Diversity of color vision: not all Australian marsupials are trichromatic

Color vision in marsupials has recently emerged as a particularly interesting case among mammals. It appears that there are both dichromats and trichromats among closely related species. In contrast to primates, marsupials seem to have evolved a different type of trichromacy that is not linked to the X-chromosome. Based on microspectrophotometry and retinal whole-mount immunohistochemistry, four trichromatic marsupial species have been described: quokka, quenda, honey possum, and fat-tailed dunnart. It has, however, been impossible to identify the photopigment of the third cone type, and genetically, all evidence so far suggests that all marsupials are dichromatic. The tammar wallaby is the only Australian marsupial to date for which there is no evidence of a third cone type. To clarify whether the wallaby is indeed a dichromat or trichromatic like other Australian marsupials, we analyzed the number of cone types in the "dichromatic" wallaby and the "trichromatic" dunnart. Employing identical immunohistochemical protocols, we confirmed that the wallaby has only two cone types, whereas 20-25% of cones remained unlabeled by S- and LM-opsin antibodies in the dunnart retina. In addition, we found no evidence to support the hypothesis that the rod photopigment (rod opsin) is expressed in cones which would have explained the absence of a third cone opsin gene. Our study is the first comprehensive and quantitative account of color vision in Australian marsupials where we now know that an unexpected diversity of different color vision systems appears to have evolved.     

Genetic basis of spectral tuning in the violet-sensitive visual pigment of African clawed frog, Xenopus laevis

Ultraviolet (UV) and violet vision in vertebrates is mediated by UV and violet visual pigments that absorb light maximally (lambdamax) at approximately 360 and 390-440 nm, respectively. So far, a total of 11 amino acid sites only in transmembrane (TM) helices I-III are known to be involved in the functional differentiation of these short wavelength-sensitive type 1 (SWS1) pigments. Here, we have constructed chimeric pigments between the violet pigment of African clawed frog (Xenopus laevis) and its ancestral UV pigment. The results show that not only are the absorption spectra of these pigments modulated strongly by amino acids in TM I-VII, but also, for unknown reasons, the overall effect of amino acid changes in TM IV-VII on the lambdamax-shift is abolished. The spectral tuning of the contemporary frog pigment is explained by amino acid replacements F86M, V91I, T93P, V109A, E113D, L116V, and S118T, in which V91I and V109A are previously unknown, increasing the total number of critical amino acid sites that are involved in the spectral tuning of SWS1 pigments in vertebrates to 13.     

Dichromatic colour vision in an Australian marsupial, the tammar wallaby

Despite earlier assertions that most mammals are colour blind, colour vision has in recent years been demonstrated in a variety of eutherian mammals from a wide range of different orders. This paper presents the first behavioural evidence from colour discrimination experiments, that an Australian marsupial, the tammar wallaby (Macropus eugenii), has dichromatic colour vision. In addition, the experiments show that the wallabies readily learn the relationship between the presented colours rather than the absolute hues. This provides a sensitive method to measure the location of the neutral-point, which is the wavelength of monochromatic light that is indistinguishable from white. This point is a diagnostic feature for dichromats. The spectral sensitivity of the wallabies' middle-wavelength-sensitive photoreceptor is known (peak: 539 nm) and the behavioural results imply that the sensitivity of the short-wavelength-sensitive receptor must be near 420 nm. These spectral sensitivities are similar to those found in eutherian mammals, supporting the view that the earliest mammals had dichromatic colour vision. Accepted: 18 July 1999     

Spectral differentiation of blue opsins between phylogenetically close but ecologically distant goldfish and zebrafish

Zebrafish and goldfish are both diurnal freshwater fish species belonging to the same family, Cyprinidae, but their visual ecological surroundings considerably differ. Zebrafish are surface swimmers in conditions of broad and shortwave-dominated background spectra and goldfish are generalized swimmers whose light environment extends to a depth of elevated short wavelength absorbance with turbidity. The peak absorption spectrum (lambdamax) of the zebrafish blue (SWS2) visual pigment is consistently shifted to short wavelength (416 nm) compared with that of the goldfish SWS2 (443 nm). Among the amino acid differences between the two pigments, only one (alanine in zebrafish and serine in goldfish at residue 94) was previously known to cause a difference in absorption spectrum (14-nm lambdamax shift in newt SWS2). In this study, we reconstructed the ancestral SWS2 pigment of the two species by applying likelihood-based Bayesian statistics and performing site-directed mutagenesis. The reconstituted ancestral photopigment had a lambdamax of 430 nm, indicating that zebrafish and goldfish achieved short wavelength (-14 nm) and long wavelength (+13 nm) spectral shifts, respectively, from the ancestor. Unexpectedly, the S94A mutation resulted in only a -3-nm spectral shift when introduced into the goldfish SWS2 pigment. Nearly half of the long wavelength shift toward the goldfish pigment was achieved instead by T116L (6 nm). The S295C mutation toward zebrafish SWS2 contributed to creating a ridge of absorbance around 400 nm and broadening its spectral sensitivity in the short wavelength direction. These results indicate that the evolutionary engineering approach is very effective in deciphering the process of functional divergence of visual pigments.     

Genetic analyses of visual pigments of the pigeon (Columba livia)

We isolated five classes of retinal opsin genes rh1(Cl), rh2(Cl), sws1(Cl), sws2(Cl), and lws(Cl) from the pigeon; these encode RH1(Cl), RH2(Cl), SWS1(Cl), SWS2(Cl), and LWS(Cl) opsins, respectively. Upon binding to 11-cis-retinal, these opsins regenerate the corresponding photosensitive molecules, visual pigments. The absorbance spectra of visual pigments have a broad bell shape with the peak, being called lambdamax. Previously, the SWS1(Cl) opsin cDNA was isolated from the pigeon retinal RNA, expressed in cultured COS1 cells, reconstituted with 11-cis-retinal, and the lambdamax of the resulting SWS1(Cl) pigment was shown to be 393 nm. In this article, using the same methods, the lambdamax values of RH1(Cl), RH2(Cl), SWS2(Cl), and LWS(Cl) pigments were determined to be 502, 503, 448, and 559 nm, respectively. The pigeon is also known for its UV vision, detecting light at 320-380 nm. Being the only pigments that absorb light below 400 nm, the SWS1(Cl) pigments must mediate its UV vision. We also determined that a nonretinal P(Cl) pigment in the pineal gland of the pigeon has a lambdamax value at 481 nm.     

Elephants and human color-blind deuteranopes have identical sets of visual pigments

Being the largest land mammals, elephants have very few natural enemies and are active during both day and night. Compared with those of diurnal and nocturnal animals, the eyes of elephants and other arrhythmic species, such as many ungulates and large carnivores, must function in both the bright light of day and dim light of night. Despite their fundamental importance, the roles of photosensitive molecules, visual pigments, in arrhythmic vision are not well understood. Here we report that elephants (Loxodonta africana and Elephas maximus) use RH1, SWS1, and LWS pigments, which are maximally sensitive to 496, 419, and 552 nm, respectively. These light sensitivities are virtually identical to those of certain "color-blind" people who lack MWS pigments, which are maximally sensitive to 530 nm. During the day, therefore, elephants seem to have the dichromatic color vision of deuteranopes. During the night, however, they are likely to use RH1 and SWS1 pigments and detect light at 420-490 nm.     

A novel spectral tuning in the short wavelength-sensitive (SWS1 and SWS2) pigments of bluefin killifish (Lucania goodei)

The molecular bases of spectral tuning in the UV-, violet-, and blue-sensitive pigments are not well understood. Using the in vitro assay, here we show that the SWS1, SWS2-A, and SWS2-B pigments of bluefin killifish (Lucania goodei) have the wavelengths of maximal absorption (lambda(max)'s) of 354, 448, and 397 nm, respectively. The spectral difference between the SWS2-A and SWS2-B pigments is largest among those of all currently known pairs of SWS2 pigments within a species. The SWS1 pigment contains no amino acid replacement at the currently known 25 critical sites and seems to have inherited its UV-sensitivity directly from the vertebrate ancestor. Mutagenesis analyses show that the amino acid differences at sites 44, 46, 94, 97, 109, 116, 118, 265, and 292 of the SWS2-A and SWS2-B pigments explain 80% of their spectral difference. Moreover, the larger the individual effects of amino acid changes on the lambda(max)-shift are, the larger the synergistic effects tend to be generated, revealing a novel mechanism of spectral tuning of visual pigments.     

Isolation and characterization of marsupial IL5 genes

The genomic nucleotide sequence and chromosomal position of the interleukin 5 (IL5) gene has been described for the model marsupial Macropus eugenii (tammar wallaby). A 272 base pair genomic IL5 polymerase chain reaction (PCR) product spanning exon 3, intron 3, and exon 4 was generated using stripe-faced dunnart (Sminthopsis macroura) DNA. This PCR product was used to isolate a genomic lambda clone containing the complete IL5 gene from a tammar wallaby EMBL3 lambda library. Sequencing revealed that the tammar wallaby IL5 gene consists of four exons separated by three introns. Comparison of the marsupial coding sequence with coding sequences from eutherian species revealed 61 to 69% identity at the nucleotide level and 48 to 63% identity at the amino acid (aa) level. A polymorphic complex compound microsatellite was identified within intron 2 of the tammar wallaby IL5 gene. This microsatellite was also found in other marsupials including the swamp wallaby, tree kangaroo, stripe-faced dunnart, South American opossum, brushtail possum, and koala. Fluorescence in situ hybridization using DNA from the IL5 clone on tammar wallaby chromosomes indicated that the IL5 gene is located on Chromosome 1.     

Epistatic Adaptive Evolution of Human Color Vision

Establishing genotype-phenotype relationship is the key to understand the molecular mechanism of phenotypic adaptation. This initial step may be untangled by analyzing appropriate ancestral molecules, but it is a daunting task to recapitulate the evolution of non-additive (epistatic) interactions of amino acids and function of a protein separately. To adapt to the ultraviolet (UV)-free retinal environment, the short wavelength-sensitive (SWS1) visual pigment in human (human S1) switched from detecting UV to absorbing blue light during the last 90 million years. Mutagenesis experiments of the UV-sensitive pigment in the Boreoeutherian ancestor show that the blue-sensitivity was achieved by seven mutations. The experimental and quantum chemical analyses show that 4,008 of all 5,040 possible evolutionary trajectories are terminated prematurely by containing a dehydrated nonfunctional pigment. Phylogenetic analysis further suggests that human ancestors achieved the blue-sensitivity gradually and almost exclusively by epistasis. When the final stage of spectral tuning of human S1 was underway 45–30 million years ago, the middle and long wavelength-sensitive (MWS/LWS) pigments appeared and so-called trichromatic color vision was established by interprotein epistasis. The adaptive evolution of human S1 differs dramatically from orthologous pigments with a major mutational effect used in achieving blue-sensitivity in a fish and several mammalian species and in regaining UV vision in birds. These observations imply that the mechanisms of epistatic interactions must be understood by studying various orthologues in different species that have adapted to various ecological and physiological environments.     

Dichromatic Colour Vision in Wallabies as Characterised by Three Behavioural Paradigms

Despite lacking genetic evidence of a third cone opsin in the retina of any Australian marsupial, most species tested so far appear to be trichromatic. In the light of this, we have re-examined colour vision of the tammar wallaby which had previously been identified as a dichromat. Three different psychophysical tests, based on an operant conditioning paradigm, were used to confirm that colour perception in the wallaby can be predicted and conclusively explained by the existence of only two cone types. Firstly, colour-mixing experiments revealed a Confusion Point between the three primary colours of a LCD monitor that can be predicted by the cone excitation ratio of the short- and middle-wavelength sensitive cones. Secondly, the wavelength discrimination ability in the wallaby, when tested with monochromatic stimuli, was found to be limited to a narrow range between 440 nm and 500 nm. Lastly, an experiment designed to test the wallaby’s ability to discriminate monochromatic lights from a white light provided clear evidence for a Neutral Point around 485 nm where discrimination consistently failed. Relative colour discrimination seemed clearly preferred but it was possible to train a wallaby to perform absolute colour discriminations. The results confirm the tammar wallaby as a dichromat, and so far the only behaviourally confirmed dichromat among the Australian marsupials.     

Estimated absorbance spectra of the visual pigments of the North Atlantic right whale (Eubalaena glacialis)

To assess the spectral sensitivities of the retinal visual pigments from the North Atlantic right whale (Eubalaena glacialis), we have cloned and sequenced two exons from the rod opsin gene and two exons from the middle‐wavelength sensitive (MWS) cone opsin gene in order to determine the amino acids at positions known to be key regulators of the spectral location of the absorbance maximum (λ_max). Based on previous mutagenesis models we estimate that the right whale possesses a rod visual pigment with a λ_max of 499 nm and a MWS cone visual pigment with a λ_max of 524 nm. Although the MWS cone visual pigment from the right whale is blue‐shifted in its spectral sensitivity like those from odontocetes, the spectral sensitivity of the right whale rod visual pigment is similar to those from terrestrial mammals.     

Molecular basis of spectral tuning in the newt short wavelength sensitive visual pigment

Previously we reported the sequence of the member of the short wavelength sensitive 2 (SWS2) family of vertebrate visual pigments from the retina of the Japanese common newt, Cynops pyrrhogaster[Takahashi, Y. et al. (2001) FEBS Lett. 501, 151-155]. Now we have expressed the apopigment and regenerated it with A1 retinal. Its absorption maximum, 474 nm, is greatly red shifted compared to other known SWS2 pigments (418-455 nm). To determine the amino acid residues that control its spectral tuning, we replaced the residues that were near the chromophore and which differed between the newt and the bullfrog (lambda(max) = 430 nm) wild-type SWS2 pigments: Pro91Ser, Ser94Ala, Ile122Met, Cys127Ser, Ser211Cys, Tyr261Phe, and Ala292Ser. Each of these site-directed mutants led to blue shifts of the newt pigment with five of them causing substantial shifts; their sum was about equal to the difference between the absorption maximum of the bullfrog and newt pigments, 44 nm. The 32 nm shift of the absorption maximum of the multiple seven-residue mutant to 442 nm is fairly close to that of the wild-type bullfrog pigment. Thus, the seven amino acid residues that we replaced are the major cause of the red shift of the newt SWS2 pigment's spectrum. Two of the residues, 91 and 94, have not previously been identified as wavelength regulating sites in visual pigments. One of these, 91, probably regulates color via a new mechanism: altering of a hydrogen bonding network that is connected via a water to the chromophore, in this case its counterion, Glu113.     

Cone topography and spectral sensitivity in two potentially trichromatic marsupials, the quokka (Setonix brachyurus) and quenda (Isoodon obesulus)

The potential for trichromacy in mammals, thought to be unique to primates, was recently discovered in two Australian marsupials. Whether the presence of three cone types, sensitive to short- (SWS), medium- (MWS) and long- (LWS) wavelengths, occurs across all marsupials remains unknown. Here, we have investigated the presence, distribution and spectral sensitivity of cone types in two further species, the quokka (Setonix brachyurus) and quenda (Isoodon obesulus). Immunohistochemistry revealed that SWS cones in the quokka are concentrated in dorso-temporal retina, while in the quenda, two peaks were identified in naso-ventral and dorso-temporal retina. In both species, MWS/LWS cone spatial distributions matched those of retinal ganglion cells. Microspectrophotometry (MSP) confirmed that MWS and LWS cones are spectrally distinct, with mean wavelengths of maximum absorbance at 502 and 538 nm in the quokka, and at 509 and 551 nm, in the quenda. Although small SWS cone outer segments precluded MSP measurements, molecular analysis identified substitutions at key sites, accounting for a spectral shift from ultraviolet in the quenda to violet in the quokka. The presence of three cone types, along with previous findings in the fat-tailed dunnart and honey possum, suggests that three spectrally distinct cone types are a feature spanning the marsupials.     

The molecular genetics and evolution of red and green color vision in vertebrates.

To better understand the evolution of red-green color vision in vertebrates, we inferred the amino acid sequences of the ancestral pigments of 11 selected visual pigments: the LWS pigments of cave fish (Astyanax fasciatus), frog (Xenopus laevis), chicken (Gallus gallus), chameleon (Anolis carolinensis), goat (Capra hircus), and human (Homo sapiens);and the MWS pigments of cave fish, gecko (Gekko gekko), mouse (Mus musculus), squirrel (Sciurus carolinensis), and human. We constructed these ancestral pigments by introducing the necessary mutations into contemporary pigments and evaluated their absorption spectra using an in vitro assay. The results show that the common ancestor of vertebrates and most other ancestors had LWS pigments. Multiple regression analyses of ancestral and contemporary MWS and LWS pigments show that single mutations S180A, H197Y, Y277F, T285A, A308S, and double mutations S180A/H197Y shift the lambda(max) of the pigments by -7, -28, -8, -15, -27, and 11 nm, respectively. It is most likely that this "five-sites" rule is the molecular basis of spectral tuning in the MWS and LWS pigments during vertebrate evolution.     

The molecular genetics of red and green color vision in mammals.

To elucidate the molecular mechanisms of red-green color vision in mammals, we have cloned and sequenced the red and green opsin cDNAs of cat (Felis catus), horse (Equus caballus), gray squirrel (Sciurus carolinensis), white-tailed deer (Odocoileus virginianus), and guinea pig (Cavia porcellus). These opsins were expressed in COS1 cells and reconstituted with 11-cis-retinal. The purified visual pigments of the cat, horse, squirrel, deer, and guinea pig have lambdamax values at 553, 545, 532, 531, and 516 nm, respectively, which are precise to within +/-1 nm. We also regenerated the "true" red pigment of goldfish (Carassius auratus), which has a lambdamax value at 559 +/- 4 nm. Multiple linear regression analyses show that S180A, H197Y, Y277F, T285A, and A308S shift the lambdamax values of the red and green pigments in mammals toward blue by 7, 28, 7, 15, and 16 nm, respectively, and the reverse amino acid changes toward red by the same extents. The additive effects of these amino acid changes fully explain the red-green color vision in a wide range of mammalian species, goldfish, American chameleon (Anolis carolinensis), and pigeon (Columba livia).     

A novel amino acid substitution is responsible for spectral tuning in a rodent violet-sensitive visual pigment

Cone short-wave (SWS1) visual pigments can be divided into two categories that correlate with spectral sensitivity, violet sensitive above 390 nm and ultraviolet sensitive below that wavelength. The evolution and mechanism of spectral tuning of SWS1 opsins are proving more complex than those of other opsin classes. Violet-sensitive pigments probably evolved from an ancestral ultraviolet-sensitive opsin, although in birds ultraviolet sensitivity has re-evolved from violet-sensitive pigments. In certain mammals, a single substitution involving the gain of a polar residue can switch sensitivity from ultraviolet to violet sensitivity, but where such a change is not involved, several substitutions may be required to effect the switch. The guinea pig, Cavia porcellus, is a hystricognathous rodent, a distinct suborder from the Sciurognathi, such as rats and mice. It has been shown by microspectrophotometry to have two cone visual pigments at 530 and 400 nm. We have ascertained the sequence of the short-wave pigment and confirmed its violet sensitivity by expression and reconstitution of the pigment in vitro. Moreover, we have shown by site-directed mutagenesis that a single residue is responsible for wavelength tuning of spectral sensitivity, a Val86Phe causing a 60 nm short-wave shift into the ultraviolet and a Val86Tyr substitution shifting the pigment 8 nm long wave. The convergent evolution of this mammalian VS pigment provides insight into the mechanism of tuning between the violet and UV.     

Identification of tammar wallaby SIRH12, derived from a marsupial-specific retrotransposition event

In humans and mice, there are 11 genes derived from sushi-ichi related retrotransposons, some of which are known to play essential roles in placental development. Interestingly, this family of retrotransposons was thought to exist only in eutherian mammals, indicating their significant contributions to the eutherian evolution, but at least one, PEG10, is conserved between marsupials and eutherians. Here we report a novel sushi-ichi retrotransposon-derived gene, SIRH12, in the tammar wallaby, an Australian marsupial species of the kangaroo family. SIRH12 encodes a protein highly homologous to the sushi-ichi retrotransposon Gag protein in the tammar wallaby, while SIRH12 in the South American short-tailed grey opossum is a pseudogene degenerated by accumulation of multiple nonsense mutations. This suggests that SIRH12 retrotransposition occurred only in the marsupial lineage but acquired and retained some as yet unidentified novel function, at least in the lineage of the tammar wallaby.     

Statistical and molecular analyses of evolutionary significance of red-green color vision and color blindness in vertebrates

Red-green color vision is strongly suspected to enhance the survival of its possessors. Despite being red-green color blind, however, many species have successfully competed in nature, which brings into question the evolutionary advantage of achieving red-green color vision. Here, we propose a new method of identifying positive selection at individual amino acid sites with the premise that if positive Darwinian selection has driven the evolution of the protein under consideration, then it should be found mostly at the branches in the phylogenetic tree where its function had changed. The statistical and molecular methods have been applied to 29 visual pigments with the wavelengths of maximal absorption at approximately 510-540 nm (green- or middle wavelength-sensitive [MWS] pigments) and at approximately 560 nm (red- or long wavelength-sensitive [LWS] pigments), which are sampled from a diverse range of vertebrate species. The results show that the MWS pigments are positively selected through amino acid replacements S180A, Y277F, and T285A and that the LWS pigments have been subjected to strong evolutionary conservation. The fact that these positively selected M/LWS pigments are found not only in animals with red-green color vision but also in those with red-green color blindness strongly suggests that both red-green color vision and color blindness have undergone adaptive evolution independently in different species.     

Identification, characterization and expression of cathelicidin in the pouch young of tammar wallaby (Macropus eugenii)

Antimicrobial peptides, such as cathelicidin, are an evolutionarily old defense system. However they have more complex actions than just simply their antimicrobial effects, including immunoregulation and interaction with the adaptive immune system. In this study we have characterized several novel cathelicidin-like peptides from the tammar wallaby (Macropus eugenii). The tammar cathelicidin-like (MaeuCath) mRNA were isolated based on the conservation of the cathelin-like amino terminus. Mature MaeuCath peptides were positively charged with hydrophobic carboxyl tails, features that are fundamental for antimicrobial function. MaeuCath1 was induced in tammar leukocytes in response to pathogen-associated molecular patterns from both gram positive and negative bacteria. In addition, we also examined the expression of MaeuCath1 in the primary and secondary lymphoid organs of the tammar neonate throughout early pouch life. The results from this study demonstrate the importance that MaeuCath1 may play in innate defense of the marsupial young, especially in the mucosal organs. Such expression of antimicrobial peptides may form part of the immune strategies of marsupials for neonatal survival during their post-partum development.     

Isolation of major histocompatibility complex Class I genes from the tammar wallaby (Macropus eugenii)

The major histocompatibility complex (MHC) plays an essential role in the adaptive immune system of vertebrates through antigen recognition. Although MHC genes are found in all vertebrates, the MHC region is dynamic and has changed throughout vertebrate evolution, making it an important tool for comparative genomics. Marsupials occupy an important position in mammalian phylogeny, yet the MHC of few marsupials has been studied in detail. We report the isolation and analysis of expressed MHC Class I genes from the tammar wallaby, a model marsupial used extensively for the study of mammalian reproduction, genetics, and immunology. We determined that there are at least 11 Class I loci in the tammar genome and isolated six expressed Class I sequences from spleen and testes cDNA libraries, representing at least four loci. Two of the Class I sequences contain substitutions at sites known to be important for antigen binding, perhaps impacting their ability to bind peptides, or the types of peptide to which they bind. Phylogenetic analysis of tammar wallaby Class I sequences and other mammalian Class I sequences suggests that some tammar wallaby and red-necked wallaby loci evolved from common ancestral genes.