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group other than the y-Proteobacteria. Therefore, this species was used as the outlier
group in the phylogeny to root the tree. The multiple sequence alignment from PileUp
was transferred into ClustalX 1.64b (Higgins et al., 1996). which was used to create a
Phylip file for the production of the phylogenetic tree using the default weight parameters
to produce a sequence distance matrix, which estimated the evolutionary distance of a
particular pair of sequences based on differences in base pair substitutions. GeneDoc
(Nicholas and Nicholas, 1997) was then used to examine and demonstrate that the
alignment was correct by directly comparing the aligned sequences. Tree Puzzle version
4 (Strimmer and von Haeseler, 1996) was then used to analyse the phylogeny and
compute the maximum likelihood tree, i. e. the tree that best fits the sequence data. The
maximum likelihood algorithm corrects for the superposition of multiple mutations at a
single sequence position, so that the observed number of nucleotide differences do not
underestimate the number of mutational events that may have occurred since the
evolutionary separation of the genes (Olsen et al., 1986). The tree was created using the
assumption that all the genes used in the alignment are mutating at a similar rate. The
maximum likelihood tree was then viewed using TreeView 1.6.6 (Page, 2001) (Fig 5.4).
It must be stressed that due to time limitations this may not be the most optimal tree, i. e.
it may not be the best representation of the data. However, it is a good approximation of
the phylogenetic relatedness of the taxa.
The phylogenetic tree (Fig 5.4) shows a similar pattern of relatedness to the
ARDRA analysis (Fig 5.2a & b). Both isolates 302 and 33 cluster together in the
Pseudomonas group. Isolate 732 clusters with the Enterobacteriaceae indicating its near
identical similarity with Enterobacter agglomerans (syn. Pantoea agglomerans). also
being closely related to Escherichia coll. Both isolates 86 and 39 cluster with
Pseudoalteromonas sp. (Alteromonadaceae) as the most closely related group to the
Enterobacteriaceae. The next group to join this clustering is the Idiomarina loihiensisf
isolate 53 group (Alteromonadeceae). The Alteromonadeceae / Enterobacteriaceae group
then joins on to the rest of the phylogeny, which comprises the Moraxellaceae family
(Psychrobacter, Moraxella
and isolate 583), Oceanospirillium group family
(Marinotnonas protea taxon) and the Halomonadaceae family (Halomonas sp. and Arctic
seawater bacterium). The Halomonadaceae family represent isolate 213 which could not
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