The spectral clustering of visual pigments

Vision Res. Vol. 5, pp. 81-100.

Pergamon Press 1965. Printed in Great Britain.

THE SPECTRAL

CLUSTERING

OF VISUAL

PIGMENTS

H. J. A. DARTNALL and J. N. LYTHGOE M.R.C. Vision Research Unit, Institute of Ophthalmology, Judd Street, London W.C.1 (Received 26 October 1964) INTRODUCTION

THE VISUALpigment molecule consists of a protein (opsin) that bears a prosthetic group in which is located the chromophore or site of absorption of a quantum. The prosthetic group can be of two kinds, i.e. related either to vitamin AI or to vitamin As or, more precisely, to the aldehydes (retinenes) of these vitamins. The principal chemical linkage between prosthetic group and opsin is a nitrogen+arbon bond formed by the elimination of the elements of water between an amino-group of the opsin and the aldehydic group of retinener or retinenes. In addition, the prosthetic group is of such a configuration that it fits the shape of the opsin, a circumstance that is undoubtedly accompanied by a play of attractive forces between them. It is on account of this close association between its two parts that the visual pigment molecule has an absorption band in the visible spectrum; for when, through photochemical change or thermal degradation, the configuration of prosthetic group or opsin is changedso that the two parts, though still joined by the principal bond, no longer correspond-then the characteristic absorption band is lost. In recent years it has become evident that the spectral location (A,,,) of the characteristic absorption band of visual pigments can extend over a wide spectral range. Thus in the retinenel series of pigments the 1maz ranges from about 430 to 562 nm ; while in the retinenes series the range is similarly broad, the shortest known A,,, being 510 nm, and the longest 620 nm (the synthetic pigment “cyanopsin”). If we accept that every visual pigment molecule possesses but one prosthetic group, and that this group is always in the 1I-cis configuration, then it seems that the only possible basis for the variability of visual pigments lies in variability of opsin and/or the subsidiary forces that bind opsin and prosthetic group together. In order to develop this theme we decided to try to determine how the visual pigments are distributed in the spectrum; i.e. to discover whether the 1 maz can occur at any wavelength within the range, or whether there is a tendency for clustering about discrete spectral positions. Either case, it seemed to us, would have important implications. For if the I,,, is continuously variable, this would suggest a continuously variable property, e.g. molecular weight of the opsin, as the important factor in determining Iz,,, location; while a discontinuous distribution would suggest discrete structural differences as the main cause. Fishes are an obvious choice of taxon for the study of A,,,a2 variation. First, the widely different optical characteristics of their various environments are reflected in the exceptional range of their visual pigments; second, of all the vertebrates, fishes are probably the easiest to obtain in variety; third, about forty different species have already been adequately examined, thus laying a useful groundwork. 81

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H. J. A. DARTNALL AND

J. N. LYTHGOE

Accordingly, in the early part of 1963 we embarked on a series of fish-collecting expeditions, a project that still continues. The purpose of this paper is to report the data obtained from the 1963 expeditions and to discuss their significance. Conclusions drawn from the data were communicated verbally to the Ciba symposium on “Physiology and Experimental Psychology of Colour Vision” in London and to the Fourth International Photobiology Congress in Oxford, both held during July, 1964 (DARTNALL and LYTHGOE, 1964, 1965). METHODS

Collection, treatment and analysis of material

Fishes were obtained from the Marseilles and Malta areas of the Mediterranean, and from the Plymouth, Isle of Man and Aberdeen regions of the British coasts. Deep-sea fishes were caught on a Bay of Biscay cruise in the Marine Biological Association’s research vessel Sarsia. The fishes were collected by a variety of methods including trawling, underwater shooting and trapping, and purchase alive from fishermen. Only rarely was it possible to follow the ideal procedure of dark-adapting the living fish for several hours, and then making an immediate extract of visual pigment from its retinae. Facilities for such a course were available in some places, but we found in practice that if a fish were allowed to die in the dark, and was left there, a good enough yield of its pigment could be obtained from the retinae when removed one or two hours later. Moreover, once the retinae had been removed they could be stored frozen in darkness for ultimate extraction on return to the base laboratory in London. These modifications, frequently dictated by necessity, thus became a virtue, for we were able thereby to concentrate when abroad on the business of collecting and identifying. There is one aspect of the work where we are aware of no short cuts, and that is the question of identification. Even professional taxonomists make occasional mistakes, and we, as amateurs-though with the frequent assistance of local experts-are hardly likely to do better. Where field identifications were made the level of certainty was very high, and in all doubtful cases the fish was preserved for subsequent identification by the British Museum. The frozen retinae, in separate, labelled polythene tubes packed in vacuum flasks, were carried or despatched to this laboratory, where they were thawed out in darkness within three weeks of removal. The thawed retinae were washed in buffer solution of pH 4.6 and then extracted with 2 % aqueous digitonin solution. Fifteen parts of saturated sodium borate solution were then added to one hundred parts of each extract to bring the pH to about 8*5. The visual pigment solutions so prepared were analysed spectrophotometrically by the method of partial bleaching. In order to exploit the analytic power of this method to the utmost it is necessary to expose extracts to bleaching lights of the longest wavelength that is compatible with a measurable bleaching effect within a reasonable time. In this way that pigment in a mixture which has the longest Amazis bleached away with least interference through the concurrent bleaching of the shorter lmaz constituents. Our procedure was to use lights of nominal wavelengths between 750 and 700 nm for the initial bleachings, and to move reluctantly to shorter wavelengths only when, through denudation of the longer I,,, constituent in the extract, these lights ceased to make any further reasonable impression. Interpretation of the diflerence spectra

After all photosensitive pigment had been bleached away, the difference spectrum for each instalment of bleaching was calculated, plotted on graph paper, and a curve drawn

83

The Spectral Ciustering of Vista4 Pigments

carefully through the points. One method of determining the Intcrz of the portion of photosensitive material bleached in each stage is to estimate the wavelength position of the maximum by eye and, as an allowance for distortion caused by the negative photoproduct limb of the difference spectrum, to subtract 2 nm from the result. This method has two drawbacks. First, it is not always easy to find to nearer than 2 or 3 nm the position of the maximum in this flattish region of the curves, and secondly the 2 nm correction is somewhat arbitrary, being correct for frog “rhodopsin” at a pH of 8.5 but not necessarily so for pigments with different A,,,.

SO

580

600

620

b40

b&4

FIG. 1. Chart for determining i,oz from difference spectra data. The lines relate I,,,,,, with the wavelengths at which various heights (expressed as percentages of the maximum and labelled 10.1. 100) are located in the long-wave arm of the visual pigment template curve. From the long-wave arm of any experimental difference spectrum the wavelengths at which 10 . . .90 per cent heights occur are noted, and these are translated by means of this chart into nine estimates for Ima, the mean of which is taken.

We have devised a method of overcoming these difficulties. It is based on the visual pigment nomogram (DARTNALL, 1953) and our experience that the yellow photoproducts, when produced in mildly alkaline solution, have a quite negligible effect on the long-wave arm of the difference spectrum. While the photoproduct absorption may have a slight effect on the position of the difference spectrum maximum, the effect rapidly diminishes at longer wavelengths so that the rest of the arm accurately corresponds with a nomogram curve. It thus suffices to find by trial what nomogram curve accurately fits the long-wave arm of the experimental difference spectrum. The I maz of this nomogram curve is then the A,,, of the instalment of pigment bleached. This could be a laborious procedure, but is made easy by the chart shown as Fig. 1. This chart, which was calculated from the nomogram, relates A,,, with the wavelengths at which various ordinate heights (10 . . . 90 per cent) on the long-wave arm of the template curve are located. Thus the wavelengths at which the 10. . , 90 per cent heights of the experimental difference spectrum occur are noted. These waveiengths are translated by Fig. 1 to give nine estimates of the AmaE,each of which should not differ from the mean by more than 1 or 2 nm. The mean value is an accurate measure of the Anaas,reflecting the accuracy with which the nomo~am reproduces the spectra of retinenel pigments.

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H. J. A. DARTNALL

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It needs to be emphasized that agreement of this nature between an individual experimental difference spectrum and a nomogram curve is no guarantee of the homogeneity of the single instalment of bleaching. (Thus for any mixture of the difference spectra of pigments 4941 and 5061 (for example) a nomogram curve of intermediate i,,, could be found that would be a very close match.) All that the procedure described accomplishes is an accurate estimate of the overall i.ma2 of the portion bleached. The test of homogeneity comes from a consideration of how the i.mQz varies from instalment to instalment in a sequence of partial bleachings. If the i,,, is invariant in a sequence, then the extract must clearly have contained only one photosensitive pigment. In other cases, the extract must have contained a mixture of pigments. The elucidation of composition in such instances has been described elsewhere, as also have the limitations of the method of partial bleaching in genera1 (DARTNALL, 1962a). RESULTS

About forty different teleost species were satisfactorily analysed by the method of partial bleaching, and the results obtained are listed in Table 1 (“present work”) in the Appendix. The great majority of these fishes had single or mixed pigments based on retinener, although As pigments were also present in a few instances. The poor representation of the Aa pigments is not unexpected, for all our species were from marine or brackish water environments. The R,,, of the A1 pigments, however, were found to extend from 468 to 528 nm, a result that significantly widens the known range of this class in fishes (previous range 478520 nm), and it is our purpose to report that these i.,,, positions are not dispersed uniformly in the range but are clustered about discrete spectral positions. Thus when i.na, is plotted versus frequency (number of species) there is clear evidence of a clustering of pigments at positions near 487, 494 and 500 nm (Fig. 2, histogram labelled “present work”). When these results are added to those already published in the literature (listed in Table 1, and plotted in the Fig. 2 histogram labelled “other authors”) the total histogram reveals clustering at two additional positions, namely 506 and 5 12 nm. The present investigation provides 57 Ai pigments from 41 species; the work of previous authors gives 45 Ar pigments from an additional 42 species. Thus the total survey includes 102 Ai pigments (all of which have been tested for homogeneity by the method of partial bleaching-a criterion for inclusion in Table 1) from 83 different species of teleost fishes obtained from widely separated parts of the world, and from various depths in the oceans. Although this total is minute in comparison with the total number of teleost species, the representation of families (37) is a significant fraction of the whole. The immediate question, of course, is whether the data listed in Table 1 and plotted in Fig. 2 are really sufficient to support our view that the pigments are clustered about certain positions in the spectrum. A useful way to determine whether a set of data is periodic, even though the periodicity may be masked to a degree by random variation, is provided by the method of autocorrelation. This was first worked out in a biological context by MERCER (1960) in order to discover periodic rhythms in the activity of animals. In this method the data are considered as a waveform with amplitude plotted as ordinate and time as abscissa. The waveform is then delayed by time r and compared with the original waveform. If the two sets of data (original and delayed) are then multiplied together, the average of the products of amplitude over a long time is called the autocorrelation function, (p, for the delay r. If the data are essentially periodic cpwill be repetitive, since a

The Spectral CIustering of Visual Pigments

85

PRESENT WORK

IO t

a

6 .c ti 5t

4 2

(r0

INNING

IO

AVERAGE

a 6

470

x max positions

500

490

480

in

nm

of

A,

pigments

510

in

teleost

520

fishes

FIG. 2. The distribution of AI visual pigments in teleost fishes (Table I). The histograms give the plot of pigment I,oz vs. frequency of occurrence (number of species). F+reviously published data, present work, and the total data are separately shown. The “‘running average” curve was obtained from the total histogram by averaging the frequencies over three consecutive wavelengths, and gives the positions (figures) about which clustering is centred.

530

86

H. J. A.

DARTNALL

AND

J. N. LYTHGOE

displacement equal to one period will reproduce the original conditions. 9 thus has the same period as the original wave. In our case the number of fish species possessing an Ai pigment of particular irn,, is taken as the ordinate and wavelength is taken as abscissa. The “delay” r then becomes a wavelength displacement expressed in nanometers (nm). The products of original and delayed waveforms cannot usefully be averaged, for the wavelength range of i.,,, is so short (460-530 nm) that as displacement proceeds we run out of overlapping data and the small number of products would result in large random fluctuations in (o. Instead, therefore, we

FIG. 3. Autocorrelation diagram for the data of Fig. 2 (total distribution of A1 pigments in teleost fishes). For explanation, see text. The diagram indicates that the data of Fig, 2 are periodic, with a spectral interval of about 6.7 nm (13.3 ~2).

have considered the sum of the products as a function of displacement, with the result that the autocorrelation diagram for our data (Fig. 3) approaches zero at either end. Nevertheless it is clear that our data are periodic, for a “normal” distribution of i,,,, centred at, say, 500 nm would not yield the shoulders seen in Fig. 3. From Fig. 3 it appears that the “period” of our data is about 6.7 nm. However, this should not be interpreted too literally as signifying that the cluster positions are separated by equal wavelength intervals of this magnitude, for the occurrence of such a large number of pigments near 500 nm and at the neighbouring positions of 494 and 506 nm (Fig. 2) so weights the results as to mask any (small) progressive change of interval there may be throughout the range. Having thus established that the Al pigments of teleost fishes are clustered about certain positions in the spectrum, it becomes necessary to know where these positions are. Since even the most accurate I,,, determinations can seldom be closer than & 1 nm to the true positions, the averages of the frequencies (number of species) over three consecutive wavelengths have been determined in order to remove some of the random error and These running averages (bottom quarter of personal predilection of the experimenters.

The Spectral Clustering of Visual Pigments

87

Fig. 2) show five well-defined positions at 4865, 494, 5005, 506 and 511.5 nm, and two vaguely suggested positions at about 478 and 519 nm. DISCUSSION

Relation between the I,,,

of AI and A2 pigments

in identical-opsin pairs

All the visual pigments so far discussed have been based on vitamin Al ; those based on vitamin As generally have &, situated at longer wavelengths. Thus, WALD et al. (cited by WALD, 1953) found that the opsin of an Al pigment could be induced to combine with a suitable isomer of retinenea to form a synthetic analogue. The opsin used was derived from the 498 nm visual pigment of cattle. The synthetic A2 pigment so formed had I maz=517 nm, i.e. 19 nm higher than the natural Al pigment. WALD et al. (1953) also performed a similar experiment using the opsin of chicken iodopsin (A,,,= 562 nm). The As analogue (“cyanopsin”) formed in this case had IZmaz=620 nm, i.e. 58 nm higher than the natural Al pigment. These two examples are the only direct substitution experiments that have been carried out. There are several instances, however, where an Al/As pair of pigments occur naturally together in a retina, and where there are good reasons for believing that both pigments are based on a common opsin. Thus DARTNALL et al. (1961) found that the rudd, a freshwater fish, possesses an A1 pigment of irnaz= 510 nm and an As pigment of Ima%= 543 nm. Under natural conditions the proportions of these two pigments vary according to season, the 5432 pigment preponderating in the winter (short days) and the other pigment in the summer (long days). Moreover, the pigment composition of the retina can be varied in the laboratory by putting the fishes either into darkness, which causes an increase in the proportion of the As pigment, or into light, when the reverse change occurs. For these reasons it is considered that the two rudd pigments are based on the same opsin. Since this work, other fishes possessing an Al/As pair have been found to behave similarly (DARTNALL, 1962b, p. 411; BRIDGES, 1964a). In general, whenever a species contains only an Al/A2 pair it may be argued that both pigments are necessarily based on the same opsin. For otherwise, since both retinenes could combine with the different opsins, one would expect not two but four pigments to be present. In the pigment list of Table 1 there are eighteen teleost species shown as possessing only an Al/As pair. In Fig. 4 the &,,az of the Ai pigment is plotted against that of its A2 counterpart for sixteen of these pairs (empty circles). The two species omitted from this plot are the tenth (4671 and 5332) and the Scarid, Sparisoma cretense (4861 and 5202). The tenth pair are omitted because it is not absolutely certain that the 467 pigment is based on retinener (DARTNALL, 1952), and the scarid pair because we could not exclude from the results of our partial bleaching experiments the possibility that a third pigment was present having A,,,,,, intermediate between those reported. The results of the two substitution experiments of Wald et al (mentioned above) are shown in Fig. 4 by the filled circles. The figure shows that these results are consistent with those of the natural pairs, and that the relation between the I,,,,,, of pigments in the Al and AZ.series is approximately linear (cf. also DARTNALL, 1962b, p. 420). The clustering phenomenon

in the A2 pigments of fishes

From the relationship between the rZmaz of pigments in the Ar and As series (Fig. 4) it can be calculated that the As analogues to our A1 series (486.5,494,500-5,506 and 51 l-5 nm) would have their A,,,,, at 501,513,524,533 and 543 nm (DARTNALL and LYTHGOE, 1964).

H. J. A. DARTNALLAND

88

J. N. LYTHGO~

By a fortunate coincidence Bridges has been studying the distribution of the natural A? pigments. He has examined the visual pigments of eighteen North American freshwater and freshwater/marine fishes and reports (BRIDGES, 1964b, c) that when his results are added to those for the twelve British freshwater species, and for three marine labrids already in the literature, the R,,, are found to cluster around the positions 511.5 (this should read 512), 523.5, 534 and 543 nm.

520

SC0 i 480 480

I

I 500

I

I

520

I

f

540

560

A ng, of A,

Pipents

/

I

!

580

(A,),

600

620

nm

FIG. 4. The relationship between the i, m(2zof Al and A2 pigments in identical-opsin pairs. Open circles represent the sixteen naturally-occurring pairs; filled circles, the data obtained in Wald, Brown and Smith’s direct substitution experiments (see text). Equation to the line is %I= 04ol.2f 186.

These four positions are at, or very close to, four of the five calculated positions for AP analogues in our A1 series. This correspondence, in our view, gives additional substance to the hypothesis that %maz occur at discrete positions in both series. On the other hand one might expect some bias towards such a correspondence on account of the fact that the naturally paired Al/A2 pigments form a significant portion of both lists of pigments (particularly that of Bridges) and that these self-same pairs provide most of the data used for establishing the relationship between pigments in the two series (Fig. 4). To answer this distribution in fishes that possess on/~? A1 pigments objection we need to consider the I,,, in the one case, and only AZ pigments in the other. Twenty of the species listed in Table 1 possess AZ as well as AI pigments. Removal of these still leaves 63 species having A1 pigments only (81 pigments). These, when plotted in histogram form show clustering at almost exactly the same positions as before (actually at 486.5, 494, 500.5, 506.5 and 512 nm; see Fig. 5). In other words removal of the “paired pigment” species from the list (Table 1) has no material effect on the distribution pattern of A1 pigments. The list of AZ pigments (thirty-three species) is more seriously depleted by removal of “paired pigment” species. Thus Bridges’ eighteen new species (BRIDGES, 1964c) include nine such members, the fifteen AZ-containing species in the literature harbour another seven, including the only two 5432 examples (rudd and chub with 5432 and 5101), while our three new examples in Table 1 are of course paired with A1 pigments. The seventeen species

89

The Spectral Clustering of Visual Pigments

only AZ pigments that remain after this operation are listed in Table 2. They still show evidence of clustering, around the positions 513 nm (two examples), 524 nm (ten examples) and 534 nm (five examples). Thus we may conclude that the positions about which clustering of A,,, is observed in fishes that contain only AI pigments on the one hand and only A2 on the other are related in the same way as are the R,,, of naturally paired pigments (Fig. 4), which are based on the same opsin. possessing

A 480

490

501

positions of

A2 pigments in

510

5.20

53G

nm 540

550

I

rr

470

480 A ux

490

positions

500

of

510

520

530

A, pigments in nm

FIG. 5. The relationship between the spectral distributions of A1 and A2 pigments in teleost fishes. The data for the AI pigments are derived from those fishes that possess only AI pigments; the data for the As pigments are not restricted: i.e. they include all fishes that possess A2 pigments, some of which (see text) have A1 pigments as well. Note that the cluster positions for the AI pigments, when translated (sloping continuous lines) by means of Fig. 4, indicate theoretical positions for AP pigments (upper set of vertical dotted lines), some of which agree closely with those actually found (vertical continuous lines).

Fig. 5 displays the relationships between cluster points in the A1 and As series for fishes. The running-average curves shown are derived from frequency histograms. The curve for the A1 pigments is the same as in Fig. 2 except that the twenty species (out of eighty-three) in which there is pairing with As pigments have been excluded. The curve for the As pigments, on the other hand, includes data from all thirty-six species (in nineteen of which there is pairing with AI pigments). The sloping lines in the figure connect the observed La, positions in the AI series with analogous As positions as derived from Fig. 4. Four of these theoretical positions (vertical dotted lines in the top half of Fig. 5) agree to 1 nm with actual positions (vertical continuous lines). Distribution

qf visual pigments in animals other than jishes

So far we have been concerned only with the visual pigments of fishes. A study of the

visual pigments of other animals, however, suggests that clustering occurs in these also.

H. J. A.

90

DARTNALL

J. N. LYTHGOE

AND

The pigment I.,,, for these other animals are given in Table 3, which is based on the list given by DARTNALL (1962~) and augmented by a few recently reported values and some new ones of our own. The spectral distribution 0f2,,~ for the AI pigments in Table 3 are shown in histogram form in the upper part of Fig. 6. The running-average curve derived from the histogram (lower part of the figure) shows that although most of the pigments have i~mazcentred at 501.5 nm, there is also evidence of clustering at 492, 519 and 528 nm.

RUNNING

N -

AVERAGE

_

I25ol5

IO8642-

-m_*_/-h 460

410

480 x rn."

490

500

510

520

533

540

550

560

positions in nm of A, pigments in animals other than fishes

FIG. 6. The distribution of AI visual pigments in animals other than fishes. The histogram gives the plot of pigment kaz vs. frequency of occurrence (number of species). The “running average” curve was obtained by averaging the frequencies over three consecutive wavelengths, and gives the positions (figures) about which clustering is centred.

Three of these four cluster points are quite close to values found in fishes, the fourth (528 nm)-exhibited by some geckoes and the Nile crocodile-being just outside the teleost range. If we assume that there are two unfilled “places” in the large interval between the 5015 and 519 nm positions the values correspond quite we11with those for fishes, as shown below. Ima, positions (nm) for AI pigments Fishes 418.5 486.5 494 500~5 506 511.5 519 -

Other animals 492 501.5 519 528

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The Spectral Clustering of Visual Pigments

This near~ongruence of A1 cluster points suggests that they are not specific to different classes of animals. Some additional support for this idea is provided by the fact that in “other animals” even the As pigments (which are but poorly represented, being restricted almost entirely to the amphibia and more primitive animals) are positioned near 522 nm, thus showing close agreement with one of the AZ positions in fishes.

I1IIIIIIlIIIIIII 550

500

450 h -x

fasitiom

of

A,

piwn%

EJII

FIG. 7. The spectral locations of cluster points for A1 pigments plotted as a function of steps from the 500 nm position. The open circies indicate the seven consecutive positions found in teleost fishes (Fig. 2); the filled circIes the four positions found in animals other than fishes (Fig. 6), and placed so as to correspond by leaving two unfiRed positions in the middle.

Structural implications of clustering

In Fig. 7 the cluster positions for Al pigments in all animals are plotted in sequence, those for the fishes being shown by open circles and those for other animals as filled circles. For extensions in either direction of this plot we shall have to wait for future results, but if, as seems probable to us, the clustering phenomenon does extend throughout the whole spectrum “places” will have to be found eventually for such well-defined pigments as 5441 of the pigeon, the 5621 of chicken and turkey, and, in the other direction, perhaps, for the pigments 4621 (euphausiid shrimp), 4571 (gecko) and 4301 frog. In a previous paper in which passing reference was made to the clustering phenomenon (DARTNALL, 1964)it was suggested that the interval between successive I,,,,, positions was regular. The more extensive data we now possess seem to show, however, that the interval between successive positions tends to widen in both directions away from 500 nm, thus indicating that the plot is S-shaped (Fig. 7). On a wave number (frequency) basis, instead of the wavelength basis used in Fig. 6 the data likewise give an S-shaped plot. On the other hand the positions 486-5 nm at one end and 519 and 528 nm at the other are probably not so precisely located (through sparsity of examples) as the central five, and even now we cannot certainly exclude the possibility that the plot is linear. What are the implications of the clustering phenomenon? As already mentioned, since the prosthetic groups of all visual pigments are presumed to be of identical shape (1 I-&), the only way open for pigment variability would seem to be by variations in opsin or in the

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AND J. N.

LYTHGOE

linkages between opsin and prosthetic group that are additional to the main carbon-nitrogen bond. We incline to the view that clustering has a chemical basis, by which we mean that the Ima%positions are not destinations of convergent evolution in a system that intrinsically can vary continuously but rather that they relate to discrete permitted forms of the opsins or their linkages to prosthetic groups.

FIG. 8. Proposed schematic formulae for Ai visual pigments. The filled and open circles on the prosthetic groups represent dipoles that are “frozen” in position by oppositely charged atoms on the protein moiety. Thirty such forms are possible, according as either the filled or open circks are considered to be +ve or -ve. Formulae 1, 6, 10, 13 and 15 are considered less probable than the others, leaving twenty more-probable forms (see text). In these formulae the prosthetic groups are drawn without prejudice to their configuration, which is probably 114s.

It would be tempting to explain the stepwise variability of visual pigments by supposing that there can be greater or less extension of the prosthetic group conjugation into the protein part of the molecule via the nitrogen-carbon bond Iinking the two. Unfortunately for the simple application of this idea the photoproducts of the pigments have similar absorptive properties irrespective of those of the parent pigments. For example, the photoproducts of the pigments 6202 (“cyanopsin”) and 5232 (carp “porphyropsin”) are both centred at about 405 nm and similarly the photoproducts of the pigments 5621 (“iodopsin”) and 5021 (frog “rhodopsin”) are both centred near 385 nm. Similar remarks apply also to the earlier photoproducts, the meta-pigments. If there were extension of conjugation via the N-C bond one would expect there to be a stepwise variation in the products to correspond with that of the parents.

The Spectral Clustering of Visual Pigments

93

Because of this difficulty we tentatively propose the range of “formulae” shown in Fig. 8 as a working basis to account for pigment variability. They are really a development of the formula for “visual purple” proposed by one of us in which the nitrogen is supposed to be in the tervalent state, and an electric tension holds prosthetic group and protein together (DARTNALL, 1957). HUBBARD(1958) has suggested a very similar formula but with nitrogen in the quadrivalent condition. The subsequent work of BRIDGES (1962b) favours the alkaline (tervalent nitrogen) and not the acid (quadrivalent nitrogen) form of N-retinylidene opsin as that nearer to the structure of the visual pigment. In the formulae of Fig. 8 we are suggesting that dipoles, which normally might exist only transitorily in the conjugated chain of the prosthetic group, may be stabilised by electrostatic attraction to oppositely-charged atoms in the opsin molecule. In Fig. 8 fifteen possible positions for a prosthetic-group dipole are shown. There are another fifteen in which the charges are reversed. According to this suggestion the polarized condition into which the 1I-cis prosthetic group is “frozen” in a particular instance would be determined by the distance between the positive and negative charges on the opsin moiety. After partial disengagement of the prosthetic group from opsin (e.g. through isomerism following exposure to light) no trace of the “frozen” dipole would remain; possibly the opsin dipole would disappear also. The disappearance of these charges in viva might initiate the excitation process in the photoreceptor, and would also leave the prosthetic group of any one pigment in a state indistinguishable from that of any other. Not all of the structures shown in Fig. 8 are equally likely. It seems to us that those dipoles on the prosthetic group that are separated by only a single carbon+arbon distance (1, 6, 10, 13 and 15 in Fig. 8) are not likely to be “interested” in charges on the relatively remote opsin molecule. In other words twenty (not thirty) forms seem more probable to us. In this connection it may be noted that the spectral separation between the shortest (430 nm) and longest (562 nm) known I,,, in the retinener series is 132 nm, and this figure, divided by nineteen (spaces between twenty positions) gives 7 nm, or about the right size for the average interval observed in the retinenel series (Fig. 7). Acknowledgements-A survey of this kind would not be possible without the co-operation and active assistance of many people, among whom we wish particularly to mention the following: Mr. G. PALMER of the British Museum (Natural History); Prof. J. M. PARTSand Dr. J. VACELETof the Station Marine d’Endoume, Marseilles; Dr. N. S. JONESand Dr. J. M. JONESof the Marine Biological Station, Isle of Man; Dr. F. S. RUSSELL,Dr. E. J. DENTON, Mr. N. A. HOLMEand Mr. G. R. FORSTERof the Marine Biological Association, Plymouth; Mr. J. FRENDO, Mr. J. J. BARBARAand Mr. J. A. GALEA of the Department of Fisheries, Malta; Prof. E. BORG-COSTANZI,Prof. 0. L. THOMASand Dr. A. P. AUSTIN of the Royal University of Malta; Prof. C. P. LUCK of Makerere College, Uganda; Mr. B. B. PARRISHand Mr. C. C. HEMMINGSof the Department of Agriculture and Fisheries for Scotland; diving-partners Dr. A. D. BADDELEY,Dr. A. DOREYand Mr. J. D. WOODS,and the members of the Oxford, Cambridge and Imperial College Underwater Exploration Groups.

REFERENCES BLISS, A. F. (1946). The chemistry of daylight vision. J. gen. Physiol. 29, 277-297. BRIDGES,C. D. B. (1956). The visual pigments of the rainbow trout (Sulmo irideus). J. Physiol. 134,620-629. BRIDGES, C. D. B. (1959). The visual pigments of some common laboratory mammals. Nature, Land. 184, 1727-1728. BRIDGES,C. D. B. (1962a). Visual pigments of the pigeon (Columba livia). Vision Res. 2, 125-137. BRIDGES, C. D. B. (1962b). Studies on the flash photolysis of visual pigments. III. Interpretation of the slow thermal reactions following flash-irradiation of frog rhodopsin solutions. Vision Res. 2,201-214. BRIDGES,C. D. B. (1964a). Effect of season and environment on the retinal pigments of two fishes. Nature, Land. 203, 191-192.

H. J. A. DARTNALLAND J. N. LYTHGOE

94

(1964b). The dis~ibution ofvisual pigments in freshwater fishes. Abstr. Fourth ~nter~ut. Cangr. Photobio~ogy, Oxford, p. 53. The Beacon Press, Bucks. BRIDGES,C. D. B. (1964c). Periodic&y of absorption properties in pigments based on vitamin Aa from fish retinae. Nature, Land. 203, 303-304. BRIDGES, C. D. B.

Cottr~s, F. D. and MORTON,R. A. (1950). Studies on rhodopsin. 1. Methods of extraction and the absorption spectrum. B&hem. J. 47,3-10. CRESCXTELLI, F. (1956a). The nature of the lamprey visual pigment. J. gen. !%y&?!. 39,423-435. CRESCITELLI,F. (1956b). The nature of the gecko visual pigment. J. gen. Physiol. 40, 217-231. CRESCITELLI,F. (1958). The natural history of visual pigments. Ann. N. Y. AC&. Ski. 74,230-255. See also “The natural history of visual pigments” in Photobiology, pp. 3&51. Oregon State ColIege, Corvaliis, f958.

CRESCITELLI,F. (1963a). The duplicity theory: a phylogenetic view. In The general physiology of cell specialization (Ed. D. MAZIA and A. TYLER),pp. 367-392. McGraw-Hill, New York. CRESCITELLI,F. (I963b). The photosensitive retinal pigment system of Gekko gekko. J. gen. Physiol, 47, 33-52.

CRESCITELLI,F. and DARTNALL,H. J. A. (19.53). Human visuai purple. Nature, Land. 172, 195-196. CRESCITELLI,F. and DARTNALL,H. 3. A. (1954). A photosensitive pigment of the carp retina. J. P!z,vsio!. 125,607-627. CRESCIIELLI,F,, WILSON,B. W. and LILYBLADE,A. L. (1964). The visual pigments of birds. I. The turkey. Vision Res. 4275-280. DARTNALL, H. J. A. (1952). Visual pigment 467, a photosensitive pigment present in tenth retinae. J. Phy.dol. 116, 257-289. DAR~NALL, H. J. A. (1953). The interpretation of spectral sensitivity curves. &it. med. Bull. 9, 24-30. DARTNALL.H. J. A. (1955). Visual nimnents of the bleak (Alburnus lucidus). J. Phvsiol. 128. 131-156. DARTNALL;H. J. A. (i956): Further bbservations on the vi&al pigments of the clawed toad, X’enopus la&s. J, Physiol. 134, 327-338. DARTNALL,H. J. A. (1957). The VisuaiPigments. Methuen, London; John Wiiey, New York. DARTNALL, H. J. A. f19@). Viiual pigment from a pure-cone retina. Nature, Lwd. 188,475-479.

DARTNALL,H. 3. A. (1962a). Extraction, measurement and analysis of visual photopi~ent. In The Eye, Vol. 2 (Ed. H. DA~sx$, pp. 323-365. Academic Press, New York and London. DARTNALL,H. J. A. (1962b). The identity and ~~t~but~on of visual pigments in the animal kingdom. In The Eye, Vol. 2 (Ed. H. DAVSON), pp. 367-426, Academic Press, New York and London. DARTNALL, H. J. A. (1962c). Tables of visual pigments. In The Eye, Vol. 2 (Ed. H. DAVSON),pp. 523-533. Academic Press, New York and London. DARTNALL,H. J. A. (1964). The visual pigments: a photobiological study. The Edridge Green memorial lecture. Ann. roy. Colt. Surg. 35, 131-150. DARTNALL,H. J. A., LANDER, M. R. and MUNZ, F. W. (1961). Periodic changes in the visual pigment of a fish. In Progress in Photobiology. (Ed. B. C. CHRISTENSEN and B. BUCHMAN),pp. 203-213. Elsevier, Amsterdam. DARTNALL,H. J. A. and LYTHGOE.J. N. (1964). The distribution of visual pigments in fishes. Abstr. Fourth Ittternat. Congr. Photobiology, 0x&d,

pp. 71-72. The Beacon Press, Bucks.

DARTNALL,H. J. A. and LYTHGOE, J. N. (1965). The clustering of fish visual pigments around discrete spectral positions, and its bearing on chemical structure. Ciba Symposium on Ph.wiology and Experimental Psychology of Coiour Vision. J. and A. Churchill, London (in press). GOLDSMITH,T. H. (1958). On the visual system of the bee (Apis meltifra). Ann. N. Y. Acad. Sci. 74,223-229. HUBBARD, R. (1958). On the chromopho~s of the visual pigments. In VisscalFrobhzms ofCoIour, Nationat Physical Lab, Stasis, No. 8, pp. 153-169. H.M.S.O., London. HUBBARD, R, and Wn~n, G. (1960). Visual pigment of the horseshoe crab, ~j~~u~uspolyphemus. Nature, Land. M&212-215. KAMPA, E. M. (1955). Euphausiopsin, a new photosensitive pigment from the eyes of euphausiid crustaceans. Nature, Land. 175,996-997. LYTHGOE, R. J. (1937). The absorption spectrum of visual purple and of indicator yellow. 1. Physiol. 89, 331-358. MERGER, D. (1960). Analytical methods for the study ofperiodic phenomena obscured by randomfluctuations. Cold Spring Harbor Symposia on Quantitative Biology 25, 73-86. MUNZ, F. W. (1956). A new photosensitive pigment of the euryhaline teleost, GiMchthys mirabilis. J. gen. .Physia/. 4@, 233-249. h$UNZ,F. W. (1957). ~pkotosensjtive retinalpigments of marine a~euryhaiine teleostfishes. Ph.D. thesis,

University of CaEfornia, Los Angeles.

MUNZ, F. W. (1958a). F~t~~~tjve pigments from the retinae of certain deep-sea fishes, f. PhysioL 140, 2%235. MUNZ, F. W. (1958b). The photo~nsitive retinal pigments of fishes from relatively turbid coastal waters. J. gen. Physiol. 42,445-459.

95

The Spectral Clustering of Visual Pigments

MUNZ, F. W. (1958~). Retinal pigments of a labrid fish. Nature, Land. 181, 1012-1013. WALD, G. (1953). Vision. Fed. Proc. 12,606-611. WALD, G. (1957). The metamorphosis of visual systems in the sea lamprey. J. gen. Physiol. 40, 901-914. WALD, G. (1960). The distribution and evolution of visual systems. In Comparative Biochemistry, Vol. 1. Academic Press, New York and London. WALD, G. and BROWN, P. K. (1958). Human rhodopsin. Science 127, 222-226. WALD, G., BROWN, P. K. and KENNEDY, D. (1957). The visual system of the alligator. J. gen. Physiol. 40,

703-713.

WALD, G., BROWN, P. K. and SMITH, P. H. (1953). Cyanopsin, a new pigment of cone vision. Science 118, 505-508. WALD, G., BROWN, P. K. and SMITH, P. H. (1955). Iodopsin. J. gen. Physiol. 38, 623-681. WALD, G. and HUBBARD, R. (1957). Visual pigment of a decapod crustacean: the lobster. Nature, Lond. 180,278-280.

WALKER, M. A. (1956). Homogeneity tests on visual pigment solutions from two sea fish. J. Physiol. 133,56~. YOSHIZAWA, T. and WALD, G. (1964). Transformations of squid rhodopsins at low temperatures. Nature, Lond. 201, 340-345.

Abstract-Evidence is presented that the 1 maz of visual pigments are not distributed uniformly throughout the spectrum but, on the contrary, are clustered around certain discrete positions. Eight of these positions for the Al pigments have been determined from a consideration of published data and a comparably large amount of new data presented here for the first time. These positions are 478.5, 486.5, 493, 501, 506, 511.5, 519 and 528 nm. The relationship between A1 and AZ pigments, and the structural implications of the clustering phenomenon, are discussed. R&urn&On prksente les arguments en faveur d’une distribution non uniforme du lmzz des pigments visuels g travers le spectre et d’une accumulation autour de certaines positions disc&es. On d&ermine huit de ces positions pour les pigments Al en considdrant les donnCes publiCs ainsi qu’un ensemble assez vaste de don&es nouvelles p&sent&s ici pour la premiere fois. Ces positions sont 478,5,486,5,493,501,506, 511,5,519 et 528 run. On discute la relation entre les pigments A1 et A2 et les implications du ph&nom&ne d’accumulation avec leur structure. Zusammenfassung-Es wird gezeigt, dass die rltRoz von Sehstoffen nicht gleichm&ssig iiber das ganze Spektrum verteilt sind, sondern an bestimmten Stellen im Spektrum HIufungspunkte haben. Acht solche Punkte werden fiir Sehstoffe vom Al-Typ aufgnmd bisheriger Literaturangaben und einer vergleichbar grossen Anzahl neuer Daten hier zum erstenmal angegeben. Diese Punkte liegen bei 478,5, 486,5, 493, 501, 506, 511,5, 519 und 528 run. Die Beziehung zwischen Al und AZ-Sehstoffen und Ursachen fiir das Hgufungs-Phlnomen werden diskutiert. Pe!NOMe--npHBCACHbI

pacnpenennroxrr OKOJIO

OI’I~A’2JEHHbIX

AJIll Al-IIHrMeHTOB BaHHbIX

.QOKa3aTeJIbCTBa

paeHoMepH0

PaHe

AHCKpeTHO

6bmw

AaHHbIX

II~ACTi=lBJI~t?MbIX

3AeCb

CTpyKTj’pOii

TOTO,

paCIlOJIO~‘2HHbIX

U Ha OCHOBaHUH BIiepBblC.

3PWTeJIbHbIX

YTO

1 maz

BceMy cnerTpy,

YCTaHOBlIeHbI

519, U 528 HaHOMeTpOB (lMl= MemAy A1 H Az-lIsrMeHTaMu CKOfi

no

BTU

Ha

3PHTeJ’IbHblX

a,

WHKTOB.

OCHOBaHWW

Hanponle, B=Mb

paCCMOT~AuSl

IlHrMeHTOB

He

rpyrrmipyrox~ TaKUX ymt?

YHKTOB

ony6nuro-

6onbmoro KOJIHYeCTBB APHHbIX, 478,5, 486,5, 493, 501, 506, 511,5, B pa6oTe 06CymqaIoTCx B3aWMOOTHOmeHWR

OTHOCSiTCJIbHO WHKTbI:

lo-km). H 3HaYeHHe IItil-MCHTOB.

SBJIeHUZt

rpymMpOBaH?Ul

B CBR3U

C XUMBYe-

Sciaenidae

Carangidae

Serrarddae

Caproidae

Zeidae

Melamphaidae

Gadidae

Congridae

Ang~~llida~

Poeciliidae

Cyprinidae

Myctophidae

Argentidae

Salmonidae3

Sternaptychiidae

Clupeidae

FISWES

POSSESSING

Golden

Chub

Dory

Drumfish Queen&h

Scad Jack Mackerel

Grouper Grouper Sea Bass Painted Comber Comber

Boar fish

John

Haddock Whiting Poor Cod Codling

Conger

Eel

Bluntnose Minnow Rudd Green Tenth

Southeastern

Bleak Hornyhead Chub

Coho Salmon King Salmon Rainbow Trout

Hatchet fish Hatchet fish

Sprat

English name

OF TELEDST

Xenodermichthys copei Gilf Clupea spraltus I... Argyropelecus afinis Garman S~ernoptyx obscura Garman Oncorhyncus kisurch (Walbaum) Oncorhyncus rskawytscha (Walbaum) Salmo gairdneri Richardson 3a~hyIagus microp~ius Norman Barhyfagus weset4i 18olin Naasenia groenlandica (Reinhardt) Sears& koefoedi Parr Lampany~tus mexicanus Gilbert ~mpanyctus sp. Alburnus ~u~idus Day Hybopsis biguttata (Kirtiand] Leuciscus cephalus Day Noremigonus crysoleucas aura~us (Rafinesyuel Notemigonus crysoleucas boscii (Vafenciennes) No~ropis cornaius frontalis (Agassizj Pimepkales nolotus (Rafinesquel Scardinius eryrhrophrhalmus (L.) Tinca rinca (L.) Belonesax be&anus her Anguilta anguilta (t.) Conger conger (I..) Cadus aegiiifinus t, Gadus merfangus L. Gadus minurus L. Gadas morrhua Day Anrimora rostrata Giinther Meiamphaes bispinosas Gilbert Zeus faber L. Capros aper fi..) Epinephelus alexaudrinas (Cuvier and Valenciennes) Epinephelus guava (t.) Morone labrax (L.) Paracentropristis scriba (L.) Serranus cabrilla (L.) Caranx rrachurus Day Trachuris symmerricus (Ayres) Corvina nigra = Sciaena mgra Bloch Seriphus poiirus Ayes

PIGMENTS

Alepocephalidae

VISUAL

Species

1.

Family

TABLE

LEAST

Shiner

RI__

AT

Ax

5332

PtONENT

5332, c. 5502 5352 5432 5101, $32~ 5041, 5292 5021, 5362 5051, 53.52 5051, 5432 5101, 5332 4671, 5212 4981, 523% 5021, 4871, 4871 49511”) 5Ofi 494t 5001 4861 4881 493t 4941 5001, 49SI 5021,495r 5348 5031, 4991,4931 4941 4991 4951 5011 5041

505,;

49% 4861 5101.

480ll’) 501 l&Q 478, 4851 5071 5071 5071, 4981,4681 5011,4781 492, 41781

Pigments

ONE

c’t d.,

1964~

1964C

1958a

work work work work work

1956

present work MUNZ, 19SfYi

present work MUNZ, 1957

work work work work work

present work present present present present present

1961

and B~ow?-1 ex WAI.I>,

present work

MUNZ,

present present present present present

WALKER,

BROWN

DARTNALL,

1952 BRZMIES, 1964~

DARTNALL

BRIDGES,

hIDGLS,

DARTNALL, 1955 BRIDGES, 1964c DAKTNALL, 1962~ BRIDGES, 1964c BRIDGES, 19f%C

MUNZ, f958a present work

present work MUNZ, 1958a pmsent work present work

1956

195-l

ibUDGES,

1957

MUNZ,

1958a 1958a

MUNZ,

MUNZ,

MUNZ,

present work

present work

Reference

1960

Oblada sp. Obiada melanura (L.) Page&s erythrinus (L.) Sarpa salpa (L.) Spondytiosoma canrharus (L.) Merolepis chryselis (Cuvier and Valenciennes) Merolepis maena (L.) Embiotoca jacksoni Agassiz Hyperprosopon orgenteum Gibbons Chromis punctipinnis (Cooper) Labrus me&a Labrus mixfus L. Pimeiometopon pulchrum (Ayres) Sparisomn cretense (L.) Axoclinus carminalis (Jordan and Gilbert) Scomberomorus concolor (Lockington) Scomber scombrus L. Pneumarophorus japonicus diego (Ayres) Coryphoplerus nicholsii (Bean) Cievelandia ios (Jordan and Gilbert) Eucyclogobius newberryi (Girard) Gillichrhys mirobilis Cooper Caltionymus lyra L. Mugil cephalus L. Mugil curema Cuvier and Valenciennes Atherinops afinis (Ayres) Scorpaeno gurrara Girard Triglo ruculus L. Trigla gurnardus L. Corrus bubalis Euphrasen cortussp. Leptocottus armams Girard Gasmwsteus aculeacus L. Arnoglossus megasioma Day Rhombus laevis Day Rhombus maximus Day Pfeuronectes flesus Day Pleuronecfes limanaiz Day Pieuronecres microcephalus Day Solea vulgaris Day Sphaeroides annularus (Jenyns)

Spanish

Mackerel

Red Gurnard Grey Gurnard Sea Scorpion Sclllpin Staghorn sculpin Three-spined Stickleback Megrim Brilt Turbot Flounder Dab Lemon sole Dover sole Network puffer

Crested Goby Arrow Goby Tidewater Goby Mudsucker Dragonet Striped Mullet White Mullet Topsmelt

Monterey Mackerel

Bay Black Perch Wall-Eye Surf Perch Blacksmith Brown Wrasse Cuckoo Wrasse Californian Sheep Head Parrot fish

Black Bream Blotched Picarel

Saddle Bream Pandora

4air

4981 5181, 5001 5111,5081 so-h, 5001~‘) 5021.4931 5021 5001,

5011. 4941,4871

:t: 5111

SO01 4931’6’ 5121.4921

4991, 4991, SO81

4911 5001 5121 5121 5121 5121, 4861

s2zr,

SO61 5011 5oor, 4941 5011 5191,4991 5121,5011 SOI t SO61 SO61 4951(s) 4921, 5281, 4871 5201.4971 4861, SO01 4851

5222

5222

5222 522e

5202

5102

5222

1957

present work MUNZ, 1957 MUNZ, 1958b MUNZ, l9S8b MUNZ, 19SSb MUNZ. 1956 present work BRIDGES, l964c BRIDOES, 1964~ MUNZ. 19S8b MUNZ, 1957 present work present work present work MUNZ, 1958b MUNZ, 1958b MIJNZ, I957 present work present work present work present work present work present work present work MUNZ, l9S8b

MUNZ,

present work present work present work present work present work present work present work MUNZ, 19SEb MUNZ, 19S8b MUNZ. I957 BROWN and BROWN ex WALL, 1960 present work MUNZ, 1958~ present work MUNZ, 1957

(1)The extract also contained a minor amount of a rctinenel based pigment of km,1 in the region of 472 nm. (2)The extract also contained a minor amount of a pigment of undetermined category and or A,,* in the region of 506 nm. 13)MUNZ (privatecommunication). however, will report that in five species of salmon (Oncor/~ynrurgorbuscha(Walbaum). 0. kero (Walbaumf, 0. kirulrh (Watbaum). 0. nsrka (Wrrlbaum) and 0. rshawytrrlm(Walbaum) ) and in four species of trout (S&m ciarkii Richardson, S. wirdneri Richardson. S. s&r L., and S. lrulm L.) the retinal pigments are mixtures of 5031 and 527%. fat The extract also contained P minor ~moont of a pigment of undetermined category and of 1,,, in the region of 499 nm. 6%).&,., (with 13 nm corrcclion) re rted as 496 nm. Mean Amoi of difference spcctrom reported aa 497 nm. 1‘) WAt.KER(1957) gives A,.*=49 4”nm. w Laation of A,.% at 507 nm onty approximate.

Soleidae Tetraodontidae

Pleuronectidae

Gasterosteidae Bothidae

Cottidae

Atherinidae Scorpaenidae Triglidae

Callionymidae Mugilidae

Gobiidae

Scaridae Tripterygiidae Scombridae

Pomacentridae Labridae

Embiotocidae

Centracanthidae

Sparidae

PETROMYZONTIA

AMPHIBIA

CAUDATA

SALIENTIA

REPTIIJA

CROCODILIA

SAURIA

Class

Order

Order

Class

Order

Order

CYCLOSTOMATA

Order

Class

Phylum CHORDATA

ma&us

Coleonyx variegatus Gray Sphaerodactylus parkeri Grant Aristelliger praesignis (Hallowell) Oedura lesueuri (Dum et Bib) 0. monilis 0. robusta Phyllurus milii (White) Gehyra mutilata (Wiegmann) G. variegatus Dum et Bib Gekko gekko (L.)

Crocodylus niloticus Alligator mississipiensis

Xenopus Iaevis Rana temporaria R. esculenta R. aurora R. clantitans R. muscosa R. pipiens Bufo boreas halophilus B. morinus B. terrestris Hyla regilla Microhyla olivacea

Taricha torosa Necturus macuIosus

Petromyzon

Euphousia pacifca Homarus americanus Apis mellifera Limulus polyphemus

Phylum ARTHROPODA

Phylum MOLLUSCA

Species

5182

Nile Crocodile Mississipi Alligator

Pacific Tree Frog

CRESCITELLI, 1958, 1963a CRESCITELLI,1958, 1963a CRESCITELLI,1958, 1963a CRESCITELLI,1958 CRESCITELLI,1958, 1963a CRESCITELLI, 1956b. 1958, l963a CRESCITELLI, 1958, 1963a CRESCITELLI,1958, 1963a CRESCITELLI,1958, 1963a CRESCITELLI, 1963b

C, 5161 e. 5281, c 5001(e) 5301(Q) 5181 5181, 4571 c. 4901 5241 518I(Q’ 5281’~’ 5211, 4781

DARTNALL, 1956 LYTHGOE, 1937 DARTNALL. 1957 CRESCITELLI,1958 DARTNALL, 1962~ CRESCITELLI,1958 CRESCITELLI,1958 CRESCITELLI,1958 CRESCITELLI,1958 CRESCITELLI,1958 CRESCITELLI,1958 CRESCITELLI,1958 present work CRESCITELLI,l956b, 1958 WALo er al., 1957

c. 4301

5232

CRESCITELLI,1958 CRESCITELLI,1958

CRESCITE~LI, 1956a; WALD, 1957

5271, c. 5071, 4991

5021, 5021 5021, 5011 5021 5011 5021 5021 5011 5031 5011 5041

Clawed Toad Common Frog Edible Frog

Leopard Frog Western Toad

5021 5222

Pacific Coast Newt Mud Puppy

Lamprey 4971,

YOSHIZAWA and WALD, 1964

4871

Squid VERTEBRATES

KAMPA, 1955 WALD and HUBBARD, 1957 GOLDSMITH, 1958 HUBBARD and WALD, 1960

Reference

4621 5151 e. 4401(e) 5201

Pigments

Lobster Honey Bee Horseshoe Crab

1NVERTEBRATES

English Name

TABLE 3. VISUAL PIGMENTSOF ANIMALS OTHER THAN FISHES

Family

(Table 2 appears on p.100)

Aims bosehas

Bubo virginianus Oius asio

Lurus occidentalis

Pelicanus occidentalis Columba livia

AVES

GALLIFORMS

ANSBRIFORMES

STRIGIFORMES

CHARADRIIFORMES

PELECANIFORMES

Class

Order

Order

Order

Order Order

Galago crassicaudutus agisymbanus Macaca mulatta Pan sp. Homo sapiens Homo sapiens

Oryctolagus caniculus

Perognathus longimembris Sciurus carolinensis ieucotis Dipodomys deserti D. merriami D. mohavensis Cricetus auratus Onychomys torridus Neotoma lepida Rattus rattus Mus rnus~n~nsdomesticus Cavia porcelius Megaptera nodosa

Procyon Jotor psora

Hippopotamus amphibius Bos taurus

EDENTATA

PRIMATES

LAGOMORPHA

RODENTIA

CETACEA

CARNIVORA

ARTIODACTYLA

Order

Order

Order

Order

Order Order

Order

Bush

Baby

Hippopotamus OX

Hump-backed Racoon

Wood Rat Rat Mouse Guinea Pig

Rat Rat

Whale

Mouse

Kangaroo Hamster

Mohave Golden Grasshopper

Kangaroo

Merriam

Little Pocket Mouse Grey Squirrel Desert Kangaroo Rat

Man Rabbit

Man

Owl

Bronze Turkey

Sloth

monkey

Tailed

Chimpanzee

Rhesus

Bushy

Opossum Three-Toed

Pelican Pigeon

GUI1

Great Horned Screech Owt

Chicken Chicken Broad Breasted Duck

Rattlesnake BLISS,

CRESCITELLI,

1958

1958

1958

present work COLLINS and MORTON, DARTNALL, 1962~

4991 4991

1958

1962~

4991

CRESCITELLI,

DARTNALL.,

1959 1959

BRIDGES, BRIDGES,

CRESCITELLI. 1958 BRIDGES, 1959

1958 1958

CRESCITEL~I. CRESCITELLI,

1959 1958 1960

BRIDGES,

CRESCITELLI. DARTNALL.

4981 4971

5OO$(‘OI

1964

1950;

CRESCITELLI and DARTNALL, WALD and BROWN, 1958

BRIDGES, 1959 CRESCITELLI. 1958

present work

CRESCITELLI,

1958

1962a

CR~SCITELLI,

BRIDGES,

CRESCITELLI,

1958 1958

1962a

GRESCITELLI, CRESCITELLI,

BRIRGES,

et al.,

1955

CRESCITELLI, 1958 CRESCITELLI, 1958 BRIDGES, 1959

4921

1963a

1946 (see DARTNALL,

et al.,

CRESCITELLI

WALD

1958

1958.

CRESCITELLI. CRESCITELLI.

1958. 1963a 1958, 1963a

CRESCITELLI, CRESCITELLS

4981

z:

5011 5021

5021 SO11 5011

502t 501;(‘0)

5021 4971 4911 4971 493r

4941 4931

5021 5021 5031wJ~

5631, 5001 5601,5001 5621, c. 5051

5001

520& c. 4901 5201~5231@. I” 528x(“)

1953

1952)

(” The author reporti the actual 1 mo= for the diflerence rpcctmm to be at 450 nm but suggcrts true Lst of the visual pigmcn:ntat c. 440 nm. 0) Where the relevant data exists for Geekoes there seems (a be no ditTerence ia A,.# bstween difference spectra obtained with and without hydroxylamine. In these cases, therefore. the “2 nm correction” normrrlly subtracted from the observed difference spectra obtained in the absence of hydroxylamine (DARTNALL. 1962h) has not been applied. (1~ In contrast to footnote 9. where birds and mammals are concerned the prcscncc of hydroxylaminc does indeed lower the Ama2 of Ihe differsnce spectra. Thus a 2 nm correction has been applied to results obtained in the absence of hydroxylaminc. 111)Plotted PI 521 nm in Fie. 6.

Dideiphis virginiana

3radypus trhiactylus

MARSUPIALA

Class Order

MAMMALIA

COLUMBIFORMES

Gollus gallus Gallus gallus Mekagris sp.

SERPENTES

Hemtdactylu..frenatus Dum et Bib ii. turcicus (L.) Tarentola mauritanica CL.) Crotalns viridis helleri

Order

R I ~--continued

s Au

Order

Southern Brown Bullhead Whitesucker Warmouth Bluegill Shellcracker Florida Largemouth

Utnbra limi (K&land)

Carassius carassius (L.) Cyprinus carpio I.,. Gobio gobio (L.) Rutilus rutilus (L.) Tim-a tinca (L.) var. aurattu

Am&us

Catostomus commersonii (Lacepede)

Chaenobryttus corcmarius (Bartram) Lepomis macrochirus purpurescens Cope Lepomis microlophus (Gunther) Micropterus salmoides .floridcmrts (Le Sueur)

Crenilabrus festivus Crenilabrus massa

Umbridae

Cyprinidae

lctaluridae

Catostomidae

Centrarchidae

Labridae

nebulosrts mnrmoratus (Holbrook)

Crucian Carp Carp Gudgeon Roach Golden Tenth

.%0x Ilicius L.

Esocidae

Bass

5138 5132 >

5242 5252 5252 5252

5242

534:

5232 5232 5352 5352 5332

5262

1962~ and DARINALL, 1962~ 1962~ 1962~

1964~ 1964~ 1964~ 1964~ BROWN and BROH.N ex Wnr.n,

BRIDGES, BRIDGES, BRIDGES, BRIDGES,

BRIDGES, 1964~

BRIDGES, 1964~

DARTNALL, CRESCITELLI DARTNALL, DARTNALL, DARTNALL,

BRIDGES, 1964~

1952

DARTNALL,

5332 Mudminnow

Pike Centrai

BRIDGES, 19646

Reference

BRIDGES, 1964~

5252

Pigment

5232

Florida

Spotted Car

Bowfin

De Kay

Lepisosteidae

I...

Amia c&a

Lepisos~eus platyrhincus

Amiidae

English name

PIGMENTS or FISHES POSSESSING ONLY AS PIGMENTS

Species

VISUAL

Family

TABLE 2.

1960

1954