Evidence of concurrent local adaptation and high phenotypic plasticity in a polar microeukaryote

Here we investigated whether there is evidence of local adaptation in strains of an ancestrally marine dinoflagellate to the lacustrine environment they now inhabit (optimal genotypes) and/or if they have evolved phenotypic plasticity (a range of phenotypes). Eleven strains of Polarella glacialis were isolated and cultured from three different environments: the polar seas, a hyposaline and a hypersaline Antarctic lake. Local adaptation was tested by comparing growth rates of lacustrine and marine strains at their own and reciprocal site conditions. To determine phenotypic plasticity, we measured the reaction norm for salinity. We found evidence of both, limited local adaptation and higher phenotypic plasticity in lacustrine strains when compared with marine ancestors. At extreme high salinities, local lake strains outperformed other strains, and at extreme low salinities, strains from the hyposaline lake outperformed all other strains. The data suggest that lake populations may have evolved higher phenotypic plasticity in the lake habitats compared with the sea, presumably due to the high temporal variability in salinity in the lacustrine systems. Moreover, the interval of salinity tolerance differed between strains from the hyposaline and hypersaline lakes, indicating local adaptation promoted by different salinity.     

Characterization of halotolerant Bicosoecida and Placididea (Stramenopila) that are distinct from marine forms,and the phylogenetic pattern of salinity preference in heterotrophic stramenopiles

Recent culture‐based studies demonstrate the distinctiveness of the microbial eukaryote biota of very hypersaline environments. In contrast, microscopy‐based faunistic studies suggest that the biota of habitats of more moderate hypersalinity (60–150‰) overlaps substantially with that of marine environments, but this has barely been studied with modern techniques. To investigate the diversity and salinity tolerance range of these organisms, eight cultures of heterotrophic stramenopiles were established from (or from nearby) moderately hypersaline locations. These isolates represent five independent groups; Groups A, B and C are bicosoecids; Groups D and E belong to Placididea. One isolate (Group A) is a strain of the widespread marine species Cafeteria roenbergensis, and cannot grow above 100‰ salinity. The other isolates – Groups B–E – can all grow at 150–175‰ salinities and are probably moderate halophiles. Groups B–E all represent previously unsequenced species or even genera, although Group B is the sister group of the borderline extreme halophile Halocafeteria. The high level of novelty en countered suggests that moderately hypersaline environments may harbour a heterotrophic stramenopile biota distinct from that of marine environments. Interestingly, our new isolates are all most closely related to marine or halophilic forms, and our phylogenies show large clades defined by saline/non‐saline habitats within bicosoecids, placidomonads and related lineages. In particular, most freshwater/soil bicosoecids form one well‐supported clade. The sole major exception is Bicosoeca, which is intermixed with marine environmental sequences originally referred to as ‘MAST‐13’, which are from brackish water, not typical seawater. It seems that the freshwater/marine barrier has been crossed very few times in the evolutionary history of these heterotrophic stramenopile flagellates.     

Halotolerant and halophilic fungi

Extreme environments have for long been considered to be populated almost exclusively by prokaryotic organisms and therefore monopolized by bacteriologists. Solar salterns are natural hypersaline environments characterized by extreme concentrations of NaCl, often high concentrations of other ions, high uv irradiation and in some cases extremes in pH. In 2000 fungi were first reported to be active inhabitants of solar salterns. Since then many new species and species previously known only as food contaminants have been discovered in hypersaline environments around the globe. The eukaryotic microorganism most studied for its salt tolerance is Saccharomyces cerevisiae. However, S. cerevisiae is rather salt sensitive and not able to adapt to hypersaline conditions. In contrast, some species like Debaryomyces hansenii, Hortaea werneckii, and Wallemia ichthyophaga have been isolated globally from natural hypersaline environments. We believe that all three are more suitable model organisms to study halotolerance in eukaryotes than S. cerevisiae. Furthermore, they belong to different and distant taxonomic groups and have developed different strategies to cope with the same problems of ion toxicity and loss of water.     

Prokaryotic and viral community structure in the singular chaotropic salt lake Salar de Uyuni

Salar de Uyuni (SdU) is the largest hypersaline salt flat and the highest lithium reservoir on Earth. In addition to extreme temperatures and high UV irradiance, SdU has high concentrations of chaotropic salts which can be important factors in controlling microbial diversity. Here, for the first time we characterize the viral diversity of this hypersaline environment during the two seasons, as well as the physicochemical characteristics and the prokaryotic communities of the analysed samples. Most of the selected samples showed a peculiar physicochemical composition and prokaryotic diversity, mostly different from each other even for samples from locations in close proximity or the same season. In contrast to most hypersaline systems Bacteria frequently outnumbered Archaea. Furthermore, an outstanding percentage of members of Salinibacter sp., likely a species different from the cosmopolitan Salinibacter ruber, was obtained in most of the samples. Viral communities displayed the morphologies normally found in hypersaline environments. Two seasonal samples were chosen for a detailed metagenomic analysis of the viral assemblage. Both viral communities shared common sequences but were dominated by sample-specific viruses, mirroring the differences also observed in physicochemical and prokaryotic community composition. These metaviromes were distinct from those detected in other hypersaline systems analysed to date.     

Experimental adaptation to marine conditions by a freshwater alga

The marine‐freshwater boundary has been suggested as one of the most difficult to cross for organisms. Salt is a major ecological factor and provides an unequalled range of ecological opportunity because marine habitats are much more extensive than freshwater habitats, and because salt strongly affects the structure of microbial communities. We exposed experimental populations of the freshwater alga Chlamydomonas reinhardtii to steadily increasing concentrations of salt. About 98% of the lines went extinct. The ones that survived now thrive in growth medium with 36 g?L^?1 NaCl, and in seawater. Our results indicate that adaptation to marine conditions proceeded first through genetic assimilation of an inducible response to relatively low salt concentrations that was present in the ancestors, and subsequently by the evolution of an enhanced inducible response to high salt concentrations. These changes appear to have evolved through reversible and irreversible modifications, respectively. The evolution of marine from freshwater lineages is an example that clearly indicates the possibility of studying certain aspects of major ecological transitions in the laboratory.     

Prokaryotic and viral community of the sulfate-rich crust from Peñahueca ephemeral lake,an astrobiology analogue

Peñahueca is an athalassohaline hypersaline inland ephemeral lake originated under semiarid conditions in the central Iberian Peninsula (Spain). Its chemical composition makes it extreme for microbial life as well as a terrestrial analogue of other planetary environments. To investigate the persistence of microbial life associated with sulfate-rich crusts, we applied cultivation-independent methods (optical and electron microscopy, 16S rRNA gene profiling and metagenomics) to describe the prokaryotic community and its associated viruses. The diversity for Bacteria was very low and was vastly dominated by endospore formers related to Pontibacillus marinus of the Firmicutes phylum. The archaeal assemblage was more diverse and included taxa related to those normally found in hypersaline environments. Several ‘metagenome assembled genomes’ were recovered, corresponding to new species of Pontibacillus, several species from the Halobacteria and one new member of the Nanohaloarchaeota. The viral assemblage, although composed of the morphotypes typical of high salt systems, showed little similarity to previously isolated/reconstructed halophages. Several putative prophages of Pontibacillus and haloarchaeal hosts were identified. Remarkably, the Peñahueca sulfate-rich metagenome contained CRISPR-associated proteins and repetitions which were over 10-fold higher than in most hypersaline systems analysed so far.     

Extremotolerance in fungi: evolution on the edge

Our planet offers many opportunities for life on the edge: high and low temperatures, high salt concentrations, acidic and basic conditions and toxic environments, to name but a few extremes. Recent studies have revealed the diversity of fungi that can occur in stressful environments that are hostile to most eukaryotes. We review these studies here, with the additional purpose of proposing some mechanisms that would allow for the evolutionary adaptation of eukaryotic microbial life under extreme conditions. We focus, in particular, on life in ice and life at high salt concentrations, as there is a surprising similarity between the fungal populations in these two kinds of environments, both of which are characterized by low water activity. We propose steps of evolution of generalist species towards the development of specialists in extreme habitats. We argue that traits present in some fungal groups, such as asexuality, synthesis of melanin-like pigments and a flexible morphology, are preadaptations that facilitate persistence and eventual adaptation to conditions on the ecological edge, as well as biotope switches. These processes are important for understanding the evolution of extremophiles; moreover, they have implications for the emergence of novel fungal pathogens.     

Ecology and molecular adaptations of the halophilic black yeast Hortaea werneckii

Molecular studies on halophilic adaptations have focused on prokaryotic microorganisms due to a lack of known appropriate eukaryotic halophilic microorganisms. However, the black yeast Hortaea werneckii has been identified as the dominant fungal species in hypersaline waters on three continents. It represents a new model organism for studying the mechanisms of salt tolerance in eukaryotes. Ultrastructural studies of the H. werneckii cell wall have shown that it synthesizes dihydroxynaphthalene (DHN) melanin under both saline and non-saline growth conditions. However, melanin granules in the cell walls are organized in a salt-dependent way, implying the potential osmoprotectant role of melanin. At the level of membrane structure, H. werneckii maintains a sterol-to-phospholipid ratio significantly lower than the salt-sensitive Saccharomyces cerevisiae. Accordingly, membranes of H. werneckii are more fluid over a wide range of NaCl concentrations, indicating high intrinsic salt stress tolerance. Even H. werneckii grown in high NaCl concentrations maintains very low intracellular amounts of potassium and sodium, demonstrating the sodium-excluder character of this organism. The salt-dependent expressions of two HwENA genes suggest roles for them in the adaptation to changing salt concentrations. The high similarity of these ENA ATPases to other fungal ENA ATPases involved in Na⁺/K⁺ transport indicates their potential importance in H. werneckii ion homeostasis. Glycerol is the main compatible solute which accumulates in the cytoplasm of H. werneckii at high salinity, although it seems that mycosporines may also act as supplementary compatible solutes. Salt dependent increase in glycerol synthesis is supported by the identification of two copies of a gene putatively coding for glycerol-3-phosphate-dehydrogenase. Expression of only one of these genes is salt dependent.     

Patterns of population structure and complex haplotype sharing among field isolates of the green alga Chlamydomonas reinhardtii

The nature of population structure in microbial eukaryotes has long been debated. Competing models have argued that microbial species are either ubiquitous, with high dispersal and low rates of speciation, or that for many species gene flow between populations is limited, resulting in evolutionary histories similar to those of macroorganisms. However, population genomic approaches have seldom been applied to this question. Here, we analyse whole‐genome resequencing data for all 36 confirmed field isolates of the green alga Chlamydomonas reinhardtii. At a continental scale, we report evidence for putative allopatric divergence, between both North American and Japanese isolates, and two highly differentiated lineages within N. America. Conversely, at a local scale within the most densely sampled lineage, we find little evidence for either spatial or temporal structure. Taken together with evidence for ongoing admixture between the two N. American lineages, this lack of structure supports a role for substantial dispersal in C. reinhardtii and implies that between‐lineage differentiation may be maintained by reproductive isolation and/or local adaptation. Our results therefore support a role for allopatric divergence in microbial eukaryotes, while also indicating that species may be ubiquitous at local scales. Despite the high genetic diversity observed within the most well‐sampled lineage, we find that pairs of isolates share on average ~9% of their genomes in long haplotypes, even when isolates were sampled decades apart and from different locations. This proportion is several orders of magnitude higher than the Wright–Fisher expectation, raising many further questions concerning the evolutionary genetics of C. reinhardtii and microbial eukaryotes generally.     

Protozoa populations are ecosystem engineers that shape prokaryotic community structure and function of the rumen microbial ecosystem

Unicellular eukaryotes are an integral part of many microbial ecosystems where they interact with their surrounding prokaryotic community—either as predators or as mutualists. Within the rumen, one of the most complex host-associated microbial habitats, ciliate protozoa represent the main micro-eukaryotes, accounting for up to 50% of the microbial biomass. Nonetheless, the extent of the ecological effect of protozoa on the microbial community and on the rumen metabolic output remains largely understudied. To assess the role of protozoa on the rumen ecosystem, we established an in-vitro system in which distinct protozoa sub-communities were introduced to the native rumen prokaryotic community. We show that the different protozoa communities exert a strong and differential impact on the composition of the prokaryotic community, as well as its function including methane production. Furthermore, the presence of protozoa increases prokaryotic diversity with a differential effect on specific bacterial populations such as Gammaproteobacteria, Prevotella and Treponema. Our results suggest that protozoa contribute to the maintenance of prokaryotic diversity in the rumen possibly by mitigating the effect of competitive exclusion between bacterial taxa. Our findings put forward the rumen protozoa populations as potentially important ecosystem engineers for future microbiome modulation strategies.Subject terms: Microbial ecology, Food webs     

ALGAL COMMUNITY DYNAMICS AND HALOTOLERANCE IN A TERRESTRIAL,HYPERSALINE ENVIRONMENT1

We studied the algal community of the Great Salt Plains (GSP), an expansive (65 km²) salt flat situated in north‐central Oklahoma, USA that has been designated as the Salt Plains Microbial Observatory (SPMO) by the National Science Foundation. The GSP offered a unique opportunity to study a terrestrial, hypersaline algal community that experiences wide‐ranging environmental conditions. We were able to show that ammonium‐N, rather than salinity, was the most important predictor of total algal biomass. However, salinity was found to be a significant controlling variable in diatom distribution at the GSP, where diatom abundance was negatively correlated with porewater salinity concentrations. Overall, chlorophytes (likely dominated by Dunaliella spp.) were the most abundant algal group at the consistently hypersaline (>300 ppt) south site. Diatom and cyanobacterial biomass were on average highest at the central site, which experienced greater fluctuations in salinity. While taxonomic diversity was limited to three algal groups (chlorophytes, diatoms, and cyanobacteria), the salinity preferences and halotolerance ranges of isolated strains were quite variable. Although porewater salinities at the GSP are commonly near saturation (>300 ppt), the large majority of isolates had halotolerance ranges below 150 ppt. This suggests that algae at the GSP rarely achieve maximum growth rates, and could only do so when intermittent rain events reduce salinity to optimal levels. Because the vast majority of our strains were isolated from salt‐saturated soil samples, maintaining viability (rather than growth efficiency) appears to be the most successful adaptation to the extreme conditions at the GSP.     

Paleoecology of Neoproterozoic hypersaline environments: Biomarker evidence for haloarchaea,methanogens, and cyanobacteria

While numerous studies have examined modern hypersaline ecosystems, their equivalents in the geologic past, particularly in the Precambrian, are poorly understood. In this study, biomarkers from ~820 million year (Ma)‐old evaporites from the Gillen Formation of the mid‐Neoproterozoic Bitter Springs Group, central Australia, are investigated to elucidate the antiquity and paleoecology of halophiles. The sediments were composed of alternating laminae of dolomitized microbial mats and up to 90% anhydrite. Solvent extraction of these samples yielded thermally well‐preserved hydrocarbon biomarkers. The regularly branched C₂₅ isoprenoid 2,6,10,14,18‐pentamethylicosane, the tail‐to‐tail linked C₃₀ isoprenoid squalane, and breakdown products of the head‐to‐head linked C₄₀ isoprenoid biphytane, were particularly abundant in the most anhydrite‐rich sediments and mark the oldest current evidence for halophilic archaea. Linear correlations between isoprenoid concentrations (normalized to n‐alkanes) and the anhydrite/dolomite ratio reveal microbial consortia that fluctuated with changing salinity levels. Halophilic archaea were the dominant organisms during periods of high salinity and gypsum precipitation, while bacteria were prevalent during stages of carbonate formation. The irregularly branched C₂₅ isoprenoid 2,6,10,15,19‐pentamethylicosane (PMI), with a central tail‐to‐tail link, was also abundant during periods of elevated salinity, highlighting the activity of methanogens. By contrast, the irregularly branched C₂₀ isoprenoid 2,6,11,15‐tetramethylhexadecane (crocetane) was more common in dolomite‐rich facies, revealing that an alternate group of archaea was active during less saline periods. Elevated concentrations of isotopically depleted heptadecane (n‐C₁₇) revealed the presence of cyanobacteria under all salinity regimes. The combination of biomarkers in the mid‐Neoproterozoic Gillen Formation resembles lipid compositions from modern hypersaline cyanobacterial mats, pointing to a community composition that remained broadly constant since at least the Neoproterozoic. However, as a major contrast to most modern hypersaline environments, the Gillen evaporites did not yield any evidence for algae or other eukaryotes.     

Rapid evolutionary adaptation to elevated salt concentrations in pathogenic freshwater bacteria Serratia marcescens

Rapid evolutionary adaptions to new and previously detrimental environmental conditions can increase the risk of invasion by novel pathogens. We tested this hypothesis with a 133‐day‐long evolutionary experiment studying the evolution of the pathogenic Serratia marcescens bacterium at salinity niche boundary and in fluctuating conditions. We found that S. marcescens evolved at harsh (80 g/L) and extreme (100 g/L) salt conditions had clearly improved salt tolerance than those evolved in the other three treatments (ancestral conditions, nonsaline conditions, and fluctuating salt conditions). Evolutionary theories suggest that fastest evolutionary changes could be observed in intermediate selection pressures. Therefore, we originally hypothesized that extreme conditions, such as our 100 g/L salinity treatment, could lead to slower adaptation due to low population sizes. However, no evolutionary differences were observed between populations evolved in harsh and extreme conditions. This suggests that in the study presented here, low population sizes did not prevent evolution in the long run. On the whole, the adaptive potential observed here could be important for the transition of pathogenic S. marcescens bacteria from human‐impacted freshwater environments, such as wastewater treatment plants, to marine habitats, where they are known to infect and kill corals (e.g., through white pox disease).     

Genomic characterization of three novel Desulfobacterota classes expand the metabolic and phylogenetic diversity of the phylum

We report on the genomic characterization of three novel classes in the phylum Desulfobacterota. One class (proposed name Candidatus ‘Anaeroferrophillalia’) was characterized by heterotrophic growth capacity, either fermentatively or utilizing polysulfide, tetrathionate or thiosulfate as electron acceptors. In the absence of organic carbon sources, autotrophic growth via the Wood–Ljungdahl (WL) pathway and using hydrogen or Fe(II) as an electron donor is also inferred for members of the ‘Anaeroferrophillalia’. The second class (proposed name Candidatus ‘Anaeropigmentia’) was characterized by its capacity for growth at low oxygen concentration, and the capacity to synthesize the methyl/alkyl carrier CoM, an ability that is prevalent in the archaeal but rare in the bacterial domain. Pigmentation is inferred from the capacity for carotenoid (lycopene) production. The third class (proposed name Candidatus ‘Zymogenia’) was characterized by fermentative heterotrophic growth capacity, broad substrate range and the adaptation of some of its members to hypersaline habitats. Analysis of the distribution pattern of all three classes showed their occurrence as rare community members in multiple habitats, with preferences for anaerobic terrestrial, freshwater and marine environments over oxygenated (e.g. pelagic ocean and agricultural land) settings. Special preference for some members of the class Candidatus ‘Zymogenia’ for hypersaline environments such as hypersaline microbial mats and lagoons was observed.     

The first metazoa living in permanently anoxic conditions

Background  

Several unicellular organisms (prokaryotes and protozoa) can live under permanently anoxic conditions. Although a few metazoans can survive temporarily in the absence of oxygen, it is believed that multi-cellular organisms cannot spend their entire life cycle without free oxygen. Deep seas include some of the most extreme ecosystems on Earth, such as the deep hypersaline anoxic basins of the Mediterranean Sea. These are permanently anoxic systems inhabited by a huge and partly unexplored microbial biodiversity.     

Halorhabdus tiamatea: proteogenomics and glycosidase activity measurements identify the first cultivated euryarchaeon from a deep‐sea anoxic brine lake as potential polysaccharide degrader

Euryarchaea from the genus Halorhabdus have been found in hypersaline habitats worldwide, yet are represented by only two isolates: Halorhabdus utahensis AX‐2^T from the shallow Great Salt Lake of Utah, and Halorhabdus tiamatea SARL4B^T from the Shaban deep‐sea hypersaline anoxic lake (DHAL) in the Red Sea. We sequenced the H. tiamatea genome to elucidate its niche adaptations. Among sequenced archaea, H. tiamatea features the highest number of glycoside hydrolases, the majority of which were expressed in proteome experiments. Annotations and glycosidase activity measurements suggested an adaptation towards recalcitrant algal and plant‐derived hemicelluloses. Glycosidase activities were higher at 2% than at 0% or 5% oxygen, supporting a preference for low‐oxygen conditions. Likewise, proteomics indicated quinone‐mediated electron transport at 2% oxygen, but a notable stress response at 5% oxygen. Halorhabdus tiamatea furthermore encodes proteins characteristic for thermophiles and light‐dependent enzymes (e.g. bacteriorhodopsin), suggesting that H. tiamatea evolution was mostly not governed by a cold, dark, anoxic deep‐sea habitat. Using enrichment and metagenomics, we could demonstrate presence of similar glycoside hydrolase‐rich Halorhabdus members in the Mediterranean DHAL Medee, which supports that Halorhabdus species can occupy a distinct niche as polysaccharide degraders in hypersaline environments.     

Salt to conserve: a review on the ecology and preservation of hypersaline ecosystems

When it comes to the investigation of key ecosystems in the world, we often omit salt from the ecological recipe. In fact, despite occupying almost half of the volume of inland waters and providing crucial services to humanity and nature, inland saline ecosystems are often overlooked in discussions regarding the preservation of global aquatic resources of our planet. As a result, our knowledge of the biological and geochemical dynamics shaping these environments remains incomplete and we are hesitant in framing effective protective strategies against the increasing natural and anthropogenic threats faced by such habitats. Hypersaline lakes, water bodies where the concentration of salt exceeds 35 g/l, occur mainly in arid and semiarid areas resulting from hydrological imbalances triggering the accumulation of salts over time. Often considered the ‘exotic siblings’ within the family of inland waters, these ecosystems host some of the most extremophile communities worldwide and provide essential habitats for waterbirds and many other organisms in already water-stressed regions. These systems are often highlighted as natural laboratories, ideal for addressing central ecological questions due to their relatively low complexity and simple food web structures. However, recent studies on the biogeochemical mechanisms framing hypersaline communities have challenged this archetype, arguing that newly discovered highly diverse communities are characterised by specific trophic interactions shaped by high levels of specialisation. The main goal of this review is to explore our current understanding of the ecological dynamics of hypersaline ecosystems by addressing four main research questions: (i) why are hypersaline lakes unique from a biological and geochemical perspective; (ii) which biota inhabit these ecosystems and how have they adapted to the high salt conditions; (iii) how do we protect biodiversity from increasing natural and anthropogenic threats; and (iv) which scientific tools will help us preserve hypersaline ecosystems in the future? First, we focus on the ecological characterisation of hypersaline ecosystems, illustrate hydrogeochemical dynamics regulating such environments, and outline key ecoregions supporting hypersaline systems across the globe. Second, we depict the diversity and functional aspects of key taxa found in hypersaline lakes, from microorganisms to plants, invertebrates, waterbirds and upper trophic levels. Next, we describe ecosystem services and discuss possible conservation guidelines. Finally, we outline how cutting-edge technologies can provide new insights into the study of hypersaline ecology. Overall, this review sheds further light onto these understudied ecosystems, largely unrecognised as important sources of unique biological and functional diversity. We provide perspectives for key future research avenues, and advocate that the conservation of hypersaline lakes should not be taken with ‘a grain of salt’.     

Communities of halotolerant micromycetes from the areas of natural salinity

The halophilic community of natural hypersaline soils has been isolated. Species of the genera Penicillium, Aspergillus, Cladosporium, as well as dark-colored yeast-like organisms, are most frequently isolated on selective media from all saline soil samples that we have studied. It has been shown that the community of micromycetes of hypersaline habitats is less dependent on geographical position than on the physicochemical parameters of habitat.     

A New Halophilic Heterolobosean Flagellate,Aurem hypersalina gen. n. et sp. n., Closely Related to the Pleurostomum‐Tulamoeba Clade: Implications for Adaptive Radiation of Halophilic Eukaryotes

Halophilic protozoa are independently scattered across the molecular phylogeny of eukaryotes; most of which are assigned to Heterolobosea. Here, we isolated a biflagellate from a hypersaline water of 342‰ salinity. This isolate shared several morphological features with typical halophilic heterolobosean flagellates. In addition, molecular phylogenetic trees of the 18S rRNA gene sequences clearly indicated flagellate is a heterolobosean species closely related to the halophilic Tulamoebidae. However, the flagellate was not accommodated to any described genus. Cells were ovoid‐shaped, and no amoebae were observed. The two unequal flagella beat heterodynamically. An ear‐like bulge at the margin of a cytostomal groove was observed. Flagellates could grow at 100–200‰ salinity, suggesting an obligately halophilic species. Currently, it appears that the new halophilic Aurem hypersalina forms a strong clade with Tulamoebidae, and is sister to the Tulamoebidae, indicating that this new clade is composed almost entirely of obligate halophilic taxa. Thus, A. hypersalina and the Tulamoebidae clade currently represent a unique adaptive radiation of halophilic eukaryotes.     

Characterization of Tulamoeba bucina n. sp., an Extremely Halotolerant Amoeboflagellate Heterolobosean Belonging to the Tulamoeba–Pleurostomum Clade (Tulamoebidae n. fam.)

Most protozoans that have been cultivated recently from high salinity waters appear to be obligate halophiles. Phylogenetic analyses indicate that these species mostly represent independent lineages. Here, we report the cultivation, morphological characterization, and phylogenetic analysis of two strains (XLG1 and HLM‐8) of a new extremely halotolerant heterolobosean amoeboflagellate. This species is closely related to the obligate halophiles Tulamoeba peronaphora and Pleurostomum flabellatum, and more specifically to the former. Like Tulamoeba, the new species has a monopodial limax amoeba stage, however, its cyst stage lacks an intrusive pore plug. The flagellate stage bears a combination of a planar spiral feeding apparatus and unequal heterodynamic flagella that discriminates it from described Pleurostomum species. Strain XLG1 grows at salinities from 35‰ to 225‰. This degree of halotolerance is uncommon in protozoa, as most species showing growth in seawater are unable to grow at 200‰ salinity. The unrelatedness of most halophilic protozoa suggested that independent colonization of the hypersaline environment is more common than speciation within it. However, this study supports the idea that the Tulamoeba–Pleurostomum clade underwent an adaptive radiation within the hypersaline environment. A new species Tulamoeba bucina n. sp. is described, with Tulamoebidae n. fam. proposed for the Tulamoeba–Pleurostomum clade.