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Correspondence were isolated by 5′- and 3′-RACE- species Petromyzon marinus and PCR and the complete coding Lampetra japonica are most sequence of each gene was closely related to the G. australis reconstructed. The sequence of RhA opsin gene, rather than to the Ancient colour the G. australis opsin genes was gnathostome Rh1 gene, with vision: multiple confirmed by amplification of which they have previously been each cDNA as a single fragment grouped [5]. We therefore suggest opsin genes in the using a proof reading DNA that the northern hemisphere polymerase. The sequences of lamprey genes be renamed P. ancestral the PCR primers and sequence marinus RhA opsin and L. japonica alignments of the five G. australis RhA opsin. vertebrates opsin genes are provided as Other investigators have supplementary material (Genbank speculated that the ancestors of Shaun P. Collin1*, Maree A. accession numbers: all vertebrates must have Knight1, Wayne L. Davies1, Ian C. AY366491–AY366495). possessed the five major types of Potter2, David M. Hunt3 and Ann The five G. australis opsin cDNA opsin genes: LWS, SWS1, SWS2, E.O. Trezise1 sequences were compared with Rh1 and Rh2 [5]. However, this the major groups of opsin genes study is the first to demonstrate Molecular investigation of the from a broad range of unequivocally that the molecular origin of colour vision has representative vertebrate species. events giving rise to four of the discovered five visual pigment A codon-matched nucleotide five classes of vertebrate opsin (opsin) genes, all of which are sequence alignment provided the genes must have already taken expressed in an agnathan (jawless) basis for estimating the place by the early Cambrian fish, the lamprey Geotria australis. evolutionary distances between (approximately 540 mya [1]), prior Lampreys are extant the gnathostome (jawed to the evolution of the jawed representatives of an ancient group vertebrate) and G. australis opsins vertebrates. Uniquely, our results of vertebrates whose origins are and the neighbour-joining method suggest that the duplication of the thought to date back to at least the [4] was used to infer phylogenetic ancestral Rh gene, to give rise to early Cambrian, approximately 540 relationships. The resulting the true rod (Rh1) and the green million years ago [1]. Phylogenetic phylogenetic tree (Figure 1C) cone (Rh2) opsins of the jawed analysis has identified the visual shows that the G. australis LWS, vertebrates, occurred specifically pigment opsin genes of G. australis SWS1 and SWS2 opsin genes are within the gnathostomes and as orthologues of the major orthologous to the LWS, SWS1 independently of the agnathan classes of vertebrate opsin genes. and SWS2 opsin genes of the lineage. This finding provides Therefore, multiple opsin genes jawed vertebrates, with the insight into previous work, which must have originated very early in branching order within each gene identified rod-like photoreceptors vertebrate evolution, prior to the group largely following the in some jawless fishes. However, separation of the jawed and accepted order of appearance of these rod-like cells also show jawless vertebrate lineages, and the vertebrate species. The two some morphological [6,7] and thereby provided the genetic basis remaining G. australis opsin genes, physiological [8] characteristics of for colour vision in all vertebrate RhA and RhB, are most closely cone photoreceptors. As this species. related to the gnathostome Rh1 study suggests that the true rod The southern hemisphere and Rh2 genes. However, both Rh1 gene did not appear before lamprey Geotria australis (Figure RhA and RhB are equally distantly the evolution of the jawed 1A,B) possesses a predominantly related to the gnathostome Rh1 vertebrates, the agnathan lineage cone-based visual system gene as they are to the Rh2 gene. probably does not possess a true designed for photopic (bright This suggests that the most recent rod photoreceptor and retains light) vision [2,3]. Previous work common ancestor of the jawed cone-like features in all identified multiple cone types and jawless vertebrates most likely photoreceptors. suggesting that the potential for possessed a single ancestral Rh Therefore, the ancestral colour vision may have been opsin gene, as well as LWS, SWS1 vertebrates possessed four major present in the earliest members of and SWS2 opsin genes. After the types of vertebrate cone opsins, this group. In order to trace the separation of the agnathan and each presumably sensitive to molecular evolution and origins of gnathostome groups, two different parts of the spectrum. vertebrate colour vision, we have independent gene duplication Although the selection pressure examined the genetic events occurred, one in the underlying the origins of colour complement of visual pigment Agnatha to yield the RhA and RhB vision is still unknown, the early opsins in G. australis. opsins, and the other in the jawed vertebrates lived in a shallow We used RT-PCR with a set of vertebrates to produce the Rh1 water environment [9,10], where a degenerate primers to amplify (rod) and Rh2 (green cone) opsin wide light spectrum could be and clone partial cDNA genes. Our analysis also shows exploited by the evolution of sequences from each of the G. that the previously isolated multiple visual pigments. The australis opsin genes. rhodopsin sequences from the presence of multiple cone opsins Overlapping cDNA sequences northern hemisphere lamprey provides the potential for colour Magazine R865

References 1. Shu, D.G., Morris, S.C., Han, J., Zhang, Z.F., Yasui, K., Janvier, P., Chen, L., Zhang, X.L., Liu, J.N., Li, Y., et al. (2003). Head and backbone of the early Cambrian vertebrate Haikouichthys. Nature 421, 526–529. 2. Collin, S.P., Potter, I.C., and Braekevelt, C.R. (1999). The ocular morphology of the southern hemisphere lamprey Geotria australis Gray, with special reference to optical specializations and the characterisation and phylogeny of photoreceptor types. Brain Behav. Evol. 54, 96–111. 3. Collin, S.P., Hart, N.S., Shand, J., and Potter, I.C. (2003). Morphology and spectral absorption characteristics of retinal photoreceptors in the southern hemisphere lamprey (Geotria australis). Vis. Neurosci. 20, 119–130. 4. Saitou, N., and Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. 5. Yokoyama, S. (2000). Molecular evolution of vertebrate visual pigments. Prog. Retin. Eye Res. 19, 385–419. 6. Dickson, D.H., and Graves, D.A. (1979). Fine structure of the lamprey photoreceptors and retinal pigment epithelium (Petromyzon marinus L.). Exp. Eye Res. 29, 45–60. 7. Collin, S.P., and Potter, I.C. (2000). The ocular morphology of the southern hemisphere lamprey Mordacia mordax Richardson with special reference to a single class of photoreceptor and a retinal tapetum. Brain Behav. Evol. 55, 120–138. 8. Govardovskii, V.I., and Lychakov, D.V. (1984). Visual cells and visual Figure 1. pigments of the lamprey, Lampetra The head (A) and suctorial mouth (B) of the southern hemisphere lamprey Geotria aus- fluviatilis L. J. Comp. Physiol. [A] tralis (reproduced from [3] with permission from Cambridge University Press). Scale bars, 154, 279–286. 1mm (A), 10 mm (B). (C) Phylogenetic tree showing the relationships between the opsin 9. Parker, A.R. (1998). Colour in genes of G. australis, the northern hemisphere lampreys Lampetra japonica and Petromy- Burgess Shale animals and the zon marinus, representative jawed vertebrates and an invertebrate outgroup. The black effect of light on evolution in the vertical line indicates the predicted genetic complement of opsins present in the most Cambrian. Proc. R. Soc. Lond. B recent common ancestor of the jawed and jawless vertebrates, approximately 540 million Biol. Sci. 265, 967–972. years ago [1]. A codon-matched nucleotide sequence alignment was used to estimate 10. Maximov, V.V. (2000). evolutionary distances between sequences and the tree was generated using the neigh- Environmental factors which may bour joining method [4]. The number at each branch point reflects the robustness of that have led to the appearance of branch point (maximum 100). The scale bar is calibrated in nucleotide substitutions per colour vision. Philos. Trans. R. site. Details of the sequences and methods are provided as Supplemental Data. Soc. Lond. B Biol. Sci. 355, 1239–1242. discrimination and thus a dependent on the evolution of the significant selective advantage for Rh1 opsin gene probably only 1School of Biomedical Sciences, The these early vertebrates. Based on within the jawed vertebrate University of Queensland, Brisbane 2 our findings, we suggest that lineage. 4072, Queensland, Australia. School of Biological Sciences and Biotechnology, photopic (bright light, cone-based) Murdoch University, Murdoch, Western colour vision originated first in the Supplemental Data Australia 6150, Australia. 3Institute of ancestral vertebrates, while full Supplemental data are available at Ophthalmology, University College scotopic (dim light, rod-based) http://www.current- London, Bath Street, London EC1V 9EL, vision developed later, being biology.com/supplemental UK.*E-mail: [email protected]