Adding A. spinosum sequences to the existing C-terminal dataset did not significantly alter the overall topology present in the N-terminal dataset, despite weaker phylogenetic support.
This theoretical expectation was empirically corroborated very recently using a 218-terminal dataset [ 29].
The terminal.pen2 dataset is expected to contain less false-positive HGT candidates than terminal.pen1 (due to the increased gain/loss penalty ratio, stronger support is required before a gene is considered to result from HGT): as the fraction of false positives decreases, the strength of clustering grows.
Alignments were performed manually with Se-Al v2.0a11 http://tree.bio.ed.ac.uk/software/seal/, and after removal of N-terminal and C-terminal unconserved residues, the dataset contained 56 taxa and 73 characters.
The 0.03 threshold segregates 70% (versus 46% false negative with 0.06) of the terminal divergences in the dataset of Faivovich et al. [69].
For transparency and to avoid circularity we only included actual occurrences of the terminal taxa in the dataset, regardless of whether the material is known to be invasive in the area of occurrence.
No changes in Trx redox status were observed among the cells with endogenous TR1, cells with TR1 knocked-down, and cells with endogenous TR1 knocked-down plus induction of the Sec-deficient C-terminal mutant TR1 (example dataset in Figure 2 and summary of multiple experiments in Table 1).
Three P450s that had single terminals in the 454 dataset, and two P450s derived from our previous cDNA library, were extended by RACE PCR.
To minimize the number of missing entries in the dataset, composite terminals were constructed with individual partitions from different species of a higher taxon, usually a nominal "family" [see Additional File 4].
Based on our findings, we recommend that future studies utilizing BiSSE that have fewer than 300 terminals and/or have datasets where high tip ratio bias is observed (i.e., fewer than 10% of species are of one character state) should be extremely cautious with the interpretation of hypothesis testing results.
