receptor family [14], was found in the phylum Nematoda (Caenorhabditis elegans) [15]. Further, the cAMP receptor family, predicted to be the ancestor to the main families (Class A, B and F) is present only in invertebrates within the Metazoan lineage [8,16]. Recently, a multi-taxon genome sequencing initiative endorsed by National Human Genome Research Institute (NHGRI) shed light on how multicellularity evolved. The study suggests that the two main eukaryotic kingdoms- Metazoa and Fungi along with unicellular relatives like Choanoflagellata and Filasterea share a common origin or group together under a eukaryotic supergroup known as Opisthokonta [17]. Over the years, GPCR repertoires have been particularly well studied in Metazoa as compared to Fungi. The five main GPCR families have previously not been found in Fungi, despite the presence of Adhesion, Frizzled and Glutamate family members in D. discoideum [8]. Literature reports that GPCR homologues already identified in Fungi can be classified into at least six classes or families that do not belong to the mammalian GRAFS families. The six classes include pheromone receptors sensing peptide pheromones (Ste2), phero- mone receptors sensing lipid modified peptide pheromones (Ste3), nutrient sensors (Gpr1), Stm1-like nitrogen sensors (Stm1), microbial opsins (Nop-1 and Orp-1) and cAMP like receptors [18]. Until today, a few GPCR homologues have been identified in Saccharomyces cerevisiae, Aspergillus nidulans, Cryptococcus neoformans, Magnaporthe grisea and in Neurospora crassa, but they are categorized under the known six classes of fungal GPCRs and not among the GRAFS families [18–21]. GPCRs in Fungi were proposed to have a role in fungal-plant interactions. They are involved in recognizing various signal molecules or ligands from plant cells paving the way for the Fungi to destroy valuable crops [22]. Pathogenic Fungi are also responsible for causing diseases in humans like Histoplasmosis and severe Pneumonia in people with immune system problems [23,24]. Recent advances in sequencing technologies led to a remarkable increase in the number of fully sequenced fungal genomes. The Fungal genome initiative (FGI) endorsed by Broad institute at MIT (http://www.broad.mit.edu/annotation/fgi) and DOE Joint Genome Institute (JGI; http://www.jgi.doe.gov/), sequenced several fungal genomes that are pathogenic to humans and plants. In this study, we have mined for GPCRs in a complete proteome dataset of Fungi from UniProt and over 18 genomes of Fungi that are available at FGI (Broad institute). We have also investigated genomes belonging to eukaryotic lineages like Choanoflagellata (Salpingocea rosetta), Filasterea (Capsaspora owczar- zaki), which share a common origin with Metazoa and from Alveolata (Paramecium tetraurelia and Tetrahymena thermophila). Over- all, we systematically mined for GPCRs in 83 genomes to obtain a nearly complete set of GPCRs in Fungi and unicellular Metazoan relatives. Results Identification of novel homologues of the Rhodopsin, Adhesion, Glutamate and Frizzled like receptors in Fungi and major eukaryotic lineages We searched for GPCRs in a comprehensive protein sequence dataset that comprise proteomes of several species representing the eukaryotic lineages like Fungi (79 species), Choanoflagellata (S. rosetta), Filasterea (C. owczarzaki), Alveolata (P. tetraurelia, T. thermophila) and from collection of proteins (3465 proteins) from several species that are from the phylum Porifera, which is one of the most basal Metazoan lineages. The complete dataset was searched using Hidden Markov Models (HMM) models for the Origin of GPCRs 11912 families of the Pfam database (version 24), which revealed that the characteristic Metazoan GPCR families of the GRAFS classification are wide spread in the eukaryotic domain. This study identified 142 novel sequences distributed among the Rhodopsin, Adhesion, Glutamate and Frizzled families that have not previously been reported in the Fungi kingdom. In S. rosetta, 8 novel members of the Adhesion and 1 member of the Glutamate family were found. In C. owczarzaki, 7 novel sequences of the Adhesion and 20 for the Glutamate family were found. Also, one sole representative was found for the Glutamate family in Porifera. In addition to the GRAFS families, our study identified 57 cAMP receptor family sequences in Fungi, 8 in Alveolata, and more surprisingly 1 cAMP receptor each in Branchiostoma floridae and Lottia gigantea. All identified novel sequences in individual species are listed with their accession numbers (Table S1); FASTA sequences are provided in Dataset S1 and the list of species investigated is given in Table S2. The Rhodopsin and cAMP receptor family The Rhodopsin family of GPCRs constitutes the largest family of GPCRs in vertebrates with 683 members in humans, classified into four main groups, termed a-, b-, c-, and d-group, and 13 major subfamilies [10]. The Rhodopsin family is very well represented in both vertebrates and in invertebrates with the ancient members (343 receptors) was found in T. adhaerens. On the other hand, the cAMP receptor family in D. discoideum is found in plants and in Alveolata [8]. Also, they are present in Fungi kingdom, but found in very few invertebrate species and subsequently lost in the vertebrates within the Metazoan lineages [8,17]. Novel homologues. This study identified 12 novel sequences of the Rhodopsin family in Fungi. However, the sequences were only found in 3 species: Allomyces macrogynus (3 sequences) from Phylum Blastocladiomycota, Batrachochytrium dendrobatidis (1 sequence) and Spizellomyces punctatus (8 sequences) which belong to the phylum Chytridiomycota, known to be a basal fungal lineage and found to have split prior to the Dikarya subkingdom. Similar to the previous reports, our search did not identify Rhodopsin family members in Dikarya that includes the two major phyla Ascomycota and Basidiomycota of Fungi. Further, search for the Rhodopsin family members in Choanoflagellata, Filasterea, Alveolata and Porifera failed to find homologues. Conserved features. The 12 novel Fungi members of the Rhodopsin family share many motifs characteristic for the Rhodopsin family in Metazoa, such as aspartate (D) in TM2, D/ERY in TM3, NP in TM7 and cysteine (C) in ELC1 and 2 (Figure 1). In addition, there are motifs that provide a link between the cAMP receptor family and the Rhodopsin family like: arginine (R)/lysine (K) in ICL1, aspartate (D) in TM2, tyrosine (Y) in TM3 & 5, cysteine (C) in ELC1 & 2. Further, we found 8 sequences which were initially identified as putative members of the Rhodopsin family in the Pfam search. They are distributed as 2 sequences in the phylum Alveolata, 4 in Ascomycota and 1 each in Basidiomycota and Chytridiomycota. These 8 sequences lack the D/ERY motif that is common for the Rhodopsin family but instead has motif NxY that are present among the cAMP receptor sequences (Figure 1). Phylogenetic analysis. The phylogenetic relationships for the Rhodopsin and cAMP receptor family were investigated using the Bayesian approach implemented in MrBayes 3.1.2. Two preliminary trees were constructed: the first tree included the novel Rhodopsin family members identified in Fungi (Chytridiomycota and Blastocladiomycota) with the representative sequences from 13 subgroups of the Rhodopsin family in humans (Figure S1): the second tree included Fungi (12 sequences) and the representative PLoS ONE | www.plosone.org 2 January 2012 | Volume 7 | Issue 1 | e29817