Differences in Temperature and Water Chemistry Shape Distinct Diversity Patterns in Thermophilic Microbial Communities

Abstract

This report describes the biodiversity and ecology of microbial mats developed in thermal gradients (20 to 65°C) in the surroundings of three drillings (Chiraleu [CH], Ciocaia [CI], and Mihai Bravu [MB]) tapping a hyperthermal aquifer in Romania. Using a metabarcoding approach, 16S rRNA genes were sequenced from both DNA and RNA transcripts (cDNA) and compared. The relationships between the microbial diversity and the physicochemical factors were explored. Additionally, the cDNA data were used for in silico functionality predictions, bringing new insights into the functional potential and dynamics of these communities. The results showed that each hot spring determined the formation of distinct microbial communities. In the CH mats (40 to 53°C), the abundance of Cyanobacteria decreased with temperature, opposite to those of Chloroflexi and ProteobacteriaEctothiorhodospira, Oscillatoria, and methanogenic archaea dominated the CI communities (20 to 65°C), while the MB microbial mats (53 to 65°C) were mainly composed of Chloroflexi, Hydrogenophilus, Thermi, and Aquificae Alpha-diversity was negatively correlated with the increase in water temperature, while beta-diversity was shaped in each hot spring by the unique combination of physicochemical parameters, regardless of the type of nucleic acid analyzed (DNA versus cDNA). The rank correlation analysis revealed a unique model that associated environmental data with community composition, consisting in the combined effect of Na⁺, K⁺, HCO₃^-, and PO₄^3- concentrations, together with temperature and electrical conductivity. These factors seem to determine the grouping of samples according to location, rather than with the similarities in thermal regimes, showing that other parameters beside temperature are significant drivers of biodiversity.IMPORTANCE Hot spring microbial mats represent a remarkable manifestation of life on Earth and have been intensively studied for decades. Moreover, as hot spring areas are isolated and have a limited exchange of organisms, nutrients, and energy with the surrounding environments, hot spring microbial communities can be used in model studies to elucidate the colonizing potential within extreme settings. Thus, they are of great importance in evolutionary biology, microbial ecology, and exobiology. In spite of all the efforts that have been made, the current understanding of the influence of temperature and water chemistry on the microbial community composition, diversity, and abundance in microbial mats is limited. In this study, the composition and diversity of microbial communities developed in thermal gradients in the vicinity of three hot springs from Romania were investigated, each having particular physicochemical characteristics. Our results expose new factors that could determine the formation of these ecosystems, expanding the current knowledge in this regard.

Keywords: hot spring; metabarcoding; microbial diversity; microbial ecology.

FIG 1

Quantification of 16S rRNA gene and transcript abundances by qPCR in each of the investigated hot spring microbial mats.

FIG 2

Relative abundances of prokaryotic classes and phyla encountered in the hot spring microbial mats. The sequencing results from both the DNA- and cDNA-based approaches are shown side by side. Different colors are used to represent the three sampling sites: yellow, Chiraleu (CH); pink, Ciocaia (CI); and blue, Mihai-Bravu (MB).

FIG 3

Shannon and Simpson diversity indices, the Chao1 richness estimator, and the number of observed OTUs in the samples from Chiraleu, Ciocaia, and Mihai Bravu, together with other microbial mats from similar sites (China, SRX206469, SRX206467, SRX206466, SRX206468, SRX206459, SRX206460, and SRX206456 [7]; India, SRS932137 and SRS932073 [89]; and Malaysia, PRJEB7059 [61]). Colors show the temperature measured at each sampling site, the shapes correspond to different nucleic acids, and the sizes are proportional to the pH values.

FIG 4

Principal-coordinate analysis (PCoA) based on weighted (a) and unweighted (b) UniFrac distances of microbial community structure. The first two components were used for plotting, along with the percent variation explained by them. Colors show the temperature specific to each microbial mat, and the shapes correspond to different sampling locations. The strength of statistical significance (P < 0.05) for the environmental parameters as explanatory variables is represented by solid arrows.

FIG 5

UPGMA tree constructed based on Bray-Curtis distances for a comparative analysis of beta-diversity of the Romanian samples and other hot spring microbial mats reported in the literature. Sample codes correspond to the following locations: Qucai, Tibetan Plateau, China (samples QC9 [SRX206469], QC7 [SRX206468], and QC2 [SRX206467]); Naqu, Tibetan Plateau, China (sample NQ4 [SRX206466]); Gulu, Tibetan Plateau, China (samples GL3.4 [SRX206459], GL9 [SRX206460], and GL20 [SRX206456]) (7); Jakrem, India (SRS932137); Yumthang, India (SRS932073) (89); and Sungai Klah, Malaysia (PRJEB7059) (61).

FIG 6

(A) Microbial mats formed in thermal gradients surrounding the Chiraleu drilling (arrows indicate the sampling sites); (B) detailed view of the green, slimy mat developed at 53°C, without a clear vertical stratification; (C) green to yellow microbial mat formed at 46°C, with a typical laminated structure; (D) thin microbial mat developed at 40°C, with a smooth orange top layer followed by green and brown layers; (E) microbial mats in the proximity of the Ciocaia drilling; (F) green, mucilaginous stratified microbial mat developed at 20°C; (G) red and slimy microbial mat formed at 35°C that does not show a clear stratification and which is thinner than the mat at 20°C; (H) white microbial mat developed at 65°C, more consolidated and firm near the substrate and with a mucilaginous, translucent surface layer; (I) microbial mats developed in thermal gradient in proximity of the Mihai Bravu drilling; (J) green, compact, and smooth microbial mat at 53°C, with thin layers; (K) stratified microbial mat at 59°C, with an orange top layer and green layers underneath; (L) long, flexible structures forming cream-colored streamers at 65°C along the flowing hot water.