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Analyses of Cytb sequence data revealed saturation of third codon transitions for Kimura-2-parameter distances greater than 0.40, a pattern confirmed by a statistical test [25] which indicated substantial saturation at third codon positions (Iss
In the case of third codon positions only, substantial saturation could be observed (Iss significantly higher than Iss.c).
For Echinodermata, Hexapoda, Tunicata, Chilopoda and Heterobranchia, the ISS of was notably larger than the asymmetrical ISSc, suggesting substantial saturation in these alignments.
If the index of substitution saturation (Iss) approaches 1 or if the Iss is not smaller than the critical Iss value (Iss.c), then sequences are considered to contain substantial saturation.
The plot of transition and transversion rates at each codon position against the Kimura's 2-parameter distances for pairwise comparisons of cytochrome b gene sequences of our sample showed evidence for substantial saturation only at third-position transitions, and evidence for moderate saturation at third-position transversions and first-position transitions.
Furthermore, to assess whether there was substantial saturation among the amino acid substitutions, we also used the software AsaturA [ 44] to visualize the accumulation of amino acid substitutions with genetic distance, which showed that there was only very slight saturation of even the most frequent types of amino acid substitutions in these data.
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Sequence divergence is expected to be neither too conservative nor too diverged as to experience substantial substitution saturation; this saturation decreases phylogenetic information [ 57].
Saturation plots revealed substantial substitution saturation in the third codon positions of 13 PCGs and rrnS (Additional file 13).
No substantial substitutional saturation was found in the COI and 16S datasets upon testing with DAMBE version 5.2.73 [ 42].
In contrast, the concatenated 4D-sites of 13 mitochondrial genes showed substantial substitution saturation (two tailed P value >0.05) and were excluded for further analysis.
Substantial content saturation was reached at the end of session three as indicated by recurrent identification of common themes and emergence of limited new information.
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