Spectral tuning of the long wavelength-sensitive cone pigment in four Australian marsupials

Abstract

The molecular basis for the spectral tuning of longwave-sensitive (LWS) visual pigments in mammals have been described in a wide range of placental species, including the primates. However, little is known about the molecular mechanisms in marsupial LWS pigments. Here, we have studied and compared the LWS opsins in four Australian marsupials: two diprotodonts and two polyprotodonts. Phylogenetic analysis establishes that all LWS marsupial sequences form a distinct clade from the placental mammals that is subdivided into diprotodont and polyprotodont groups. Amino acid sequences reveal that substitutions at sites 277 and 285 are largely responsible for the spectral shifts in marsupial LWS pigments and species comparison indicates that the ancestral gene most likely encoded Tyr277 and Ala180. Amino acid substitutions are discussed in the context of spectral shifts in marsupial LWS and in relation to the mechanisms in primate pigments.

Introduction

Most placental mammals possess dichromatic colour vision based on two opsin genes encoding a shortwave-sensitive pigment with peak absorbances (λ_max) varying from around 360 nm to 430 nm (reviewed in Hunt et al., 2001) and a longwave-sensitive (LWS) pigment with λ_max varying from approximately 510 to 565 nm (Dartnall et al., 1983, Bowmaker et al., 1991, Sun et al., 1997). In Old World primates however, trichromacy is present as a result of a duplication of the LWS gene to give “red” (L) and “green” (M) variants that specify pigments with λ_max around 563 and 535 nm respectively (Dartnall et al., 1983, Bowmaker et al., 1991). The λ_max of a pigment is determined by interactions between the opsin protein and the chromophore to which it is linked (reviewed in Bowmaker and Hunt, 1999) and the molecular basis for the spectral shift between the L and M pigments is well established. The key amino acid differences that are responsible for pigment tuning are at sites 180, 277 and 285, with polar Ser180, Tyr277 and Thr285 in the L pigment and non-polar Ala180, Phe277 and Ala285 in the M pigment (Merbs and Nathan, 1992, Asenjo et al., 1994). Variation at the same sites also underlies the spectral differences between the different LWS alleles in New World primates (Neitz et al., 1991, Williams et al., 1992). In addition, Yokoyama and Radlwimmer, 1999, Yokoyama and Radlwimmer, 2001 have proposed that the ancestral mammalian LWS pigment was red-shifted in λ_max with Ser180, Tyr277 and Thr285 respectively.

Recent microspectrophotometric studies of photoreceptors in Australian marsupials have revealed the presence of two longwave-sensitive photoreceptor classes, thereby providing the potential for trichromacy in these species (Arrese et al., 2002, Arrese et al., 2005a). The pigment in one class is shortwave-tuned to 502–509 nm and has yet to be identified at the molecular level whereas the pigment in the other class shows a range of λ_max values similar to those seen in Old and New World primates. Amino acid sequence identifies this latter pigment as encoded by the orthologue of the LWS gene in placental mammals (Deeb et al., 2003, Strachan et al., 2004) but whether the same residues are utilised for these shifts is presently unknown.

In this paper, we present the LWS opsin coding sequences for four species, the quokka (Setonix brachyurus), Western pygmy possum (Cercartetus concinnus), numbat (Myrmecobius fasciatus) and quenda or bandicoot (Isoodon obesulus). The first two species are diprotodonts and the latter two are polyprotodonts; these species represent therefore the major marsupial taxonomic subdivision. For the quokka and quenda, microspectrophotometric measurements indicate a 13 nm difference in λ_max of their LWS pigments. We discuss the causative substitutions for the spectral shifts in these pigments in the light of known mechanisms in primate M and L pigments.