Patterns of Limnohabitans Microdiversity across a Large Set of Freshwater Habitats as Revealed by Reverse Line Blot Hybridization

Hahn MW (2013) Patterns of Limnohabitans Microdiversity across a Large Set of Freshwater Habitats as Revealed by Reverse Line Blot Hybridization. PLoS ONE 8(3): e58527. doi:10.1371/journal.pone.0058527 Patterns of Limnohabitans Microdiversity across a Large Set of Freshwater Habitats as Revealed by Reverse Line Blot Hybridization Jan Jezbera 0 Jitka Jezberova 0 Vojte ch Kasalicky 0 Karel S imek 0 Martin W. Hahn 0 Jonathan H. Badger, J. Craig Venter Institute, United States of America 0 1 Biology Centre of the ASCR, v.v.i., Institute of Hydrobiology, C eske Bud ejovice, Czech Republic, 2 University of Innsbruck, Research Institute for Limnology , Mondsee , Austria Among abundant freshwater Betaproteobacteria, only few groups are considered to be of central ecological importance. One of them is the well-studied genus Limnohabitans and mainly its R-BT subcluster, investigated previously mainly by fluorescence in situ hybridization methods. We designed, based on sequences from a large Limnohabitans culture collection, 18 RLBH (Reverse Line Blot Hybridization) probes specific for different groups within the genus Limnohabitans by targeting diagnostic sequences on their 16 S-23 S rRNA ITS regions. The developed probes covered in sum 92% of the available isolates. This set of probes was applied to environmental DNA originating from 161 different European standing freshwater habitats to reveal the microdiversity (intra-genus) patterns of the Limnohabitans genus along a pH gradient. Investigated habitats differed in various physicochemical parameters, and represented a very broad range of standing freshwater habitats. The Limnohabitans microdiversity, assessed as number of RLBH-defined groups detected, increased significantly along the gradient of rising pH of habitats. 14 out of 18 probes returned detection signals that allowed predictions on the distribution of distinct Limnohabitans groups. Most probe-defined Limnohabitans groups showed preferences for alkaline habitats, one for acidic, and some seemed to lack preferences. Complete niche-separation was indicated for some of the probe-targeted groups. Moreover, bimodal distributions observed for some groups of Limnohabitans, suggested further niche separation between genotypes within the same probe-defined group. Statistical analyses suggested that different environmental parameters such as pH, conductivity, oxygen and altitude influenced the distribution of distinct groups. The results of our study do not support the hypothesis that the wide ecological distribution of Limnohabitans bacteria in standing freshwater habitats results from generalist adaptations of these bacteria. Instead, our observations suggest that the genus Limnohabitans, as well as its R-BT subgroup, represent ecologically heterogeneous taxa, which underwent pronounced ecological diversification. - Funding: This study was financially supported by the Czech Grant Agency projects P504/10/0566 and EEF/10/E011 awarded to Jan Jezbera. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Betaproteobacteria usually represent a major fraction and frequently even the numerically dominating fraction of bacterioplankton in lentic (standing) freshwater systems [1,2]. Eighteen distinct lineages of Betaproteobacteria have been identified as typical constituents of freshwater bacterioplankton [1], however, only few lineages were found to be of ecological as well as numerical importance in lentic freshwater habitats [1,2]. These lineages are represented by two genera; Polynucleobacer and Limnohabitans [2]. The recently described genus Limnohabitans (Comamonadaceae, [3]) currently comprises four described species [35] in four tribes (Lhab-A1 to Lhab-A4) [1]. Both betaproteobacterial taxa have been described as abundant parts of freshwaters bacterioplankton, responding fast to changing environmental conditions, frequently forming together a vast majority of Betaproteobacteria and in case of Limnohabitans usually also its biomass [2]. Just recently, a refined Limnohabitans taxonomy has been suggested, based on 35 new isolates retrieved from various standing freshwater habitats and new revised and refined lineages have been proposed [6]. In particular, the R-BT subgroup or subcluster [7] embedded in the genus Limnohabitans, which contains at least two species [5], received serious scientific attention enabled by the introduction of a specific FISH (R-BT065) probe targeting this phylogenetically defined group [7]. This included studies aiming at explaining the genus abundance and distribution [8,9], ecophysiology [3,6], grazing vulnerability [10], and niche separation [11]. Manipulation experiments [12] suggested that Limnohabitans bacteria exhibit an opportunistic life strategy. Furthermore, it was suggested that they are able to utilize algal exudates [8,11]. R-BT cluster bacteria can in general be characterized by large cell volumes, fast growth rates, and pronounced vulnerability to protozoan predation; [2] and references therein. More than 700 publicly available (GenBank) 16 S rRNA gene sequences of bacteria affiliated with the Limnohabitans genus have been retrieved from very different freshwater habitats located on at least three continents. This high number probably suggests a unique role of these bacteria among other typical freshwater microbes [1]. Habitats, in which Limnohabitans were detected, span from oligotrophic [13] to hypertrophic lakes, include both Arctic [14] and tropical habitats [15], high mountain lakes (Yuhana, unpubl data), lowlands [7], brackish waters [13] and even lower courses of rivers [16,17]. On the other hand, the natural distribution of Limnohabitans bacteria seems to be restricted to running and standing freshwater systems, since reports on detection of this taxon in marine and terrestrial systems are lacking [36]. The broad spectrum of standing freshwater habitats occupied by Limnohabitans bacteria probably suggests an ecological diversification within the Limnohabitans genus and accommodation of distinct ecotypes in various ecosystems. Recently [18], such ecological diversification turned out to be the main reason for apparent ubiquity of another important, more narrowly defined, taxon Polynucleobacter necessarius ssp. asymbioticus across lentic freshwater habitats. In this previous study, Reverse Line Blot Hybridization (RLBH) probes were used to distinguish 13 groups within the subspecies P.n. asymbioticus. The genus Limnohabitans, however, represents a much broader group, including probably many more than the currently described four species [6] and is thus expected to show strong intragenus diversification. To address such intriguing question we designed 18 RLBH probes targeting the 16 S23 S spacer (ITS) region of 35 available isolates of Limnohabitans bacteria plus a sm (...truncated)