BMC Genomics 2009, 10(Suppl 1):S14 http://www.biomedcentral.com/1471-2164/10/S1/S14 FMiagjuoreG1PCR pathways, Gs, Gi/o, Gq/11, G12/13 Major GPCR pathways, Gs, Gi/o, Gq/11, G12/13. peculiar [16]. We also excluded 23 human non-olfactory GPCRs that were not classified by the GRAFS system [14], 11 GPCRs in the delta group of Rhodopsin that were likely misclassified, and several predicted GPCR genes that were no longer supported by current genome annotations. In total, our GPCR data set covered 52 human GPCR families containing 302 human non-olfactory GPCRs. Vertebrate and invertebrate homologues of the human GPCRs were identified and linked to the families (see Methods). Estimating the duplication ages of human GPCRs and the "downstream genes" We estimated the ages of the duplication events of human GPCRs and "downstream genes" using the "nearest neigh- bor clock" approach we introduced in a previous work [2]. Briefly, a phylogenetic tree was constructed for each fam- ily. The age of a divergence event was estimated based on its distances to the nearest bracketing species-split times in the phylogenetic tree under the molecular clock hypothe- sis [17,18]. Shown in Figure 2 was an example of the proc- ess for the Adenosine Receptor A subfamily (ADORA), which is a subfamily in the alpha group of the Rhodopsin class. The ADORA subfamily has four human genes and 25 homologous sequences from other species as distant as fly. We first reconstructed the phylogenetic tree (Neigh- bor-Joining) based on the protein sequences and calcu- lated the linearized tree [18]. Among the four human genes in the ADORA family, there were three duplication events T1–T3 (always one less than the number of human genes, in bifurcate trees), which were marked in the phyl- ogenetic tree in Figure 2. For T3 as an example, the nearest bracketing species-split times were 830 Myrs (million years before present, same below) of the vertebrate-fly split and 430 Myrs of the tetrapod-teleost split. The age of T3 should satisfy (830-T3)/(T3-430) = d1/d2, where d1 was the distance from T3 to the vertebrate-fly split and d2 was the average distance from T3 to the two subordinate tetrapod-teleost splits in the linearized tree. Distances d1 and d2 were directly measurable from the linearized tree. T3 was thus estimated as 487.95 Myrs. The ages of other duplication events were calculated likewise. The duplica- tion ages of all the GPCRs in our dataset was summarized in Additional file 1. For the "downstream genes", the process was essentially the same. The ages were estimated using the "nearest neighbor clock" approach as well for each family and were summarized in Additional file 2. Differences in age distribution between GPCRs and the "downstream genes" We first compared the age distributions of GPCRs and the "downstream genes" in general. We focused on the diver- gence events happened after 1000 Myrs (before present, same below), as we were mainly interested in the evolu- tion of vertebrates. Single homologues in fly were identi- fied in most of the families, suggesting most of the gene families emerged early in evolution and stayed in single copy in the genomes up at least to the fly. As was shown in Figure 3A, however, both GPCRs and the "downstream genes" families had an explosive increase in duplication events which caused strong expansions of the families during the period of about 400–800 Myrs, peaked at around 600 Myrs. This was a time close to the emergence of early vertebrates. This pattern recaptured the general pattern of human gene families expansion that has been shown in one of our previous studies [2]. We have sug- gested that this rapid increase in paralogous genes at the early stage of vertebrate evolution were likely caused by genome duplications. However, the age distributions of GPCRs and the "downstream genes" differed greatly after 400 Myrs. Continued expansion of gene families was only observed for the GPCRs, while few families of "down- stream genes" saw expansion after 400 Myrs. We further examined the age distributions of GPCRs and their direct downstream partners, G proteins. As was shown in Figure 3B, the age distributions of G proteins were close to the "downstream genes" in general, which also saw few dupli- cation events after 400 Myrs. Taken together, we found that GPCRs families did experience more expansion than the "downstream genes" in more recent evolutionary stages when most gene duplications have been shown to be local small-scale duplication events [2]. However, at the time of early vertebrate emergence, when large scale genome duplication happened, both GPCRs and the "downstream genes" experienced similar explosive expan- sions. Page 3 of 10 (page number not for citation purposes)