Supplementary materials
Microbial Ecology
Faunal Burrows Alter the Diversity and Structure of AOA, AOB, Anammox and n-Damo Bacterial Communities in Coastal Mangrove Sediments
Jing Chen†, and Ji-Dong Gu*
Laboratory of Environmental Microbiology and Toxicology, School of Biological Sciences, Faculty of Science, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, P.R. China
*Corresponding author
Mailing address: School of Biological Sciences, Faculty of Science, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, P.R. China
Tel: 852-2299-0605; Fax: 852-2559-9114; E-mail:
† Current Address: School of Life Sciences, The University of Warwick, Coventry, United Kingdom
Table S1 PCR primer sets and reaction conditions in this study
Primers sets / Sequences5’-3’ / Targeting gene / Expected product size
(bp) / Thermal profile / References
Arch-amoAF / STA ATG GTC TGG CTT AGA CG / Archaeal amoA
(AOA) / 635 / 95 °C, 5 min; 32×(95 °C, 30 s; 56 °C, 30 s; 72 °C, 45 s); 72 °C, 10 min / Francis, et al. [1]
Arch-amoAR / GCG GCC ATC CAT CTG TAT GT
amoA-1F / GGG GTT TCT ACT GGT GGT / Bacterial amoA
(AOB) / 491 / 95 °C, 5 min; 32×(95 °C, 30 s; 54 °C, 30 s; 72 °C, 45 s); 72 °C, 10 min* / Rotthauwe, et al. [2]
amoA-2R / CCC CTC KGS AAA GCC TTC TTC
A438f / GTC RGG AGT TAD GAA ATG / Anammox
16S rRNA / 246 / 95 °C, 5 min; 32×(95 °C, 45 s; 54 °C, 30 s; 72 °C, 50 s); 72 °C, 10 min / Humbert, et al. [3]
A684r / ACC AGA AGT TCC ACT CTC
A189_b / GGN GAC TGG GAC TTY TGG / n-damo pmoA / 530 / 94 °C, 5 min; 34×(94 °C, 60 s; gradient 53-63 °C, 60 s; 72 °C, 90 s); 72 °C, 10 min** / Luesken, et al. [4]
cmo682 / AAA YCC GGC RAA GAA CGA
cmo182 / TCA CGT TGA CGC CGA TCC / 386
cmo568 / GCA CAT ACT CCA TCC CCA TC
qP1F / GGG CTT GAC ATC CCA CGA ACC TG / n-damo
16S rRNA / 459 / 95 °C, 5 min; 40×(95 °C, 15 s; 54 °C, 30 s; 72 °C, 45 s; 78 °C, 15 s); 95 °C, 15 s; 72 °C, 1 min; 95 °C, 15 s / Ettwig, et al. [5]
qP2R / GGG GAA CTG CCA GCG TCA AG
* For AOB-Y, gradient annealing temperatures of 50-60 °C was applied;
**For n-damo-Wall, gradient annealing temperatures of 50-60 °C was applied in the nest PCR step 1.
The selection of PCR primer sets were based on the previous studies and qPCR primers for n-damo bacteria were adjusted to the current one after trail experiment. In brief, primer pairs of Arch-amoAF + Arch-amoAR [1] and amoA-1F + amoA-2R [2] were used for detecting AOA and AOB, respectively, according to our previous studies in the non-burrowed sediments of coastal wetlands [6, 7]. For anammox bacteria, primers A438f and A684r were applied according to the findings in the previous report, which highly recommended the above primer pairs for screening anammox bacteria from various environments [8]. The primers detecting AOA, AOB and anammox bacteria were used for both PCR and qPCR analyses. Primers specific for n-damo pmoA gene were selected for a better comparison with the marine n-damo pmoA gene clusters identified previously [9]. For the PCR amplification of n-damo pmoA gene, a nested PCR approach with primers A189_b + cmo682 and cmo182 + cmo568 was applied according to a previous investigation [4].
The quantification of n-damo bacteria was conducted using a different primer pair targeting n-damo 16S rRNA gene. The n-damo pmoA gene primers in the above are based on nest PCR approach with a temperature gradient as annealing condition [4, 10]. The application of either pair of primer sets could not result satisfied PCR product for all samples in the present work. The only qPCR primers specific for n-damo pmoA gene [11] also failed to generate single band product in gel for all samples in this study, which means that there were potential over estimation of the signal obtained using this primer set. The 16S rRNA gene primers qP1F and qP2R in table S1 were previously designed for qPCR detection of n-damo enrichment in the combination of primer sets of qP1F+qP1R and qP2F + qP2R [5]. However, neither of the two pairs generated good result. The current combination of primers qP1F + qP2R can quantify the samples in the present work with single band product in gel after PCR. Therefore, primers qP1F +qP2R were applied for n-damo bacterial quantification.
Table S2 Summary and analysis of reference n-damo bacteria from different habitats based on pmoA gene sequences*
Source/Environment / N-damo related clone / OTU / Shannon-Wiener / Chao1 / Type / Reference**Mangrove Sm / 25 / 4 / 0.8605 / 4.5 / C/R / Chen, et al. [12]
Mangrove Bm / 25 / 7 / 1.3168 / 15 / C/R / Chen, et al. [12]
Mudflat S / 25 / 6 / 1.4317 / 8 / C/R / Chen, et al. [12]
Mudflat B / 18 / 3 / 0.9338 / 1 / C/R / Chen, et al. [12]
Reed bed / 27 / 6 / 1.2421 / 1 / C/R / Chen, et al. [12]
CF5S / 21 / 2 / 0.6365 / 1 / M/R / Chen, et al. [9]
CF5B / 4 / 1 / 0 / 1 / M/R / Chen et al. (2014)
E201S / 8 / 2 / 0.3768 / 1 / M/R / Chen et al. (2014)
E407S / 6 / 2 / 0.4506 / 1 / M/R / Chen et al. (2014)
E407B / 24 / 2 / 0.3768 / 1 / M/R / Chen et al. (2014)
E510S / 16 / 2 / 0.2338 / 1 / M/R / Chen et al. (2014)
E525S / 24 / 1 / 0 / 1 / M/R / Chen et al. (2014)
E525B / 1 / 1 / -- / -- / M/R / Chen et al. (2014)
SCS E704S / 24 / 3 / 1.0055 / 1 / M/R / Chen, et al. [13]
SCS E401 / 13 / 3 / 0.9110 / 1 / M/R / Chen, et al. [13]
Lake Constance / 15 / 6 / 1.4878 / 10.5 / F / Deutzmann and Schink [14]
Lake Biwa / 21 / 1 / 0 / 1 / F / Kojima, et al. [15]
Paddy soil / 14 / 1 / 0 / 1 / F / Wang, et al. [16]
Paddy field / 20 / 2 / 0.1985 / 1 / F / Zhu, et al. [17]
Jingshan paddy field / 27 / 1 / 0 / 1 / F / Hu, et al. [18]
Xixi wetland / 21 / 3 / 0.3805 / 1 / F / Hu, et al. [18]
Xiazhuhu wetland / 4 / 1 / 0 / 1 / F / Hu, et al. [18]
FreshwaterWetlands
(Jingshan Paddy;
Xixi wetland;
Xiazhuhu wetland) / 52 / 5 / 1.0575 / 1 / F / Hu, et al. [18]
Baiyangdian Lake / 24 / 1 / 0 / 1 / F / Zhu, et al. [17]
Bosten Lake / 16 / 2 / 0.4826 / 1 / F / Zhu, et al. [17]
Chaohu Lake / 19 / 6 / 1.1513 / 1 / F / Zhu, et al. [17]
Dongting Lake / 21 / 2 / 0.6920 / 1 / F / Zhu, et al. [17]
Jiaxing Constructed wetland-winter / 13 / 8 / 1.8393 / 26 / F / Zhu, et al. [17]
Jiaxing Constructed wetland-summer / 11 / 8 / 2.0198 / 12.1667 / F / Zhu, et al. [17]
Subsurface North canal / 18 / 2 / 0.6870 / 1 / U / Zhu, et al. [17]
North canal-12 m / 3 / 1 / 0 / 1 / U / Zhu, et al. [17]
North canal-15 m / 8 / 1 / 0 / 1 / U / Zhu, et al. [17]
Panjin swamp / 21 / 1 / 0 / 1 / F / Zhu, et al. [17]
Peat land China / 23 / 6 / 1.3944 / 8 / F / Zhu, et al. [17]
Poyang Lake / 21 / 2 / 0.5983 / 1 / F / Zhu, et al. [17]
Shahe River / 18 / 5 / 1.2094 / 1 / F / Zhu, et al. [17]
Songhuajiang River / 18 / 7 / 1.6715 / 9.25 / F / Zhu, et al. [17]
Tarim River / 19 / 2 / 0.6918 / 1 / F / Zhu, et al. [17]
Tiaoxi River / 20 / 3 / 0.9973 / 1 / F / Zhu, et al. [17]
Tibetan Lake / 10 / 1 / 0 / 1 / S/R / Yang, et al. [19]
Tulufan River / 18 / 3 / 0.8487 / 3 / F / Zhu, et al. [17]
Wuliangshuhai Lake / 24 / 4 / 0.9366 / 4.5 / F / Zhu, et al. [17]
Yellow River / 15 / 1 / 0 / 1 / F / Zhu, et al. [17]
Yuanmingyuan Lake / 12 / 3 / 0.7215 / 3.5 / F / Zhu, et al. [17]
Pearl River-winter / 20 / 5 / 1.0098 / 6 / F / Zhu, et al. [17]
Pearl River-summer / 13 / 2 / 0.6172 / 1 / F / Zhu, et al. [17]
Donghu Lake / 15 / 3 / 0.8033 / 1 / F / Zhu, et al. [17]
Honghaitan Tidal Land / 17 / 4 / 1.1151 / 4.5 / C / Zhu, et al. [17]
Shangqiu Reservoir / 17 / 7 / 1.6100 / 8.5 / F / Zhu, et al. [17]
Tianchi Lake / 18 / 9 / 1.8121 / 18 / F / Zhu, et al. [17]
Qiantang River / 50 / 16 / 2.1437 / 29.5 / F / Shen, et al. [20]
West Lake / 24 / 8 / 1.5359 / 12 / F / Zhu, et al. [21]
Jiaojiang estuary / 80 / 20 / 2.3787 / 30.1250 / C / Shen, et al. [22]
*based on percentage sequence identity of 95% for pmoA gene using Fastgroup Ⅱ[23];
Abbreviations: NA-not applicable; M-marine; F-freshwater; S-saline lake; C-costal; R-representative sequences available in the Genbank database only;
**All the isolation sources/environments are from sediments.
Table S3 Correlations between physicochemical parameters and alpha diversity/abundance*
Parameter / OTU / Shannon-Wiener / Chao1 / Abundance1 / 2** / 3 / 4 / 1 / 2 / 3 / 4 / 1 / 2 / 3 / 4 / 1 / 2 / 3 / 4
pH / -0.3134 / 0.8046 / 0.8743 / 0.6038 / -0.3652 / 0.7225 / 0.7851 / 0.8336 / -0.3367 / 0.8720 / 0.7414 / 0.9163 / 0.5018 / 0.5993 / -0.6314 / 0.8032
Redox / 0.8337 / 0.2089 / 0.2453 / 0.5332 / 0.8749 / 0.3072 / 0.2016 / 0.3774 / 0.7678 / -0.2087 / 0.4895 / 0.2141 / 0.5437 / 0.5683 / -0.1023 / 0.2655
Temperature / 0.9239 / 0.6303 / 0.5329 / 0.2705 / 0.8892 / 0.7259 / 0.6460 / 0.4368 / 0.9183 / 0.3311 / 0.4284 / 0.3898 / 0.1787 / 0.3289 / -0.6841 / 0.1213
Salinity / -0.8469 / -0.4049 / -0.4297 / -0.5985 / -0.8697 / -0.4962 / -0.3985 / -0.5230 / -0.7850 / 0.0034 / -0.6069 / -0.3833 / -0.5775 / -0.6388 / 0.2966 / -0.3660
NH4+ / 0.0724 / 0.6736 / 0.8250 / 0.9794 / 0.0805 / 0.6452 / 0.6399 / 0.9643 / -0.0128 / 0.4706 / 0.9910 / 0.9048 / 0.9366 / 0.9813 / -0.3742 / 0.9789
NO2- / -0.0828 / 0.4552 / 0.6513 / 0.9834 / -0.0500 / 0.4149 / 0.4141 / 0.8604 / -0.1806 / 0.2685 / 0.9194 / 0.7785 / 0.9817 / 0.9709 / -0.1143 / 0.9725
NO3- / 0.8830 / 0.4697 / 0.4706 / 0.5528 / 0.8942 / 0.5636 / 0.4675 / 0.5271 / 0.8308 / 0.0733 / 0.5933 / 0.4035 / 0.5157 / 0.5978 / -0.3903 / 0.3328
NH4+/NOx- / -0.7080 / 0.3471 / 0.3283 / -0.1205 / -0.7781 / 0.2383 / 0.3472 / 0.1691 / -0.6617 / 0.6918 / 0.0208 / 0.3469 / -0.2003 / -0.1419 / -0.3679 / 0.1873
*Based on Pearson moment correlation, Pearson’s r is given by
;
Numbers in bold are regarded as significant (P < 0.05, detailed data not shown). The significance is computed using a two-tailed t test with n-2 degrees of freedom and
.
**1-AOA; 2-AOB; 3-Anammox bacteria; 4-n-damo bacteria;
(a) (b)
Fig. S1 Map of Mai Po Nature Reserve (a) (Adapted from WWF_HongKong [24]) and the bioturbated areas rich with burrows in the mangrove sediments there (b). The exact locations were as followings.
Site 1 / N22° 29.704' / E114° 818'Site 2 / N22° 29.709' / E114° 01.814'
Site 3 / N22° 29.709' / E114° 01.812'
Site 4 / N22° 29.722' / E114° 01.863'
Site 5 / N22° 29.760' / E114° 01.869'
Fig. S2 The PCoA scree plots of n-damo community after qualitative (a) and quantitative (b) analyses using Fast Unifrac algorithm
Fig. S3 Jackknife sample clusters of n-damo bacterial communities. Results are generated by Fast Unifrac using no weight (a) and normalized abundance (b) analyses. The number of permutations is 1000. Scale: 1 dash, slash, backslash ~ 0.0050 (a) and 0.0020 (b) branch length units. Samples in the present work are bold and italic. Other sample names refer to those n-damo communities listed in Table S2.
When the statistical resampling of Jackknifing was performed using normalized abundance weights, the confidence of most subclusters lowered to grey color of <50% (Fig. S3-b). However, the n-damo bacterial communities of Wall and Surface were more reasonably clustered with the samples River Pearl-summer and winter. Meanwhile, the Black and Yellow clustered with previously revealed n-damo bacterial communities in the sediments of the Mai Po wetlands and the South China Sea.
Fig. S4 Plots of Principal Coordinate Analyses based on weighted Unifrac algorithm and amplified sequences in the present work. (a)-AOA; (b)-AOB; (c)-Anammox bacteria; (d)-n-damo bacteria. Mothur [20] was applied to conduct the calculation. The results were visualized in Excel and regenerated using Adobe Illustrator CS3 (www.adobe.com).
Fig. S5 Schematic of sampling site and classification of sample types.
Fig. S6-a Reconstructed phylogenetic tree (AOA)
Fig. S6-b Reconstructed phylogenetic tree (AOB)
Fig. S6-c Reconstructed phylogenetic tree (anammox bacteria)
Fig. S6-d Reconstructed phylogenetic tree (n-damo bacteria)
References
1. Francis CA, Roberts KJ, Beman JM, Santoro AE, Oakley BB (2005) Ubiquity and diversity of ammonia-oxidizing archaea in water columns and sediments of the ocean. Proc Natl Acad Sci USA 102: 14683-14688.
2. Rotthauwe JH, Witzel KP, Liesack W (1997) The ammonia monooxygenase structural gene amoA as a functional marker: Molecular fine-scale analysis of natural ammonia-oxidizing populations. Appl Environ Microbiol 63: 4704-4712.
3. Humbert S, Zopfi J, Tarnawski SE (2012) Abundance of anammox bacteria in different wetland soils. Env Microbiol Rep 4: 484-490.
4. Luesken FA, Zhu BL, van Alen TA, Butler MK, Diaz MR, Song B, den Camp HJMO, Jetten MSM, Ettwig KF (2011) pmoA Primers for Detection of Anaerobic Methanotrophs. Appl Environ Microbiol 77: 3877-3880.
5. Ettwig KF, van Alen T, van de Pas-Schoonen KT, Jetten MSM, Strous M (2009) Enrichment and Molecular Detection of Denitrifying Methanotrophic Bacteria of the NC10 Phylum. Appl Environ Microbiol 75: 3656-3662.
6. Wang YF, Feng YY, Ma XJ, Gu J-D (2013) Seasonal dynamics of ammonia/ammonium-oxidizing prokaryotes in oxic and anoxic wetland sediments of subtropical coastal mangrove. Appl Microbiol Biotechnol 97: 7919-7934.
7. Wang YF, Gu J-D (2013) Higher diversity of ammonia/ammonium-oxidizing prokaryotes in constructed freshwater wetland than natural coastal marine wetland. Appl Microbiol Biotechnol 97: 7015-7033.
8. Han P, Huang YT, Lin JG, Gu J-D (2013) A comparison of two 16S rRNA gene-based PCR primer sets in unraveling anammox bacteria from different environmental samples. Appl Microbiol Biotechnol 97: 10521-10529. doi: 10.1007/s00253-013-5305-z
9. Chen J, Zhou ZC, Gu J-D (2014) Occurrence and Diversity of Nitrite-dependent Anaerobic Methane Oxidation Bacteria in the Sediments of the South China Sea Revealed by Amplification of both 16S rRNA and pmoA Genes. Appl Microbiol Biotechnol 98: 5685-5696.