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Vision Research 41 (2001) 1–12 www.elsevier.com/locate/visres

Retinal photoreceptors of paleognathous : the ostrich (Struthio camelus) and rhea (Rhea americana)

Mathew W. Wright, James K. Bowmaker *

Department of Visual Science, Institute of Ophthalmology, Uni6ersity College London, Bath Street, London EC1V 9EL, UK

Received 25 January 2000; received in revised form 16 May 2000

Abstract

Microspectrophotometry was used to determine the absorbance spectra of both rod and cone visual pigments and oil droplets from the retinae of the ostrich (Struthio camelus) and rhea (Rhea americana). Light and fluorescence microscopy of whole fresh tissue mounts were used to determine the relative numbers and distribution of oil droplets in the retinae. Both possessed u rods, double cones and four classes of single cone identified by their oil droplets. The rods had max at about 505 nm, whereas u three cone pigments were recorded with max at 570, 505 and 445 nm. The P570 pigment was located in both members of the u double cones and in a class of single cone containing an R-type oil droplet ( cut at 555 nm). The P505 and P445 cone pigments u were found in populations of single cones containing Y-type and C-type oil droplets ( cut of 500 and 420 nm, respectively). The u fourth class of single cone contained a T-type droplet and in the ostrich contained a visual pigment with max at about 405 nm. Double cones possessed a P-type droplet in the principal member and an A-type droplet in the accessory member. The complement of visual pigments and oil droplets, and the ratio of cone types in the ostrich and rhea, are remarkably similar to those found in many groups of neognathous birds. © 2000 Elsevier Science Ltd. All rights reserved.

Keywords: Visual pigment; Oil droplet; Photoreceptor; Paleognathous; Avian

1. Introduction (Rheinae) of South America and (Tinamidae) of the neotropics, ranging from north-eastern Mexico Studies of the cone visual pigment complement from to Tierra del Fuego (Blake, 1979). The extinct groups a wide range of representatives of the vertebrate classes include moas (Dinornithidae) of New Zealand, elephant have provided data on the origins and evolution of birds (Aepyornithidae) of Africa and Madagascar and vertebrate colour vision, including the tetrachromacy of mihirung birds (Dromornithidae) of Australia (Rich, many diurnal fish, and neognathous birds, and 1979). Except for the , these birds are entirely the more limited dichromacy of the majority of mam- flightless and are known collectively as . The mals and the trichromacy of primates (for reviews, see tinamou is capable of only semi-sustained flight. Goldsmith, 1991; Jacobs, 1993; Bowmaker, 1998). Based purely upon anatomical analyses, early evolu- However, little data are available for the paleognathous tionary biologists failed to derive a consensus opinion birds, a group that may provide an insight into the on paleognathous phylogeny. However, more recent evolution of avian visual systems. studies of molecular genetics have indicated that pale- There are nine main groups of paleognathous birds, ognathous birds are likely to be monophylous and to but only six of them have living representatives. These have evolved from the oldest offshoot of the Class are the cassowaries (Casuariinae) of New Guinea, emus Aves, with the Galliformes and Anseriformes as their (Dromiceinae) of Australia, kiwis (Apterygidae) of New closest relatives (for historical review see Sibley & Zealand, ostriches (Struthioniformes) of Africa, rheas Ahlquist, 1990). Contrary evidence from comparisons of mitochondrial DNA sequences indicate that the Passeriformes branched off from the main avian stem * Corresponding author. Tel: +44-20-7608-6832; fax: +44-20- 7608-6850. prior to the paleognathous offshoot (Ha¨rlid, Janke, & E-mail address: [email protected] (J.K. Bowmaker). Arnason, 1997, 1998; Ha¨rlid & Arnason, 1999), though

0042-6989/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S0042-6989(00)00227-3 2 M.W. Wright, J.K. Bowmaker / Vision Research 41 (2001) 1–12 these findings have been questioned (Groth & Barrow- and the accessory member possesses an A-type droplet clough, 1999). If the Paleognathae are indeed the first that has a low carotenoid concentration. Both members offshoot of the Aves, it would suggest that the visual contain the LWS cone pigment. systems of paleognathous birds may be more closely The first microspectrophotometric analysis of the related to an ancestral avian form than those identified visual pigment and oil droplet complement of pale- in the neognathous birds. ognathous birds were from the emu (Dromiceius no6ae- Regardless of their origins, paleognathous birds, be- hollandiae), ( c. cause of their flightless existence, are regarded as one of cinerascens) and Chilean tinamou (Nothoprocta perdi- the more interesting avian orders and yet with respect caria sanborni ) (Sillman, Bolnick, Haynes, Walter, & to their visual systems are probably one of the least Loew, 1981). These species were shown to have a observed. Only the visual fields of the ostrich have been typical rhodopsin in their rods, whereas in the cones, examined (Martin & Katzir, 1995). The monocular field there was only clear evidence for a LWS pigment. The of each extends over about 155° and probably analysis of oil droplets revealed evidence of only R-type functions to alert the ostrich to dangers on the horizon and Y-type droplets. Sillman et al. (1981) also sug- (Martin & Katzir, 1999). They also have a binocular gested that there was a class of single cone that lacked area in front of the bill that extends vertically through oil droplets, a cone type not found in neognathous 80° and up to a width of about 20°. This small binocu- birds, and this is supported by electron microscopy of lar field is used for foraging food on the ground and cones in the emu (Braekevelt, 1998). These observations Martin and Katzir (1995, 1999) suggest that foraging imply that paleognathous birds have a complement of behaviour is the principal determinant of the character- visual pigment and oil droplets different from the typi- istics of a ’s visual field. Both ostriches and rheas cal four spectrally distinct cone visual pigments and are primarily herbivorous foragers and feed via ballistic associated oil droplets found in neognathous birds. If pecking of leaves, flowers and seeds of a wide variety of the inference is correct, then the different retinal com- plants. position of paleognathous birds may reflect either a In the ostrich, the area centralis extends as a visual specific visual adaptation to their lifestyle, so distinct streak with the central fovea formed as a depression from that of most neognathous birds, or could repre- within the ribbon. The horizontal streak, as with the sent a more ‘primitive’ ancestral avian . small frontal binocular field (Martin & Katzir, 1995), We have addressed the question with a detailed survey probably represents an adaptation to the ostrich’s vi- of the retinal photoreceptors of two further species of sual requirements. Horizontal streaks are mainly seen paleognathous birds, the ostrich (Struthio camelus) and in birds that live in open spaces (Duijm, 1958) and are rhea (Rhea americana). characteristic of birds that procure their food from the ground (Duke-Elder, 1958). The retinal photoreceptors of a number of avian 2. Methods species have been characterized using microspectropho- tometry (e.g. Bowmaker, Heath, Wilkie, & Hunt, 1997 2.1. Subjects and see Table 4). These findings have established that most neognathous birds have four spectrally distinct Ostrich (S. camelus) and rhea (R. americana) chicks, classes of single cone that give the birds the potential aged between 2 and 14 days, were obtained from com- for a tetrachromatic colour vision system. Each cone mercial farms in England and Wales, where the birds class contains an intra-ocular filter, an oil droplet con- are reared for their meat and, in the case of ostriches, taining a high concentration of carotenoids that is also for oil, feathers and leather. The chicks were located within the inner segment of the cone and is thus housed (normally for about 2 weeks) in environmen- interposed between the visual pigment and the incident tally controlled conditions, with a 12-h light/dark ratio light. The relative proportions of the different cone and fed a standard diet. Individuals were dark adapted classes are similar in the retinae of many neognathous for at least 1 h prior to being sacrificed by humane avian species and the type of oil droplet associated with methods (cervical dislocation followed by decapitation). each cone class is normally the same (Bowmaker, 1991; The sacrifice and all following procedures were carried Bowmaker et al., 1997). In single cones, there is typi- out under dim red illumination (Kodak Safelight No. cally an R-type droplet with a long-wave-sensitive 2). Both were enucleated, with one being wrapped (LWS or red) cone pigment, a Y-type droplet with a in foil and stored in a light-tight box at 4°C until the middle-wave-sensitive (MWS or green) cone pigment, a following day. The other eye was hemisected and the C-type droplet with a short-wave-sensitive (SWS or anterior half and vitreous humour discarded. The re- blue) cone pigment and a T-type with an ultraviolet- or sulting eyecup was placed in a solution of chilled cal- violet-sensitive (UVS/VS) cone pigment. In the double cium-free avian saline solution (154 mM NaCl; 5 mM cones, the principal member contains a P-type droplet KCl; 17.5 mM Na2HPO4; 7.5 mM NaH2PO4;10mM M.W. Wright, J.K. Bowmaker / Vision Research 41 (2001) 1–12 3 glucose; pH 7.1). A small piece of retinal tissue (approx. pigments were to compensate for the decreased short- 1mm2) was taken and prepared for microspectrophoto- wave output from the tungsten light source. After metric examination as described previously (Mollon, bleaching, the outer segments were recorded again us- Bowmaker, & Jacobs, 1984). ing exactly the same protocol as before. This equalises the weighting of data for pre- and post-bleach spectra 2.2. Microspectrophotometry for subsequent averaging and calculation of difference spectra. Measurements from individual photoreceptors were made using a modified Liebman dual beam microspec- 2.3. Analysis of 6isual pigment spectra trophotometer under computer control (Liebman & u Entine, 1964; Mollon et al., 1984; Bowmaker, Astell, The wavelength of maximum absorbance ( max) for Hunt, & Mollon, 1991). By using an infrared converter, each outer segment was estimated using a standardized the measuring beam (approx. 2 mm2 cross-section) was computer program. For each cell, the two spectra were aligned to pass transversely through a given structure, averaged and then a 2-nm averaged curve of the out- either an outer segment or an oil droplet, while the ward and return traces was created. These mean curves reference beam passed through a clear space adjacent to were analysed by fitting an A1 visual pigment template the photoreceptor. The beams were polarized so that (Knowles & Dartnall, 1977) to the data. This template the e-vector of the beams was perpendicular to the long is expressed on an abscissal scale of log frequency, since axis of the outer segment, thus utilizing the dichroism visual pigment absorbance curves have an almost iden- of the visual pigment molecules. The monochromator tical shape when expressed in this way (Mansfield, u of the microspectrophotometer scanned from 750 to 1985; Bowmaker et al., 1991). The max of each ab- 350 nm in 2 nm steps, returning from 351 to 749 nm at sorbance spectrum and difference spectrum was calcu- the interleaving wavelengths. To minimize the effects of lated using two methods: first from 20 absorbance bleaching, each outer segment was normally only mea- values on the long wavelength limb of the curve and sured once. Since SWS and UVS/VS photoreceptor secondly from 50 points centred on the peak (see cells are few in number and are less prone to bleaching, Mollon et al., 1984; Bowmaker et al., 1991). The aver- u these cells were measured three times. In addition, two age of these two estimates provided the calculated max independent estimates of the baseline absorbance spec- value for each measured absorbance curve. trum for all cell types were usually obtained by arrang- Since SWS and VS cells were scarce, all recorded cells ing both beams to pass outside the cell. of these types were used for further analysis. However, All putative outer segments were bleached by expo- for the other photoreceptor types stringent selection sure to white light from the monochromator passing criteria were employed. The criteria for the LWS and u through the measuring beam of the microspectrophoto- MWS cells were (i) a transverse density at the max meter. The exposure time was varied according to the greater than 0.009; (ii) a standard deviation from the u visual pigment type. Rod, LWS and MWS pigments right-hand limb estimate of max of less than 7 nm; and u were exposed for 2–3 min, whereas SWS and UVS/VS (iii) a difference between the two max estimates of less pigments were exposed for 5 and 10 min, respectively. than 5 nm. As a result of these criteria, around 40% of The increased bleaching times for shorter wavelength measured LWS and MWS cells were selected. For the rods more stringent selection criteria were applied, with u Table 1 no cells selected with a transverse density at the max u cut of oil droplets for the ostrich, Struthio camelus, and rhea, Rhea less than 0.016 and/or a standard deviation from the americanaa u right-hand limb estimate of max more than 5 nm. The u for each spectral class was determined from the Struthio camelus Rhea americana max mean spectrum for each class of cell (Fig. 2) derived Oil droplet A B AB from the selected data (Table 2). A second estimate of u max for each cell type was also determined (mean of R-type555.3 552.293.1 554.5 555.093.4 u u max in Table 2) from the mean of the individual max (16) (16) for all the selected cells. Y-type 507.1 506.492.4 503.8 504.893.6 (16) (16) C-type 417.1 416.593.4 416.7 417.692.9 2.4. Analysis of oil droplet spectra (14) (10) P-type 498.1 497.992.5 496.4 498.194.5 The oil droplets in avian cones have diameters of (14) (6) about of 2–4 mm and may contain high concentrations a u of carotenoids (Liebman & Granda, 1975; Goldsmith, A, cut of mean absorbance spectrum (nm); B, mean of individual u cut (nm). Numbers in parentheses are the numbers of each droplet Collins, & Licht, 1984). As a consequence, light leakage type. around the droplet becomes significant during spectral 4 M.W. Wright, J.K. Bowmaker / Vision Research 41 (2001) 1–12

2.6. Analysis of oil droplet ratios

Eyes from both ostrich and rhea were hemisected and placed in chilled calcium-free saline solution. The reti- nae were cut into four quadrants and the pieces imme- diately flat mounted onto slides under saline, with the photoreceptor layer uppermost. The preparations were viewed with a light microscope under standard white light and with UV illumination (340–380 nm with a 430-nm cut-off). Most oil droplet types are easily classified according to their colour under natural light, for instance R- and Y-type oil droplets can be clearly observed as red and yellow, respectively. C- and T-type droplets show no observable colouration under natural light. However, under UV illumination, C-type droplets exhibit a bright fluorescence which makes them easily discernible from T-type droplets which lack fluores- cence (Ohtsuka, 1984). Photomicrographs were taken of the preparations and counts were made of the overall numbers of oil droplets, and hence cone photoreceptor classes. Obviously, since rod photoreceptors have no oil droplets, they cannot be counted using this technique.

3. Results Fig. 1. The mean absorbance spectra of the cone oil droplets from ostrich (upper panel) and rhea (lower panel). Letters indicate droplet type. Red, Yellow, Clear and Transparent are all found in single 3.1. Oil droplets cones. Pale droplets (dotted lines) were located in the principal members of double cones and were clearly divided into two popula- Five oil droplet types were characterised in both the tions according to the relative size of the 480-nm shoulder on the ostrich and rhea: red (R-type), yellow (Y-type), pale absorbance spectra. (P-type), clear (C-type) and transparent (T-type). The R-, Y-, C- and T-type droplets were found in single cones, whereas the P-type droplets were located in the principal member of double cones. The mean ab- measurements and accounts for the featureless ‘flat- sorbance spectra of each oil droplet type are presented u topped’ spectra normally recorded from such droplets, in Fig. 1 and the cut for the droplets are summarised in especially the R- and Y-types. Essentially, the droplets Table 1. act as long-pass cut-off filters and were classified ac- The data from the two species were almost identical. u u cording to their ‘cut-off’ wavelength ( cut) which is The R-, Y- and C-type oil droplets had mean cut defined as the wavelength of the intercept at the value around 555, 505 and 417 nm, respectively (Table 1). of maximum measured absorptance by the line tangent The P-type droplets, characterized by a shoulder or to the oil droplet absorptance curve at half measured peak close to 480 nm, could be divided into two absorptance (Lipetz, 1984). populations in both species according to the relative absorbance of the shoulder (Fig. 1). In the ostrich, the 2.5. Measurement of ocular media droplets had either the 480-nm peak or a prominent shoulder close to 480 nm, whereas in the rhea, the The anterior segment of the eye (cornea and lens) difference was more significant with either a peak at was dissected from the intact eye and immediately 480 nm or a diminished shoulder. The droplets typically placed in avian saline solution. A central strip was cut had a maximum absorbance of around 0.2, except for out and placed in a 10-mm black-walled cuvette, such those with the reduced shoulder in the rhea, which had u that the measuring beam of the spectrophotometer a low absorbance around 0.1. The mean cut for the (Perkin Elmer Lambda 2) passed through the cornea, P-type droplets (assuming these droplets act as cut-off aqueous humour and lens. Vitreous humour was col- filters) was around 498 nm. T-type droplets showed no lected and measured separately. Three independent significant absorbance over the measured range. The measurements were taken and averaged to produce an accessory members of the double cones contained a estimate of absorbance for the ocular media. small A-type droplet with a triple peaked absorbance M.W. Wright, J.K. Bowmaker / Vision Research 41 (2001) 1–12 5 spectrum (not shown) that had very low absorbance, pigment contained in a fourth single cone class, which indicating low levels of carotenoid in the droplet. These to date has only been characterized in the ostrich, was u A-type droplets are typical of other avian species (Bow- violet-sensitive with a max around 405 nm and con- maker & Knowles, 1977; Jane & Bowmaker, 1988; tained a T-type droplet. The mean absorbance and Maier & Bowmaker, 1993; Bowmaker et al., 1997; difference spectra and spectral distribution for all the Hart, Partridge, & Cuthill, 1998). visual pigments are presented in Figs. 2 and 3. Table 2 represents a summary of the analysis of all the spectra. 3.2. Visual pigments 3.3. Histology Again, the data from the two species were almost identical. A rod and four spectrally distinct classes of Fig. 4 presents photomicrographs from fluorescent cone visual pigment were characterized. The rods had and light microscopy of retinal whole tissue mounts u and highlights the individual oil droplet types. The max at about 505 nm, whereas three cone pigments u R-and Y-type droplets occur in approximately equal were recorded with max at about 570, 505 and 445 nm. The 570-nm pigment is located in both members of the numbers, each accounting for about 24% of the total double cones and in a class of single cone containing an cone population (Table 3). The C- and T-type droplets R-type oil droplet. The 505-nm and 445-nm cone pig- account for a further 11 and 2%, respectively, and the ments were found in populations of single cones con- P-type droplets form the remaining 40%. No detailed taining Y- and C-type oil droplets, respectively. The systematic survey was carried out across the four retinal quadrants, but no gross differences in the percentages Table 2 of each cone type were observed. u max of visual pigments for the ostrich, Struthio camelus, and rhea, Rhea americana 3.4. Ocular media

Struthio camelus Rhea americana The spectral absorbance of the lens and cornea and Rods: vitreous humour from the ostrich are shown in Fig. 5. u 9 9 Mean of max 504.3 2.3 503.9 2.2 The lens and cornea are virtually transparent at longer u 9 9 max of difference 505.6 1.6 505.0 1.8 wavelengths, with a small increase in absorbance appar- spectrum u 9 9 ent below 450 nm and a substantial increase below 380 max of mean spectrum 504.2 1.2 504.4 1.8 Maximum absorbancea 0.02/0.018 0.02/0.017 nm. The vitreous humour is transparent throughout the Number of cells9 4 measured spectral range. For comparison, the ab- LWS Cones: sorbance spectra of the lens from the canary (Serinus u 9 9 Mean of max 569.9 2.9 570.7 5.5 canaria) (Das, Wilkie, Hunt, & Bowmaker, 1999), Mal- u of difference 570.692.9 570.192.0 max lard duck (Anas platyrhynchos) (Jane & Bowmaker, spectrum u 9 9 1988) and human (Wyszecki & Stiles, 1982) are also max of mean spectrum 569.9 1.8 569.9 2.1 Maximum absorbance0.01/0.009 0.01/0.009 shown (Fig. 5). Number of cells 98 MWS cones: u 9 9 Mean of max 505.5 1.8 505.7 4.4 u 9 9 4. Discussion max of difference 506.7 3.4 506.9 1.8 spectrum u 9 9 6 max of mean spectrum 505.2 2.1 505.7 0.9 4.1. Cone spectral sensiti ity Maximum absorbance 0.011/0.012 0.015/0.013 Number of cells10 8 Since avian cones contain coloured oil droplets that SWS cones: u 9 9 interpose between the incident light and the visual Mean of max 445.0 2.2 446.7 2.1 u 9 9 pigments, the effective spectral sensitivity of each class max of difference 451.0 4.3 456.4 5.8 spectrum of cone will reflect the transmission of the droplet and u 9 9 max of mean spectrum444.4 2.9 445.5 3.5 the spectral sensitivity of the photosensitive pigment. In Maximum absorbance0.008/0.007 0.009/0.007 addition, at short wavelengths, the transmission of the Number of cells4 5 ocular media also has to be considered. The derived VS cones: u cone spectral sensitivities (normalized to log 2.0) for the Mean of max 404.9 – u 9 max of difference408.0 6.5 – four classes of single cone in the ostrich and rhea are spectrum shown in Fig. 6. In the cases of the LWS, MWS and u 9 max of mean spectrum404.9 4.2 – SWS cones, the cut-offs of the oil droplets not only Maximum absorbance0.013/0.010 – remove short-wave sensitivity, but also displace the Number of cells1 – spectral sensitivity of the visual pigments to longer a Maximum absorbance: unbleached spectrum/difference spectrum. wavelengths, such that the three cone classes have 6 M.W. Wright, J.K. Bowmaker / Vision Research 41 (2001) 1–12

Fig. 2. Mean absorbance spectra of visual pigments from the ostrich (Struthio camelus). LWS, MWS, SWS and VS are long-wave-, middle-wave-, short-wave- and violet-sensitive cone visual pigments, respectively. In each case the upper panel represents the mean pre-bleach spectrum (open symbols) and post-bleach spectrum (solid symbols). The lower panels are difference spectra and have been fitted with an A1 visual pigment u template (solid line). The template curves have a max at 571, 507, 451, 408 and 506 nm, respectively. The histograms (bottom right) show the u spectral distribution of the max of individual cones (upper panel) and rods (lower panel). M.W. Wright, J.K. Bowmaker / Vision Research 41 (2001) 1–12 7

Fig. 3. Mean absorbance spectra of visual pigments from the rhea (Rhea americana). LWS, MWS and SWS are long-wave-, middle-wave- and short-wave-sensitive cone visual pigments, respectively. In each case the upper panel represents the mean pre-bleach spectrum (open symbols) and post-bleach spectrum (solid symbols). The lower panels are difference spectra and have been with an A1 visual pigment template (full line). The u u template curves have a max at 570, 507, 456,and 506 nm, respectively. The histograms (bottom right) show the spectral distribution of the max of individual cones (upper panel) and rods (lower panel). 8 M.W. Wright, J.K. Bowmaker / Vision Research 41 (2001) 1–12

Fig. 4. Photomicrographs of sections of retina from ostrich (Struthio camelus) (left panels) and rhea (Rhea americana) (right panels) taken under visible light (upper panels) and UV illumination (lower panels). The R- and Y-type (labelled R and Y) oil droplets are clearly seen as red and yellow under natural light, whereas the P-type droplets (P) appear yellowish green and the C- and T-type (labelled C and T) are colourless. Under UV illumination, C-type droplets exhibit a bright fluorescence that makes them easily discernible from T-type droplets that do not fluoresce. Scale bar represents 20 mm.

Table 3 Relative proportions of oil droplet typesa

P-type R-type Y-type C-type T-type

Ostrich (Struthio camelus) 40.5% (291)24.0% (172) 23.0% (164) 11.0% (81) 1.5% (12) Rhea (Rhea americana) 39.0% (91) 26.0% (61) 21.5% (50) 11.0% (26) 2.5% (6) Chicken1 (Gallus gallus) 50–60% 15–20% 15–20% 10% – Duck2 (Anas platyrhynchos) 52.0% 22.0% 21.0% 5% – Budgerigar3 (Melopsittacus undulatus) 40.0% 20.0% 21.0% 10.0% 9.0% European Starling4 (Sturnus 6ulgaris) 52–57%15–20% 17–18% 4–10% 3–7%

a Figures in parentheses indicate number of each droplet type counted. 1Bowmaker and Knowles (1977); 2Jane and Bowmaker (1988); 3Wilkie et al. (1998); 4Hart et al. (1998).

u effective max at about 615, 545, 470 nm (cf. 570, 505 the spectrum and probably subserve tetrachromatic and 445 nm, respectively, for the visual pigments). The colour vision (Maier & Bowmaker, 1993; Bowmaker et T-type droplet of the VS cones will have little or no al., 1997). effect, but the decreasing transmission of the lens at wavelengths below about 450 nm will lead to a small 4.2. Ocular media u displacement of the max by a few nanometers to longer wavelengths at about 410 nm (Fig. 6). The effective The cornea and lens of the ostrich (Fig. 5) are spectral sensitivities of the four classes of single cone relatively transparent, but show increased absorbance thus have maxima roughly equally spaced throughout below about 450 nm with a steep rise below about 370 M.W. Wright, J.K. Bowmaker / Vision Research 41 (2001) 1–12 9 nm. The absorbance characteristics are more similar to 4.3. Oil droplets that for the lens of the duck (A. platyrhynchos) (Jane & u Bowmaker, 1988), which has a VS cone, than to the The oil droplet complement (Fig. 1) and the cut for more transparent lens of the canary (Das et al., 1999) each specific droplet type in the ostrich and rhea (Table and starling (Hart et al., 1998) which both possess true 1) are remarkably similar to each other and are typical UVS cones. The marked increase in absorbance of the of those seen in most neognathous birds. The relatively lens in the near ultraviolet correlates with the finding high absorbances of the R-, Y-, and C-type droplets that the ostrich (and rhea) has a fourth class of single strongly suggest that they act as long-pass cut-off cone that is violet-sensitive and not ultraviolet-sensitive. filters, whereas this is probably not the case with the Although the VS cone pigment in humans is spectrally P-type droplets of the double cones that have a rela- similar to that of the galliforms, sensitivity at wave- tively low absorbance. lengths below about 390 nm is precluded by the yellow Although the C-type droplets probably act as cut-off human lens (Fig. 5) filters in the ostrich and rhea, this is not the case in all avian species. It has been suggested that the level of u absorbance of these droplets is related to the max of the UVS/VS cones and the spectral separation between the UVS/VS visual pigment and the SWS pigment (Bow- maker et al., 1997). In species with UVS cones, where the separation is great (about 70 nm), the C-type droplets generally have low absorbance, but in species with VS cones, where the spectral separation is small (only about 30–40 nm), the droplets have high ab- sorbance and probably act as cut-off filters. This is the most likely situation in the two paleognathous species, although only a single VS cone outer segment was recorded in the ostrich with a P405. The high ab- sorbance of the C-type droplets is possibly designed to increase the spectral separation between the maximum sensitivities of the SWS and VS cones. The cut-off of the C-type droplets associated with SWS pigments will Fig. 5. Absorbance spectra of ocular media. (1) vitreous humour from cause a narrowing of the bandwidth of the spectral ostrich (Struthio camelus); (2) lens of canary (Serinus canaria) (Das et sensitivity of the cone and displace the maximum sensi- al., 1999); (3) lens and cornea of ostrich (Struthio camelus); (4) lens of tivity to longer wavelengths, around 470 nm and main- duck (Anas platyrhynchos) (Jane & Bowmaker, 1988); (5) lens of tain a more even spread of sensitivity functions human (Wyszecki & Stiles, 1982). throughout the spectrum (Fig. 6). The P-type droplets in both ostrich and rhea fall into two distinct populations according to the relative ab- sorbance of the 480-nm shoulder. Variation in the absorbance of this droplet type has also been reported in most neognathous birds (Bowmaker et al., 1997). An extensive study on variations of P-type droplets in the European starling (Hart et al., 1998) indicated a gradi- ent across the retina in the absorbance of the 480-nm shoulder. It is possible that similar gradients are present in the ostrich and rhea, but no detailed systematic survey of the retinal distribution of oil droplets in these species was carried out. Nevertheless, from general observations of retinal quadrants, no gross differences were noted across the retina. Sillman et al. (1981) reported single cones without oil droplets in the emu and tinamou, but these were not Fig. 6. Relative spectral sensitivities of the four single cone classes observed in the ostrich and rhea. It is possible that from ostrich, Struthio camelus. The sensitivities are derived from the these single cones represent the accessory members of spectral sensitivities of the visual pigments modified for the transmis- the double cones that do not contain a droplet and sion of the R-, Y- and C-type droplets acting as cut-off filters and by u which often become separated from the principal mem- the effect of the lens. The approximate max of the four classes of single cones are 615, 545, 470 and 408 nm. ber in the preparation of retinal tissue for microspec- 10 M.W. Wright, J.K. Bowmaker / Vision Research 41 (2001) 1–12

Table 4 u max of visual pigments from the ostrich, Struthio camelus and rhea, Rhea americana, and neognathous birds, as determined by microspectrophotometrya

UVS VS SWS MWS LWS ROD

Paleognathae Ostrich (Struthio camelus)– 405 444 505 570 504 Rhea (Rhea americana) –– 445 506 570 504 Galliformes Chicken (Gallus gallus)1 –418 455 507 569 509 Japanese quail (Coturnix japonica)2 –419 456 505 569 505 Turkey (Meleagris galopar6o)3 –420 460 505 564 504 Peacock (Pa6o cristatus)4 –421 457 505 566 504 Anseriformes Mallard duck (Anas platyrhynchos)6 – 420 452 502 570 505 Columbiformes Pigeon (Columba li6ia)1 –409 453 507 568 506 Passeriformes Zebra Finch (Taeniopygia guttata)1 360–380– 430 505 567 507 Pekin Robin (Leiothrix lutea)7 355 – 453 501 567 500 Canary (Serinus canaria)8 366– 442 505 569 506 European Starling (Sturnus 6ulgaris)9 362 – 449 504 563 503 Blackbird (Turdus merula)5 373– 454 504 557 504 Blue tit (Par6us caeruleus)5 374 – 449 503 563 503 Psittaciformes Budgerigar (Melopsittacus undulatus)1 371– 444 508 564 509 Sphenisciformes Penguin (Spheniscus humboldti)10 – 403 450 – 543 504 Strigiformes Tawny Owl (Strix aluco)11 – – 463 503 555 503

a1Bowmaker et al. (1997); 2Bowmaker, Kovach, Whitmore, and Loew (1993); 3Hart, Partridge, and Cuthill (1999); 4Hart (1998); 5Hart, Partridge, Cuthill and Bennett (2000); 6Jane and Bowmaker (1988); 7Maier and Bowmaker (1993); 8Das et al. (1999); 9Hart et al. (1998); 10Bowmaker and Martin (1985); 11Bowmaker and Martin (1978). trophotometry. Nevertheless, since Braekevelt (1998) they were so difficult to locate by also suggests that single cones without oil droplets microspectrophotometry. occur in the emu, the possibility of a real species difference cannot be ignored. 4.5. Visual pigments

4.4. Cone type ratios The absorbance spectra of all the visual pigments

could be fitted with an A1 template (Figs. 2 and 3), There is a high similarity between the ratios of the implying that the visual pigments of paleognathous different cone types in the ostrich and rhea (Table 3). birds are, like their neognathous cousins, typically Double cones account for about 40% of the total cone rhodopsins rather than porphyropsins. This is not an population, whereas the LWS and MWS single cones idle observation, since although porphyropsins are nor- are in roughly equal numbers making up a further mally found only in freshwater species, some terrestrial 20–25% each. The remaining 10–15% of cones is com- reptiles are known to possess them (Provencio, Loew, & prised of the SWS and VS cones. These values are Foster, 1992; Bowmaker, Loew, & Ott, 2000). u remarkable similar to many neognathous birds (Table The max of the visual pigments from the ostrich and 3), though in the two paleognathous species, the VS rhea (Table 2) are almost identical and reflect either the cones account for only about 2% of the total, which is close phylogenetic relationship or the similar visual considerably lower than reported in some neognathous requirements of the two species. Recent molecular stud- birds. UVS cones account for about 9% in the budgeri- ies indicate that the ostrich belongs to a closely related gar (Wilkie et al., 1998) and 3–7% in the starling (Hart sister group of the rhea (Ha¨rlid et al., 1997; Ha¨rlid et et al., 1998) (Table 3). Given the extreme scarcity of VS al., 1998; Ha¨rlid & Arnason, 1999). However, it is u cones in the ostrich and rhea, it is not surprising that remarkable that the max of the visual pigments of the M.W. Wright, J.K. Bowmaker / Vision Research 41 (2001) 1–12 11 rod, LWS, MWS and SWS cones of the paleognathous (lack of ultraviolet sensitivity) in both galliforms and birds are so strikingly similar to those of many paleognaths may simply reflect a shared ancestral diver- neognathous species (Table 4). It is apparent that these gence from other avian species. Whilst molecular stud- visual pigments (and their associated oil droplets) are ies may disagree with one another on the basal highly conserved across avian species, irrespective of divergence of Paleognathae and Neognathae, almost all the very different life styles and visual requirements of agree that the galliforms are the closest relatives of the different species, ranging, for example, from those of paleognathous birds (Sibley & Ahlquist, 1990; Caspers, finches to that of the ostrich. This fundamental avian Uit de Weerd, & Wattel, 1997; Ha¨rlid et al., 1997; photoreceptor system appears to be highly modified Ha¨rlid et al., 1998; Ha¨rlid & Arnason, 1999). only in species such as nocturnal birds and penguins that may be considered to occupy ‘extreme’ photic environments. Acknowledgements In contrast to the longer-wave pigments, notable differences are found in the spectral sensitivity of the M.W. Wright was supported by an MRC stu- fourth class of single cone that possesses a T-type oil dentship. We thank Juliet Parry and Stuart Peirson for droplet. The visual pigment of these cells is usually u experimental help and useful discussion. either violet-sensitive, with max close to either 420 or u 405 nm, or ultraviolet-sensitive with max near 370 nm (Table 4). In this study, the only cell of this class, measured from the ostrich, contained a visual pigment References u with max around 405 nm. The reasons for the possible ‘clustering’ of the VS Andersson, S. (1996). Bright ultraviolet coloration in the Asian and UVS cone pigments are not immediately apparent. whistling thrushes (Myiophonus spp.). Proceedings of the Royal u Society of London B, 263, 843–848. UVS cones with max close to 370 nm have been found Andersson, S., & Amundsen, T. (1997). Ultraviolet colour vision and in passeriforms and psittaciforms (Table 4) and hum- ornamentation in bluethroats. Proceedings of the Royal Society of ming birds have also been shown to be ultraviolet-sensi- London B, 264, 1587–1591. tive (Goldsmith, 1980). Specific behavioural functions Andersson, S., O8rnborg, J., & Andersson, M. (1998). Ultraviolet have been attributed to this sensitivity (see Bennett & sexual dimorphism and assortative mating in blue tits. Proceed- ings of the Royal Society of London B, 265, 445–450. Cuthill, 1994; Maier, 1994). These include orientation Bennett, A. T. D., & Cuthill, I. C. (1994). Ultraviolet vision in birds; and navigation, foraging (Goldsmith, 1980; Burkhardt, what is its function? Vision Research, 34, 1471–1478. 1982; Cooper, Charles-Dominique, & Vie´not, 1986) and Bennett, A. T. D., Cuthill, I. C., Partridge, J. C., & Maier, E. J.: inter- and intra-specific communication such as sexual (1996). Ultraviolet vision and mate choice in zebra finches. Na- displays and mate choice (e.g. Burkhardt, 1989; ture, 380, 433–435. Bennett, A. T. D., Cuthill, I. C., Partridge, J. C., & Lunau, K. (1997). Burkhardt & Finger, 1991; Bennett, Cuthill, Partridge, Ultraviolet plumage predict mate preference in starlings. & Maier, 1996; Andersson, 1996; Andersson & Amund- Proceedings of the National Academy of Sciences USA, 94, 8618– sen, 1997; Bennett, Cuthill, Partridge, & Lunau, 1997; 8621. Andersson, O8rnborg, & Andersson, 1998; Church, Ben- Blake, E. R. (1979). Checklist of birds of the world. In Museum of 6 nett, Cuthill, & Partridge, 1998; Hunt, Bennett, Cuthill, Comparati e Zoology 1 (pp. 12–47). Cambridge, MA: Museum of Comparative Zoology. & Griffiths, 1998). However, it may be misleading to Bowmaker, J. K. (1991). Visual pigments, oil droplets and photore- consider visual behaviour limited to specific regions of ceptors. In P. Gouras, Vision and 6isual dysfunction, 6, the percep- spectral sensitivity in isolation, since these visual func- tion of colour (pp. 108–127). London: Macmillan. tions may equally require responses from all or some of Bowmaker, J. K. (1998). Evolution of colour vision in vertebrates. the other cone classes. The potential tetrachromacy of Eye, 12, 541–547. Bowmaker, J. K., Astell, S., Hunt, D. M., & Mollon, J. D. (1991). birds and chromatic discrimination at short wave- Photosensitive and photostable pigments in the retinae of Old lengths will be determined by the neural opponency of World monkeys. Journal of Experimental Biology, 156, 1–19. all four spectral classes of cone. Bowmaker, J. K., Heath, L. A., Wilkie, S. E., & Hunt, D. M. (1997). In contrast to the ‘truly’ ultraviolet-sensitive birds, Visual pigments and oil droplets from six classes of photoreceptor the ostrich and rhea, as well as the galliforms and in the retinas of birds. Vision Research, 37, 2183–2194. Bowmaker, J. K., & Knowles, A. (1977). The visual pigments and oil anseriforms, are less sensitive to such short wave- droplets of the chicken Gallus gallus. Vision Research, 17, 755– lengths, with their fourth spectral cone class having 764. maximum sensitivity above 400 nm. Why these birds Bowmaker, J. K., Kovach, J. K., Whitmore, A. V., & Loew, E. R. lack high sensitivity in the ultraviolet remains unan- (1993). Visual pigments and oil droplets in genetically manipu- swered. It is notable however, that birds which possess lated and carotenoid deprived quail: a microspectrophotometric study. 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