Denitrifiers and dissimilatory nitrate reducers detected via narG in

A. gracilis

For gut contents and soils of G. paulistus and A. gracilis, genes were amplified from extracted DNA (2.5.7.2.1) and used for barcoded amplicon pyrosequencing (2.5.11).

Coverage (2.5.12.6) and diversity (2.5.12.7, 2.5.12.8) were calculated with gene sequences (2.5.12.3) whereas phylogenetic analyses (2.5.12.9) were calculated with in silico translated amino acid sequences.

3.1.1.3.2.1. Barcoded amplicon pyrosequencing and diversity analysis of narG

For narG, altogether 7,809 valid sequences were retrieved, yielding 15, 28, 27, and 17 OTUs at a species-level cutoff of 67 % (i.e., on nucleotide sequence level) for the libraries from gut contents of G. paulistus, gut contents of A. gracilis, soil of G. paulistus, and soil of A. gracilis, respectively (Table 16). The species-level cutoff value for narG of 67 % is a very conservative estimate; the real number of species represented by narG sequences is assumed to be higher. However, the cutoff value used displays the minimum amount of OTUs and was the standard of comparison between the analyzed samples. Coverages ranged between 99.0 % and 100 %, indicating that sampling was sufficient. Estimated richness was highest in gut contents of A. gracilis with 34 OTUs, whereas for the other libraries, estimated richness was only slightly higher (gut of G. paulistus and soil of A. gracilis) or the same (soil of G. paulistus) as already sampled (Table 16). Diversity indices (i.e., Shannon-Weaver index, Evenness, and reciprocal Simpson index) of gut-derived narG sequences were always higher than those of the corresponding soil. This is indicative of a more broad than selective stimulation of soil-derived nitrate reducers in the earthworm gut.

Table 16: Sequence qualities, OTUs, coverages, and diversity indices of narG sequences from gut contents and soils of G. paulistus and A. gracilis.

Sequences Diversity indices

Library^a Valid^b Invalid^c OTUs^d C^e (%) Chao1^f H´^g E^h 1/Dⁱ

Gut GP 405 16 15 99.0 17 1.66 0.61 3.85

Gut AG 1,486 57 28 99.7 34 1.78 0.53 3.07

Soil GP 4,985 45 27 100 27 1.49 0.45 2.79

Soil AG 933 22 17 99.6 19 1.39 0.49 2.79

a GP, G. paulistus; AG, A. gracilis.

b Sequences encoding for the desired gene as verified by BLAST analysis. Potential chimeras were excluded.

c Sequences that were discarded for further analyses as encoding not for the desired gene or representing potential chimeras.

d Species-level OTUs of valid sequences. Cutoff value was 67%.

e Coverage.

f Chao1 richness estimator.

g Shannon diversity index.

h Evenness.

i Reciprocal Simpson diversity index.

See the methods part for further information (2.5.12).

Modified from Depkat-Jakob et al. (2013).

3.1.1.3.2.2. Phylogenetic analysis of narG

The narG sequences of OTU 1 were most closely affiliated with Methylobacterium sp.

4-46 and Oligotropha carboxidovorans OM5, both members of the Bradyrhizobiaceae within the Rhizobiales (Garrity et al. 2005, Sadowsky & Graham 2006). Related sequences were abundantly detected in the libraries derived from the gut contents of G. paulistus, gut contents of A. gracilis, and soil of G. paulistus. Sequences derived from the soil of A. gracilis were predominantly affiliated with OTU 4 that was distantly related to Anaeromyxobacter sp. K (Figure 15, Figure 16). Sequences related to Mycobacterium sp. D9-7 (OTU 2) and Saccharopolyspora erythraea NRRL2338 (OTU 5), both members of the Actinobacteria, were dominant in both gut content and soil-derived libraries of G. paulistus, but did not exceed 1 % relative abundance in the libraries from gut content and soil of A. gracilis.

Sequences related to Micromonospora aurantiaca (OTU 7) were abundantly detected in the gut contents of A. gracilis but were virtually absent in all other libraries (Figure 15, Figure 16).

Most narG sequences occurred in OTUs that contained both soil-derived and gut-derived sequences, except for the quantitatively minor OTUs 10 and 13 (Figure 15). This indicates that the majority of gut-derived sequences originated from ingested soil Bacteria.

Altogether, gut and soil-derived narG sequences of G. paulistus were more similar to each other than to gut and soil-derived sequences of A. gracilis (Figure 16, Figure 17).

Despite of the detected differences in narG diversity, differences between narG libraries were not significant except for the soil library of A. gracilis that showed significant differences to all other libraries (Table A 1). NC 010473 Escherichia coli str. K12 substr. DH10B AM419046 Dechloromonas denitrificans ED1

Figure 15: Phylogenetic neighbor-joining tree of representative narG sequences from gut contents and soils of G. paulistus and A. gracilis, and related sequences.

The phylogenetic tree is based on in silico translated amino acid sequences. OTUs that accounted for at least 1 % in at least one library are shown with one representative sequence (bold, with accession numbers in parentheses). The percentage of replicate trees the associated taxa clustered together in the bootstrap test (10,000 replicates), are shown at the node of two branches (values below 80 % are not displayed). The table shows the relative distribution of sequences in each OTU (>0 indicates that at least one sequence was detected but its relative abundance was below 1%). Numbers at the bottom of the table indicated the sums of percentages of sequences for each library covered by the OTUs shown in the tree. Differences between the sum and the combined percentage of the individual OTU percentages for one library are due to the rounding off of values. GP, G. paulistus; AG, A. gracilis. The libraries Gut GP, Gut AG, Soil GP, and Soil AG contain 405, 1,486, 4,985, and 933 sequences, respectively. The enumeration of OTUs corresponds with those in the figure displaying the relative distribution of narG OTUs. The bar indicates a 0.05 estimated change per amino acid. Modified from Depkat-Jakob et al. (2013).

Figure 16: Relative distribution of narG OTUs from gut contents of G. paulistus and A. gracilis, and from their corresponding soils.

G. paulistus and A. gracilis were sampled from pasture soil and grassland soil, respectively. OTUs were calculated from narG sequences. OTU numbers at the right correspond with those in the phylogenetic tree of in silico translated narG sequences; OTUs below 5 % relative abundance were combined and are displayed as white OTUs (i.e., non-shaded wedges) in the pies. Based on Depkat-Jakob et al. (2013).

Figure 17: FastUniFrac principle coordinate analysis of narG sequences from gut contents of G. paulistus and A. gracilis, and from their corresponding soils.

This analysis was used to display relative differences between the four different narG gene libraries.

G. paulistus and A. gracilis were sampled from pasture soil and grassland soil, respectively.

A variance of 94.9 % is covered by x-axis (PC 1, 74.7 %) and y-axis (PC 2, 20.2 %). GP, G. paulistus;

AG, A. gracilis. Modified from Depkat-Jakob et al. (2013).

3.1.1.3.3. Nitrite reducers detected via nirK in gut contents and