craspedid choanoflagellates and ecological experiments revealed that these species were able to tolerate a broad range of varying salinities. Molecular data of the SSU and LSU rDNA pointed towards high genetic distances to their closest marine relatives and phylogenetic analyses underlined their isolation-driven speciation derived from biogeographical restrictions. Further multigene analyses based on protein level revealed high mutation rates of aquatic organisms from this area. The biological divergence estimates correlated with geological events, e.g. the formation of the salt flats, and help to understand colonization and radiation processes. The establishment of choanoflagellate cultures and their transcriptomes from this area will be the basis to get further insights into the adaptation strategies also from a molecular perspective. By studying one of the salt flats, a new lorica-bearing species, Enibas tolerabilis gen. et sp. nov. could be isolated and described. This species is euryoecious regarding salinity, able to survive freshwater and hypersaline conditions, a characteristic never observed before for acanthoecids. The newly established genus Enibas clustered within a separated sister clade to all previously identified nudiform reproducing species in the family of Acanthoecidae, which comprised so far only four genera. Using video microscopy, its nudiform lorica reproduction could be proven by life observation of the cell division. In a targeted resampling approach, a phylogenetic closely related amplicon from molecular surveys was identified and a morphological description to this prior deposited sequence provided. By including other environmental eukaryotic sequences, it became obvious that the species richness within this family was underestimated. To summarize, several choanoflagellate species belonging to the order of Craspedida and Acanthoecida from diverse aquatic habitats were isolated and described. In particular, the amount of comprehensive transcriptomes of choanoflagellates, which will be the basis for further studies on gene functionality, was doubled. The use and combination of different methodologies gave detailed species descriptions for a comprehensive integrative taxonomy which can elucidate the ecological and evolutionary role of choanoflagellates. The present thesis emphasizes the urgent need of taxonomy to generate reliable reference data for further molecular meta-analyses investigating the biodiversity of protists

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. (C) 2020 Elsevier GmbH. All rights reserved

Morphological and molecular reinvestigation of acanthoecid species I. - Enibas urnula (Thomsen, 1973) comb. nov. (=Stephanoeca urnula Thomsen, 1973)

In the course of the last decades, several acanthoecid choanoflagellates have been assigned to the tectiform reproducing genus Stephanoeca based on lorica morphological comparability. The recent description of a Stephanoeca-like nudiform genus, Enibas Schiwitza, Arndt and Nitsche, 2019, has emphasized the possibility that some species assigned to Stephanoeca (based solely on lorica morphological features) might in the end have to be relegated to other genera. Stephanoeca urnula Thomsen, 1973, shows a striking morphological resemblance to E. thessalia Schiwitza and Nitsche, 2020. Also, the inability to document from microscopy any bundles of supernumerary costal strips in the collar region of this species, makes it an obvious Stephanoeca outcast candidate. We have revisited the S. urnula type locality and established this species in crude culture. A detailed SEM morphological analysis of cultured specimens, and a molecular analysis of the SSU and LSU rRNA, has unveiled its undisputed affiliation to the nudiform genus Enibas. Here, we redescribe S. urnula Thomsen, 1973 as E. urnula (Thomsen, 1973) comb. nov. Based on microscopical examinations of species of Stephanoeca and Enibas we have been able to pinpoint anterior lorica chamber construction details, that in the future renders possible a morphological distinction between the two genera. (c) 2022 Elsevier GmbH. All reserved

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 choanofiagellates 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 Ascotan 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 Glade 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. (C) 2018 Elsevier GmbH. All rights reserved

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. (C) 2018 Elsevier GmbH. All rights reserved

Biogeographical distribution of Hartaetosiga strains (Choanoflagellatea, Craspedida, Salpingoecidae) including morphological and transcriptomic data from a transect across the Atlantic Ocean

The genus Hartaetosiga Carr, Richter and Nitsche, 2017 comprised up to now only three species, H. gracilis (Kent) Carr, Richter, Nitsche, 2017, H. balthica (Wylezich and Karpov) Carr, Richter and Nitsche, 2017 and H. minima (Wylezich and Karpov) Carr, Richter and Nitsche, 2017. Based on distinct molecular data these species were relocated from the strictly freshwater genus Codosiga (Ehrenberg) Butschli, 1878 to a new genus comprising brackish and marine species. During the cruise MSM82/2 across the Atlantic Ocean in 2019, surface water samples were taken from 15 stations along a transect ranging from 35 degrees S to 23 degrees N. We were able to isolate and cultivate 14 strains of the genus Hartaetosiga. Morphometric data showed no distinct morphological traits allowing for a species delineation, indicating a cryptic species complex within the genus. Based on cultivation, morphological data, and molecular analyses, we recorded H. gracilis for the first time from off-shelf waters of the Atlantic Ocean and could describe a new species, H. australis n. sp. This new species was recorded from sampling stations in the Southern Hemisphere only, which may indicate a potential biogeographic restriction likely caused by the Equatorial Counter Current (ECC), dividing the northern and southern surface waters

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 franscriptome data. In addition, we give extended redescriptions of S, amphoridium and S. angulosa, based on SSU and ESU 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 dales 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 choanollagellates is broadly underestimated. (C) 2020 Elsevier Gmbh. All rights reserved

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 Llamara, a hypersaline basin within the Central Depression of the Atacama Desert, reflects colonisation and divergence events which correspond to geological data of the paleohydrology. (C) 2021 Elsevier GmbH. All rights reserved

Morphological and molecular reinvestigation of acanthoecid species II. - Pseudostephanoeca paucicostata (Tong et al., 1998) gen. et comb. nov. (= Stephanoeca diplocostata var. paucicostata Throndsen, 1969) including also the description of Pseudostephanoeca quasicupula sp. nov. and Stephanoeca ellisfiordensis sp. nov.

The present study has revealed morphological differences of species assigned to the acanthoecid choanoflagellate genus Stephanoeca based on the relative position of transverse and longitudinal costae in the anterior lorica chamber. A detailed re-examination of published material of S. paucicostata Tong et al., 1998 combined with new data has shown that the species characteristic, a double transverse costa, is clearly located outside of the longitudinal costae excluding this taxon from an affiliation to Stephanoeca sensu stricto, confirmed also by molecular data of a clonal culture from Kos Islands, Greece. Two specimens of S. paucicostata with only a single, interiorly positioned transverse costa, namely from Marchant et al. (1987) and Nitsche et al. (2011) were erroneously assigned to this species. These facts have here led to the establishment of a new genus, Pseudostephanoeca with its type species P. paucicostata, and the description of S. ellisfiordensis for the two specimens mentioned above, corroborated by new material from Antarctica. In addition, two specimens previously assigned to S. cupula have been shown to share the core characteristic of Pseudostephanoeca with exteriorly positioned transverse elements. This has led (c) 2022 Elsevier GmbH. All rights reserved