ing the E-INS_I version (optimal for sequences with conserved motifs and carrying multiple domains) with default parameters [44]. All the alignments and the phylogenetic analyses were constructed based only on the seven transmembrane spanning regions and, for the phylogenetic analysis, unaligned regions were removed. Consensus sequences for each of the subgroups reported in Figure 1 were generated from all available human GPCR sequences for each of the subfamilies. The sequences were obtained from our human GPCR repertoire [4]. Similarly, a consensus sequence for the cAMP receptor family homologues in Fungi (Figure 1) were generated from the identified sequences in this study. First, the sequences that belong to each of the subgroups were aligned and separate HMM profiles were built from those alignments. Each HMM profiles that are respective to the groups serves as an input for the HMMEMIT program and a consensus sequence were obtained using option ‘‘-C’’ as implemented in the HMMER3 package. The consensus sequence is formed using a plurality rule that selects the maximum probability residue at each match state from the HMM profiles. The alignments were inspected and edited using Jalview. The phylogenetic analysis was performed using a Bayesian approach as implemented in MrBayes version 3.1.2 [45]. Markov Chain Monte Carlo (MCMC) analysis was used to approximate the posterior probabilities of the trees. Analysis was run using a gamma shaped model for the variation of evolutionary rates across sites (rates = gamma) and the mixed option (aamodelpr = mixed) was used to estimate the best amino acid substitution model. Each analysis was set to run for 3000000 generations and every hundredth tree was sampled. A stop rule was applied to determine when to terminate the MCMC generations as recommended in the MrBayes manual (standard deviation of split frequencies ,0.01). If the MCMC analysis doesn’t hit the stop value within the default number of generations, additional generations were run for it to reach the minimum. The first 25% of the sampled trees were discarded (burnin = 0.25) to reassure a good sample from the posterior probability distribution. A consensus tree was built from the remaining 75% of the sampled trees with the MrBayes sumt command using the 50% majority rule method. The sump command was used to control so that an adequate sample of the posterior probability distribution was reached during the MCMC procedure. The topology of all the phylogenetic trees supported by the posterior probability (PP) of the Bayesian approach was cross verified with bootstrap analysis (500 replicates) using maximum likelihood (ML) approach as implemented in PhyML (version 3.0) program [46]. Bootstrap values were indicated for the nodes that received good support in ML approach. The phylogenetic tree was drawn in FigTree 1.3.1 (http://tree.bio.ed.ac.uk/software/ figtree/). Domain search The N-terminal domains for the identified Adhesion and Glutamate family members were identified with Pfam search and also verified using RPS-blast with a cutoff e-value of 0.01 against the Conserved Domain Database (CDD) version 2.29 position- specific scoring matrixes (PSSMs). Supporting Information Dataset S1 Amino acid sequences of all novel sequences identified in this study in standard FASTA format. (TXT) Accession numbers of novel sequences identified in Table S1 this study. (XLS) Origin of GPCRs Table S2 (DOC) List of species investigated. Table S3 Accession numbers of all Fungi specific GPCRs identified in this study. (XLS) Figure S1 Phylogenetic relationship between the Rhodopsin family sequences in Fungi and human. (TIF) Figure S2 Phylogenetic relationship between the Rhodopsin family sequences in Fungi and T. adhaerens. (TIF) Figure S3 Phylogenetic relationship between the novel Adhesion family sequences in Fungi, Filasterea, Choanoflagellata and Alveolata with the representatives from Metazoa. The tree is rooted with D. discoideum. The node that is highlighted in red clustered the Adhesion receptor from Alveolata with the fungal members (PP.90%). They share about 50% identity within the 7TM regions (see Figure S8). (TIF) Figure S4 Alignment of the Glutamate family sequences in diverse eukaryotic lineages. The alignment shows the conserved regions within the 7TM region between the novel sequences in diverse eukaryotic lineages and human. The consensus sequence for each species was obtained from separate alignments. The number of sequences aligned to emit the consensus sequences for each species is given in the parenthesis. Regions which show .50% conservation are highlighted. (TIF) Figure S5 Phylogenetic relationship between the Glutamate family sequences in Fungi, Choanoflagellata, Porifera and Alveolata with human. (TIF) Figure S6 Alignment of the novel Frizzled receptor sequences in Fungi and Porifera with the representative consensus sequences of the Frizzled receptors from human, N. vectensis (NV), T. adhaerens (TA) and D. discoideum (dicty). Regions which show .50% conservation are highlighted. Multiple cysteine residues that are characteristic for the Frizzled family are mostly conserved in the novel sequences. (TIF) Figure S7 Phylogenetic relationship between the Frizzled family sequences in Fungi and sponges (Porifera) with the representatives from Metazoa. (TIF) Figure S8 Alignment showing the conservation in the 7TM region between the Adhesion receptor sequences in Fungi and Alveolata. Regions which show more than 50% conservation are highlighted. (TIF) Acknowledgments We thank Dr. Michael Williams, Uppsala University for valuable comments on the manuscript. Author Contributions Conceived and designed the experiments: AK MSA. Performed the experiments: AK MSA. Analyzed the data: AK MSA. Contributed reagents/materials/analysis tools: RF HBS. Wrote the paper: AK MSA RF HBS. PLoS ONE | www.plosone.org 14 January 2012 | Volume 7 | Issue 1 | e29817