Frizzled receptor family We establish that the Frizzled family evolved from the cAMP receptor family before the divergence of Unikonts from a common ancestor of eukaryotes. This is supported by our extensive search in genomes that diverged before the eukaryotic supergroup Unikonts, which showed that they are absent in plant kingdom and Alveolata. Our earlier report showed that the Frizzled share an HHsearch homology probability of 99.4% with the cAMP receptor family [8]. However, we cannot trace which of the Adhesion and the Frizzled family diverged first; as they evolved within the same timeframe from the cAMP receptor family (see Figure 6). In addition, here we provided the first identification of 6 novel Frizzled family receptors in Fungi and Porifera. Interestingly, they are present only in the basal fungal lineage Chytridiomycota, which also have Rhodopsin, Adhesion and Glutamate families that all together account to 112 novel receptors. This suggests that Chytridiomycota phylum have remarkable necessity for a broader signaling system than the other 3 major phyla of Fungi that resembles the Metazoan GPCR repertoire. Fungi specific GPCRs Our results suggest that the pheromone sensing receptors (Ste2, Ste3) that are mainly involved in exchanging mating signals across the individuals of the same species, emerged after the split of Dikarya subkingdom. Interestingly, the Chytridiomycota has only Metazoan GPCRs (except the presence of Fungi nutrient sensing GPCRs) while Dikarya, which evolved later, has only fungal specific GPCRs. This could suggest that the basal fungal lineages that had the Metazoan like GPCR signaling system could have evolved later to become fungal specific GPCRs in Ascomycota and Basidiomycota. This observation finds support from our previous report which suggests that these pheromone sensing GPCRs were closely related to cAMP receptors with an HHsearch homology probability of 96.1% [8]. Considering the fact that the Dictyostelium cAMP receptors are involved in sexual chemotaxis and develop- ment [27,28], it is thus tempting to speculate that Dictyostelium cAMP like receptors in Chytridiomycota may perform similar roles in finding mates and that these functions could be analogous to the function of pheromone receptors in Dikarya. Novel mammalian like GPCRs: a possible antifungal drug targets and candidates for deorphanization? GPCRs mediated signaling is known to be involved in fungal pathogenic morphogenesis and mycotoxin production in filamen- tous Fungi [22,29]. Despite the role of GPCRs in fungal pathogenesis, there is no current development of potential antifungal drugs that target GPCRs. There are several recent reports of infectious diseases caused by Fungi that affect humans and wild life [30,31]. To highlight a few, Cryptococcus gattii (Basidiomycota) causes infectious diseases in immunocompromised individuals in United States and B. dendrobatidis that belong to the group Rhizophydiales under the phylum Chytridiomycota is a major cause for the decline of amphibians [32,33]. As novel fungal pathogens are emerging, there is an increasing need to develop novel drugs in this field. One of the possible reasons for the lack of development of the drugs is there are only few fungal GPCRs known and these are not strongly related to the well characterized mammalian like GPCR families that are major drug targets [1,34]. Here, we have identified the first mammalian like GPCRs in the basal fungal phyla Chytridiomycota and Blastocladiomycota, creating an opportunity to delineate potential antifungal targets and aid deorphanization of these receptors by comparison with the well studied mammalian counterparts that serve as targets for 36% of the available drugs. Origin of GPCRs Materials and Methods Genomes and proteomes Complete proteome and genome dataset for 79 fungal species were downloaded. The dataset consists of proteomes for 61 species from UniProt (http://www.uniprot.org), which are distributed in phylum Ascomycota (48 species), Basidiomycota (9 species), Microsporidia (4 species). Other 18 Fungi proteomes were downloaded from FGI endorsed by Broad institute at MIT (http://www.broad.mit.edu/annotation/fgi), which are distribut- ed in Ascomycota (13 species), Basidiomycota (1 species), Zygomycota (1 species) and in Chytridiomycota (3 species). In addition to the fungal proteome dataset, the study investigated proteomes for unicellular species with key evolutionary positions within the eukaryotic tree: S. rosetta [35], C. owczarzaki [36], P. tetraurelia [37] from NCBI (http://www.ncbi.nlm.nih.gov/), and T. thermophila [38] from UniProt. Furthermore, a collection of proteins (3465 proteins from several species) were obtained from NCBI that represents the phylum Porifera, a basal group of animals [39]. Sequence retrieval, mining and removal A) GRAFS families: Full-length Rhodopsin, Adhesion, Glutamate and Frizzled family sequences for representative Metazoan genomes and the cAMP receptor family sequences from D. discoideum and C. intestinalis were retrieved from previously published GPCR repertoires to compare the common motifs of individual families and to perform phylogenetic analysis [3–8]. GPCRs in 79 fungal and the remaining genomes from other eukaryotic lineages were mined using HMM searches performed using the recent version of profile HMM software HMMER3 (http://hmmer.janelia.org/) with the Pfam_scan.pl script available at the Pfam homepage [40]. The Pfam_scan.pl script aligns sequences with the HMMs corresponding to the Pfam domains and only keeps the best aligned Pfam domain for each region. The putative dataset was aligned to the complete Pfam database version 24, which has 11912 families with sensitive HMM models built using HMMER3 software [41]. For the search against the complete Pfam database, the standard settings were utilized as provided by Pfam_scan.pl. We retrieved only the sequences which received the Pfam domains 7tm_1 (PF00001), 7tm_2 (PF00002) 7tm_3 (PF00003), Frizzled (PF01534) and Dicty_CAR (PF05462) from each proteome and classified them into GPCR families based on the Pfam result. The sequences with a 7TM domain predicted to have fewer than six or more than nine transmembrane segments in Phobius [42] were excluded from further analysis in order to reduce the number of incomplete sequences. Furthermore, the program CD-HIT [43] was run within the sequences of each genome separately with 90% cutoff to reduce the size of the dataset. B) Fungi specific GPCRs: Similar to the methodology mentioned above, we retrieved the sequences that received the Pfam domains STE2 (PF02116), STE3 (PF02076), Git3 (PF11710)/Git3_c (PF11970). For nutrient receptors, we constructed an HMM profile from the sequences retrieved from [18]. The HMM profile for the nutrient receptors was then used as a query for the HMMSEARCH program (with strict e-value cutoff of 1e225) to search against our complete proteome dataset analyzed in this study. We identified 619 Fungi specific GPCRs from the 79 fungal genomes analyzed in this study. The accession numbers for all the identified sequences were given in Table S3. Alignments and phylogenetic analysis The selected GPCR candidates were aligned using MAFFT version 6 (MAFFT, (http://mafft.cbrc.jp/alignment/server/) us- PLoS ONE | www.plosone.org 13 January 2012 | Volume 7 | Issue 1 | e29817