BMC Genomics 2009, 10(Suppl 1):S14 http://www.biomedcentral.com/1471-2164/10/S1/S14 FAinguexraem2ple of how the ages of duplication events were estimated in the ADORA family An example of how the ages of duplication events were estimated in the ADORA family. The neighbor-Joining phylogenetic tree (A) was constructed based on the protein sequences, and the correspondent linearized tree (B) was also cal- culated. The protein sequence distances (mutation per amino acid residue) were marked on the graph as the scales. The dupli- cation events, T1–T3, were marked on the trees, and the age of the duplication events were estimated based on the linearized trees (see Methods). We further examined the age distribution of duplication events of different subclasses of GPCRs to see if they con- tributed equally to the age distribution of GPCRs. Shown in Figure 3C were the age distributions of the GRAFS sub- classes of GPCRs (and the "downstream genes (D)" in a dashed line as reference). The densities were multiplied by the number of genes in each class to reflect the differences in sizes among classes. Different classes of GPCRs contrib- uted differently to the overall distribution of GPCRs. Rho- dopsins, which makes up the majority of GPCRs, had a similar age distribution to GPCRs overall, with the peak slightly moved right to over 600 Myrs. The Secretin and Glutamate receptor classes had only an obvious expan- sion between 400 to 600 Myrs. In contrast, duplication events in the Adhesion and Frizzled/Taste2 receptor classes happened more recently and were mostly after 300 Myrs. Actually, the continued expansion of GPCR families after 400 Myrs were largely contributed by several small classes such as Adhesion and Frizzled/Taste2 receptors and the chemokine receptors of the Rhodopsin class (see Additional file 1 for the actually ages). This result showed that within the GPCR superfamily, and even within the Rhodopsin subclass, the expansion of different sub- families was asymmetric. In a recent report [19], GPCRs with non-peptide ligands were reported to have a significantly higher retention rate than GPCRs with peptide ligands after a lineage-specific whole genome duplications in the pufferfish Tetraodon nigroviridis more than 230 Myrs ago. We examined if the same was true for GPCRs in general. Based on the report [19], GPCRs that bind non-peptide ligands included GPCRs in the Glutamate receptor class and A1-A4 sub- classes of the Rhodopsin class (see Additional file 1), while the rest GPCRs bind peptide ligands. As was shown in Figure 3D, it was obvious that more duplication events were found among the GPCRs that bind peptide ligands than those bind non-peptide ligands in more recent evo- lutionary stages (after 400 Myrs). This was in direct con- trast to the results of the lineage-specific GPCRs in pufferfish by the report [19]. This may have reflected that the selective pressure driving the fixation of different types of duplicated GPCRs were different in different species and at different evolutionary stages. Actually, it was also shown in Figure 3D that, before 600 Myrs, more dupli- cated events were observed in GPCRs with non-peptide ligands than GPCRS with peptide-ligands. This was not anti-intuitive as many of the ancestor species emerged at that evolutionary stage were simple marine invertebrates. Page 4 of 10 (page number not for citation purposes)