VOLUME
65, No. 3
SEPTEMBER
THE QUARTERLY
1990
REVIEW
of B IOLOGY OPTIMIZATION, CONSTRAINT, AND HISTORY IN THE EVOLUTION OF EYES TIMOTHY
H.
GOLDSMITH
ofBiology,Yale University, Department 06511 USA New Haven, Connecticut ABSTRACT
toexplore in an effort areexamined ofeyesandphotoreceptors oftheevolution Severalfeatures shows constraints. Opticaldesign andhistorical anddevelopmental therelative roles ofadaptation from physics. optima predictable whichinsomerespects approaches clearevidence ofadaptation, bydevelopmental howadaptation canbechanneled ontheother Theprimatefovea, hand,illustrates heritage. within both Drosophila lineages evolutionary reveal multiple ofopsins Theprimary structures relationwhose rods a subset Thepigments evolutionary ofopsins comprise andhumans. ofvertebrate rodpigTheevolutionary reasons forwhymost species. oftheparent shipsmapontothephylogeny based as there is noconvincing explanation areobscure, at500 + 10 nmr maximally ments absorb onadaptation alone. conesonthebasisofwhichopsingeneis expressed. Rodsareappropriately distinguishedfrom lines(eg, geckos, inphyletic snakes) is likely tobeinconflict withother definitions Thiscriterion histories opsin genes, followed ornocturnal bylossofoneormore that havelongdiurnal accompanied tolifein a different photicenvironment. adaptation bya secondary - is - a generalizable withthespectral associated composition oflight Colorvision perception vibehaviors. Thelatter arealsobasedonmultiple wavelength-specific distinguishedfrom usefully bealtered The butcannot classofreceptors bylearning. andmore thanonespectral sualpigments bothkindsofbehavior. in bees,whichexhibit isparticularly distinction forceful anextensive colorvision hasbeen involving shapedbyhistoricalfactors Theevolution ofprimate elaborate cones anda richer havemore mammalian nocturnality. Birds,bycontrast, period ofearly in a tetrahedron. Aviancolorspacecanberepresented setofvisualpigments. seems, selection, . .. couldhavebeen bynatural formed contrivances Tosuppose thattheeye,withall itsinimitable in thehighest degree. absurd confess, Ifreely HESE WORDS fromTheOriginofSpecies in this fashion, the eye has continued to perSelection wereDarwin'sintroduc- plex those who are determinedto be confused byNatural tion to a general outline of the selectiveforces by evolution (forboth historicaland contemthatcould have led to theevolutionofeyes.Per- poraryexamples, see Dawkins, 1986). Recent haps partlybecause Darwin pointed to the eye work on eyes, however,provides a number of ? 1990 by the StonyBrookFoundation,Inc. All rightsreserved.
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282
THE QUARTERLY REVIEW OF BIOLOGY
interestinginsightsabout theevolutionofphotoreceptorsand visual systemsin both thevertebrates and the invertebrates.Because little ofthisworkwas eitherperformedor presented with an explicit evolutionaryperspective,the present account may be useful both to biologists who work on eyes but tend not to think about explanations involving evolutionary cause, as well as to biologistswho are conversant with evolutionarytheorybut are less familiar withrecentfindingson visual systems. EVOLUTIONARY
ASPECTS
OF OPTICAL
DESIGN
Optimization and OpticalPerformance The idea thatevolutionoptimizes is subject to a sizeable caveat, but it remains a usefulhypothesis if one is going to be able to identify the relevantconditions and constraints(Maynard Smith,1978). In analyses ofbehaviorthis can presenta dauntingchallenge, identification for unanticipated circumstances in the real world of animals have a way of confounding the assumptions and boundary conditions in hypothesizedmodels of optimal behavior. In dealing with the interrelatedsensorytasks of maximizing spatial acuity and contrastsensitivity,however,both the "camera"eyes of Old World primatesand birds, as well as the compound eyesofdiurnal insects,presentclear examples of evolutionaryoptimization. The examples are clear forthe simple reason thatthe thingsthatshouldbe optimizedare determined by some straightforward rules of physics.The investigator'stask in examining the hypothesis of optimization is thereforeto ask how closelytheopticalperformanceofeyesofdifferent optical design approaches the limitsset by physics.This question has been posed and answered in several cases, albeit without direct referenceto optimization theory. Eyes representimages ofthe externalworld on two-dimensional sheetsofphotoreceptors, analogous to pieces offilm.It is usefulto recognize that evolution has produced two fundamentallydifferent optical systems,depending on whetherthe retinal sheet is concaveor convex (Kirschfeld,1969; Wehner,1981).When the photoreceptorsurfaceis concave, an optical image can be produced witha singlerefractile lens suitably placed in frontof the retina (Fig. 1A). This is the optical design ofthe vertebrateeyeas wellas ofthelargeeyesofcephalopod molluscs.On theotherhand, whenthereti-
VOLUME
65
nal surface is convex, it can forman image if the individualphotoreceptorelementsare sensitive only to a narrow cone of light incident perpendicularlyto theretinalsurface(Fig. 1B). This is theoptical principleon whichthe compound eyes of arthropods is based. Despite the verydifferentmodes of design thatunderlietheconstructionofthesingle-lens eyes of vertebratesand the compound eyes of arthropods,similarconsiderationsdetermine theircapacities to resolveimages (Kirschfeld, 1976; Snyder,1979; Land, 1981;Wehner,1981). An importantfactorin visual acuity (angular resolution)is theangularseparation (cI) ofthe individual receptors.Resolution is proportional to 1/4c.For a single-lenseye, 1/4c = fis, where f is the focallengthofthelens and s is the separationbetweenreceptors(Fig. 1C). The longer the focallength,the greaterthe magnification of the image in the focal plane. For a diurnal compound eye (apposition type), on the other hand, simple geometricalconsiderationsshow that 1/4c = rldl,where r is the radius of curvature of the eye and dl is the diameter of the ommatidial lens (Fig. 1E). In eithercase, angular resolutioncan be improved by increasingthe numerator(f or r) or decreasingthe denominator(s or di). In singlelens eyes,decreasings means makingthereceptors narrower(decreasing drin Fig. 1D) and packing them closer together.But there are limitsto how farthisprocess can be carried to advantage, fora second physicalconsideration intrudes. In resolving spatial detail, the responsesofadjacent photoreceptors mustbe distinguishednot only fromeach other but also fromthe noise that is due to the random natureofthe absorptionofsmall numbersofphotons. Put anotherway,receptorsmust be able to detectreasonablysmall differences in intensity,i.e.,theymustpossess adequate contrast senWhen only a fewquanta are absorbed sitivity. by a photoreceptor,the signal-to-noiseratio (S/N) is poor. From physical principles, S/N shouldimprovein proportionto thesquare root ofthe number ofquanta absorbed. The probabilityofphotoncapture(sensitivity)is proportional to the cross sectional area presentedby the photoreceptor,so making the diametersof the receptors(drin Fig. 1D) very small leads to a decrease in S/N and loss ofcontrastsensitivity,particularlydisadvantageous ifthe animal must optimize performancein conditions
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SEPTEMBER
EVOLUTION
1990
OF EYES
283
/1T
C 2
\
E
,V
~~~~d r
-
D FIG. 1. THE Two EYES OPTICAL SYSTEMS OF IMAGE-FORMING Image-formingeyes differ,depending on whetherthe retina is concave (A, single-lenseyes, as forexample in vertebrates)or convex (B, compound eyes of arthropods). AfterKirschfeld, 1969. C-E: As described in the text,several common parameters contributeto the optical performanceof eyes. 0J,angular separation of receptors;f, focal length; s, spacing of receptors; dr,diameter of receptor; dl, diameter of lenslet; r, radius of curvature.
of dim light. Furthermore,receptors act as wave-guidesforlightpropagating along their length. For waveguides less than about a ,um in diameter,a significantfractionoftheenergy (ofwavelengthsin thevisibleregionofthespectrum) is in a boundary region externalto the photoreceptor.Consequently,ifthe spacing of
receptorsis too close therewillbe optical crosstalkbetweennear neighbors.For thesereasons the outer segmentsofvertebratereceptorsare not smaller than about 1 ,umin diameter and are spaced at least 2 ,umapart. The obvious wayto increase thefocallength is to increase the diameter of the eye, but the
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284
THE QUARTERLY REVIEW OF BIOLOGY
size of the animal imposes an upper limit on the size of eye that can be accommodated in the skull. Larger eyes also permita larger entrancepupil, whichis also necessaryto preserve sensitivity,because the quantum flux on the image plane (the retina) is inverselyproportional to the square of the focal length. The size of the entrance pupil is important angleof an for another reason. The acceptance individual receptor(the angle throughwhich the receptorcollects light) is as importantfor between visual acuity as the angularseparation the acceptance receptors.If diffraction-limited, angle e is proportional to lid, where d is the diameter of the pupil, and Ais the wavelength of light (Snyder, 1979). In other words, if the pupil diameter becomes too small the image is broadened by diffractioneffects. In thehuman eyethesize oftheentrancepupil can adjust so thatoptical performanceapproaches what is theoreticallypossible. Fig. 2 showsthemodulationtransferfunction(MTF) forthe human eye withpupil diameters of 1.5 and 2.0 mm,comparedwiththetheoreticalperformanceofa lens ofthe same aperture.Imagine an eyeviewinga grating(the object) whose stripesmodulate sinusoidallyin intensity.The MTF is the ratio of image contrastto object contrast, plotted as a functionof spatial frequency of the grating. For the ideal lens, the fall in MTF with spatial frequencyis due to diffraction,and at its best, the human eye aplimit.Making the puproaches the diffraction and ifthe pupil smaller increases diffraction, pil dilates under conditions of dim light, the image is degraded by lens aberrations. How well is the retina designed to take advantageofthisopticalperformance?The finest sinusoidallymodulated gratingsthatcan be detected by the human eye have a frequencyof about 50 cycles/degree,which requires sampling intervalsat about 36 sec ofarc. The spacing of cones in the central fovea (see below), where visual acuity is highest is about 2 pm, which correspondsto a samplingintervalof25 sec if all cones participateor 35 sec forcones of one spectralclass. The densityof receptors in the foveais thereforeappropriate to utilize fullytheoptical performanceofthefrontofthe eye (Woodhouse and Barlow, 1982). Similar considerations determinethe optical performanceofcompound eyes.If thefacet diameter di is made too small, the visual field
VOLUME
65
1.0 0
E
N
ideallens
l.5mm 0
2.0mm
0 0 FIG.
2. EYE
NormalizedSpatialFrequency OPTICAL APPROACHES
PERFORMANCE
1.0
OF THE HUMAN
THE DIFFRACTION
LIMIT
describesthe function The modulationtransfer degradationofcontrastin theimagewithincreasmeasurements In thisfigure, ingspatialfrequency. for two pupil diametersare compared with a (Camplensofthesameaperture diffraction-limited bell and Gubisch,1966). The objectswereseries spatialfreof lightand dark stripesof different Frein intensity. quency,eachvaryingsinusoidally about50 cyquenciesarenormalizedtothecut-off, that In an ideallensthelossofcontrast cles/degree. See is describedin thiswayis due to diffraction. details. the textforfurther
oftheommatidiumis broadenedbydiffraction. In practice,however,facetsare notdiffractionlimited. The need forabsolute sensitivityand the preservationof S/N and contrastsensitivity imposes a lower limit of facet diameter of about 10 ,um, significantlylarger than the diffractionlimit. Absolute sensitivityand the concomitantmaintenance ofcontrastsensitivityhas thereforebeen the determiningfactor in the evolution of facet size. The obvious way to increase angular resolutionis thereforeto increasetheradius ofcurvature of the eye. As with the single-lenseyes of vertebrates,however,there are anatomical limitationsto the size of eye that can be carried on an insect'shead. This restrictionis partially circumventedin a veryinterestingway. Many insects have local regions of the eye in which the cornea is quite flatand the radius of curvatureof the eye is relativelylarge. The ommatidial axes in such a "fovealregion"are
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285
EVOLUTION OF EYES
SEPTEMBER 1990
,..
A
A,1.~~~~~~~~~~
X
Y
iF ,~~~~~~~~~~~~X
FIG. 3. THE OPTICAL PROPERTIES OF COMPOUND EYES VARY LOCALLY showstheheadofwhatappearstobe a tabanidfly(WehThisdrawingfromHooke's1665Micrographia regionsoftheeye. ner,1981).Note thevariationin facetdiameterand radiusofcurvaturein different Large facetsare foundwherethe radiusof curvatureis greatest;theseare regionsof small interomSee thetext matidialangle,smallacceptanceangle,highspatialacuity,and highcontrastsensitivity. details. forfurther
bysmallangles.The visual separated therefore approachesin size the(small) fieldtherefore angle,andvisualacuityislointerommatidial callyhigh.But becauseofthelargeradiusof facetdiametersare also large.Abcurvature, are and contrastsensitivity solutesensitivity thusalso kepthigh,and smallobjectsthatlie withinthevisualfieldofa singleommatidium
aremorelikelytogeneratea detectablesignal. Fig. 3 is RobertHooke's 1665 drawingof a tabanidflywitha so-calleddividedeyeinwhich and inthedorsalregionboththefacetdiameter arerelatively large.Fig. theradiusofcurvature 4 showsthatin a largenumberofarthropods facetdiameteris about twiceas largeas the limit. diffraction
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THE QUARTERLY REVIEW OF BIOLOGY
286
VOLUME
1.8
__
o
1.6
65
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EvOLUTION
REVEAL
ONE Focus
OF COMPOUND
2
OF NATURAL
EYES
In a number of insects,facetdiameters are about twice as large as theywould need to be at the diffraction limit,indicatingthat absolute quantum catch and contrastsensitivityare criticalparameters in natural selection. Axes: d, facetdiameter; r,radius of curvature.Other symbols indicate species fromwhich the measurements were made. Solid line: diffractionlimit. From Wehner, 1981.
in theFace of Optimization Constraint Embryological From optical considerations,thevertebrate retina has a curious design. Because the eye arises fromtheinvaginationand collapse ofan outpocketingof the brain (Fig. 5), lightmust pass throughthe neural retinabeforeit strikes the photoreceptorcells. This is equivalent to placing a thindiffusingscreendirectlyoverthe filmin your camera; it can only degrade the quality of the image. The eyes of cephalopod molluscsare not so compromisedin design;the vertebrateeyeis burdened withthisfeaturebecause of the mechanics of its ontogeny. Birds and primateshave regions of the retina specialized for high visual acuity called foveasin whichthecone cellsoccurin highden-
sityand convergelittleifat all on retinalinterneurons. In fovealregionsthe optical problem ofscatterhas been alleviatedbylateraldisplacement ofthe cell bodies ofthe neural retina,insofaras possibleclearingan opticalpathin front of the fovealcones (Fig. 6). This morphological example showsthatevolutionaryhistorycan shape both the need forand the reach ofadaptation, and it therebysets the stage forwhat follows. OF VISUAL
EVOLUTION
PIGMENTS
OpsinsHave SeveralAncient Lineages Evolutionary The visual pigmentsare intrinsicmembrane proteins
-
opsins
-
located in the photorecep-
tor organelles in the receptors. Opsins bind
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DevelopingRetina Ventricular Space PigmentEpithelium )
//
_
Optic Cup Wallof Forebrain
LenVece ChorioidFissure Optic Stalk
_
covalentlywith 11-cisretinoidsto formthe visual pigments.The retinoidchromophoresand thenatureoftheattachmentto opsin are shown in Fig. 7. Light causes an isomerization from the 11-cisto the all-transconfiguration,and ensuing changes in the conformationof the opsin are instrumentalin triggeringa biochemical cascade that leads to excitation of the photoreceptorcell. At thiswriting,the primarystructuresof 13 opsins have been reported,based, in 12 cases, on the nucleotide sequence in the encoding gene. With the exception of the opsin of cephalopod molluscs, the polypeptide chains
Space Ventricular FIG. 5.
REVERSAL ARISES
OF THE VERTEBRATE
287
OF EYES
EVOLUTION
1990
SEPTEMBER
RETINA
IN DEVELOPMENT
The vertebrateeye develops by invagination of the optic vesicle, formingthe optic cup. The cells lining the ventricularspace along the back wall of the optic cup become the pigment epithelium. The frontwall differentiatesinto the retina, with the
receptorsfacingthe old ventricularcavity,and theneuralretinafacingthevitrealsurface.Light musttherefore pass throughtheretinalinterneuronsbeforereachingthe receptors.The chorioid fissureis an extensionoftheoriginalinvagination oftheopticvesiclealongtheopticstalk.It eventuallypinchesclosedwithinthestalk,housingtheoptic nerve(axons oftheretinalganglioncells) and the blood supplyto the retina.
PigmentEpithelium
r
tReceptor
~outer Segments Cell Nuclei
t?InnerNuclearLayer 0
100
0
til.I
II
0 10 25 FIG. 6.
THE
CENTRAL
1i i..U.
FOVEA THE
OF THE
HUMAN
DISADVANTAGE
300 pm
200
150
100
50
EYE
SHOWS
OF A REVERSED
GanglionCell Layer
ADAPTATIONS
200 pm TO AMELIORATE
RETINA
In this view of the fovea,incident lightwould come frombelow. The inner nuclear layer and ganglion cell layer of the neural retina are evident at the edges of the fovea; in the center of the fovea the cell bodies are displaced in order to provide a clearer path for light to reach the photoreceptor cells. The fibersrunning fromthe central foveato make synaptic connection with bipolar cells and horizontal cells in the neural retina are the basal ends of the fovealcones. Xanthophylls deposited in the cell membranes of these fibersare responsible for the macula lutea,the yellow filterthat covers the foveal region of the human retina. Note that the receptors become wider and less densely packed with distance from the center of the fovea (lower, expanded scale). Modified from Polyak, 1941. This content downloaded from 128.143.023.241 on August 15, 2016 09:29:19 AM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c).
THE QUARTERLY REVIEW OF BIOLOGY
288
VOLUME
65
acids associated withthe C-terminal,cytoplasmic tail. When the polypeptide chains of the pigretinol(VitaminA) all-trans mentsare aligned-with judicious recourseto hypotheticalinsertionsand deletions(O'Tousa et al., 1985; Applebury and Hargrave, 1986), CH=O a process facilitatedby recognitionofthe helical domains -it is possible to see similarities retinal all-trans betweenpigmentsat theamino acid level.Identitiesofcodon or of amino acid are frequently referredto casuallyas homologies;however,hoCH=O -,, W NNN mology refersto descent froma common ancestor,and the assertion of homology in this 3,4-dehydroretinal all-trans situationrequires additional evidence (Reeck et al., 1987). Two analyses have been performedon these CH = O sequence data. First,fromthe comparisons of amino acids at correspondingpositions,a ma3-hydroxyretinal all-trans HO trixwas constructedof the number of differences betweeneach pair ofopsins, and thematrix used to calculate evolutionarydistances betweenopsins. Fig. 9 is an unrootedtree,calculated using FITCH, one ofJ. Felsenstein's programsin PHYLIP. The sum ofthelengths 11-cis retinal of the internodesbetween any pair of opsins CH =O is proportional to the number of amino acid substitutionsrequired to make the transition between opsins, and the nodes representhypothetical ancestral proteins. Startingwith this analysis we can begin to Schiff'sbase linkageof C=NH-opsin discuss homology.(a) The vertebraterod pig11-cis retinalwith opsin ments (human, mouse, cow, sheep, chicken) forma clusterwhose branchingpatternreflects INVOLVED STRUCTURES OF THE RETINOIDS FIG. 7. the phyleticrelatedness of the animals themIN VISUAL EXCITATION Threealdehydesare commonas chromophores selves. (b) Evidence to be discussed further ofvisualpigments;in each,lightcausesan isomer- below indicates that the long- and middlecon- wavelengthhuman cone pigment genes stem isomerto theall-trans izationoftheeleven-cis The retinoidsare attachedbya Schiff's froma gene duplication withinthelast 60 milfiguration. base linkageto the?-aminogroupofa lysinein he- lion years. Within each of these two branches at the of the tree (rod opsins, long- and mid-wavelix 7 oftheopsin,as shownby thestructure bottomof the figure. lengthhuman cones), opsinsare clearlyhomologous. There are reasons to believe that the long branches in Fig. 9 also reflectevolutionary are similarin length,varyingbetween348 and divergencewithin a larger familyof homolo382 amino acid residues. Analyses designed to gous proteins. (c) If the C-terminal tail of all identifyhydrophobicregions ofthe chain and the opsinsis "excised"and thetreerecalculated, (e.g., rhodopsin bovine to applied originally the branch to the octopus opsin seems quite Argos et al., 1982) suggestthatall thesevisual pigmentsfoldso as to formseven membrane- unremarkable(Fig. IOA). The extradivergence spanninghelices (Fig. 8). The octopus protein in Fig. 9 is thereforedue largelyto theaddition of severalscore amino acids to the C-terminal fitsthis model as well as the other opsins, but end. it differsin having about 90 additional amino A
,,
CH20H
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ALA
ALA
TAR
A
TAR SIR
L~~~~~~~~~~~~~I RU PRO
ASNL
R
v
L
289
E VOLUTION OF EYES
1990
SEPTEMBER
T
GIA GIN~~PHETH
A
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GA
LE~~~~~~~~~~~~~AL
L
AL ALA
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I
ITS A
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FIG.
L
~~~~~~~~~~~~~~~~~~ALA 0~~~~~~~~~~~~~~~GL
VAL1,A
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PR
A
ITS~~~~~~~~~~~~~~~~~~~
ERAAAN
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Ro
A
OPSINS
SHARE
A SIMILAR
STRUCTURE
CONTAINING
SEVEN
HELICES
The primary structureof opsins suggest a common secondary structureconsisting of seven a-helices spanning the membrane. This amino acid sequence is forcattle opsin and is fromHargrave et al., 1983. A version of the model that indicates similarities and differencesbetween species and is published in color can be found in Applebury and Hargrave, 1986.
(d) There are local regions of particularly highsimilaritybetweenproteinswhichsuggests conservationof homologous domains; forexample, the cytoplasmic loop between helices 1 and 2 and withinhelix 7 (which contains the binding siteofthechromophore)(Fig. 11).Appleburyand Hargrave(1986) have summarized extensivelythe evidence forlocal similarityin the subset of opsins that had been sequenced at thattime.But can we argue thattheseregions ofhighsimilaritydo notreflectconvergentevolution?Ifevolutionarydistancesare calculated usingjust the amino acids associated withhelix 7 (Fig. lOB), the relationshipsbetween opsinsare qualitativelysimilarto thosein thebulk of the molecule (Fig. 10A). Helix 7 therefore appears to be a region ofthe molecule thathas
been relativelyconserved during a process of divergentevolutionarychange. (e) The treesin Figs. 9 and 10were obtained with an algorithmthat minimizes the sum of whered is theobservedand d' is the (d - d')2/d2, expected distance between opsins. Alternatively,one can utilize informationin the sequencesof amino acids and calculate the minimum number of nucleotide changes in the geneticcode requiredto generatethemostparsimonious phylogenies (Felsenstein, 1982). Presented with the sequences (less the C-terminal amino acids that extend into the cytoplasm) forall 13 opsins, thePHYLIP program PROTPARS findsthat there are two equally only parsimonioustreeswhose topologiesdiffer in whetherthe branch to Cbh arises closer to
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290
THE QUARTERLY REVIEW OF BIOLOGY Ocp
oCp
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EVOLUTIONARY THIRTEEN
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65
VOLUME
DISTANCES
Crh
0~~~~~~~~~~~~~~~~~~~~~~~0
BETWEEN
OPSINS
This tree was calculated fromnumbers of differences in amino acids between pairs of opsins by using the distance matrixprogram FITCH (an implementation of the method of Fitch and Margoliash, 1967) in the PhylogenyInference Package (PHYLIP, version3.1) (Felsenstein,1985). Dl, Doc, D7a, and D7b are the pigments of retinular cells 1 to 6, the ocellus (Feiler et al., 1988; Pollock and Benzer, 1988), and two subsets of retinular cell 7 ofDrosophila; Ocp, theopsin ofoctopus; Rch, chicken rod pigment;Rbo, Rov, Rmo, Rhu are mammalian rod pigments from cattle, sheep, mouse, and human retinas; Cbh, Cgh, and Crh are the human cone pigments. [These three cone pigments are sometimesreferredto as blue, green, and red; later in thebody ofthe texttheyare called short(S), middle (M), and long (L), referring to theirrelativespectral positions.] Original referencesto the sequences are as follows: O'Tousa et al., 1985; Zuker et al., 1985 (Dl6); Cowman et al., 1986 (Doc); Zuker et al., 1987 (R7a); Montell et al., 1987 (R7b); Ovchinnikov et al., 1988 (Ocp); Takao et al., 1988 (Rch); Ovchinnikov et al., 1982; Hargrave et al., 1983; Nathans and Hogness, 1983 (Rbo); Findlay, 1986 (Rov); Baehr et al., 1988 (Rmo); Nathans and Hogness, 1984 (Rhu); and Nathans, Thomas, and Hogness, 1986 (human cone pigments). The vertebrateand most of the invertebrateop-
FIG.
10.
EVOLUTIONARY
REGIONS
DISTANCES
OF THE OPSIN
FOR
Two
MOLECULE
(A) Evolutionary relationships of 13 opsins calculated as for Fig. 9, but without including the C-terminal, cytoplasmicend of the molecule. This makes the mollusc (octopus) opsin seem less different from the arthropod and vertebratepigments, and emphasizes that the opsin of the short-wavelength sensitive human cone (Cbh) is about as closely related to the opsins of rods as it is to the other human cone pigments. (B) Evolutionary tree calculated for the stretch of amino acids in helix 7 and displayed on a 10 x expanded scale relative to the tree in A. The relationshipsof the opsins are approximatelythe same in A and B, suggestingthat helix 7 is a conserved region in a familyofhomologous proteins,and that the similaritiesbetween opsins in Fig. 11 are due to conservation rather than convergence.
sins differin length by several percent and were aligned for maximal similarity. The opsins of cephalopod molluscs have about 90 more amino acids on the C-terminal end, which accounts for the long branch to Ocp (compare with Fig. IOA).
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SEPTEMBER
helix7
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291
EVOLUTION OF EYES
1990
AAAAAA A
A conservative substitutions SIMILARITY
IN
12
OPSINS
loop betweenhelices Two regionsthatappear to be highlyconservedare loop 1-2,thecytoplasmic 1 and 2, and helix7, theregioninwhichthechromophore indicatesitesat which binds.The arrowheads The amino substitutions. conservative thereare manyaminoacid identities and numerousfunctionally acids are indicatedby thesingle-letter code. The double arrowheadunderhelix7 indicatesthelysine binds. (code k) to whichthe chromophore
rod opsins or to the othercone pigments(Fig. 12). In all otherrespectsthebranchingpatterns ofthesetwotreesare the same as thosein Figs. 9 and IOA. This parsimonymethod therefore agrees with the analysis based on pairwise differencematrices.In both methodsthequestion ofwhetherthe opsin ofblue-sensitivehuman cones is closerto rod or otherhuman cone pigmentsremains unresolvedby the data currentlyavailable. (f) There is a relativeconservationof gene structureimpliedby thepatternofintronsand exons betweenotherwisedistantlyrelatedproteins (Applebury and Hargrave, 1986), although this patternis not conserved between all members of the ensemble. (g) Recent workon the P-adrenergicreceptor(Dixon et al., 1986) and themuscarinicacetylcholinereceptor(Kubo et al., 1986) suggest that opsins are part of a still larger group of relatedproteinsthatfunctionby activatingsecond messengercascades. This interestingob-
servationalso indicatestheneed forcaution in concluding,solelyon thebasis ofhybridization of genomic fragmentswith cDNA forbovine rhodopsin(Martin et al., 1986), thatorganisms such as the alga Chlamydomonas or the bacterium Halobacterium halobiumhave rhodopsins engaged in sensory transduction. Other evidence for Halobacteriumdoes exist, however (Spudich and Bogomolni, 1984). The well-known"bacteriorhodopsin"from Halobacterium raisesan interestingevolutionary issue. This molecule is a proton pump rather than a visual pigment, the retinal cycles beand 13- ratherthan 11-cis tweenall-trans (Stoeckenius and Bogomolni, 1982), and there is no significantsimilaritywith cattle rhodopsin at theamino acid level(Hargrave et al, 1983). On theotherhand, bacteriorhodopsinsharessome conformationalfeatureswith the visual pigments, making seven helical traversesof the membrane (Henderson, 1977; Engelman and Zaccai, 1980). The question thereforearises
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THE QUARTERLY REVIEW OF BIOLOGY
292 D7a
D7b
Doc D1-6
OcP
Rch Roy
D7a
D7b
Doc D j6
Ocp
Rch Rov
FIG.
12.
Rbo Rmo Rhu Cbh
Rbo
Cgh Crh
Rmo Rhu C h Cgh
Crh
THE Two CLADISTIC TREES GENERATED BY A PARSIMONY
ANALYSIS
The analysis (PROTPARS) takes account of only aminoacid sequencedata.The twotreesdiffer in theplacementofthepigmentforhumanbluesensitivecones.
whethersimilaritiesin conformationare an example ofconvergentevolution,or whetherthe secondaryand perhapssome aspectsoftertiary structurehave been the only common foci of natural selection,the divergenceof visual opsins and bacteriorhodopsinhavingtakenplace so long ago thatthereis no trace of homology remaining in the primary structures.Lysoare thoughtto share zymesfromseveral-sources structuralsimilaritiesthroughdivergentevolutionfroma common precursordespite a loss of sequence homology (Weaver et al., 1985), but convincingexamples ofconvergentevolution of protein conformations(not just catalytic sites) are harder to identify(Creighton, 1984). The separation of human and bovine rhodopsinshas been used to suggest a rate of divergenceof about 1 percent every 107 years (Nathans, Thomas, and Hogness, 1986), but the divergencewithinthe familyBovidae indicatestheratecan be about twiceas fast.Such ratesare well withinthe range ofevolutionary rates of change of other proteins (Creighton, 1984). Furthermore,fishof the genus Salveli-
VOLUME
65
withmaximalabsorbance nushaverhodopsins (Amax) at 503,508,and 512nm(Bridges,1972). Each oftheseformsmustinvolveat leastone and theevolutionary aminoacid substitution, changeshavealmostcertainlytakenplace in time.In fact,twoofthepopupost-Pleistocene makeboth and hybrids lationsareinterfertile, parent pigments(McFarland and Munz, suggestthatunder 1965).These observations changes evolutionary thepropercircumstances in visualpigmentscan be tentimesas rapid as inferredfromthe data on mammalian rhodopsins. It is easyto maketoo muchofanyestimachangebased on tionofrateof evolutionary thesenumbers.First,thedatabaseisthin.Second,membersofa familyofproteinsmayditimeat a ratethatcorrevergeinevolutionary spondsto theaveragerateofaccumulationof mutationsthatproducelittleor no alteration in function ("neutral"mutations).The diverrodopsinsmaybe largely genceofvertebrate ofthisnature.On theotherhand,somesubset betweenthered-and greenofthedifferences sensitive conepigmentsis due tochangesthat havealteredfunction (i.e.,at leasttheabsorptionspectrum).Mutationsthatalterfunction can be theobjectofvigorousselectionat rates fardifferent fromthepassiveaccumulationof The longneutralsubstitutions. functionally cone pigmentsofthe and middle-wavelength seem to have dihuman retinanevertheless vergedfromeach othersometimeduringthe past65 millionyears,duringtheadaptiveradiationofmammals.But ofthis,morebelow. and regardlessofhowbacteIn summary, thevisualpigfitsintothepicture, riorhodopsin ofproa singlefamily mentsseemtorepresent teins.We now have data on twoorganisms, more thatsynthesize ourselvesand Drosophila, thanonekindofopsin.In eachofthesespecies thereareopsinsthatdivergedfromeachother classes, beforetheappearanceofcontemporary ifnotphyla,and stillotheropsinsthatdiverged at aboutthesametimeas theseparationofextantordersorfamilies.Andas wasmentioned above,thereis spectralevidencefortheevolutionofopsinat thelevelofspecies.The evolutionaryhistoryofopsinsis thusmultidimensional,and each ofthemajorbrancheson the treesin Figs.9, 10and 12(as wellas othermajor branchesthatremaintobe drawn)willeach awaitingexhaveitsownphyletic substructure
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SEPTEMBER
EVOLUTION OF EYES
1990
PALEOZOIC 500
I
400
300
293 MESOZOIC 200
CENOZOIC 100
0
Millions of years
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65, No. 3
SEPTEMBER
THE QUARTERLY
1990
REVIEW
of B IOLOGY OPTIMIZATION, CONSTRAINT, AND HISTORY IN THE EVOLUTION OF EYES TIMOTHY
H.
GOLDSMITH
ofBiology,Yale University, Department 06511 USA New Haven, Connecticut ABSTRACT
toexplore in an effort areexamined ofeyesandphotoreceptors oftheevolution Severalfeatures shows constraints. Opticaldesign andhistorical anddevelopmental therelative roles ofadaptation from physics. optima predictable whichinsomerespects approaches clearevidence ofadaptation, bydevelopmental howadaptation canbechanneled ontheother Theprimatefovea, hand,illustrates heritage. within both Drosophila lineages evolutionary reveal multiple ofopsins Theprimary structures relationwhose rods a subset Thepigments evolutionary ofopsins comprise andhumans. ofvertebrate rodpigTheevolutionary reasons forwhymost species. oftheparent shipsmapontothephylogeny based as there is noconvincing explanation areobscure, at500 + 10 nmr maximally ments absorb onadaptation alone. conesonthebasisofwhichopsingeneis expressed. Rodsareappropriately distinguishedfrom lines(eg, geckos, inphyletic snakes) is likely tobeinconflict withother definitions Thiscriterion histories opsin genes, followed ornocturnal bylossofoneormore that havelongdiurnal accompanied tolifein a different photicenvironment. adaptation bya secondary - is - a generalizable withthespectral associated composition oflight Colorvision perception vibehaviors. Thelatter arealsobasedonmultiple wavelength-specific distinguishedfrom usefully bealtered The butcannot classofreceptors bylearning. andmore thanonespectral sualpigments bothkindsofbehavior. in bees,whichexhibit isparticularly distinction forceful anextensive colorvision hasbeen involving shapedbyhistoricalfactors Theevolution ofprimate elaborate cones anda richer havemore mammalian nocturnality. Birds,bycontrast, period ofearly in a tetrahedron. Aviancolorspacecanberepresented setofvisualpigments. seems, selection, . .. couldhavebeen bynatural formed contrivances Tosuppose thattheeye,withall itsinimitable in thehighest degree. absurd confess, Ifreely HESE WORDS fromTheOriginofSpecies in this fashion, the eye has continued to perSelection wereDarwin'sintroduc- plex those who are determinedto be confused byNatural tion to a general outline of the selectiveforces by evolution (forboth historicaland contemthatcould have led to theevolutionofeyes.Per- poraryexamples, see Dawkins, 1986). Recent haps partlybecause Darwin pointed to the eye work on eyes, however,provides a number of ? 1990 by the StonyBrookFoundation,Inc. All rightsreserved.
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282
THE QUARTERLY REVIEW OF BIOLOGY
interestinginsightsabout theevolutionofphotoreceptorsand visual systemsin both thevertebrates and the invertebrates.Because little ofthisworkwas eitherperformedor presented with an explicit evolutionaryperspective,the present account may be useful both to biologists who work on eyes but tend not to think about explanations involving evolutionary cause, as well as to biologistswho are conversant with evolutionarytheorybut are less familiar withrecentfindingson visual systems. EVOLUTIONARY
ASPECTS
OF OPTICAL
DESIGN
Optimization and OpticalPerformance The idea thatevolutionoptimizes is subject to a sizeable caveat, but it remains a usefulhypothesis if one is going to be able to identify the relevantconditions and constraints(Maynard Smith,1978). In analyses ofbehaviorthis can presenta dauntingchallenge, identification for unanticipated circumstances in the real world of animals have a way of confounding the assumptions and boundary conditions in hypothesizedmodels of optimal behavior. In dealing with the interrelatedsensorytasks of maximizing spatial acuity and contrastsensitivity,however,both the "camera"eyes of Old World primatesand birds, as well as the compound eyesofdiurnal insects,presentclear examples of evolutionaryoptimization. The examples are clear forthe simple reason thatthe thingsthatshouldbe optimizedare determined by some straightforward rules of physics.The investigator'stask in examining the hypothesis of optimization is thereforeto ask how closelytheopticalperformanceofeyesofdifferent optical design approaches the limitsset by physics.This question has been posed and answered in several cases, albeit without direct referenceto optimization theory. Eyes representimages ofthe externalworld on two-dimensional sheetsofphotoreceptors, analogous to pieces offilm.It is usefulto recognize that evolution has produced two fundamentallydifferent optical systems,depending on whetherthe retinal sheet is concaveor convex (Kirschfeld,1969; Wehner,1981).When the photoreceptorsurfaceis concave, an optical image can be produced witha singlerefractile lens suitably placed in frontof the retina (Fig. 1A). This is the optical design ofthe vertebrateeyeas wellas ofthelargeeyesofcephalopod molluscs.On theotherhand, whenthereti-
VOLUME
65
nal surface is convex, it can forman image if the individualphotoreceptorelementsare sensitive only to a narrow cone of light incident perpendicularlyto theretinalsurface(Fig. 1B). This is theoptical principleon whichthe compound eyes of arthropods is based. Despite the verydifferentmodes of design thatunderlietheconstructionofthesingle-lens eyes of vertebratesand the compound eyes of arthropods,similarconsiderationsdetermine theircapacities to resolveimages (Kirschfeld, 1976; Snyder,1979; Land, 1981;Wehner,1981). An importantfactorin visual acuity (angular resolution)is theangularseparation (cI) ofthe individual receptors.Resolution is proportional to 1/4c.For a single-lenseye, 1/4c = fis, where f is the focallengthofthelens and s is the separationbetweenreceptors(Fig. 1C). The longer the focallength,the greaterthe magnification of the image in the focal plane. For a diurnal compound eye (apposition type), on the other hand, simple geometricalconsiderationsshow that 1/4c = rldl,where r is the radius of curvature of the eye and dl is the diameter of the ommatidial lens (Fig. 1E). In eithercase, angular resolutioncan be improved by increasingthe numerator(f or r) or decreasingthe denominator(s or di). In singlelens eyes,decreasings means makingthereceptors narrower(decreasing drin Fig. 1D) and packing them closer together.But there are limitsto how farthisprocess can be carried to advantage, fora second physicalconsideration intrudes. In resolving spatial detail, the responsesofadjacent photoreceptors mustbe distinguishednot only fromeach other but also fromthe noise that is due to the random natureofthe absorptionofsmall numbersofphotons. Put anotherway,receptorsmust be able to detectreasonablysmall differences in intensity,i.e.,theymustpossess adequate contrast senWhen only a fewquanta are absorbed sitivity. by a photoreceptor,the signal-to-noiseratio (S/N) is poor. From physical principles, S/N shouldimprovein proportionto thesquare root ofthe number ofquanta absorbed. The probabilityofphotoncapture(sensitivity)is proportional to the cross sectional area presentedby the photoreceptor,so making the diametersof the receptors(drin Fig. 1D) very small leads to a decrease in S/N and loss ofcontrastsensitivity,particularlydisadvantageous ifthe animal must optimize performancein conditions
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SEPTEMBER
EVOLUTION
1990
OF EYES
283
/1T
C 2
\
E
,V
~~~~d r
-
D FIG. 1. THE Two EYES OPTICAL SYSTEMS OF IMAGE-FORMING Image-formingeyes differ,depending on whetherthe retina is concave (A, single-lenseyes, as forexample in vertebrates)or convex (B, compound eyes of arthropods). AfterKirschfeld, 1969. C-E: As described in the text,several common parameters contributeto the optical performanceof eyes. 0J,angular separation of receptors;f, focal length; s, spacing of receptors; dr,diameter of receptor; dl, diameter of lenslet; r, radius of curvature.
of dim light. Furthermore,receptors act as wave-guidesforlightpropagating along their length. For waveguides less than about a ,um in diameter,a significantfractionoftheenergy (ofwavelengthsin thevisibleregionofthespectrum) is in a boundary region externalto the photoreceptor.Consequently,ifthe spacing of
receptorsis too close therewillbe optical crosstalkbetweennear neighbors.For thesereasons the outer segmentsofvertebratereceptorsare not smaller than about 1 ,umin diameter and are spaced at least 2 ,umapart. The obvious wayto increase thefocallength is to increase the diameter of the eye, but the
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284
THE QUARTERLY REVIEW OF BIOLOGY
size of the animal imposes an upper limit on the size of eye that can be accommodated in the skull. Larger eyes also permita larger entrancepupil, whichis also necessaryto preserve sensitivity,because the quantum flux on the image plane (the retina) is inverselyproportional to the square of the focal length. The size of the entrance pupil is important angleof an for another reason. The acceptance individual receptor(the angle throughwhich the receptorcollects light) is as importantfor between visual acuity as the angularseparation the acceptance receptors.If diffraction-limited, angle e is proportional to lid, where d is the diameter of the pupil, and Ais the wavelength of light (Snyder, 1979). In other words, if the pupil diameter becomes too small the image is broadened by diffractioneffects. In thehuman eyethesize oftheentrancepupil can adjust so thatoptical performanceapproaches what is theoreticallypossible. Fig. 2 showsthemodulationtransferfunction(MTF) forthe human eye withpupil diameters of 1.5 and 2.0 mm,comparedwiththetheoreticalperformanceofa lens ofthe same aperture.Imagine an eyeviewinga grating(the object) whose stripesmodulate sinusoidallyin intensity.The MTF is the ratio of image contrastto object contrast, plotted as a functionof spatial frequency of the grating. For the ideal lens, the fall in MTF with spatial frequencyis due to diffraction,and at its best, the human eye aplimit.Making the puproaches the diffraction and ifthe pupil smaller increases diffraction, pil dilates under conditions of dim light, the image is degraded by lens aberrations. How well is the retina designed to take advantageofthisopticalperformance?The finest sinusoidallymodulated gratingsthatcan be detected by the human eye have a frequencyof about 50 cycles/degree,which requires sampling intervalsat about 36 sec ofarc. The spacing of cones in the central fovea (see below), where visual acuity is highest is about 2 pm, which correspondsto a samplingintervalof25 sec if all cones participateor 35 sec forcones of one spectralclass. The densityof receptors in the foveais thereforeappropriate to utilize fullytheoptical performanceofthefrontofthe eye (Woodhouse and Barlow, 1982). Similar considerations determinethe optical performanceofcompound eyes.If thefacet diameter di is made too small, the visual field
VOLUME
65
1.0 0
E
N
ideallens
l.5mm 0
2.0mm
0 0 FIG.
2. EYE
NormalizedSpatialFrequency OPTICAL APPROACHES
PERFORMANCE
1.0
OF THE HUMAN
THE DIFFRACTION
LIMIT
describesthe function The modulationtransfer degradationofcontrastin theimagewithincreasmeasurements In thisfigure, ingspatialfrequency. for two pupil diametersare compared with a (Camplensofthesameaperture diffraction-limited bell and Gubisch,1966). The objectswereseries spatialfreof lightand dark stripesof different Frein intensity. quency,eachvaryingsinusoidally about50 cyquenciesarenormalizedtothecut-off, that In an ideallensthelossofcontrast cles/degree. See is describedin thiswayis due to diffraction. details. the textforfurther
oftheommatidiumis broadenedbydiffraction. In practice,however,facetsare notdiffractionlimited. The need forabsolute sensitivityand the preservationof S/N and contrastsensitivity imposes a lower limit of facet diameter of about 10 ,um, significantlylarger than the diffractionlimit. Absolute sensitivityand the concomitantmaintenance ofcontrastsensitivityhas thereforebeen the determiningfactor in the evolution of facet size. The obvious way to increase angular resolutionis thereforeto increasetheradius ofcurvature of the eye. As with the single-lenseyes of vertebrates,however,there are anatomical limitationsto the size of eye that can be carried on an insect'shead. This restrictionis partially circumventedin a veryinterestingway. Many insects have local regions of the eye in which the cornea is quite flatand the radius of curvatureof the eye is relativelylarge. The ommatidial axes in such a "fovealregion"are
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285
EVOLUTION OF EYES
SEPTEMBER 1990
,..
A
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Y
iF ,~~~~~~~~~~~~X
FIG. 3. THE OPTICAL PROPERTIES OF COMPOUND EYES VARY LOCALLY showstheheadofwhatappearstobe a tabanidfly(WehThisdrawingfromHooke's1665Micrographia regionsoftheeye. ner,1981).Note thevariationin facetdiameterand radiusofcurvaturein different Large facetsare foundwherethe radiusof curvatureis greatest;theseare regionsof small interomSee thetext matidialangle,smallacceptanceangle,highspatialacuity,and highcontrastsensitivity. details. forfurther
bysmallangles.The visual separated therefore approachesin size the(small) fieldtherefore angle,andvisualacuityislointerommatidial callyhigh.But becauseofthelargeradiusof facetdiametersare also large.Abcurvature, are and contrastsensitivity solutesensitivity thusalso kepthigh,and smallobjectsthatlie withinthevisualfieldofa singleommatidium
aremorelikelytogeneratea detectablesignal. Fig. 3 is RobertHooke's 1665 drawingof a tabanidflywitha so-calleddividedeyeinwhich and inthedorsalregionboththefacetdiameter arerelatively large.Fig. theradiusofcurvature 4 showsthatin a largenumberofarthropods facetdiameteris about twiceas largeas the limit. diffraction
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THE QUARTERLY REVIEW OF BIOLOGY
286
VOLUME
1.8
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EvOLUTION
REVEAL
ONE Focus
OF COMPOUND
2
OF NATURAL
EYES
In a number of insects,facetdiameters are about twice as large as theywould need to be at the diffraction limit,indicatingthat absolute quantum catch and contrastsensitivityare criticalparameters in natural selection. Axes: d, facetdiameter; r,radius of curvature.Other symbols indicate species fromwhich the measurements were made. Solid line: diffractionlimit. From Wehner, 1981.
in theFace of Optimization Constraint Embryological From optical considerations,thevertebrate retina has a curious design. Because the eye arises fromtheinvaginationand collapse ofan outpocketingof the brain (Fig. 5), lightmust pass throughthe neural retinabeforeit strikes the photoreceptorcells. This is equivalent to placing a thindiffusingscreendirectlyoverthe filmin your camera; it can only degrade the quality of the image. The eyes of cephalopod molluscsare not so compromisedin design;the vertebrateeyeis burdened withthisfeaturebecause of the mechanics of its ontogeny. Birds and primateshave regions of the retina specialized for high visual acuity called foveasin whichthecone cellsoccurin highden-
sityand convergelittleifat all on retinalinterneurons. In fovealregionsthe optical problem ofscatterhas been alleviatedbylateraldisplacement ofthe cell bodies ofthe neural retina,insofaras possibleclearingan opticalpathin front of the fovealcones (Fig. 6). This morphological example showsthatevolutionaryhistorycan shape both the need forand the reach ofadaptation, and it therebysets the stage forwhat follows. OF VISUAL
EVOLUTION
PIGMENTS
OpsinsHave SeveralAncient Lineages Evolutionary The visual pigmentsare intrinsicmembrane proteins
-
opsins
-
located in the photorecep-
tor organelles in the receptors. Opsins bind
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DevelopingRetina Ventricular Space PigmentEpithelium )
//
_
Optic Cup Wallof Forebrain
LenVece ChorioidFissure Optic Stalk
_
covalentlywith 11-cisretinoidsto formthe visual pigments.The retinoidchromophoresand thenatureoftheattachmentto opsin are shown in Fig. 7. Light causes an isomerization from the 11-cisto the all-transconfiguration,and ensuing changes in the conformationof the opsin are instrumentalin triggeringa biochemical cascade that leads to excitation of the photoreceptorcell. At thiswriting,the primarystructuresof 13 opsins have been reported,based, in 12 cases, on the nucleotide sequence in the encoding gene. With the exception of the opsin of cephalopod molluscs, the polypeptide chains
Space Ventricular FIG. 5.
REVERSAL ARISES
OF THE VERTEBRATE
287
OF EYES
EVOLUTION
1990
SEPTEMBER
RETINA
IN DEVELOPMENT
The vertebrateeye develops by invagination of the optic vesicle, formingthe optic cup. The cells lining the ventricularspace along the back wall of the optic cup become the pigment epithelium. The frontwall differentiatesinto the retina, with the
receptorsfacingthe old ventricularcavity,and theneuralretinafacingthevitrealsurface.Light musttherefore pass throughtheretinalinterneuronsbeforereachingthe receptors.The chorioid fissureis an extensionoftheoriginalinvagination oftheopticvesiclealongtheopticstalk.It eventuallypinchesclosedwithinthestalk,housingtheoptic nerve(axons oftheretinalganglioncells) and the blood supplyto the retina.
PigmentEpithelium
r
tReceptor
~outer Segments Cell Nuclei
t?InnerNuclearLayer 0
100
0
til.I
II
0 10 25 FIG. 6.
THE
CENTRAL
1i i..U.
FOVEA THE
OF THE
HUMAN
DISADVANTAGE
300 pm
200
150
100
50
EYE
SHOWS
OF A REVERSED
GanglionCell Layer
ADAPTATIONS
200 pm TO AMELIORATE
RETINA
In this view of the fovea,incident lightwould come frombelow. The inner nuclear layer and ganglion cell layer of the neural retina are evident at the edges of the fovea; in the center of the fovea the cell bodies are displaced in order to provide a clearer path for light to reach the photoreceptor cells. The fibersrunning fromthe central foveato make synaptic connection with bipolar cells and horizontal cells in the neural retina are the basal ends of the fovealcones. Xanthophylls deposited in the cell membranes of these fibersare responsible for the macula lutea,the yellow filterthat covers the foveal region of the human retina. Note that the receptors become wider and less densely packed with distance from the center of the fovea (lower, expanded scale). Modified from Polyak, 1941. This content downloaded from 128.143.023.241 on August 15, 2016 09:29:19 AM All use subject to University of Chicago Press Terms and Conditions (http://www.journals.uchicago.edu/t-and-c).
THE QUARTERLY REVIEW OF BIOLOGY
288
VOLUME
65
acids associated withthe C-terminal,cytoplasmic tail. When the polypeptide chains of the pigretinol(VitaminA) all-trans mentsare aligned-with judicious recourseto hypotheticalinsertionsand deletions(O'Tousa et al., 1985; Applebury and Hargrave, 1986), CH=O a process facilitatedby recognitionofthe helical domains -it is possible to see similarities retinal all-trans betweenpigmentsat theamino acid level.Identitiesofcodon or of amino acid are frequently referredto casuallyas homologies;however,hoCH=O -,, W NNN mology refersto descent froma common ancestor,and the assertion of homology in this 3,4-dehydroretinal all-trans situationrequires additional evidence (Reeck et al., 1987). Two analyses have been performedon these CH = O sequence data. First,fromthe comparisons of amino acids at correspondingpositions,a ma3-hydroxyretinal all-trans HO trixwas constructedof the number of differences betweeneach pair ofopsins, and thematrix used to calculate evolutionarydistances betweenopsins. Fig. 9 is an unrootedtree,calculated using FITCH, one ofJ. Felsenstein's programsin PHYLIP. The sum ofthelengths 11-cis retinal of the internodesbetween any pair of opsins CH =O is proportional to the number of amino acid substitutionsrequired to make the transition between opsins, and the nodes representhypothetical ancestral proteins. Startingwith this analysis we can begin to Schiff'sbase linkageof C=NH-opsin discuss homology.(a) The vertebraterod pig11-cis retinalwith opsin ments (human, mouse, cow, sheep, chicken) forma clusterwhose branchingpatternreflects INVOLVED STRUCTURES OF THE RETINOIDS FIG. 7. the phyleticrelatedness of the animals themIN VISUAL EXCITATION Threealdehydesare commonas chromophores selves. (b) Evidence to be discussed further ofvisualpigments;in each,lightcausesan isomer- below indicates that the long- and middlecon- wavelengthhuman cone pigment genes stem isomerto theall-trans izationoftheeleven-cis The retinoidsare attachedbya Schiff's froma gene duplication withinthelast 60 milfiguration. base linkageto the?-aminogroupofa lysinein he- lion years. Within each of these two branches at the of the tree (rod opsins, long- and mid-wavelix 7 oftheopsin,as shownby thestructure bottomof the figure. lengthhuman cones), opsinsare clearlyhomologous. There are reasons to believe that the long branches in Fig. 9 also reflectevolutionary are similarin length,varyingbetween348 and divergencewithin a larger familyof homolo382 amino acid residues. Analyses designed to gous proteins. (c) If the C-terminal tail of all identifyhydrophobicregions ofthe chain and the opsinsis "excised"and thetreerecalculated, (e.g., rhodopsin bovine to applied originally the branch to the octopus opsin seems quite Argos et al., 1982) suggestthatall thesevisual pigmentsfoldso as to formseven membrane- unremarkable(Fig. IOA). The extradivergence spanninghelices (Fig. 8). The octopus protein in Fig. 9 is thereforedue largelyto theaddition of severalscore amino acids to the C-terminal fitsthis model as well as the other opsins, but end. it differsin having about 90 additional amino A
,,
CH20H
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ALA
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289
E VOLUTION OF EYES
1990
SEPTEMBER
T
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SHARE
A SIMILAR
STRUCTURE
CONTAINING
SEVEN
HELICES
The primary structureof opsins suggest a common secondary structureconsisting of seven a-helices spanning the membrane. This amino acid sequence is forcattle opsin and is fromHargrave et al., 1983. A version of the model that indicates similarities and differencesbetween species and is published in color can be found in Applebury and Hargrave, 1986.
(d) There are local regions of particularly highsimilaritybetweenproteinswhichsuggests conservationof homologous domains; forexample, the cytoplasmic loop between helices 1 and 2 and withinhelix 7 (which contains the binding siteofthechromophore)(Fig. 11).Appleburyand Hargrave(1986) have summarized extensivelythe evidence forlocal similarityin the subset of opsins that had been sequenced at thattime.But can we argue thattheseregions ofhighsimilaritydo notreflectconvergentevolution?Ifevolutionarydistancesare calculated usingjust the amino acids associated withhelix 7 (Fig. lOB), the relationshipsbetween opsinsare qualitativelysimilarto thosein thebulk of the molecule (Fig. 10A). Helix 7 therefore appears to be a region ofthe molecule thathas
been relativelyconserved during a process of divergentevolutionarychange. (e) The treesin Figs. 9 and 10were obtained with an algorithmthat minimizes the sum of whered is theobservedand d' is the (d - d')2/d2, expected distance between opsins. Alternatively,one can utilize informationin the sequencesof amino acids and calculate the minimum number of nucleotide changes in the geneticcode requiredto generatethemostparsimonious phylogenies (Felsenstein, 1982). Presented with the sequences (less the C-terminal amino acids that extend into the cytoplasm) forall 13 opsins, thePHYLIP program PROTPARS findsthat there are two equally only parsimonioustreeswhose topologiesdiffer in whetherthe branch to Cbh arises closer to
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290
THE QUARTERLY REVIEW OF BIOLOGY Ocp
oCp
|Octopus
D7a
J|
octopusD7a |humlan
Drosophila D7b
cones
Cb|
t
j
c?
9.
EVOLUTIONARY THIRTEEN
)/
Rchl
C
A N-ternminal throughhelix 7
Rch
C h
o
FIG.
|
r
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Dl76
Cgh
|?
Doc
65
VOLUME
DISTANCES
Crh
0~~~~~~~~~~~~~~~~~~~~~~~0
BETWEEN
OPSINS
This tree was calculated fromnumbers of differences in amino acids between pairs of opsins by using the distance matrixprogram FITCH (an implementation of the method of Fitch and Margoliash, 1967) in the PhylogenyInference Package (PHYLIP, version3.1) (Felsenstein,1985). Dl, Doc, D7a, and D7b are the pigments of retinular cells 1 to 6, the ocellus (Feiler et al., 1988; Pollock and Benzer, 1988), and two subsets of retinular cell 7 ofDrosophila; Ocp, theopsin ofoctopus; Rch, chicken rod pigment;Rbo, Rov, Rmo, Rhu are mammalian rod pigments from cattle, sheep, mouse, and human retinas; Cbh, Cgh, and Crh are the human cone pigments. [These three cone pigments are sometimesreferredto as blue, green, and red; later in thebody ofthe texttheyare called short(S), middle (M), and long (L), referring to theirrelativespectral positions.] Original referencesto the sequences are as follows: O'Tousa et al., 1985; Zuker et al., 1985 (Dl6); Cowman et al., 1986 (Doc); Zuker et al., 1987 (R7a); Montell et al., 1987 (R7b); Ovchinnikov et al., 1988 (Ocp); Takao et al., 1988 (Rch); Ovchinnikov et al., 1982; Hargrave et al., 1983; Nathans and Hogness, 1983 (Rbo); Findlay, 1986 (Rov); Baehr et al., 1988 (Rmo); Nathans and Hogness, 1984 (Rhu); and Nathans, Thomas, and Hogness, 1986 (human cone pigments). The vertebrateand most of the invertebrateop-
FIG.
10.
EVOLUTIONARY
REGIONS
DISTANCES
OF THE OPSIN
FOR
Two
MOLECULE
(A) Evolutionary relationships of 13 opsins calculated as for Fig. 9, but without including the C-terminal, cytoplasmicend of the molecule. This makes the mollusc (octopus) opsin seem less different from the arthropod and vertebratepigments, and emphasizes that the opsin of the short-wavelength sensitive human cone (Cbh) is about as closely related to the opsins of rods as it is to the other human cone pigments. (B) Evolutionary tree calculated for the stretch of amino acids in helix 7 and displayed on a 10 x expanded scale relative to the tree in A. The relationshipsof the opsins are approximatelythe same in A and B, suggestingthat helix 7 is a conserved region in a familyofhomologous proteins,and that the similaritiesbetween opsins in Fig. 11 are due to conservation rather than convergence.
sins differin length by several percent and were aligned for maximal similarity. The opsins of cephalopod molluscs have about 90 more amino acids on the C-terminal end, which accounts for the long branch to Ocp (compare with Fig. IOA).
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SEPTEMBER
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Two
REGIONS
OF HIGH
A
D7a D7b Doc Dl Ocp
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p v iy -v f m p v iy -v f m p i iy -c b m
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291
EVOLUTION OF EYES
1990
AAAAAA A
A conservative substitutions SIMILARITY
IN
12
OPSINS
loop betweenhelices Two regionsthatappear to be highlyconservedare loop 1-2,thecytoplasmic 1 and 2, and helix7, theregioninwhichthechromophore indicatesitesat which binds.The arrowheads The amino substitutions. conservative thereare manyaminoacid identities and numerousfunctionally acids are indicatedby thesingle-letter code. The double arrowheadunderhelix7 indicatesthelysine binds. (code k) to whichthe chromophore
rod opsins or to the othercone pigments(Fig. 12). In all otherrespectsthebranchingpatterns ofthesetwotreesare the same as thosein Figs. 9 and IOA. This parsimonymethod therefore agrees with the analysis based on pairwise differencematrices.In both methodsthequestion ofwhetherthe opsin ofblue-sensitivehuman cones is closerto rod or otherhuman cone pigmentsremains unresolvedby the data currentlyavailable. (f) There is a relativeconservationof gene structureimpliedby thepatternofintronsand exons betweenotherwisedistantlyrelatedproteins (Applebury and Hargrave, 1986), although this patternis not conserved between all members of the ensemble. (g) Recent workon the P-adrenergicreceptor(Dixon et al., 1986) and themuscarinicacetylcholinereceptor(Kubo et al., 1986) suggest that opsins are part of a still larger group of relatedproteinsthatfunctionby activatingsecond messengercascades. This interestingob-
servationalso indicatestheneed forcaution in concluding,solelyon thebasis ofhybridization of genomic fragmentswith cDNA forbovine rhodopsin(Martin et al., 1986), thatorganisms such as the alga Chlamydomonas or the bacterium Halobacterium halobiumhave rhodopsins engaged in sensory transduction. Other evidence for Halobacteriumdoes exist, however (Spudich and Bogomolni, 1984). The well-known"bacteriorhodopsin"from Halobacterium raisesan interestingevolutionary issue. This molecule is a proton pump rather than a visual pigment, the retinal cycles beand 13- ratherthan 11-cis tweenall-trans (Stoeckenius and Bogomolni, 1982), and there is no significantsimilaritywith cattle rhodopsin at theamino acid level(Hargrave et al, 1983). On theotherhand, bacteriorhodopsinsharessome conformationalfeatureswith the visual pigments, making seven helical traversesof the membrane (Henderson, 1977; Engelman and Zaccai, 1980). The question thereforearises
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THE QUARTERLY REVIEW OF BIOLOGY
292 D7a
D7b
Doc D1-6
OcP
Rch Roy
D7a
D7b
Doc D j6
Ocp
Rch Rov
FIG.
12.
Rbo Rmo Rhu Cbh
Rbo
Cgh Crh
Rmo Rhu C h Cgh
Crh
THE Two CLADISTIC TREES GENERATED BY A PARSIMONY
ANALYSIS
The analysis (PROTPARS) takes account of only aminoacid sequencedata.The twotreesdiffer in theplacementofthepigmentforhumanbluesensitivecones.
whethersimilaritiesin conformationare an example ofconvergentevolution,or whetherthe secondaryand perhapssome aspectsoftertiary structurehave been the only common foci of natural selection,the divergenceof visual opsins and bacteriorhodopsinhavingtakenplace so long ago thatthereis no trace of homology remaining in the primary structures.Lysoare thoughtto share zymesfromseveral-sources structuralsimilaritiesthroughdivergentevolutionfroma common precursordespite a loss of sequence homology (Weaver et al., 1985), but convincingexamples ofconvergentevolution of protein conformations(not just catalytic sites) are harder to identify(Creighton, 1984). The separation of human and bovine rhodopsinshas been used to suggest a rate of divergenceof about 1 percent every 107 years (Nathans, Thomas, and Hogness, 1986), but the divergencewithinthe familyBovidae indicatestheratecan be about twiceas fast.Such ratesare well withinthe range ofevolutionary rates of change of other proteins (Creighton, 1984). Furthermore,fishof the genus Salveli-
VOLUME
65
withmaximalabsorbance nushaverhodopsins (Amax) at 503,508,and 512nm(Bridges,1972). Each oftheseformsmustinvolveat leastone and theevolutionary aminoacid substitution, changeshavealmostcertainlytakenplace in time.In fact,twoofthepopupost-Pleistocene makeboth and hybrids lationsareinterfertile, parent pigments(McFarland and Munz, suggestthatunder 1965).These observations changes evolutionary thepropercircumstances in visualpigmentscan be tentimesas rapid as inferredfromthe data on mammalian rhodopsins. It is easyto maketoo muchofanyestimachangebased on tionofrateof evolutionary thesenumbers.First,thedatabaseisthin.Second,membersofa familyofproteinsmayditimeat a ratethatcorrevergeinevolutionary spondsto theaveragerateofaccumulationof mutationsthatproducelittleor no alteration in function ("neutral"mutations).The diverrodopsinsmaybe largely genceofvertebrate ofthisnature.On theotherhand,somesubset betweenthered-and greenofthedifferences sensitive conepigmentsis due tochangesthat havealteredfunction (i.e.,at leasttheabsorptionspectrum).Mutationsthatalterfunction can be theobjectofvigorousselectionat rates fardifferent fromthepassiveaccumulationof The longneutralsubstitutions. functionally cone pigmentsofthe and middle-wavelength seem to have dihuman retinanevertheless vergedfromeach othersometimeduringthe past65 millionyears,duringtheadaptiveradiationofmammals.But ofthis,morebelow. and regardlessofhowbacteIn summary, thevisualpigfitsintothepicture, riorhodopsin ofproa singlefamily mentsseemtorepresent teins.We now have data on twoorganisms, more thatsynthesize ourselvesand Drosophila, thanonekindofopsin.In eachofthesespecies thereareopsinsthatdivergedfromeachother classes, beforetheappearanceofcontemporary ifnotphyla,and stillotheropsinsthatdiverged at aboutthesametimeas theseparationofextantordersorfamilies.Andas wasmentioned above,thereis spectralevidencefortheevolutionofopsinat thelevelofspecies.The evolutionaryhistoryofopsinsis thusmultidimensional,and each ofthemajorbrancheson the treesin Figs.9, 10and 12(as wellas othermajor branchesthatremaintobe drawn)willeach awaitingexhaveitsownphyletic substructure
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SEPTEMBER
EVOLUTION OF EYES
1990
PALEOZOIC 500
I
400
300
293 MESOZOIC 200
CENOZOIC 100
0
Millions of years
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