Nutrient Stoichiometry Shapes Microbial Community Structure in an Evaporitic Shallow Pond

Abstract

Nutrient availability and ratios can play an important role in shaping microbial communities of freshwater ecosystems. The Cuatro Ciénegas Basin (CCB) in Mexico is a desert oasis where, perhaps paradoxically, high microbial diversity coincides with extreme oligotrophy. To better understand the effects of nutrients on microbial communities in CCB, a mesocosm experiment was implemented in a stoichiometrically imbalanced pond, Lagunita, which has an average TN:TP ratio of 122 (atomic). The experiment had four treatments, each with five spatial replicates - unamended controls and three fertilization treatments with different nitrogen:phosphorus (N:P) regimes (P only, N:P = 16 and N:P = 75 by atoms). In the water column, quantitative PCR of the 16S rRNA gene indicated that P enrichment alone favored proliferation of bacterial taxa with high rRNA gene copy number, consistent with a previously hypothesized but untested connection between rRNA gene copy number and P requirement. Bacterial and microbial eukaryotic community structure was investigated by pyrosequencing of 16S and 18S rRNA genes from the planktonic and surficial sediment samples. Nutrient enrichment shifted the composition of the planktonic community in a treatment-specific manner and promoted the growth of previously rare bacterial taxa at the expense of the more abundant, potentially endemic, taxa. The eukaryotic community was highly enriched with phototrophic populations in the fertilized treatment. The sediment microbial community exhibited high beta diversity among replicates within treatments, which obscured any changes due to fertilization. Overall, these results showed that nutrient stoichiometry can be an important factor in shaping microbial community structure.

Keywords: algae; bacteria; beta diversity; community structure; growth rate hypothesis; rRNA gene copy number; stoichiometry.

FIGURE 1

Principal coordinates analysis (PCoA) plot of Bray–Curtis distances for bacterial communities in the water column. Black = unenriched, orange = P-only, light blue = NP16, dark blue = NP75.

FIGURE 2

Box and whisker plots for beta diversity distances of 16S rRNA gene libraries within and between treatments for (A) water and (B) sediment samples. The plot was constructed using the lower and upper quartile of the data (“whiskers” extending from either end of the box; one going from the first quartile to the smallest non-outlier and the other going from third quartile to the largest non-outlier), the inter-quartile range (width of the “box”; the bottom and the top being the lower and upper quartile, respectively), median (red line) and outliers (+ sign). Trt, treatment.

FIGURE 3

Taxonomic heat map of the 50 most abundant phylotypes demonstrating the effect of nutrient application on the composition of bacterial communities in the water column and sediment of the mesocosms. The color scale represents relative abundance as the average percent read totals of five replicates within a treatment, shown in log scale.

FIGURE 4

Fold change (log₂FC) in bacterial phylotype abundance between U and each of the fertilized treatments (orange = P-only, light blue = NP16, dark blue = NP75). Only genera with fold change with p-value ≤ 0.05 are represented in the figure. ^∗Represents change with false discovery rate (FDR) ≤ 0.05. B0088: Actinobacteria; Actinobacteria; PeM15 B0127: Bacteroidetes; VC2.1 Bac22 B0172: Bacteroidetes; Cytophagia; Algoriphagus B0234: Bacteroidetes; Flavobacteriia; Owenweeksia B0330: Bacteroidetes; Sphingobacteriia; Lewinella B0335: Bacteroidetes; Sphingobacteriia; uncultured Saprospiraceae B0800: Proteobacteria; Alphaproteobacteria; Brevundimonas B0802: Proteobacteria; Alphaproteobacteria; Phenylobacterium B0816: Proteobacteria; Alphaproteobacteria; DB1-14 B0978: Proteobacteria; Alphaproteobacteria; Roseovarius B0980: Proteobacteria; Alphaproteobacteria; Rubribacterium B0992: Proteobacteria; Alphaproteobacteria; Tabrizicola B1001: Proteobacteria; Alphaproteobacteria; uncultured Rhodobacteraceae B1019: Proteobacteria; Alphaproteobacteria; Roseococcus B1096: Proteobacteria; Alphaproteobacteria; Candidatus Captivus B1130: Proteobacteria; Alphaproteobacteria; Erythrobacter B1131: Proteobacteria; Alphaproteobacteria; Porphyrobacter B1162: Proteobacteria; Betaproteobacteria; GKS98 freshwater bacteria B1195: Proteobacteria; Betaproteobacteria; Polaromonas B1202: Proteobacteria; Betaproteobacteria; Variovorax B1556: Proteobacteria; Gammaproteobacteria; Vibrio

FIGURE 5

Maximum-likelihood phylogenetic tree of planktonic Alphaproteobacteria B1001.

FIGURE 6

Principal coordinates analysis plot of Bray–Curtis distances for eukaryote communities in the water column. Black = unenriched, orange = P-only, light blue = NP16, dark blue = NP75.

FIGURE 7

Box and whisker plot for beta diversity distances of 18S rRNA gene libraries within and between treatments for (A) water and (B) sediment samples. The plot was constructed using the lower and upper quartile of the data (“whiskers” extending from either end of the box; one going from the first quartile to the smallest non-outlier and the other going from third quartile to the largest non-outlier), the inter-quartile range (width of the “box”; the bottom and the top being the lower and upper quartile, respectively), median (red line) and outliers (+ sign). Trt, treatment.

FIGURE 8

Taxonomic heat map of the 50 most abundant phylotypes demonstrating the effect of nutrient application on the composition of eukaryotic communities in the water column and sediment of the mesocosms. The color scale represents relative abundance as the average percent read totals of five replicates within a treatment, shown in log scale.

FIGURE 9

Fold change (log₂FC) in eukaryotic genera abundance between the control treatment (U) and each of the fertilized treatments (orange = P-only, light blue = NP16, dark blue = NP75). Only genus with fold change that has p-value ≤ 0.05 is represented in the figure. ^∗Represents change with FDR ≤ 0.05. E064: Archaeplastida; Chloroplastida; Tetranephris E090: Archaeplastida; Chloroplastida; unclassified E260: Opisthokonta; Metazoa; Flosculariacea E261: Opisthokonta; Metazoa; Ploimida E395: SAR; Alveolata; A31