Journal of Oceanology and Limnology Vol. 37 No. 4, P. 1211-1228, 2019 https://doi.org/10.1007/s00343-019-8200-3

Spatiotemporal distributions and environmental drivers of diversity and community structure of nosZ -type denitrifi ers and in sediments of the Bohai Sea and North Yellow Sea, China*

CAI Youjun1 , ZHANG Xiaoli 2 , LI Guihao2, 3 , DONG Jun 3, 4 , YANG Anjing 2, 3 , WANG Guangyu 5 , ** , ZHOU Xiaojian1 , ** 1 College of Environmental Sciences and Engineering, Yangzhou University, Yangzhou 225000, China 2 Key Laboratory of Coastal Environmental Processes and Ecological Remendation, Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China 3 University of Chinese Academy of Sciences, Beijing 100049, China 4 Shenzhen Lightsun Technology Co. Ltd., Shenzhen 518000, China 5 Department of Bioengineering, School of Marine Science and Technology, Harbin Institute of Technology, Weihai 264209, China

Received Jul. 26, 2018; accepted in principle Sep. 5, 2018; accepted for publication Oct. 23, 2018 © Chinese Society for Oceanology and Limnology, Science Press and Springer-Verlag GmbH Germany, part of Springer Nature 2019

Abstract Denitrifi cation and anammox processes are major removal processes in coastal . However, the spatiotemporal dynamics and driving factors of the diversity and community structure of involved functional bacteria have not been well illustrated in coastal environments, especially in human-dominated ecosystems. In this study, we investigated the distributions of denitrifi ers and anammox bacteria in the eutrophic Bohai Sea and the northern Yellow Sea of China in May and November of 2012 by constructing clone libraries employing nosZ and 16S rRNA gene biomarkers. The diversity of nosZ -denitrifi er was much higher at the coastal sites compared with the central sites, but not signifi cant among basins or seasons. Alphaproteobacteria were predominant and prevalent in the sediments, whereas Betaproteobacteria primarily occurred at the site near the Huanghe (Yellow) River . Anammox bacteria was predominant in the sediments, and besides, Candidatus Brocadia and Candidatus Kuenenia were also detected at the site near the Huanghe River estuary that received strong riverine and anthropogenic impacts. Salinity was the most important in structuring communities of nosZ - denitrifi er and anammox bacteria. Additionally, anthropogenic perturbations (e.g. nitrogen overloading and consequent high primary productivity, and heavy metal discharges) contributed signifi cantly to shaping community structures of denitrifi er and anammox bacteria, suggesting that anthropogenic activities would infl uence and even change the ecological function of coastal ecosystems.

Keyword : nosZ -denitrifi er; anammox; community structure; distribution; anthropogenic perturbations

1 INTRODUCTION * Supported by the National Natural Science Foundation of China (Nos. Nitrogen (N) pollution in coastal ecosystems due 41206155, 41676154), the Strategic Priority Research Program of the to excessive anthropogenic N inputs has become a Chinese Academy of Sciences (No. XDA11020702), the Science and serious environmental issue on regional and global Technology Development Program of Yantai, China (No. 2015ZH074), the Special Program for Basic Research of the Ministry of Science and scales, which leads to eutrophication and associated Technology, China (No. 2014FY210600), and the Discipline Construction deleterious ecological changes. These changes Guide Foundation in Harbin Institute of Technology at Weihai (No. included hypoxia and anoxia (Camargo and Alonso, WH20160205) ** Corresponding authors: [email protected]; zhouxiaojian@ 2006), increased harmful algal blooms (Anderson et yzu.edu.cn al., 2002), alteration of community structure (Bürgi CAI Youjun and ZHANG Xiaoli contributed equally to this work . 1212 J. OCEANOL. LIMNOL., 37(4), 2019 Vol. 37 and Stadelmann, 2002), and loss in biotic diversity extensive investigations of genetic diversity and (Bürgi and Stadelmann, 2002). Therefore, increasing community composition of denitrifi ers and anammox concerns regarding pathways for N loss in coastal bacteria in various coastal habitats, little is known environments have been raised for decades (Galloway about their spatial and seasonal patterns in margin et al., 2008). basins, where the spatiotemporal heterogeneity in Microbial mediated denitrifi cation and anammox hydrology, sedimentary characteristics, and are two major pathways of N removal in marine anthropogenic infl uences determined complex environments (Ward, 2013). Denitrifi cation reduces compositions of denitrifi ers and anammox bacteria. (NO ˉ3 ) sequentially to dinitrogen gas (N2 ) Various environmental factors have been suggested coupling to oxidizing organic matters, while anammox to aff ect distributions of denitrifying and anammox + combines (NH 4 ) and (NOˉ 2 ) to yield bacterial communities, including availability of

N 2 . These two pathways account for about 70% of nitrogen, temperature, oxygen, trace metals, salinity fi xed N loss in the marine N cycle (Codispoti, 2007; and organic matters (Dang et al., 2010; Hou et al., Ward, 2013), whereas their contributions vary widely 2013; Babbin et al., 2014; Zhang et al., 2014). over space and time (Hietanen and Kuparinen, 2008; Recently, Lipsewers et al. (2016) found that seasonal Brin et al., 2014). Considering the importance of hypoxia and elevated sulfi de concentration in coastal denitrifi cation and anammox for nitrogen removal, it bottom waters impacted distributions of denitrifi ers is critical to understand the dynamics and distributions and anammox bacteria. In the Mai Po nature reserve, of the relative functional microbes in coastal a higher diversity of anammox bacteria was observed ecosystems. during summer due to numerous anthropogenic and Denitrifi cation is performed by a diverse terrestrial inputs bringing in Kuenenia (Li et al., assemblage of microorganisms, during which 2011a). However, the seasonality and environment diff erent types of metabolic enzymes are produced drivers of these functional groups have not been well

(Zumft, 1997). The reduction of N2 O to N2 is catalyzed illustrated in coastal ecosystems. by reductases (Nos), and this is an The Bohai Sea (BS) is the innermost basin of important step in the denitrifi cation process because China, with an average depth of 18 m and a very long greenhouse gas N 2 O is converted into N2 and complete water exchange half-life of 17 to 21 months (Wei et denitrifi cation is performed during this step (Zumft, al., 2002). The BS and its coast are known as a “golden 1997). Therefore, the nosZ gene is usually used as a necklace” in North China. Because of rapid biomarker to study the ecological behavior of developments, the BS environment is aff ected denitrifying microorganisms in coastal environments increasingly by human activities, especially inorganic (Scala and Kerkhof, 1999; Magalhães et al., 2008; N inputs. It receives roughly 2.5×104 t per year of Wyman et al., 2013; Wang et al., 2014; Yang et al., dissolved inorganic nitrogen (DIN) from more than 2015). 40 tributary rivers, mainly the Huanghe (Yellow) The diversity of anammox bacteria has been River (SOA, 2016), of which the fl uxes in the fl ood explored using specifi c 16S rRNA, hzo (hydrazine season (July–September) account for 70%–80%. Up oxidoreductase), Annirs (anammox nitrite reductase), to 1/3 area of the BS was eutrophicated in 2015, and and hzsA (hydrazine synthase) genes as molecular the average seawater N:P ratio reached 67:1 (SOA, markers (Li et al., 2010, 2011b; Hou et al., 2013; Bale 2016), was signifi cantly higher than the Redfi eld et al., 2014; Shehzad et al., 2016). The known Ratio (16:1) of clean seawaters (Redfi eld, 1958). anammox bacteria are affi liated to the order Heavy eutrophication mostly occurred in the coastal Candidatus Brocadiales within the phylum regions and (Wang et al., 2009). From 2015 and include fi ve candidate genera: to 2017, more than 20 algal blooms occurred in the Ca . Brocadia , Ca . Kuenenia , Ca . Anammoxoglobus , BS during May to September, with impact on an area Ca . Jettenia , and Ca . Scalindua (Schmid et al., 2003; of over 1 500 km2 (SOA, 2015–2017). Through the Kartal et al., 2007, 2008; Humbert et al., 2010). narrow Bohai Strait, the BS connects with the outer Scalindua typically dominates in marine settings basin North Yellow Sea (NYS). Relative to the BS, (Schmid et al., 2007; Woebken et al., 2008), while the NYS is opener and cleaner, with an average depth non- Scalindua mainly appeared in freshwater, of 40 m. The most sediments of the two basins are reactors, estuarine and coastal environments (Dale et composed of clayey silt and silt sediments and deeply al., 2009; Dang et al., 2013; Hou et al., 2013). Despite infl uenced by sediment inputs from the Huanghe No.4 CAI et al.: Denitrifying and anammox bacteria in marginal seas 1213

3 Luanhe River and PO 4 ˉ i n pore waters were determined with a

-10 nutrient AutoAnalyser (Seal, Germany). The N Yalu River geographic distance from the sampling site to the -20 Tianjin coast (off shore distance, L-dist), bottom water Bohai Sea Dalian

39° Haihe River 50- Chlorophyll a (Chl- a), dissolved oxygen (DO) and -20 North Yellow Sea -10 B41 -30 pH, as well as sediment grain size (GS), total organic B66 B24 carbon (TOC) and nitrogen (TN), and trace metals Yantai Huanghe River BF01 (Pb, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, and Cd) was -10 -70 measured according to methods described previously Qingdao -30 -80 by Zhang et al. (2018).

36° 040 80 160 km

120° 123° E 2.2 DNA extraction, amplifi cation, and cloning Fig.1 Location of sampling sites in the Bohai Sea and North Approximate 0.5 g of sediment was used to extract Yellow Sea total genomic DNA by the FastDNA spin kit for soil River (Qiao et al., 2017). The hydrographic conditions (MP Biomedical, USA) according to the of the two basins are governed by coastal currents and manufacturer’s protocol with slight modifi cations. the Yellow Sea Warm Current (YSWC). The YSWC The concentration of extracted DNA from sediment transports warm and saline waters into the BS through samples was measured by a NanoDrop 2000 the NYS, which prevails in winter and weakens in Spectrophotometer (Thermo Scientifi c, Wilmington, summer (Xu et al., 2009). DE, USA). In the present study, we focus on (1) the The nosZ gene was amplifi ed using the primers heterogeneities in diversity and composition of nosZF/nosZR (Throbäck et al., 2004) to generate denitrifi ers and anammox bacteria in basins (BS and about 700-bp fragments. Anammox 16S rRNA gene NYS), seasons (May and November), and regions was used to amplify the desired gene fragments (about (coastal and central), and (2) the key environmental 477 bp) by a nested PCR technique described factors aff ecting the distribution of denitrifi ers and previously (Hou et al., 2013) using the primers anammox bacteria. The results highlight the PLA46f/1390r-AMX368f/820r. The obtained comparative ecological roles of the two functional products were separated by electrophoresis on a 1% microbes in complex and heterogeneous coastal agarose gel and purifi ed using the Agarose Gel DNA environments. Recovery Kit (Tiangen, Beijing, China). Three replicates of each site were mixed well, ligated into 2 MATERIAL AND METHOD the pTZ57R/T vector (Thermo, USA) and transformed into Escherichia coli DH5α competent cells (Tiangen, 2.1 Sampling and physicochemical analysis China). Finally, 8 clone libraries were constructed for nosZ and anammox 16S rRNA gene, respectively. Four sampling sites (B66, B41, BF01, and B24) in The positive clones were carefully selected using the BS and NYS (Fig.1) were selected during the R/V X-Gal-IPTG LB indicator plates supplemented with Dong - Fang - Hong 2 cruises on May 2–24 and 100-μg/mL ampicillin, and re-amplifi ed using November 1–20, 2012. Sediments were box-cored M13F/R primers. and three random replicated surface sediments (top 0–5 cm) were collected at each site during each cruise. 2.3 Sequencing and phylogenetic analysis At last, 24 sediment samples were obtained. The sediment samples were homogenized in sterile plastic Approximate 100 positive clones were randomly bags, and aliquots were put into cryo-vials and stored selected from each gene library for sequencing immediately in liquid nitrogen for subsequent DNA (Sangon Biotech, Shanghai, China). Analyses of nosZ extraction and physicochemical analysis. sequences were carried out by translating into amino Physicochemical parameters such as salinity (Sal), acid sequences using the BioEdit software (Hall, temperature (Temp), and water depth were estimated 2011). The nosZ amino acid sequences and anammox in situ by Seabird 911 Conductivity-Temperature- 16S rRNA gene sequences were aligned by BioEdit Depth (CTD). Sediment pore waters were obtained and grouped into operational taxonomic units (OTUs) + by centrifuging at 6 000 r/min, and NOˉ 3 , N O ˉ 2 , N H 4 at 80% and 95% identity, respectively, using the 1214 J. OCEANOL. LIMNOL., 37(4), 2019 Vol. 37

DOTUR program (Schloss and Handelsman, 2005). 3 RESULT The closest matches of each OTU identifi ed by 3.1 Environmental factors of the BS and NYS BLASTn were retrieved from the GenBank. The phylogenetic trees were constructed by the maximum The environmental conditions of the studied area likelihood method using the program RAxML 8.0 have been described in a recent study of Zhang et al. (Stamatakis, 2014). The optimum substitution models (2018). In brief, the water depth of our sampling sites for nosZ and anammox 16S rRNA were determined was distinctly deeper in the NYS (42.4±5.10 m) than in by the program ProtTest 2.4 (Abascal et al., 2005) and the BS (19.4±4.05 m) (P =0.01; Table S1). Infl uenced Modeltest 3.7 (Posada and Crandall, 1998), by freshwater and nutrient discharges from the Huanghe respectively, and WAG+I+G and GTR+I+G models River, the site B66 held the lowest salinity and highest were the best fi ts for nosZ and anammox 16S rRNA, Chl-a in bottom waters. Porewater nutrients showed separately. We determined the confi dence in tree signifi cant seasonal and regional variations (P ≤0.05; topology using a bootstrap analysis with 1 000 Table S1). Higher NO ˉ3 and NOˉ 2 concentrations were restarts. Sequences were deposited in GenBank under observed in May, especially at the coastal sites BF01 the accession numbers: MH152718–MH153503 + and B66, but on the contrary, the NH 4 concentration ( nosZ gene) and MH121704–MH122515 (anammox was higher in November, particularly in the BS. The 16S rRNA gene). 3 concentration of PO 4ˉ was signifi cantly higher in the central regions (5.54±1.70 μmol/L) than in the coastal 2.4 Statistical Analysis regions (1.15±0.65 μmol/L) (P =0.05). However, N:P 3 Coverage of each gene library was calculated as (the molar ratio of DIN to PO 4ˉ ) showed no any spatial C =[1–(n / N )]×100, where n is the number of unique or seasonal diff erence. No signifi cant diff erence in OTUs and N is the total number of clones in a library. sediment grain size (GS) was observed across seasons, By using DOTUR, the numbers of observed OTUs basins, and regions, but the fi nest sediment was found and alpha diversity indices (Shannon H , Simpson 1/D at the site B66. Sediment TOC% and TN% were and Evenness J ) were calculated for each gene library. signifi cantly higher in the NYS than those in the BS To compare the spatiotemporal heterogeneities in (P ≤0.03), but C:N (the mass ratio of the total organic diversity and composition of denitrifi ers and anammox carbon to total nitrogen), in the range of 2.18 to 5.54, bacteria, all samples were divided into BS and NYS was similar in the two basins. Trace metals in sediments groups based on the geographic separation between showed strong seasonal trends (P <0.01) with higher the two basins, the May and November groups based values in November, except for metal Mn, which was on seasonality, as well as coastal and central groups much higher in the BS compared with the NYS based on anthropogenic disturbance intensity. (P <0.01) (Table S1). Student’s t test was conducted to test the diff erences 3.2 Spatiotemporal variations in the alpha diversity in environmental factors and alpha diversity of denitrifi ers and anammox bacteria estimators between groups. Pearson’s correlations were conducted to investigate the associations The obtained 786 nosZ sequences were 30.5%– between alpha diversity estimators or relative 98.6% identical with each other and 77.4%–98.7% abundance of a specifi c cluster and environmental identical to the top-hit GenBank sequences at the parameters. All of the analyses were conducted using amino acid scale. The obtained 812 anammox 16S SPSS v.19.0 (Chicago, USA). To visualize the rRNA sequences were 68.6%–99.7% identical with diff erences of denitrifying and anammox bacterial each other and 88.1%–100% identical to the top-hit community in all samples, nonmetric multidimensional GenBank sequences at the DNA scale. Finally, 112 scaling (NMDS) was conducted based on a Bray- nosZ and 51 anammox 16S rRNA OTUs were Curtis similarity matrix using the PRIMER (v.6) identifi ed by DOTUR and the coverages of all libraries software package (Primer-E, UK), and ANOSIM was ranged from 76.6% to 100.0% (Table 1), indicating used to test pairwise community structure diff erences that the majority of the nosZ and anammox 16S rRNA among groupings of samples. To investigate the sequence types was captured. relationship between environmental parameters and Shannon, Simpson and Evenness indices showed denitrifying or anammox bacterial assemblages, CCA that the greatest nosZ gene biodiversity was observed or RDA analysis was performed in R v.3.4.3 with the at the site B66 near the Huanghe River estuary, while vegan package. the lowest value occurred at the central NYS site B24, No.4 CAI et al.: Denitrifying and anammox bacteria in marginal seas 1215

Table 1 Diversity characteristics of nosZ and anammox 16S rRNA gene clone libraries

nosZ gene Anammox 16S rRNA gene Sample ID Season Basin Region (80% amino acid identity) (95% nucleotide identity) OTUs/clones C H 1 / D J OTUs/clones C H 1 / D J B66S May BS Coastal 43/116 76.7 3.20 0.93 0.84 16/99 91.9 1.78 0.71 0.64 B41S May BS Central 27/85 84.7 2.62 0.87 0.79 6/104 98.1 0.46 0.18 0.26 BF01S May NYS Coastal 28/87 82.8 2.83 0.92 0.83 3/100 98 0.11 0.04 0.10 B24S May NYS Central 18/120 98.3 2.21 0.82 0.75 14/100 94 1.75 0.70 0.66 B66W November BS Coastal 42/135 84.4 3.23 0.94 0.87 6/104 89 2.15 0.77 0.70 B41W November BS Central 16/79 89.9 2.16 0.84 0.78 1/101 100 0 0 NA BF01W November NYS Coastal 35/118 88.1 3.03 0.91 0.85 4/103 99 0.73 0.39 0.53 B24W November NYS Central 16/110 94.5 1.90 0.73 0.66 2/104 99 0.05 0.02 0.08 NYS 90.9 2.49 0.85 0.77 97.5 0.66 0.29 0.34 BS 83.9 2.80 0.90 0.82 94.8 1.10 0.42 0.53 P 0.16 0.43 0.36 0.36 0.37 0.53 0.63 0.41 May 85.6 2.7 0.89 0.80 95.5 1.03 0.41 0.42 November 89.2 2.6 0.86 0.79 96.8 0.73 0.30 0.44 P 0.50 0.74 0.59 0.82 0.69 0.67 0.67 0.93 Coastal 83 3.07 0.93 0.82 94.5 1.19 0.48 0.53 Central 91.9 2.22 0.82 0.77 97.8 0.57 0.23 0.34 P 0.06 <0.01 0.01 0.36 0.27 0.35 0.32 0.41

Signifi cant diff erences ( P <0.05) are highlighted in bold. NA means no data available. especially in November (Table 1). Generally, the dominant genotype identifi ed (44.1% of all nosZ diversity of the nosZ gene was signifi cantly higher at clones), exhibited low overlap with known denitrifi ers coastal sites than at central sites (P ≤0.01). However, in the database, suggesting that they might be unique no obvious seasonal and basinal diff erence in the nosZ to the BS and NYS sediments (Fig.2). gene diversity was observed (P >0.05) (Table 1). All nosZ sequences putatively derived from Alpha-, Overall, the diversity of anammox bacteria was Beta-, and Gamaproteobacteria, and much lower than that of nosZ -denitrifi ers. The greatest Alphaproteobacteria-related sequences were highly anammox 16S rRNA gene biodiversity was also represented in our samples, accounting for 78.5% of found at the site B66 during November, while central all nosZ sequences detected. The most abundant OTU, sites B41 and B24 held the lowest anammox 16S E119 (19.7% of all clones), with a sequence identity of rRNA gene diversities, where only 1 or 2 OTUs were 89% to the nosZ amino acid sequence of Nitratireductor found (Table 1). indicus , was frequently detected at the central sites B24 and B41 (Fig.2). The second abundant OTU, H92 3.3 Community compositions of nosZ - denitrifi ers (10.3% of all clones), with a sequence identity of 92% and anammox bacteria to the nosZ amino acid sequence of Ruegeria pomeroyi , A wide range of nosZ sequence divergence was represented higher relative abundance in May than in observed in the BS and NYS sediments. The maximum November (Fig.2). However, Betaproteobacteria and likelihood phylogenetic tree showed that all nosZ Gamaproteobacteria-related sequences were sequences fell into 10 (I–X) clusters (Fig.2). These exclusively retrieved from coastal sites BF01 and B66 sequences were closely affi liated with other (Fig.2), and among Betaproteobacteria, the dominant environmental nosZ clones retrieved from the South OTU, C37, with 90% similarity to the nosZ amino acid China Sea sediments (Yu et al., 2018), Arable Land sequence of Thiobacillus denitrifi cans , occurred only coastal marine sediments, New Jersey marine at the site B66 (Fig.2). sediments (Scala and Kerkhof, 1998), Laizhou Bay Three known anammox bacterial genera including sediments, wastewater treatments, and soils. However, C a . Scalindua , C a . Brocadia and C a . Kuenenia were the sequences from cluster V, the most numerically detected in the studied area (Fig.3). C a . Scalindua 1216 J. OCEANOL. LIMNOL., 37(4), 2019 Vol. 37

82 Wastewater (AIW00730) 97 D39 (1) 91 Dinoroseobacter shibae (WP_012179854) 0.1 88 A9 (1) Coastal marine sediment (ACJ02269) BF01 B66 B24 B41 C53 (1) I Summer 57 Pannonibacter indicus (WP_055457241) A54(2),E7(2),E52(2), H92(81) 12 7 25 13 21 12 Winter 100 D109(4),D6(10) 97 Paracoccus sp. BW001 (ABW74158) 72 Rhodobacter sphaeroides (WP_002725070) 52 Acivated sludge (CBM40993) 73 Acivated sludge (AHB18492) C58 (2) Pseudogulbenkiania sp. NH8B (ADZ68997) Coastal marine sediment (ACJ02272) C54 (4) C35 (3) Leisingera (WP_019294814) II 60 Ruegeria pomeroyi (WP_011241834) Marine sediment (AAC38340) G51(7),E49(2),A140(11) A1 (18) 92 Sulfitobacter mediterraneus (WP_025046722) 90 G49 (8) 89 Mesotidal sediment (CBI71154) Hoeflea sp. BAL378 (WP_035525942) C88 (3) III 79 Coastal marine sediment (ACJ02242) C65 (10) 60 Coastal marine sediment (ACJ02247) Sedimentitalea nanhaiensis (WP_027261814) 98 Coastal marine sediment (ACJ02283) D56 (3) 62 Laizhou Bay sediment (AFS65242) C71 (1) IV 100 Laizhou Bay sediment (AFS65234) G7 (3) F62(2),D25(1) 82 E17(13),E13(2) 69 A39(2),G74(2),A121(5),H139(33),B89(1),B108(1), F33(6),A3(1),C42(14) G82(7),B50(5),E110(5),G63(1),F93(1),H58(1),D148(3), V A86(1),B77(2),A107(7),G28(8),E85(12),B93(2),C61(2),D87(47) α-proteobacteria B112(1),F152(2),A127(1),F91(1), E119(155) 18 16 1 3 42 54 20 16 87 Coastal marine sediment (ACJ02252) 92 B59 (4) 96 Coastal marine sediment (ACJ02236) 53 B67 (1) B143 (3) B131 (7) Coastal marine sediment (ACJ02281) 55 C85(1),E156(3),B114(1),B91(6),B32(1) B21 (1) VI 89 B6 (11) Laizhou Bay sediment (AFS65254) 75 Marine sediment (AAC38341) Coastal marine sediment (ACJ02241) A98 (10) 89 E43(17),G25(8) F31 (12) VII A22 (1) Shewanella sp. cp20 (WP_041509851) γ D142 (3) 86 Mesotidal sediment (CBI71156) 66 Reinekea blandensis (WP_008045430) γ A35 (2) 70 C30(1),D29(1),D139(2) 98 Treatment plant (ADF30166) C56 (2) VIII α+γ-proteobacteria 93 Soil (ADR10847) 60 D7 (5) Microvirga vignae (WP_047189240) α Soil (ACC76944) D134 (1) 75 Terasakiella pusilla (WP_028877987) α 100 Azoarcus sp. CIB (AAY16303) γ C14 (1) 84 Soil(AAY16287) B138 (3) 69 63 C89 (3) B139(2),B73(40),D15(8) IX 99 Candidatus Magnetobacterium casensis (WP_040335623) 95 G61 (1) D72 (1) Coastal marine sediment (ACJ02295) D13 (1) C28 (1) 74 C44 (1) C9 (1) 66 86 C97 (4) Coastal marine sediment (AEO94574) C20 (1) C37 (29) 15 14 60 Rhizosphere (ABY19453) 93 Municipal wastewater (AHB18456) 52 Simplicispira psychrophila (WP_051603276) 69 99 Wastewater treatment (AHZ56339) D52 (2) D28(1),D88(1) 99 C99 (2) 57 Lake sediment (ABX52726) Pseudogulbenkiania sp. NH8B (BAK78389) D91 (1) D16 (4) Polaromonas glacialis (WP_029528411) Soil (AHN63240) 98 81 C7 (3) Soil (BAJ61387) D113 (1) β-proteobacteria C11 (2) 98 Treatment plant (ADF30135) D128 (6) X 73 89 Candidatus Competibacter denitrificans (WP_048676714) Wastewater treatment (AHZ56333) 99 C4 (3) Earthworm alimentary canal (CBM40807) Wastewater treatment (AHZ56336) D68 (3) 58 D31 (1) 53 Rhizosphere (ABY19431) C70 (15) 52 Wastewater treatment (AHZ56311) 60 98 Thauera humireducens (WP_048706496) D36 (2) 57 61 Ralstonia solanacearum (WP_016725390) 99 Soil (ACI48871) F89 (5) 53 D120 (1) Wastewater treatment (AHZ56385) Acidovorax sp. SD340 (WP_055393513) Wastewater treatment (AHZ56350) Lake water (ADV91909) D97 (4) D18 (3) Anaeromyxobacter dehalogenans (AFB35544) δ-proteobacteria Fig.2 Maximum-likelihood phylogenetic analysis of nosZ amino acid sequences with the nosZ sequence from Anaeromyxobacter dehalogenans (AFB35544) used as an outgroup The scale bar represents 0.1 substitution per amino acid position. The phylogenetic positions of pure cultures based on 16S ribosomal DNA genes are indicated by α, β, and γ for the α, β, and γ subclasses of the Proteobacteria, respectively. The values in parentheses are the number of sequences. Some abundant OTUs appeared in blue with its sequence number recovered from each library. No.4 CAI et al.: Denitrifying and anammox bacteria in marginal seas 1217

82 D87(1) Candidatus Scalindua marina (KM925249) E1(1) Candidatus Scalindua marina (KM925481) B36(80) Scalindua marina 56 Candidatus Scalindua marina (KM925481) (SI) 69 C97(3) 59 Candidatus Scalindua marina (KM925461) Candidatus Scalindua marina (KM925833) C57(2) Candidatus Scalindua marina (KM925461) 92 A44(1) Scalindua wagneri A4(112) (SII) 57 Candidatus Scalindua sp. (JX945930) Candidatus Scalindua brodae (KM925762) Candidatus Scalindua brodae (KM926292) Scalindua brodae Candidatus Scalindua sp. (KM266370) H100(369) 55 F9(1) Scalindua marina 52 Candidatus Scalindua marina (JX243729) (SIII) Candidatus Scalindua marina (KM925727) E14(11) E50(3) E6(3) 84 C70(2) Scalindua 85 C21(19) 66 Candidatus Scalindua sp. (FM992881) Candidatus Scalindua wagneri (KM925317) 72 Candidatus Kuenenia stuttgartiensis (KM925543) Candidatus Scalindua wagneri (JX243224) Candidatus Scalindua wagneri (KM926188) Candidatus Scalindua wagneri (KM925351) Scalindua wagneri Candidatus Scalindua marina (KM925870) (SIV) Candidatus Scalindua wagneri (JX243493) Candidatus Scalindua sp. (JX945927) E10(11) E17(4) E54(4) G29(2) Candidatus Scalindua wagneri (KM925919) C54(1) 59 Candidatus Scalindua wagneri (KM925197) Candidatus Scalindua marina (KM926216) E81(1) C52(3) 84 Candidatus Brocadia caroliniensis (JX243476) Brocadia (BI) Candidatus Brocadia fulgida (JX243147) 71 D6(1) Scalindua marina 55 C58(2) Scalindua Candidatus Scalindua marina (KM925500) (SV) D11(2) 100 Candidatus Anammoxoglobus propionicus (DQ317601) 58 Candidatus Anammoxoglobus propionicus (EU478694) Anammoxoglobus Candidatus Jettenia asiatica (KJ002641) Candidatus Jettenia asiatica (KT230392) Jettenia D25(1) C3(7) D88(1) Candidatus Brocadia fulgida (KM925804) Brocadia D31(1) (BII) 100 D48(1) Uncultured Planctomycete (KM095468) Candidatus Brocadia caroliniensis (JX243560) Candidatus Kuenenia stuttgartiensis (KM926004) D44(2) 50 Candidatus Brocadia fulgida (KM925686) Uncultured Planctomycetales bacterium (JQ918937) D100(4) D27(2) D39(5) 58 Candidatus Kuenenia stuttgartiensis (JX243572) Kuenenia 68 Candidatus Kuenenia stuttgartiensis (KM926037) (KI) Candidatus Kuenenia stuttgartiensis (JX243427) 69 Uncultured Planctomycetales bacterium (JN051583) D62(1) Anaerobic ammonium-oxidizing Planctomycete (AJ250882) Candidatus Kuenenia stuttgartiensis (AF375995) 5690 Candidatus Kuenenia stuttgartiensis (KF429801) Candidatus Brocadia anammoxidans (JX243388) Candidatus Brocadia anammoxidans (JX243602) Candidatus Brocadia anammoxidans (JX243601) 94 D89(1) Brocadia Candidatus Brocadia anammoxidans (JX243662) (BIII) 97 Candidatus Brocadia anammoxidans (JX243654) 74 E64(1) 93 Candidatus Kuenenia stuttgartiensis (KM925250) 57 E98(1) Kuenenia (KII) D26(2) Candidatus Brocadia fulgida (JX243329) Candidatus Brocadia fulgida (JX243136) Brocadia C1(65) Uncultured Planctomycete (GQ504007) (BIV) Candidatus Brocadia fulgida (JX243635) Candidatus Brocadia anammoxidans (JX243328) C16(1) B55(1) 56 C33(1) C71(9) Candidatus Kuenenia sp. (JX243323) Kuenenia Candidatus Kuenenia sp. (JX243201) (KIII) 54 Candidatus Kuenenia stuttgartiensis (JX243553) Candidatus Kuenenia sp. (JX243520) 77 D63(4) 67 Candidatus Kuenenia stuttgartiensis (KM925908) Candidatus Brocadia caroliniensis (JQ889546) Candidatus Brocadia fulgida (JQ889388) Candidatus Brocadia sp. (KM884880) D38(4) Candidatus Brocadia fulgida (JX243451) Brocadia 100D74(43) Candidatus Brocadia fulgida (JX243133) (BV) C35(4) 0.1 Candidatus Brocadia fulgida (JX243618) 100 Uncultured Planctomycete (AB645290) B59(7) 87 C49(1) 52 D33(1) Potential Anammox C94(1) (P) Planctomycetales bacterium (HQ675572) Pelotomaculum thermopropionicum (NR_0746855) 100 Isosphaera pallida (NR_028892) Pirellula sp. (X86388) Fig.3 Maximum-likelihood phylogenetic analysis of anammox 16S rRNA gene sequences Isosphaera pallida (NR_028892) and Pirellula sp. (X86388) were used as the outgroup. The values in parentheses are the number of sequences. The scale bar represents 0.1 substitution per nucleotide position. 1218 J. OCEANOL. LIMNOL., 37(4), 2019 Vol. 37

Summer Winter B66W 2D Stress: 0.01 2D Stress: 0.01 BS B66S NYS B41W BF01W B24W B41W B24S B41S B66W B41S B24S BF01W B24W BF01S B66S

BF01S

a b Fig.4 NMDS plots showing the distribution patterns of nosZ -denitrifi ers (a) and anammox bacteria (b) Table 2 ANOSIM testing seasonal, basinal and regional 3.4 Distributions of nosZ - denitrifi ers and anammox diff erences of benthic nosZ -denitrifi er and anammox bacteria bacterial structures based on Bray-Curtis metrics The NMDS plots (Fig.4) showed that the whole nosZ Anammox 16S rRNA gene Grouping nosZ -denitrifi er community was divided into two R P R P distinctive groups and the assemblages of B66 were BS vs. NYS 0.188 0.2 0.021 0.486 clearly separated from those of other sites. Similarly, May vs. November -0.073 0.6 0.135 0.257 the anammox bacterial assemblages of B66S, B66W, Coast vs. central 0.385 0.057 0.438 0.029 and BF01S were distinctive from those of other samples. The ANOSIM results (Table 2) verifi ed that Signifi cant P -values (<0.05) are highlighted in bold. the seasonal and basin-wise diff erences of both nosZ - denitrifi ers and anammox bacteria were not signifi cant was predominant (78.3% of all clones) in the ( P >0.05), but the regional diff erence was distinct (for anammox bacterial libraries, and fi ve (SI–SV) nosZ , P =0.057 and for anammox 16S rRNA, distinctive Scalindua clusters were identifi ed. Clusters P =0.029). SI, SIII, and SV are affi liated with Scalindua marina The distributions of specifi c clusters of nosZ - with 96.4%–99.8% sequence identity, and clusters SII denitrifi ers and anammox bacteria identifi ed in the and SIV show 98.5%–99.4% identity to the sequences phylogenetic analysis are demonstrated in the of Scalindua wagneri . The most abundant OTU, heatmap plots (Fig.5). The nosZ sequences from H100, has an identity of 99.8% to the sequence of cluster V are shared in all samples and occur in the Scalindua marina from the sediments of the central sites more frequently than at coastal sites. In Changjiang (Yangtze) River estuary (Hou et al., addition, Cluster I also presents in all samples and 2013). Most Brocadia clusters belonged to Brocadia less in B66. Cluster VII occurred most at B24 in fulgida (98.3%–98.7% identity), including BI, BII, November. On the contrary, sequences from Clusters BIV, and BV, while cluster BIII shows 95.8% identity II, VI, IX, and X present higher relative abundance at to the sequence of Brocadia anammoxidans . The coastal sites than at central sites (Fig.5a). As for dominant Brocadia OTU, C1, and D74 has an identity anammox bacteria (Fig.5b), diff erent Scalindua of 100% to the sequences of anammox clones in the clusters have their respective niches. Sequences from suspended sediments of Huanghe (Yellow) River, and SIII were primarily retrieved from B24 and B41, 99.9% to the sequences of Brocadia fulgida in the representing 75.5% and 95.2% of the sequences in the sediments of the Changjiang River estuary (Hou et two sites, respectively. Cluster SII was found al., 2013). Altogether 34 sequences are affi liated to prevalent in the sample BF01S, while Cluster SI is Kuenenia , with 96.2%–98.9% identity to the prevalent in the sample BF01W. However, Cluster sequences of Kuenenia stuttgartiensis . In addition, SIV presents frequently in samples B66S and B24S. the phylogenetic analysis showed a potential The Brocadia cluster BV is highly represented in the anammox cluster, sharing less than 93% similarity sample B66W, while Cluster BIV mainly presents in with sequences from all the other clades, which only the sample B66S. The Kuenenia clusters are restricted occurred at the site B66. to the site B66. No.4 CAI et al.: Denitrifying and anammox bacteria in marginal seas 1219

a I 0.6 II 0.5 a-proteobacteria III IV 0.4 V VI 0.3

VII 0.2 a+γ-proteobacteria VIII IX 0.1

β-proteobacteria X 0

1 b SI SII SIII Scalindua 0.8 SIV

SV

BI 0.6 BII BIII Brocadia BIV 0.4 BV

KI 0.2 Kuenenia KII KIII Potential Anammox P 0 BF01S BF01W B66S B66W B24S B24W B41S B41W Fig.5 The heatmap showing distributions of specifi c nosZ -denitrifi er (a) and anammox bacterial (b) clusters identifi ed in the phylogenetic analysis The color bar indicates the relative abundance of each specifi c cluster in percentage.

Table 3 Pearson’s correlation coeffi cients ( R ) between alpha diversities of nosZ -denitrifi ers or anammox bacteria and environmental factors across all samples (n =8)

+ 3 L-dist Depth Temp Sal DO pH Chl-a N O ˉ3 N O ˉ2 NH 4 DIN PO 4ˉ N : P G S T OC% TN% C:N Pb Cr Mn Fe Co Ni Cu Zn As Cd H -0.93 -0.75 -0.72 -0.84 0.72 nosZ 1/ D -0.90 -0.77 -0.78 0.73 J -0.88 -0.74 -0.70

H Anammox 1/ D 16S rRNA J

Only the signifi cant correlations (P <0.05) are shown and P values (<0.01) were highlighted in bold.

3 3.5 Factors driving variations in nosZ - denitrifi ers -0.88, P ≤0.004), depth ( R ≤-0.74, P< 0.04), and PO 4 ˉ and anammox bacterial community diversity and ( R ≤-0.70, P <0.05), and positively with the ratio of structure N:P ( R ≥0.72, P ≤0.04; Table 3). However, no any environmental factor showed signifi cant correlations All alpha diversity estimators of nosZ -denitrifi ers with the alpha diversity of anammox bacteria ( P >0.05; negatively correlated with the off shore distance ( R ≤ Table 3). 1220 J. OCEANOL. LIMNOL., 37(4), 2019 Vol. 37

Summer Winter 1.0 BS B66W NYS Pb Environemental factor 4 B66S

B66W 0.5 2 Sal BF01S B41W B41S BF01W 0.0 Sal B41W B24W 0 DO B24W B41S B24S B24S

BF01W RDA2 (variation explained 11.2%) -0.5 -2

B66S

abBF01S -4 -1.0 -0.5 0.0 0.5 -1012345 RDA1 (variation explained 64.1%) CCA1 (variation explained 35.1%) Fig.6 Canonical correspondence or redundancy analysis showed that the changes in the nosZ -denitrifi er community (a) was mainly driven by salinity, DO and sediment metal Pb, whereas the anammox bacterial community (b) was structured only by salinity

The whole nosZ -denitrifi er community structure ( R ≥0.88, P ≤0.004), and Cluster SIV showed high was signifi cantly co-varied with bottom water salinity correlation with the concentration of metal Pb ( P =0.005), DO ( P =0.015), and the concentration of ( R =0.82, P =0.01). Salinity seemed to be the most Pb in sediment (P =0.005) (Fig.6a). These factors important factor aff ecting the relative abundance of explained 84.1% of the total variance of the nosZ - Brocadia and Kuenenia clusters (R ≤-0.74, P ≤0.03). denitrifi er community-environment relationship. Apart from that, Clusters BIV and KIII had a high Nevertheless, only salinity ( P =0.002) was identifi ed correlation with the concentration of Chl-a in bottom as the signifi cant environmental factor in correlation waters ( R ≥0.71, P <0.05) and high levels of tolerance with the variation of the whole anammox bacterial to metal As in sediments (R ≥0.85, P ≤0.007) (Table 4). community structure and spatial distribution (Fig.6b), providing 35.1% of the total variance of the anammox 4 DISCUSSION bacterial community-environment relationship. 4.1 Distribution and environmental drivers of For cluster-specifi c correlation with environmental benthic nosZ -denitrifi ers factors, the nosZ cluster V responded positively to 3 elevated off shore distance, depth, salinity, and PO 4ˉ Sequencing of nosZ clones revealed 112 OTUs at ( R ≥0.77, P ≥0.02); Cluster I positively correlated with 80% amino acid identity in the BS and NYS sediments. DO ( R =0.79, P =0.02) and negatively correlated with This inherent diversity of nosZ gene was much higher temperature (R =-0.80, P =0.02). Nevertheless, Cluster than those in other coastal and marine environments,

II showed a signifi cant correlation with NOˉ 3 and NOˉ 2 such as Douro River estuary (Magalhães et al., 2008), ( R ≥0.81, P <0.02). Higher sedimentary TOC% and Goa mangrove forest (Fernandes et al., 2012), and TN% seemed to favor the relative abundance of Pacifi c Ocean (Scala and Kerkhof, 1999, 2000). The Cluster VII ( R ≥0.80, P <0.02), while Cluster X result suggested that the anthropogenic perturbation- decreased with increasing of salinity, depth and dominated setting of the BS and NYS might promote sediment grain size (R ≤-0.71, P <0.05). Within the biodiversity of the nosZ gene to adapt to the anammox clusters, Cluster SIII was highly correlated complex environments. Another possible reason was with the off shore distance ( R =0.91, P =0.002), Cluster the PCR bias of the primer set nosZF/nosZR, which

SII was most strongly correlated with NOˉ 3 and NOˉ 2 would likely recover more diverse environmental No.4 CAI et al.: Denitrifying and anammox bacteria in marginal seas 1221

Table 4 Pearson’s correlations between relative proportions of nosZ -denitrifi ers and anammox bacterial clusters and environmental variables

Specifi c L-dist Depth Temp Sal DO pH Chl- a N O ˉ N O ˉ NH+ DIN PO3 ˉ N : P G S T OC% TN% C:N Pb Cr Mn Fe Co Ni Cu Zn As Cd cluster 3 2 4 4 I -0.80 0.79 I I 0.86 0.81 -0.81 III 0.86 0.82 -0.71 0.74 -0.70 0.86 I V -0.83 0.71 -0.85 0.86 V 0.89 0.78 0.86 0.77

nosZ V I VII 0.76 0.80 0.80 VIII -0.81 IX X -0.74 -0.96 -0.71 0.74

S I SII 0.91 0.88 SIII 0.91 SIV 0.82 SV -0.79 -0.74 B I 0.80 0.82 BII -0.91 -0.73 0.75 BIII -0.75 BIV -0.74 0.74 0.88 Anammox 16S rRNA Anammox 16S rRNA B V -0.80 KI -0.75 KII KIII -0.69 -0.78 0.71 0.85 P -0.73 -0.82 0.87

Only the signifi cant correlations (P <0.05) are shown and P values (<0.01) were highlighted in bold.

nosZ phylotypes than the primer sets (e.g. nosZ661F/ In addition, the alpha diversity and composition of nosZ1773R and nosZ1211F/nosZ1897R) used in our nosZ -denitrifi ers presented a clear heterogeneity other coastal environments, although these primers between coastal and central sites but no signifi cant captured a similar range of cultrue-based phylotypes diff erence between seasons and basins (Table 1 and (Throbäck et al., 2004). This possibility should be Table 2). Several studies have monitored the verifi ed in further studies. distribution of denitrifi ers in marine sediments. Although certain nosZ sequences appeared to be Magalhães et al. (2008) found a similar result to us, ubiquitous in coastal and marine sediments, for that the composition of nosZ assemblages in the example, some of our sequences were closely Douro River estuary sediments showed a site-specifi c affi liated with those in the South China Sea sediments diff erence but was stable over time. On the contrary, (Yu et al., 2018), Arable Land coastal marine Scala and Kerkhof (2000) found that geographic sediments, New Jersey continental shelf sediments distance (centimeters to kilometers) had a major (Scala and Kerkhof, 1998), and Laizhou Bay infl uence on the structure of nosZ -denitrifi ers in sediments, a big nosZ group (including more than continental shelf sediments. In the Pacifi c coast of 40% of all nosZ clones) was restricted in the BS and Mexico, Liu et al. (2003) observed that the distribution NYS setting (Fig.2), suggesting that nosZ -denitrifi ers of nirS and nirK -denitrifi ers was controlled by could evolve inhabit-specifi cally not geographically. geographic location and biogeochemical conditions. 1222 J. OCEANOL. LIMNOL., 37(4), 2019 Vol. 37

Collectively, local environmental properties and the organism is prevalent in that season. In contrast to geographic separation could have a combined eff ect other works (Magalhães et al., 2008; Mills et al., on structuring marine denitrifi er communities. The 2008; Fernandes et al., 2012; Yang et al., 2015; Yu et coastal sites, especially near the Huanghe River al., 2018), we observed that the Betaproteobacteria- estuary, held higher diversity and distinct community related nosZ group comprised a substantial fraction of structure of nosZ gene from central sites, probably the overall nosZ community detected in the Huanghe because freshwater and sediment inputs from the river River estuary site. Betaproteobacteria is commonly brought in terrigenous nosZ phylotypes, and on the detected in the freshwater and estuarine systems, such other hand, some specifi c nosZ phylotypes could be as Columbia River estuary (Crump et al., 1999), evolved in the anthropogenic perturbation-dominated Changjiang River estuary (Feng et al., 2009), and coastal setting. Zhujiang (Pearl) River estuary (Liu et al., 2015). In Similar to previous studies of coastal and marine this study, most Betaproteobacteria-related nosZ nosZ -denitrifi ers (Magalhães et al., 2008; Mills et al., sequences were similar to the terrigenous nosZ clones 2008; Fernandes et al., 2012; Yang et al., 2015; Yu et retrieved from wastewater treatments, lake waters al., 2018), all nosZ sequences detected are related to and soils (Fig.2), suggesting that Huanghe (Yellow) proteobacteria, and Alphaproteobacteria formed the River diluted water contributed profoundly to the most dominant and ubiquitous putative nosZ group in composition of nosZ -denitrifi er in the region. The the BS and NYS sediments, suggesting that this group most abundant Betaproteobacteria nosZ phylotypes was well adapted to coastal marine sediments and were closely related (about 90% similarity) to a likely contributed importantly to nitrogen removal in known autotrophic denitrifi er Thiobacillus denitrifi cans . the environments. Within Alphaproteobacteria, Thiobacillus denitrifi cans is also a well-known sulfur- N . indicus -related (89% similarity) nosZ sequences oxidizing bacterium (Shao et al., 2010) and its primarily occurred in the central sites B24 and B41. predominance may suggest the frequent occurrence In addition to denitrifi cation (Labbé et al., 2004), of sulfur oxidization process in the coastal region. some members of Nitratireductor spp. have been Based on multiple analyses, salinity had the most reported to be capacity of degrading crude oil, signifi cant impact on nosZ -denitrifi er diversity, hydrocarbon or complex organic matters in marine community structure, and distribution. Separation of environments (Lai et al., 2011a, b; El Hanafy et al., nosZ genes according to salinity was also observed in 2016). Site B41 located nearby the drilling platform estuarine sediments along a salinity gradient Penglai 19-3, where a large oil spill incident occurred (Magalhães et al., 2008; Yang et al., 2015). in November 2011 (Pan et al., 2015), resulting in Additionally, at the global scale, salinity was the serious crude-oil pollution in the sea. Site B24 located major driver of nirS and nirK -denitrifi er communities in the center of the NYS, in which large amounts of in aquatic environments (Jones and Hallin, 2010). old and recalcitrant organic matters with high C:N The ecological mechanism of how salinity governs ratio (0.73%–0.76%) existed, owing to the denitrifi er community remains unclear. A study in the hydrodynamic forces constrained by cyclonic Douro estuarine water column, in which NOˉ 3 circulations (Hu et al., 2016). Responding to these concentrations co-varied with salinity (Magalhäes et complex organic matter compositions in sediments, al., 2005), and previous NO ˉ3 and salt addition N . indicus may be selected and enriched in these sites. experiments (Magalhäes et al., 2005) revealed that

R . pomeroyi -related (92% similarity) nosZ sequences denitrifi cation rates could be a function of NOˉ 3 were mainly detected during May. R . pomeroyi was a availability and that salinity does not have a direct model marine Roseobacter bacterium, with the eff ect on the denitrifi cation process. In this study, the capacity not only to denitrify (Wyman et al., 2013; El nosZ cluster V (Alphaproteobacteria) preferred high Hanafy et al., 2016) but also to demethylate salinity, whereas Cluster X (Betaproteobacteria) was dimethylsulfoniopropionate (DMSP) (Gonzalez- exclusively detected in the low-salinity site, which is Silva et al., 2017), an important compatible solute of in line with the patterns of the whole bacteria along a marine algae. This organism often dominates in the salinity gradient with a shift in the dominance of phycosphere microenvironment and utilizes DMSP as Betaproteobacteria in ecosystems infl uenced by a carbon source (Rink et al., 2007; Goecke et al., freshwater inputs into a predominance of 2013). In the BS and NYS, algal blooms generally Alphaproteobacteria in the high-salinity conditions peak during May to July (Wei et al., 2004), and thus (Bouvier and del Giorgio, 2002; Piao et al., 2012). No.4 CAI et al.: Denitrifying and anammox bacteria in marginal seas 1223

4.2 Distribution and environmental drivers of (Amano et al., 2007). This assumption could be benthic anammox bacteria supported by the recent investigation of Zhang et al. (2018), in which the high anammox activity was It has been reported that functional gene marker detected using 15 N tracing technology at the same site. hzo can describe anammox bacterial more Salinity was evidently a signifi cant factor governing comprehensively than 16S rRNA because of its anammox bacterial distributions in this study. The linking with anammox activity (Li et al., 2011a; Dang results are not surprising given that previous studies et al., 2013), however, hzo products are usually have demonstrated that salinity infl uenced the diffi cult to be obtained due to lower ratios of anammox geographical distribution of anammox bacteria in bacteria compared with the whole bacteria. In the estuarine sediments (Dale et al., 2009; Hou et al., present study, no target hzo product was obtained, so 2013). Recently, Sonthiphand et al. (2014) concluded only anammox bacterial 16S rDNA libraries were that salinity drove the distribution of anammox shown. Similar to nosZ -denitrifi ers, the anammox bacteria at the global scale. The important role of bacterial community also exhibited signifi cant salinity may be related to the diff erent salinity regional patterns. At the central sites, the anammox tolerance of anammox bacteria. Nevertheless, the bacterial community was dominated by Ca . Scalindua , direct infl uence of salinity on anammox bacterial while at the coastal sites, besides Ca . Scalindua , community is diffi cult to be confi rmed in natural Ca . Brocadia and Ca . Kuenenia were also detected. environments due to the covariation of salinity and In the BS sediments, Dang et al. (2013) have found other factors (e.g. NO ˉ , N O ˉ and NH+ ) (Sonthiphand Ca . Jettenia , but no Ca . Brocadia and Ca . Kuenenia . 3 2 4 et al., 2014). Scalindua adapts better to high-salinity Other previous studies have also found the coexistence habitats and can also attribute to its higher affi nity of of non- Scalindua anammox bacteria along with NO ˉ and NH+ that is commonly limited in high- Scalindua in coastal environments that received 2 4 salinity habitats (Sonthiphand et al., 2014). strong terrestrial infl uences (Amano et al., 2007; Dang et al., 2010; Li et al., 2010; Shehzad et al., 4.3 Responses of nosZ -denitrifi ers and anammox 2016). Even so, the non-Scalindua clades generally bacteria to coastal anthropogenic perturbations accounted for a small fraction of anammox bacteria in marine environments (Dang et al., 2010, 2013; Li et In the eutrophic coastal ecosystems, in addition to al., 2010; Shehzad et al., 2016), and were considered natural factors, such as freshwater dilution, currents, to exist allochthonously without activities (Amano et tides, waves, upwelling, lateral transport, and water al., 2007). In this study, most Brocadia sequences mixing, anthropogenic perturbations contributed were similar to those in the suspended sediments of importantly to the dynamics of marine denitrifying the Huanghe River, indicated that these anammox and anammox bacterial community diversity and bacteria might be introduced from river runoff s. composition (Dang et al., 2013; Babbin et al., 2016). However, inconsistent with other results (Amano et Highest primary productivity (represented as Chl- al., 2007; Dang et al., 2010, 2013; Li et al., 2010; a ) and highest contents of sediment heavy metal Pb, Shehzad et al., 2016), Brocadia fulgida accounted for As, and Cd were found at the coastal site near the more than 60% of total clones of the Huanghe River Huanghe River estuary, due to excessive anthropogenic estuary site in this study, suggested that this phylotype nitrogen loading and wastewater discharge (Table was likely gradually adapted to the dynamic habitat, S1). These factors were found signifi cant in where river-sea interaction was intensive with lower constraining the distributions of nosZ -denitrifi ers and salinity and richer nutrient and became a prominent anammox bacteria (Table 4; Fig.6). The primary group. Brocadia fulgida is generally hypothesized to productivity can supply carbon source for be freshwater-adapted anammox bacteria and has heterotrophic denitrifi ers, and a few denitrifi er extremely low tolerance to salinity (Gonzalez-Silva et phylotypes respond quickly to labile organic matter al., 2017). Recently, nevertheless, Malovanyy et al. pulses and suit to highly productive conditions + (2015) found that Brocadia fulgida was the major (Babbin et al., 2016). Anammox bacteria gain NH 4 anammox phylotype and active in removing nitrogen and NOˉ 2 from oxidizing organic materials, and some when slowly adapt to the salinity of 15 in a wastewater special species can directly utilize small organic + bioreactor. Therefore, Brocadia fulgida would be compounds for alternative sources of NH 4 (Van De highly possible to perform active nitrogen removing Vossenberg et al., 2013). The biogeochemical cycling in the marine habitat not only survive and hibernate of heavy metals is coupled to nitrogen cycling in 1224 J. OCEANOL. LIMNOL., 37(4), 2019 Vol. 37 environments, and the signifi cance of heavy metals to the R/V Dong -Fang - Hong 2 for their assistance in denitrifi ers and anammox bacteria was also observed sample collection during the expedition. in other coastal ecosystems (Dang et al., 2010, 2013; References Yang et al., 2015). Metal Pb, As, and Cd, no biological role, are potentially toxic to microorganisms. Abascal F, Zardoya R, Posada D. 2005. ProtTest: selection of Interestingly, the non-Scalindua clusters were best-fi t models of protein evolution. 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LIMNOL., 37(4), 2019 Vol. 37 k g ) (mg/ k g ) (mg/ (mg/kg ) (mg/kg k g ) (mg/ k g ) (mg/ <0.01 <0.01 <0.01 <0.01 <0.01 0.33 0.13 <0.01 1 1 0.91 0.57 0.79 0.09 0.1 (g/kg ) (g/kg ) (mg/kg ) ) (mg/kg ) (g/kg (g/kg k g ) (mg/ <0.01 <0.01 0.49 <0.01 0.03 0.81 0.84 0.99 GS TOC TN C:N Pb Cr Mn Fe Co Ni Cu Zn As Cd

ˉ N : P 4

3 =3) of the bottom waters and sediments collected from the BS and NYS =3) of the bottom waters and sediments collected from n 0.05 0.1 0.61 0.51 0.6 0.31 0.83 0.61 0.87 0.61 0.61 0.66 0.48 0.39 0.2 0.82 DIN PO 4 4 + 2 ˉ NH 0.03 0.1 0.12 0.38 0.17 0.96 0.77 0.16 1 3 ˉ NO N O (μmol/L) (μmol/L) (μmol/L) (μmol/L) (μmol/L) (μmol/L) (μmol/L) (μmol/L) (μmol/L) (μm) % % ) (mg/kg

a ) 3 m (mg/

L) (mg/ Table S1 Physical and chemical properties (mean values, S1 Physical and chemical properties Table <0.05) are highlighted in bold. P 0.01 0.65 0.12 0.73 0.46 0.08 0.53 0.56 0.51 0.54 0.4 0.3 0.71 Bottom water Porewater Sediment (m) (m) C) (° L-dist Depth Temp Temp Depth L-dist Sal DO pH Chl- P 0.72 P <0.01 0.16 0.53 0.1 0.68 0.46 0.77 0.2 0.39 0.39 0.47 P 1 0.78 0.11 0.51 0.09 0.57 0.19 0.27 BS 41.7 19.4 11.1 29.4 9.1 8.09 3.9 6.8 0.72 327.9 335.3 2.3 15.2 378.8 0.4 0.1 4.1 7.8 11.5 0.51 10.58 6.16 13.9 11.7 26.3 2.23 0.17 May 47.2 29.3 8.5 30.7 9.5 8.09 3.6 18.8 1.77 208.3 228.9 2.2 16.4 406.2 0.51 0.11 4.21 13.9 2.6 0.37 3.6 2.9 4.8 6.9 16 3.9 0.11 NYS 52.6 42.4 9.9 31.2 8.9 8.07 1.1 15.7 1.23 254.2 271.2 4.4 17.2 188.1 0.66 0.14 4.3 6.6 11.7 0.3 10.6 6.17 12.9 9.6 29.3 2.42 0.1 B24S 93.3 51 B41S 4.5 31.8 71.6 9.57 8.12 0.95 25 1.33 B66S 8.08 5.09 10.33 7.79 31.26 0.17 11.8 1.3 11 28.25 8.42 0.78 13.92 8.05 7.01 20.96 327.5 196.3 330.2 1.64 197.2 7.74 152.3 0.49 42.7 174.9 15.7 17.3 402.5 0.73 0.44 0.13 0.09 5.54 0.29 5.05 14.59 10.75 2.93 603 2.4 11.4 0.17 0.37 0.09 0.53 4.06 3.57 19.3 3.65 2.8 2.3 2.68 5.07 4.06 8.29 0.41 6.3 21.34 3.18 2.51 14.26 2.98 0.1 3.01 0.09 5.83 7.65 11.12 6.39 0.16 B24W B24W 93.3 50.5 31.23 8.17 13.45 8.04 1.06 6.27 B41W 71.6 27.5 30.76 8.48 0.16 12.78 8.11 1.9 B66W 2.93 244.3 11.8 14 250.7 0.2 10.04 27.2 9.23 1.58 8.11 6.58 3.01 583.6 38.1 10.8 0.76 586.7 0.27 0.16 4.87 0.44 379.3 7.36 23.49 79.7 382.6 26.3 0.3 0.35 0.11 19.54 3.08 0.89 11.05 0.58 24.44 14.74 429.9 46.23 5.8 25.8 1.45 0.45 0.14 0.57 0.11 4.2 21.84 11.17 0.74 26.74 18.3 15.69 48.08 0.52 3.51 13.63 0.19 7.77 18.78 14.4 31.92 4.05 0.23 BF01S 11.9 30 7.79 31.57 9.59 8.09 1.39 52.76 3.34 157.2 213.3 0.37 576.4 21 0.5 0.11 2.18 10.82 2.73 0.35 4.07 3 4.33 5.46 17.31 3.68 0.07 Central 82.5 38.5 9.6 31.3 9.14 8.09 2.25 2.7 0.61 337.9 341.2 5.54 17.5 140.8 0.57 0.12 4.64 6.59 13.7 0.39 12.2 6.9 15.1 11.9 32.5 2.61 0.13 Coastal 11.9 23.3 11.4 29.3 8.88 8.07 2.77 19.8 1.35 244.2 265.3 1.15 14.9 426.2 0.49 0.11 3.78 7.88 9.5 0.41 9 5.4 11.7 9.3 23.1 4.04 0.14 Sample BF01W BF01W 11.9 38 30.26 8.27 13.69 8.01 1.09 2.52 0.13 287.9 290.5 3.05 95.3 21.3 0.63 0.14 4.66 0.64 17.45 0.36 15.21 7.84 17.92 9.87 32.23 2.04 0.1 November 47.2 32.5 12.5 29.9 8.5 8.07 1.4 3.7 0.19 373.8 377.6 4.5 16.1 160.8 0.55 0.13 4.2 0.6 20.6 0.44 17.6 9.5 22 14.3 39.6 2.8 0.17 Signifi cant diff erences ( cant diff Signifi