Broad Habitat Range of the Phylogenetically Narrow R-BT065 Cluster,Representing a Core Group of the Betaproteobacterial Genus Limnohabitans

The distribution of the phylogenetically narrow R-BT065 cluster (Betaproteobacteria) in 102 freshwater lakes, reservoirs, and various ponds located in central Europe (a total of 122 samples) was examined by using a cluster-specific fluorescence in situ hybridization probe. These habitats differ markedly in pH, conductivity, trophic status, surface area, altitude, bedrock type, and other limnological characteristics. Despite the broad ecological diversity of the habitats investigated, the cluster was detected in 96.7% of the systems, and its occurrence was not restricted to a certain habitat type. However, the relative proportions of the cluster in the total bacterioplankton were significantly lower in humic and acidified lakes than in pH-neutral or alkaline habitats. On average, the cluster accounted for 9.4% of the total bacterioplankton (range, 0 to 29%). The relative abundance and absolute abundance of these bacteria were significantly and positively related to higher pH, conductivity, and the proportion of low-molecular-weight compounds in dissolved organic carbon (DOC) and negatively related to the total DOC and dissolved aromatic carbon contents. Together, these parameters explained 55.3% of the variability in the occurrence of the cluster. Surprisingly, no clear relationship of the R-BT065 bacteria to factors indicating the trophic status of habitats (i.e., different forms of phosphorus and chlorophyll a content) was found. Based on our results and previously published data, we concluded that the R-BT065 cluster represents a ubiquitous, highly active segment of bacterioplankton in nonacidic lakes and ponds and that alga-derived substrates likely form the main pool of substrates responsible for its high growth potential and broad distribution in freshwater habitats.Heterotrophic bacterioplankton assemblages found in a broad variety of freshwater ecosystems are frequently dominated by representatives of a few phylogenetic clusters of Betaproteobacteria and Actinobacteria (1, 7, 9, 17, 21, 23, 34, 40). One of these clusters is the phylogenetically defined R-BT065 group (34), which is currently represented by >700 environmental ribosomal sequences deposited in the GenBank database. These sequences were obtained in several nonquantitative diversity studies of many European and North American freshwater habitats (5, 10, 18, 27, 30, 42). The R-BT065 cluster is characterized by a minimum level of intragroup 16S rRNA sequence similarity of 97.3%, and investigations of recently cultivated strains demonstrated that members of the group are heterotrophic bacteria (V. Kasalický, J. Jezbera, K. Šimek, and M. W. Hahn, submitted for publication). This cluster forms a phylogenetically distinct subgroup of the so-called “Rhodoferax sp. BAL47” cluster (42). The new genus Limnohabitans was recently established for some strains affiliated with the “Rhodoferax sp. BAL47” cluster (11), and it has to be assumed that bacteria belonging to the R-BT065 cluster are also affiliated with this genus (Kasalický and coworkers, submitted for publication).Bacteria belonging to the R-BT065 cluster in natural systems can be quantified using a homonymous fluorescence in situ hybridization (FISH) probe (34). This probe enabled intensive investigations of the ecology of R-BT065 bacteria; however, only a few freshwater systems (mainly representing larger pH-neutral lakes and reservoirs) have been quantitatively investigated to determine the presence of this bacterial group so far (2, 13, 24, 27, 30, 30). In the few habitats investigated, R-BT065 bacteria comprised 3 to 50% of the total bacteria. Importantly, these bacteria consistently had the highest net growth rates and showed high levels of substrate uptake in a freshwater reservoir (12, 13, 31, 32), as well as in an alpine lake (27). These investigations indicated that the ecology of the R-BT065 group is rather uniform and is characterized by (i) the potential for a rapid response to environmental changes, (ii) a high level of vulnerability to flagellate predation (14, 35), and (iii) a strong link with phytoplankton-derived organic material as the key growth substrate for the bacteria (24, 25, 33). However, these findings were based on quantitative investigations of only a few freshwater systems, and we can only speculate that the findings could be generalized for populations of this bacterial group in systems with different limnological characteristics. In particular, investigations of the habitat range, as well as investigations of environmental factors modulating the abundance of R-BT065 bacteria, have not been performed. Therefore, we set out to conduct a comprehensive survey of a broad variety of freshwater habitats that could support our hypothesis concerning the potential general importance of the cluster.The specific aims of this study were (i) to quantify the R-BT065 bacteria in the surface waters of a large variety of central European freshwater ecosystems (102 habitats) spanning broad ranges of system type and size (from large and deep alpine lakes to small and shallow ponds or various fishponds), type of bedrock, altitude, trophic status, pH, and humic substance content; (ii) to determine the proportion of R-BT065 bacteria in the total bacteria and in the total Betaproteobacteria in these habitats using FISH probes; and (iii) to suggest major physical, chemical, and biological factors that influence the occurrence of the R-BT065 phylotypes in the bacterioplankton.