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.
Highlights
The genus Halorubrum was proposed in order to accommodate the species Halobacterium saccharovorum, Halobacterium sodomense, Halobacterium trapanicum, and Halobacterium lacusprofundi (McGenity and Grant, 1995)
Using samples obtained from the hypersaline lake Aran-Bidgol (Iran) and solar salterns in Namibia and Spain we were able to isolate 21, two, and two strains respectively (Table S1) that were affiliated with the genus Halorubrum according to their 16S rRNA and housekeeping gene sequences
multilocus sequence analysis (MLSA) and average nucleotide identity (ANI) group Aran-Bidgol, Spanish and Namibian strains, as well as Hrr. chaoviator Halo-G∗T/DSM 19316T and Hrr. ezzemoulense DSM 17463T into a single species, it is clear that the DNA-DNA hybridization (DDH) results are in agreement with the polar lipid profiles that indicated Aran-Bidgol group 1 strains are homogeneous, but clearly different from Hrr. chaoviator HaloG∗T/DSM 19316T and Hrr. ezzemoulense DSM 17463T strains
Summary
The genus Halorubrum was proposed in order to accommodate the species Halobacterium saccharovorum, Halobacterium sodomense, Halobacterium trapanicum, and Halobacterium lacusprofundi (McGenity and Grant, 1995). Its highly conserved nature does not allow relevant discernable differentiation among closely related species (for example, 99.4% sequence similarity between Halorubrum californiense and Halorubrum chaoviator [Pesenti et al, 2008; Mancinelli et al, 2009]); the rRNA operons experience intragenic recombination (Boucher et al, 2004), resulting in reticulated evolutionary histories It was the most frequently transferred gene among closely related but otherwise distinct lineages (Papke, 2009), making haloarchaeal phylogeny and taxonomy difficult to interpret. A set of five housekeeping genes, i.e., atpB, EF2, glnA, ppsA, and rpoB’, have been suggested as recommended markers for the genus Halorubrum (Fullmer et al, 2014; Ram Mohan et al, 2014) The use of this MLSA approach instead of the employment of 16S rRNA gene sequence analysis has been endorsed by the ICSP-Subcommittee on the taxonomy of Halobacteria (Oren and Ventosa, 2013, 2016)
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