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DNA sequence analysis
The helicosporidial 18S rDNA
region sequence was aligned with 138 other sequences from
representative eukaryotic taxa obtained from the Ribosomal
Database Project (RDP; Maidak et al., 2000). Downloaded
sequences were pre-aligned based on the secondary structure
of the rDNA. An additional 18S sequence from the
pathogenic alga Prototheca wickerhamii was downloaded
from GenBank (accession no. X56099) and incorporated in
the small-subunit (SSU) RNA dataset. Additionally, 28S
sequences were downloaded from GenBank and aligned
with the helicosporidial 28S sequence using clustal x
(Thompson et al., 1997). Eventually, the SSU- and large
subunit (LSU)-rDNA datasets were combined to infer one
single ribosomal phylogeny. Both actin and β-tubulin
sequences were aligned with homologous sequences downloaded
from GenBank. Alignments were obtained using
clustal x software with default parameters. All datasets
were checked by eye before further analyses and are available
upon request. They can also be obtained from TreeBase
(Morell, 1996; http://www.treebase.org/treebase/)
with the study accession number S604. The 18S algal
alignment was kindly provided by V. A. R. Huss (University
of Erlangen, Germany).
Aligned datasets were subjected to a partition-homogeneity
test using the program paup*, version 4.0b4a (Swoåord,
2000), in order to assess the extent of character incongruence
between the datasets (Farris et al., 1994). Phylogenies
were then reconstructed using neighbour-joining (NJ) as
implemented in the paup* program version 4.0b4a. NJ
analyses were based on the Paralinear/LogDet model of
nucleotide substitution (Lockhart et al., 1994). This method
allows for non-stationary changes in base composition and
has been shown to reduce support for spurious resolutions, such as long-branch attraction. Monophyly of groups was
assessed with the bootstrap method (100 replicates). Additionally,
maximum-parsimony analyses, including jackknifing
(100,000 replicates ; Farris et al., 1996), were also
performed using paup*. We chose the latter, conservative
approach for its ability to search rapidly a large amount of
tree space and to estimate support for an unambiguously
resolved group (Lipscomb et al., 1998).
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