Distribution, abundance, and diversity patterns of the thermoacidophilic "deep-sea hydrothermal vent euryarchaeota 2"

Abstract

Cultivation-independent studies have shown that taxa belonging to the "deep-sea hydrothermal vent euryarchaeota 2" (DHVE2) lineage are widespread at deep-sea hydrothermal vents. While this lineage appears to be a common and important member of the microbial community at vent environments, relatively little is known about their overall distribution and phylogenetic diversity. In this study, we examined the distribution, relative abundance, co-occurrence patterns, and phylogenetic diversity of cultivable thermoacidophilic DHVE2 in deposits from globally distributed vent fields. Results of quantitative polymerase chain reaction assays with primers specific for the DHVE2 and Archaea demonstrate the ubiquity of the DHVE2 at deep-sea vents and suggest that they are significant members of the archaeal communities of established vent deposit communities. Local similarity analysis of pyrosequencing data revealed that the distribution of the DHVE2 was positively correlated with 10 other Euryarchaeota phylotypes and negatively correlated with mostly Crenarchaeota phylotypes. Targeted cultivation efforts resulted in the isolation of 12 axenic strains from six different vent fields, expanding the cultivable diversity of this lineage to vents along the East Pacific Rise and Mid-Atlantic Ridge. Eleven of these isolates shared greater than 97% 16S rRNA gene sequence similarity with one another and the only described isolate of the DHVE2, Aciduliprofundum boonei T469(T). Sequencing and phylogenetic analysis of five protein-coding loci, atpA, EF-2, radA, rpoB, and secY, revealed clustering of isolates according to geographic region of isolation. Overall, this study increases our understanding of the distribution, abundance, and phylogenetic diversity of the DHVE2.

Keywords: acidophile; archaea; biogeography; deep-sea; hydrothermal vents; multi-locus sequence analysis; thermophile.

Figure 1

Photograph of deep-sea hydrothermal vent mineral deposits from the Eastern Lau Spreading Center. A series of horizontal flanges are shown in the foreground while two vertical chimneys can be seen in the background. Photo was taken from ROV Jason II (courtesy of Woods Hole Oceanographic Institution).

Figure 2

Percentage of DHVE2 16S rRNA gene sequences in the archaeal communities of hydrothermal vent deposits from several different vent fields as determined using qPCR. →, Indicates a chimney sample with a high proportion of DHVE2 sequences while ⋆ indicates two samples collected from a single flange deposit and illustrates the spatial heterogeneity of the DHVE2. Abbreviations are: Rb, Rainbow; TaM, Tahi Moana; TC, Tow Cam; TuM, Tui Malila; LS, Lucky Strike; Guay09, Guaymas Basin 2009; KM, Kilo Moana; EPR07, East Pacific Rise 2007.

Figure 3

Results of local similarity analysis showing OTUs that are negatively (red lines) and positively (green lines) correlated with the DHVE2 (yellow circles). Circles represent OTUs and are colored according to family level taxonomic classification by the RDP-classifier when possible. Color key is as follows: white, unclassified Archaea; pink, unclassified Euryarchaeota; red, unclassified Crenarchaeota; orange, Nanoarchaeota; purple, unclassified Desulfurococcales; lavender, Thermoplasmatales; light green, Archaeoglobaceae; light blue, Methanococcaceae; gray, Thermococcaceae; brown, Thermofilaceae; black, Pyrodictaceae; dark blue, Desulfurococcaceae; dark green, Thermoproteaceae.

Figure 4

Neighbor-joining tree based on 16S rRNA gene sequence comparisons of novel DHVE2 isolates and other archaeal families. Note that strains MAR08-276, MAR08-307, and MAR08-361 were not included in MLSA analysis as ambiguities were observed in protein-coding genes but not in 16S rRNA gene sequences. Bootstrap percentages above 50% are shown for the neighbor-joining analysis (based on 500 replicates) and for the maximum-likelihood analysis (based on 100 replicates). The phylogenetic tree was generated considering only unambiguously aligned nucleotide positions for a diversity of Archaea (n = 789). New isolates are shown in bold. The scale bar represents 0.01 changes per nucleotide position. All 16S rRNA gene sequences from the DHVE2 isolates were deposited in the European Nucleotide Archive database under accession numbers FR865176 to FR865190.

Figure 5

Maximum-likelihood bootstrapped phylogenetic trees of various protein-coding genes from DHVE2 isolates. (A) atpA consensus tree constructed using the Kimura 2-parameter model (Kimura, 1980) taking into account a gamma distribution for the substitution rate. (B) rpoB consensus tree constructed using the Tamura-Nei model (Tamura and Nei, 1993). (C) secY consensus tree generated using Tamura 3-parameter model (Tamura, 1992). (D) EF-2 consensus tree generated as in (C). (E) radA consensus tree generated as in (A). Bootstrap values greater than 60 are shown at nodes in each tree. Branch lengths are the number of substitutions per site. Gene sequences from Thermoplasma acidophilum were used as outgroups in all trees [Gene ID’s (A) = 1455676, (B) = 1456006, (C) = 1456739, (D) = 1456055, (E) = 1456613].

Figure 6

Regional G + C% differences among protein-coding loci from DHVE2 isolates. Average shown, bars indicate range of observed values.

Figure 7

Synonymous and non-synonymous nucleotide positions and substitutions in protein-coding loci among DHVE2 isolates from different regions. (A) Isolates from the MAR compared to isolates from the ELSC (B) isolates from the ELSC compared to isolates from the East Pacific Rise; (C) isolates from the MAR compared to the EPR. Mean values shown on graph, range given with bars. Abbreviations are: Pos, possible synonymous or non-synonymous nucleotide positions; Dif, actual synonymous or non-synonymous differences.