Metazoan opsin evolution reveals a simple route to animal vision

Results

Common problems with previous studies (1⇓⇓–4) were the use of under-sampled data sets, substitution models that did not fit the data [precomputed empirical time reversible (GTR) matrices], and inadequate outgroup selection (as detailed earlier). To avoid such problems, we assembled three GPCR and opsin alignments scoring hundreds of sequences (Methods), and estimated alignment-specific GTR matrices. Our matrices differ from available, precomputed GTR matrices (SI Appendix, Table S2 and Fig. S2), with the Akaike information criterion and Bayesian cross validation showing that they fit the data significantly better than any precomputed GTR matrix, and at least as well as any precomputed site-heterogeneous model (SI Appendix, Tables S3 and S4).

Fig. 1A represents the phylogeny derived from our all opsin master (AOM) alignment (Methods). AOM includes only neuralian opsins (no outgroups), and Fig. 1A is thus an unrooted phylogeny of our opsin data set (SI Appendix, Table S1). Fig. 1A (see also SI Appendix, Fig. S3) is consistent with the monophyly of the traditionally recognized bilaterian opsin subfamilies (C, R, and Go/RGR). In contrast, the Cnidarian opsins are split into three clans (hereafter referred to as groups A, B, and C). This is in agreement with the results of Suga et al. (4), but in disagreement with others (1⇓–3). Group A includes only two sequences and sits on the branch separating the R opsins from all the other sequences in our dataset [posterior probability (PP) of 0.84]. The sequences in group A are from the study of Suga et al. (4), in which they were named group 3. These sequences were not included in the other three studies (1⇓–3). Group B forms a relatively poorly supported clan with the Go/RGR opsins (PP = 0.69), whereas group C is found in a polytomy with the C opsins and the Go/RGR plus group B clans (Fig. 1A). Group C includes both the sequences that, in the study of Suga et al. (4), emerged as the sister group of the R opsins (their group 2 opsins) and the single sequence that Plachetzki et al. (1) classified as a C opsin. The phylogeny shown in Fig. 1A rejects the possibility that Suga et al.’s (4) group 2 opsins could be related to the R opsins. However, it could neither confirm nor reject the C opsin nature of Plachetzki et al.’s (1) putative C opsin. This is because Fig. 1A shows that all the aforementioned sequences belong to group C: a group that could not be placed with confidence with reference to the C and the Go/RGR plus group B opsins.

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Fig. 1.

Phylogeny of the opsin family. (A) Unrooted phylogeny of the neuralian opsins. (B) Rooted phylogeny of the neuralian opsins and of other GPCRs showing that the Placopsins are members of the opsin family (Ore, orexin; Lys, lysosphingolipid). (C) Opsin phylogeny rooted by using only the MLT receptors, and showing that cnidarians have orthologues of each bilaterian opsin subfamily: the C, R, and Go/RGR subfamilies. Support values (Bayesian PPs) are reported only for key nodes (SI Appendix shows all support values). The ancestral RBD of the LOCA and of the LOCNA are reported and are identified, respectively, by a black star and a black circle (SI Appendix, Fig. S7). The red position in the logos identifies position 296.

Posterior predictive analysis (SI Appendix, Table S5) showed that some of the sequences in AOM were compositionally heterogeneous. Because of their skewed amino acid composition, these sequences can mislead phylogenetic analyses (27). Heterogeneous sequences were included in AOM for the purposes of testing to which major opsin clan they belong. However, most of these sequences were excluded from further analyses (Methods and SI Appendix) to avoid their potentially biasing effect. Other sequences, such as short expressed sequence tags (ESTs) that, in Fig. 1A, were unequivocally identified as members of one of the opsin clans, were also excluded from further analyses.

We analyzed the GPCR and opsin master alignment (G&OM; Methods) to test what GPCR family is most closely related to the opsin family. These analyses (Fig. 1B and SI Appendix, Fig. S4) shown that the neuralian opsins form a monophyletic group. Importantly, the relationships among the neuralian opsins in Fig. 1B are consistent with those of Fig. 1A. That is, the tree in Fig. 1B is a rooted resolution of Fig. 1A in which the polytomy from which the C opsins, the Go/RGR plus group B opsins, and the group C opsins stem is resolved according to one of its possible resolutions. Fig. 1B also shows that the neuralian opsins are most closely related to a set of placozoan “opsin-like” sequences (PP = 0.98). By turn, the neuralian opsins and the placozoan opsin-like sequences are most closely related to the melatonin (MLT) receptors (PP = 0.89). Fig. 1B shows that both the placozoans and the cnidarians have MLT receptors, and, most importantly, that the placozoan opsin-like receptors are orthologues of the neuralian opsins. This implies that from an evolutionary point of view, the placozoan opsin-like receptors are members of the opsin family, even though they lack a retinal binding domain (RBD) with a K296 residue and might thus be unable to detect light. Neither an opsin nor an MLT receptor could be identified in Oscarella and Amphimedon, and we can thus conclude that both these protein families are eumetazoan specific. This confirms recent results showing that light sensitivity in Amphimedon is mediated by a cryptochrome, rather than an opsin (28). Fig. 1B shows that the MLT-plus-opsin clade is most closely related to a group including the lysosphingolipid and the orexin receptors (albeit with very low support; PP = 0.46; Fig. 1B and SI Appendix, Fig. S4). Oscarella and Amphimedon have sequences belonging to the latter (PP = 0.94; Fig. 1B), further confirming the eumetazoan nature of the opsin family.

We tested whether distant outgroups in the G&OM data set could have caused tree-reconstruction artifacts with reference to the opsin phylogeny. To do so, we analyzed the opsins and outgroups (O&O) alignment (Methods). The MLT receptors are the sole outgroups of O&O, which also include the placozoan opsin-like receptors. The Bayesian O&O phylogeny is reported in Fig. 1C (SI Appendix, Fig. S5), and the O&O maximum likelihood (ML) phylogeny is reported in SI Appendix, Fig. S6. Analyses of O&O confirmed the results obtained using G&OM (compare Fig. 1B vs. Fig. 1C). Both data sets show that the Cnidarian opsins can be classified in three groups (A, B, and C). These groups represent, respectively, the cnidarian orthologue of the bilaterian R opsins [group A; GTR PP = 0.89 and ML bootstrap proportion (BP) under an LG plus Γ model = 62%], the cnidarian orthologue of the bilaterian Go/RGR opsins (group B; PP = 0.81 and LG BP < 50), and the cnidarian orthologue of the bilaterian C opsins (group C; PP = 0.71 and LG BP < 50). ML bootstrap support values for the opsin internal relationships are low. Therefore, we used the approximately unbiased (AU) test (29) to evaluate whether the data, under the best-fitting GTR plus Γ model, can discriminate between alternative opsin phylogenies. The results of the AU test (Table 1) confirm that the data are informative and that the trees in Fig. 1C fit the O&O data set significantly better than the trees of the aforementioned previous publications (1⇓⇓–4).

To provide further insights into opsin evolution, we carried out Bayesian and ML ancestral character state reconstruction of the RBD at key internal nodes. Results of the Bayesian reconstruction are reported as logos in Fig. 1C (SI Appendix, Fig. S7), and indicate that the last opsin common ancestor (LOCA) most likely did not have the key K296 residue (PP for K296 = 0.0034). Instead, position 296 was either occupied by an asparagine (PP for N296 = 0.51) or by a methionine (PP for M296 = 0.37). Absence of K296 in LOCA is confirmed by ML, which suggests with reasonable probability (P) that asparagine was the most likely amino acid in position 296 (P-N296 = 0.81 and P-K296 = 0.054). K296 is necessary to link the chromophore, and our results suggest that K296-mediated chromophore binding was not a feature of LOCA: it evolved within the opsin family. Indeed, even in the case of the last opsin common neuralian ancestor (LOCNA), the Bayesian reconstruction suggests that the RBD might not have had a K296 residue (PP for K296 = 0.15; Fig. 1C). However, ML contradict this result, as it finds a P-K296 value of 0.99. This incongruence leaves the question of occupancy of position 296 in LOCNA unresolved. No matter what the amino acid in LOCNA was, our results strongly suggest that a K296-based RBD was not a feature of LOCA.