BACKGROUND
environmental conditions create selective pressures driving the evolutionary process and creating, over long timescales, a plethora of new species, genera, families and orders  <cit> .
elucidating mechanisms and environmental factors generating and maintaining biodiversity is one of the major challenges in microbial ecology.
we examine evidence for environmental filtering of protistan plankton communities driven by environmental constraints in marine water columns with unique chemistries.
as model organisms we targeted the signatures of the ciliated protists  because this group was found in earlier studies to be a major component of the protistan community, representing 45% of the major taxonomic groups with high alpha diversity  <cit> .
other taxonomic groups with smaller proportions were dinoflagellates, fungi and radiolaria   <cit> .
as study sites, we chose four hypersaline anoxic deep-sea basins  located in the eastern mediterranean sea .
dhabs in the eastern mediterranean sea, located at depths of more than 3000 m below sea level, result from the dissolution of outcropping ancient subterranean salt deposits from the messinian salinity crisis .
due to the high densities of the brines , mixing of these water masses with overlying deep-sea water  is restricted, resulting in anoxic conditions in these brines.
an interface  separates the anoxic brine from the normoxic and normsaline deep-sea water.
due to the dissolution of different strata of the evaporites from the messinian salinity crisis, the hydrochemistries of the eastern mediterranean sea dhabs differ significantly.
for example, while salinity in some basins  ranges between  <dig> and 352 g l- <dig> , others exhibit a much lower salinity .
potassium ions range between  <dig> and 300 mmol l- <dig>  magnesium ions between  <dig> and 792 mmol l- <dig> sulfate between  <dig> and 323 mmol l- <dig>  sulfide between  <dig>  and 15 mmol l- <dig> <cit>  and methane between  <dig>  and  <dig>  mmol l- <dig> <cit> .
because of their unique hydrochemistries and physical separation for thousands of years, the dhabs may serve as island habitats and provide an ideal scenario to test the hypothesis that species sorting of planktonic ciliate communities results from environmental filtering through niche separation.
molecular diversity surveys of protists, employing domain-specific pcr primers for the amplification of taxonomic marker genes , clone library construction and sanger sequencing revealed, that ciliates are among the most diverse and abundant plankton taxa thriving in some of the eastern mediterranean dhabs  <cit> .
ciliates, through their grazing activities on bacteria, archaea and smaller eukaryotes are central players in the marine microbial loop  <cit>  and species composition of ciliates can serve as an indicator of environmental health  <cit> .
they have been used extensively as model organisms to develop and test ideas about microbial biodiversity and biogeography .
one major reason for this is that compared to amoeboid and flagellated organisms, they are morphologically diverse  <cit>  and there is a long history of their taxonomic and phylogenetic study .
the extensive foundation of knowledge on ciliate species and their inferred relationships facilitates data evaluation and hypothesis testing for studies that aim to explore ciliate biodiversity, evolution and biogeography.
none of the previous taxon samplings of ssu rrna signatures in initial dhab protistan diversity surveys reached saturation  <cit> , as is generally the case in cloning and sanger sequencing-based strategies  <cit> .
therefore, it was not possible to observe many patterns of diversity in previous studies of dhabs, nor was it possible to perform comparative statistical analyses of specific taxa in the dhabs.
in this study we applied a high-throughput next generation sequencing strategy  and a ciliate-specific primer set in order to recover a comprehensive dataset on this target group.
the resulting data from deep sequencing enabled us to address basic ecological questions.
our first hypothesis was that the distinct chemistries of the different basins would drive species sorting in planktonic ciliate communities in the brines and interfaces of each basin.
if this hypothesis is true, we would expect  that interface communities will differ decisively from brine communities  and  that ciliate communities in interfaces are more similar to each other than in the brines .
the brines of the different basins are isolated from one another due to the sharp density gradient that exists between these hypersaline basins and overlying mediterranean seawater.
in contrast, exchange may be possible between interface populations in different dhabs since some exchange is possible between seawater and the typically ca.
2 m-thick interfaces .
our second hypothesis was that ciliate community composition in the brines and interfaces of these four dhabs, separated by up to 500 km, would not be significantly affected by distance between basins.
if this hypothesis is true, we would expect no significant correlation between pairs of samples and geographic distance between the respective sampling sites, therefore, no isolation with distance.
RESULTS
data overview
in total, we obtained between  <dig>   and  <dig>   v4-amplicons .
after quality filtering of the data , between  <dig>   and  <dig>   ciliate v4-amplicons remained for further analyses .
the resulting number of ciliate otus called at 95% sequence similarity ranged between  <dig>  and  <dig> .
after normalization to the smallest dataset  the resulting number of ciliate otus ranged between  <dig>  and  <dig> .
sampling saturation curves are presented in additional file 1: figure s <dig>  the proportion of rare versus abundant ciliate taxa can be found in additional file 2: figure s <dig>  sequences have been deposited in the genbank short read archive .
ciliate diversity in the dhabs
hierarchical clustering of sampling sites based on bray-curtis distance  identified two clusters, one of which unites the brine ciliate communities of the basins tyro , thetis  and urania , and, distantly related to these brine communities, the ciliate community from tyro interface .
the parametric estimator ace predicted highest ciliate richness in tif .
tyro brine, thetis brine and urania brine shared most ciliate amplicons.
the shannon index  indicated the highest ciliate diversity in these three samples .
the second cluster included the interface ciliate communities from thetis , urania  and medee .
the medee brine  ciliate community was distinct from all other ciliate communities analyzed in this study.
the shannon diversity index of medee brine was the lowest of all communities analyzed , and also richness estimates were distinctively lower than for all other samples .
putative taxonomy of ciliate amplicons
the v4-amplicons analyzed in this study were related to a total of  <dig> identified ciliate genera and one unclassified ciliate taxon .
the unique character of the medee brine ciliate community can be inferred from figure 2b, which displays the taxonomy assigned to the ciliate amplicons obtained from each sampling site.
medee brine was dominated by amplicons , which were all related to the genus anoplophrya  as closest blast match in ncbis genbank nr database.
the sequence similarities of these amplicons to anoplophrya ranged between  <dig> and 89% .
the remaining  <dig> ciliate amplicons from medee brine were related to a few other taxon groups belonging predominantly to the peniculida , other astomatida , and pleuronematida .
thetis brine and tyro brine had a relatively similar ciliate community composition, both of which were dominated by amplicons that have strombidium as the closest blast match in the genbank nucleotide database .
other abundant taxon groups shared by these two samples were novistrombidium , and pseudotontonia .
while laboea accounted for 11% of all amplicons in thetis brine, this taxon group was absent in tyro brine.
a tintinnid ciliate taxon related to salpingella as closest database relative occured exclusively in tyro brine , but not in thetis .
the ciliate community composition in urania brine was dissimilar to the brines in tyro and thetis basins.
one striking quantitative difference was the high proportion of pseudotontonia-related amplicons  in urania brine.
however, while most of the relatively abundant taxon-groups were shared between these three brine samples , most qualitative differences between tyro, thetis and urania brines were attributed to taxon groups with lower abundances.
medee brine was distinct in its ciliate composition from other brines.
tyro interface stood out from the other interface samples.
the most significant difference was the occurrence of  <dig>  amplicons , with apocoleps  as the best blast match.
the proportion of amplicons in thetis, urania and medee interfaces related to this taxon was less than  <dig> %.
also the proportion of strombidium-like amplicons in tyro interface  was decisively higher compared to the other interfaces .
thetis interface and urania interface had a very similar taxon composition, dominated by amplicons most closely related to pleuronema  .
this taxon was also highly represented in medee interface .
the second most abundant taxon group in medee interface were clevelandellids, represented with 43%.
this taxon was underrepresented in the interfaces of other basins .
four taxa occured in all eight samples analyzed .
four taxa were exclusive to all interfaces .
interestingly, not a single taxon occured exclusively in all brines simultaneously.
however,  <dig> taxon groups were absent from interfaces but present in at least one of the brines.
the same number of taxon groups was absent from all brines but occured in at least one of the interfaces.
the majority of taxon groups had abundances accounting for less than 5% of all amplicons obtained within a sample.
relating community patterns to environmental variables
we used a canonical correspondence analysis  to extract environmental gradients from the observed partitioning of ciliate amplicon diversity.
in the resulting ordination diagram , environmental variables with arrows close to the canonical axes may explain a large proportion of the variation accounted for by this axis.
the longer the arrow, the more variation may be explained by this factor.
the best model in our cca explained  <dig> % of the total variation within the ciliate amplicon profiles with the first two axes  accounting for  <dig> % and the first two canonical axes explaining  <dig> % of the variation of the species-environment relation.
eigenvalues of axis  <dig> and axis  <dig> were similar .
while all interface samples  were at the left part  of axis  <dig>  all brine samples were distributed along its positive scale of values.
even though only sodium concentration was significantly correlated with the second axis  also oxygen concentration and salinity described the differential habitat preferences of the communities distributed along the second canonical axis.
thus, these factors can be identified as main explainable environmental selection factors for interface and brine ciliate community composition .
the ciliate communities in the dhab interfaces showed only small variation along the first axis, while brine samples spread across a wider range of this first axis, with medee brine and thetis brine defining the longest distance.
none of the ccas conducted found a meaningful correlation of this axis with any environmental variable that we have measured and tested explaining this first axis.
however, it must be a factor that only separates niches for the brine communities, but not for interface communities.
distance effect on dhab ciliate community profiles
distance dependence was low , and very little of the overall variability in ciliate community similarity was accounted for by the regression model .
a correlation between distance and community similarity was insignificant .
a permutation mantel test between the geographic distance and the bray curtis distance showed also a non-significant correlation .
fluorescent in situ hybridization and scanning electron microscopy
scanning electron microscopy performed on samples collected from urania halocline revealed abundant ciliates  present at a concentration of  <dig>   ×  <dig> cells l-1), all of which hosted bacterial epibionts approximately 2– <dig>  μm long that .
these results supported the decision to focus on ciliates only in this work.
sem was not performed on brine or interface samples from the other basins, however fish hybridizations with the general eukaryotic probe euk <dig> confirmed the presence of ciliates  in urania brine.
discussion
deep hypersaline anoxic basins  in the eastern mediterranean sea are ideally suited for testing the effect of historical contingencies on the evolution of protist communities.
the distance between individual basins is variable, and each basin is characterized by hydrochemical gradients , and slightly different origins, leading to differences in physicochemical factors of the brines and interfaces in each of the different basins.
due to the steep density gradients along the interfaces of these basins, there is little connectivity between basin brines and overlying seawater, and therefore, between basin brines.
first insights into the ciliate communities in the mesopelagic realm above the brine basins came from a sanger sequencing-based approach  <cit> .
because of the relatively small amount of data  it is not a reliable dataset for comparison to the high throughput sequencing data from this study.
however, the data from that preliminary study did indicate a significant community shift between the water column and the basin brines.
we assessed ciliate community structures in the interfaces and brines of several basins in order to determine the degree to which these environmental barriers and basin chemistries influenced the ciliate plankton.
the proportion of rare versus abundant ciliate taxa in this study  is comparable to previous findings reporting protistan communities with few abundant and many rare taxa  <cit> .
several molecular diversity surveys over different spatial scales ranging from centimeters to tens of thousands of kilometers have supported distance-decay relationships  for microbial organisms, including bacteria , archaea , fungi  and also protists .
even organisms with large population sizes and the potential to spread globally using spores, which were assumed to be cosmopolitan  <cit> , show significant non-random spatial distribution patterns  <cit> .
however, in our study of ciliate communities in these dhabs, a similar distance-decay relationship was not observed .
a potential explanation could be that the small number of compared locations may have masked true patterns.
alternatively, the presence of a metacommunity  <cit>  within the mediterranean sea could cause the absence of a significant heterogeneous distribution  <cit> .
in limnic systems geographic distance has been found to influence asymmetric latitudinal genus richness patterns between 42° s and the pole  <cit> .
however, this seems to be a fundamental difference between marine and “terrestrial”  systems.
furthermore, on a global scale, historical factors were significantly more responsible for the geographic patterns in community composition of diatoms than environmental conditions  <cit> .
in other marine studies ciliates showed variations in taxonomic composition between closely related samples, which were explained by environmental factors rather than distance  <cit> .
similarly, in our study geographic distance could not explain the variations observed between the ciliate communities.
instead, hydrochemistry explained some of the variation in observed ciliate community patterns, and there was a strong separation of halocline interface and brine communities .
the dhab interfaces are characterized by extremely steep physicochemical gradients on a small spatial scale typically less than a couple of meters .
the concentrations of salt and oxygen are the most prominent environmental factors that change dramatically along the interfaces into the brines.
in a recent metadata-analysis of environmental sequence data, these two factors were identified as strong selection factors for ciliates  <cit> .
also for bacterial communities, salt concentration emerged as the strongest factor influencing global distribution  <cit> .
likewise, the bacterioplankton community composition in coastal antarctic lakes was weakly related with geographical distance, but strongly correlated with salinity  <cit> .
accordingly, logares et al.
assumed that a long-term salinity change ‘promoted the diversification of bacterioplankton communities by modifying the composition of ancestral communities and by allowing the establishment of new taxa’  <cit> .
salinity shifts characterize a boundary which is one of the most difficult barriers to cross for organisms in all three domains of life  <cit> .
while mechanisms to cope with high salt concentrations are relatively well studied in prokaryotes, they are still largely unknown in protists .
while there is evidence that many protists have narrow ranges of salt tolerance  <cit> , some taxa are known to occur under a wide range of salinities, from freshwater to hypersaline  <cit> .
one example is the ciliate cyclidium glaucoma <cit> , which may explain the occurrence of some of the same phylotypes in haloclines and brines of specific dhabs.
other examples are likely to exist.
in contrast, adaptations to anoxia in ciliates are well known.
ciliates are one of the most successful eukaryotic taxon groups in hypoxic and anoxic habitats.
in their long evolutionary history, they have acquired several strategies that allow for an anaerobic lifestyle, including hydrogenosomes  <cit> , anaerobic mitochondria  <cit> , and/or symbiotic networks  <cit> .
the high taxonomic diversity of anaerobe ciliates includes taxa such as nyctotherus, loxodes, pleuronema, strombidium, trimyema, cyclidium and metopus, some of which were also detected in our genetic diversity survey.
electron microscopy and fluorescence in situ hybridization assays provide unbiased evidence that the genetic signatures we detected in our rrna-targeted gene survey can be assigned to ciliates living in the dhabs rather than reflecting ancient nucleic acids.
.
taking advantage of phylotypes that we detected exclusively in specific habitats and phylotypes that can be found in several habitats with distinct hydrochemical characteristics, we may assume that the latter have a character of more generalist taxa compared to the more locally restricted phylotypes.
the total number of observed taxon groups is  <dig> distributed over eight different datasets  .
in those eight samples there are  <dig> generalist taxonomic groups that appeared simultaneously in at least six of the datasets.
only four taxonomic groups appeared in all of the eight datasets.
specialists, i.e.
taxa that are restricted to a single unique habitat account for  <dig> different taxonomic groups.
this results in a specialist/generalist ratio of  <dig>  to  <dig>  indicating a high specialization of taxa in the habitats under study.
however, there is a limitation to infer the autecology of specific evolutionary lineages based on sequence data and microscopy evidence  <cit> .
we do not make any attempt to explain the presence or absence of specific phylotypes in individual samples, and we instead focus only on community level ciliate diversity.
hydrochemical gradients have been identified as environmental barriers in previous protistan diversity surveys including the cariaco basin  <cit> , framvaren fjord  <cit> , mariager fjord  <cit> , baltic sea  <cit>  and the black sea  <cit>  and also stratified lakes  <cit> .
therefore, the clear distinction of halocline ciliate communities from brine communities is not an unexpected result.
however, it is surprising that the environmental variables we measured had a minor contribution to differences among the individual brine ciliate communities.
in the cca analyses  the different brine communities were spread out along the y-axis.
this axis, however, does not represent an environmental gradient.
this is surprising, considering that different types of salts may have different physiological effects  <cit>  and therefore, should require different adaptation strategies in halophiles.
basically, we can assume two scenarios: first, for isolated evolution as described in  <cit> , the scenario starts with a seed taxon.
after physical separation of the original habitat into two habitats neutral mutations are changing the seed taxon in these habitats independently.
these neutral mutations are of minor nature considering the time scale of the basins’ geological histories.
from this event we would expect similar taxon groups with only minor genetic changes in both habitats.
as mentioned above, each eighth taxon recorded in our study  falls into this category.
in the second scenario  we have the same ‘seed bank’ community for different basins.
through environmental filtering  some taxa may go extinct, others have the genomic potential to adapt to some specific hydrochemistries, while others are genomically equipped for adaptation to other environmental conditions.
in this case we would find taxa differing on higher taxonomic  hierarchies.
this is the case for  <dig> of  <dig> detected taxon groups .
we cannot rule out all environmental factors from causing differences between the ciliate communities because we did not measure all possible environmental factors, but only the hydrogeochemical factors that account for the most pronounced and obvious differences.
this suggests that  other hydrochemical variables we did not measure are leading to this separation, or  that biotic interactions may explain some of the differences between brine ciliate communities.
even though interactions of top-down and bottom-up factors in shaping community structures of aquatic microbes are still poorly understood  <cit>  some well known biotic interactions could be considered.
such biotic interactions may be, for example, parasitic relationships between organisms like amoeboid parasitic forms that can shape the composition of cyanobacterial species in lakes .
furthermore, symbiotic relationships include ciliates, associated with epibiontic prokaryotes, were found to be the dominant eukaryotic morphotypes in the haloclines of dhabs in the mediterranean sea  <cit> .
biotic interaction between protists and viruses are also known and have been shown  <cit> .
viruses specifically infect protists, e.g.
the coccolithovirus and it’s host, the calicifying haptophyte emiliania huxleyi <cit> .
additionally, viruses can also have an an indirect influence on protists by infecting the bacteria on which the protistan grazers feed or protistan grazers can even feed directly on viruses even though the carbon transfer to the higher trophic level is of minor importance  <cit> .
furthermore, different bacterioplankton communities can produce a bottom-up control on grazing protists.
namely, the growth efficiency of protists can relate strongly to the available bacterial prey  <cit> .
this is highly likely because differences in bacterial community composition in dhabs have been shown before  <cit> .
that leads to the assumption that different bacterial communities support different phagotrophic protists that show strong preferences for particular prey species  <cit>  or morphotypes  <cit> .
other possible explanations are founder effects, which describe a genetic deviation of an isolated population or founder population  compared to the original population based on a low number of alleles within the founders individuals  <cit> , random effects or genetic drift is the change in the frequency of a gene in a population due to random sampling  <cit>  and random extinctions that describe when a gene causes its carriers to have a deviating fitness from unity, its frequency will be determined by selection  <cit>  in different basins.
for protists in particular there is no literature available on this topic to our knowledge.
at last, the monoplization hypothesis by de meester et al.
<cit>  could be relevant to protist biogeography stating that a fast population growth and local adaptation and colonization of a new habitat result in the monopolization of resources, which yields a strong priority effect.
the effect is even enhanced when a locally adapted population can provide a ‘large resting propagule bank’ as a strong buffer against new genotypes invading.
this holds true especially for species that reproduce asexually and form resting stages.
even though mass effect and dispersal  <cit>  cannot be ruled out, these are unlikely alternatives to explain the observed community patterns.
the habitats of the water column above the dhabs represent a potential source habitat with ‘high quality’.
in comparison, the narrow interphase and the brine show ‘low quality’ conditions because these habitats harbor high gradients of change, anoxia, high salt concentration up to saturation and therefore require a high degree of physiological adaptation for microbial colonization.
chances for highly specialized organisms to cross environmental barriers outside their habitat and to disperse beyond their specific habitat are very low.
evidence for this hypothesis comes, for example, from the very rare and infrequent dispersal of protists between marine and freshwater habitats  <cit> .
one possibility may be the dispersal of spores and/or cysts , however, our knowledge about the number of ciliates that can form such resting stages in nature is very limited  <cit> .
furthermore, physical mechanisms of transport for resting stages between different basins are difficult to imagine, considering the lack of fluid flow, high density, and lack of animal vectors in the brines.
in contrast, this scenario may be more plausible for cysts/spores in halocline/interphase habitats.
physical transport of resting stages between haloclines at different basin sites could explain the observed similarities in ciliate interphase communities .
the deep basins in the eastern mediterranean sea may have recruited their protistan seed communities from atlantic sea water during the zenclean flood , when the strait of gibraltar opened permanently and refilled the mostly dried out mediterranean sea  <cit> .
subsequently, due to the dissolution of evaporites and the rise of anoxia in deep basins the water masses became physically separated from each other.
anoxia and hydrochemistry likely exerted an increased pressure on the original protistan communities.
species sorting may have been driven through environmental filtering  <cit> .
this is a predictable and fundamental process of community assembly  <cit> , that allows only those taxa with the genomic and physiological potential to cope with each specific set of environmental conditions.
this has been evidenced for recent ciliate communities  <cit> .
the normsaline and normoxic deep-sea water separating the different hypersaline anoxic basins from each other then became an environmental barrier for most protists , with the consequence that genetic exchange among the different brines was no longer likely.
changes in the ssu are presumably neutral, therefore, these changes would be due to random mutations.
however, it is reasonable to assume that changes in the ssu rdna are occurring in congruency with whole genome changes and not independent of evolutionary genome processes.
evolution over geological time may have resulted in significantly different ciliate communities in the brines.
divergence of species occurring in isolation through adaptive shifts that occurs in common seed species populations has been demonstrated for a number of taxa, including several macro- and microinvertebrates using molecular as well as taxonomic studies  <cit> .
based on our data, it is not unreasonable to assume that protists are also subjected to such evolutionary processes.
our study strongly suggests that evolutionary time scales combined with physical and hydrochemical isolation can explain, in part, the observed evolutionary differences in the ciliate communities in the different dhabs studied here.
CONCLUSIONS
the data presented here suggests that ancient isolated habitats, like deep-sea brine lakes, which also occur in the red sea  <cit>  and the gulf of mexico  <cit>  or habitats like aquifers and cave systems will represent ‘hot spots’ for the discovery of as-of-yet unstudied, possibly highly divergent, and endemic microbial eukaryotes.
financial competing interests
in the past five years we did not receive reimbursements, fees, funding, or salary from an organization that may in any way gain or lose financially from the publication of this manuscript, either now or in the future.
we do not hold any stocks or shares in an organization that may in any way gain or lose financially from the publication of this manuscript, either now or in the future.
we neither hold nor apply for any patents relating to the content of the manuscript.
we did not receive reimbursements, fees, funding, or salary from an organization that holds or has applied for patents relating to the content of the manuscript.
we, the authors, do not have any other financial competing interests.
non-financial competing interests
there are no non-financial competing interests  to declare in relation to this manuscript.

 environmental conditions create selective pressures driving the evolutionary process and creating, over long timescales, a plethora of new species, genera, families and orders. elucidating mechanisms and environmental factors generating and maintaining biodiversity is one of the major challenges in microbial ecology. we examine evidence for environmental filtering of protistan plankton communities driven by environmental constraints in marine water columns with unique chemistries. as model organisms we targeted the signatures of the ciliated protists because this group was found in earlier studies to be a major component of the protistan community, representing 45% of the major taxonomic groups with high alpha diversity. other taxonomic groups with smaller proportions were dinoflagellates, fungi and radiolaria. as study sites, we chose four hypersaline anoxic deep-sea basins located in the eastern mediterranean sea. dhabs in the eastern mediterranean sea, located at depths of more than 3000 m below sea level, result from the dissolution of outcropping ancient subterranean salt deposits from the messinian salinity crisis. due to the high densities of the brines, mixing of these water masses with overlying deep-sea water is restricted, resulting in anoxic conditions in these brines. an interface separates the anoxic brine from the normoxic and normsaline deep-sea water. due to the dissolution of different strata of the evaporites from the messinian salinity crisis, the hydrochemistries of the eastern mediterranean sea dhabs differ significantly. for example, while salinity in some basins ranges between and 352 g l-, others exhibit a much lower salinity. potassium ions range between and 300 mmol l- magnesium ions between and 792 mmol l- sulfate between and 323 mmol l- sulfide between and 15 mmol l- and methane between and  mmol l-. because of their unique hydrochemistries and physical separation for thousands of years, the dhabs may serve as island habitats and provide an ideal scenario to test the hypothesis that species sorting of planktonic ciliate communities results from environmental filtering through niche separation. molecular diversity surveys of protists, employing domain-specific pcr primers for the amplification of taxonomic marker genes, clone library construction and sanger sequencing revealed, that ciliates are among the most diverse and abundant plankton taxa thriving in some of the eastern mediterranean dhabs. ciliates, through their grazing activities on bacteria, archaea and smaller eukaryotes are central players in the marine microbial loop and species composition of ciliates can serve as an indicator of environmental health. they have been used extensively as model organisms to develop and test ideas about microbial biodiversity and biogeography. one major reason for this is that compared to amoeboid and flagellated organisms, they are morphologically diverse and there is a long history of their taxonomic and phylogenetic study. the extensive foundation of knowledge on ciliate species and their inferred relationships facilitates data evaluation and hypothesis testing for studies that aim to explore ciliate biodiversity, evolution and biogeography. none of the previous taxon samplings of ssu rrna signatures in initial dhab protistan diversity surveys reached saturation, as is generally the case in cloning and sanger sequencing-based strategies. therefore, it was not possible to observe many patterns of diversity in previous studies of dhabs, nor was it possible to perform comparative statistical analyses of specific taxa in the dhabs. in this study we applied a high-throughput next generation sequencing strategy and a ciliate-specific primer set in order to recover a comprehensive dataset on this target group. the resulting data from deep sequencing enabled us to address basic ecological questions. our first hypothesis was that the distinct chemistries of the different basins would drive species sorting in planktonic ciliate communities in the brines and interfaces of each basin. if this hypothesis is true, we would expect that interface communities will differ decisively from brine communities and that ciliate communities in interfaces are more similar to each other than in the brines. the brines of the different basins are isolated from one another due to the sharp density gradient that exists between these hypersaline basins and overlying mediterranean seawater. in contrast, exchange may be possible between interface populations in different dhabs since some exchange is possible between seawater and the typically ca. 2 m-thick interfaces. our second hypothesis was that ciliate community composition in the brines and interfaces of these four dhabs, separated by up to 500 km, would not be significantly affected by distance between basins. if this hypothesis is true, we would expect no significant correlation between pairs of samples and geographic distance between the respective sampling sites, therefore, no isolation with distance.  data overview in total, we obtained between and v4-amplicons. after quality filtering of the data, between and ciliate v4-amplicons remained for further analyses. the resulting number of ciliate otus called at 95% sequence similarity ranged between and. after normalization to the smallest dataset the resulting number of ciliate otus ranged between and. sampling saturation curves are presented in additional file 1: figure s the proportion of rare versus abundant ciliate taxa can be found in additional file 2: figure s sequences have been deposited in the genbank short read archive. ciliate diversity in the dhabs hierarchical clustering of sampling sites based on bray-curtis distance identified two clusters, one of which unites the brine ciliate communities of the basins tyro, thetis and urania, and, distantly related to these brine communities, the ciliate community from tyro interface. the parametric estimator ace predicted highest ciliate richness in tif. tyro brine, thetis brine and urania brine shared most ciliate amplicons. the shannon index indicated the highest ciliate diversity in these three samples. the second cluster included the interface ciliate communities from thetis, urania and medee. the medee brine ciliate community was distinct from all other ciliate communities analyzed in this study. the shannon diversity index of medee brine was the lowest of all communities analyzed, and also richness estimates were distinctively lower than for all other samples. putative taxonomy of ciliate amplicons the v4-amplicons analyzed in this study were related to a total of identified ciliate genera and one unclassified ciliate taxon. the unique character of the medee brine ciliate community can be inferred from figure 2b, which displays the taxonomy assigned to the ciliate amplicons obtained from each sampling site. medee brine was dominated by amplicons, which were all related to the genus anoplophrya as closest blast match in ncbis genbank nr database. the sequence similarities of these amplicons to anoplophrya ranged between and 89%. the remaining ciliate amplicons from medee brine were related to a few other taxon groups belonging predominantly to the peniculida, other astomatida, and pleuronematida. thetis brine and tyro brine had a relatively similar ciliate community composition, both of which were dominated by amplicons that have strombidium as the closest blast match in the genbank nucleotide database. other abundant taxon groups shared by these two samples were novistrombidium, and pseudotontonia. while laboea accounted for 11% of all amplicons in thetis brine, this taxon group was absent in tyro brine. a tintinnid ciliate taxon related to salpingella as closest database relative occured exclusively in tyro brine, but not in thetis. the ciliate community composition in urania brine was dissimilar to the brines in tyro and thetis basins. one striking quantitative difference was the high proportion of pseudotontonia-related amplicons in urania brine. however, while most of the relatively abundant taxon-groups were shared between these three brine samples, most qualitative differences between tyro, thetis and urania brines were attributed to taxon groups with lower abundances. medee brine was distinct in its ciliate composition from other brines. tyro interface stood out from the other interface samples. the most significant difference was the occurrence of amplicons, with apocoleps as the best blast match. the proportion of amplicons in thetis, urania and medee interfaces related to this taxon was less than %. also the proportion of strombidium-like amplicons in tyro interface was decisively higher compared to the other interfaces. thetis interface and urania interface had a very similar taxon composition, dominated by amplicons most closely related to pleuronema. this taxon was also highly represented in medee interface. the second most abundant taxon group in medee interface were clevelandellids, represented with 43%. this taxon was underrepresented in the interfaces of other basins. four taxa occured in all eight samples analyzed. four taxa were exclusive to all interfaces. interestingly, not a single taxon occured exclusively in all brines simultaneously. however, taxon groups were absent from interfaces but present in at least one of the brines. the same number of taxon groups was absent from all brines but occured in at least one of the interfaces. the majority of taxon groups had abundances accounting for less than 5% of all amplicons obtained within a sample. relating community patterns to environmental variables we used a canonical correspondence analysis to extract environmental gradients from the observed partitioning of ciliate amplicon diversity. in the resulting ordination diagram, environmental variables with arrows close to the canonical axes may explain a large proportion of the variation accounted for by this axis. the longer the arrow, the more variation may be explained by this factor. the best model in our cca explained % of the total variation within the ciliate amplicon profiles with the first two axes accounting for % and the first two canonical axes explaining % of the variation of the species-environment relation. eigenvalues of axis and axis were similar. while all interface samples were at the left part of axis all brine samples were distributed along its positive scale of values. even though only sodium concentration was significantly correlated with the second axis also oxygen concentration and salinity described the differential habitat preferences of the communities distributed along the second canonical axis. thus, these factors can be identified as main explainable environmental selection factors for interface and brine ciliate community composition. the ciliate communities in the dhab interfaces showed only small variation along the first axis, while brine samples spread across a wider range of this first axis, with medee brine and thetis brine defining the longest distance. none of the ccas conducted found a meaningful correlation of this axis with any environmental variable that we have measured and tested explaining this first axis. however, it must be a factor that only separates niches for the brine communities, but not for interface communities. distance effect on dhab ciliate community profiles distance dependence was low, and very little of the overall variability in ciliate community similarity was accounted for by the regression model. a correlation between distance and community similarity was insignificant. a permutation mantel test between the geographic distance and the bray curtis distance showed also a non-significant correlation. fluorescent in situ hybridization and scanning electron microscopy scanning electron microscopy performed on samples collected from urania halocline revealed abundant ciliates present at a concentration of  ×  cells l-1), all of which hosted bacterial epibionts approximately 2–  μm long that. these results supported the decision to focus on ciliates only in this work. sem was not performed on brine or interface samples from the other basins, however fish hybridizations with the general eukaryotic probe euk confirmed the presence of ciliates in urania brine. discussion deep hypersaline anoxic basins in the eastern mediterranean sea are ideally suited for testing the effect of historical contingencies on the evolution of protist communities. the distance between individual basins is variable, and each basin is characterized by hydrochemical gradients, and slightly different origins, leading to differences in physicochemical factors of the brines and interfaces in each of the different basins. due to the steep density gradients along the interfaces of these basins, there is little connectivity between basin brines and overlying seawater, and therefore, between basin brines. first insights into the ciliate communities in the mesopelagic realm above the brine basins came from a sanger sequencing-based approach. because of the relatively small amount of data it is not a reliable dataset for comparison to the high throughput sequencing data from this study. however, the data from that preliminary study did indicate a significant community shift between the water column and the basin brines. we assessed ciliate community structures in the interfaces and brines of several basins in order to determine the degree to which these environmental barriers and basin chemistries influenced the ciliate plankton. the proportion of rare versus abundant ciliate taxa in this study is comparable to previous findings reporting protistan communities with few abundant and many rare taxa. several molecular diversity surveys over different spatial scales ranging from centimeters to tens of thousands of kilometers have supported distance-decay relationships for microbial organisms, including bacteria, archaea, fungi and also protists. even organisms with large population sizes and the potential to spread globally using spores, which were assumed to be cosmopolitan, show significant non-random spatial distribution patterns. however, in our study of ciliate communities in these dhabs, a similar distance-decay relationship was not observed. a potential explanation could be that the small number of compared locations may have masked true patterns. alternatively, the presence of a metacommunity within the mediterranean sea could cause the absence of a significant heterogeneous distribution. in limnic systems geographic distance has been found to influence asymmetric latitudinal genus richness patterns between 42° s and the pole. however, this seems to be a fundamental difference between marine and “terrestrial” systems. furthermore, on a global scale, historical factors were significantly more responsible for the geographic patterns in community composition of diatoms than environmental conditions. in other marine studies ciliates showed variations in taxonomic composition between closely related samples, which were explained by environmental factors rather than distance. similarly, in our study geographic distance could not explain the variations observed between the ciliate communities. instead, hydrochemistry explained some of the variation in observed ciliate community patterns, and there was a strong separation of halocline interface and brine communities. the dhab interfaces are characterized by extremely steep physicochemical gradients on a small spatial scale typically less than a couple of meters. the concentrations of salt and oxygen are the most prominent environmental factors that change dramatically along the interfaces into the brines. in a recent metadata-analysis of environmental sequence data, these two factors were identified as strong selection factors for ciliates. also for bacterial communities, salt concentration emerged as the strongest factor influencing global distribution. likewise, the bacterioplankton community composition in coastal antarctic lakes was weakly related with geographical distance, but strongly correlated with salinity. accordingly, logares et al. assumed that a long-term salinity change ‘promoted the diversification of bacterioplankton communities by modifying the composition of ancestral communities and by allowing the establishment of new taxa’. salinity shifts characterize a boundary which is one of the most difficult barriers to cross for organisms in all three domains of life. while mechanisms to cope with high salt concentrations are relatively well studied in prokaryotes, they are still largely unknown in protists. while there is evidence that many protists have narrow ranges of salt tolerance, some taxa are known to occur under a wide range of salinities, from freshwater to hypersaline. one example is the ciliate cyclidium glaucoma, which may explain the occurrence of some of the same phylotypes in haloclines and brines of specific dhabs. other examples are likely to exist. in contrast, adaptations to anoxia in ciliates are well known. ciliates are one of the most successful eukaryotic taxon groups in hypoxic and anoxic habitats. in their long evolutionary history, they have acquired several strategies that allow for an anaerobic lifestyle, including hydrogenosomes, anaerobic mitochondria, and/or symbiotic networks. the high taxonomic diversity of anaerobe ciliates includes taxa such as nyctotherus, loxodes, pleuronema, strombidium, trimyema, cyclidium and metopus, some of which were also detected in our genetic diversity survey. electron microscopy and fluorescence in situ hybridization assays provide unbiased evidence that the genetic signatures we detected in our rrna-targeted gene survey can be assigned to ciliates living in the dhabs rather than reflecting ancient nucleic acids.. taking advantage of phylotypes that we detected exclusively in specific habitats and phylotypes that can be found in several habitats with distinct hydrochemical characteristics, we may assume that the latter have a character of more generalist taxa compared to the more locally restricted phylotypes. the total number of observed taxon groups is distributed over eight different datasets. in those eight samples there are generalist taxonomic groups that appeared simultaneously in at least six of the datasets. only four taxonomic groups appeared in all of the eight datasets. specialists, i.e. taxa that are restricted to a single unique habitat account for different taxonomic groups. this results in a specialist/generalist ratio of to indicating a high specialization of taxa in the habitats under study. however, there is a limitation to infer the autecology of specific evolutionary lineages based on sequence data and microscopy evidence. we do not make any attempt to explain the presence or absence of specific phylotypes in individual samples, and we instead focus only on community level ciliate diversity. hydrochemical gradients have been identified as environmental barriers in previous protistan diversity surveys including the cariaco basin, framvaren fjord, mariager fjord, baltic sea and the black sea and also stratified lakes. therefore, the clear distinction of halocline ciliate communities from brine communities is not an unexpected result. however, it is surprising that the environmental variables we measured had a minor contribution to differences among the individual brine ciliate communities. in the cca analyses the different brine communities were spread out along the y-axis. this axis, however, does not represent an environmental gradient. this is surprising, considering that different types of salts may have different physiological effects and therefore, should require different adaptation strategies in halophiles. basically, we can assume two scenarios: first, for isolated evolution as described in, the scenario starts with a seed taxon. after physical separation of the original habitat into two habitats neutral mutations are changing the seed taxon in these habitats independently. these neutral mutations are of minor nature considering the time scale of the basins’ geological histories. from this event we would expect similar taxon groups with only minor genetic changes in both habitats. as mentioned above, each eighth taxon recorded in our study falls into this category. in the second scenario we have the same ‘seed bank’ community for different basins. through environmental filtering some taxa may go extinct, others have the genomic potential to adapt to some specific hydrochemistries, while others are genomically equipped for adaptation to other environmental conditions. in this case we would find taxa differing on higher taxonomic hierarchies. this is the case for of detected taxon groups. we cannot rule out all environmental factors from causing differences between the ciliate communities because we did not measure all possible environmental factors, but only the hydrogeochemical factors that account for the most pronounced and obvious differences. this suggests that other hydrochemical variables we did not measure are leading to this separation, or that biotic interactions may explain some of the differences between brine ciliate communities. even though interactions of top-down and bottom-up factors in shaping community structures of aquatic microbes are still poorly understood some well known biotic interactions could be considered. such biotic interactions may be, for example, parasitic relationships between organisms like amoeboid parasitic forms that can shape the composition of cyanobacterial species in lakes. furthermore, symbiotic relationships include ciliates, associated with epibiontic prokaryotes, were found to be the dominant eukaryotic morphotypes in the haloclines of dhabs in the mediterranean sea. biotic interaction between protists and viruses are also known and have been shown. viruses specifically infect protists, e.g. the coccolithovirus and it’s host, the calicifying haptophyte emiliania huxleyi. additionally, viruses can also have an an indirect influence on protists by infecting the bacteria on which the protistan grazers feed or protistan grazers can even feed directly on viruses even though the carbon transfer to the higher trophic level is of minor importance. furthermore, different bacterioplankton communities can produce a bottom-up control on grazing protists. namely, the growth efficiency of protists can relate strongly to the available bacterial prey. this is highly likely because differences in bacterial community composition in dhabs have been shown before. that leads to the assumption that different bacterial communities support different phagotrophic protists that show strong preferences for particular prey species or morphotypes. other possible explanations are founder effects, which describe a genetic deviation of an isolated population or founder population compared to the original population based on a low number of alleles within the founders individuals, random effects or genetic drift is the change in the frequency of a gene in a population due to random sampling and random extinctions that describe when a gene causes its carriers to have a deviating fitness from unity, its frequency will be determined by selection in different basins. for protists in particular there is no literature available on this topic to our knowledge. at last, the monoplization hypothesis by de meester et al. could be relevant to protist biogeography stating that a fast population growth and local adaptation and colonization of a new habitat result in the monopolization of resources, which yields a strong priority effect. the effect is even enhanced when a locally adapted population can provide a ‘large resting propagule bank’ as a strong buffer against new genotypes invading. this holds true especially for species that reproduce asexually and form resting stages. even though mass effect and dispersal cannot be ruled out, these are unlikely alternatives to explain the observed community patterns. the habitats of the water column above the dhabs represent a potential source habitat with ‘high quality’. in comparison, the narrow interphase and the brine show ‘low quality’ conditions because these habitats harbor high gradients of change, anoxia, high salt concentration up to saturation and therefore require a high degree of physiological adaptation for microbial colonization. chances for highly specialized organisms to cross environmental barriers outside their habitat and to disperse beyond their specific habitat are very low. evidence for this hypothesis comes, for example, from the very rare and infrequent dispersal of protists between marine and freshwater habitats. one possibility may be the dispersal of spores and/or cysts, however, our knowledge about the number of ciliates that can form such resting stages in nature is very limited. furthermore, physical mechanisms of transport for resting stages between different basins are difficult to imagine, considering the lack of fluid flow, high density, and lack of animal vectors in the brines. in contrast, this scenario may be more plausible for cysts/spores in halocline/interphase habitats. physical transport of resting stages between haloclines at different basin sites could explain the observed similarities in ciliate interphase communities. the deep basins in the eastern mediterranean sea may have recruited their protistan seed communities from atlantic sea water during the zenclean flood, when the strait of gibraltar opened permanently and refilled the mostly dried out mediterranean sea. subsequently, due to the dissolution of evaporites and the rise of anoxia in deep basins the water masses became physically separated from each other. anoxia and hydrochemistry likely exerted an increased pressure on the original protistan communities. species sorting may have been driven through environmental filtering. this is a predictable and fundamental process of community assembly, that allows only those taxa with the genomic and physiological potential to cope with each specific set of environmental conditions. this has been evidenced for recent ciliate communities. the normsaline and normoxic deep-sea water separating the different hypersaline anoxic basins from each other then became an environmental barrier for most protists, with the consequence that genetic exchange among the different brines was no longer likely. changes in the ssu are presumably neutral, therefore, these changes would be due to random mutations. however, it is reasonable to assume that changes in the ssu rdna are occurring in congruency with whole genome changes and not independent of evolutionary genome processes. evolution over geological time may have resulted in significantly different ciliate communities in the brines. divergence of species occurring in isolation through adaptive shifts that occurs in common seed species populations has been demonstrated for a number of taxa, including several macro- and microinvertebrates using molecular as well as taxonomic studies. based on our data, it is not unreasonable to assume that protists are also subjected to such evolutionary processes. our study strongly suggests that evolutionary time scales combined with physical and hydrochemical isolation can explain, in part, the observed evolutionary differences in the ciliate communities in the different dhabs studied here.  the data presented here suggests that ancient isolated habitats, like deep-sea brine lakes, which also occur in the red sea and the gulf of mexico or habitats like aquifers and cave systems will represent ‘hot spots’ for the discovery of as-of-yet unstudied, possibly highly divergent, and endemic microbial eukaryotes. financial competing interests in the past five years we did not receive reimbursements, fees, funding, or salary from an organization that may in any way gain or lose financially from the publication of this manuscript, either now or in the future. we do not hold any stocks or shares in an organization that may in any way gain or lose financially from the publication of this manuscript, either now or in the future. we neither hold nor apply for any patents relating to the content of the manuscript. we did not receive reimbursements, fees, funding, or salary from an organization that holds or has applied for patents relating to the content of the manuscript. we, the authors, do not have any other financial competing interests. non-financial competing interests there are no non-financial competing interests to declare in relation to this manuscript. 
the different basins have recruited their original biota from the same source, but their geological evolution eventually constituted sharp environmental barriers, restricting genetic exchange between the individual basins. we specifically focused on ciliated protists as a major component of protistan dhab plankton by pyrosequencing the hypervariable v fragment of the small subunit ribosomal rna. however, none of the analyzed hydrochemical parameters explained the significant differences between brine ciliate communities in different basins. ciliate communities in the brines of four investigated dhabs are distinctively different from ciliate communities in the interfaces immediately above the brines. while the interface ciliate communities from different sites are relatively similar to each other, the brine ciliate communities are significantly different between sites. we found no distance-decay relationship, and canonical correspondence analyses identified oxygen and sodium as most important hydrochemical parameters explaining the partitioning of diversity between interface and brine ciliate communities. our data indicate a frequent genetic exchange in the deep-sea water above the brines. the “isolated island character” of the different brines, that resulted from geological events and contemporary environmental conditions, create selective pressures driving evolutionary processes, and with time, lead to speciation and shape protistan community composition. therefore, dhabs are unique model systems to assess the effect of geological events and environmental conditions on the evolution and diversification of protistan plankton. deep hypersaline anoxic basins are isolated habitats at the bottom of the eastern mediterranean sea, which originate from the ancient dissolution of messinian evaporites.
 deep hypersaline anoxic basins are isolated habitats at the bottom of the eastern mediterranean sea, which originate from the ancient dissolution of messinian evaporites. the different basins have recruited their original biota from the same source, but their geological evolution eventually constituted sharp environmental barriers, restricting genetic exchange between the individual basins. therefore, dhabs are unique model systems to assess the effect of geological events and environmental conditions on the evolution and diversification of protistan plankton. here, we examine evidence for isolated evolution of unicellular eukaryote protistan plankton communities driven by geological separation and environmental selection. we specifically focused on ciliated protists as a major component of protistan dhab plankton by pyrosequencing the hypervariable v fragment of the small subunit ribosomal rna. geospatial distributions and responses of marine ciliates to differential hydrochemistries suggest strong physical and chemical barriers to dispersal that influence the evolution of this plankton group.  ciliate communities in the brines of four investigated dhabs are distinctively different from ciliate communities in the interfaces immediately above the brines. while the interface ciliate communities from different sites are relatively similar to each other, the brine ciliate communities are significantly different between sites. we found no distance-decay relationship, and canonical correspondence analyses identified oxygen and sodium as most important hydrochemical parameters explaining the partitioning of diversity between interface and brine ciliate communities. however, none of the analyzed hydrochemical parameters explained the significant differences between brine ciliate communities in different basins.  our data indicate a frequent genetic exchange in the deep-sea water above the brines. the “isolated island character” of the different brines, that resulted from geological events and contemporary environmental conditions, create selective pressures driving evolutionary processes, and with time, lead to speciation and shape protistan community composition. we conclude that community assembly in dhabs is a mixture of isolated evolution and species sorting. ciliateshypersalinedeep-sea anoxic basinsdhabsbrinespecies sortingenvironmental filteringniche separation 