Niphargus-Thiothrix associations may be widespread in sulphidic groundwater ecosystems: evidence from southeastern Romania.

Niphargus is a speciose amphipod genus found in groundwater habitats across Europe. Three Niphargus species living in the sulphidic Frasassi caves in Italy harbour sulphur-oxidizing Thiothrix bacterial ectosymbionts. These three species are distantly related, implying that the ability to form ectosymbioses with Thiothrix may be common among Niphargus. Therefore, Niphargus-Thiothrix associations may also be found in sulphidic aquifers other than Frasassi. In this study, we examined this possibility by analysing niphargids of the genera Niphargus and Pontoniphargus collected from the partly sulphidic aquifers of the Southern Dobrogea region of Romania, which are accessible through springs, wells and Movile Cave. Molecular and morphological analyses revealed seven niphargid species in this region. Five of these species occurred occasionally or exclusively in sulphidic locations, whereas the remaining two were restricted to nonsulphidic areas. Thiothrix were detected by PCR on all seven Dobrogean niphargid species and observed using microscopy to be predominantly attached to their hosts' appendages. 16S rRNA gene sequences of the Thiothrix epibionts fell into two main clades, one of which (herein named T4) occurred solely on niphargids collected in sulphidic locations. The other Thiothrix clade was present on niphargids from both sulphidic and nonsulphidic areas and indistinguishable from the T3 ectosymbiont clade previously identified on Frasassi-dwelling Niphargus. Although niphargids from Frasassi and Southern Dobrogea are not closely related, the patterns of their association with Thiothrix are remarkably alike. The finding of similar Niphargus-Thiothrix associations in aquifers located 1200 km apart suggests that they may be widespread in European groundwater ecosystems.

Methanobacterium movilense sp. nov., a hydrogenotrophic, secondary-alcohol-utilizing methanogen from the anoxic sediment of a subsurface lake.

A novel strain of methanogenic archaea, designated MC-20(T), was isolated from the anoxic sediment of a subsurface lake in Movile Cave, Mangalia, Romania. Cells were non-motile, Gram-stain-negative rods 3.5-4.0 µm in length and 0.6-0.7 µm in width, and occurred either singly or in short chains. Strain MC-20(T) was able to utilize H2/CO2, formate, 2-propanol and 2-butanol as substrate, but not acetate, methanol, ethanol, dimethyl sulfide, monomethylamine, dimethylamine or trimethylamine. Neither trypticase peptone nor yeast extract was required for growth. The major membrane lipids of strain MC-20(T) were archaeol phosphatidylethanolamine and diglycosyl archaeol, while archaeol phosphatidylinositol and glycosyl archaeol were present only in minor amounts. Optimal growth was observed at 33 °C, pH 7.4 and 0.08 M NaCl. Based on phylogenetic analysis of 16S rRNA gene sequences, strain MC-20(T) was closely affiliated with Methanobacterium oryzae FPi(T) (similarity 97.1%) and Methanobacterium lacus 17A1(T) (97.0%). The G+C content of the genomic DNA was 33.0 mol%. Based on phenotypic and genotypic differences, strain MC-20(T) was assigned to a novel species of the genus Methanobacterium for which the name Methanobacterium movilense sp. nov. is proposed. The type strain is MC-20(T) ( = DSM 26032(T) = JCM 18470(T)).

Methanosarcina spelaei sp. nov., a methanogenic archaeon isolated from a floating biofilm of a subsurface sulphurous lake.

A novel methanogenic archaeon, strain MC-15(T), was isolated from a floating biofilm on a sulphurous subsurface lake in Movile Cave (Mangalia, Romania). Cells were non-motile sarcina-like cocci with a diameter of 2-4 µm, occurring in aggregates. The strain was able to grow autotrophically on H2/CO2. Additionally, acetate, methanol, monomethylamine, dimethylamine and trimethylamine were utilized, but not formate or dimethyl sulfide. Trypticase peptone and yeast extract were not required for growth. Optimal growth was observed at 33 °C, pH 6.5 and a salt concentration of 0.05 M NaCl. The predominant membrane lipids of MC-15(T) were archaeol and hydroxyarchaeol phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol as well as hydroxyarchaeol phosphatidylserine and archaeol glycosaminyl phosphatidylinositol. The closely related species, Methanosarcina vacuolata and Methanosarcina horonobensis, had a similar composition of major membrane lipids to strain MC-15(T). The 16S rRNA gene sequence of strain MC-15(T) was similar to those of Methanosarcina vacuolata DSM 1232(T) (sequence similarity 99.3%), Methanosarcina horonobensis HB-1(T) (98.8%), Methanosarcina barkeri DSM 800(T) (98.7%) and Methanosarcina siciliae T4/M(T) (98.4%). DNA-DNA hybridization revealed 43.3% relatedness between strain MC-15(T) and Methanosarcina vacuolata DSM 1232(T). The G+C content of the genomic DNA was 39.0 mol%. Based on physiological, phenotypic and genotypic differences, strain MC-15(T) represents a novel species of the genus Methanosarcina, for which the name Methanosarcina spelaei sp. nov. is proposed. The type strain is MC-15(T) ( = DSM 26047(T) = JCM 18469(T)).

Competition-cooperation in the chemoautotrophic ecosystem of Movile Cave: first metagenomic approach on sediments.

BACKGROUND: Movile Cave (SE Romania) is a chemoautotrophically-based ecosystem fed by hydrogen sulfide-rich groundwater serving as a primary energy source analogous to the deep-sea hydrothermal ecosystems. Our current understanding of Movile Cave microbiology has been confined to the sulfidic water and its proximity, as most studies focused on the water-floating microbial mat and planktonic accumulations likely acting as the primary production powerhouse of this unique subterranean ecosystem. By employing comprehensive genomic-resolved metagenomics, we questioned the spatial variation, chemoautotrophic abilities, ecological interactions and trophic roles of Movile Cave's microbiome thriving beyond the sulfidic-rich water. RESULTS: A customized bioinformatics pipeline led to the recovery of 106 high-quality metagenome-assembled genomes from 7 cave sediment metagenomes. Assemblies' taxonomy spanned 19 bacterial and three archaeal phyla with Acidobacteriota, Chloroflexota, Proteobacteria, Planctomycetota, Ca. Patescibacteria, Thermoproteota, Methylomirabilota, and Ca. Zixibacteria as prevalent phyla. Functional gene analyses predicted the presence of CO2 fixation, methanotrophy, sulfur and ammonia oxidation in the explored sediments. Species Metabolic Coupling Analysis of metagenome-scale metabolic models revealed the highest competition-cooperation interactions in the sediments collected away from the water. Simulated metabolic interactions indicated autotrophs and methanotrophs as major donors of metabolites in the sediment communities. Cross-feeding dependencies were assumed only towards 'currency' molecules and inorganic compounds (O2, PO43-, H+, Fe2+, Cu2+) in the water proximity sediment, whereas hydrogen sulfide and methanol were assumedly traded exclusively among distant gallery communities. CONCLUSIONS: These findings suggest that the primary production potential of Movile Cave expands way beyond its hydrothermal waters, enhancing our understanding of the functioning and ecological interactions within chemolithoautotrophically-based subterranean ecosystems.

Microbial eukaryotes in the suboxic chemosynthetic ecosystem of Movile Cave, Romania.

Movile Cave is a small system of partially inundated galleries in limestone settings close to the Black Sea in Southeast Romania. Isolated from the surface for 6 million years, its sulfidic, methane and ammonia-rich waters harbour unique chemosynthetic prokaryotic communities that include sulphur and ammonium-metabolizing chemolithotrophs, methanogens, methanotrophs and methylotrophs. The cave also harbours cave-dwelling invertebrates and fungi, but the diversity of other microbial eukaryotes remained completely unknown. Here, we apply an 18S rRNA gene-based metabarcoding approach to study the composition of protist communities in floating microbial mats and plankton from a well-preserved oxygen-depleted cave chamber. Our results reveal a wide protist diversity with, as dominant groups, ciliates (Alveolata), Stramenopiles, especially bicosoecids, and jakobids (Excavata). Ciliate sequences dominated both, microbial mats and plankton, followed by either Stramenopiles or excavates. Stramenopiles were more prominent in microbial mats, whereas jakobids dominated the plankton fraction of the oxygen-depleted water column. Mats cultured in the laboratory were enriched in Cercozoa. Consistent with local low oxygen levels, Movile Cave protists are most likely anaerobic or microaerophilic. Several newly detected OTU clades were very divergent from cultured species or environmental sequences in databases and represent phylogenetic novelty, notably within jakobids. Movile Cave protists likely cover a variety of ecological roles in this ecosystem including predation, parasitism, saprotrophy and possibly diverse prokaryote-protist syntrophies.

Cultivable microscopic fungi from an underground chemosynthesis-based ecosystem: a preliminary study.

Movile Cave, a unique groundwater ecosystem in southern Romania, was discovered in 1986. This chemoautotrophic cave contains an abundant and diverse fauna with terrestrial and aquatic invertebrate communities, including 33 endemic species. Since its discovery, studies have focused mainly on cave chemoautotrophic bacteria, while the microfungal community has been largely neglected. In this study, we determined the microfungal species living on various substrates in Movile Cave and compared this spectrum with the mycobiota detected outside the cave (outside air-borne and soil-borne microfungi). To investigate all of the niches, we collected samples for two consecutive years from the dry part of the cave (cave air and sediment, corroded limestone walls, isopod feces, and isopod and spider cadavers) and from the post-siphon part of the cave, i.e., Airbell II (sediment and floating microbial mat). A total of 123 microfungal species were identified from among several hundred isolates. Of these, 96 species were only detected in the cave environment and not outside of the cave, while 90 species were from the dry part of the cave and 28 were from Airbell II. The most diverse genera were Penicillium (at least 18 species) and Aspergillus (14 species), followed by Cladosporium (9 species). Surprisingly, high CFU counts of air-borne microfungi were found inside the cave; they were even higher than outside the cave during the first year of investigation.

Fungi in perennial ice from Scarișoara Ice Cave (Romania).

Screening of 1,000-years old ice layers from the perennial ice block of Scarișoara Ice Cave (NW Romania) revealed the presence of fungal communities. Using culture-dependent methods and molecular techniques based on DGGE fingerprinting of 18S rRNA gene fragments and sequencing, we identified 50 cultured and 14 uncultured fungi in presently-forming, 400 and 900 years old ice layers, corresponding to 28 distinct operational taxonomic units (OTUs). The dominant ice-contained fungal OTUs were related to Ascomycota, Basidiomycota and Cryptomycota phyla. Representatives of Mucoromycota and Chytridiomycota were also isolated from recent and 400 years old ice samples. The cryophilic Mrakia stokesii was the most abundant fungal species found in the cave ice samples of all prospected ages, alongside other cryophilic fungi also identified in various glacial environments. Ice deposits formed during the Little Ice Age (dated between AD 1,250 and 1,850) appeared to have a higher fungal diversity than the ice layer formed during the Medieval Warm Period (prior to AD 1,250). A more complex fungal community adapted to low temperatures was obtained from all analyzed ice layers when cultivated at 4 °C as compared to 15 °C, suggesting the dominance of cold-adapted fungi in this glacial habitat. The fungal distribution in the analyzed cave ice layers revealed the presence of unique OTUs in different aged-formed ice deposits, as a first hint for putative further identification of fungal biomarkers for climate variations in this icy habitat. This is the first report on fungi from a rock-hosted cave ice block.

Aerobic proteobacterial methylotrophs in Movile Cave: genomic and metagenomic analyses.

BACKGROUND: Movile Cave (Mangalia, Romania) is a unique ecosystem where the food web is sustained by microbial primary production, analogous to deep-sea hydrothermal vents. Specifically, chemoautotrophic microbes deriving energy from the oxidation of hydrogen sulphide and methane form the basis of the food web. RESULTS: Here, we report the isolation of the first methane-oxidizing bacterium from the Movile Cave ecosystem, Candidatus Methylomonas sp. LWB, a new species and representative of Movile Cave microbial mat samples. While previous research has suggested a prevalence of anoxic conditions in deeper lake water and sediment, using small-scale shotgun metagenome sequencing, we show that metabolic genes encoding enzymes for aerobic methylotrophy are prevalent in sediment metagenomes possibly indicating the presence of microoxic conditions. Moreover, this study also indicates that members within the family Gallionellaceae (Sideroxydans and Gallionella) were the dominant taxa within the sediment microbial community, thus suggesting a major role for microaerophilic iron-oxidising bacteria in nutrient cycling within the Movile Cave sediments. CONCLUSIONS: In this study, based on phylogenetic and metabolic gene surveys of metagenome sequences, the possibility of aerobic microbial processes (i.e., methylotrophy and iron oxidation) within the sediment is indicated. We also highlight significant gaps in our knowledge on biogeochemical cycles within the Movile Cave ecosystem, and the need to further investigate potential feedback mechanisms between microbial communities in both lake sediment and lake water.

Draft Genome Sequence of the Methane-Oxidizing Bacterium "Candidatus Methylomonas sp. LWB" Isolated from Movile Cave.

We describe the draft genome sequence of "Candidatus Methylomonas sp. LWB" isolated from Movile Cave microbial mat samples. The genome contains both the soluble and particular methane monooxygenase; however, one of the putative particulate methane monooxygenase gene clusters is ordered pmoABC rather than in the canonical gene arrangement of pmoCAB.

The evolutionary and phylogeographic history of woolly mammoths: a comprehensive mitogenomic analysis.

Near the end of the Pleistocene epoch, populations of the woolly mammoth (Mammuthus primigenius) were distributed across parts of three continents, from western Europe and northern Asia through Beringia to the Atlantic seaboard of North America. Nonetheless, questions about the connectivity and temporal continuity of mammoth populations and species remain unanswered. We use a combination of targeted enrichment and high-throughput sequencing to assemble and interpret a data set of 143 mammoth mitochondrial genomes, sampled from fossils recovered from across their Holarctic range. Our dataset includes 54 previously unpublished mitochondrial genomes and significantly increases the coverage of the Eurasian range of the species. The resulting global phylogeny confirms that the Late Pleistocene mammoth population comprised three distinct mitochondrial lineages that began to diverge ~1.0-2.0 million years ago (Ma). We also find that mammoth mitochondrial lineages were strongly geographically partitioned throughout the Pleistocene. In combination, our genetic results and the pattern of morphological variation in time and space suggest that male-mediated gene flow, rather than large-scale dispersals, was important in the Pleistocene evolutionary history of mammoths.

Draft Genome Sequences of Facultative Methylotrophs, Gemmobacter sp. Strain LW1 and Mesorhizobium sp. Strain 1M-11, Isolated from Movile Cave, Romania.

Facultative methylotrophs belonging to the genera Gemmobacter and Mesorhizobium were isolated from microbial mat and cave water samples obtained from the Movile Cave ecosystem. Both bacteria can utilize methylated amines as their sole carbon and nitrogen source. Here, we report the draft genome sequences of Gemmobacter sp. strain LW1 and Mesorhizobium sp. strain IM1.

Bacterial metabolism of methylated amines and identification of novel methylotrophs in Movile Cave.

Movile Cave, Romania, is an unusual underground ecosystem that has been sealed off from the outside world for several million years and is sustained by non-phototrophic carbon fixation. Methane and sulfur-oxidising bacteria are the main primary producers, supporting a complex food web that includes bacteria, fungi and cave-adapted invertebrates. A range of methylotrophic bacteria in Movile Cave grow on one-carbon compounds including methylated amines, which are produced via decomposition of organic-rich microbial mats. The role of methylated amines as a carbon and nitrogen source for bacteria in Movile Cave was investigated using a combination of cultivation studies and DNA stable isotope probing (DNA-SIP) using (13)C-monomethylamine (MMA). Two newly developed primer sets targeting the gene for gamma-glutamylmethylamide synthetase (gmaS), the first enzyme of the recently-discovered indirect MMA-oxidation pathway, were applied in functional gene probing. SIP experiments revealed that the obligate methylotroph Methylotenera mobilis is one of the dominant MMA utilisers in the cave. DNA-SIP experiments also showed that a new facultative methylotroph isolated in this study, Catellibacterium sp. LW-1 is probably one of the most active MMA utilisers in Movile Cave. Methylated amines were also used as a nitrogen source by a wide range of non-methylotrophic bacteria in Movile Cave. PCR-based screening of bacterial isolates suggested that the indirect MMA-oxidation pathway involving GMA and N-methylglutamate is widespread among both methylotrophic and non-methylotrophic MMA utilisers from the cave.