Bacterioplankton communities in a high-altitude freshwater wetland

Abstract

Microbial communities play a crucial role in various biogeochemical processes in aquatic ecosystems. However, existing knowledge on microbial communities in the waters of wetlands is still very scant. The objective of the present study was to investigate the bacterioplankton community in the Luoshijiang Wetland, a high-altitude freshwater wetland in the Yunnan-Kweichow Plateau. Water samples were collected from different sites. The bacterioplankton community was characterized using 16S rRNA gene clone library analysis. A spatial variation of the diversity and composition of the bacterioplankton community was observed. Verrucomicrobia and Proteobacteria were the most abundant components. Proteobacteria might play an important role in water self-purification, but the significance of Verrucomicrobia remained unclear. Moreover, Pearson’s correlation analysis showed that Actinobacteria and Gemmatimonadetes were positively correlated with nitrite nitrogen in waters, while Alphaproteobacteria with dissolved phosphorous.

Introduction

Microbial communities play vital roles in decomposition of organic matter, remineralization of nutrients, and biogeochemical cycling in terrestrial and aquatic environments. Wetlands are unique ecosystems, and can act as ecotones between terrestrial and aquatic systems (Menon et al. 2013). Wetlands are important players in nutrient cycling, sediment accretion, pollution filtration, and erosion control (Qin and Mitsch 2009). Knowledge of microbial communities can greatly contribute to our understanding of the complex processes within wetland ecosystems. To date, little is known about microbial communities in wetland ecosystems. A few previous studies indicated that microbial community structure in wetlands was dependent on soil type (Tang et al. 2012; Peralta et al. 2013), soil depth (Tang et al. 2012), carbon and nitrogen sources (Morrissey et al. 2013), vegetation (Akiyama et al. 2010), and successional stage (Tang et al. 2011). These previous studies focused on soil microorganisms in wetlands. However, the bacterioplankton community is also a key component in aquatic ecosystems, due to its efficient nutrient uptake, high abundance, and large growth potential (Parveen et al. 2013). Unfortunately, information on bacterioplankton communities in the waters of wetlands is still very limited (Dorador et al. 2013).

The Luoshijiang Wetland, a typical freshwater wetland in the Yunnan-Kweichow Plateau, is located in Dali City, Yunnan Province. The fresh wetland is adjacent to the Rrhai Lake, the second largest high-altitude lake in Yunnan Province. The wetland covers an area of 1 km² with an elevation of about 2,056 m and is surrounded by farmlands. Annual mean air temperature and annual precipitation in the local region were 15.7 °C, and 1000–1200 mm, respectively. To date, information on microbial communities in high-altitude wetlands is still very scant. Tang et al. (2012) investigated soil bacterial communities in the Zoige Wetland of the Qinghai-Tibetan Plateau in China. Dorador et al. (2013) reported on microbial diversity of five high altitude wetlands from the Chilean Altiplano. Unfortunately, there has been no report on microbial communities in other high-altitude wetlands. Therefore, the main objective of the current study was to investigate the bacterioplankton community structure of the Luoshijiang Wetland.

Materials and methods

Study sites and water sampling

Surface water samples (0–20 cm) in triplicate from four different sites of the Luoshijiang Wetland were collected in March 2013: A (25°57′25″N–100°06′06″E, no vegetation zone), B (25°57′12″N–100°05′59″E, reed-planted zone), C (25°57′4″N–100°06′00″E, densely water-lily-planted zone), and D (25°56′55″N–100°05′59″E, sparsely water-lily planted zone) (Fig. 1). At the time of sample collection, the temperature and pH of the four samples were about 15 °C and 7.5, respectively. The chemical parameters of the water samples are shown in Table 1.

Fig. 1

Schematic representation of the Luoshijiang Wetland and sampling sites.

Table 1 Chemical parameters of water samples

Bacterial clone library analysis

For analysis of the bacterial community, water samples (250 mL) were filtered through 0.22-μm-pore-size membranes (diameter 50 mm; Millipore). The membrane filter was cut into quarters with a sterile scalpel and was used for further molecular analysis. DNA was extracted using the E.Z.N.A.® Water DNA kit (Omega, USA) according to the manufacturer’s protocol. Bacterial clone libraries were constructed according to the literature (Zhang et al. 2012; Lu et al. 2013). Briefly, bacterial 16S rRNA genes were amplified using primers 27 F (5′-GAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′). The purified PCR products were cloned into pGEM-T-easy Vector (Takara Corp, Japan), and clones containing an insert of the correct size were sequenced. Chimera-free bacterial sequences with ≥97 % identity were grouped as operational taxonomic units (OTUs). OTUs, rarefaction curves, and Shannon diversity index were determined using the DOTUR program (Schloss and Handelsman 2005). The Ribosomal Database Project analysis tool “classifier” ( http://rdp.cme.msu.edu/classifier/classifier.jsp) was used to determine the taxonomic identities of the bacterial sequences (Wang et al. 2007). In addition, Pearson’s correlation analysis of the bacterial community structure with the determined chemical parameters was performed using SPSS 20.0 software.

Nucleotide sequence accession numbers

The sequences obtained in this study were submitted to GenBank under accession numbers KF443412–KF443504 for Sample A, KF443505 –KF443596 for Sample B, KF443597–KF443691 for Sample C, and KF443692 –KF443782 for Sample D, respectively.

Results

Bacterial diversity

A total of 91– 95 bacterial sequences were retrieved from each sample. Based on the threshold of 3 % difference, OTUs, rarefaction curves, and Shannon diversity index were obtained using the DOTUR program (Schloss and Handelsman 2005). Clone libraries with Samples A, B, C, and D were composed of 26, 35, 9, and 20 OTUs, respectively (Table 2). The Shannon diversity index of bacterial community in Sample C was only 0.7, much lower than that in the other three samples (1.7– 3.2).

Table 2 OTU-based community richness and diversity indices for Samples A–D

The rarefaction curve for Sample C approached a plateau, indicating that the community was well sampled but had a very low bacterial diversity (Fig. 2). However, the rarefaction curves for Samples A, B, and D did not level off completely, suggesting that further sequencing would have resulted in more OTUs in each sample.

Fig. 2

Rarefaction curves of OTUs in Samples A–D evaluated by 3 % sequence variation

Bacterial community composition

The bacterial phylum composition of the four water samples is shown in Fig. 3. In this study, six known phyla were identified in these samples including Proteobacteria, Actinobacteria, Bacteroidetes, Cyanobacteria, Gemmatimonadetes, and Verrucomicrobia. However, only Proteobacteria, Bacteroidetes, and Verrucomicrobia were shared among all the four water samples. A shift in the composition of major bacterial groups was found in the different sampling sites in the Luoshijiang Wetland. For example, the major bacterial groups (with relative abundance no less than 10 %) in both Sample A and Sample D were Verrucomicrobia (64.5 % or 65.9 %) and Gammaproteobacteria (12.9 % or 15.4 %), while Verrucomicrobia (88.4 %) predominated in Sample C. Sample B was mainly represented by Verrucomicrobia (43.5 %), Betaproteobacteria (25 %), and Gammaproteobacteria (17.4 %).

Fig. 3

Comparison of the quantitative contribution of the sequences affiliated with different phyla and classes to the total number of sequences from Samples A–D. Sequences not classified to any known phylum are included as unclassified bacteria.

Table 3 shows the Pearson’s correlation coefficients for the relationship between the proportion change of the major bacterial groups and water chemical properties. Actinobacteria and Gemmatimonadetes had a significant positive correlation with nitrite nitrogen (p < 0.05). Alphaproteobacteria also showed a significant positive correlation with dissolved phosphorus (p < 0.05). However, other major bacterial groups did not show any significant correlation with the determined water chemical parameters. Correlation analyses were limited by the very low number of samples analyzed (n = 4).

Table 3 Pearson’s correlation coefficients describing the relationship between water characteristics and the change of the relative abundance of major bacterial groups

Table 4 illustrates the abundance and distribution of the 24 known genera detected in the four samples. At the genus level of taxonomic classifications, variations of bacterial community structure among the four samples were more evident. For Samples A and B, a large portion of sequences (50/93 or 61/92) could be classified at the genus level. In contrast, for Samples C and D, only a small portion of sequences could be related to known bacterial genera. A total of 14 genera were present only in a single sample. Members of genus Luteolibacter were dominant in Samples A and B, but were detected with much lower abundance in Samples C and D. Haloferula was the third largest component in Sample A, while the minor one in the other samples. These results indicate a large spatial variation of verrucomicrobial communities in the investigated wetland. Moreover, the abundance and composition of proteobacterial communities also differed greatly among the four different samples.

Table 4 Distribution of the sequences affiliated with the identified genera in Samples A–D

Discussion

Bacterial community in the water body of freshwater ecosystems is usually highly diverse (Humbert et al. 2009; Kwon et al. 2011; Kadnikov et al. 2012). However, to the authors’ knowledge, this was among the first reports on diversity of bacterioplankton communities in freshwater wetlands. A spatial variation of bacterioplankton community diversity was observed in the Luoshijiang Wetland.

Verrucomicrobia is a universally distributed phylum and can be present in terrestrial and aquatic habitats (Arnds et al. 2010; Freitas et al. 2012; Parveen et al. 2013) and manmade bioreactors (Feng et al. 2013; Liao et al. 2013a, b). The reported isolates from phylum Verrucomicrobia could utilize various carbon compounds (Arnds et al. 2010). Little attention has been paid to the abundance of aquatic Verrucomicrobia, although a few previous studies have shown the dominance of Verrucomicrobia in the waters of freshwater lakes (Lindström et al. 2004; Arnds et al. 2010; Kolmonen et al. 2011). The abundance of Verrucomicrobia in lakes might be positively correlated with nutrient-richness and phosphorus availability, and could vary between seasons and between more and less humic basins (Arnds et al. 2010). However, de Figueiredo et al. (2007) found that Verrucomicrobia was associated with the most oligotrophic aquatic ecosystems and low pH values. To date, the existing information on the abundance and distribution of Verrucomicrobia in the waters of wetlands is very scant. Dorador et al. (2013) revealed a low abundance of Verrucomicrobia in high-altitude wetlands of the Chilean Altiplano. To the authors’ knowledge, this was the first report on the dominance of Verrucomicrobia in the waters of wetlands. In this study, Verrucomicrobia was the largest component in the bacterial communities, but its relative abundance varied among the different sampling sites. Surprisingly, no significant correlation was observed between Verrucomicrobia and the determined water chemical parameters. Therefore, further efforts are necessary in order to elucidate the links between Verrucomicrobia and the environmental parameters in aquatic ecosystems.

Luteolibacter (Verrucomicrobia) was the largest genus detected in the Luoshijiang Wetland. To the authors’ knowledge, this was the first report on the dominance of Luteolibacter in a freshwater ecosystem. Members of genus Luteolibacter have been isolated from activated sludge (Park et al. 2013), Arctic tundra soil (Jiang et al. 2012), marine environments (Yoon et al. 2008), and leek rhizosphere (da Rocha et al. 2011). Unfortunately, the role of Luteolibacter species in the environment remains largely unclear. Only a recent study indicates Luteolibacter algae H18 could assimilate fucoidan as a sole carbon source (Ohshiro et al. 2012). Moreover, information on the other detected verrucomicrobial genera Haloferula, Prosthecobacter, and Opitutus is still very limited, and their ecological roles also remain unclear. Therefore, further study is necessary in order to elucidate the significance of the dominance of phylum Verrucomicrobia in wetland.

Phylum Proteobacteria might play active roles in biodegradation of organic pollutants and carbon cycling, and various biogeochemical processes in aquatic ecosystems (Cheng et al. 2013). Proteobacteria (mostly Alpha-, Beta-, and Gammaproteobacteria) usually predominate in freshwater habitats (Kwon et al. 2011). There have also been a few reports on the abundance and composition of Proteobacteria in the waters of high-altitude lakes. Sommaruga and Casamayor (2009) found that Betaproteobacteria commonly predominated in high-altitude lakes in the Mount Everest region (Nepal). Betaproteobacteria was also the dominant group in Lake Namco, the largest Tibetan lake (Liu et al. 2013a). Wu et al. (2006) revealed that, in 16 high-mountain lakes located on the Tibetan Plateau (China), Betaproteobacteria was abundant in all freshwater lakes, while Alpha- and Gammaproteobacteria gained much higher abundance in saline lakes. However, very limited information exists on the abundance and composition of Proteobacteria in the waters of wetlands. Dorador et al. (2013) reported the abundance of Proteobacteria (alpha, beta, gamma and delta subgroups) in high-altitude wetlands of the Chilean Altiplano. In this study, Proteobacteria, composed of alpha, beta and gamma classes, was the second largest phylum in the Luoshijiang Wetland, but a marked shift in its relative abundance occurred in the different sampling sites. Alphaproteobacteria was detected with low abundance in all the water samples and was positively correlated with dissolved phosphorus (p < 0.05). Betaproteobacteria was one of the major components in Sample B (25 %), but became much less abundant in the other sampling sites. Gammaproteobacteria was abundant in Samples A, B and D. However, Betaproteobacteria and Gammaproteobacteria did not show significant correlation with the determined water chemical parameters.

Microorganisms from alphaproteobacterial genera (Rhodobacter and Sphingomonas), betaproteobacterial genera (Hydrogenophaga and Acidovorax), and gammaproteobacterial genera (Pseudomonas, Shewanella and Aeromonas) are known for biodegradation of a variety of environmental organic pollutants (Zhang et al. 2011; Ogugbue et al. 2012; Wang et al. 2012; Cheng et al. 2013; Johnson et al. 2013; Liao et al. 2013a; Liu et al. 2013b). Therefore, the presence of these microorganisms might play important roles in reduction of organic pollutants and water self-purification in the Luoshijiang Wetland.