We found that the cone opsins of guppies are highly polymorphic, both within and between populations, resulting in at least two different green-sensitive opsins and two highly differentiated LWS opsin isoforms. While RH1, RH2-1 and SWS1 appear to be single copy genes, LWS is found in at least two copies per individual. The 15 different LWS opsin haplotypes identified from nine strains can encode seven different proteins, which can clearly be grouped into three distinct forms. Only variant 1 was found in populations from both Trinidad and Venezuela, while variants 2 and 3 were restricted to Trinidad and Venezuela, respectively ( and ). The prevalence of non-synonymous substitutions known to change maximum absorbance of visual pigments, along with the high ratio of non-synonymous to synonymous substitutions, suggests strong diversifying selection of these proteins, especially in the functionally important TM domain 4 ().
Our finding of five different cone opsins is compatible with microspectrophotometric (MSP) studies of the guppy retina, which suggest the presence of UV, blue/violet, green and variable red/orange-sensitive cone opsins. These long wavelength-sensitive cones are by far the most polymorphic class, in which three different absorption peaks at 533, 548 and 572
nm can be distinguished (Archer et al. 1987
; Archer & Lythgoe 1990
). Although we found two different RH2 isoforms, MSP data did not indicate the presence of functionally different green sensitive cones, suggesting that the RH2 differences are too subtle to be detected. Alternatively, expression of some visual pigments could be developmentally regulated, as has been found for green opsins in cichlids (Spady et al. 2006
Archer & Lythgoe (1990)
suggested the existence of two LWS opsins with distinct absorption maxima, with co-expression of both causing a third absorption peak, to account for individuals with a single, two or three absorption peaks for long wavelength light. Our data provide a possible alternative explanation, since at least some individuals have three different LWS forms ( and ). The MSP data indicated that about half of the individuals contain two different LWS cones, with most of the other individuals having either one or another. Our genomic data are in broad agreement with this conclusion, as most individuals have a repertoire of LWS genes that should allow for at least two different proteins. While the maximum number of isoforms observed within one individual was four, a quarter of individuals seemed to have only one type of LWS protein (). There was only partial overlap between the populations studied by Archer & Lythgoe (1990)
and the strains included in our survey, and the percentage of individuals with a single LWS opsin could differ between populations.
Our estimates of LWS haplotype number are conservative, although a low frequency of artefacts owing to template switching during PCR cannot be excluded. Several factors may have resulted in an underestimate of the number of haplotypes per individual. For example, we could not directly select for LWS alleles represented by cDNA OR6-3 (variant 1) with allele-specific primers. In addition, point mutations in the regions covered by the consensus primers might have prevented detection of some isoforms.
Long-wave sensitive opsin genes have been compared between cichlid species that have only recently evolved in East African rift lakes (Terai et al. 2002
; Carleton et al. 2005
; Parry et al. 2005
; Spady et al. 2005
). Although DNA blot experiments did not indicate multiple LWS copies, genomic PCR analysis revealed 14 different LWS alleles in cichlids from Lakes Victoria and Nabugabo, with two alleles found in most species (Terai et al. 2002
). In cichlids, differentiation of colour patterns and of the visual system have been associated with adaptation to different habitats (Parry et al. 2005
), and both of these factors have been proposed as essential components of speciation.
The situation in cichlids, in which allelic diversity is found predominantly between species (Terai et al. 2002
), contrasts with the one in guppies, in which eight strains of the same species contain at least 15 different haplotypes encoding seven different proteins, with obvious geographical differentiation. Importantly, much of the variation in LWS opsins occurred within populations and even within individuals in guppies. Guppies can live in different photic environments that are distinguished by degree of transparency and amount of canopy (Endler 1991
), but the main forces driving differentiation of male colour patterns are thought to be sexual selection (Houde 1997
) and predation (Olendorf et al. 2006
). Further detailed analysis of a greater number of specimens from upper and lower ranges of several northern Trinidad river systems might reveal whether a correlation exists between opsin gene pools and male ornamentation. How this colour variation, one of the highest in vertebrates (Haskins et al. 1961
), is maintained continues to be an important question in evolutionary biology (Olendorf et al. 2006
). The colour diversity is due to as many as 40 genes with polymorphic alleles for male coloration, most of which are linked to the sex chromosomes (reviewed in Lindholm & Breden 2002
In contrast to the highly variable patterns of guppies, five distinct male colour morphs coexist in Poecilia parae
and female mate choice may counteract loss of the less frequent morphs (Lindholm et al 2004
). In this context, it would be interesting to know to what extent opsin gene diversification has occurred in closely related poeciliids such as P. parae
Guppies also stand out among species studied for sexual selection in that female preference functions are variable within and between populations (Houde & Endler 1990
; Endler & Houde 1995
; Brooks & Endler 2001
). Sensitivity to UV light and the detection of black and iridescent ornaments play a role in female preferences in guppies (Houde 1997
; Smith et al. 2002
). However, sensitivity to red and orange wavelength is likely to be particularly important for differentiation because red and orange ornaments are the most consistent targets of female choice (Endler & Houde 1995
; Houde 1997
; Brooks & Endler 2001
). Further, sensitivity to red wavelengths shows heritable variation in guppies (Endler et al. 2001
), and changes in wavelength sensitivity are expected to alter female preferences. Hence, differentiation in LWS opsins could select for variation in male nuptial colour patterns (Endler 1992
; Endler et al. 2001
). Alternatively, the variation in red and orange ornaments themselves might select for diversification of red colour perception. Either way, the highly polymorphic nature of opsins, and the partitioning of some of this variation between individuals within populations, provides a potential molecular framework for future studies of coevolution between visual perception and male ornamentation.