1 Appendix S1. Experimental procedures.
2 Sediment collection and chemical analyses
3 The Qiantang River is a major river system in Zhejiang Province in southeastern China (Fig. 1) between
4 117.62° and 121.87° east longitude and 28.17° and 30.48° north latitude. Its total length is 688 km, with a
5 watershed area of 55,600 km2. Seven sediment samples investigated in this study were collected from
6 Lanxi City (upstream) to Hangzhou City (downstream) in September 2009 (Fig. 1). Sediments were
7 obtained using box-cores, and the top 3 cm of sediment was carefully collected. All the samples were
8 sectioned into two subsamples: one was stored anaerobically at 4 °C for analysis of physical and chemical
9 properties; the other was frozen at -80 °C for subsequent DNA extraction and molecular analysis. Sediment
10 pH was measured in situ. Other environmental factors were measured in the laboratory. Sediment organic
11 carbon and nitrogen contents were measured with a PE 2400 Series II CHNS/O analyzer (Perkin Elmer,
12 USA). The concentrations of NO3--N and NH4+-N were measured by ion chromatography.
13 DNA extraction and PCR amplification
14 DNA was extracted from Qiantang River sediments using the Power Soil DNA Kit (Mo Bio Laboratories,
15 Carlsbad, California, USA), following the manufacturer’s instructions. The extracted DNA was examined
16 in 1.0% agarose gels by electrophoresis.
17
Different combinations of primers were used for the amplification of anammox bacterial 16S rRNA
18 genes, and a nested PCR approach was finally chosen based on the amplification yield and without
19 nonspecific PCR products with positive controls. In the first PCR round, Planctomycetales 16S rRNA
20 genes were amplified with Pla46f (E. coli positions 46-63; Schmid et al., 2000) as the forward primer and
21 1545r (E. coli positions 1529-1545; Juretschko et al., 1998) as the reverse primer. In the second round,
22 anammox-bacteria-specific PCR was performed using anammox-specific primers, Amx368f (E. coli
1
23 positions 368-385; Schmid et al., 2003) and Amx820r (E. coli positions 820-841; Schmid et al., 2000). The
24 PCR reaction mixture (25 μl in total) contained 2.5 µl 10×PCR buffer (containing 2 mM MgCl2), 20 mM
25 each deoxyribonucleoside triphosphate, 1 mM each primer, 1 U Taq polymerase and 1 μl DNA template (126 10 ng). The PCR thermal cycle programs were performed as previously described (Humbert et al., 2010).
27 The amplified products were examined in 1.0% agarose gels by electrophoresis.
28 Cloning and sequencing
29 PCR products were cloned using the pMD19-T vector (TaKaRa, Japan) according to the manufacturer’s
30 instructions. Plasmid DNA was isolated with the Gene JET™ Plasmid Miniprep kit (Fermentas Life
31 Sciences, Germany). Plasmids were digested with 5U EcoRI enzyme in EcoRI buffer for 1.5 h at 37°C. The
32 digestion products were examined for an insertion of the expected size by agarose (1.0%) gel
33 electrophoresis. The different numbers of positive clones in each library were randomly selected for
34 sequencing on an ABI3100 automated sequencer (Applied Biosystems, California, USA).
35 Phylogenetic analysis
36 Phylogenetic analysis of the sequences was conducted with ARB software as previously described (Schmid
37 et al., 2003). Phylogenetic analysis was performed with neighbour-joining method with 50% sequence
38 conservation filters for Planctomycetes. Bootstrap values were 1000 replicates.
39 Real-time quantitative PCR (qPCR)
40 A primer set (Amx694f-Amx960r) (Ni et al., 2010) targeting 16S rRNA genes was used to quantify the
41 abundances of anammox bacteria in different sediment samples collected from Qiantang River. The qPCR
42 was performed with an iCycler iQ5 thermocycler and real-time detection system (Bio-Rad, California,
43 USA). The qPCR was conducted as previously described (Ni et al., 2010). The specificity of the PCR
2
44 amplification was determined by the melting curve and gel electrophoresis.
45 Statistical analysis
46 Operational taxonomic units (OTUs) for 16S rRNA gene diversity of anammox bacteria were defined by
47 3% differences in nucleotide sequences, as determined by using the furthest neighbour algorithm in
48 DOTUR program (Schloss and Handelsman, 2005). DOTUR was also used to generate Chao, Shannon, and
49 Simpson index for each clone library. The coverage of clone libraries was calculated as C = [1 - (n1/N)]
50 ×100, where n1 is the number of unique OTUs and N is the total number of clones in a library. The values
51 of library coverage (C) ranged from 90.6 to 100% (Table 1), indicated that the anammox bacteria detected
52 in Qiantang River sediments were sufficiently represented in the clone libraries. The ecological distribution
53 of anammox bacterial communities and their correlations with environmental factors were determined by
54 the principal components analysis and the redundancy analysis respectively using the software CANOCO
55 (ter Braak and Šmilauer, 2002). In addition, Pearson correlation analyses (significance level ɑ=0.05) were
56 used to test for correlations between the anammox bacterial diversity, abundance and environmental factors
57 (Li et al., 2010).
58 Nucleotide sequence accession numbers
59 The sequences obtained in this study are available in GenBank under accession numbers HM537194 to
60 HM537228.
61 References
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71 Schloss, P. D., and Handelsman, J. (2005) Introducing DOTUR, a computer program for defining
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73 Schmid, M.C., Twachtmann, U., Klein, M., Strous, M., Juretschko, S., Jetten, M.S.M., et al. (2000)
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4