To assess the diversity of Archaea
present in each water sample, cells were collected by centrifugation, their DNA extracted, and 16S rRNA genes PCR amplified using haloarchaeal-specific primers. The products were cloned into TOPO plasmids and sequenced (see Methods), and about 40, near-complete 16S rRNA gene sequences (average of 1,440 nt) were determined for each sample. Table summarizes the numbers of clones sequenced, the number of 97% sequence similarity OTUs, indices of community variation and estimates of species richness. Sequences were imported into the ARB phylogeny package, aligned to the Greengenes pre-aligned dataset (http://greengenes.lbl.gov
), and compared by percentage similarity (Fig. ) and phylogenetic tree reconstructions (Fig. ).
Fig. 2 Diversity of archaeal clone library sequences present in saltern crystalliser ponds at the three sites. Cloned 16S rRNA genes were sequenced, and the aligned sequences were collected into OTU at the 97% sequence similarity level (furthest neighbor, MOTHUR). (more ...)
Fig. 3 Phylogenetic tree reconstruction using cloned 16S rRNA gene sequences derived from three saltern crystallizer ponds (see “Materials and methods” for details). Shown is a representative Maximum Likelihood tree (AxML, ARB phylogeny package). (more ...)
As expected from the initial microscopy, sequences related to Haloquadratum
dominated the clone libraries of all three samples, representing just over 40% of the sequences at each site (Fig. ). Halorubrum
-related sequences were also significant at all sites (8–20%), while Halonotius
-related sequences were present at two sites (10–15%). The latter group was identified in a previous study (Burns et al. 2004a
), and has recently been formally described (Burns et al. 2009a
). The number of 97%-similarity groups (97%-OTUs) varied between 9 and 16, a relatively low number compared to the microbial communities found in non-extreme environments (Janssen et al. 2002
), but typical for crystallizer ponds (Benlloch et al. 2002
; Pašić et al. 2007
). Each site possessed sequences representing novel taxa, and these were highly variable between the sample sites, with many showing very low levels of similarity to classified taxa of the Halobacteriaceae.
The relationships between the archaeal sequences recovered from the three sites are more clearly seen when presented as a phylogenetic tree reconstruction, as shown in Fig. , which includes all the clone sequences as well as some isolate sequences from this study (see below) along with representatives of 27 genera of the Halobacteriaceae. For convenience, closely branching sequences have been collapsed into numbered phylogroups, most of which consist of sequences belonging to the same 97%-OTU. Where a phylogroup contains more than one OTU, this is indicated under the name. Single sequences that branch separately from the others are named according to site and clone number (e.g., LDS clone 35). 51 clone sequences were closely similar to each other and to the rRNA gene sequence of Hqr. walsbyi C23T (analyzed in more detail below), and they branched tightly together forming Haloquadratum-related phylogroup 1. By comparison, the next most dominant phylogroup, Halorubrum-related phylogroup 4, was considerably more divergent, as denoted in Fig. by differences in their rightwards-extending peaks, and the number of 97%-OTUs. Several minor groups are scattered throughout the tree, and show varying affinities to currently classified taxa. A particularly deeply branching group of related sequences (bottom of Fig. ) was designated the MSP-clade (discussed further below), and contained sequences from all three sites.
In parallel to the culture-independent assessment of archaeal diversity, cultivation was also used in order to confirm, and possibly extend, the diversity captured by the PCR-generated clone library sequences. Using the methods described in our previous studies, water samples were plated onto solid media (MGM), and also titrated in DBCM2 liquid medium (extinction cultures), and incubated for at least 12 weeks (plates) or 8 weeks (liquid). A total of 201 pure isolates of haloarchaea were recovered, and a random selection of 132 of these had near-complete 16S rRNA gene sequences determined. Due to the biases involved in cultivation, and that many isolates had nearly identical 16S rRNA gene sequences, only a few of these sequences were included in the tree reconstruction shown in Fig. (see legend). Cultivation results are summarized at the right side of the tree, where asterisks indicate the phylogroups for which isolates were recovered. Representatives of five phylogroups were cultivated, including the major common clades related to Haloquadratum, Halorubrum, and Halonotius. The sequences of isolates closely matched (≥99% identity) clone library sequences of their corresponding phylogroup, excepting phylogroup 9, for which only isolates were recovered. The closest full-length matches in Genbank to the two phylotype 9 sequences showed only 95% identity, e.g., accession GQ282621 (isolate TNN28, saltern crystallizer, China). For many of the phylogroups and deeply branching clone sequences, isolates were not recovered, including members of the MSP8-clade. Seven phylogroups and four clones showed only ≤90% sequence identity to classified taxa, and so appear to represent novel genera.
A Venn diagram shown in Fig. summarizes the shared and site-specific diversity of 97% OTUs for the three sites, using all the sequence data from both clones and isolates. Apart from the two clades found at all sites, there are 4–10 97%-OTUs that are found only at a single site, and 1–3 that are shared between two of the three sites.
Fig. 4 Venn diagram showing the shared and unique 97%-OTUs between the three sample sites. The same clone and isolate sequences from this study that were included in Fig. were also used in this figure. Analysis and diagram generation were performed (more ...)
Looking more closely at the Haloquadratum-related sequences of phylogroup 1 (Fig. ), the 52 sequences determined in this study differed from each other and from the type strain, Hqr. walsbyi C23T, by ≤2%. A more closely related subgroup of these sequences that differed from each other by ≤0.5% included members originating from all three sites, while even more similar sequences (99.7% identity) were shared between the Bajool and Dry Creek sites, and between the Bajool and Lara sites. Since the Hqr. walsbyi genome contains two 16S rRNA genes that differ by 3 positions (0.2% divergence, strain C23T, unpublished data), at least some of the divergence observed between very similar clone sequences could derive from differences in paralogous genes. Indeed, in an alignment of these clone sequences, the same 3 positions were hypervariable, and two displayed the same base variation as seen between the C23T copies, i.e., C↔T at E. coli positions 198 and 845.
To compare the sequences derived from this study with those from other sites, Hqr. walsbyi
-related 16S rRNA gene sequences were retrieved from Genbank by a BLASTN search using the C23T
sequence. 28 sequences of at least 1400 nt, of good quality and non-chimeric (Bellerophon checked), and that were deposited as part of a published study, were retained and aligned with C23T
sequence (Table ). Within this group of sequences were members that differed from Hqr. walsbyi
by only 2–5 nt, and had been recovered from saltern crystallizer ponds in Spain (Bolhuis et al. 2006
) (Legault et al. 2006
), Israel (Sorensen et al. 2005
), Turkey (Mutlu et al. 2008
), Tunisia (Baati et al. 2008
) and Peru (Maturrano et al. 2006
) (see Fig. for locations). Except for the high-altitude Peruvian saltern, all of the Hqr. walsbyi
-related sequences recovered at the other sites differed by less than 1% from that of the (Australian) type strain. Data from the studies cited in Table , as well as other, previous published studies, indicate that Hqr. walsbyi
was numerically significant at all of these sites (e.g., Oren et al. 2006
), and at most sites it was the dominant archaeal clade.
Haloquadratum sequences reported by other published studies
By comparison, the Halorubrum
-related sequences (phylogroup 4) displayed 7% sequence divergence, and this value was unchanged if the sequences of the 78 cultured isolates that branched with this phylogroup were included (data not shown). Similar divergence values have been reported in previous studies of natural isolates of Halorubrum
spp. (Legault et al. 2006
), and even within the classified species of this genus, the 16S rRNA gene sequence divergence is 7–8%.
Phylogroup 2 is a tight cluster of 4 OTUs, each consisting of a single clone sequence. The sequences branched closely to each other and to phylogroup 1. Two sequences shared 97% sequence similarity to the 16S rRNA gene of Hqr. walsbyi
, and may represent novel species of Haloquadratum
. The other two sequences were 96.4% similar to each other but only 93% similar to Hqr. walsbyi
, and so probably represent different species of a separate, as yet uncultivated, genus (Fry et al. 1991
The closest matching sequences in Genbank to the other phylogroups and phylotypes are given in supplementary Table 2. Phylogroup 3 was represented by two clone sequences, and these were 97% identical to an isolate (CSW 2.24.4) recovered previously in this laboratory from the Moolap saltern crystallizer, 10 km from the Lara site (Burns et al. 2004a
). Phylogroup 5 includes clones and isolates, and represents a new genus within the Halobacteriaceae, with the type species being Halonotius pteroides
strain 1.15.5 (Burns et al. 2009a
). The latter strain was also isolated from the Moolap site. Phylogroup 8 sequences are closely related to Natronomonas pharaonis
, a haloalkaliphile isolated from an African soda lake with a pH around 10. Nmn. pharaonis
has an optimum pH of 8.5 but the sites examined in the current study were all around pH 7. A new species of this genus has recently been proposed to accommodate such neutrophilic members (Burns et al. 2009b