Chamber monitoring
The chamber maintained a constant 5 °C, 50% relative humidity, and -15 °C in the lower permafrost. Before starting the experiment, Lake Hoare soils presented a moisture content of 0.4 ± 0.3%, pH of 8.75 ± 0.2, and electrical conductivity of 268 ± 267 µS. A power failure affected the chamber and interrupted the permafrost freezing process 29 days into the experiment leading to an increase in the chamber's temperature and consequent alteration of the relative humidity and defrosting of the permafrost layer for three consecutive days (Supplementary material Fig. S2). This event was recorded and likely contributed to the variance observed at T4 and T5 in the dry control samples. We did not identify other impacts in samples submitted to wetting (Fig. 2).
Sequencing analysis
After quality control steps, we identified 2,779 ASVs. Chimera check identified 431 bimeric sequences, which were subsequently removed, generating 2,348 unique ASVs and 1,339,818 reads (average length 215 bp) across 50 samples. Filtering out low abundant ASVs (< 0.007% cutoff across the entire dataset) removed 59% of the initial ASVs. The additional removal of ASVs classified as mitochondria (0 ASVs), chloroplast (2 ASVs), eukaryotes (7 ASVs), not classified at the Phylum level (204 ASVs), not present or with only one sequence at T0 (48 ASVs), removed an additional 27% of the ASVs. The remaining dataset comprised 1,108,012 sequences representing 82% of the initial sequencing data and 706 ASVs.
Temporal dynamics of community diversity during the wetting treatment
Microbial α-diversity, as measured by phylogenetic diversity (Faith's Phylogenetic Diversity), decreased over time in both the wetting treatment and wetting/re-drying treatment but not within the dry control (one-way ANOVA, p < 0.05, Supplementary material Fig. S3). The decrease in α-diversity was most prominent during the wetting treatment after five weeks (T4) (Supplementary material Fig. S3, Table S1). Additional α-diversity measures supported this trend: the observed number of ASVs, Chao1 richness, and Shannon index (data not shown).
Microbial community β-diversity changed with time and treatment (Fig. 2). Samples were clustered by sampling time point across the primary axis (55% of the variability within the dataset) and treatment across the secondary axis (22% of the variability within the dataset) (Fig. 2). During the four weeks of constant wetting (T0-T3), β-diversity increased between communities from the dry control and the two wetting treatments (Fig. 3; one-way ANOVA, p < 0.05). However, once the wetting ceased (T3), β-diversity decreased between the control and communities submitted to the re-drying process (T5) (Fig. 3).
Communities from the dry control samples were compositionally and structurally similar to field samples between T0 and T3. We also did not observe any significant initial variance in β-diversity amongst dry control samples until T3 (Fig. 2). Nonetheless, one month since the start of the experiment, β-diversity increased amongst the control samples becoming significant between time-points T4/T5 and T1/T2 (Supplementary material Fig. S4). We suggest the latter could be associated with the chamber breakdown (occurring between T3 and T4), which led to an increase in temperature and changes in relative humidity (Supplementary material Fig. S2). While changes in β-diversity were significant between the control samples taken at T4 and T5 and the control samples at the beginning of the experiment (T1/T2), the dissimilarity distance between those communities was still smaller than when comparing the dissimilarity distances between samples subjected to wetting and the dry control (Fig. 3, Supplementary material Fig. S4). Therefore, we do not expect that the increase in temperature and relative humidity caused by the chamber power failure significantly impacted the communities already subjected to the wetting/re-drying treatment. This observation also highlights the more predominant effect of changes in moisture on microbial communities from ice-free regions than alterations in temperature.
Microbial compositional changes during the wetting treatment
Temporal changes in the relative abundances of microbial taxa were analyzed by aggregating all taxa at the phylum (Fig. 4) and family (Fig. 5) levels. Four major bacterial phyla dominated dry soil communities alongside the field samples: Actinobacteriota (39%), mainly composed of the family Solirubrobactereaceae; Bacteroidota (20%), primarily consisting of the family Chitinophagaceae; Acidobacteriota (11%), mainly represented by the family Blastocatellaceae, and Proteobacteria (10%) primarily consisting of the Alphaproteobacteria family Sphingomonadaceae. This structure remained consistent in the dry control soils throughout the entirety of the experiment (Fig. 4).
During the wetting treatment and the initial 4-week wet phase of the wetting/re-drying treatment, the phylum Proteobacteria (representing 10% of the initial community) doubled in relative sequence abundance to 23% from T0 to T3. By the end of the experiment (T5), the relative sequence abundance of this phyla was three times higher (from 10% to 31%) compared to the initial sample (T0) in the wetting treatment (Fig. 4). The Proteobacteria family Sphingomonadaceae (Alphaproteobacteria) dominated the community apart from T3 when it was succeeded by taxa belonging to the Oxalobacteraceae family (Fig 5A). After one week of constant wetting (T2 onwards), we observed a significant increase in the Comamonadaceae and Xanthomonadacea, both belonging to the subdivision Gammaproteobacteria (Fig. 5A). Alongside Proteobacteria, members of the low abundant phylum Plactomycetota increased steadily by 2.5% in their relative sequence abundance throughout the wetting treatment (Fig. 4). Bacteroidota phylum had an overall increase of 5% with constant wetting treatment compared to the relative abundance at the beginning of the experiment (T0) (Fig. 4). However, throughout the wetting treatment, different families showed an asynchronous response toward this perturbation (Fig. 5D). In contrast, the relative sequence abundance of the Actinobacteroidota phylum declined from 39% to 11% in response to wetting (Fig. 4). A 23% reduction in relative sequence abundance across all detected Actinobacteroidota families signs the negative impact of wetting in this phylum (Fig. 5B). Taxa within the Acidobacteriota and Chloroflexi also decreased relative sequence abundance by 3% after eight weeks of continuous wetting (Fig. 4). Such decline was more pronounced in the Blastocatellaceae family (Fig. 5C). Low abundant phyla, such as Deinococcota, despite an initial increase by 0.02% in relative sequence abundance with wetting, decreased by 0.05% from T2 to T5 (Fig. 4).
Microbial compositional changes during the wetting/re-drying treatment
Four weeks after the wetting ceased, the relative sequence abundance of Proteobacteria decreased by 3%. By the end of the experiment (T5), it was 10% less abundant than the samples that were kept wet (Fig. 4). The response to the re-establishment of dry conditions was particularly evident for the dominant families Sphingomonadaceae, Comamonadaceae, and Oxalobacteraceae (Fig. 5A). In contrast, after four weeks of drying (T5), the relative sequence abundance of Actinobacteriota was 14% higher than those in the wetting treatment but still comparatively 9% lower than the dry control samples (Fig. 4). The family Solirubrobactereacea in particular, showed a slow recovery after the drying process started (Fig. 5B). Acidobacteria were 5% more abundant than those in the wetting treatment and reached a similar relative sequence abundance to those observed in the dry control samples (15%) (Fig. 4). Blastocatellaceae and Pyrinomonadaceae families showed signs of a quick recovery one week after the drying process started (T4) (Fig. 5C). Despite the positive response to wetting, the Bacteroidota phylum also increased relative sequence abundance once the drying process started, being 3% more abundant when compared to the dry control (Fig. 4). The latter was mainly driven by the 8% increase in relative sequence abundance of the family Chitinophagaceae compared to the beginning of the experiment (T0). All other Bacteroidota families declined in relative sequence abundance with the re-establishment of dry soil conditions (Fig. 5D).
Differentially abundant ASVs during wetting and wetting/re-drying periods
We used DESeq2 to identify the ASVs that were differentially abundant between the treated samples and the control after 24 hours (T1), one week (T2), four weeks (T3), five weeks (T4), and eight weeks (T5) (Fig. 6). At T1, T2, and T3, we identified 96, 144, and 116 ASVs significantly different between the wetting treatment and the control. The number of ASVs continued to increase at T4 and T5, with 202 and 194 ASVs significantly different from the control. Of the identified ASVs, those that became less abundant than the control (log2 fold change, LFC < 0) continuously increased with the exposure to wet conditions, from 36% (T1) to 58% (T5), while those that became more abundant with wetting decreased from 64% (T1) to 42% (T5) (Fig. S5, Table S2).
In the wetting/re-drying treatment, the number of ASVs significantly different from the control was similar to those identified in the wetting treatment, with 105, 139, and 119 ASVs found at T1, T2, and T3, respectively. However, one week after the wetting ceased (T4), we only identified 54 ASVs as significantly different from the control, decreasing to 48 ASVs one month after (T5) (Fig. 6, Table S2). By the end of the experiment, communities submitted to an initial wetting disturbance followed by a re-drying event resembled the dry control communities more than the wet counterparts (Fig. 6). Of the identified ASVs, those that became less abundant than the control during the wetting phase (T1 to T3) represented 41% of the identified ASVs. During the re-drying phase, the percentage of ASVs that became more abundant than the control increased from 59% (T3) to 65% (T5) (Fig. S5, Supplementary material Table S2).
Most ASVs affiliated to the phyla Actinobacteroidota, Bacteroidota (family Chitinophagacea), Chloroflexi, and Acidobacteriota decreased relative sequence abundance with wetting. In contrast, the relative sequence abundance of ASVs affiliated with Proteobacteria, Bacteroidota, and Planctomycetota increased during wetting (Fig. 6, Supplementary material Table S3).
Phylogenetic structure of the communities during the wetting treatment
Microbial communities displayed significant phylogenetic clustering in dry soils (Dry control NTI mean values: 2.13 ± 0.20) during the experiment. This pattern was significantly affected by prolonged wetting (one-way ANOVA, p < 0.05), depicted by a shift towards a stochastic assembly of the community after four weeks of daily wetting (T3) (wetting treatment NTI mean values: 1.38 ± 0.70; wetting/re-drying treatment mean values: 1.75 ± 0.2) (Fig. 7). We observed a second shift after T3 once wetting ceased and the re-drying phase started (Fig. 7).