To demonstrate the capabilities of RT-PCR/ESI-MS to track the evolution of circulating influenza viruses, we created a tree representation of the H3N2 influenza virus sequences from Genbank ( Figure 3 , black) as described in Methods. The 104 experimentally determined H3N2 base compositions were mapped onto this tree (Figure 3 , blue). Analysis revealed a distribution very similar to the sequence-derived clades of Holmes et al. [12] . The branch terminating in clade A represents the dominant branch between years 1999-2004, with a clade B branch that co-circulates in the same time period. Strikingly, however, clade-A isolates were not detected after this time and Table S3 ) and confirmed the findings described above. A visual display of the most likely relationships among the isolates is shown in Figure 4 . Collectively, these results demonstrate the richness of the genetic information provided by direct RT-PCR/ESI-MS analysis of human clinical materials. Figures 5A and B show the spectra from samples containing a mixture of the ''parent'' BCtype AADFAA and single-nucleotide variations AAHFAA and AADFBB, respectively, providing a snapshot of enduring ''fit'' quasispecies contributors and of potential viral evolution in action.