Population structure of P. jirovecii msg sequences present in a single infected host. Sequences were aligned and neighbor joining phylogenetic trees constructed as described in the methods using a rat msg sequence (black box; MSG100, GenBank # D31909) to root the tree. Bootstrap analysis (*=bootstrap values≥ 99) identified two large distinct branches (Groups A and B), each of which consisting of several smaller groups of distinct sequences.

Analysis of indels reveals evidence of recombination. Sequences were arranged according to the phylogenetic analysis shown in with groups A and B color coded and with bootstrap values for branches indicated (left). Sequences in the nucleotide region ~1340 and ~2920 with obvious indels (boxed) showed strong but not exclusive segregation according to groups. An insertion at position 1340 in group A was absent in 12/13 of group B sequences, but the same 3 nucleotide insertion was present in one group B sequence (Cl-9). Similar segregation of indels at position 2910 and 2926 revealed strong evidence of recombination between groups A and B.

A. Simplot analysis of msg sequences. Sequences were subjected to Simplot analysis comparing all other msg sequences to sequence Cl-5 of group A. Each line represents a single clone. All group A sequences (blue lines) are relatively closely related to Cl-5 (similarity to Cl-5 score 70-100), while group B sequences (orange lines) diverged sharply between nucleotides ~1200-2300 (similarity to Cl-5 score < 60), suggesting recombination at the divergance site. Similarity scores of group B sequences increased near the end of the msg sequence, suggesting additional recombination events.
B. Bootscanning detects evidence of frequent recombination. msg sequences were subjected to bootscanning analysis using Cl-14 as the query sequence and Cl -7, Cl -21, and Cl -25 as comparator sequences. For simplicity, only the comparisons between Cl-14 and Cl-21 (in black), and that between Cl-14 and Cl-25 (in red) are shown. The X axis depicts the nucleotide position within the msg sequence, and the Y axis, the percent of trees grouping with the query sequence. The number of permuted trees grouping Cl -21 with Cl -14 using a sliding window of 200 nucleotides showed sharp switches in frequency, with demarcations suggesting a recombination event approximately at nucleotides 700 and 1520. In contrast the number of Cl-14 permuted trees grouping with Cl-25 show reciprocal switch in homology at approximately the same locations. Additional potential switches in homology were seen in both comparisons, indicating multiple recombination events.

A: RFLP analysis of msg of P. jirovecii. msg was amplified from DNA prepared from the pulmonary samples of P. jirovecii infected patients (1-6: pulmonary samples collected between 1985 and 1999). By ITS1 and UCS typing, the 6 isolates were respectively E and 3 repeats; E and G and 3, 4, and 6 repeats; E and 6 repeats; E and 4 repeats; E and 3 and 4 repeats; E and 3 repeats. The PCR products were digested with Mbo I, Hind III, or Dra I restriction enzymes and analyzed using 1% agarose gels in 1X TBE buffer and then stained with SYBR green.
B-C. Southern blot analysis of msg of P. jirovecii. The DNA fragments from the gel (RFLP analysis, Fig. 4A) were blotted onto a membrane and hybridized successively with oligonucleotides GK195 (B) and GK576 (C). The blot was stripped between hybridizations. GK195 is from a highly conserved region near the 3’ end of msg and GK576 is specific for a single msg clone (msg 32).
D. Southern blot analysis of DNA from P. jirovecii-infected pulmonary samples. Genomic DNA extracted from the pulmonary samples of P. jirovecii infected patients (1-4) was digested with Mbo I, Hind III, or Dra I restriction enzymes. The blot was hybridized with a probe spanning nucleotides 1576-2974 of HuMSG14.

A: RFLP analysis of msg of P. murina. msg was amplified from DNA prepared from the lungs of infected scid mice collected over a 5 year period (1-6: lungs collected from 1999 to 2004, respectively). The PCR products were digested with the restriction enzymes Hind III or Dra I, analyzed using 1% agarose gels in 1X TBE buffer and stained with SYBR green.
B: RFLP analysis of msg of P. carinii. msg was amplified from the DNA preparations from the lungs of P. carinii infected rats. Lanes 1-4: rats from one facility; lungs were collected from 1998 to 2001; lanes 5-7: rats from a second facility; the lungs were collected from 1993 to 1995. The PCR products were digested with Hind III or Dra I restriction endonucleases, analyzed on 1% agarose gels in 1X TBE buffer and stained with SYBR green.
C. Southern blot analysis of DNA from P. carinii-infected lung samples. Genomic DNA was digested with Hind III or Dra I restriction enzymes. The blot was hybridized with a probe generated by amplifying genomic DNA from P. carinii with primers GK524 and GK526.