Origin of GPCRs red (Rhodopsin) and green (cAMP) rectangular boxes respectively. The text above the alignment denotes the transmembrane (TM) passage, intracellular loops (ICL) or extra-cellular loops (ECL). Schematic representation on the left indicates the evolution of major eukaryotic lineages from a unicellular common ancestor. Nodes defining relationships across the eukaryotic tree are marked with dotted circles (black), common eukaryotic ancestor (green), the same representation applies to Figure 4 and 5. The dotted lines pointing to the sequence id indicate to which phylum it belongs to, within the Fungi kingdom. The overall schematic representation of the eukaryotic tree was adapted from [17]. doi:10.1371/journal.pone.0029817.g001 Rhodopsin family members from T. adhaerens, which belong to the most basal lineage of Metazoa and therefore containing the evolutionarily most distant members of the Rhodopsin family (Figure S2). Both these preliminary trees demonstrated that the Fungi Rhodopsin family members cluster together with more than 90% posterior probability (PP) and clearly separate the cluster from its homologues in humans and T. adhaerens, respectively. Further, the 8 sequences (identified as Rhodopsin family members in Pfam search) that do not have the D/ERY motif and instead have motifs corresponding to the cAMP receptor family, cluster (PP.95%, 367/500) separate from the Rhodopsin family members identified in Fungi. Based on this inference, a separate phylogeny was constructed that included all newly identified homologues of the cAMP receptor family and the Rhodopsin family with the representative sequences from previous repertoires of both the families (Figure 2). The unrooted tree demonstrated two clusters, one grouping the cAMP receptor family and the other grouping the Rhodopsin family. All newly identified cAMP receptor family sequences in Fungi, Alveolata, Metazoa, Choanoflagellata and the representative sequences in D. discoideum and homologues from plants cluster together with a posterior probability greater than 90% (466/500). Furthermore, the tree demonstrated that the ancient cAMP receptor family members identified from Alveolata (ciliates) cluster together (PP.80%) with the cAMP receptors from plants. The 8 sequences identified as Rhodopsin family in Pfam search cluster with the cAMP receptor family homologues. Of the 8 sequences, Q23WQ7_TETTS and XP_001447229.1 from ciliates were placed in the branch with the other cAMP receptor family members from ciliates (PP.90%) and D. discoideum (PP.70%), respectively (Figure 2). The rest of the sequences were from Fungi (BDEG_02994, FOXG_08466, FVEG_06210, FGSG_05239, B6HSY3_PENCW) and unambiguously clustered with the putative cAMP receptors from Fungi (.80%) with the exception of Q4P8Z0_USTMA, which was placed basal to the node clustering all cAMP receptor family sequences. Instead, we classified the sequence Q4P8Z0_USTMA as a member of the cAMP receptor family based on the characteristic NxY motif in TM3 (Figure 1). All the Rhodopsin family members in Fungi cluster (PP.90%) with the sequences in human. However, the sequences in Fungi form a separate cluster (PP.90%) that is distinct from the human sequences, which are representative for 13 Metazoan subgroups of the Rhodopsin family. Scatter plot. The similarity between the Rhodopsin (PF00001) and the Dictyostelium cyclic AMP receptor: Dicty_CAR (PF05462) was tested using the HMM models downloaded from Pfam database. A scatter plot on a logarithmic scale was made using the e-value for each sequence (used in the phylogeny) aligned with both Rhodopsin and Dicty_CAR HMM models using HMMSEARCH (Figure 3). The plot demonstrates that there are uncertain sequences which receive very similar scores but were classified as Rhodopsin family based on a negligible difference in e- value which over scored the Dicty_CAR HMM model. However, based on the strong support from phylogeny and sequence motifs, we classified these sequences as cAMP receptor homologues. The Adhesion receptor family The Adhesion family of GPCRs is the second largest according to the GRAFS GPCR classification with 33 members in humans [3]. The family is characterized with long N-termini containing multiple functional domains and having numerous sites for glycosylation events [1]. Comprehensive phylogenetic analysis on the TM regions of human Adhesion receptors classified the family into 8 main groups (I–VIII). The phylogenetic classification was also supported by the composition of the functional domains in the N-termini which differs between the subgroups. Novel homologues. This study identified 32 novel Adhesion family members in Fungi. They are distributed as 30 sequences within 22 species of the phylum Ascomycota and 2 sequences in A. macrogynus from the phylum Blastocladiomycota. Also, 8 sequences in S. rosetta, 7 in C. owczarzaki and 1 in P. tetraurelia were found. Interestingly, the Adhesion family members were not found in the phylum Basidiomycota despite being grouped with the phylum Ascomycota, which has 30 members of the Adhesion family. Conserved features. The 48 novel Adhesion family members found in major eukaryotic lineages shared motifs of the Adhesion family common to known Metazoan sequences. The common motifs/conserved residues were adapted from our previous study on the overall relationship between the GPCR families [8]. Residues cysteine (C) in ECL 1 & 2, tryptophan (W) in TM3, proline (P) in TM4 & 5 and glycine (G) in TM7 were found to be .90% conserved between all eukaryotic lineages in which the Adhesion family receptors were identified. Phylogenetic analysis. A phylogenetic tree of the Adhesion family was built with the novel members of the family identified from Fungi, Choanoflagellata, Filasterea and Alveolata with the formerly classified sequences from human (Group I–VIII and VLGR1) and representative sequences from C. intestinalis, B. floridae, N. vectensis and T. adhaerens, which have key evolutionary position within the Metazoan lineages (Figure S3). The phylogenetic analysis revealed that the Adhesion family sequences from Fungi (Ascomycota and Chytridiomycota) clustered separately (PP 99%, 443/500) indicating that were not orthologous to any of the Metazoan receptors, though they have diverged from a common ancestor to all Metazoan and Fungi genes of the Adhesion family. Within the largest cluster of Fungi Adhesion family members, the sequences from the phylum Ascomycota split into two groups. Furthermore, the sequences from the phylum Blastocladiomycota (AMAG_09540, AMAG_ 13158 from A. macrogynus) clustered with the group of Ascomycota. Interestingly, the lone representative (XP_001450983) of the Adhesion family from T. thermophila (Alveolata) clustered with the Adhesion family members in Fungi with a posterior probability of .95% (272/500). The novel putative members of the Adhesion family from S. rosetta and C. owczarzaki clustered with the representative sequences of the Adhesion family from the Metazoan lineage, as they all share a common origin. VLGR1 from human and its orthologue in N. vectensis (NV_242264) were placed basal to the node that clustered the Metazoan sequences. N-terminal domain architecture. The N-terminal domains of all newly identified Adhesion family members were searched and presented together with abbreviations in Figure 4. The N-terminal PLoS ONE | www.plosone.org 4 January 2012 | Volume 7 | Issue 1 | e29817