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Appendix S2 Detailed description of PCR-DGGE results
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Bacterial 16S rRNA gene fragments. The bacterial PCR-DGGE profiles of Sarcotragus
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spinosulus were characterized by 9 dominant and 11 to 36 fainter bands (Fig. S1a).
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Most of the prevailing bands were present in all sponge specimens, whereas the fainter
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ones showed varied patterns of abundance or presence/absence across the profiles (Fig.
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S1a). Overall, 23.4% of the total PCR-DGGE band data variation could be attributed to
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the factor “year of sampling”, with specimens collected in 2010 and 2012 found to
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significantly influence band variation in the profiles (p<0.05, Fig. S1d). PCR-DGGE
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band richness and diversity measures of 2010 (35.33 ± 6.35 and 3.55 ± 0.17,
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respectively) were significantly different (p<0.05) from the values observed for 2011
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(26.21 ± 4.87 and 3.25 ± 0.19, respectively) and 2012 (20.92 ± 1.49 and 3.03 ± 0.07
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respectively), revealing a slight decrease in these two parameters over time.
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Archaeal 16S rRNA gene fragments. The archaeal PCR-DGGE profiles of S. spinosulus
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consisted of two conspicuous bands observed in all S. spinosulus specimens along with
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few (1 to 12) other detectable bands across the samples (Fig. S1b). Here, 45.8% of the
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whole PCR-DGGE variation was attributed to the factor “year of sampling”. In spite of
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the high degree of conservation of the two major bands in the gel, ordination via RDA
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revealed that 2010 and 2012 replicates possessed different archaeal community
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structures (p<0.05, Fig. S1e). No significant difference (p>0.05) was found in the PCR-
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DGGE band richness and diversity measurements across sampling years 2010 (3.39 ±
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0.96 and 1.18 ± 0.33, respectively), 2011 (5.47 ± 1.52 and 1.67 ± 0.27, respectively) and
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2012 (5.24 ± 1.16 and 1.64 ± 0.22, respectively).
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1
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Bacterial and Archaeal amoA gene fragments. The bacterial ammonia-oxidizing PCR-
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DGGE profiles of S. spinosulus revealed two dominant and few fainter bands in almost
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all S. spinosulus patterns (Fig. S1c). The independent variable “sampling year” was
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responsible for 23.8% of the variation in PCR-DGGE profiles, with replicates from
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2012 significantly affecting band abundance data across samples (p<0.05, Fig. S1f).
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PCR-DGGE band richness and diversity measures were not significantly different
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across the sampling years 2010 (8.05 ± 3.94 and 1.98 ± 0.53, respectively), 2011 (10.71
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± 2.16 and 2.35 ± 0.22, respectively) and 2012 (10.68 ± 4.17 and 2.30 ± 0.43,
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respectively). For the archaeal amoA gene, no amplification was obtained from any of
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the S. spinosulus replicates.
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2