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Dataset (Supplemental)
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The supplemental dataset contains information for the source of each sequence, the major lineage
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and subgroup designation based on our phylogenetic analysis, the G-protein binding partner, cell
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type, tissue expression, and counter-ion location, as well as the associated references for each
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piece of information. All of the sequences acquired from genome data represent manually
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curated sequences, which can be found on the UCSC genome browser
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(http://genomewiki.ucsc.edu/index.php/Opsin_evolution:_update_blog) or in the supplemental
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alignment. For records without an associated Genbank accession number (i.e. 123 sequences),
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the source has been listed as ‘Genome’ or ‘EST library’. Because genome assemblies are
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unstable and change as improvements are made, the accessions used at the time of making the
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gene models are transient. Therefore, these labels indicate that the gene model was obtained by a
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BLAST of close homologs to GenBank genomic sequences or ESTs rather than to trace reads or
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transcripts or extracted from published literature. To find the source for these gene models,
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BLAST the sequence supplied in our supplemental alignment against the raw genomic and EST
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data at GenBank for the species in question to find the current assembly, scaffold, contig, trace
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or transcript number. To validate the model for consistency, compare visually to known
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orthologs in our compilation of manually curated sequences
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(http://genomewiki.ucsc.edu/index.php/Opsin_evolution:_update_blog) for exon phase and
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length matching, perform a simple multiple alignment for consistency with orthologs using
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Multalin (http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_multalin.html),
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and examine the gene model for conservation at known conserved sequence motifs. For more
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details on the methods used, see below.
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METHODS (Supplemental)
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Sequence Acquisition
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Opsin sequences were acquired from Genbank using one of two methods. To include sequences
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where expression data are available, opsin transcript sequences were mined from Genbank for
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major taxonomic and known opsin groups using queries of either the nucleotide or the
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‘transcriptome shotgun assmebly’ databases. To generate the dataset of genomically-derived
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opsins, we used conventional transcript-derived entries from GenBank as tBlastn queries against
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the wgs division of GenBank (where all genome assemblies are stored). Because opsins have a
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minimum of ~ 20% protein identity to bovine rhodopsin, as determined by the degree of
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conservation across all GPCRs, and because other opsins in the collection will outperform
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bovine rhodopsin as a query in specific cases, this method has more than enough sensitivity to
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detect even short homologous exons in the bovine rhodopsin K296 region. Thus the complete
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opsin repertoire can be recovered from each species - even new homology classes - provided it is
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present in the assembly. The recovered sequences using this methodology often need curation in
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regions of weak alignment against the original data for a particular species and in the case of
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misassembly stutter. Missing exons were often locatable at the NCBI trace archive in the form of
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isolated reads that were omitted from the assembly, taking into account the risk of gathering
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inappropriate exons from unsuspected gene duplications. Recovered opsin sequences were
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quality checked for the presence of a lysine in the 7th transmembrane helix (bovine rhodopsin site
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296), conservation of invariant opsin residues and motifs, and better back-blast to opsins than
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any annotated non-opsin GPCR at the GenBank nucleotide division. With the exception of
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species too closely related to one already represented in the dataset, all metazoan genomes at
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GenBank as of 1 Dec 2010 are represented by the dataset used in this study. No species
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diverging earlier than the ctenophore (notably neither the sponge Amphimedon queenslandica
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nor the choanoflagellate Monosiga brevicollis) contains a GPCR with a lysine in a position
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alignable with the K296 motif of bovine rhodopsin. A curated set of recovered opsins from
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genome trace files, updated monthly, is available at the UCSC genome browser
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(http://genomewiki.ucsc.edu/index.php/Opsin_evolution:_update_blog).
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For each opsin extracted from a genome project, the location and phase of each intron
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within the coding region was determined by alignment to genomic contigs. All opsins to date
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utilize standard GT-AG splice donors and acceptors. We parsimoniously resolved each
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significant sequence change (i.e. intron change and indels) as a gain or loss by determination of
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its ancestral status via multiple outgroups. In bilaterans, notably the tunicate Ciona and insects
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including Drosophila, very rapid turnover of introns (both gain and loss) occurs; however these
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limitations do not interfere with opsin classification because enough species with conservatively
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evolving introns are available to reconstruct the evolutionary history.
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Phylogenetic Analyses
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For phylogenetic analyses, sequences that spanned less than half of the transmembrane regions
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of the protein were discarded from the analyses, resulting in 889 transcript plus genome trace
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opsin sequences. In order to root our opsin phylogenetic analyses, 22 non-opsin GPCRs from the
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human genome were used as outgroups: somatostatin receptor, opioid receptor mu 3, galanin
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receptor, chemokine (C-C motif) receptor, bradykinin receptor 1, uracil/cys-leukotriene dual
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receptor, cys-leukotriene receptor 1, purinergic receptor, orexin receptor, tachykinin receptor,
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neuromedin U receptor, pyroglutamylated RFamide peptide receptor, human orphan receptor 19,
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pancreatic polypeptide receptor, neuropeptide Y receptor, prolactin releasing hormone, human
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orphan receptor 161, alpha-1D-adrenergic receptor, thyrotropin-releasing hormone receptor,
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thyrotropin receptor, adenosine A3 receptor, and opiate receptor-like 1. This set of sequences
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was selected as outgroups based on previous phylogenetic studies of opsin and GPCR evolution
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(Davies et al. 2010; Fredriksson et al. 2003; Plachetzki et al. 2010 Porter et al. 2007; Suga et al.
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2008) as well as based on a rigorous procedure of BLASTing human opsins against the all other
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human GPCRs (for description of detailed BLAST procedure see:
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http://genomewiki.ucsc.edu/index.php/Opsin_evolution#GPCR_outgroup_sequences).
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Furthermore, rerunning the phylogeny reconstruction without outgroup sequences does not
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significantly change the sequences within each of the major clades nor the relationships among
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them, with the exception of three sequences (Platynereis dumerilii TMT1 and TMT2;
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Stronglocentrus purpuratus encephalopsin) placed at the base of the ‘C-type’ clade when rooted,
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which are placed at the base of the ‘Cnidops’ clade when unrooted (data not shown).
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Amino acid sequences of the 889 opsins mined from Genbank and the 22 human
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outgroup GPCR sequences were aligned using the online MAFFT v6.0 server
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(http://mafft.cbrc.jp/alignment/server/) (Katoh et al. 2005a; Katoh et al. 2005b; Katoh et al.
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2002). The aligned dataset was then trimmed to remove the N- and C-terminal sequences,
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leaving only the transmembrane and loop regions of the protein for further phylogenetic
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analyses. The resulting alignment has been provided as a supplemental FASTA data file.
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The aligned and trimmed dataset was used to reconstruct a maximum likelihood
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phylogeny using Randomized Axelerated Maximum Likelihood (RAxML) v.7.2.7 with rapid
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bootstrapping as implemented on the Cyberinfrastructure for Phylogenetic Research (CIPRES)
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Portal v.3.1 (Miller et al. 2010; Stamatakis 2006; Stamatakis et al. 2008; Stamatakis et al. 2005).
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Using the resulting phylogeny, character mapping of amino acid residues at particular counterion
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sites was accomplished in Mesquite v2.72 (Maddison & Maddison 2010) using unordered
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parsimony reconstruction.
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References
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