1
Sequence Alignments
For the 18S rRNA, the alignment of the Piroplasmorida initially followed the secondarystructure model of Gutell et al. (1994), obtained from the Comparative RNA Website (Cannone
et al., 2002), where a model and an associated alignment of almost-complete rRNA sequences is
available for the piroplasms. The alignment was refined using the model for the variable area V4
of Wuyts et al. (2000) (which did not have any structure indicated in most of the initial models).
The alignment was then manually optimized for best fit to the consensus structure, taking
particular note of compensatory base changes where there was length variation in stems. For
regions with variable lengths, hypotheses of base pairing were evaluated using the Mfold
program (Zucker, 2003), which folds RNA based on energy minimizations. All structure
nomenclature (for stems and regions) follows Wuyts et al. (2000).
The remaining almost-complete database sequences (defined as >1500 nt long) were then
added to this initial alignment using the pairwise alignment feature of the MacClade v. 4.08
program (Maddison and Maddison, 2000), and then manually optimized for fit to the consensus
structure. Most of the new sequences were obtained from the SILVA database (Pruesse et al.,
2007), although direct searches of the main sequence databases were also undertaken. The
alignment and consensus structure were modified, if necessary, to accommodate sequences that
deviated strongly from those already in the alignment.
Sequences FJ668368 and HM538196 were excluded from the alignment because the
sequencing seemd to be very poor, and sequence HM538255 proved to be difficult to align,
possibly due to sequencing errors. The final alignment consisted of 603 sequences and 2085
aligned positions. Cardiosporidium cionae (EU052685) was used as the outgroup, based on the
tree shown by Morrison (2009).
For the two protein-coding genes, the DNA sequences were translated to amino acids and
then the amino acid sequences were aligned. For regions with variable lengths, hypotheses of
alignment were evaluated using the Promals3D program (Pei et al., 2008), which aligns the
sequences based on the secondary sructure of the protein.The final alignment consisted of 36
sequences and 1980 aligned positions for the hsp70 gene, and 17 sequences and 1323 positions
for the beta-tubulin gene. In neither case was a suitable outgroup sequence available, and so the
trees were rooted based on the root location of the 18S rRNA tree.
2
Phylogenetic Analyses
For the rRNA, sequence locations in which positional homology was ambiguous across
all taxa were identified as regions of ambiguous alignment (RAA) or regions of expansion and
contraction (REC) (Gillespie, 2004). The REC notably included stems 10, E10-1, 12, E23-2,
E23-7, 43, 46 and 49, all of which are widely recognized as variable regions in 18S rRNA.
Indeed, it proved to be impossible to derive a consensus secondary-structure model for stems
E23-1 to E23-7 (in the V4 region). These RAA and REC regions were excluded from the
phylogenetic analyses. Excluded from all analyses were autapomorphies, as many of these
appeared to be sequencing errors. All sequence locations were included in the analyses of the
protein-coding genes.
Phylogenetic analyses were performed using bayesian analyses via the MrBayes v3.2.1
program (Ronquist and Huelsenbeck 2003). Following Beiko et al. (2006), analyses consisted of
multiple, relatively short MCMC runs each with a small number of chains. The Tracer v. 1.4
program (Rambaut and Drummond, 2007) was used to check for convergence of the pooled data
from the multiple runs. The number of runs and the length of the burnin for each analysis were
calculated so that the expected sample size (ESS) values for all of the model parameters were
>100, and with the standard deviation of the split frequencies <0.05. The default priors were
used in all analyses.
The analysis model for the rRNA used two partitions, one for the unpaired rRNA
positions with the GTR+G+I model, and one for the paired (stem) positions with the
doublet+G+I model. All model parameters were unlinked across the partitions, except for the
tree topology. This analysis consisted of 17 runs of 2 chains each, with 1,200,000 generations per
run and a burnin of 200,000 generations. This yielded 170,000 trees for the consensus tree, with
a final standard deviation of the split frequencies = 0.064. The chains sampled from two islands
of trees, with somewhat different parameter values for the likelihood models. The composition of
the clades with large posterior probabilities was the same for both islands, but with some
differences in branch order both between and within those clades.
The analyses for the protein-coding genes used the M3 codon model. For the hsp70 gene,
the analysis consisted of 6 runs of 2 chains each, with 1,025,000 generations per run and a burnin
of 125,000 generations. This yielded 54,000 trees for the consensus tree, with a final standard
deviation of the split frequencies = 0.010. For the beta-tubulin gene, the analysis consisted of 8
3
runs of 2 chains each, with 2,000,000 generations per run and a burnin of 750,000 generations.
This yielded 100,000 trees for the consensus tree, with a final standard deviation of the split
frequencies = 0.024.
The phylogenetic trees were drawn with either FigTree v. 1.3.1 (Rambaut, 2009) or
Dendroscope v. 2.6.1 (Huson et al., 2007).
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