several times through the viscera and foot; thehemolymph, after being strained through 250->mnylon mesh, was add~ to an approximatcly equalvolume of collection fluid consistin of0SM NaCI,0.05M Hepes, 5 mM EGTA, 1 mMptosyl ar*uiinemethyl ester, Pepstatin A (3 I^g/ml, Boehringer-Mannheim), and leupeptin (3 p.g/ml, Boehringer-Mannheim) (pH 7.5). After the hemolymph prepa-ration was centifuged at 10,400g m a Sorvallcentrifiuge with an fB4 rotor, fluid between thepcllet and the floating lipid pad was removed andrecentrifuged. The supematant was then spun for 2hours at 80,000g in an SW-39 rotor with an L-2Bcckman ultracentrifuge; the resulting pellet wasused for gel electrophoresis.

9. U. K. Laemmli, Nature (Lond) 227, 680(1970).

10. Energy-dispersive spectroscopy was performed in aPhilips 400 analytic transmission dectron micro-scope fitted with an x-ray energy-dispersive spec-trometer. Thin sections (60 to 80 nm) of auriculartissue that had been prepared for standard electronmicroscopy were mounted on aluminum grids. Theanalyses were done on clusters ofhemocyanin mole-cules found within the hemocoelic spaces at 20-kVaccelerating voltage, 150 specimen tilt, and a probesize of 100 nm for 300 live seconds. Ratios ofenergy peaks for copper K- and L-shell electrons tobackground were 4.8 and 2.2, respectively.

11. A. C. Redfidd, Biol. Re. 9, 175 (1934).

12. R. C. Terwiliiger, N. B. Terwilliger, E. Schabtach,ConV. Biochem. Ph. 59, 9 (1978).

13. C. M. Yonge, Phios. Trans. R. Soc. London S6r. B.230, 79 (1941).

14. Most ofthis research was done at the Friday HarborMarine Laboratories, University ofWashington. Wethank S. A. Woodin, P. I11s, T. Schroeder, and D.Willows for critically rev*wi the manuscript.Spedal thanks go to D. Hcnry for editorial assist-ance. This rsearch was partially funded by DOEcontract DE-ACOZ-77EV04580. This is North-eastern University Marine Science Laboratory con-tribution No. 142.

9 September 1985; accepted 20 November 1985

Flying Primates? Megabats Have the AdvancedPathway from Eye to Midbrain

JOHN D. PETrIGREW

The pattern ofconnections between the retina and midbrain has been determined withdelctrophysiological and neuroanatomical methods in bats representing the two majorsubdivisions of the Chiroptera. Megachiropteran fiuit bats (megabats), Pter spp.,were found to have an advanced retinotectal pathway with a vertical hemidecussationof the kind previously found only in primates. In contrast, the microchiropteran batMaerodcwagsas has the "ancestral" or symplesiomorphous pattern of retinotectalconnections so far found in all vertebrates except primates. In addition to linkingprimates and megachiropteran bats, these findings est that flight may have evolvedtwice among the mammals.

T HE PATrERN OF CONNECrIONS BE-tween the retina and the midbrainsuperior colliculus (or tectum) dis-

tinguishes primates from all other mammalsso far studied (1). In strepsirhine and ha-plorhine primates (2), the pattern of cross-over of retinotectal fibers is like that of theretinothalamic fibers, with the result that thesuperior colliculus on one side of the brainsubserves both eyes but only the oppositehemifield of visual space (3). In contrast, inall other vertebrate groups so far examined,the crossover pattern of retinotectal fibersdiffers from that ofthe retinothalamic fibers,with the result that the superior colliculussubserves the whole visual field ofthe oppo-site eye (4-10). Bats have not previouslybeen studied in this regard, although thequestion is of some interest because somescholars have placed bats together with pri-mates, dermopterans, and tree shrews in thesuperorder Archonta (11). Moreover, thereare two distinct bat assemblages, one ofwhich has an advanced visual organizationwith similarities to that ofthe primates. Thelatter are megabats, suborder Megachirmp-tera, a uniform Old World group of large,vegetarian bats reliant on their highly devel-oped vision for foraging and obstacle avoid-ance (12). The microbats, suborder Micro-cbinptfera, are, by contrast, diverse, world-wide, small, predominantly insectivorousbats, all of which use ultrasonic emissionsfor echolocation (13). I now report that

fruit bats of the genus Pteropus have theadvanced pattern of retinotectal fiber con-nections like that of primates. In contrast,the microbat, Macrodrma gigas, has theplesiomorphous pattem of retinotectal pro-jection found in most vertebrates. The find-ings lend support to the older dassificationslinking primates and bats, with the newqualification that this applies only to theMe,gaciroptera. A corollary of this phyloge-netic hypothesis is that mammalian flighthas evolved independently more than once(14).The megabats used in this study were

three grey-headed flying foxes, Pteropus po-liocphalus, two black flying foxes, Pteropusalecto, and one little red flying fox, Pteropsscapulatus, taken from the wild near Bris-bane, Australia, and maintained in an out-door aviary. The microbats were two Aus-tralian ghost bats, Macrodermagiqas, takenfrom a colony of 400 breeding females atPine Creek, Northern Territory. Macro-dema was chosen because, in comparisonwith most other microchiropterans, it has arelatively well-developed and experimentallytractable visual system, with large eyes and atemporal retinal area of increased ganglioncell density which "looks" forward like thatfound in Pterops (Fig. 1). Two methodswere used to determine the pattern of reti-notectal fiber connections: electrophysio-logical and neuroanatomical. In the first,microelectrodes were used to record the

visual responses ofindividual neurons in thesuperior colliculus of bats anesthetized withintramuscular injections of ketamine andxylazine (15). The locations of visual recep-tive fields were plotted with respect to thezero vertical meridian for each eye, the latterhaving been established from the projectionof the ophthalmoscopically visible opticnerve head and data from retinal wholemounts giving the relation between the areaof increased ganglion cell density and thenerve head (16). A check on this estimatewas provided by binocular fields recorded inoverlying visual cortex (17). For the neuro-anatomical studies, injections of horserad-ish peroxidase (HRP), in some cases conju-gated to wheat germ agglutinin (WGA-HRP), were made into the superficial layersof the superior colliculus receiving retinalinput to enable retrograde labeling ofretinalganglion cells (18). The previous electro-physiological determination of the locationof the superior colliculus was used to guidethe placement of the HRP injections inthree cases. In five other cases, the overlyingvisual cortex was removed by suction abla-tion to expose the superior colliculus so thatinjections could be directed visually to itswhole retinal projection area. After survivaltimes of 24 to 72 hours, the animals werekilled with an overdose of anesthetic, per-fused with fixative, and the brain and eyesremoved. Retinal whole mounts were pre-pared to show the pattern of labeled retino-tectal ganglion cells and the brain sectionedand reacted to verify the injection site (19).

All three Pteropus spp. examined electro-physiologically had the primate pattern ofretinotopic organization in the superior col-liculus. At the caudal edge of the superiorcolliculus, neurons had receptive fields inthe far contralateral field, whereas at therostral edge receptive fields were locatedclose to the zero vertical meridian. No re-ceptive fields were found more than 50 (theaccuracy inherent in the method of plottinglandmarks) into the ipsilateral hemifield. A

Neuroscience Laboratory, Department of Physiologyand Pharnacology, University ofQueensland, St. Lucia,4067 Australia.

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majority of neurons could be driven indc-pendently by both eyes. InMacrodma, thetopographical arrangement in the superiorcolliculus was with the lateral edge repre-senting the lower visual field, the medialedge representing the upper field and thecaudal edge representing the extreme con-tralateral periphery as in Pterps, but re-sponses at the rostral edge could be obtainedfrom points in visual space which were as faras 300 across the zero vertical meridian inthe ipsilateral visual field. Moreover, fromnone of the 30 sites studied in the rostralpart of the colliculus ofMacrodema couldresponses be elicited from the ipsilateral eye.These electrophysiological differences be-

tween the retinotectal organization of thetwo groups ofbats were graphically illustrat-ed by the results of the retrograde labelingexperiments. Pteropus showed a vertical de-cussation line passing through the special-ized area of both retinas, with labeled reti-notectal ganglion cells on one side ofthe linebut not the other. Densities of labeled reti-notectal ganglion cells were low (2 to4 x 102'mm2) compared with the densi-ties (2 to 4 x 103 mm-2) of the total popu-lation of neurons in the retinal ganglion celllayer (20), although both ipsilateral andcontralateral densities were comparable. Thesame pattem was observed in all five Ptero-pus (three species) studied. Macrodermashowed no decussation line, with labelingacross the whole extent of the contralateralretina and no labeled ganglion cells at all inthe ipsilateral retina. The density of labeledcells reached 2.5 x 103 mm-2 in the contra-lateral retina, a significant fraction of thetotal population of neurons in the retinalganglion cell layer, which peaked at3.5 x 103mm-2 (Fig. 1).These results show that a major, repre-

sentative genus of megabats has the ad-vanced or synapomorphous pattern of reti-notectal organization found in primates. Incontrast, one of the most highly visual mi-crochiropteran bats known does not havethis pattern, but shares the primitive orplesiomorphous condition with othergroups of mammals (and all other verte-brates). Apart from the musculoskeletal ad-aptations associated with the wing itself,there are no known synapomorphous char-acters which unequivocally link megabatsand microbats (21), so a natural questionarises as to the relative weighting to be givento these two conflicting synapomorphies:the wing and the hemidecussated retinotec-tal pathway. Is it more likely that flightevolved in parallel in two separate lines ofmammals, one of them ancestral primates,or that megabats have evolved an advancedmode of retinotectal organization indepen-dently of primates (22)?

L4 MARCH I986

Separate origins for the wings of mega-bats and microbats are supported by thepresence of a number of small, but consist-ent skeletal differences between them (23),the presence of other synapomorphies link-ing megabats to primates but not to micro-bats (24), the appearance of sustained flightin at least three separate nonmammalianlines (25), the numerous appearances ofgliding flight with three separate "inven-tions" in the marsupials alone (26), and afossil record indicating an origin for micro-bats more than 50 million years ago (27)compared with the recent fossil megachirop-terans which have even been classified asprimates (28). Separate origins for the ad-vanced retinotectal organization in fruit bats

and primates are not supported by the closeidentity between the complex details of thepathways involved in each group (29), norby their absence in such highly visual andarboreal mammals as squirrels, cats, treeshrews, and phalangers (5-10). Serologicalevidence linking dermopterans and primates(30), the homologous structure and innerva-tion of the patagium in dermopterans andmegachiropterans (11), the present evidencelinking primates and megachiropterans, plusthe previous morphological evidence linkingdermopterans, primates, and megachirop-terans (23) all taken together, suggest that afruitful line of future investigation will bethe evolutionary relations of these threegroups of mammals. In the meantime it

N -

All cells

b

Contralateralretinotectal

d

Ipsilateralretinotectal

Fig. 1. Differing patterns ofretinotectal organization in a megachiropteran bat, Pteropuspolcphalus (a,c, and e) and a microchiropteran bat, Maodemagigas (b, d, and f). Retinal whole mounts are shownwith contours of isodensity for cells in the retinal ganglion cell layer (x 103 mm-2 in a, b, d, and f;x 10 mm-2 in c and e), either stained with cresyl violet (AU cels, a and b), or reacted to show thepresence ofHRP in ganglion cells contralateral (c and d) and ipsilateral (c and f) to the injection site inthe right superior colliculus. Abbreviations: N, nasal; T, temporal. The ipsilateral retinas (from righteyes, e and f) have been mirror-reversed to facilitate comparison with the others. Stippling schematicindicates only the extent ofthe labeled retinal area. Both kinds of bats have a similar topography in theretinal ganglion cell layer (a and b) with a horizontal "streak" and a dearly defined area centralis intemporal retina subserving the region of frontal visual space. Despite the similarities in overalltopography in the ganglion cell layer, there are dramatic differences in the topography ofthe retinotectalganglion cells. In Pteropus retinotectal ganglion cells are found in relatively low density compared withthe total in the ganglion cell layer, but both ipsilateral and contralateral retinas have comparabledensities. There is a sharp decussation line, dose to the vertical meridian of both ipsi- and contralateralretinas. No labeled ganglion cells could be detected (despite a long search at high power for any weaklylabeled ones) temporal to the dotted line marked (0) in the contralateral retina (c) or nasal to the dottedline marked (0) in the ipsilateral retina (e). InMaacdema, retinotectal ganglion cells were found onlycontralateral to the injection site, with no indication of a decussation at the zero vertical meridian.

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seems appropriate to propose that an earlybranch of the primate tree may have devel-oped the power of flight long before thehominid branch even dreamed of it.

REFERENCES AND NOTES

1. J. M. Aliman, Prog. Physiol. Psychol. 7, 1 (1977).2. By this test and others, members of the Menotyphla,

which indudes the tree shrew, Tupaia, are nowexcluded from the primates, although they are theprimates' dosest "sister group" (1, 9).

3. Proimian Galgo (R. H. Lane, J. M. Alman, J. H.Kaas, F. M. Miezin, Brain Res. 60, 335 (1973)];new world primates, Sais [S. Kadoya, L. R.Wolin, L. C. Massopust,J. Conwp. Neurd. 142,495(1972)]; and Aotis [R. H. Lane et at., ibid.]; oldworld monkey, Maca [M. Cynader and N. Ber-man,J. Neurphsiol. 35, 187 (1972)].

4. This is the "primitive" or plesiomorphous pattern[E. 0. Wiley, Phykejn (Wiley, New York,1981)].

5. Anurans [R. M. Gaze, Q. J. Exp. Physiol. 43, 209(1958)]; teleosts [H. Schwassman and L. Kruger,J.Comp. Neurl. 124, 113 (1965)]; birds [H. Bravoand D. Pettigrew, ibid. 199, 419 (1981)]; lizard[B. S. Stein and N. S. Gaither, ibid. 202, 69(1981)].

6. Rodents: rat [K. S. Lashley, J. Conm. Neumrl. 59,341 (1934)], squirrel [W. C. Hall, . H. Kaas, H.Killackey, I. T. Diamond, J. Neurhysiol. 34, 437(1971)], and ground squirrel [C. N.Woolsey,T. G.Carlton, J. H. Kaas, F. J. Earls, VisionRes. 11, 115(1971)].

7. Rabbit [A. Hughes, Docum. Ophthahmol. (DenHa) 30, 33 (1971)].

8. Cat jM. Straschili and K. P. Hoffinan,BrainRes. 13,274 (1972)].

9. Opossum [C. Rocha-Miranda, R. Mendez-Otero,A. S. Ramoa, E. Volchan, L. G. Gawryszewsli, inDevelopment of Visual Pathways in Mammals, J.Stone, B. Dreher, D. Rapaport, Eds. (Liss, NewYork, 1984), pp. 179-198].

10. Tree shrew [J. H. Kaas, J. K. Harting, R. W.Guillery, Brain Res. 65, 343 (1974)].

11. W. K. Gregory, Bull. Am. Mus. Nat. Hist. 27, 332(1910).

12. W. A. Wimsatt, Biogy of Bats (Academic Press,New York, 1970).

13. M. B. Fenton, Rev. Biol. 59, 33 (1984).14. J. D. Smith, in Biolo of Bats of the New World

Family Phyllostomatidae, R. J. Baker, J. K. Jones, Jr.,D. C. Carter, Eds. (Texas Tech Press, Lubbock,1976), part 1, pp. 49-69; in Major Pattern inVertebrate Evolution, M. K. Hecht, P. C. Goody, B.M. Hecht, Eds. (Plenum, New York, 1977), pp.427-438; in Proc. Fifth Interationa Bat ResearebConference, D. E. Wilson and A. L. Gardner, Eds.(Texas Tech Press, Lubbock, 1980), pp. 233-244.

15. In two animals supplementation with Nembutal(pentobarbitone sodium) was used.

16. In Pterops and Macdma the vertical meridianwas horizontally displaced from the blind spot ap-proximately 18 and 200, respectively. These valuescan be obtained from Fig. 1, given that in Pteropm1 mm = 4.30 and inMacrderma 1 mm = 80 on theretina.

17. T. Nikara, P. O. Bishop, J. D. Pettigrew, &p. BrasnRus. 6, 353 (1968).

18. In Pterops, six to ten separate injections were made,totaling 1 to 1.5 pl of 20 percent HRP or 1 percentWGA-IIRP solution, to involve the complete retinluprojection area ofthe superior colliculus. In the caseof WGA-HRP, which is colorless at the concentra-tion used, fast-green dye was added to help gaugcthe degree of diffusion. InMacrdorma, whose supe-rior colliculus is only 2 mm across, two injections of0.3 pl each were sufficient to involve the wholestructure.

19. M. L. Cooper and J. D. Pettigrew,J. Comp. Neurol.184,1 (1979).

20. Most of the remaining unlabeled retinal ganglioncells are retinothalamic ganglion cells projecting tothe lateral geniculate nudeus (J. D. Pettigrew, M. L.Graydon, P. Giorgi, in preparation).

21. Most of the characters usually advanced to linkmegabats and microbats are associated with theiht adaptation [for example, characters 51 to 60

of M. J.Novacek, in Macrmoecular Sequences inSysematic and Evolutiona Biv, M.Goodman,f1d. (Plenum, New York, 1982)], pp. 3-41. Other

'.9

characters are contestable, having evolved in otherunrelated mammalian orders (for example, fetalmembrane characters 61 to 63, ibid.) or possiblyrepresenting plesiomorphous rather than synapo-morphous characters (for example, 48 to 50, ibid.).

22. A third possibility is that the advanced mode ofretinotectal organuzation arose first in megachirop-teran bats, some of which later lost their powers offlight and gave rise to the primates. The extensivcand fairly continuous fossil record ofprimates makesthis scenario highly unlikely [M.Archer, in Verte-brate And E i MA ia, M.Archer and CA. Clayton, Eds. (Hesperian Press,Perth, 1983), pp. 949-993; F. Szalay and E. Del-son, Evolution ind Hitoy ofthe Primate (AcademicPress, New York, 1979)].

23. J. D. Smith and A. Starrett, in Bislr ofBats oftheNew World Famiy Phyllostomatidae, part 3, R. J.Baker, J. K. Jones, Jr., D. C. Carter, Eds. (TexasTech Press, Lubbock, 1979), pp. 229-316; J. D.Pettigrew, K. S. Robson, K. I.McAnalIy, in prepa-ration.

24. J. D. Smith and G. Madkour, in Proceed#g of theFirh I1te 1imal Bat R;esea* Conference, D. E.Wilson and A. L. Gar r, E&. (Txas Tech Press,Lubbock, 1980), pp. 347-365; J. E. Hill and J. D.Smith, Bat: A Nieural H (British Museum,London, 1984).

25. K. Padian, Paleobolqy 9, 218 (1983); J. M. V.Rayner, Symp. Zool. Soc. London 48, 137 (1981).

26. M. Archer, in Archer and Clayton [in (22), pp. 633-807].

27. M. Novacek, Nature (ondon) 315, 140 (1985).

28. S. Hand, in Archer and Clayton [in (22), p. 851-904]; A. Walker,Nature (Londn) 223, 647(1969).

29. There may be corollary developments in the tdelnce-phalic visual processing of movement by primatesaccompanying the advances in retinotectalorganiza-tion, such as the MT (middle-temporal) visual corti-cal area found in primates, but in no other mammalsso far studied (1). By the diagnostic criteria for MTthat it be located in the temporal lobe and receive amajor direct input from the prnmary visual cortex,this cortical area seems to be present in Pterpu [M.B. Calford et al., Nature (London) 313, 477 (1985)]but not inMardena (unpublished observations).The similarity of so many different and complexdetails of visual orguzation in primates and ptero-pids strengthens the argument against parallel ap-pearance of the two systems.

30. J. E. Cronin and V. M. Sarich, in RecentAdvances inPnmadoloy, vol. 3, Evolution, D. J. Chivers and K.A. Joysey, Eds. (Academic Press, New York, 1978),pp. 287-288.

31. Supported bjgrants from the Australian ResearchGrants Scheme and the Naponal Health and Medi-cal Council of Australia. L. Wise and M. Calfordhelped with some ofthe electrophysiological record-ing expenments and provided critical comments onthe manuscript. R. Collins provided expert technicalassistance. Staff of the Conservation Commission ofNorthern Territory gave invaluable assistance in thecollection ofMacro&rma, which were obtained un-der permit D85-5633.

8 July 1985; accepted 5 December 1985

Structure of Ribosomal Subunits ofM. vannielii:Ribosomal Morphology as a Phylogenetc Marker

MARINA STOFFLER-MEILICKE, CLAUDIA BOHME, OIAF STROBEL,AUGUST BMcK, GEORG STOFFLER

On the basis of ribosomal morphology, it has been proposed that the sulfur-metabolizing archaebacteria constitute a group (the eocytes) with a phylogeneticimportance equal to that ofthe eubacteria, archaebacteria, and eukaryotes. It has beenfiuther proposed that cocytes should be given kingdom status. Ribosomal subunitsfrom the methanogenic archaebacterium Matbancoccus vannklii were amined byelectron micscopy, and tdeir stctrs were compared to those ofother archaebac-teria, cubacteraland ukaryotic ribosmet. 30S subunits from M. vannielii showedthe elongated contour and the one-third to two-thirds partition characteristic of suchsubunits. In addition, the angled asynmetric projections of those subunits showed asquarish base and a beak on the head. 50S subunits fromM. vannielii were seen in bothcrown and kidney views. In crown views, the Li protuberance was fiequentlypronounced and split; an incision below this protuberance and a protrusion at the baseof the partidle were also observed. Although prevous studies suggested that certain ofthese structural features were found exclusively inribosomes from sulfur-metabolizingarchaebacteria, these new results indicate that such features also occur in ribosomesfrom a typical methanogenic arhabacterium and thus may not be reliable phylogenet-ic markes.

IT HAS BEEN PROPOSED THAT ANALYSISof the structure of ribosomal subunitsby electron microscopy is a rapid and

accurate method for the classification oforganisms (1). Five different ribosomestructures have been described, and havebeen assigned to four independent evolu-tionary lineages (2-4). We present hereelectron micrographs of ribosomal subunitsfrom Methawcoccus vannidii that are incon-sistent with the assignment of one of theselineages (2).

The ribosomes used for this study wereprepared as described (5, 6), and their activi-ties in poly(U)-dependent polyphenylala-nine synthesis were assessed (6). The sub-

M. Stoffler-Meilicke and C. Bdhme, Max-Planck-Institutfir Molekularc Genetik (Abt. Wittmann), Ihnestrasse63-73, D-1000 Berlin 33 (Dahlem), Germany.0. Strobel and A. Bock, Lehrstuhl flir Mikrobiologie derUnivcrsitat Miinchen, Maria-Ward-Strasse la, D-8000Miinchen 19, Germany.G. Stoffler, Institut mor Mikrobiologie, MedizinischeFakultit, Universitat Innsbruck, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria.

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