Community Structure of Archaea in the Water Column above Gas Hydrates in the Gulf of Mexico

Microorganisms play fundamental roles in the ecosystem of the Gulf of Mexico (GOM), yet their vertical distributions along the depth continuum of water column are not well known. In this study, we presented the 16S rDNA sequences and lipid profiles in the context of water chemistry to characterize the archaeal community structure above a gas hydrate mound (MC 118) in GOM. Our results showed that all archaeal sequences were related to unknown species of Crenarchaeota or Euryarchaeota. Phylogenetically, group II –β Euryarchaeota dominated the surface water and mid-depth (400-m) water (74% and 58% of total archaeal species, respectively) whereas the marine group I-γ Crenarchaeota dominated the bottom (869 m) water (61% of total archaeal species). Estimates of the Shannon index showed the highest diversity of planktonic Archaea at the 400 m depth. Glycerol dialkyl glycerol tetraether (GDGT) lipids were detected from the 400- and 869-m depths only and characterized by relatively high abundances of GDGT-5 (crenarchaeol) and GDGT-0. Our studies suggested a possible zonation of archaeal community in the water column, which did not seem to be affected by the possible venting of hydrocarbons from the hydrate location in GOM.     

Contribution of Crenarchaeota and Euryarchaeota to the prokaryotic plankton in the coastal northwestern Black Sea

The NW Black Sea is an area with peculiar hydrological, hydrochemicaland biological features (river plume dynamics, coastal upwelling,intense eutrophication, blooms algae). We determined the abundanceand distribution of the two major groups of Archaea (Crenarchaeotaand Euryarchaeota) in three contrasting sites of the NW BlackSea from May to August 2004 to estimate the contribution ofnon-extremophilic Archaea to the coastal prokaryotic plankton.Using catalyzed reporter deposition-fluorescence in situ hybridization,Euryarchaeota were found to be particularly prominent (11–22%of total prokaryotic plankton) in the low-salinity waters duringlate-spring and summer, whereas Crenarchaeota contributed only5% to the prokaryotic community in these waters. The abundanceof Crenarchaeota was highest in nutrient- and particle-richwaters and lowest in offshore Black Sea surface waters. Theseresults confirm recent studies on the distribution of planktonicArchaea with Euryarchaeota dominating the archaeal communityin surface waters. Statistical analyses indicated significantcorrelations between crenarchaeotal abundance, ammonia and nitrateconcentrations at each sites. Our data show that the archaealplankton constitute an important and dynamic component of theprokaryotic plankton in the coastal oxygenated waters of theNW Black Sea and might play a significant role in the nitrogencycle.     

Phylogenetic analysis of Group I marine archaeal rRNA sequences emphasizes the hidden diversity within the primary group Archaea

Archaea form one of the three primary groups of extant life and are commonly associated with the extreme environments which many of their members inhabit. Currently, the Archaea are classified into two kingdoms, Crenarchaeota and Euryarchaeota, based on phylogenetic analysis of ribosomal RNA (rRNA) sequences. Molecular techniques allowing the retrieval and analysis of rRNA sequences from diverse environments are increasing our knowledge of archaeal diversity. This report describes the presence of marine Archaea in north-east Atlantic waters. Quantitative estimates indicated that the marine Archaea constitute 8 per cent of the total prokaryotic rRNA in Irish coastal waters. Phylogenetic analysis of the archaeal rRNA gene sequences revealed sufficient genetic diversity within Archaea to indicate that the current two-kingdom classification of Crenarchaeota and Euryarchaeota is restrictive.     

Variations in spatial and temporal distribution of Archaea in the North Sea in relation to environmental variables

The spatial and temporal distribution of pelagic Archaea was studied in the southern North Sea by rRNA hybridization, sequencing and quantification of 16S rRNA gene and membrane lipid analyses and related to physical, chemical and biological parameters to determine the factors influencing archaeal biogeography. A clear temporal variability was observed, with marine Crenarchaeota (Group I.1a) being relatively more abundant in winter and Euryarchaeota dominating the archaeal assemblage in spring and summer. Spatial differences in the lateral distribution of Crenarchaeota were also evident. In fact, their abundance was positively correlated with the copy number of the gene encoding the alpha subunit of crenarchaeotal ammonia monooxygenase (amoA) and with concentrations of ammonia, nitrate, nitrite and phosphorus. This suggests that most Crenarchaeota in the North Sea are nitrifiers and that their distribution is determined by nutrient concentrations. However, Crenarchaeota were not abundant when larger phytoplankton (>3 microm) dominated the algal population. It is hypothesized that together with nutrient concentration, phytoplankton biomass and community structure can predict crenarchaeotal abundance in the southern North Sea. Euryarchaeotal abundance was positively correlated with chlorophyll a concentrations, but not with phytoplankton community structure. Whether this is related to the potential of Euryarchaeota to perform aerobic anoxygenic phototrophy remains to be shown, but the conspicuous seasonal distribution pattern of Crenarchaeota and Euryarchaeota suggests that they occupy a different ecological niche.     

High archaeal richness in the water column of a freshwater sulfurous karstic lake along an interannual study

We surveyed the archaeal assemblage in a stratified sulfurous lake (Lake Vilar, Banyoles, Spain) over 5 consecutive years to detect potential seasonal and interannual trends in the free-living planktonic Archaea composition. The combination of different primer pairs and nested PCR steps revealed an unexpectedly rich archaeal community. Overall, 140 samples were analyzed, yielding 169 different 16S rRNA gene sequences spread over 14 Crenarchaeota (109 sequences) and six Euryarchaeota phylogenetic clusters. Most of the Crenarchaeota (98% of the total crenarchaeotal sequences) affiliated within the Miscellaneous Crenarchaeota Group (MCG) and were related to both marine and freshwater phylotypes. Euryarchaeota mainly grouped within the Deep Hydrothermal Vent Euryarchaeota (DHVE) cluster (80% of the euryarchaeotal sequences) and the remaining 20% distributed into three less abundant taxa, most of them composed of soil and sediment clones. The largest fraction of phylotypes from the two archaeal kingdoms (79% of the Crenarchaeota and 54% of the Euryarchaeota) was retrieved from the anoxic hypolimnion, indicating that these cold and sulfide-rich waters constitute an unexplored source of archaeal richness. The taxon rank-frequency distribution showed two abundant taxa (MCG and DHVE) that persisted in the water column through seasons, plus several rare ones that were only detected occasionally. Differences in richness distribution and seasonality were observed, but no clear correlations were obtained when multivariate statistical analyses were carried out.     

Evolution of the Archaea

Archaea, members of the third domain of life, are bacterial-looking prokaryotes that harbour many unique genotypic and phenotypic properties, testifying for their peculiar evolutionary status. The archaeal ancestor was probably a hyperthermophilic anaerobe. Two archaeal phyla are presently recognized, the Euryarchaeota and the Crenarchaeota. Methanogenesis was the main invention that occurred in the euryarchaeal phylum and is now shared by several archaeal groups. Adaptation to aerobic conditions occurred several times independently in both Euryarchaeota and Crenarchaeota. Recently, many new groups of Archaea that have not yet been cultured have been detected by PCR amplification of 16S ribosomal RNA from environmental samples. The phenotypic and genotypic characterization of these new groups is now a top priority for further studies on archaeal evolution.     

Contribution of Archaea to total prokaryotic production in the deep Atlantic Ocean

Fluorescence in situ hybridization (FISH) in combination with polynucleotide probes revealed that the two major groups of planktonic Archaea (Crenarchaeota and Euryarchaeota) exhibit a different distribution pattern in the water column of the Pacific subtropical gyre and in the Antarctic Circumpolar Current system. While Euryarchaeota were found to be more dominant in nearsurface waters, Crenarchaeota were relatively more abundant in the mesopelagic and bathypelagic waters. We determined the abundance of archaea in the mesopelagic and bathypelagic North Atlantic along a south-north transect of more than 4,000 km. Using an improved catalyzed reporter deposition-FISH (CARD-FISH) method and specific oligonucleotide probes, we found that archaea were consistently more abundant than bacteria below a 100-m depth. Combining microautoradiography with CARD-FISH revealed a high fraction of metabolically active cells in the deep ocean. Even at a 3,000-m depth, about 16% of the bacteria were taking up leucine. The percentage of Euryarchaeota and Crenarchaeaota taking up leucine did not follow a specific trend, with depths ranging from 6 to 35% and 3 to 18%, respectively. The fraction of Crenarchaeota taking up inorganic carbon increased with depth, while Euryarchaeota taking up inorganic carbon decreased from 200 m to 3,000 m in depth. The ability of archaea to take up inorganic carbon was used as a proxy to estimate archaeal cell production and to compare this archaeal production with total prokaryotic production measured via leucine incorporation. We estimate that archaeal production in the mesopelagic and bathypelagic North Atlantic contributes between 13 to 27% to the total prokaryotic production in the oxygen minimum layer and 41 to 84% in the Labrador Sea Water, declining to 10 to 20% in the North Atlantic Deep Water. Thus, planktonic archaea are actively growing in the dark ocean although at lower growth rates than bacteria and might play a significant role in the oceanic carbon cycle.     

一个新的古菌类群———奇古菌门(Thaumarchaeota)

基于16S rRNA基因的系统发育关系,古菌域(Archaea)被分为两个主要类群:广古菌门(Euryarchaeota)和泉古菌门(Crenarchaeota)。近20年来,微生物分子生态学技术的快速发展和应用显示,在中温环境中广泛存在着大量的未培养古菌,而且它们可能在自然界重要元素(N、C)的生物地球化学循环中发挥着重要作用。最初,这些未培养古菌因在16S rRNA基因系统发育上与泉古菌关系较密切而被称作中温泉古菌(non-thermophilic Crenarchaeota)。而近年来,对更多新发现的中温古菌核糖体RNA基因序列和其它分子标记物进行的分析均不支持中温泉古菌由嗜热泉古菌进化而来的假设,而揭示其可能代表古菌域中一个独立的系统发育分支。基因组学、生理生态特征等分析也显示中温泉古菌与泉古菌具有明显不同的特征。因而专家建议将这些古菌(中温泉古菌)划分为一个新的门,成为古菌域的第三个主要类群—Thaumarchaeota(意译为奇古菌门)。这一新古菌门提出后得到其他研究证据的支持和认可。本文对目前已知的奇古菌门的分类地位演化、基因组学、多样性和生理代谢特征等作一简要综述。     

Diverse archaeal community of a bat guano pile in Domica Cave (Slovak Karst,Slovakia)

The molecular diversity of Archaea in a bat guano pile in Cave Domica (Slovakia), temperate cave ecosystem with significant bat colony (about 1600 individuals), was examined. The guano pile was created mainly by an activity of the Mediterranean horseshoe bat (Rhinolophus euryale) and provides a source of organic carbon and other nutrients in the oligotrophic subsurface ecosystem. The upper and the basal parts of guano surface were sampled where the latter one had higher pH and higher admixture of limestone bedrock and increased colonization of invertebrates. The relative proportion of Archaea determined using CARD-FISH in both parts was 3.5–3.9 % (the basal and upper part, respectively). The archaeal community was dominated by non-thermophilic Crenarchaeota (99 % of clones). Phylogenetic analysis of 115 16S rDNA sequences revealed the presence of Crenarchaeota previously isolated from temperate surface soils (group 1.1b, 62 clones), deep subsurface acid waters (group 1.1a, 52 clones) and Euryarchaeota (1 clone). Four of the analyzed sequences were found to have little similarity to those in public databases. The composition of both archaeal communities differed, with respect to higher diversity of Archaea in the upper part of the bat guano pile. High diversity archaeal population is present in the bat guano deposit and consists of both soil- and subsurface-born Crenarchaeota.     

Insights into the evolution of Archaea and eukaryotic protein modifier systems revealed by the genome of a novel archaeal group

The domain Archaea has historically been divided into two phyla, the Crenarchaeota and Euryarchaeota. Although regarded as members of the Crenarchaeota based on small subunit rRNA phylogeny, environmental genomics and efforts for cultivation have recently revealed two novel phyla/divisions in the Archaea; the 'Thaumarchaeota' and 'Korarchaeota'. Here, we show the genome sequence of Candidatus 'Caldiarchaeum subterraneum' that represents an uncultivated crenarchaeotic group. A composite genome was reconstructed from a metagenomic library previously prepared from a microbial mat at a geothermal water stream of a sub-surface gold mine. The genome was found to be clearly distinct from those of the known phyla/divisions, Crenarchaeota (hyperthermophiles), Euryarchaeota, Thaumarchaeota and Korarchaeota. The unique traits suggest that this crenarchaeotic group can be considered as a novel archaeal phylum/division. Moreover, C. subterraneum harbors an ubiquitin-like protein modifier system consisting of Ub, E1, E2 and small Zn RING finger family protein with structural motifs specific to eukaryotic system proteins, a system clearly distinct from the prokaryote-type system recently identified in Haloferax and Mycobacterium. The presence of such a eukaryote-type system is unprecedented in prokaryotes, and indicates that a prototype of the eukaryotic protein modifier system is present in the Archaea.     

Population structure and phylogenetic characterization of marine benthic Archaea in deep-sea sediments.

During the past few years Archaea have been recognized as a widespread and significant component of marine picoplankton assemblages and, more recently, the presence of novel archaeal phylogenetic lineages has been reported in coastal marine benthic environments. We investigated the relative abundance, vertical distribution, phylogenetic composition, and spatial variability of Archaea in deep-sea sediments collected from several stations in the Atlantic Ocean. Quantitative oligonucleotide hybridization experiments indicated that the relative abundance of archaeal 16S rRNA in deep-sea sediments (1500 m deep) ranged from about 2.5 to 8% of the total prokaryotic rRNA. Clone libraries of PCR-amplified archaeal rRNA genes (rDNA) were constructed from 10 depth intervals obtained from sediment cores collected at depths of 1,500, 2,600, and 4,500 m. Phylogenetic analysis of rDNA sequences revealed the presence of a complex archaeal population structure, whose members could be grouped into discrete phylogenetic lineages within the two kingdoms, Crenarchaeota and Euryarchaeota. Comparative denaturing gradient gel electrophoresis profile analysis of archaeal 16S rDNA V3 fragments revealed a significant depth-related variability in the composition of the archaeal population.     

The vertical distribution of bacterial and archaeal communities in the water and sediment of Lake Taihu

This study was conducted to characterize the vertical distribution of bacterial and archaeal communities in the water and sediment of Lake Taihu, which underwent a change in trophic status from oligotrophic to hypertrophic in last half of the 20th century. The results revealed that the bacterial communities in different layers of sediment sample were very similar, and were related to Alpha -, Beta -, Gamma - and Deltaproteobacteria, Nitrospira, Bacteroidetes, Firmicutes, Gemmatimonadetes, Verrucomicrobia, Chlorobi, Actinobacteria and Acidobacteria . In contrast, the archaeal communities varied greatly with depth. The archaeal communities were primarily related to Euryarchaeota and Crenarchaeota , with methanogenic Archaea accounting for approximately 2–35% of the total Archaea. Additionally, sequences related to putative ammonia-oxidizing Archaea and ammonia-oxidizing Bacteria were detected in different layers of sediment samples. The abundance of Archaea, Bacteria, methanogenic Archaea and Nitrospira was further characterized by real-time PCR.     

Characterization of Archaeal Community in Contaminated and Uncontaminated Surface Stream Sediments

Archaeal communities from mercury and uranium-contaminated freshwater stream sediments were characterized and compared to archaeal communities present in an uncontaminated stream located in the vicinity of Oak Ridge, TN, USA. The distribution of the Archaea was determined by pyrosequencing analysis of the V4 region of 16S rRNA amplified from 12 streambed surface sediments. Crenarchaeota comprised 76% of the 1,670 archaeal sequences and the remaining 24% were from Euryarchaeota. Phylogenetic analysis further classified the Crenarchaeota as a Freshwater Group, Miscellaneous Crenarchaeota group, Group I3, Rice Cluster VI and IV, Marine Group I and Marine Benthic Group B; and the Euryarchaeota into Methanomicrobiales, Methanosarcinales, Methanobacteriales, Rice Cluster III, Marine Benthic Group D, Deep Sea Hydrothermal Vent Euryarchaeota 1 and Eury 5. All groups were previously described. Both hydrogen- and acetate-dependent methanogens were found in all samples. Most of the groups (with 60% of the sequences) described in this study were not similar to any cultivated isolates, making it difficult to discern their function in the freshwater microbial community. A significant decrease in the number of sequences, as well as in the diversity of archaeal communities was found in the contaminated sites. The Marine Group I, including the ammonia oxidizer Nitrosopumilus maritimus, was the dominant group in both mercury and uranium/nitrate-contaminated sites. The uranium-contaminated site also contained a high concentration of nitrate, thus Marine Group I may play a role in nitrogen cycle.     

Archaeal diversity in tidal flat sediment as revealed by 16S rDNA analysis

During the past ten years, Archaea have been recognized as a widespread and significant component of marine picoplankton assemblages. More recently, the presence of novel archaeal phylogenetic lineages has been discovered in coastal marine environments, freshwater lakes, polar seas, and deep-sea hydrothermal vents. Therefore, we conducted an investigation into the archaeal community existing in tidal flat sediment collected from Ganghwa Island, Korea. Phylogenetic analysis of archaeal 16S rDNA amplified directly from tidal flat sediment DNA revealed the presence of two major lineages, belonging to the Crenarchaeota (53.9%) and Euryarchaeota (46.1%) phyla. A total of 102 clones were then sequenced and analyzed by comprehensive phylogenetic analysis. The sequences determined in our samples were found to be closely related to the sequences of clones which had been previously obtained from a variety of marine environments. Archaeal clones exhibited higher similarities (83.25-100%) to sequences from other environments in the public database than did those (75.22-98.46%) of previously reported bacterial clones obtained from tidal flat sediment. The results of our study suggest that the archaeal community in tidal flat sediment is remarkably diverse.     

Response of Archaeal communities in beach sediments to spilled oil and bioremediation

While the contribution of Bacteria to bioremediation of oil-contaminated shorelines is well established, the response of Archaea to spilled oil and bioremediation treatments is unknown. The relationship between archaeal community structure and oil spill bioremediation was examined in laboratory microcosms and in a bioremediation field trial. 16S rRNA gene-based PCR and denaturing gradient gel analysis revealed that the archaeal community in oil-free laboratory microcosms was stable for 26 days. In contrast, in oil-polluted microcosms a dramatic decrease in the ability to detect Archaea was observed, and it was not possible to amplify fragments of archaeal 16S rRNA genes from samples taken from microcosms treated with oil. This was the case irrespective of whether a bioremediation treatment (addition of inorganic nutrients) was applied. Since rapid oil biodegradation occurred in nutrient-treated microcosms, we concluded that Archaea are unlikely to play a role in oil degradation in beach ecosystems. A clear-cut relationship between the presence of oil and the absence of Archaea was not apparent in the field experiment. This may have been related to continuous inoculation of beach sediments in the field with Archaea from seawater or invertebrates and shows that the reestablishment of Archaea following bioremediation cannot be used as a determinant of ecosystem recovery following bioremediation. Comparative 16S rRNA sequence analysis showed that the majority of the Archaea detected (94%) belonged to a novel, distinct cluster of group II uncultured Euryarchaeota, which exhibited less than 87% identity to previously described sequences. A minor contribution of group I uncultured Crenarchaeota was observed.     

Vertical structure of archaeal communities and the distribution of ammonia monooxygenase A gene variants in two meromictic High Arctic lakes

The distribution of archaeal amoA and 16S rRNA genes was evaluated in two marine-derived, meromictic lakes in the Canadian High Arctic: Lake A and Lake C1 on the northern coast of Ellesmere Island. The amoA gene was recorded in both lakes, with highest copy numbers in the oxycline. Sequence analysis showed that amoA from the two lakes shared 94% similarity, indicating at least two phylogenetically distinct clusters. Clone libraries of archaeal 16S rRNA genes from Lake A revealed strong vertical differences in archaeal community diversity and composition down the water column. The oxic layer was dominated by one group of Euryarchaeota affiliated to the Lake Dagow Sediment (LDS) cluster. This group was absent from the oxycline, which had an extremely low archaeal diversity of two phylotypes. Both belonged to the Crenarchaeota Marine Group I (MGI), the marine group that has been linked to archaeal amoA ; however, there was a low ratio of amoA to MGI copy numbers, suggesting that many MGI Archaea did not carry the amoA gene. The anoxic zone contained representatives of the RC-V (Rice Cluster-V) and LDS clusters of Euryarchaeota. These results show the strong vertical differentiation of archaeal communities in polar meromictic lakes, and they suggest archaeal nitrification within the oxycline of these highly stratified waters.     

The origin and evolution of Archaea: a state of the art

Environmental surveys indicate that the Archaea are diverse and abundant not only in extreme environments, but also in soil, oceans and freshwater, where they may fulfil a key role in the biogeochemical cycles of the planet. Archaea display unique capacities, such as methanogenesis and survival at temperatures higher than 90 degrees C, that make them crucial for understanding the nature of the biota of early Earth. Molecular, genomics and phylogenetics data strengthen Woese's definition of Archaea as a third domain of life in addition to Bacteria and Eukarya. Phylogenomics analyses of the components of different molecular systems are highlighting a core of mainly vertically inherited genes in Archaea. This allows recovering a globally well-resolved picture of archaeal evolution, as opposed to what is observed for Bacteria and Eukarya. This may be due to the fact that no rapid divergence occurred at the emergence of present-day archaeal lineages. This phylogeny supports a hyperthermophilic and non-methanogenic ancestor to present-day archaeal lineages, and a profound divergence between two major phyla, the Crenarchaeota and the Euryarchaeota, that may not have an equivalent in the other two domains of life. Nanoarchaea may not represent a third and ancestral archaeal phylum, but a fast-evolving euryarchaeal lineage. Methanogenesis seems to have appeared only once and early in the evolution of Euryarchaeota. Filling up this picture of archaeal evolution by adding presently uncultivated species, and placing it back in geological time remain two essential goals for the future.     

Archaeal chromatin proteins

Archaea, along with Bacteria and Eukarya, are the three domains of life. In all living cells, chromatin proteins serve a crucial role in maintaining the integrity of the structure and function of the genome. An array of small, abundant and basic DNA-binding proteins, considered candidates for chromatin proteins, has been isolated from the Euryarchaeota and the Crenarchaeota, the two major phyla in Archaea. While most euryarchaea encode proteins resembling eukaryotic histones, crenarchaea appear to synthesize a number of unique DNA-binding proteins likely involved in chromosomal organization. Several of these proteins (e.g., archaeal histones, Sac10b homologs, Sul7d, Cren7, CC1, etc.) have been extensively studied. However, whether they are chromatin proteins and how they function in vivo remain to be fully understood. Future investigation of archaeal chromatin proteins will lead to a better understanding of chromosomal organization and gene expression in Archaea and provide valuable information on the evolution of DNA packaging in cellular life.     

Not so old Archaea – the antiquity of biogeochemical processes in the archaeal domain of life

Since the archaeal domain of life was first recognized, it has often been assumed that Archaea are ancient, and harbor primitive traits. In fact, the names of the major archaeal lineages reflect our assumptions regarding the antiquity of their traits. Ancestral state reconstruction and relaxed molecular clock analyses using newly articulated oxygen age constraints show that although the archaeal domain itself is old, tracing back to the Archean eon, many clades and traits within the domain are not ancient or primitive. Indeed many clades and traits, particularly in the Euryarchaeota, were inferred to be Neoproterozoic or Phanerozoic in age. Both Eury- and Crenarchaeota show increasing metabolic and physiological diversity through time. Early archaeal microbial communities were likely limited to sulfur reduction and hydrogenotrophic methanogenesis, and were confined to high-temperature geothermal environments. However, after the appearance of atmospheric oxygen, nodes containing a wide variety of traits (sulfate and thiosulfate reduction, sulfur oxidation, sulfide oxidation, aerobic respiration, nitrate reduction, mesophilic methanogenesis in sedimentary environments) appear, first in environments containing terrestrial Crenarchaeota in the Meso/Neoproterozoic followed by environments containing marine Euryarchaeota in the Neoproterozoic and Phanerozoic. This provides phylogenetic evidence for increasing complexity in the biogeochemical cycling of C, N, and S through geologic time, likely as a consequence of microbial evolution and the gradual oxygenation of various compartments within the biosphere. This work has implications not only for the large-scale evolution of microbial communities and biogeochemical processes, but also for the interpretation of microbial biosignatures in the ancient rock record.     

A hotspot for cold crenarchaeota in the neuston of high mountain lakes

We have surveyed the first 1 m of 10 oligotrophic high mountain lakes in the Central Pyrenees (Spain) for both abundance and predominant phylotypes richness of the archaeaplankton assemblage, using CARD-FISH and 16S rRNA gene sequencing respectively. Archaea inhabiting the air-water surface microlayer (neuston) ranged between 3% and 37% of total 4,6-diamidino-2-phenylindole (DAPI) counts and were mainly Crenarchaeota of a new freshwater cluster distantly related to the Marine Group 1.1a. Conversely, most of the Archaea from the underlying waters (the remaining first 1 m integrated) were mainly Euryarchaeota of three distantly related branches ranging between 0.4% and 27% of total DAPI counts. Therefore, a consistent qualitative and quantitative spatial segregation was observed for the two main archaeal phyla between neuston and underlying waters at a regional scale. We also observed a consistent pattern along the lakes surveyed between lake area, lake depth and water residence time, and the archaeal enrichment in the neuston: the larger the lake the higher the proportion of archaea in the neuston as compared with abundances from the underlying waters ( n  = 10 lakes; R ² > 0.80; P  < 0.001, in all three cases). This is the first report identifying a widespread non-thermophilic habitat where freshwater planktonic Crenarchaeota can be found naturally enriched. High mountain lakes offer great research opportunities to explore the ecology of one of the most enigmatic and far from being understood group of prokaryotes.