Items with insufficient variation across response options were considered for deletion.
The trees were constructed with the following settings: Tree Inference as Neighbor-Joining; Include Sites as pairwise deletion option for total sequences analysis and complete deletion option for each class analysis; Substitution Model: Poisson correction; and Bootstrap test of 1,000 replicates for internal branch reliability.
NJ tree was based on the distance calculation with JTT matrix [ 46] after removing position containing gaps (complete deletion option) for genes other than Zic genes, or after removing only in pairwise sequence comparisons (Pairwise deletion option) for Zic genes.
Alignment gaps were treated by choosing pairwise deletion option for the NJ method.
Phylogenetic trees were calculated in MEGA with the neighbor-joining method and with the pairwise deletion option for gaps.
When we used the pairwise deletion option for neighbour joining (NJ) in MEGA4 we obtained the same tree topology.
Phylogenetic analyses were conducted using the Neighbour-Joining method with the pairwise deletion option for handling alignment gaps and the Poisson correction model for distance computation.
Analysis was conducted with complete deletion option for gaps and missing data and with the Poisson correction model for distance computation.
UPGMA trees were constructed with MEGA (version 1.0; [ 14]) using p-distances and the pair-wise deletion option for missing data.
Phylogenetic and molecular evolutionary analyses were conducted using the neighbor-joining (NJ) method [ 30] implemented in MEGA, with the pair-wise deletion option for handling alignment gaps, and the Poisson correction model for computing distance.
NJ algorithm was based on the number of amino acid substitutions per site with the Poisson-correction distance method and pairwise-deletion option for gap sites, and bootstrap support values were obtained with 5,000 pseudoreplicates.
