Toupoint 2012 Effect of biofilm age on settlement of Mytilus edulis Supplemental

Supplementary material

Table S1. List of the parameters and the associated lengths of the PP bands (1.3 cm width) sampled for biofilmcharacterization.

Parameters Analysis

Polypropylene bands

Length (cm) Side Surface (cmo)

(a) Biomass dry weight 91.4 Recto 116.1(b) Concentrations FCM 22.9 Recto-verso 58.1(c) Diversity rRNA gene 10.2 Recto-verso 25.8(d) Lipid quality FAME The same as in (a), performed after weighing(e) Structure CLSM 2.5 Recto 3.2(f) Imaging SEM 2.5 Recto 3.2

FCM ¼ flow cytometry; rRNA ¼ ribosomal ribonucleic acid; FAME ¼ fatty acids methyl esters; CLSM ¼ confocal laser scanning microscopy;SEM ¼ scanning electronic microscopy.

Table S2. Results from univariate PERMANOVAs investigating the differences in bacterial (a) and eukaryotic (b) communitiesbetween biofilm ages (T1, T2, and T3) during the S4 session.

df SS MS Pseudo-F/t P (perm) Unique perms

a- BacteriaAge 2 5 664.1 2 832.1 1.6747 0.0107 280

Global Residuals 6 10 147.0 1 691.1Total 8 15 811.0

T1 vs T2 1.2667 0.1055 10Pairwise T1 vs T3 1.5030 0.0979 10

T2 vs T3 1.0875 0.2988 10

b- EukaryotesAge 2 937.1 468.5 1.0067 0.4766 280

Global Residuals 6 2 792.3 465.4Total 8 3 729.4

Bold value indicates a significant effect (p 5 0.05).

Table S3. Bacterial composition of the biofilms identified at each age through 16S rRNA gene sequences analysis.

Phylum Class Order Genus

T1 T2 T3

a b c a b c a b c

Unclas. Bacteria 0 0 0 1 2 0 1 1 2Actinobacteria Actinobacteria Acidimicrobiales Unclas. Acidimicrobineae 0 0 0 0 0 0 0 0 1

Iamia 0 5 1 3 4 2 5 1 4Ilumatobacter 0 0 1 1 0 0 0 0 0

Actinomycetales Unclas. Frankineae 0 0 0 0 0 0 0 0 1Firmicutes Erysipelotrichi Erysipelotrichales Erysipelothrix 0 0 0 0 0 0 1 0 0Bacteroidetes incertae sedis incertae sedis Fulvivirga 0 0 0 0 0 0 0 1 0

Sphingobacteria Sphingobacteriales Lewinella 0 1 0 0 0 0 0 0 0Haliscomenobacter 0 0 0 0 0 2 0 0 0Salisaeta 0 0 0 0 0 0 0 1 0

Flavobacteria Flavobacteriales Unclas. Cryomorphaceae 0 0 0 0 0 0 1 0 1Aestuariicola 0 0 0 0 1 0 0 0 0Winogradskyella 0 1 0 0 0 0 0 1 0

Proteobacteria Unclas. 1 0 1 0 1 0 0 1 3Deltaproteobacteria Unclas. 1 0 0 1 1 0 3 1 5

Bdellovibrionales Bdellovibrio 0 0 0 0 0 1 0 0 0Unclas. Bacteriovoracaceae 0 0 0 0 0 0 0 0 1Peredibacter 1 0 0 0 0 0 0 0 0

Desulfobacterales Desulfosarcina 0 0 0 0 0 0 1 0 0Myxococcales Sorangium 1 0 0 0 0 0 0 0 0

Kofleria 0 1 0 0 0 0 0 0 0Unclas. Nannocystaceae 0 0 0 0 1 0 0 0 0

Betaproteobacteria Burkholderiales Achromobacter 0 0 0 0 1 0 0 0 0Alphaproteobacteria Unclas. 0 0 0 0 0 1 0 0 1

Rhizobiales Unclas. Phyllobacteriaceae 0 1 0 0 0 0 0 0 0Pseudaminobacter 1 0 0 0 0 0 0 0 0Hoeflea 0 0 0 0 0 0 0 0 1Anderseniella 0 0 0 0 1 0 0 0 0Unclas. Hyphomicrobiaceae 0 0 0 0 2 2 0 0 0Filomicrobium 0 0 0 0 0 0 0 0 1Devosia 0 0 0 0 0 0 0 1 0

Parvularculales Parvularcula 0 0 0 0 1 0 0 0 0Caulobacterales Unclas. Hyphomonadaceae 1 1 0 0 0 0 0 0 0

Hellea 0 0 0 0 0 0 0 1 0Hirschia 1 0 0 0 0 0 0 0 0Hyphomonas 1 0 1 0 1 0 1 0 0Maricaulis 1 0 0 0 0 0 0 0 0

Rickettsiales Orientia 0 0 0 0 1 0 0 0 0Pelagibacter 1 0 0 0 0 0 0 0 0

Rhodospirillales Unclas. Rhodospirillaceae 1 1 0 0 0 0 0 0 0Sphingomonadales Sphingopyxis 0 0 1 0 0 0 1 0 0

Erythrobacter 1 0 0 0 0 0 0 0 0Croceicoccus 1 0 0 0 0 0 0 0 0

Kordiimonadales Kordiimonas 0 1 0 0 0 0 0 0 0

(continued)

Table S3. (Continued).

Phylum Class Order Genus

T1 T2 T3

a b c a b c a b c

Rhodobacterales Unclas. Rhodobacteraceae 6 2 6 3 6 9 1 7 2Wenxinia 0 2 0 0 0 0 3 1 0Octadecabacter 0 2 0 0 0 0 0 0 0Oceanicola 1 0 0 0 0 1 0 0 0

Rhodobacterales Sulfitobacter 1 5 2 0 1 1 1 0 0Thalassobacter 2 1 0 0 0 0 0 0 0Tateyamaria 0 0 1 0 0 0 0 0 0Pelagicola 0 2 0 0 0 0 0 2 0Loktanella 2 4 2 0 0 1 0 0 0Thalassobius 0 0 1 0 0 0 0 0 0Ruegeria 1 0 0 0 0 0 0 0 0Roseisalinus 0 0 0 0 0 0 0 0 1Jannaschia 0 1 0 1 0 3 0 0 0Shimia 0 0 0 0 0 0 0 1 0Albimonas 0 1 0 0 0 0 0 0 0Ahrensia 0 0 0 0 0 2 1 0 0Roseovarius 0 1 5 1 0 1 0 0 0

Proteobacteria Gammaproteobacteria Unclas. 0 0 0 0 2 0 0 0 0Sedimenticola 0 0 0 1 0 0 0 0 0

Chromatiales Unclas. Ectothiorhodospiraceae 0 0 0 0 0 0 0 1 0Oceanospirillales Kangiella 1 0 0 0 0 0 0 1 0

Neptunomonas 0 0 0 0 0 1 0 0 0Neptuniibacter 1 0 0 0 0 0 0 0 0

Enterobacteriales Unclas. Enterobacteriaceae 1 0 0 0 0 1 0 0 0Escherichia/Shigella 0 0 5 0 5 2 0 1 1Rahnella 0 0 0 1 1 0 1 1 3Yersinia 0 0 0 0 0 0 0 0 1Serratia 0 0 0 0 1 1 0 2 1

Vibrionales Vibrio 1 0 0 0 0 0 0 0 0Listonella 1 0 0 0 0 0 0 0 0

Alteromonadales Colwellia 0 0 1 0 0 0 0 0 0Teredinibacter 2 2 3 0 0 0 0 0 0Saccharophagus 0 0 0 0 1 0 0 0 0Haliea 0 0 0 0 0 1 0 0 0Glaciecola 0 0 1 0 0 0 0 0 0

Thiotrichales Francisella 0 0 0 0 0 0 2 0 0Fangia 1 0 0 0 0 0 0 0 1

Gemmatimonadetes Gemmatimonadetes Gemmatimonadales Gemmatimonas 1 0 0 0 0 0 0 0 0Acidobacteria Acidobacteria Gp10 Gp10 0 0 0 0 0 0 1 0 0Deinococcus-Thermus Deinococci Deinococcales Truepera 0 0 0 0 0 0 0 0 1Verrucomicrobia Verrucomicrobiae Verrucomicrobiales Unclas. Verrucomicrobiaceae 0 0 1 0 0 0 1 0 1

Luteolibacter 0 0 0 0 0 0 1 0 1Persicirhabdus 0 0 0 1 0 0 0 0 0

(continued)

Table S3. (Continued).

Phylum Class Order Genus

T1 T2 T3

a b c a b c a b c

Planctomycetes Planctomycetacia Planctomycetales Unclas. Planctomycetaceae 2 6 6 6 0 0 6 0 1Schlesneria 1 0 0 1 1 0 0 0 0Planctomyces 8 3 3 21 3 10 19 8 8Rhodopirellula 4 0 0 9 2 0 5 3 3Blastopirellula 3 2 3 4 0 1 6 3 2Pirellula 1 0 0 0 0 0 0 1 2

Cyanobacteria Cyanobacteria GpIIa 0 3 1 0 0 0 0 0 0Lentisphaerae Lentisphaeria Unclas. 0 0 1 0 0 0 0 0 0Chloroflexi Anaerolineae Anaerolineales Unclas. Anaerolineaceae 1 0 0 0 0 0 0 0 0

Longilinea 0 0 0 0 0 0 0 1 0Caldilineae Caldilineales Caldilinea 0 0 0 1 0 1 0 0 1

Chloroplast Chlorophyta 1 1 0 1 0 0 0 0 0Bacillariophyta 0 1 0 2 1 0 1 2 0

Total 56 51 47 59 41 44 63 44 51

Note : Values are number of sequences (a, b, c ¼ replicates).

Table S4. Eukaryote composition of the biofilms identified at each age through 18S rRNA gene sequences analysis.

Phylum Class Order Genus

T1 T2 T3

a b c a b c a b c

Alveolata Unclas. Unclas. 1 1 0 3 0 3 10 3 1Ciliophora Intramacronucleata Zoothamnium 55 51 73 54 30 59 35 24 37

Pseudovorticella 3 0 2 0 2 0 0 0 0Intramacronucleata 1 1 5 4 2 1 7 6 0

Postciliodesmatophora Unclas. 2 0 0 2 2 0 3 0 1Dinophyceae Suessiales Pelagodinium 0 0 0 1 0 0 0 0 0

Gymnodiniales Unclas. 0 0 0 1 0 0 0 0 0Viridiplantae Chlorophyta Trebouxiophyceae Nannochloris 0 0 0 0 0 0 1 0 0

Unclas. 1 0 0 0 0 0 0 0 0Rhizaria Cercozoa Unclas. 0 0 0 1 0 0 0 0 0

Unclas. 0 0 0 0 0 0 3 0 0Metazoa Rotifera Encentrum 0 0 0 0 0 0 0 0 1

Cnidaria Scyphozoa Haliclystus 1 0 0 0 0 0 0 0 0Arthropoda Crustacea Tisbe 3 0 1 1 0 0 0 1 0

Harpacticus 0 0 0 0 1 2 3 7 2Unclas. 0 0 0 0 4 2 0 1 0

Unclas. 0 0 1 0 0 0 1 0 0

Total 67 53 82 67 41 66 64 42 42

Note: Values are number of sequences (a, b, c ¼ replicates).

Figure S1. CLSM images of a biofilm surface.

Figure S2. SEM images of biofilm surfaces.

Figure S2. (Continued).

Figure S2. (Continued).

Figure S2. (Continued).

Figure S2. (Continued).

Figure S2. (Continued).

Figure S2. (Continued).