AAP bacteria are able to supplement their aerobic respiration with photosynthesized ATP, thus conserving organic carbon (Kolber et al., 2000, 2001). Because of such a unique role in global carbon cycling, AAP bacteria have increasingly drawn researchers' attention since the last decade. However, little is known about the abundance and diversity of AAP bacteria in high-elevation saline lakes.
AAP bacteria abundance
The abundance of AAP bacteria varies considerably in aquatic environments (Kolber et al., 2001; Schwalbach & Fuhrman, 2005; Cottrell et al., 2006; Sieracki et al., 2006; Lami et al., 2007), with estuaries and mountain lakes having a high abundance (Schwalbach & Fuhrman, 2005; Waidner & Kirchman, 2007). The reasons for such a large variation are not clear, although environmental nutrient status (Kolber et al., 2001; Waidner & Kirchman, 2007; Masin et al., 2008), light intensity (Waidner & Kirchman, 2007), associations with sediment particle (Waidner & Kirchman, 2007), and chlorophyll a concentration (Jiao et al., 2007) have been hypothesized to be some variables responsible for this variation. In the three Tibetan lakes studied, the fractions of AAP bacteria (i.e. the percentage of AAP bacteria in total bacteria) were comparable to the levels detected in the Indian (3.79 ± 1.72%), the Atlantic (1.57 ± 0.68%), and the Pacific (1.08 ± 0.74%) oceans (Table 2), but much lower than those in freshwater lakes of an oligotrophic to a mesotrophic nature (7–80% depending on seasonality and nutrient levels) (Masin et al., 2008) and in estuaries (Waidner & Kirchman, 2007, 2008). The lakes with the higher DOC content (i.e. Erhai and Gahai) exhibited lower AAP bacteria abundance, consistent with results from a previous study (Masin et al., 2008).
AAP bacterial diversity
The overall diversity of AAP bacteria was low in the Tibetan lakes. The AAP bacterial diversity decreased as the salinity and pH increased (Fig. 3, Table 3). Among the three lakes studied, Gahai Lake with the highest salinity and pH exhibited the lowest AAP bacteria diversity, Erhai Lake with the lowest salinity and pH had the highest diversity, and Qinghai Lake with an intermediate salinity and pH showed an intermediate diversity. Of the limited environmental factors examined, salinity and pH appeared to be important in controlling AAP bacteria diversity. Interestingly, the sediment sample at the 52 cm depth of Qinhai Lake had a salinity similar to the Erhai water column (c. 8 PSU) and, correspondingly, the AAP bacteria diversity for the QL-52-S sample was similar to that for Erhai (Table 3 and Fig. 3), further demonstrating that salinity might be one of the important factors in controlling AAP bacteria diversity in these lakes. However, this salinity–diversity relationship did not appear to hold true across different ecosystems. For example, the salinity in all three lakes studied was lower than that of seawater, but the AAP bacteria diversity was qualitatively lower than that in oceans. This complication suggests that a number of other factors could drive AAPB diversity (such as light availability, N and P availability, and UV irradiance) (Jiao et al., 2007 and references therein). In addition, ecosystem size and evolution time may be important factors accounting for this complication, but they were not examined in this study.
Despite recent advances in studies of AAP bacteria in many different environments, the relationship between environmental controlling factors and AAP bacterial community composition is still poorly understood. It appears that in saline water bodies (such as oceanic and coastal waters, lakes, and the saline side of estuaries), AAP bacteria belonging to Alphaproteobacteria and Gammaproteobacteria are dominant (Yurkova et al., 2002; Allgaier et al., 2003; Koblížek et al., 2003; Oz et al., 2005; Waidner & Kirchman, 2005, 2007, 2008; Cho et al., 2007; Jiao et al., 2007). In freshwater lakes and the freshwater side of estuaries, Betaproteobacteria are predominant (Karr et al., 2003; Waidner & Kirchman, 2005, 2008). This distribution pattern of AAB bacteria is similar to that defined by the 16S rRNA gene, i.e. with increased salinity, the relative abundance of Alphaproteobacteria and Gammaproteobacteria increases, but that of Betaproteobacteria decreases (Wu et al., 2006; Dong & Yu, 2007; references therein).
Consistent with this distribution pattern, our phylogenetic analysis revealed that the AAP bacterial community structure in the Tibetan saline lakes was dominated by Alpha- and Gammaproteobacteria, broadly similar to the AAP bacterial community structure in oceans. Indeed, Yurkova et al. (2002) observed a close phylogenetic relation of some strains purified from a meromictic lake (Mahoney Lake in British Columbia) to species obtained from marine environments. This observation implies that the similar environmental conditions of saline lakes (including Mahoney Lake) and the oceans (i.e. pH, salinity, and water chemistry) may have been responsible for the similar AAP bacterial community structure. Interestingly, Jiang et al. (2008) observed archaeal biogeography where similar environmental conditions between Qinghai Lake and the world oceans resulted in a similar archaeal community structure. However, a closer examination at a finer phylogenetic resolution reveals differences in the AAP bacterial community structure between the oceans and the Tibetan lakes studied. For example, Roseobacter- and Erythrobacter-related AAP bacterial clone sequences of Alphaproteobacteria were ubiquitous in the major ocean regimes (Jiao et al., 2007), but Loktanella-like sequences of Alphaproteobacteria were predominant in the Tibetan lakes (this study). Considering that AAP bacteria require light for energy, it is not surprising to observe such a difference, because of drastically different elevations and thus light intensities between Qinghai Lake and the oceans.
It is remarkable to note that the AAP bacterial community structure of Erhai Lake was completely different from that of either Qinghai or Gahai Lake. This difference could not be solely ascribed to salinity, because the salinity contrast between Erhai Lake and Qinghai Lake/Gahai Lake was not as great as between freshwater and seawater in the Delaware estuary (Waidner & Kirchman, 2008). The AAP bacterial community structure from Erhai Lake (with the lowest salinity, 9.8 PSU) was not dominated by freshwater Betaproteobacteria-like, but by saline water Gammaproteobacteria-like AAP bacterial sequences. An important difference between Erhai Lake and Qinghai/Gahai Lake was turbidity (Table 1). Whereas Erhai Lake was turbid, Qinghai and Gahai Lakes were clear. Thus, it was reasonable to speculate that the AAP bacteria detected in Erhai may largely be associated with sediment particles, whereas AAP bacteria in Qinghai/Gahai Lakes were mostly free-living. Previous studies observed that particle-attached and free-living bacterial community composition is fundamentally different (DeLong et al., 1993; Crump et al., 1999; Phillips et al., 1999; Schweitzer et al., 2001; Acinas et al., 2005). It has been shown that most AAP bacteria are associated with particles, and this attachment apparently increases their competitiveness in estuary environments (Yutin et al., 2007; Waidner & Kirchman, 2008).
Although the Qinghai Lake water column was vertically mixed with only slight stratification of geochemical variables (Table 1) and there was no systematic decline in the AAP bacteria abundance with increased depth, the AAP bacteria community composition did vary with depth. This variation may be partly caused by decreased light intensity with depth. It is well established that light intensity affects pigment formation necessary for photosynthesis (Aagaard & Sistrom, 1972; Drews & Golecki, 1995). Blue light inhibits photosynthetic pigment accumulation in Roseobacter denitrificans (Iba & Takamiya, 1989; Takamiya et al., 1992). The fact that the Roseobacter-like pufL-M sequences at the bottom were more abundant than the top of the water column in Qinghai Lake (Fig. 3) appeared to be consistent with this inhibition effect. Because of high elevation, UV light intensity is expected to be much stronger than at sea level (Askew, 2002).
AAP bacteria in anoxic sediments: fossil or light-independent AAP bacteria?
Retrieval of AAP bacteria pufL-M gene sequences from the anoxic and dark sediments of Qinghai Lake was unexpected (Fig. 2). Although the surface sediment of Tokyo Bay was used previously for AAP bacteria isolation work (Yurkov & Beatty, 1998b), AAP bacteria were never isolated successfully. We postulated that two possibilities could explain the presence of the AAP bacteria pufL-M genes in the sediments: (1) they were derived from dead AAP bacteria cells deposited from the overlying water column (fossil DNA); (2) they were derived from active cells (of unknown metabolic pathways) that may have the pufL-M genes. The water column and sediments of Qinghai Lake had some AAP bacteria groups in common, such as the Loktanella-like, Roseobacter-like, Porphyrobacter/Erythrobacter-like, and unknown group-1 sequences. Excluding the unknown group-1, none of the known AAP bacteria in the Loktanella-like, Roseobacter-like, and Porphyrobacter/Erythrobacter-like groups has either been cultured from sediments or shown the ability to survive in anoxic environments, because they all require light for growth. Thus, the AAP bacteria-originated pufL-M gene sequences in the sediments might have been of fossil origin. The fossil DNA of obligate phototrophic algae and their lipid derivatives have been well studied in the context of paleoecology, archaeology, and paleontology (Coolen & Overmann, 1998; Coolen et al., 2004, 2006). However, no similar studies have been performed on AAP bacteria-derived fossil DNA.
Despite the similarity of some cloned gene sequences between the water column and the sediments of Qinghai Lake, multiple AAP bacterial pufL-M gene sequences retrieved from the water–sediment interface (i.e. the unknown group-2, Figs 2 and 3) were not found in the water column. This observation also indicated two possibilities. First, these sequences may have been present in the water column but were overlooked using our sampling scheme. In this scenario, these sequences may represent fossil DNA from the water column (see the discussion above). Second, these AAP bacteria pufL-M gene sequences might be unique to the sediments and possibly derived from indigenous cells. Xiong et al. (2000) and Beatty (2002) proposed that certain purple photosynthetic bacteria might have lost their photosynthetic functions in evolution. According to such a theory, Jiao et al. (2007) speculated that some AAP bacteria species could have lost their photosynthetic genes and gradually developed heterotrophic metabolic pathways. If this evolution pathway were true, AAP bacteria at the transition might have first developed heterotrophic metabolic pathways before they became completely independent of light (i.e. loss of pufL-M genes). The AAP bacteria cells, from which the pufL-M gene sequences in the unknown group-2 originated, could have represented this transition stage. Although these AAP bacteria could still possess the pufL-M genes because they may be deposited from the oxic water column within years (according to a sedimentation rate of 1.25 mm year−1, CAS, 1979), the anoxic and dark environments in the Qinghai Lake sediments would not permit them to depend on light for growth. Such AAP bacteria could have developed a distinct heterotrophic pathway to cope with the conditions in the sediments. However, future work would be necessary for verification of this evolution hypothesis.