Bacterial and fungal communities in a degraded ombotrophic peatland undergoing natural and managed re-vegetation

Key to zone codes

U.OV Unmanaged - original vegetation
U.Gu Unamanaged - gully
M.YH Managed - young heather
M.RG Managed - restored grass
M.25 Managed - 25 year old heather
D.BP Degraded - bare peat

Source code

Analyses were performed in R. Commented source code is provided in file peat-microbiome.Rmd.

Source data

There were 94627 bacterial and 84552 fungal raw reads off the sequencer. UPARSE clustered OTUs and BLAST assigned taxonomy were imported and analysed using the phyloseq package for R. The source data for bacteria and fungi can be found in folders data/hb_uparse and data/hf_uparse respectively. Those folders also contain a record of commands used to prepare the raw sequences to this stage.

print(expt.bacteria)

## phyloseq-class experiment-level object
## otu_table()   OTU Table:         [ 441 taxa and 18 samples ]
## sample_data() Sample Data:       [ 18 samples by 27 sample variables ]
## tax_table()   Taxonomy Table:    [ 441 taxa by 7 taxonomic ranks ]

print(expt.fungi)

## phyloseq-class experiment-level object
## otu_table()   OTU Table:         [ 300 taxa and 18 samples ]
## sample_data() Sample Data:       [ 18 samples by 27 sample variables ]
## tax_table()   Taxonomy Table:    [ 300 taxa by 7 taxonomic ranks ]

Number of quality controlled reads per sample:

colSums(otu_table(expt.bacteria))

##   W1   W2   W3   W4   W5   W6   M1   M2   M3   M4   M5   M6   E1   E2   E3 
## 1469  373 2233 3150 1695 2072 2046 2398 3968 1714 3435  992 1489 1954 1169 
##   E4   E5   E6 
##  398 1117 1088

colSums(otu_table(expt.fungi))

##   W1   W2   W3   W4   W5   W6   M1   M2   M3   M4   M5   M6   E1   E2   E3 
## 2825  705 2114 3464  724 1333 3319 1688 2096  328 2072 1369 1136  708 1799 
##   E4   E5   E6 
## 1859  932 1491

Number of quality controlled reads per zone:

rowSums(otu_table(expt.bacteria.zone))

## D.BP M.RG M.YH M.25 U.Gu U.OV 
## 3940 4641 6599 5004 7702 4874

rowSums(otu_table(expt.fungi.zone))

## D.BP M.RG M.YH M.25 U.Gu U.OV 
## 4192 2801 6069 7280 6468 3152

Figures and Tables

pdf 2

pdf 2 pdf 2 pdf 2

## [1] TRUE

Figure 2. [chem]. Plant nutrients, soil properties, and heavy metals.

Figure 3. [cfu]. Cultivable numbers of bacteria and fungi detected in each zone.

Figure 4. [rankf]. Rank abundance of bacterial and fungal OTUs in each zone.

Figure 5. [phyla]. Relative abundance of bacterial and fungal phyla (within each kingdom) in the six zones.

Figure 6. [ord]. Correspondence analysis of bacterial and fungal communities, constrained by vegetation zone.

Kingdom Phylum D.BP M.25 M.RG M.YH U.Gu U.OV

B2 Bacteria Acidobacteria 33.92 38.57 19.80 19.82 43.49 28.72

B1 Bacteria Proteobacteria 49.57 49.93 55.49 48.95 44.88 51.32

B37 Bacteria Bacteroidetes 0.79 3.32 2.89 2.22 2.72 4.49

B443 Bacteria Firmicutes 0.03 0.09 0.00 0.00 0.00 0.06

B310 Bacteria OD1 0.00 0.05 0.05 0.00 0.00 0.00

B10 Bacteria Actinobacteria 9.47 5.55 11.63 12.04 4.70 10.82

B92 Bacteria TM7 0.00 0.11 0.10 0.08 0.07 0.29

B166 Bacteria Spirochaetes 0.00 0.29 0.02 0.03 0.09 0.00

B7 Bacteria AD3 2.91 0.22 5.62 11.43 1.53 2.29

B50 Bacteria Verrucomicrobia 1.17 0.73 1.82 3.26 0.96 0.43

Table was truncated to 10 rows for display. Click link to see whole table
Table S3. [all_phyla]. Mean relative abundance of all phyla in each zone. Relative abundances are expressed as a percentage within each kingdom (i.e. columns add up to 200 %)

	Kingdom	Phylum	D.BP	M.25	M.RG	M.YH	U.Gu	U.OV
B2	Bacteria	Acidobacteria	33.92	38.57	19.80	19.82	43.49	28.72
B1	Bacteria	Proteobacteria	49.57	49.93	55.49	48.95	44.88	51.32
B37	Bacteria	Bacteroidetes	0.79	3.32	2.89	2.22	2.72	4.49
B443	Bacteria	Firmicutes	0.03	0.09	0.00	0.00	0.00	0.06
B310	Bacteria	OD1	0.00	0.05	0.05	0.00	0.00	0.00
B10	Bacteria	Actinobacteria	9.47	5.55	11.63	12.04	4.70	10.82
B92	Bacteria	TM7	0.00	0.11	0.10	0.08	0.07	0.29
B166	Bacteria	Spirochaetes	0.00	0.29	0.02	0.03	0.09	0.00
B7	Bacteria	AD3	2.91	0.22	5.62	11.43	1.53	2.29
B50	Bacteria	Verrucomicrobia	1.17	0.73	1.82	3.26	0.96	0.43

Phylum D.BP U.OV D.BP.expected U.OV.expected Xsq_p.value Xsq_p.value.corrected D.BP_pc U.OV_pc diff

F20 Zygomycota 58.00 557.00 351.05 263.95 0.00 0.00 1.38 17.67 -16.29

F1 Ascomycota 2121.00 1882.00 2284.94 1718.06 0.00 0.00 50.60 59.71 -9.11

B37 Bacteroidetes 27.00 204.00 103.26 127.74 0.00 0.00 0.69 4.19 -3.50

B10 Actinobacteria 380.00 546.00 413.94 512.06 0.02 0.04 9.64 11.20 -1.56

B1 Proteobacteria 1980.00 2483.00 1995.03 2467.97 0.65 0.65 50.25 50.94 -0.69

B165 WPS-2 29.00 27.00 25.03 30.97 0.29 0.33 0.74 0.55 0.18

B81 TM6 29.00 7.00 16.09 19.91 0.00 0.00 0.74 0.14 0.59

B7 AD3 115.00 112.00 101.47 125.53 0.07 0.09 2.92 2.30 0.62

B39 Verrucomicrobia 44.00 19.00 28.16 34.84 0.00 0.00 1.12 0.39 0.73

F64 Glomeromycota 41.00 5.00 26.26 19.74 0.00 0.00 0.98 0.16 0.82

Table was truncated to 10 rows for display. Click link to see whole table
Table S4. [phyla-xsq]. Pairwise comparisons of phylum abundance in D.BP and U.OV. Note that p-values of 0 in the linked file actually mean <2.2e-16.

	Phylum	D.BP	U.OV	D.BP.expected	U.OV.expected	Xsq_p.value	Xsq_p.value.corrected	D.BP_pc	U.OV_pc	diff
F20	Zygomycota	58.00	557.00	351.05	263.95	0.00	0.00	1.38	17.67	-16.29
F1	Ascomycota	2121.00	1882.00	2284.94	1718.06	0.00	0.00	50.60	59.71	-9.11
B37	Bacteroidetes	27.00	204.00	103.26	127.74	0.00	0.00	0.69	4.19	-3.50
B10	Actinobacteria	380.00	546.00	413.94	512.06	0.02	0.04	9.64	11.20	-1.56
B1	Proteobacteria	1980.00	2483.00	1995.03	2467.97	0.65	0.65	50.25	50.94	-0.69
B165	WPS-2	29.00	27.00	25.03	30.97	0.29	0.33	0.74	0.55	0.18
B81	TM6	29.00	7.00	16.09	19.91	0.00	0.00	0.74	0.14	0.59
B7	AD3	115.00	112.00	101.47	125.53	0.07	0.09	2.92	2.30	0.62
B39	Verrucomicrobia	44.00	19.00	28.16	34.84	0.00	0.00	1.12	0.39	0.73
F64	Glomeromycota	41.00	5.00	26.26	19.74	0.00	0.00	0.98	0.16	0.82

Figure S5. [ord_unconstrained]. Unconstrained correspondence analyses.

Figure S6. [scree]. Scree plots for constrained and unconstrained ordinations

variable factor W P

1 pH bare peat and early stage restoration/other 31 0.42582

2 Moisture bare peat and early stage restoration/other 81 0.00004

3 OM bare peat and early stage restoration/other 81 0.00004

4 Ammonium bare peat and early stage restoration/other 18 0.05031

5 Nitrate bare peat and early stage restoration/other 30 0.38651

6 P bare peat and early stage restoration/other 8 0.00276

7 K bare peat and early stage restoration/other 16 0.03147

8 Pb bare peat and early stage restoration/other 1 0.00008

9 Cd bare peat and early stage restoration/other 6 0.00123

10 Cu bare peat and early stage restoration/other 2 0.00016

Table was truncated to 10 rows for display. Click link to see whole table
Table S7. [stats]. Mann-Whitney-Wilcoxon test results for various comparisons of chemistry and cultivable microbe data.

	variable	factor	W	P
1	pH	bare peat and early stage restoration/other	31	0.42582
2	Moisture	bare peat and early stage restoration/other	81	0.00004
3	OM	bare peat and early stage restoration/other	81	0.00004
4	Ammonium	bare peat and early stage restoration/other	18	0.05031
5	Nitrate	bare peat and early stage restoration/other	30	0.38651
6	P	bare peat and early stage restoration/other	8	0.00276
7	K	bare peat and early stage restoration/other	16	0.03147
8	Pb	bare peat and early stage restoration/other	1	0.00008
9	Cd	bare peat and early stage restoration/other	6	0.00123
10	Cu	bare peat and early stage restoration/other	2	0.00016

Figure S8. [diversity]. Diversity metrics for each zone, per-sample and per-zone

Calculations for manuscript text

## $`n archaeal observations`
## [1] 2
## 
## $`n bacterial observations`
## [1] 32760
## 
## $`n fungal observations`
## [1] 29962
## 
## $`n archael OTUs`
## [1] 1
## 
## $`n bacterial OTUs`
## [1] 441
## 
## $`n fungal OTUs`
## [1] 300
## 
## $`n bacterial phyla`
## [1] 21
## 
## $`n fungal phyla`
## [1] 5
## 
## $`mean seqs per sample fungi`
## [1] 1665
## 
## $`mean seqs per sample bacteria`
## [1] 1820
## 
## $`mean seqs per sample per kingdom`
## [1] 1742
## 
## $`mean seqs per zone fungi`
## [1] 4995
## 
## $`mean seqs per zone bacteria`
## [1] 5460
## 
## $`mean seqs per zone per kingdom`
## [1] 5226
## 
## $`mean pH all samples`
## [1] 3.878

R package versions

print(sessionInfo(), locale = FALSE)

## R version 3.1.1 (2014-07-10)
## Platform: x86_64-w64-mingw32/x64 (64-bit)
## 
## attached base packages:
## [1] grid      stats     graphics  grDevices utils     datasets  methods  
## [8] base     
## 
## other attached packages:
## [1] car_2.0-21      vegan_2.0-10    lattice_0.20-29 permute_0.8-3  
## [5] xtable_1.7-3    reshape2_1.4    ggplot2_1.0.0   phyloseq_1.9.11
## 
## loaded via a namespace (and not attached):
##  [1] ade4_1.6-2              annotate_1.40.1        
##  [3] AnnotationDbi_1.24.0    ape_3.1-4              
##  [5] Biobase_2.22.0          BiocGenerics_0.8.0     
##  [7] biom_0.3.12             Biostrings_2.30.1      
##  [9] cluster_1.15.2          codetools_0.2-8        
## [11] colorspace_1.2-4        data.table_1.9.2       
## [13] DBI_0.2-7               DESeq2_1.4.5           
## [15] digest_0.6.4            evaluate_0.5.5         
## [17] foreach_1.4.2           formatR_0.10           
## [19] genefilter_1.44.0       geneplotter_1.40.0     
## [21] GenomicRanges_1.14.4    gtable_0.1.2           
## [23] htmltools_0.2.4         igraph_0.7.1           
## [25] IRanges_1.20.7          iterators_1.0.7        
## [27] knitr_1.6               labeling_0.2           
## [29] locfit_1.5-9.1          MASS_7.3-33            
## [31] Matrix_1.1-4            multtest_2.18.0        
## [33] munsell_0.4.2           nlme_3.1-117           
## [35] nnet_7.3-8              parallel_3.1.1         
## [37] plyr_1.8.1              proto_0.3-10           
## [39] RColorBrewer_1.0-5      Rcpp_0.11.2            
## [41] RcppArmadillo_0.4.320.0 RJSONIO_1.2-0.2        
## [43] rmarkdown_0.2.50        RSQLite_0.11.4         
## [45] scales_0.2.4            splines_3.1.1          
## [47] stats4_3.1.1            stringr_0.6.2          
## [49] survival_2.37-7         tools_3.1.1            
## [51] XML_3.98-1.1            XVector_0.2.0          
## [53] yaml_2.1.13

Citations

citation("vegan")

## 
## To cite package 'vegan' in publications use:
## 
##   Jari Oksanen, F. Guillaume Blanchet, Roeland Kindt, Pierre
##   Legendre, Peter R. Minchin, R. B. O'Hara, Gavin L. Simpson,
##   Peter Solymos, M. Henry H. Stevens and Helene Wagner (2013).
##   vegan: Community Ecology Package. R package version 2.0-10.
##   http://CRAN.R-project.org/package=vegan
## 
## A BibTeX entry for LaTeX users is
## 
##   @Manual{,
##     title = {vegan: Community Ecology Package},
##     author = {Jari Oksanen and F. Guillaume Blanchet and Roeland Kindt and Pierre Legendre and Peter R. Minchin and R. B. O'Hara and Gavin L. Simpson and Peter Solymos and M. Henry H. Stevens and Helene Wagner},
##     year = {2013},
##     note = {R package version 2.0-10},
##     url = {http://CRAN.R-project.org/package=vegan},
##   }
## 
## ATTENTION: This citation information has been auto-generated from
## the package DESCRIPTION file and may need manual editing, see
## 'help("citation")'.

citation("phyloseq")

## 
## To cite phyloseq in publications, or otherwise credit, please use:
## 
##   phyloseq: An R package for reproducible interactive analysis and
##   graphics of microbiome census data. Paul J. McMurdie and Susan
##   Holmes (2013) PLoS ONE 8(4):e61217.
## 
## A BibTeX entry for LaTeX users is
## 
##   @Article{,
##     author = {Paul J. McMurdie and Susan Holmes},
##     journal = {PLoS ONE},
##     pages = {e61217},
##     title = {phyloseq: An R package for reproducible interactive analysis and graphics of microbiome census data},
##     volume = {8},
##     number = {4},
##     year = {2013},
##     url = {http://dx.plos.org/10.1371/journal.pone.0061217},
##   }