Extreme environments are usually considered ecosystems with reduced biological complexity due to extreme salinity, temperature and solar radiation [48]. Accordingly, the microbial mats of Guerrero Negro, Baja California Sur, Mexico, have been considered simple systems, dominated by cyanobacteria and sulphate-reducing bacteria [49]. However, recent studies [50, 51], have reported that the microbial communities of ESSA at Guerrero Negro are highly complex, with an unexpected diversity. The results obtained in this study allow for an increased knowledge of the unexplored archaeal diversity in these hypersaline microbial mats (Fig. S1).
The archaeal 16S rRNA analysis showed that six of the ten recovered phyla belonged to the recently proposed super-cluster DPANN, with a remarkable dominance of the phylum Nanoarchaeota, observed in all samples analysed (Table S2). This phylum, as well as several other of the understudied members of DPANN, are distinguished by reduced cell sizes, genes and genomes, rapidly evolving gene sequences and the absence of some primary biosynthetic core genes, such as those involved in respiration and ATP synthesis, which confer them with limited metabolic capacities [52–54]. These genetic characteristics translate into an evolved dependence as a mutualist, commensalistic or parasitic, ecto- endo-symbiotic lifestyle [53, 55]. In addition, their presence also evidenced that microbial mats from Guerrero Negro harbour symbiotic or syntrophic lifestyles and highly diverse archaeal populations with a high number of uncultivated low-abundance species with unknown physiologies.
Several phyla of the DPANN super-cluster have shown a symbiotic lifestyle, which could explain why site A5, a particular ASV signature of the order Micrarchaeales appeared in high relative abundance together with some signatures of members assigned to the class Thermoplasmata, where particular species of these taxa, as well as members of the Nanoarchaeota, have been reported to physically interact through pili-like-structures [52, 56]. Furthermore, shotgun analysis of A5 also showed the presence of both types of symbiont members, specifically the presence of Ca. Microarchaeum sp. and Ca. Mancarchaeum acidiphilum of Micrarchaeota, as well as Cuniculiplasma divulgatum of Thermoplasmata (Table S3). It is important to highlight that most of the interactions discovered so far occur between acidophilic or thermophilic members of both taxa [55, 57]. Therefore, the recovery of relatively strong signals of these archaea in a hypersaline environment (12.36%), and with a moderately alkaline pH (8.34), suggests that members of these taxa could have a wider range of tolerance to different physico-chemical conditions [55, 58], and, hence, play potentially different ecological roles in this environment.
A previous study [59] found a strong co-occurrence between Wosearchaeotales and methanogens (Methanomicrobia and Methanobacteria), proposing a syntrophic metabolic model by a consortium of H2/CO2-using and acetate-using methanogens and members of the order Woesearchaeles, phylum Nanoarchaeota. This hypothesis could explain the observed high abundances of both Nanoarchaeota and Methanomicrobiales in our study, opening new perspectives regarding the possible interactions among them.
In turn, our results exhibited a low abundance of the recently discovered archaea of the new class Lokiarcheia (superphylum Asgard). This group has been considered as one of the major achievements regarding the exploration of uncultivated diversity of this domain [60, 61]. Although all four sites had the same dominant phyla in archaeal communities, the estimated PCoA in a weighted UniFrac distance matrix showed significant differences in the community structure among the sites (Fig. S2). These differences were consistent despite the observation of a small interaction of ASVs among sites, indicating the presence of site-specific phylotypes (Table S2). The results suggest that highly diverse populations with low-abundant ASVs (rare biosphere) are important in shaping the community structure and hence, represents a reservoir of genetic diversity that actively responds to environmental perturbations [62–64]. Moreover, the results of beta diversity analysis pinpoint that archaeal populations are adapted to the specific environmental conditions of each site, specifically salinity, which was different at each site (Table 1).
Methanogenic Diversity In Hypersaline Microbial Mats
Taxonomic assignment based on 16S rRNA and mcrA sequences, and metagenomic analysis, evidenced the presence of Methanosarcinales, which is the better studied methanogenic order in hypersaline environments [5, 6, 8, 65]. However, this study also evidenced the presence of the orders Methanobacteriales, Methanomicrobiales, Methanomassiliicoccales, Ca. Methanofastidiosales, Methanocellales, Methanococcales and Methanopyrales, although some of these were present at extremely low relative abundances (Tables S2 and S3, Fig. S3), suggesting that hypersaline microbial mats of Guerrero Negro harbour a previously unexplored diversity of methanogens. It should be noted that the results may be limited by the reference databases used for the taxonomic classification of each gene. These findings highlight the necessity to use a variety of marker genes to characterise a broader spectrum of the methanogenic diversity residing in this ecosystem. Similar results have been reported for anaerobic digesters [66], but studies in extreme environments are scarce [67].
The lack of new cultures of methanogens [64] has limited the assignment of the environmental mcrA sequences at low taxonomic levels. Several mcrA environmental sequences obtained in this study were significantly different from those previously reported and did not match with any known methanogenic archaea, suggesting the presence of specific environmental clusters of methanogenic archaea in Guerrero Negro. The recovery of genomes of uncultured groups from environmental metagenomes could result in the description of several new higher taxonomic levels, such as phylum [4]. However, since in this study only a small fraction of incomplete archaeal genomes was assembled due to their low abundance, further deeper sequencing or additional strategies to recover archaeal genomes from metagenomes are needed.
Strains recovered from hypersaline lakes have resulted in a new class of archaea (Halobacteria) that are methyl-reducing methanogens that use C1 methylated compounds as electron acceptors, and H2 or formate as electron donors [68]. We presume that the unknown environmental Guerrero Negro clusters, closely related with Methanonatronarchaeia archaeon and unrelated with hydrogenotrophic, acetoclastic and methylotrophic methanogens members, could be relatives of this new class of Euryarchaeota (Fig. 2). We observed the presence of sequences belonging to the class Thermoplasmata in all sampled sites (Fig. 1B), which could be related to uncultured archaeal lineages. This group of uncultured archaea is poorly studied, and new members have been described as hydrogen-dependent methylotrophs.
No signatures belonging to methanogenic members of the groups Verstraetearchaeota, Bathyarchaeota, Hadesarchaeota or Nezhaarchaeota, were detected. Additionally, no sequences from methanotrophic groups (i.e., ANME Class Methanomicrobia, Helarchaeota or Korarchaeota) were identified. These results suggest that methanogenesis in hypersaline microbial mats is restricted to Euryarchaeota and Thermoplasmata, whereas anaerobic methane oxidation is absent [69]. However, methane oxidation by aerobic members of bacteria cannot be ruled out.
Until recently, all known species of aerobic methanotrophs belonged to the phylum Proteobacteria, in the classes Gammaproteobacteria and Alphaproteobacteria. However, thermoacidophilic methanotrophs were described that represented a distinct lineage within the bacterial phylum Verrucomicrobia. In this study, sequences with low relative abundances, assigned to the family Methylacidiphilaceae phylum Verrucomicrobia were detected which have been described in the literature as obligate aerobic methylotrophs capable of growth on methane and methanol. Draft genomic analyses have shown that the pmoCAB operon structure is the same as observed in proteobacterial methanotrophs, and phylogenetic analyses demonstrate that the proteobacterial and verrucomicrobial pmoA genes that encode to particulate methane monooxygenase evolved from a common ancestor [70]. In addition, 16S rRNA sequences were detected for the phylum Gemmatimonadota, which are potentially capable of aerobic methanotrophy as was indicated by the detection of genes that encode methane monoxygenase, pmoA, mmoA [71].
Methanogenic Metabolisms In Hypersaline Microbial Mats
Methylotrophic metabolism has been considered the only methanogenic pathway occurring within hypersaline environments [72, 73]. Accordingly, we found the presence of several methylotrophic members of the order Methanosarcinales, as well as methylotrophic genes in the metagenome. Furthermore, putative hydrogenotrophic members related to Methanomicrobiales have also been reported for this environment [8]. In this study, we hypothesised that all four of the methanogenic pathways could occur in hypersaline environments, based on functional inference and the detection of specific genes in the metagenome. Methylotrophic and acetoclastic methanogens of the Methanosarcinales order were well represented (Table S3). Moreover, hydrogenotrophic methanogenic members of the orders Methanobacteriales, Methanomicrobiales, Methanocellales, Methanococcales and Methanopyrales were also observed (Table S3). The presence of hydrogen-dependent methylotrophic methanogens was also evidenced by the detection of members related with the orders Methanomassiliicoccales and Ca. Methanofastidiosales (Table S3), and by the detection of genes in the metagenome that are related with methylotrophic metabolism, presumably lacking the Wood-Ljungdahl pathway [15].
Although the first isolates and cultures of Methanomassiliicoccales have been obtained from human faeces, termite guts and water treatment sludge [74–76], recently, it has been reported that metagenome-assembled genomes (MAGs) of this clade were recovered from natural environments [77–80]. In the current study, through 16S rRNA and mcrA amplicon sequencing approaches, members that belong to the methanogenic taxa Ca. Methanofastidiosa, and presumptive members closely related to the order Methanomassiliicoccales, were recovered from our samples. To our knowledge, this is the first report describing members of microbial communities harbouring hydrogen-dependent methylotrophic methanogenesis metabolism recovered from a hypersaline environment. These findings provide insight regarding their ecological importance and suggest that high salinity does not limit their presence.
Bacterial composition analysis (Table S2) showed that specific bacterial phyla recovered have key roles as detritus and polysaccharide degraders, and are also reported to produce extracellular proteases, glycosyl hydrolases, and lipases [81–85]. Additional members of these, and other bacterial groups, that are described to be involved in the subsequent steps of the organic matter degradation, through the fermentative, acidogenic and acetogenic pathways, which ultimately give rise to the methane formation, were also retrieved [86, 87].
In hypersaline environments methylated compounds play a central role in the production of methane [65, 73] and their occurrence might be explained by the conversion of osmolytes, such as glycine-betaine and choline up to trimethylamine by representatives of the Class Clostridia [88], Halanaerobiia [89] and by sulphate-reducing bacteria [90], while the trimethylamine N-oxide (TMAO) can be reduced to TMA by the bacterial genera Alteromonas, Flavobacterium, and halophilic archaea [88, 91]. This could be consistent with results described by [92], who pointed out the requirement of H2/methyl substrates and acetate for the growth and methanogenic activity of Methanomassiliicoccales. Furthermore, the apparent adaptation of Methanomassiliicoccales to thrive in sediments with high sulphate concentrations [93], as well as the specialisation of Ca. Methanofastidiosa in the use of methylated thiols as a methanogenic substrate [94], not only establishes a bridge between the carbon and sulphur cycles in eutrophic environments but also a potential contribution to the regulation of the H2 partial pressure in the microbial mats.
On the other hand and given that hypersaline environments exhibit a high rate of sulphate reduction [95], the finding of genes associated with all methanogenic pathways (Table 2) suggests the coexistence of not only novel methanogens, but also hydrogenotrophic and acetoclastic groups with sulphate-reducing bacteria, despite their competition for H2 and acetate [96]. Similar coexistence patterns have been observed in non-oligotrophic environments, such as estuarine and marine sediments, tropical coastal lagoons and mangroves [77, 80, 97, 98]. These results support the hypotheses of the occurrence of putative hydrogenotrophic methanogens in the ecological functioning of hypersaline microbial mats.