Diversity of Haloquadratum and other haloarchaea in three, geographically distant, Australian saltern crystallizer ponds

Abstract

Haloquadratum walsbyi is frequently a dominant member of the microbial communities in hypersaline waters. 16S rRNA gene sequences indicate that divergence within this species is very low but relatively few sites have been examined, particularly in the southern hemisphere. The diversity of Haloquadratum was examined in three coastal, but geographically distant saltern crystallizer ponds in Australia, using both culture-independent and culture-dependent methods. Two 97%-OTU, comprising Haloquadratum- and Halorubrum-related sequences, were shared by all three sites, with the former OTU representing about 40% of the sequences recovered at each site. Sequences 99.5% identical to that of Hqr. walsbyi C23(T) were present at all three sites and, overall, 98% of the Haloquadratum-related sequences displayed <or=2% divergence from that of the type strain. While haloarchaeal diversity at each site was relatively low (9-16 OTUs), seven phylogroups (clones and/or isolates) and 4 different clones showed <or=90% sequence identity to classified taxa, and appear to represent novel genera. Six of these branched together in phylogenetic tree reconstructions, forming a clade (MSP8-clade) whose members were only distantly related to classified taxa. Such sequences have only rarely been previously detected but were found at all three Australian crystallizers.

Fig. 1

Map showing the locations of sample sites in Australia that were used in this study (Bajool, Lara, Dry Creek). These are indicated as black circles on a world map, and on the enlargement of Australia shown in left, lower box. The state boundaries and names are indicated on the enlargement. The world map also indicates the sites where the Hqr. walsbyi 16S rRNA gene sequences presented in Table 2 were recovered. For the small map of Australia, the letters indicate B Bajool, L Lara, D Dry Creek

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). The sample site is stated at the left of each bar. Some OTUs were closely related to validly described members of the family Halobacteriaceae (see Fig. 3) and, where appropriate, these have been clustered together with the same fill shade/pattern, and delimited with solid vertical lines. The key below indicates the taxon to which each shade/pattern cluster corresponds. Dashed vertical lines mark off individual OTUs within clusters, and the entire bar length for each site represents 100% of the sequences from that site, as indicated by the scale below. See the text and Fig. 3 for details about the MSP8 clade. Haloarchaeal sequences not closely related to the six designated groups in the figure are collected together under other

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). Bootstrap confidence values, derived from 1000 distance matrix replications, are indicated by filled circles (>75%) at branch points. Representatives of the currently recognized members of the family Halobacteriaceae were included, and a methanogen sequence was used as an outgroup (Methanosaeta concilii). Organism names are shown with culture collection numbers and sequence database accessions. The Halonotius reference sequence in phylogroup 5 was AY498641. For convenience, the branches of sequences belonging to single or closely related 97%-OTUs have been collapsed into numbered phylogroups, prefixed either with a name of a related taxon, or with the acronym ASC (Australian Saltern Crystallizer). Where phylogroups contain more than one OTU, this is indicated below the phylogroup name. Numbers in or next to the trapeziums representing collapsed branches are the number of clone sequences. Single clone sequences that formed isolated branches are named according to site of origin and clone or isolate number (e.g., DC clone 50, indicating a clone from Dry Creek). The sites of origin of sequences are indicated in brackets at the right of the tree by: B Bajool; D Dry Creek; L Lara; or −, absent; and a nearby asterisk indicating isolates belonging to this phylogroup was recovered

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. 3 were also used in this figure. Analysis and diagram generation were performed using the MOTHUR v 1.4 suite of programs (http://schloss.micro.umass.edu/)