Figure 1.
Bryopsis sampling sites along the Pacific Mexican coast.
Bryopsis hypnoides (•) and Bryopsis pennata var. leprieurii (▴) samples were collected from following sites: Playa el Pantheon (MX19), Mazunte Beach (MX90), Acapulco (MX164), Playa las Gatas (MX263) and Playa Careyero (MX344).
Table 1.
Taxonomic affiliation of the clones representing the bacterial OTUs, sorted per Bryopsis sample.
Figure 2.
Epifluorescence microscopy images of Bryopsis sections hybridized with the universal bacterial Cy3-EUB338 probe mix (red).
DAPI (light blue) and calcofluor (dark blue) were used as counter stains to visualize algal DNA in nuclei and chloroplasts and the algal cell wall, respectively. Metabolically active bacteria (red) are present throughout the Bryopsis cytoplasm: in the outer layer (OL) next to the cell wall (CW) which contains most of the organelles like mitochondria, endoplasmic reticulum, and nuclei (A–C), and in the inner chloroplast layer (CHL) immediately adjacent to the vacuole (V) (B–C). Since the Bryopsis thalli were not surface sterilized before fixation, the red probe also hybridized with epiphytic bacteria on the calcofluor stained cell wall (B–C). The scale bar on all images is 20 µm.
Figure 3.
Normalized DGGE profiles of MX DNA extracts and their representative OTUs.
DGGE bands marked with letters A, B and C, which did not match any of the individual OTU bands, were excised from the polyacrylamide gel and sequenced. The first and last lanes contain a known molecular marker used for normalization.
Figure 4.
Endophytic diversity results (right) plotted against the Bryopsis host phylogeny (left).
The OTU diversity (1–7) displayed on the right summarizes the diversity results from the clone libraries and DGGE analyses. The concatenated chloroplast 16S rRNA gene - rbcL maximum likelihood tree on the left classifies the Bryopsis MX samples in two distinct species clades with 100% bootstrap support. The scale bar indicates 0.002 nucleotide changes per nucleotide position.
Figure 5.
A wide-range maximum likelihood tree showing the phylogenetic positions of endophytic clones and DGGE bands.
Phylogenies were inferred from 16S rRNA gene sequences determined in this study (in bold), BLAST hits (see Table 1), Bacteroidetes, Proteobacteria and Mollicutes type strains, and algae-associated bacteria described in the literature (see Table S1). The tree was generated in PhyML according the HKY + G4 algorithmic model. Bootstrap values above 50% are indicated at the branch nodes and the scale bar shows 10 nucleotide substitutions per 100 nucleotides. Asterisks denote sequences previously isolated from micro * - and macroalgae**. The same phylogenetic tree without compressed branches is depicted in Figure S1.