Prokaryotic and Viral Community Composition of Freshwater Springs in Florida, USA

Abstract

Aquifers, which are essential underground freshwater reservoirs worldwide, are understudied ecosystems that harbor diverse forms of microbial life. This study investigated the abundance and composition of prokaryotic and viral communities in the outflow of five springs across northern Florida, USA, as a proxy of microbial communities found in one of the most productive aquifers in the world, the Floridan aquifer. The average abundances of virus-like particles and prokaryotic cells were slightly lower than those reported from other groundwater systems, ranging from 9.6 × 10³ ml^-1 to 1.1 × 10⁵ ml^-1 and 2.2 × 10³ ml^-1 to 3.4 × 10⁴ ml^-1, respectively. Despite all of the springs being fed by the Floridan aquifer, sequencing of 16S rRNA genes and viral metagenomes (viromes) revealed unique communities in each spring, suggesting that groundwater microbial communities are influenced by land usage in recharge zones. The prokaryotic communities were dominated by Bacteria, and though the most abundant phyla (Proteobacteria, Cyanobacteria, and Bacteroidetes) were found in relatively high abundance across springs, variation was seen at finer taxonomic resolution. The viral sequences were most similar to those described from other aquatic environments. Sequencing resulted in the completion of 58 novel viral genomes representing members of the order Caudovirales as well as prokaryotic and eukaryotic single-stranded DNA (ssDNA) viruses. Sequences similar to those of ssDNA viruses were detected at all spring sites and dominated the identifiable sequences at one spring site, showing that these small viruses merit further investigation in groundwater systems.IMPORTANCE Aquifer systems may hold up to 40% of the total microbial biomass on Earth. However, little is known about the composition of microbial communities within these critical freshwater ecosystems. Here, we took advantage of Florida's first-magnitude springs (the highest spring classification based on water discharge), each discharging at least 246 million liters of water each day from the Floridan aquifer system (FAS), to investigate prokaryotic and viral communities from the aquifer. The FAS serves as a major source of potable water in the Southeastern United States, providing water for large cities and citizens in three states. Unfortunately, the health of the FAS and its associated springs has declined in the past few decades due to nutrient loading, increased urbanization and agricultural activity in aquifer recharge zones, and saltwater intrusion. This is the first study to describe the prokaryotic and viral communities in Florida's first-magnitude springs, providing a baseline against which to compare future ecosystem change.

Keywords: Florida; bacilladnavirus; ecology; freshwater; microbial ecology; phage; prokaryote; springs; ssDNA virus; viral; virome; virus.

FIG 1

Map showing the location of investigated springs and the estimated land usage profile around each spring based on the inferred recharge zone. Data are from the Florida Department of Environmental Protection (DEP). This figure was made using ArcGIS (85).

FIG 2

(A) Venn diagram depicting the distribution of ASVs among springs. (B) NMDS plot showing the similarity of the 16S rRNA gene community structures of each spring site, based on a Bray-Curtis dissimilarity matrix. Biological replicates of each spring are represented by points of identical colors and shapes. The ordinal ellipses represent the 95% confidence interval of the standard error. The arrows represent physiochemical parameters significantly (P < 0.05) correlating with the ordination of the communities. The direction of each arrow indicates direction, and the length denotes magnitude of influence.

FIG 3

(A) Heat map representing the percentages of 16S rRNA gene sequences in each spring belonging to the 25 most abundant phyla. Dark orange indicates a higher percentage of sequence abundance, and white represents the absence of the phylum. (B) Stacked bar chart representing the 10 most abundant families in each spring site. The spotted pattern indicates that the family was in the top 10 most abundant families in all five springs, vertical stripes indicate that it was in the 10 most abundant families in 4 springs, and horizontal stripes indicate that it was in the 10 most abundant families in 3 springs.

FIG 4

(A) Venn diagram depicting the distribution of curated viral contigs among springs. (B) NMDS plot showing the similarity of viral community structures of each spring site based on a Bray-Curtis dissimilarity matrix of the relative abundances of viral contigs measured by read coverage normalized by contig length and library size. Biological replicates of each spring are represented by points of identical shapes and colors. The ordinal ellipses represent the 95% confidence interval of the standard error. The arrows represent physiochemical parameters significantly (P < 0.05) correlating with the ordination of the communities. The direction of each arrow indicates direction, and length denotes magnitude of influence.

FIG 5

Stacked bar plot showing the ecosystem types represented by the sequences in the IMG/VR database that most closely matched the curated viral contig sequences from the springs. Shades of blue represent aquatic ecosystems.

FIG 6

Stacked bar plot of the relative abundances of reads most similar to ssDNA (green) versus dsDNA (blue) eukaryotic viruses (light colors) and prokaryotic viruses (dark colors) in each spring based on best BLASTx hit to the NCBI RefSeq virus database. Relative abundance was estimated through read coverage normalized by contig length and library size. Unassigned viruses were manually curated, and any contigs that could not be assigned to one of the four categories were excluded from analysis. Excluded contigs comprised <1% of the total contigs analyzed for each spring.

FIG 7

Heat maps showing the relative abundance of each complete circular viral genome (calculated as read coverage normalized by genome length and library size). Each row represents one viral genome with 20 dsDNA circular virus genomes on the bottom half of the map and 38 ssDNA circular genomes (including the 3 satellites) on the top. White represents absence of the genome.

FIG 8

Genome maps of the two viruses (A and B) and one satellite (C) found in four of five spring sites. The graph in the center of each map represents normalized read coverage from each spring site. nts, nucleotides.