1 software (Applied Maths, Belgium). As standard, a marker containing the V3 16S rRNA gene fragments of all bacterial endophyte and chloroplast OTUs formerly obtained from the five Bryopsis MX samples [3] was used (see additional file 2). The temporal stability of the endophytic communities was explored by visually comparing the normalized endophytic community profiles of MX sample’s DNA extracts made in October 2009 (EN-2009) versus October 2010 (EN-2010). To study the specificity of the Bryopsis-bacterial endobiosis, normalized EP, WW and CW bacterial community profiles

of each Bryopsis sample were comparatively clustered with previously obtained endophytic (EN-2009) DGGE banding patterns [15] using Dice similarity coefficients. A dendrogram was composed using the Unweighted Pair Group Method with Arithmetic #click here randurls[1|1|,|CHEM1|]# Mean Capmatinib cell line (UPGMA) algorithm in BioNumerics to determine the similarity between

the EP, WW, CW and EN-2009 samples. The similarity matrix generated was also used for constructing a multidimensional scaling (MDS) diagram in BioNumerics. MDS is a powerful data reducing method which reduces each complex DGGE fingerprint into one point in a 3D space in a way that more similar samples are plotted closer together [19]. Additionally, EP, WW and CW DGGE bands at positions of endophytic (including chloroplast) marker bands were excised, sequenced and identified as described by Hollants et al. [3]. To verify their true correspondence with Bryopsis endophytes, excised bands’ sequences were aligned and clustered with previously obtained endophytic bacterial sequences [3] using BioNumerics. Excised DGGE bands’ V3 16S rRNA gene sequences were submitted to EMBL under accession numbers :HE599189-HE599213. BCKDHA Results Temporal stability of endophytic bacterial communities after prolonged cultivation The endophytic bacterial communities showed little time variability after prolonged cultivation when visually comparing

the normalized EN-2009 and EN-2010 DGGE fingerprints (Figure 1). The band patterns of the different MX90, MX263 and MX344 endophytic extracts were highly similar, whereas Bryopsis samples MX19 and 164 showed visible differences between the community profiles of their EN-2009 and EN-2010 DNA extracts. Both the MX19 and MX164 sample had lost the DGGE band representing the Phyllobacteriaceae endophytes (black boxes in Figure 1) after one year of cultivation. Figure 1 Visual comparison of normalized endophytic DGGE fingerprints obtained from surface sterilized Bryopsis DNA extracts made in October 2009 (EN-2009) versus October 2010 (EN-2010). Differences are indicated with black boxes. The first and last lanes contain a molecular marker of which the bands correspond to known Bryopsis endophyte or chloroplast sequences (see additional file 2). This marker was used as a normalization and identification tool.