Ligase secondary structure and bursts sampled from two lineages of continuous in vitro evolution. Two lineages were evolved from a homogeneous population of 100 molecules of B16-19 ligase ribozyme (A). The secondary structure of the B16-19 ribozyme is shown in orange, in which the primer binding site for reverse transcription (3' end) and the 5' end of the promoter for forward transcription (5' end) during CE are denoted by solid rectangles. The ribozyme effects the ligation of an external substrate oligomer (black lowercase letters) that contains the T7 RNA transcriptase promoter sequence, needed for CE, to its own 5' end. The ligase ribozyme catalyzes the attack of the 3'-hydroxyl group of the substrate onto the 5'-α-phosphate of the ribozyme (dashed arrow). Mutations that occurred during CE lineage that produced new quasispecies centered around the master sequences are shown, following the color scheme used throughout this paper: MS1 (purple), MS2 (blue), and MS3 (green). All evolved master sequences contain the U62A mutation. Genotypic samples of ligases were taken at various serial transfers in both lineages (B). The lineages were started from a homogeneous population indicated by orange. As time passes, mutated forms arise and accumulate in the population generating an increased diversity, indicated by white. Based on RFLP data [Additional file 1: Supplemental Figure S1] the bursts selected for cloning and sequencing are indicated by small arrows: 5, 35 and 50 in lineage 6H; and 22, 23, 39 and 42 in lineage 6L.