ADDITIONAL STATISTICAL ANALYSES. The data were not normally distributed (Kolmogorov-Smirnov test; Legendre &

Transcription

1 DDITIONL STTISTICL NLYSES The data were not normally distributed (Kolmogorov-Smirnov test; Legendre & Legendre, 1998) and violate the assumption of equal variances (Levene test; Rohlf & Sokal, 1994) for pairwise (e.g., Student s t-test) and multiple comparisons (e.g., analysis of variance [NOV]). Thus, non-parametric tests based on the ranks of the data were used as these do not assume normality and are more robust to extreme values. Spearman rank correlation coefficients (r; non-parametric equivalent of Pearson s correlation coefficient) were calculated to test for significant correlations among selected numerical parameters. s a non-parametric equivalent of a Student s t-test, a Mann-Whitney test was used to test for significant differences between two data sets (e.g., between the number of viral bands detected by primers OP-13 and CR-22). Kruskal-Wallis test (the non-parametric equivalent of NOV) was used to test for statistically significant differences in the data among the three sampling regions. statistically significant result from a Kruskal- Wallis test was further explored using Mann-Whitney pairwise comparisons for all groups tested, as a non-parametric equivalent of post-hoc tests normally performed in conjunction with NOV. The Kruskal-Wallis and Mann-Whitney test statistics were corrected for ties, i.e., data points that when ranked according to increasing order of magnitude would receive the same rank. Kruskal-Wallis and Mann-Whitney tests still assume that the data have equal variances, which was not the case in our study. Thus, to determine p-values that can be used to either reject (p 0.0) or accept (p > 0.0) the null hypothesis (H0: there is no difference), permutation tests were performed that are independent of data distribution and variance (Manly, 2007). This procedure involves random, unrestricted permutations (10,000 times in this case) of the observed values of either one of the groups being compared (re-sampling 1

2 without replacement). The distribution of the difference between groups, for example, is then used to test whether the observed (calculated from the original data) difference is unexpectedly high. The p-value of this test is then calculated as the proportion of cases that exceed the observed difference, where low proportions (e.g., p < ) indicate that the observed difference is extremely unlikely to have occurred by chance. Thus, the probability for H0 is calculated based on the permuted data sets instead of the Χ 2 - (Kruskal-Wallis) or the t-distribution (Mann-Whitney). The results of the statistical tests were assumed significant at p-values 0.0. However, in case of multiple comparison tests, the α-level indicating significant results was corrected for the number of comparisons using the Bonferroni method. DDITIONL RESULTS Variation within and among sampling areas Geographic distance, extent of sea ice, and abiotic environmental parameters. Geographic distance between the sampling stations varied between 120 and 2600 km (Fig. 1). In the Labrador Sea and at station BB1 in Baffin Bay there was no ice cover, while at stations BB2 to BB and 1 to it ranged from 20 to 80% and 10 to 90%, respectively (Fig. 1). Sampling depth did not differ significantly among the three sampling regions (Kruskal-Wallis: H = 4.8; p = ; Fig. S2). Seawater temperature was significantly higher in the Labrador Sea than in Baffin Bay and the rctic rchipelago (Table S1, Fig. S2). Salinity was lower in the rctic rchipelago than in the Labrador Sea and Baffin Bay and was higher in the Labrador Sea than in Baffin Bay (Table S1, Fig. S2). Prokaryotic and viral abundance, relative abundance of Bacteria. Neither prokaryotic nor viral abundances differed significantly among the sampling regions (Kruskal-Wallis: 2

3 prokaryotes: H = 2.74; p = 0.220; viruses: H = 3.00; p = ; Fig. S2B). The relative abundance of Bacteria was higher in the rctic rchipelago than in the Labrador Sea and Baffin Bay (Table S1; Fig. S2B). T-RFLP analysis of Bacteria and rchaea, RPD-PCR analysis of viruses. The number of peaks for the bacterial forward and reverse primers did not differ among regions (Kruskal-Wallis: forward: H = 6.4; p = ; reverse: H = 3.23; p = 0.190; Fig. S2C). However, the number of bacterial peaks from the forward primer was significantly higher than from the reverse primer in the Labrador Sea (Mann-Whitney: p < ) and the rctic rchipelago (Mann-Whitney: p = ). No significant difference was detected in the number of archaeal peaks among regions (Kruskal-Wallis: forward: H = 3.87; p = ; reverse: H = 4.97; p = ; Fig. S2D). In the Labrador Sea, the number of archaeal peaks from the forward primer was lower than from the reverse primer (Mann-Whitney: p = ). The average combined number of viral bands for primers OP-13 and CR-22 was 21.1 in the Labrador Sea, 20.3 in Baffin Bay, and 21.1 in the rctic rchipelago. The number of viral bands from OP-13 was lower in the rctic rchipelago compared to the Labrador Sea and Baffin Bay, whereas the number of bands for CR-22 was higher in the rctic rchipelago compared to the Labrador Sea (Table S1; Fig. S2E). lso, the number of viral bands from OP-13 was lower than from CR-22 in Baffin Bay (Mann-Whitney: p = ) and the rctic rchipelago (Mann-Whitney: p < ). The combined number of viral bands for primers OP-13 and CR-22 did not differ among sampling regions (Kruskal- Wallis: H = 1.81; p = ). Variation of prokaryotic and viral community composition within sampling areas. Based on a comparison of Jaccard distance calculated among the individual samples (Table 3

4 S2), variation of bacterial community composition determined by the forward primer was significantly higher in Baffin Bay than in the Labrador Sea and the rctic (Fig. S2) but was similar in all three sampling areas based on data of the reverse primer (Fig. S2B). Variation in archaeal community composition determined by the forward primer was significantly higher in the rctic as compared to the Labrador Sea and Baffin Bay (Fig. S2C) and was similar in all three sampling areas for the reverse primer (Fig. S2D). Viral community composition determined by the primer OP-13 was significantly more variable in the rctic as compared to the Labrador Sea and Baffin Bay (Fig. S2E). Variation of viral community composition determined by the primer CR-22 was significantly higher in Baffin Bay compared to the Labrador Sea and it was highest in the rctic rchipelago (Fig. S2F). Schematics of the presence-absence matrices of bacterial, archaeal, and viral community composition are shown in Figs. S3 S8. Relationships between parameters Prokaryotic and viral abundance were correlated in the Labrador Sea (r = 0.68, p = ) and Baffin Bay (r = 0.76, p = ) but were not significantly correlated in the rctic rchipelago (r = 0.40, p = 0.013). 4

5 Table S1: Differences between sampling regions. The table gives the Kruskal-Wallis test statistic (H) and the p-value for parameters that differed significantly between the sampling areas (p 0.0). dditionally, for the pairwise Mann-Whitney tests, p-values are given. For both tests, p-values were obtained by random permutations. Results of the Mann-Whitney pairwise comparison tests are considered to be significant at p , indicated in bold. Parameter Kruskal-Wallis Mann-Whitney H p Baffin Bay rctic Temperature < Labrador Sea < < Baffin Bay Salinity < Labrador Sea < Baffin Bay Bacteria Labrador Sea Baffin Bay Viruses OP Labrador Sea Baffin Bay Viruses CR Labrador Sea Baffin Bay

6 Table S2: Differences in the variation of bacterial, archaeal, and viral community composition among the sampling areas. The table gives the Kruskal-Wallis test statistic (H) and the p-value for the variation in Jaccard distance of bacterial, archaeal, and viral community composition that differed significantly between the sampling areas (p 0.0). dditionally, for the pairwise Mann-Whitney tests, p-values are given. For both tests, p-values were obtained by random permutations. Results of the Mann-Whitney pairwise comparison tests are considered to be significant at p , indicated in bold. Parameter Kruskal-Wallis Mann-Whitney H p Baffin Bay rctic Bacteria-forward < Labrador Sea < Baffin Bay - < rchaea-forward Labrador Sea Baffin Bay Viruses OP < Labrador Sea < Baffin Bay - < Viruses CR < Labrador Sea < Baffin Bay - <0.0001

7 Table S3: Partial Mantel statistics calculated between viral RPD-PCR and bacterial as well as archaeal T-RFLP data corrected for the influence of abiotic (temperature, salinity) and biotic (prokaryotic and viral abundance, relative abundance of Bacteria) environmental parameters together. The table shows the Mantel statistic (rm) calculated between viral and bacterial as well as archaeal compositional data, the corresponding partial Mantel statistic (rpm) corrected for the influence of abiotic and biotic environmental parameters, and its p-value for the three sampling regions and the entire data set. Results are considered to be significant at p 0.012, indicated in bold. Parameters Vir-OP-13 Labrador Sea Baffin Bay rctic rchipelago Entire sampling region rm rpm p rm rpm p rm rpm p rm rpm p Bacteria-forward < Bacteria-reverse < rchaea-forward rchaea-reverse Vir-CR-22 Bacteria-forward < Bacteria-reverse rchaea-forward rchaea-reverse

8 Table S4: Partial Mantel statistics calculated between viral RPD-PCR and bacterial as well as archaeal T-RFLP data corrected for the influence of spatial distance. The table shows the Mantel statistic (rm) calculated between viral and bacterial as well as archaeal compositional data, the corresponding partial Mantel statistic (rpm) corrected for the influence of spatial distance, and its p-value for the three sampling regions and the entire data set. Results are considered to be significant at p 0.012, indicated in bold. Parameters Vir-OP-13 Labrador Sea Baffin Bay rctic rchipelago Entire sampling region rm rpm p rm rpm p rm rpm p rm rpm p Bacteria-forward Bacteria-reverse < rchaea-forward rchaea-reverse < Vir-CR-22 Bacteria-forward Bacteria-reverse rchaea-forward rchaea-reverse

9 Table S: Variation partitioning for T-RFLP data obtained with the bacterial forward primer. The matrix of environmental parameters consisted of temperature, salinity, prokaryotic and viral abundance, and the relative abundance of Bacteria. The coordinates of the sampling stations were used as co-variables representing spatial distance. The table gives the fraction (%) of variation in the bacterial T-RFLP data explained by the models and the corresponding p-value (n.a.: not applicable). Results are assumed to be significant at p 0.0. Parameters Labrador Sea Baffin Bay rctic rchipelago Entire sampling region Fraction p Fraction p Fraction p Fraction p Environmental parameters and spatial distance < < Environmental parameters not corrected for spatial distance < < < Spatial distance not corrected for environmental parameters Environmental parameters Spatially correlated environmental parameters 7 n.a. 6 n.a. n.a. 4 n.a. Spatial distance Unexplained 32 n.a. 29 n.a. 3 n.a. 68 n.a.

10 Table S6: Variation partitioning for T-RFLP data obtained with the bacterial reverse primer. The matrix of environmental parameters consisted of temperature, salinity, prokaryotic and viral abundance, and the relative abundance of Bacteria. The coordinates of the sampling stations were used as co-variables representing spatial distance. The table gives the fraction (%) of variation in the bacterial T-RFLP data explained by the models and the corresponding p-value (n.a.: not applicable). Results are assumed to be significant at p 0.0. Parameters Labrador Sea Baffin Bay rctic rchipelago Entire sampling region Fraction p Fraction p Fraction p Fraction p Environmental parameters and spatial distance < < Environmental parameters not corrected for spatial distance < < Spatial distance not corrected for environmental parameters Environmental parameters Spatially correlated environmental parameters 6 n.a. 4 n.a. 2 n.a. 3 n.a. Spatial distance Unexplained 29 n.a. 30 n.a. 9 n.a. 71 n.a.

11 Table S7: Variation partitioning for T-RFLP data obtained with the archaeal forward primer. The matrix of environmental parameters consisted of temperature, salinity, prokaryotic and viral abundance, and the relative abundance of Bacteria. The coordinates of the sampling stations were used as co-variables representing spatial distance. The table gives the fraction (%) of variation in the archaeal T-RFLP data explained by the models and the corresponding p-value (n.a.: not applicable). Results are assumed to be significant at p 0.0. Parameters Labrador Sea Baffin Bay rctic rchipelago Entire sampling region Fraction p Fraction p Fraction p Fraction p Environmental parameters and spatial distance < Environmental parameters not corrected for spatial distance < Spatial distance not corrected for environmental parameters Environmental parameters Spatially correlated environmental parameters 11 n.a. 2 n.a. 3 n.a. 0 n.a. Spatial distance Unexplained 32 n.a. 4 n.a. n.a. 7 n.a.

12 Table S8: Variation partitioning for T-RFLP data obtained with the archaeal reverse primer. The matrix of environmental parameters consisted of temperature, salinity, prokaryotic and viral abundance, and the relative abundance of Bacteria. The coordinates of the sampling stations were used as co-variables representing spatial distance. The table gives the fraction (%) of variation in the archaeal T-RFLP data explained by the models and the corresponding p-value (n.a.: not applicable). Results are assumed to be significant at p 0.0. Parameters Labrador Sea Baffin Bay rctic rchipelago Entire sampling region Fraction p Fraction p Fraction p Fraction p Environmental parameters and spatial distance < < < Environmental parameters not corrected for spatial distance < < < Spatial distance not corrected for environmental parameters Environmental parameters Spatially correlated environmental parameters n.a. 6 n.a. 1 n.a. 2 n.a. Spatial distance Unexplained 39 n.a. 36 n.a. 2 n.a. 73 n.a.

13 Table S9: Variation partitioning for viral RPD-PCR data obtained with the primer OP-13. The matrix of environmental parameters consisted of temperature, salinity, prokaryotic and viral abundance, and the relative abundance of Bacteria. The coordinates of the sampling stations were used as co-variables representing spatial distance. The table gives the fraction (%) of variation in the viral RPD-PCR data explained by the models and the corresponding p-value (n.a.: not applicable). Results are assumed to be significant at p 0.0. Parameters Labrador Sea Baffin Bay rctic rchipelago Entire sampling region Variation p Variation p Variation p Variation p Environmental parameters and spatial distance < < Environmental parameters not corrected for spatial distance 64 < < Spatial distance not corrected for environmental parameters < < Environmental parameters Spatially correlated environmental parameters 16 n.a. 10 n.a. 13 n.a. 9 n.a. Spatial distance Unexplained 23 n.a. 47 n.a. 47 n.a. 67 n.a.

14 Table S10: Variation partitioning for viral RPD-PCR data obtained with the primer CR-22. The matrix of environmental parameters consisted of temperature, salinity, prokaryotic and viral abundance, and the relative abundance of Bacteria. The coordinates of the sampling stations were used as co-variables representing spatial distance. The table gives the fraction (%) of variation in the viral RPD-PCR data explained by the models and the corresponding p-value (n.a.: not applicable). Results are assumed to be significant at p 0.0. Parameters Labrador Sea Baffin Bay rctic rchipelago Entire sampling region Variation p Variation p Variation p Variation p Environmental parameters and spatial distance < < Environmental parameters not corrected for spatial distance < Spatial distance not corrected for environmental parameters < < Environmental parameters Spatially correlated environmental parameters 11 n.a. 4 n.a. 11 n.a. 4 n.a. Spatial distance Unexplained 34 n.a. 2 n.a. 6 n.a. 78 n.a.

15 Table S11: Variation partitioning for bacterial, archaeal, and viral compositional data. Similar to Tables S S10, however, only either abiotic (temperature, salinity) or biotic (prokaryotic and viral abundance, relative abundance of Bacteria) environmental parameters constituted the matrix of explanatory variables. The table gives the fraction (%) of variation in the compositional data explained by abiotic or biotic environmental parameters. Results are assumed to be significant at p 0.0, indicated in bold. Parameters Labrador Sea Baffin Bay rctic rchipelago Entire sampling region Fraction p Fraction p Fraction p Fraction p Bacteria-forward abiotic biotic Bacteria-reverse abiotic biotic rchaea-forward abiotic biotic rchaea-reverse abiotic biotic Viruses OP-13 abiotic biotic Viruses CR-22 abiotic biotic

16 Depth (m) C E Temperature ( C) No. of peaks in T-RFLP profiles for Bacteria using the forward and reverse primer No. of bands in RPD-PCR profiles for viruses using primers OP-13 and CR vg. depth vg. temperature vg. salinity Labrador Sea vg. no. peaks Bact. forward vg. no. peaks Bact. reverse No. peaks Bact. forward No. peaks Bact. reverse Labrador Sea Labrador Sea Baffin Bay Baffin Bay Baffin Bay Depth Temperature Salinity 36 rctic rctic rctic vg. no. bands viruses, OP-13 vg. no. bands viruses, CR-22 No. bands viruses, OP-13 No. bands viruses, CR-22 Salinity B Prokaryotic (N 10 ml -1 ) and viral (N 10 6 ml -1 ) abundance D No. of peaks in T-RFLP profiles for rchaea using the forward and reverse primer vg. prokaryotes vg. viruses vg. Bacteria Labrador Sea 0 Labrador Sea Baffin Bay Baffin Bay Prokaryotes Viruses Bacteria 100 rctic rctic vg. no. peaks rchaea forward vg. no. peaks rchaea reverse No. peaks rchaea forward No. peaks rchaea reverse 0 Bacteria (% of DPI-stained prokaryotes) Figure S1: Parameters obtained in the three sampling regions. The figure shows the average and individual data points of the parameters collected in the Labrador Sea (n = 16, except for viruses OP-13 and CR-22 where n = 1), Baffin Bay (n = 18), and the rctic rchipelago (n = 24). () Depth (m), temperature ( C) and salinity, (B) prokaryotic (N 10 ml -1 ) and viral abundance (N 10 6 ml -1 ), relative abundance of Bacteria (% of DPI-stained cells), (C) number of bacterial T-RFLP peaks for the forward and reverse primer, (D) number of archaeal T-RFLP peaks for the forward and reverse primer, and (E) the number of viral RPD-PCR bands for primers OP-13 and CR-22.

17 B C Jaccard dissimilarity index (mean ± standard deviation) D Labrador S. B Baffin Bay rctic Jaccard dissimilarity index (mean ± standard deviation) E Labrador S. Baffin Bay rctic Jaccard dissimilarity index (mean ± standard deviation) F Labrador S. Baffin Bay B rctic Jaccard dissimilarity index (mean ± standard deviation) Labrador S. Baffin Bay rctic Jaccard dissimilarity index (mean ± standard deviation) Labrador S. Baffin Bay B 0.8 B rctic Jaccard dissimilarity index (mean ± standard deviation) Labrador S. Baffin Bay C rctic Figure S2: Variation of bacterial, archaeal, and viral community composition within the three sampling areas. The figure shows the Jaccard dissimilarity index (mean ± standard deviation) calculated among samples within the Labrador Sea, Baffin Bay, and rctic rchipelago. () Bacterial forward primer, (B) bacterial reverse primer, (C) archaeal forward primer, (D) archaeal reverse primer, (E) primer OP-13, and (F) primer CR-22. Letters above data points indicate statistically significant differences based on Kruskal-Wallis and Mann-Whitney tests (Table S2).

18 LS1 LS2 LS BB1 7 BB BB3 7 BB BB Figure S3: Schematic representation of the T-RFLP peak patterns obtained from the bacterial forward primer. Presence and absence of peaks is indicated by black and white squares, respectively.

19 LS1 LS2 LS BB1 7 BB BB3 7 BB BB Figure S4: Schematic representation of the T-RFLP peak patterns obtained from the bacterial reverse primer. Presence and absence of peaks is indicated by black and white squares, respectively.

20 LS1 LS2 LS BB1 7 BB BB3 7 BB BB Figure S: Schematic representation of the T-RFLP peak patterns obtained from the archaeal forward primer. Presence and absence of peaks is indicated by black and white squares, respectively. LS1 LS2 LS BB1 7 BB BB3 7 BB BB Figure S6: Schematic representation of the T-RFLP peak patterns obtained from the archaeal reverse primer. Presence and absence of peaks is indicated by black and white squares, respectively.

21 LS1 LS2 BB1 LS BB BB3 7 BB BB Figure S7: Schematic representation of the RPD-PCR banding patterns obtained from the primer OP-13. Presence and absence of bands is indicated by black and white squares, respectively. LS1 LS2 BB1 LS BB BB3 7 BB BB Figure S8: Schematic representation of the RPD-PCR banding patterns obtained from the primer CR-22. Presence and absence of bands is indicated by black and white squares, respectively.