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Keywords:

  • Polynucleobacter;
  • Limnohabitans;
  • ecological diversification;
  • competition

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

The distribution and abundance of Betaproteobacteria and three of its genera – Limnohabitans (R-BT065 lineage), Polynucleobacter (including subclusters Polynucleobacter necessarius and Polynucleobacter acidiphobus/Polynucleobacter difficilis), and Methylophilus – across the epilimnia of 72 limnologically diverse freshwater habitats were investigated using fluorescence in situ hybridization. Moreover, seasonal development of Betaproteobacteria subgroups along the longitudinal axis of a reservoir was followed. Betaproteobacteria comprised on average 29.1%, Polynucleobacter 11.6%, P. necessarius 10.1%, Pacidiphobus/difficilis 0.5%, Limnohabitans 8.9%, and Methylophilus 0.9% of total bacterioplankton cells in the investigated habitats. Polynucleobacter necessarius and Limnohabitans coexisted in the majority of habitats but showed contrasting abundance patterns along the pH gradient of habitats (pH, 3.8–8.5). The observed distribution patterns could theoretically be explained by different preferences for substrate sources, that is, substances of humic origin in acidic waters and algal-derived substances in alkaline waters. However, substrate utilization patterns observed in laboratory experiments indicate no coherent group-specific differences in substrate preferences. Interestingly, similar distribution patterns were revealed for Limnohabitans and Pacidiphobus/difficilis, suggesting similar ecological adaptations of these distantly related taxa. Our findings further emphasize that at least two taxa of freshwater Betaproteobacteria represent ecologically diversified groups. Investigations at higher phylogenetic resolution are required for obtaining further insights into their ecology.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Betaproteobacteria represent one of the key components of freshwater bacterioplankton (Hiorns et al., 1997; Glöckner et al., 2000; Barberan & Casamayor, 2010) constituting 23% (three lakes, Glöckner et al., 2000) to 70% of freshwater bacterioplankton (one habitat, Hahn et al., 2005). Freshwater bacteria affiliated with the Betaproteobacteria have formerly been divided into four (Glöckner et al., 2000), six (Zwart et al., 2002), or seven lineages (Newton et al., 2011). However, recent studies (e.g. Hahn et al., 2005; Lindström et al., 2005; Salcher et al., 2008; Jezberová et al., 2010; Šimek et al., 2010a) indicated that in a vast majority of cases, only two of these lineages, that is, BetI and BetII (Newton et al., 2011), are responsible for the overall abundance of the Betaproteobacteria in freshwater habitats. These two groups are mainly represented by the genus Limnohabitans (Hahn et al., 2010a) and especially by its R-BT065 lineage (Šimek et al., 2001; Kasalický et al., 2010) – a monophyletic cluster within this genus – as well as by the genus Polynucleobacter (Heckmann & Schmidt, 1987; Hahn et al., 2009). So far, much less is known about the other five lineages of Betaproteobacteria, of which only BetIII and BetIV have received some attention (Glöckner et al., 2000; Friedrich et al., 2003; Salcher et al., 2008; Newton et al., 2011).

The genus Limnohabitans constitutes, together with the genus Rhodoferax, the BetI lineage (Newton et al., 2011). So far, Limnohabitans harbors four validly described species (Hahn et al., 2010ab; Kasalický et al., 2010.

The genus Polynucleobacter, synonymous with PnecABCD (Wu & Hahn, 2006a), and BetII (Newton et al., 2011), is currently represented by five species, which are more or less equivalent to the previously designated subclusters (Hahn, 2003).

A wealth of information is now available on Limnohabitans and Polynucleobacter bacteria, including information on ecophysiology (Hahn et al., 2009; Kasalický et al., 2010), intraspecific ecological differentiation (Jezbera et al., 2011), seasonality (e.g. Crump et al., 2003; Hahn et al., 2005; Wu & Hahn, 2006b), abundance and distribution (e.g. Hahn, 2003; Wu et al., 2006; Buck et al., 2009; Jezberová et al., 2010), habitat preferences (Hahn, 2003; Hahn et al., 2005; Šimek et al., 2010a), vertical distribution (Wu & Hahn, 2006b; Salcher et al., 2008), niche separation (Šimek et al., 2010b; Jezbera et al., 2011), grazing vulnerability (Šimek et al., 2001; Boenigk et al., 2004; Jezbera et al., 2005), etc. On the other hand, almost all this knowledge has been generated from studies that focused on a single or only a few habitats, or on populations of microorganisms belonging to only one of the lineages, or from results gained from manipulation experiments. What is entirely missing is a comprehensive investigation into the distribution and abundance patterns of the major groups of Betaproteobacteria across a large number of freshwater systems representing a broad ecological spectrum of the habitat types that we offer here.

While several studies have focused on Polynucleobacter and Limnohabitans, considerably less is known about the other groups of freshwater Betaproteobacteria. In this comprehensive study, we intended to close this gap by investigating the abundance of major Betaproteobacteria groups: the genus Limnohabitans, the genus Polynucleobacter and two of its subgroups, and the genus Methylophilus across a large set of 72 systems representing a broad spectrum of habitats, covering for example a pH gradient ranging from 3.8 to 8.5. The major aim of the study was to convincingly document the overall numerical importance and the hypothesized contrasting ecological roles of the two key groups of Betaproteobacteria, that is Limnohabitans and Polynucleobacter, not only among Betaproteobacteria alone, but also among the whole bacterioplankton community. In addition, to cover the distribution patterns of the major Betaproteobacteria groups, we decided to additionally use other available genetic probes specific to other betaproteobacterial groups or subgroups of the two mainly studied genera. Last but not least, we intended to explore the fundamental trophic niches of the distinct isolates by following their substrate utilization patterns.

The foremost goals of the presented study thus were (1) to reveal the distribution patterns of the four key subgroups of Betaproteobacteria, (2) to identify environmental drivers that determine their distribution and abundance, (3) to identify conditions under which the two major genera, Polynucleobacter and Limnohabitans, co-occur, and (4) to investigate whether they are potentially competing for certain resources. On the basis of the presented results, we intended to create a generalizing picture on the distribution of Betaproteobacteria across ecologically diverse habitat types serving as a tool for directing future studies on Betaproteobacteria diversity.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Sampling of habitats

Distribution of taxa across environmental gradients

In total, 72 lentic freshwater habitats (Supporting Information, Table S1) representing a pH gradient from 3.8 to 8.5 were sampled once or few times during the period 2006–2008 (in the period June–November). The primary study area was represented by the Salzkammergut Lake District, a mountainous area with numerous lakes and ponds located on the northern slope of the Alps close to the city of Salzburg (Austria). Apart from this area, several other habitats located in Austria and the Czech Republic were sampled. A detailed list of a set of habitats including the ones presented here can be found elsewhere (Jezberová et al., 2010). The selected 72 habitats represented the largest possible variety of lakes, small natural and artificial ponds and puddles differing in a multitude of parameters. Almost all habitats were sampled at a depth of 0.5 m.

Temporal and spatial distribution of taxa in a single habitat

A seasonal distribution study was performed on the canyon-shaped meso-eutrophic Římov reservoir located near the city of České Budějovice (Czech Republic). The reservoir was sampled along its longitudinal axis at three sites (assigned as DAM, MIDDLE, and RIVER) in 3-week intervals between March and November 2005, as described previously (Šimek et al., 2008).

Determination of physicochemical parameters of the water samples

Temperature, pH, oxygen concentration, and conductivity were measured on site. In the habitat survey, dissolved organic carbon (DOC) and concentrations of humic substances were estimated photospectroscopically, as described previously (Table S1 and Jezberová et al., 2010). In the seasonal study of the Římov reservoir, DOC, dissolved reactive phosphorus (DRP), particulate reactive phosphorus, total phosphorus, particulate phosphorus (PP), total primary production (PPtot), primary production in 1- to 2-μL filtrate (PP1-2μl), and chlorophyll a (ChlA) were measured as described in Šimek et al. (2008).

Determination of bacterial abundance

Subsamples were fixed with formaldehyde (2% final concentration), stained with DAPI, (Sigma), filtered onto black 0.2-μm-pore-size Nuclepore filters (Osmonic Inc., Livermore) and enumerated using an epifluorescence microscope under UV excitation at a magnification of 1250×. At least 500 bacterial cells were enumerated.

Absolute and relative abundance of selected bacterial taxa

The abundances of Betaproteobacteria and of major Betaproteobacteria subgroups/species – the genus Limnohabitans (R-BT065 lineage), Polynucleobacter necessarius (PnecC), the Polynucleobacter acidiphobus/Polynucleobacter difficilis lineage (PnecB), and the genus Methylophilus – were determined by Catalyzed Reporter Deposition Fluorescence in situ Hybridization (CARD-FISH) following the protocols of Pernthaler et al. (2002) and Sekar et al. (2003). The CARD-FISH probes deployed and their specificity are listed in Table 1.

Table 1. Oligonucleotide CARD-FISH probes used in this study, their specificity and reference papers
ProbeSpecificityReference
BET42aBetaproteobacteriaManz et al. (1992)
R-BT065 R-BTLineage within the Limnohabitans genusŠimek et al. (2001)
PnecABCD-445Polynucleobacter genus (= PnecABCD)Hahn et al. (2005)
PnecC-16S-445Polynucleobacter necessarius (= PnecC)Hahn et al. (2005)
PnecB-23S-166P. acidiphobus & P. difficilis (= PnecB) Wu & Hahn (2006a)
Met1217Methylophilus genusFriedrich et al. (2003)

Incorporation of radioactively labeled substrates assessed by MAR-FISH

To track bacterial cells with active biomass synthesis and the potential production of storage matter, duplicate samples and formaldehyde-fixed blanks of 5 mL were incubated with either L-[3H]-leucine (Leu, final concentration 20 nM; specific activity 6.4 TBq mmol−1, MP Biomedicals) or D-[3H]-glucose (Glc, final concentration 20 nM, specific activity 2.22 TBq mmol−1, MP Biomedicals). Samples were incubated for 2 h at in situ temperature in the dark, preserved in formaldehyde (final concentration 2%) and filtered onto 0.2-μm polycarbonate filters, as described previously (Horňák et al., 2006). Filters were rinsed with Milli-Q water, air-dried, and kept frozen (–20 °C) until further processing. After the CARD-FISH procedure, the filters were transferred onto slides coated with autoradiography emulsion (NTB, Kodak). After 24–48 h of exposure in the dark, the cells were stained with DAPI (final concentration 1 μg mL−1). The relative abundances of hybridized cells were enumerated by epifluorescence microscopy. At least 500 DAPI-stained cells were counted per sample. MAR-FISH experiments were performed with water samples from Loipersbacher ponds 1 and 2 (acidic ponds, Hahn et al., 2005), Lake Mondsee (alkaline, deep lake, Wu & Hahn, 2006a), and the Římov Reservoir (circum-neutral canyon-shaped reservoir, this study).

Substrate assimilation analyses

The previously published as well as the unpublished data on substrate utilization by Limnohabitans and Polynucleobacter strains (Hahn et al., 2009, 2010a, b, c, 2011a,b, 2012a; Kasalický et al., 2010; V. Kasalický, unpublished data; M.W. Hahn, unpublished data) were compiled and analyzed. All the established data were obtained by following the same protocol. Briefly, growth of bacterial isolates based on utilizing specific substrates was determined by comparison of the optical density measured at 575 nm (OD575) in liquid one-tenth-strength NSY medium (0.3 g L−1, Hahn, 2003) with and without 0.5 g of the test substrate. Differences in OD575 of 10, 10–50, and 50% and more compared with growth on the medium lacking the test substance after 10 days of growth were scored as no utilization (−), weak utilization (w), and good utilization (+), respectively.

Statistical analysis

The program canoco (TerBraak & Šmilauer, 1998) was used for the multivariable analysis. Redundancy analyses (RDA) were performed, and the results were visualized by CanoDraw for Windows (TerBraak & Šmilauer, 1998).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Occurrence of Betaproteobacteria groups along the pH gradient of investigated habitats

Total bacterial numbers differed strongly between the sampled habitats (Fig. 1, upper panel). Betaproteobacteria formed almost one-third (29.1%) of all heterotrophic bacteria when averaged across all 72 habitats. Differences in Betaproteobacteria numbers were more pronounced in acidic/circum-neutral habitats (pH, 3.8–7.4), ranging there from 1.5–72% of all bacteria. In the alkaline pH range from 7.4 to 8.5, Betaproteobacteria numbers were more stable, ranging from 11.3% to 44.8% (Fig. 1). On average, Betaproteobacteria contributed less to total bacterial numbers in alkaline habitats than in acidic ones (Fig. 1, lower panel), which can be attributed to the dominant role of P. necessarius in the acidic habitats (Fig. 1). In acidic habitats, Betaproteobacteria contributed, on average, 34.3% to the total bacteria, in contrast to alkaline habitats where they constituted 23.9% of all bacteria, on average.

image

Figure 1. Upper panel, distribution of subgroups of Betaproteobacteria along the pH gradient (from 3.8 to 8.5) of 72 different habitats in absolute cell numbers. Lower panel, heat map of the relative (%) proportions of distinct Betaproteobacteria groups along the gradient of increasing pH (from 3.8 to 8.5) of the investigated 72 habitats. Relative proportion classes used are 0–1%, 1.1–5%, 5.1–10%, 10.1–20%, 20.1–30%, 30.1–40%, and over 40%.

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Limnohabitans (R-BT065) bacteria contributed on average to about 8.9% of bacterioplankton cells and showed a clear trend of increasing abundance with increasing pH. However, they were also present, although in smaller quantities, in habitats of lower or very low pH (close to 3.8). In the alkaline range of the pH gradient, at approximately pH 7.5 and higher, Limnohabitans bacteria were clearly the dominant group among Betaproteobacteria (Fig. 1).

The entire genus Polynucleobacter contributed on average to 11.6% of bacteria in the investigated samples. The vast majority of the detected Polynucleobacter cells were affiliated with the species P. necessarius (Fig. 1). This species alone accounted for approximately 10% of all bacteria. The P. acidiphobus/P. difficilis lineage comprised about 0.5% of bacteria detected across all studied habitats. A clear trend in the distribution of these two taxa was observed. The P. difficilis/P. acidiphobus lineage was more abundant exclusively in the alkaline part of the sampled habitat range, being most abundant in the large pre-alpine lakes of the Salzkammergut area (Figs 1 and 2). P. necessarius showed a completely opposite trend, displaying a clear preference for low-pH habitats, where its abundance ranged between 5% and 60% of all detected bacteria. This species was also detectable, however, in alkaline habitats, though in much lower percentages. The three species P. necessarius, P. difficilis and P. acidiphobus constituted together almost 90% of all Polynucleobacter bacteria.

image

Figure 2. Abundance of the Polynucleobacter difficilis/Polynucleobacter acidiphobus lineage, Polynucleobacter necessarius, and the entire Polynucleobacter genus as detected by respective FISH probes along the pH gradient of the 72 investigated habitats.

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In contrast to Limnohabitans and Polynucleobacter, bacteria belonging to the genus Methylophilus were never highly abundant, reaching on average only 0.9% of all bacteria and displaying no clear trend across the investigated habitats (Fig. 1).

Importantly, using the CARD-FISH probes for Polynucleobacter, Limnohabitans and Methylophilus, we were able to cover on average almost three quarters (72.3%) of all Betaproteobacteria across the wide range of sampled habitats.

Comparison of substrate assimilation patterns

No clear pattern in substrate utilization that would clearly separate Limnohabitans from Polynucleobacter bacteria was observed (Table 2). Moreover, large differences in patterns even among strains of the same genus were observed for both genera. Pronounced differences in utilization patterns for strains of the same species (e.g. P. necessarius) were also obvious.

Table 2. Substrate utilization by Polynucleobacter and Limnohabitans isolatesThumbnail image of

Despite using a large array of substances, we were unable to find a sole substrate utilized by all members of one genus and not utilized at all by all members of the other genus (Table 2). The only distinct traceable trends were higher preferences of Limnohabitans for monosaccharides (namely fructose, glucose, mannose etc.) and certain amino acids (l-alanine) contrasting with no or very weak utilization of these substances by P. acidiphobus, P. difficilis, and P. necessarius. Another important result was a positive and strong preference of Limnohabitans and P. necessarius for acetate as opposed to no utilization of acetate by the P. difficilis/P. acidiphobus lineage.

Activity measurements by MAR-FISH

Uptake of two radioactively labeled substances (leucine and glucose) by Polynucleobacter and Limnohabitans bacteria (targeted by the R-BT065 probe) was investigated in four habitats representing three limnologically contrasting habitat types. Uptake of both substrates by the two bacterial groups was observed in all four habitats (Table S2). In acidic Loiperbacher ponds 1 and 2 (habitats #11 and 12 in Table S2), as well as in large, alkaline Lake Mondsee (habitat #51 in Table S2), Polynucleobacter displayed a markedly lower affinity for leucine than did Limnohabitans bacteria. On the other hand, glucose was similarly assimilated by both groups. In the meso-eutrophic, circum-neutral Římov reservoir (Table S2), similar trends were observed. On average, 92% of Limnohabitans bacteria incorporated leucine as opposed to approximately 56% of Polynucleobacter bacteria actively incorporating leucine. A similar pattern was observed for the utilization of glucose, where approximately 81% of Limnohabitans and only 45% of Polynucleobacter bacteria incorporated this substrate.

Factors influencing the distribution of the major Betaproteobacteria groups across 72 habitats and during one season in the Římov reservoir

In 2005, temporal and spatial development of the P. difficilis/P. acidiphobus lineage, P. necessarius, and Limnohabitans (R-BT065) bacteria were followed at the DAM, MIDDLE and RIVER stations of the Římov reservoir (Fig. 3). The P. difficilis/P. acidiphobus lineage and P. necessarius showed clearly contrasting trends. In the DAM area, which is primarily supplied by autochthonous primary production, the P. difficilis/P. acidiphobus lineage markedly dominated over P. necessarius during most of the season, as opposed to the allochthonously loaded RIVER station, where P. necessarius formed the major part of the whole Polynucleobacter assemblage. In the MIDDLE station, both groups alternated. Note that on the August 23, when sampling of the RIVER station was not feasible because of a flood event, the high P. necessarius numbers, normally typical of the RIVER station, were projected as far as to the MIDDLE station.

image

Figure 3. Distribution of relative proportions of the major Betaproteobacteria subgroups as detected by FISH at the DAM, MIDDLE, and RIVER stations located along a longitudinal transect of the canyon-shaped Římov reservoir in the period from mid of March until mid of November 2005.

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RDA were performed for the set of 72 distinct habitats (Fig. 4a) as well as for the seasonal data from the Římov reservoir (Fig. 4b). Both RDA analyses confirmed contrasting roles of the P. difficilis/P. acidiphobus lineage and P. necessarius. The latter taxon was positively related to changing ChlA concentrations, primary production, extracellular primary production, and temperature and was more abundant in the ‘lake’ part of the Římov reservoir (Fig. 4b). In contrast, P. necessarius bacteria showed a positive correlation with DRP and humic substances, being more abundant in the inflow (‘river’ part) of the reservoir. Limnohabitans bacteria did not show any trend within the Římov reservoir, while their distribution patterns were clearly indicated in the highly heterogeneous set of 72 habitats (Fig. 4a). In this analysis, the P. difficilis/P. acidiphobus lineage and Limnohabitans preferred habitats with higher pH, higher conductivity, and lower amounts of humic substances, whereas P. necessarius was more abundant in habitats at higher altitude. Interestingly, P. necessarius, compared to the P. difficilis/P. acidiphobus lineage and Limnohabitans, displayed exactly opposite relationships to the investigated environmental parameters. Methylophilus bacteria showed no clear distribution trend across the 72 investigated habitats (data not shown).

image

Figure 4. (a) Seventy-two different habitats, (b) the Římov reservoir, a seasonal study. Redundancy analysis (RDA) showing only the significant parameters responsible for the distribution of the entire Polynucleobacter genus (labeled as PnecABCD); Polynucleobacter necessarius (PnecC); Polynucleobacter difficilis/Polynucleobacter acidiphobus lineage (PnecB); Limnohabitans (R-BT065) bacteria; and Methylophilus genus (MET1217). PPtot, total primary production; A250nm, absorption at 250 nm (DOC proxy); PP1–2um, primary production in the 1–2 μm size fraction; chlA, chlorophyll a; temp., temperature; EPP, extracellular primary production; ‘lake’ triangle represents pooled data for the station DAM and MIDDLE; ‘river’ triangle represents data for the RIVER stations (see Methods section); n.s., not significant.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Polynucleobacter and Limnohabitans bacteria as omnipresent components of freshwater systems

In the 72 freshwater habitats investigated, Polynucleobacter and Limnohabitans bacteria together formed the vast majority of Betaproteobacteria (on average more than 70%). However, it is important to note that the FISH probe used for the detection of Limnohabitans bacteria does not target the entire genus but only a core lineage within this diverse taxon (Table 1; Kasalický et al., 2010). Other lineages within the genus Limnohabitans, not detected by the probe, also represent bacteria frequently inhabiting the pelagic zones of freshwater systems (Zwart et al., 2002; Lindström et al., 2005; Hahn et al., 2010ab). For instance, the type species of L. curvus and some closely related strains have been isolated from Lake Mondsee, which was included in the set of habitats investigated here. Because of the lack of complete coverage of the genus by the probe, underestimation of the contributions of the entire genus Limnohabitans to total bacterioplankton is quite likely. However, because of the lack of suitable FISH probes, it is currently impossible to estimate how significant this underestimation is.

In this study, we used three independent FISH probes to cover the B and C lineages of Polynucleobacter as well as the entire genus Polynucleobacter. On the basis of the previous studies, where the numerical abundance of A and D lineages was found to be negligible (Wu & Hahn, 2006a), we decided not to deploy probes targeting these two lineages. This decision is well supported by the fact that probes specific to the B and C lineages detected on average 91.3% of all Polynucleobacter bacteria enumerated by using the genus-specific probe.

In contrast to the other two genera studied, the genus Methylophilus constituted only a small part of the bacterial communities, as well as of the whole Betaproteobacteria assemblages in the investigated habitats. However, it is important to note that we have mainly investigated epilimnetic samples that were more or less oxygen-saturated. We did not sample deeper water layers with lower oxygen concentrations, which were found in other studies to be richer in Methylophilus bacteria (Salcher et al., 2008). Potentially, this sampling strategy could have resulted in an underestimation of the importance of this bacterial taxon. On the other hand, it has been demonstrated recently that Methylophilus bacteria can be enriched on phenol or humic matter additions (Hutalle-Schmelzer et al., 2010), that might, to a certain extent, link these microorganism to direct degradation of humic substances and probably not so much to the utilization of methane produced in anoxic zones and other C1-compounds as suggested by the previous studies (Ginige et al., 2004). The study of Hutalle and colleagues cannot, however, be generalized, because only few isolates and clones were analyzed. If this assumption would be true, one would expect a tight relation of Methylophilus spp. numbers to concentration of humic matter which was not so far confirmed.

We have omitted a detailed analysis of the environmental drivers controlling distribution and abundance of Methylophilus bacteria because the cell numbers determined by FISH were frequently close to the detection limit, resulting in low accuracy of the determined data. Aside from this, these data indicate also a rather negligible role of these bacteria in the overall carbon flow of the systems studied.

Because of the broadness of ecological spectrum of standing freshwater systems sampled, we believe that the observed distribution pattern of major groups of Betaproteobacteria can be generalized, to a certain extent, for the majority of freshwater habitats worldwide.

There are inevitably other factors that may influence the distribution and detection of the investigated bacterial taxa, to name just a few: water retention time (Lindström & Bergström, 2004; Lindström et al., 2005), dispersal limitations (Whitaker et al., 2003), seasonality of occurrence (e.g. Wu & Hahn, 2006b), and biotic interactions (e.g. Eiler et al., 2011). Because of the large number of habitats sampled, none of these factors could be considered in our study. Nonetheless, especially biotic factors including predation, competition, and trophic dependencies could be of importance (Boenigk et al., 2004; Šimek et al., 2010b, 2011; Eiler et al., 2011).

Contrasting distribution patterns of bacteria along the pH gradient

We observed the coexistence of Polynucleobacter and Limnohabitans bacteria in the majority of investigated lakes, but the two groups showed also rather contrasting abundance patterns regarding pH of the habitats (Fig. 1). Limnohabitans occurred, on average, with higher relative and absolute abundances in circum-neutral and alkaline habitats, while the opposite trend was observed for the genus Polynucleobacter. However, the two Polynucleobacter subgroups considered in our study display both differences in total abundance as well as opposite abundance patterns along the pH gradient (Fig. 1, lower panel). Newton et al. (2011) also demonstrated a negative correlation of the P. necessarius occurrence and a positive correlation of the P. difficilis/P. acidiphobus lineage with lake pH. Interestingly, in this meta-analysis, the R-BT065 lineage of Limnohabitans was split in two subclusters (Lhab-A1 and Lhab-A2) for which opposite – that is positive and negative – pH correlations were revealed (Newton et al., 2011).

Several previous investigations revealed pH as one of the key drivers or even as the strongest driver influencing bacterioplankton composition (Lindström et al., 2005; Yannarell & Triplett, 2005), or unveiled significant distribution differences of related taxa based on lake pH (Schauer et al., 2005; Newton et al., 2007). Despite the well-documented role of pH as the factor strongly shaping bacterioplankton composition (Lindström et al., 2005), it is not known whether pH acts as a direct factor linked to pH adaptation of the respective bacteria or as an indirect factor influencing other growth conditions. In the case of Polynucleobacter and Limnohabitans bacteria, the distribution trends observed cannot simply be explained by differences in pH adaptation. The MAR-FISH investigations indicated that populations of both taxa, actively incorporating selected substrates, were present in all four investigated habitats, which represented a pH range of more than three units (pH, 4.7–8.3, Table S2). Other investigations performing similar MAR-FISH experiments on other habitats previously indicated that metabolically active bacteria affiliated with the genus Polynucleobacter, and especially with the species P. necessarius, are present in habitats strongly differing in pH (Alonso et al., 2009; Buck et al., 2009; Salcher et al., 2010). Additionally, recent findings suggested differences in pH preferences across subgroups of P. necessarius (Jezbera et al., 2011).

An alternative explanation for the opposite distribution trends observed could be the utilization of different major substrate sources. Previous analyses indicated the utilization of different substrate pools for Limnohabitans (R-BT065 lineage) and P. necessarius. Šimek et al. (2008) proposed that Limnohabitans bacteria mainly rely on algal-derived substrates, that is, utilize direct or indirect products of autochthonous primary production. Moreover, it has been documented that L. planktonicus and L. parvus grow well even in diluted exudates produced by axenic cultures of typical planktonic algae (Šimek et al., 2011). In contrast, bacteria affiliated with the species P. necessarius are believed to utilize mainly photooxidation products of humic substances (Watanabe et al., 2009; Jezberová et al., 2010; Hahn et al., 2012b) – that is, they utilize allochthonous organic matter that is, at least partially, of terrestrial origin. In a previous analysis by Jezberová et al. (2010), which investigated a similar set of habitats, it was revealed that pH of habitats and concentrations of humic substances were negatively correlated. On the other hand, the majority of circum-neutral and alkaline habitats studied in the current study represent nonhumic lakes with low allochthonous DOC input, which should favor bacteria utilizing autochthonous production.

A previous investigation indicated that not all free-living Polynucleobacter rely on humic substances. Wu & Hahn (2006b) proposed that bacteria of the P. difficilis/P. acidiphobus lineage mainly utilize substrates derived from algal primary production, which is supported by the fact that this taxon was found in stock cultures of various algae as accompanying bacteria (Šimek et al., 2011). Thus, utilization of similar substrate pools was suggested for Limnohabitans (R-BT065 lineage) and the P. acidiphobus/P. difficilis lineage. Interestingly, both the latter lineage and the Limnohabitans group share similar distribution patterns along the investigated pH gradient.

Both the P. difficilis/P. acidiphobus lineage and Limnohabitans appear in alkaline and circum-neutral habitats in higher relative abundances and in lower proportions in some of the acidic habitats (Fig. 1). Detection of the P. difficilis/P. acidiphobus lineage in acidic habitats is in disagreement with the previously reported lack of detection in acidic waters (Wu & Hahn, 2006b). These contradicting observations may have resulted from differences in the sensitivity of the FISH methods deployed (CARD-FISH vs. FISH).

Previously, there have been numerous studies documenting the effect of protozoan grazing on bacterial community composition. The effect of grazing on Limnohabitans and Polynucleobacter bacteria specifically can be found for instance in Boenigk et al., 2004; Jezbera et al., 2005 and Šimek et al., 2007;. It was documented that bacteria belonging to the Limnohabitans genus (partially represented by the R-BT065 cluster) are highly susceptible to grazing and are selectively grazed upon by protists. The study by Hahn et al. (2012ab) provides a thorough discussion on the effect of grazing on a specific lineage within the Polynucleobacter cluster. Generally, the above-mentioned studies on grazing on Limnohabitans and Polynucleobacter bacteria seem to suggest that the natural predation mortality of Limnohabitans bacteria is higher than that of Polynucleobacter bacteria; however, large intragenus differences in predation vulnerability can be expected for both taxa.

Environmental gradient analysis vs. single habitat analysis

Opposite distribution patterns of P. necessarius and Limnohabitans (R-BT065 lineage) along broad pH gradients (Fig. 1) were also observed in separate previous investigations (Jezberová et al., 2010; Šimek et al., 2010a). By contrast, our presented seasonal study of the Římov reservoir revealed no significant differences in the relation of the two taxa to parameters related to primary production or phytoplankton biomass. As Limnohabitans growth is expected to depend on a substrate pool derived from algal production (Šimek et al., 2011), the two taxa could be expected to reveal different relationships to these variables. However, the pH and conductivity values in the Římov reservoir remained virtually unchanged during the season, showing only small diurnal fluctuations (data not shown). This hints that other variables may be responsible for the segregation of these two groups, apart from self-correlating ones such as pH and conductivity. Interestingly, no direct significant relationship of Limnohabitans bacteria to ChlA was found, strongly contrasting with its positive relationship to certain algal groups, for example cryptophytes (Šimek et al., 2008). Jones et al. (2009), however, recently suggested a proxy, that is the ratio of water color to chlorophyll A (the CtCH ratio), which indicates the dominance of allochthonous vs. autochthonous organic carbon sources available for bacteria. This proxy showed significant correlation with the relative proportions of R-BT065 bacteria in total Betaproteobacteria (Šimek et al., 2010a), which further corroborates the finding on enhanced proportions of the Limnohabitans bacteria in nonhumic habitats where autochthonous organic carbon sources dominate. On the other hand, the P. difficilis/P. acidiphobus lineage shows a positive correlation with the above-mentioned variables related to primary production and phytoplankton biomass (Fig. 4), which agrees with the previous findings (Wu & Hahn, 2006b).

Lack of taxon-specific trends in substrate utilization patterns

The spectrum of substrates used in substrate utilization tests included several types of substances (e.g. acetate, pyruvate and other short-chain fatty acids) reported as typical products of photooxidation of humic substances (Moran & Zepp, 1997), as well as substances reported as algal exudates (e.g. carbohydrates, Giroldo et al., 2007). If P. necessarius and Limnohabitans tend to utilize different substrate pools, one could expect different preferences in the assimilation tests. Obviously, the performed tests indicated no taxon-specific trends in substrate preferences, although one has to take into account that these tests were performed at significantly higher substrate concentrations than usually inherent in the water environment. The predictive power of the utilization tests performed should thus not be overemphasized. It may be that the investigated taxa differ in substrate affinity, but not in substrate uptake potential, that is, the ability to absorb available substrate. Only the latter was tested by the substrate utilization tests.

We are aware of the fact that by using relatively high substrate concentrations for testing of the utilization in the laboratory, we might have overlooked differences between strains in substrate affinity, which could play an important role in niche partitioning. On the other hand, the revealed small genome sizes of Polynucleobacter bacteria (Vannini et al., 2007; Hahn et al., 2012ab) suggest that these bacteria can only encode a rather small number of substrate utilization pathways.

Potential limitations of ecological analyses caused by ecological diversification of taxa

Several contradictions revealed above indicate that the current pictures of the ecology of P. necessarius and Limnohabitans (R-BT065) are too simple. Several recent findings suggest intrataxon ecological diversification resulting in differently adapted ecotypes for both groups (Šimek et al., 2010b; Jezbera et al., 2011). It seems that – at least for future ecological investigations – taxonomy with a higher phylogenetic resolution as well as molecular tools enabling detection and quantification of newly defined taxa will be required. We demonstrated that the two groups P. necessarius and Limnohabitans represent together the majority of Betaproteobacteria in a broad range of habitat types. However, it seems that the postulated within-taxon diversity currently limits further insights into the ecological function of these bacterial taxa.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This study was mainly supported by GAČR projects P504/10/0566 awarded to J.J. and the Austrian Science Fund Project P19853 granted to M.W.H., and partially also by EEF/10/E011 project awarded to J.J. and GAČR project 206/08/0015 project awarded to K.Š. Anton Baer is greatly acknowledged for English proofreading.

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  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
fem1372-sup-0001-TableS1-S2.docWord document324KTable S1. List of sampled habitats sorted according to the increasing pH; showing date of sampling, measured pH, altitude, conductivity, DOC concentration, oxygen concentration, abundance of bacteria, fluorescence probe targeted groups of bacteria, Temp. – temperature. Table S2. Percentage of Polynucleobacter and Limnohabitans bacteria actively incorporating leucine and glucose, respectively as measured by MAR-FISH.

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