Surface (1 m depth) and bottom (1–2 m above sediment) waters were collected along the salinity gradient of the Pearl Estuary from 16 July to 12 August, 2012 using a Sealogger CTD (SBE 25, Sea-Bird Co.) rosette water sampler (Liu et al., 2014). A total of 16 water samples from eight sites, including P01, P03, and P07, located in the freshwater part of the estuary, C2, C3, and A08, located in the mesohaline region, and F412 and F414, located in the polyhaline region, were collected (Figure 1). One liter of water samples was pre-filtered through 3-μm-pore size filters to remove large organisms and particles, and free-living bacterioplankton cells were collected through 0.22-μm polycarbonate membranes (Millipore Corporation, Billerica, MA, USA). Filters were frozen at −80°C until DNA extraction. Water chemistries such as salinity, temperature, pH, turbidity and DO were monitored with the CTD. Samples for nutrients (NO⁻₂, NO⁻₃, NH⁺₄, and PO³⁻₄) were filtered with 0.45-μm cellulose acetate membranes and analyzed on deck with a QuAAtro (Bran-Lube) except for NO⁻₃, which was analyzed in land-based laboratory at Xiamen University with spectrophotometric and colorimetric methods using an AA3 Auto-Analyzer (Technicon) (Dai et al., 2008; Han et al., 2012; Liu et al., 2014).

The collected samples were immediately mixed with paraformaldehyde (Sigma-Aldrich, St. Louis, MO, USA; final concentration 2%, v/v) for 1 h in the dark at room temperature, and then stored in liquid nitrogen onboard. The abundances of Synechococcus, picoeukaryotes and heterotrophic bacteria were measured by flow cytometry (BD FACSVantage SE, Becton, Dickinson, USA) (Marie et al., 1997).

DNA was extracted according to Yin et al. (2013) with an additional step to facilitate cell lysis by a Fast Prep-24 Homogenization System (MP Biomedicals, Irvine, California, USA). Amplification of bacterial 16S rRNA gene fragments was conducted using barcode and adaptor added primer 28F (5′-AGAGAGTTTGATCCTGGCTCAG-3′) and 519R (5′-TTACCGCGGCTGCTGGCAC-3′) (Wu et al., 2012). Barcode sequences were ligated to the sequencing primer during the process of primer synthesis, before PCR was performed. A 20 μl PCR reaction was performed in triplicate at the following condition: an initial denaturation at 95°C for 2 min, 25 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and then a final extension at 72°C for 5 min. The triplicate PCR products were pooled and purified using an AxyPrepDNA Gel Extraction Kit (Axygen, Hangzhou, China), and then quantified using a Quant-iT PicoGreen double-stranded DNA assay (Invitrogen, Carlsbad, CA). The amplicons from each reaction mixture were pooled in equimolar ratio based on concentration and subjected to emulsion PCR to generate amplicon libraries. Sequencing was carried out on a Roche Genome Sequencer FLX Titanium platform at Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China.

The 454 sequences have been deposited at the National Center for Biotechnology Information (NCBI) Short Read Archive database under accession number SRP019932.

The raw reads were processed following the pipeline of Mothur (Schloss et al., 2009). All reads completely matching the barcodes were retained as well as reads with a maximum single mismatch to the primers. Reads were then trimmed by removing the sequencing adaptor, barcodes and primer sequences. These reads were further screened by using the following thresholds: (i) minimum average quality score of 25; (ii) minimum read length of 200 bp; (iii) sequences containing no ambiguous bases; and (iv) maximum homopolymers of 8 bp. Chimeric sequences were identified and removed using UCHIME Ref (Edgar et al., 2011). “Pre-cluster” (Huse et al., 2010) was further used to remove reads caused by sequencing error. Totally 86468 reads were left after quality control and were clustered into OTUs at a 3% distance level with the furthest neighbor algorithm. Taxonomy was assigned against the SILVA v111 ref database (http://www.arb-silva.de) at a minimum support threshold of 80%.

The distribution and abundance matrix of OTUs was randomly resampled using “daisychopper.pl” (Gilbert et al., 2009) to equalize sampling efforts. After normalizing, 4354 sequences were left for each sample. Alpha diversity measures including richness estimator Chao 1 (Chao and Bunge, 2002), diversity index Shannon (Magurran, 1988) and Good's coverage (Good, 1953) were calculated at a 3% dissimilarity level in Mothur. For the beta diversity, a multi-sample similarity dendrogram was constructed according to the OTUs composition and abundance based on Bray-Curtis dissimilarity. Pairwise analyses of similarities (ANOSIM) of bacterioplankton communities were calculated in PRIMER 5 (Plymouth Marine Laboratory, West Hoe, Plymouth, UK). A ternary plot was generated in R software (RDC Team, 2008) to compare the community composition of different groups at the genus level. Redundancy analysis (RDA) with Monte Carlo test was performed to calculate the relationship between bacterial clades and water properties at both order and genus levels, following the results of pretested detrended correspondence analysis using Canoco (Version 4.5, Microcomputer Power). A neighbor-joining phylogenetic tree comparing bacterial community compositions in different estuaries was constructed by MEGA 5 (Tamura et al., 2011).

Considering the shallow water depth of the Pearl Estuary [Table S1 (Liu et al., 2014)], only surface and bottom water samples were collected, which were designated _S (surface) and _B (bottom), respectively in the following analysis. The freshwater and saltwater end members were separated by the Humen Outlet. The physicochemical attributes of waters from both freshwater and saltwater regions have been described in detail [Table S1 (Liu et al., 2014)]. Briefly, the freshwater sites had significantly lower levels of salinity, DO and pH, and higher levels of nutrients and Chl a than the saltwater sites. There were clear vertical environmental gradients in the saltwater area, with the surface water possessing a higher level of NO⁻₃, and lower levels of turbidity and salinity compared with the bottom water. The DO content of the freshwater sites was extremely low (<1 mg L⁻¹) except for P03 (>3 mg L⁻¹), indicative of severe hypoxia.

The overall abundance of heterotrophic bacteria from flow cytometry decreased from the freshwater to saltwater sites and was positively correlated with nutrients (Figure S1). Synechococcus and picoeukaryotes displayed opposite trends and both of them peaked in surface waters of sites A08 and F412. While picoeukaryotes were positively correlated with nutrients, Synechococcus was negatively correlated with turbidity (Figure S1). In the freshwater sites, the abundances of all three groups were basically constant between water layers, whereas in the saltwater sites, higher abundances of all three groups were observed in the surface water than the bottom water.

Totally, 86468 reads, ranging from 4354 to 6504 in all samples, were obtained for further analyses. The average length of the obtained reads was 470 base pairs. After randomly resampling, a total of 5314 OTUs were assigned at the 3% dissimilarity threshold. Both of the rarified Chao1 and Shannon diversity indexes basically showed no remarkable differences (P > 0.05, Wilcoxon's rank test) across all samples with the exception of surface waters of sites F412 and F414, where lower estimators were observed (Table 1). This was further confirmed by the Shannon curves that plotted with rarefied data (Figure S2). The Good's coverage values at the 3% dissimilarity level were 84.4–94.3% among all samples indicating that the libraries could represent most of the species in the natural habitat.

Totally, 41 bacterial phyla were found. Significant variations in bacterioplankton communities at the phylum level (plus proteobacterial classes) were observed along the salinity gradient. While marked distinction in bacterial assemblages was observed between the two depths in the saltwater sites, no consistent difference was observed in the freshwater sites (Figure 2A).

Betaproteobacteria dominated the freshwater sites but its orders distributed differently along the estuary, with Burkholderiales (r = −0.937, P < 0.05; Pearson correlation coefficient with salinity) and Rhodocyclales (r = −0.851, P < 0.05), predominant in the freshwater sites whereas Hydrogenophilales in the saltwater sites. It was noticeable that the abundance of Methylophilales was markedly different among the three freshwater sites with a higher amount at site P03. Synechococcus of Cyanobacteria was the most predominant group in the surface water of saltwater sites, consolidating the result of flow cytometry, whereas Oceanospirillales of Gammaproteobacteria, SAR406 clade of Deferribacteres and SAR324 clade of Deltaproteobacteria were more prevalent in the bottom water.

There were slight increases of Alphaproteobacteria and Bacteroidetes from freshwater to saltwater samples with Alphaproteobacteria occupying both depths of the saltwater sites whereas Bacteroidetes was slightly higher in the bottom. Among Alphaproteobacteria, orders Sphingomonadales and Rhizobiales were more abundant in the freshwater sites whereas Rhodobacterales dominated the saltwater sites. In addition, a small amount of SAR11 clade was detected in the bottom water of saltwater sites. Within Bacteroidetes, the abundance of Flavobacteriales was significantly higher in saltwater samples than freshwater ones. Actinobacteria was almost consistent across all samples, within which, Frankiales and Acidimicrobiales dominated the freshwater and saltwater sites, respectively, and the abundance of Acidimicrobiales was higher in the bottom water (Figure 2B).

Eleven clades, either within the top 50 OTUs (see below) unaffiliated to any described genera in SILVA or the top 50 genera undesignated at a given family (for example, Rhodobacteraceae uncultured or Acidimicrobiaceae uncultured), were designated PE1-11 at the genus level, respectively (ranked by abundance, Table S2 and Figure 3).

The hgcI clade (Actinobacteria), followed by R.12up (Rhodocyclales) and Hydrogenophaga (Burkholderiales), as well as the newly named PE6-8 clades, were abundant in the freshwater sites. In the saltwater sites, PE1 and PE3 clades were abundant at both depths with slightly higher proportions in the surface water. Meanwhile, Synechococcus, and PE9 and PE11 dominated the surface and bottom waters, respectively. Furthermore, the proportions of genera such as Roseobacter and Thiobacillus increased. Clades affiliated to Flavobacteriaceae shifted from Cloacibacterium in the freshwater sites to NS5, NS4 marine group, Owenweeksia and Polaribacter in the saltwater sites (Figure 4). Fluviicola, affiliated to Bacteroidetes, was one member of the very few genera that commonly distributed in the sampling areas (Figure 4). However, the freshwater-dominant members of Fluviicola formed a monophyletic cluster as shown in the phylogenetic tree constructed using sequences of the top 16 Fluviicola OTUs (Figure S3).

The bacterial community compositions of the hypoxic (P01 and P07) sites were distinguished from that of the non-hypoxic site (P03) (Figure S4). In concordance with the order level result, the abundance of PE2, a prevalent genus of Methylophilaceae in the freshwater sites, was significantly higher in the non-hypoxic sites than the hypoxic sites, whereas R.12up affiliated to Rhodocyclales showed an opposite trend. In addition, the hypoxic sites harbored more unclassified reads at both the order and genus levels.

While significant phylogenetic shifts were observed at the order level, OTU level analysis facilitated discerning reads even belonging to the same genus. The abundance of OTU 5 belonging to NS5 marine group was higher in the bottom water of saltwater sites while OTU 52, also affiliated to NS5 marine group, was more abundant in the surface water. Analogously, within the OCS155 marine group, OTU 7 and OTU 14 were more abundant in the surface water while OTU 2, 8, 18, 26, and 50 showed opposite trends (Figure 3).

Based on the composition and abundance of OTUs, the multi-sample dendrogram clustered all samples into three groups: surface and bottom waters of the freshwater sites (SF+BF), surface waters of the saltwater sites (SS), and bottom waters of the saltwater sites (BS) (Figure 5). The ANOSIM R statistic (Table S3) further confirmed the grouping result, as no significant variation was observed between SF and BF (R = −0.407, P > 0.05).

The RDA across all samples was conducted to find the determinant environmental parameters shaping specific orders (Figure 6A). The first axis explained 65.6% of the total variance while the second axis explained 11.9%. Montel Carlo permutation tests showed that most of the environmental parameters except for depth significantly (P < 0.05) contributed to the heterogeneous distribution of major bacterial clades. Salinity explained most of the variation, and different clades of Betaproteobacteria and Actinobacteria were observed occupying distinct salinity ranges. N, P nutrients were also determinant factors. Sphingomonadales was found to be more related to NO⁻₃, while Rhodocyclales and Methylophilales were positively related to NO⁻₂ and PO³⁻₄. In addition, Synechococcus was negatively related to turbidity, in agreement with the result obtained by flow cytometry.

The overall comparison would obscure the impact of some important factors, due to the complex environmental features of the Pearl Estuary. Therefore, RDA analysis targeting only freshwater sites was conducted, as quite different DO levels were observed among the three sites. DO (F = 2.76, P < 0.01) and Chl a (F = 2.51, P < 0.01) were the only two significant factors accounting for the variability. Methylophilales was positively correlated to DO, while Rhodocyclales, Rhizobiales and Sphingomonadales were on the opposite correlation (Figure 6B). Although not significant, Frankiales seemed to be positively correlated with NO⁻₃, while negatively correlated with PO³⁻₄ and NH⁺₄. Taken together, these results were quite different from that revealed by the overall RDA, indicating a significant impact of hypoxia on bacterial structure.

In-depth genus distribution patterns determined by environmental factors revealed that many genera such as R.12up (Rhodocyclales), PE2 (Methylophilales) and hgcI (Actinobacteria) were inversely related to salinity. Nevertheless, PE2 and R.12up as well as Novosphingobium and Dechloromonas were also positively correlated to nutrients (Figure 6C). Specifically, Novosphingobium and Dechloromonas were positively correlated to PO³⁻₄ and NH⁺₄, while PE2 and R.12up were positively correlated to NO⁻₃ and NO⁻₂ (Figure 6C).

The RDA targeting only freshwater sites was also conducted at the genus level. We surprisingly found that NH⁺₄, PO³⁻₄, and NO⁻₃, instead of DO, became the most significant environmental factors. Polynucleobacter and CL500-29 marine group were driven by NO⁻₃/NO⁻₂, while Novosphingobium and Dechloromonas were still correlated well with PO³⁻₄ and NH⁺₄. At the same time, PE2 was positively correlated with DO, whereas R.12up was on the opposite position (Figure 6D).

Bacterial community data of the Delaware Bay (Campbell and Kirchman, 2013) and Baltic Sea (Herlemann et al., 2011) were used as proxies of temperate estuaries. These two datasets were selected because they were both carried out using the high throughput sequencing and the PCR fragments were overlapped with that of this study. The bacterial community of the Baltic Sea was used because it receives large freshwater discharges which generate far-reaching saltwater-freshwater gradients, although not being a typical estuary. The phylogenetic tree (Figure S5) showed that Verrucomicrobia was a prevalent phylum in the two temperate estuaries, but accounted for only a minor proportion in the subtropical Pearl Estuary. SAR11 clade was found in all the three locations whereas Sphingomonadales and Rhizobiales were only detected in the Pearl Estuary, which might resulted from the high nutrient inputs. Moreover, among Rhodobacterales, sequences obtained from the Baltic Sea were phylogenetically different from that from the Pearl Estuary.

Zhou et al. (2011) reported a decline trend of heterotrophic bacterial abundance from freshwater to coastal areas of the Pearl Estuary along with the decrease of nutrients. Our study confirmed this result as heterotrophic bacteria was found to be positively related to nutrients. Similar to previous results on estuaries, a significant variation in bacterioplankton community between freshwater and saltwater sites was observed (Kirchman et al., 2005; Zhang et al., 2006). However, a major finding of our study was the phylogenetic shifts in clades occupying different estuarine areas, consistent with the result in the Baltic Sea (Herlemann et al., 2011). Alphaproteobacteria was generally abundant in marine waters (Kirchman et al., 2005; Zhang et al., 2006). Nonetheless, we observed a relative small difference in the abundance of Alphaproteobacteria between the two areas of the Pearl Estuary, with Rhodobacterales (a common Alphaproteobacteria order in the polyhaline water, Campbell and Kirchman, 2013) dominating the saltwater sites while Sphingomonadales and Rhizobiales showing opposite distributions. Sphingomonadales has been documented to have wide metabolic capabilities (Miller et al., 2010) and can degrade aromatic compounds (Fredrickson et al., 1995), and its abundance, especially that of Novosphingobium and Sphingobium, appeared to be driven by high concentrations of PO³⁻₄ and NH⁺₄ (Figure 6). Additionally, Rhizobiales was active in biofilm formation under both oxic and anoxic conditions (Masuda et al., 2010). Thus, the prevalence of them in the freshwater area contributed to the comparable abundance of Alphaproteobacteria between the two end members of the Pearl Estuary and might be attributed to the highly polluted condition resulted from the increasing anthropogenic disturbances. These results indicated that the high concentration of nutrients has shifted the bacterial community components toward copiotrophic groups.

Among Actinobacteria, OCS155 marine group and hgcI were the two groups dominant in the saltwater and freshwater areas, respectively. Since its first discovery in Oregon coastal waters (Rappe et al., 2000), the OCS155 marine group has been found in various marine waters (e.g., Lu et al., 2015), but its function is still unknown. The hgcI clade, also known as acI, is common and abundant in a wide range of freshwater habitats (Warnecke et al., 2004), a recent single-cell genomic study showed that it had a strong genetic ability to take carbohydrate and N-rich organic compounds (Ghylin et al., 2014). The hgcI clade had also the potential to utilize sunlight via actinorhodopsin which might promote anaplerotic carbon fixation (Ghylin et al., 2014), indicative of both heterotrophic and autotrophic lifestyles of this clade. In this study, although the abundance of hgcI clade was constant in hypoxic and non-hypoxic samples, no apparent phylogenetic shift between these sites was observed (data not shown). This suggests that the hgcI clade could tolerant to a lower DO content and the maintaining mechanism needs to be investigated further.

It was interesting that Thiobacillus, affiliated to Hydrogenophilales of Betaproteobacteria was found prevalent in the saltwater area of the Pearl Estuary, different from the classical freshwater Betaproteobacteria such as Rhodocyclales and Burkholderiales. Thiobacillus is a well-known sulfur-oxidizing bacteria and its predominance may suggest frequent occurrence of sulfur oxidization process in the estuarine water. Additionally, some genera of Betaproteobacteria were driven by nutrients, for example, Dechloromonas of Rhodocyclales was positively correlated with PO³⁻₄ in both RDAs (Figures 6C,D). The extent to which clades of Betaproteobacteria in freshwater systems are driven by environmental variables such as nutrients worths further investigation. Furthermore, among the several genera that distributed commonly in the sampling areas (Figure 4), members of Fluviicola displayed a clear phylogenetic shift from the freshwater to saltwater sites. Our results suggest the presence of distinct Fluviicola phylotypes in saltwater environments from so far isolated species, which grow in the absence of sodium ion (O'Sullivan et al., 2005; Yang et al., 2014). Similarly, Luo et al. (2014) has demonstrated that the isolated and yet uncultivable Roseobacters had clearly distinct genetic characters. Taken together, in-depth taxonomic assignments can reveal a more resolved microbial distribution pattern, which would provide insight into the ecology of bacteria.

Although the average water depth of the Pearl Estuary was shallower (<50 m), a clear stratified variation in bacterioplankton community in the saltwater sites was observed, compared with the consistency of bacterioplankton community in the two water layers of the freshwater sites. Previous studies have reported a significant depth-related bacterial variability in estuarine plumes with a deeper water depth, where the actual influential factor resulted in this variance is elusive (Herlemann et al., 2011; Fortunato et al., 2012). In the Pearl Estuary, the difference in water masses with different chemical parameters might be one plausible interpretation for this disparity as marine water enters the estuary at the bottom and freshwater flows out of the estuary at the surface (Dong et al., 2004).

The predominance of Synechococcus in the downstream surface of the Pearl Estuary was consistent with a previous study on the Baltic Sea where light attenuation might be the determining factor (Herlemann et al., 2011). Despite the shallow water depth of the Pearl Estuary, the high turbidity in the bottom water of saltwater sites would prevent penetration of light into the bottom and thus inhibit the growth of Synechococcus. Williams et al. (2012) demonstrated a nutrient cycle in the surface water of the coastal East Antarctica, where Flavobacteria-mediated algal organic matter degradation could facilitate the growth of SAR11 clade and marine Gammaproteobacteria (mainly Oceanospirillales and Alteromonadales). Here, high abundance of picoplankton (both prokaryotic and eukaryotic), and SAR11 clade and Gammaproteobacteria was observed in surface and bottom waters of the saltwater sites, respectively. As marine water enters the estuary bottom during summer (Dong et al., 2004), it is likely that both SAR11 clade and Gammaproteobacteria (mainly Oceanospirillales in this study) are of ocean origin and come from the surface water of the South China Sea. This, together with the existence of Flavobacteria in the bottom water, led to the hypothesis that nutrients cycle using organic matter from the ocean or sinking from the surface to the bottom was prevalent in the bottom water of the Pearl Estuary.

Hypoxia is becoming one of the most serious problems in various ecosystems, and its impact on bacterial assemblages has been documented in many marine and fresh water systems (Stevens and Ulloa, 2008; Zaikova et al., 2010; Li et al., 2012; Peura et al., 2012) but rare in estuarine areas (Crump et al., 2007; Hewson et al., 2014). Microbes can alternatively use a variety of terminal electron acceptors, which enables their growth under various oxygen regimes (Eggleston et al., 2014; Hewson et al., 2014). Concomitantly, phylogenetic and functional transitions of bacterial community under different oxygen conditions have been observed in mesohaline waters of the Chesapeake Bay, and the most extensive shifts occurred during onset of hypoxic condition (Hewson et al., 2014). In the present study, the seasonal hypoxia in the bottom water of the lower Pearl Estuary was not observed, in part due to the effect of typhoon just before of our cruise. However, whole water column hypoxia, mainly induced by high organic matter and nutrient loads, was observed in two freshwater sites (P01 and P07) of the Pearl Estuary, similar to that reported by Dai et al. (2006). It was notable that the DO level of P03, in the midway of P01 and P07, was unexpectedly higher, but the mechanism and the existing time of this were uncertain. Although with a relatively few number of samples, DO accounted for most of the bacterioplankton variation in the RDA targeting only freshwater sites, remarkably different from that targeting all samples. It might be the overwhelming salinity that obscured the impact of DO in the local region. However, the most significant environmental factor in governing bacterial assemblages in the RDA targeting only freshwater sites shifted from DO (at the order level) to nutrients (at the genus level). Close correlations of some specific genera with nutrients such as Novosphingobium and Sphingobium might have diluted the effect of DO.

Rhodobacterales and Methylophilales (mainly PE2) were positively correlated to DO in the RDA targeting all samples and only freshwater sites (Figure 6), respectively. Methylophilales has been reported to be involved in the process of methylotropic metabolism (Lueders et al., 2004), and it is dominant in oxic surface waters (Wright et al., 2012) such as the Dongjiang River, another tributary of the Pearl Estuary with DO level >3 mg L⁻¹ (Liu et al., 2012), suggesting that oxygen is requisite for the methylotropic metabolism in the Pearl River. On the other hand, the abundances of Rhodocyclales (mainly R.12up), Sphingomonadales and Rhizobiales increased when oxygen was depleted. Rhodocyclales has been demonstrated its capability of pollutants degradation (Loy et al., 2005), and the phylogenetic relative of R.12up, “Candidatus Accumulibacter,” was reported to involve in the phosphorus removal from wastewater (Tsuneda et al., 2005). The prevalence of R.12up, along with Sphingomonadales and Rhizobiales as mentioned above, reflected the nearly anoxic and highly polluted condition of the Pearl Estuary. Burkholderiales, the most abundant order in the freshwater sites of the Pearl Estuary, as well as “Candidatus Accumulibacter” was reported to be involved in denitrification (Saito et al., 2008; Hesselsoe et al., 2009). Contrasting to the higher ratio of denitrifier, a relatively lower ratio of nitrifier was observed in our study. However, nitrification has been demonstrated to contribute substantially to the consumption of DO in the upper reach of the Pearl Estuary (Dai et al., 2006, 2008) in addition to aerobic respiration. The lower abundance of nitrifier might indicate a greater nitrification rate per cell in the wet season as suggested by Dai et al. (2008). Another explanation for the oxygen depletion might be attributed to the high abundance of Marine Group I, a widely known aerobic ammonia-oxidizing archaea (Mußmann et al., 2011), in the freshwater sites of the Pearl Estuary (Liu et al., 2014). Additionally, phylogenetically different Marine Group I subclades were observed between the hypoxic and non-hypoxic water samples (Liu et al., 2014). The nitrification capability of the hypoxia-adapted Marine Group I subclade is unknown and worths further investigation. In all, these results demonstrated substantial metabolic shifts between the hypoxic and non-hypoxic sites in the upper reach of the Pearl Estuary.

Although microorganisms have a cosmopolitan distribution, rare and extreme environments (e.g., hot springs) have been found to possess some special ecotypes. As a consequence, it is interesting to investigate the bacteria inhabiting geographically distant non-extreme environments. Different climate zones therefore provide a natural geographical segmentation for such surveys. The phylogenetic tree (Figure S5) showed that the Pearl Estuary harbored fundamentally different bacterial communities from the temperate Delaware Bay (Campbell and Kirchman, 2013) and Baltic Sea (Herlemann et al., 2011). It was notable that Sphingomonadales and Rhizobiales of Alphaproteobacteria were only detected in the Pearl Estuary. As mentioned above, the high nutrient loads might favor the growth of these groups, which might indicate more intensive anthropogenic activities compared with the other estuaries. In addition, Verrucomicrobia was prevalent in the two temperate estuaries, which on the contrary made up only a minor proportion in the Pearl Estuary. The high nutrients concentration sustained a high biomass of phytoplankton, consistent with the high Chl a in the water column of the Pearl River, and the high abundance of phytoplankton might reduce the availability of P for the bacterioplankton (Lindström et al., 2004). Thus, the high abundance of phytoplankton might have inhibited the growth of Verrucomicrobia in the Pearl River. Moreover, among Rhodobacterales, sequences obtained from the Baltic Sea were phylogenetically different from that from the Pearl Estuary. Despite that evident differences were observed between bacterial communities of different climate zones, to what extent temperature and other environmental factors contributed to the variation should be further solved.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.