The HIV-1 group M envelope sequence alignment was retrieved from the Los Alamos National Laboratory (LANL) HIV Sequence Database (http://hiv-web.lanl.gov/). The alignment was reduced to subtype reference sequences (3 strains for each where available), 53 CRF strains (2 strains for each where available) and finally 197 apparently unique recombinants. Recombination was analyzed using the RDP [46] , GENECONV [47] , BOOTSCAN [48] , MAXCHI [49] , CHIMAERA [50] , SISCAN [51] , and 3SEQ [52] methods implemented in the program RDP3BETA30 [53] . Default settings were used throughout except that: (1) only potential recombination events detected by four or more of the above methods, coupled with phylogenetic evidence of recombination were considered significant; (2) sequences were treated as linear; and (3) a window size of 30 variable nucleotide positions was used for the RDP method. Using the approach outlined in the RDP3 program manual (http:// darwin.uvigo.es/rdp/rdp.html), the approximate breakpoint positions and recombinant sequence(s) inferred for every potential recombination event, were manually checked and adjusted where necessary using the phylogenetic and recombination signal analysis features available in RDP3. Breakpoint positions were classified as unknown if they were (1) detected at the 59 and 39 ends of the alignment but could have actually fallen outside the analysed region; or (2) within 20 variable nucleotide positions or 100 total nucleotides of another detected breakpoint within the same sequence (in such cases it could not be discounted that the actual breakpoint might not have simply been lost due to a more recent recombination event). All of the remaining breakpoint positions were manually checked and adjusted when necessary using mainly the MAXCHI and 3SEQ methods (using three sequence scans and the MAXCHI matrix method) but also the LARD matrix method (generated by the LARD two breakpoint scan; [54] ), and the CHIMAERA method as tie breakers. The distribution of unambiguously detected breakpoint positions of all unique recombination events were analysed for evidence of recombination hot-and cold-spots with RDP3 as described by Heath et al. ([44] ; a window size of either 50 or 200 nucleotides and 10 000 permutations). A normalised version of the breakpoint distribution plot described in that study was used in which the local probability values of breakpoint numbers (determined by a permutation test that takes into account that local degrees of sequence diversity influence the delectability of recombination events) were plotted instead of absolute breakpoint numbers.