Genotypic and Lipid Analyses of Strains From the Archaeal Genus Halorubrum Reveal Insights Into Their Taxonomy, Divergence, and Population Structure

Abstract

To gain a better understanding of how divergence occurs, and how taxonomy can benefit from studying natural populations, we isolated and examined 25 closely related Halorubrum strains obtained from different hypersaline communities and compared them to validly named species and other reference strains using five taxonomic study approaches: phylogenetic analysis using the 16S rRNA gene and multilocus sequencing analysis (MLSA), polar lipid profiles (PLP), average nucleotide identity (ANI) and DNA-DNA hybridization (DDH). 16S rRNA gene sequence could not differentiate the newly isolated strains from described species, while MLSA grouped strains into three major clusters. Two of those MLSA clusters distinguished candidates for new species. The third cluster with concatenated sequence identity equal to or greater than 97.5% was comprised of strains from Aran-Bidgol Lake (Iran) and solar salterns in Namibia and Spain, and two previously described species isolated from Mexico and Algeria. PLP and DDH analyses showed that Aran-Bidgol strains formed uniform populations, and that strains isolated from other geographic locations were heterogeneous and divergent, indicating that they may constitute different species. Therefore, applying only sequencing approaches and similarity cutoffs for circumscribing species may be too conservative, lumping concealed diversity into a single taxon. Further, our data support the interpretation that local populations experience unique evolutionary homogenization pressures, and once relieved of insular constraints (e.g., through migration) are free to diverge.

The Various Roles of Fatty Acids

“…The strain, proposed to represent a novel species of a new genus, Glycocaulis abyssi , contained as main FA C18:1ω7 cis , C18:0, an unknown FA and C12:0 3-OH. Polar lipids may also differentiate Archaea organisms at the genus and sometimes at species levels [ 263 , 264 , 265 ]. The Subcommittees on the taxonomy of Halobacteriaceae and Halomonadaceae even suggest that fatty acid analysis “should be added as required rather than recommended” for describing new taxa in Halomonadaceae [ 264 ].…”

Genome-based analyses reveal a synonymy among Halorubrum distributum Zvyagintseva and Tarasov 1989; Oren and Ventosa 1996, Halorubrum terrestre Ventosa et al. 2004, Halorubrum arcis Xu et al. 2007 and Halorubrum litoreum Cui et al. 2007. Emended description of Halorubrum distributum Zvyagintseva and Tarasov 1989; Oren and Ventosa 1996

“…Alternative methodologies such as the use of the rpoB ′ gene or a multilocus sequence analysis (MLSA) based on the comparison of several single-copy housekeeping genes have been used [13, 14]. Recently, we have proposed the use of the five housekeeping genes atpB , EF-2 , glnA , ppsA and rpoB ′ for such phylogenetic analyses as an alternative to the 16S rRNA gene [15]. In this study, we determined that some well-stablished species of Halorubrum were not separate species, such as in the case of Halorubrum ezzemoulense and Halorubrum chaoviator [15, 16] and other similar situations might happen for other species of Halorubrum .…”

“…The 16S rRNA gene and MLSA phylogenetic analyses were carried out as described previously [14, 15]. Since some members of the class Halobacteria possess highly divergent 16S rRNA genes, we compared the reference 16S rRNA gene sequence (see in Table S1, available in the online verion of this article) of each of the analysed Halorubrum species with the 16S rRNA gene sequences extracted from the corresponding genome (partial 16S rRNA gene sequences <500 bp or low quality sequences were excluded).…”

“…S1) and maximum-parsimony algorithms were very similar to that of the tree reconstructed by neighbour-joining. As previously indicated, the comparison of the 16S rRNA gene sequences do not permit to determine in depth the phylogenetic relationships within the genus Halorubrum and thus an MLSA approach based on the comparison of partial sequences of the atpB , EF-2 , glnA , ppsA and rpoB ′ housekeeping genes (Table S1) has been recently recommended for this genus [15]. shows the phylogenetic tree obtained by the concatenation of these five housekeeping genes, reconstructed by the maximum-likelihood algorithm following the same methodology as aforementioned.…”

“…arcis JCM 13916 T and Hrr. litoreum JCM 13561 T ranged between 98.3–99.3 % and those of these four species with respect to the other type strains of Halorubrum were always below 95.6 % (Table S3), below the 96 % cut-off value established for species delineation within the genus Halorubrum [ 15 ].…”