Ferruginibacter paludis and Polaromonas aquatica as biosignatures of high-altitude mountain snowpacks of the Northern Caucasus, Arkhyz and Dombai
Authors: Bulat Sergey , Doronin Maxim , Anosova Oxana , Gorbova Victoria , Sumbatyan Danyl , Khablyuk Victor , Martins Jean 
Published: February 2023

The objective was to investigate the diversity, abundance, and comparative structure of the microbial communities of the multiannual snowpacks of the Northern Caucasus at two distantly (~40km apart) locations &#8211; Arkhyz (two spots &#8211; &#8216;pristine&#8217; at 2546m and &#8216;touristic&#8217; at 2826m) and Dombai (&#8216;touristic&#8217; spot at 3131m) by implementing high-throughput Oxford Nanopore sequencing. The study aims to discover microbial markers that would be useful in tracking &#8216;icy&#8217; microbial communities&#8217; structure in response to ongoing and past climate changes.Two snowpacks of Arkhyz and one snowpack of Dombai were cleanly sampled, and the snow was processed under clean room conditions. The genomic DNA was isolated, the v3-v5 (~590 bp) (Arkhyz) and v3-v4 regions (~485 bp) (Dombai) of bacterial 16S ribRNA genes were amplified, and amplicons generated were nanopore barcode sequenced using MinIon device following Trim barcodes and Fast Basecalling options. &#160;For Arkhyz samples, the following results were obtained. The &#8216;touristic&#8217; snow sample (Ark1) generated about 1140000 reads (92% accuracy) which were classified (17% of total) in 30 phylotypes (Beta-Proteobacteria (~54%), Actinobacteria (~24%), Bacteroidetes (~13%), Alpha-Proteobacteria (~9%)) at 0.5% abundance and 97% similarity levels. Of them, six phylotypes (Ferruginibacter paludis (~9.1%) of Bacteroidetes, Massilia psychrophila, Rhizobacter profundi, Polaromonas aquatica and Aquaspirillum arcticum of Beta-Proteobacteria and Novosphingobium gossypii of Alpha-Proteobacteria) dominated (in the range of 5.2-9.1%).The pristine less altitudinal snow sample (Ark2) generated about 1160000 reads (92% accuracy) which were classified (23% of total) in 27 phylotypes (Actinobacteria (~40%), Beta-Proteobacteria (~24%), Bacteroidetes (~20%), Alpha-Proteobacteria (~14%)) at 0.5% abundance and 97% similarity levels. Of them, four phylotypes (Articella aurantiaca (12.7%) and Ferruginibacter paludis of Bacteroidetes, Polaromonas aquatica of Beta-Proteobacteria and Methylobacterium goesingense of Alpha-Proteobacteria) dominated (in the range of 5.1-12.7%).It is worth noting that two dominant taxa Ferruginibacter paludis and Polaromonas aquatica, inhabiting wetlands and water, respectively, were present in both samples. At the same time, there was a surprising find - the presence of alpha-proteobacteria in both Arkhyz snow samples.For the Dombai snow sample, about 33500 reads (90% accuracy) were obtained, resulting in 22 phylotypes (Bacteroidetes (~60%), Beta-Proteobacteria (~32%), Actinobacteria (~8%)) at 0.5% abundance and 97% similarity levels (38% classified). Of them, the same two phylotypes (Ferruginibacter paludis of Bacteroidetes and Polaromonas aquatica of Beta-Proteobacteria) dominated (13.2% and 12.2%, respectively).When comparing the microbial communities of the snowpacks of Arkhyz and Dombai, the overlapping in the structure is clear &#8211; dominant Phyla Beta-Proteobacteria, Actinobacteria, and Bacteroidetes. Two species, Ferruginibacter paludis of Bacteroidetes and Polaromonas aquatica of Beta-Proteobacteria, dominate all the samples. It seems that the human presence, as well as the thickness and the age of the snowpacks, do not affect the icy microbial community&#8217;s structure. Thus, the discovered dominant bacteria could serve as a biosignature of &#8216;healthy&#8217; high-altitude terrestrial mountain snowpacks and, in the form of a specific assay (e.g., PCR-based), could be used in their monitoring. In addition, it would be worth looking for these bacteria in the nearest past &#8211; searching for them inside terrestrial glaciers.The reported study was funded by RFBR and DFG according to the research project &#8470;20-55-12006.