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The character changes were polarized a posteriori using the outgroup comparison criterion [ 42].
A posteriori polarization of character changes along this tree using the outgroup comparison criterion showed that more than 50 chromosomal rearrangements occurred during the karyotypic evolution of the diurnal birds of prey studied here (Fig. 6).
In addition to using the outgroup comparison, there is a more direct method for inferring the GC content of the common ancestor of D. melanogaster and D. willistoni; that is to calculate the GC content of the conserved sites, that is, those sites which have remained unchanged since the time of species divergence.
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To infer the ancestral categories of the AIS subfamilies, we used the outgroup comparison method [ 19], which assumes that the categories shared between the ingroup and outgroup are relatively primitive with respect to those found only in the ingroup.
To test these two hypotheses, we rooted gene sequence comparisons using the outgroup S. franciscanus (Biermann et al. 2003).
We rooted the mtDNA phylogeny using the outgroup (Plutella porrectalla).
This is illustrated using the outgroup genes hIL10 and TnIL10.
The lone exception comes when we use the lamprey as the outgroup comparison for the N-terminal region of the protein.
An asterisk * denotes the use of T. vivax as the outgroup comparison, in place of T. cruzi.
The latter study [ 10] used the outgroup approach to distinguish between exon birth and loss, and considered an exon present in the human genome but absent in the comparison (say, mouse) genome and the outgroup genome to be a new human exon created after branching out of the comparison genome (similarly for the exon loss).
Secondly, since only a single D. pseudoobscura sequence is used for outgroup comparison, some of the inferred divergence includes undetected segregating polymorphism, which could also result in an upward bias in the estimation of α [ 8].
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