Reconstruction of gene gain and loss during microsporidian evolution was performed using the phylogenetic gain loss-duplication model employed in the software packagain loss-duplication).
This does not ensure all paths in the graph correspond to LCBs because of genome gain, loss, duplications and rearrangements.
Chromosomal aberrations were identified from log2 CN ratios using the Gain and Loss Analysis of DNA (GLAD) 22. Duplications and deletions were called using log2-thresholds of +0.3 and −0.3, respectively, containing a minimum of 10 consecutive probes.
Further analyses were carried out to search for gene gain and loss events in phylogenetic subtrees using the pattern-matching module in PhyloPattern [ 61].
To gain deeper insights into the major evolutionary mechanisms that act on mycobacterial evolution, we analyzed the distribution of gene gains and losses within a phylogenetic context using the statistical framework provided by BadiRate (Librado et al. 2012).
Rates of gain, loss, and duplication were drawn from a discretized gamma distribution with four categories.
Using the mapped presence/absence of introns on the phylogenetic tree we calculated there are 105 intron gain events and only four intron loss events.
A reconciled species-domain tree was generated using the Notung program, which offers a unified framework for incorporating duplication-loss parsimony into phylogenetic analysis [ 32].
This final analysis was performed using the 'posteriors' option of Count, which analyzes and integrates several phylogenetic scenarios and calculates rates of gain, loss, expansion and reduction across all branches.
Using GO terms, GO Slim terms, and PFAM domain groupings with less than 500 members, we calculated over-representations within losses, gains, and duplications each of these groupings at each node using the hypergeometric test.
Gains and losses for each cluster were obtained using generalized parsimony with ACCTRAN in PAUP* 4.0b10 (Wilgenbusch and Swofford 2003) using the following costs for events: 10 for ortholog acquisition, 5 for ortholog loss, 1 for the first gene duplication, and 0.2 per copy for all other copy-number variations.
