Population structure and species description of aquatic Sphingomonadaceae

Prokaryotes consist of the domains of Bacteria and Archaea and exist since approximately 3.8 billion years. Prokaryotes, despite the small size of the individual cells, are regarded to represent the 'unseen majority' among the living world as they occur numerously in all types of habitats and contribute greatly to the biogeochemical cycle. They diversified strongly throughout their long evolutionary history. Prokaryotes have usually a short generation time and relatively small amount of genetic information as compared to eukaryotes, and large census population sizes. This renders them suitable test organisms for studying their evolutionary processes. The discipline of population genetics analyses the evolutionary change of the genotypic and phenotypic variants at the level of species. Most of the recent bacterial population genetic studies have focussed on pathogens. Little is known of the population structure of freshwater bacteria. Natural freshwater lakes harbor a considerably lower diversity of bacteria, this facilitating the study of the genetic variability of bacteria. Sphingomonadaceae represent typical constituents of freshwater bacterioplankton communities and therefore served as a target group for a high-resolution multilocus sequence analysis (MLSA) of nine housekeeping genes (atpD, dnaK, fusA, tufA, gap, groEL, gyrB, recA, rpoB) and a parallel phenotypic characterization. Among 95 strains recovered from two trophically different freshwater lakes (Starnberger See and Walchensee), only 19 different 16S rRNA gene sequences were found. Yet, each strain represented a unique MLSA haplotype and the population displayed extraordinary high levels of nucleotide diversity. A split decomposition analysis revealed eight genetically distinct subpopulations, three of which comprised a single phylotype G1A with 52 strains. The population recombination rate ρ was comparable to that of other bacteria but two to eight-fold lower than the population mutation rates θS. Consequently, the impact of recombination on the population structure of freshwater Sphingomonadaceae is markedly lower than in most other free-living aquatic bacteria investigated to date. This was supported by a linkage disequilibrium analysis on the allele distribution. Together with the large effective population size (estimate, ~6•108), our data suggest that the incipient sexual isolation of subpopulations is caused by natural selection rather than genetic drift or demographic effects. Since neutrality tests did not provide evidence for an effect of selective forces on the housekeeping genes and no consistent physiological differences were detected between the G1A subpopulations, alternative phenotypic traits are supposed to provide a selective advantage for individual subpopulations of Sphingomonadaceae. This conclusion is supported by discrete seasonal abundance patterns that were detected based on pyrosequencing of internal transcribed spacer sequences in the natural samples. MLSA is a widely applied genotyping tool in studies of the evolution and population structure of microbial organism and also represents a novel standard in microbial molecular systematics. Population genetic analysis of Sphingomonadaceae by MLSA revealed a distinct population substructure among individual 16S rRNA phylotypes, providing insights into the diversity within bacterial species. A 'species' is the main taxonomic unit in the systematics of prokaryotes, but the subject of the species concept of prokaryotes has always been controversial. Until now there is no prokaryotic species concept that is accepted by all scientists. But for practical reasons, bacterial strains are affiliated to different species on the basis of DNA-DNA reassociation and diagnostic phenotypes. As DNA-DNA hybridization is difficult to be compared between laboratories and time consuming, MLSA becomes a valuable alternative to it. The population genetic structure revealed by MLSA is strongly associated with the results from DNA-DNA relatedness values. When sufficient numbers of suitable loci are selected, the concatenated sequence similarity values can in principle be used for species delineation. To assess the population and subpopulation structure revealed by MLSA also from a taxonomic perspective, four Sphingomonadaceae strains belonging to four different subpopulations were chosen for new species description. Based on morphological, physiological and biochemical characterization, strain 247 from group G3B was affiliated to a species formerly named 'Caulobacter leidyi' and which was now reclassified as 'Sphingomonas leidyi'. Strain 382 from group G1A2 was proposed as type strain of a novel species 'Sphingomonas limneticum'. Strain 301 from group G2D was proposed as type strain of a novel species 'Sphingobium oligotrophica', and a strain 469 was proposed as type strain of a novel species 'Sphingobium boeckii', and the closely related species formerly names 'Sphingomonas suberifaciens' was reclassified as 'Sphingobium suberifaciens'.