In order to avoid such phylogenetic inference artifacts, especially those caused by possible saturation in the fast-evolving lineages, first and third codon positions of mt protein-coding genes, and third codon positions of nuclear protein-coding genes were excluded from the corresponding nucleotide alignments.
For the mitochondrial genes, 265 positions have been identified as fast-evolving sites (eighth relative gamma rate of 5.42) and subsequently removed.
For introns, 536 fast-evolving positions (eighth gamma rate of 2.1) were excluded representing 149 sites for MGF, 91 for PRKC, 181 for SPTBN, 97 for THY and 18 for the combined flanking-exonic regions of the introns.
This is done by either substituting slow-evolving for fast-evolving taxa, or removing third codon positions [ 12, 67].
The objective of removing fast-evolving positions was first to identify and improve the signal to noise ratio in all three different datasets (mitochondrial, exon and intron fragments) that showed different patterns of evolutionary rates.
In molecular phylogenetic investigations, a common practice to reduce long-branch effects is to exclude fast-evolving molecular loci such as third codon positions from inference analysis, based on the rationale that these loci are likely saturated or randomized [ 19, 40, 74- 80].
When fast evolving third codon positions were removed from alignments, the "supergene" tree recovers our reference species phylogeny as well as the Cytb, ND4L and ND6 genes.
Only the BI analyses yielded strong support for monophyly of this group, and this occurs only when fast evolving third positions are included.
When fast evolving third codon positions were removed from alignments, the "supergene" tree recovers our reference species phylogeny (Fig. 5) as well as the Cytb, ND4L and ND6 genes (Additional file 13).
Saturation occurs more rapidly in fast-evolving sites, such as the third codon position, and removal of these sites may increase phylogenetic accuracy [ 19].
