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Home , Insectivora, Palaeochiropteryx, Rhinolophoidea

South African Journal of Science Vol. 91 September 1995 JOHN T. ROBINSON FESTSCHRIFT 477

:Flying revisited: DNA hybridization with fractionated, GC-enriched DNA

John D. PettigreW' and John A.W. Kirsch2

'Vision, Touch and Hearing Research Centre, University of Queensland, Queensland 4072, Australia and 2Department of Zoology and Zoology Museum, University of Wisconsin, Madison 53705, USA.

Molecular studies have been brought to bear on the primates from other mammalian orders. I I The finding of all of debate over the nature of the evolutionary links between these shared derived brain characters in flying foxes and pri­ primates and . The present investigation supports the mates, but not in , resurrects the old notions about fly­ view that base composition is important in understanding ing primates. It is very hard to explain how these sophisticated phylogenetic relationships. Our discovery that some bats brain characters appeared independently in flying foxes and pri­ have extreme base compositional biases in their DNA com­ mates. The selective forces that would bring about such simila­ pared to others may help in understanding the origin of rity in functionally-separate brain systems are obscure. More­ these biases. over, with time, more and more examples of shared derived brain characters have been found linking primates and flying foxes A link between primates and bats has a long history. Linnaeus' (see review of this by Pettigrew(2). In other words, it is becoming placed bats into his Primates on the basis of anatomical increasingly unparsimonious to suggest that the brain features like the pendulous penis and axillary breasts that are characters that are also found in flying foxes have evolved inde­ shared by primates and some bats. Later debates led to the pendently. The alternative explanation, that flying foxes share a removal of bats from the primates, largely on the basis of the common ancestry with primates which is more recent than either increasing information made available about the peculiarities of shares with the microbats, is highly controversial because of its the microbats as these acoustically-specialized became implications for the origin of flight. Accepting this 'flying pri­ l more familiar to anatomists (cf. Leche2 vs. Winge ). It is interest­ mate' hypothesis has the corollary that flight has evolved in ing to note that all of the characters which were used by Winge) mammals on two separate occasions. The striking similarities to remove bats from the primates were features that we now between the flying apparatus of the two kinds of bats would then know are confined to Microchiroptera, but are not found in flying have arisen from the constraints that operate on the hand wing foxes. There are thus two independent concepts here: I) that bats system, such as the need to use all digits to support the wing .

) may have a special relationship to primates; and 2) that the membrane in the absence of structural elements like feathers or 0

1 Microchiroptera and Megachiroptera may not have had a com­ actinofibrillae that were useful in the construction of the avian 0

2 and pterosaurian hand wings, respectively. In this regard it is of mon origin from the same flying ancestor. If proposition 2 is true, d interest that a simple measurement on the digital bones of the e then primate- affinities may have been confused by trying to t a include microbats along with flying foxes. hand wings of microbats and flying foxes can separate these two d ( In more recent times, these same ideas have been debated kinds of wings unambiguously.7 Moreover, the derived state of r e heatedly. In the 1970s there was a well-known, but unpublished, this skeletal feature of the hand is shared by primates and flying h s

i foxes, but not microbats.

l debate between Carl Koopman and James Dale Smith on this b same question, with the issue remaining largely unresolved. Molecular studies have been brought to bear on this debate. At u P

Smith"·j had raised the remarkable similarities between flying present the consensus is that DNA sequence data failed to sup­ e h foxes and primates. He questioned whether the similarities in port a special link between flying foxes and primates. In contrast, t t3 y wing structure between the two suborders of bats might not rep­ the amino acid sequence data from lens alpha crystallin, b resent a case of convergent evolution, a result of the considerable haemoglobin7 and epitopes on serum proteins," all tend to sup­ d e

t constraints that are known to operate upon flight surfaces, rather port separate origins of the two kinds of bats. Possible reasons n than reflecting a common flying ancestor as was usually for this discrepancy between the interpretation of protein and a r '2 '5 g assumed. This idea has been taken seriously by some laboratories DNA data have been laid down elsewhere. . Of special interest e

c engaged in the study of flight mechanics of bats (e.g. Scholey"). in this regard is the fact that flying fox DNA has the most biased n e New evidence on this controversial question of bat-primate base composition known in . This is manifest as a dra­ c i

l matic increase in the proportion of DNA that is taken up by A or

affinities appeared in the 80s from studies of the brain. A number r T substitutions, with the DNA of all having around e of derived brain characters, thought to be uniquely shared among d 75% AT content. 16 A marked AT bias is also present in the DNA n the primates, were found in flying foxes. Such characters include u the pattern of lamination in the lateral geniculate nucleus where of some families, although none has such a strong bias y a the magno-cellular layers are well differentiated and lie external as the flying foxes. 17 In view of this large bias in the base compo­ w e to the other layers in the nucleus next to the optic tract.1 Another sition of DNA, it seems possible that work using DNA to estab­ t a set of characters is found in the projection from the retina to the lish affinities between the bats could be affected by coincidental G

similarities in AT substitutions. In support of this idea it was t mid-brain optic tectum. Of the large number of mammalian e found that the substitutions claimed in support of a link between n orders that has been studied, only in primates and flying foxes i b does one find a hemi-decussated pattern of projection to the mid­ flying foxes and microbats have a preponderance of A or T by a a 15 S 1 to factor of 4. brain tectum which mimics that to the thalamus. - Other charac­ y

b ters in the motor pathways and the organization of the multiple In the present study we aimed to address this possible AT bias

d representations of sensory fields within the cortex as well as the in the DNA of bats by fractionating DNA to enrich the content of e c disposition of sub-divisions of the hippocampus, all distinguish GC, and then undertake DNA-DNA hybridization studies with u d o r p e R 478 JOHN T. ROBINSON FESTSCHRIFT South African Journal of Science Vol. 91 September 1995

Fig. I. The flying primate hypothesis. Literal and metaphor­ ical tree of primates and relatives to illustrate the hypothesis that flying foxes (megabats, lower right) had a separate evolu­ tionary origin, via the dermopteran (gliding to the left of the flying fox), from the origin that gave rise to the other branch of flying mammals, the micro bats (far left). When this drawing was commissioned, one of us believed that the tarsier had strong affinities with anthropoids (top right branch), but new evidence suggests that the tarsier is para­ phyletic to all living primates, illustrating that primate rela­ tionships are still not agreed, even when one puts aside the controversy over the possible derivation of flying foxes from an early branch of the primate tree.

1, Bushbaby; 2, Ruffed lemur; 3, Colobus monkey; 4, Tarsier; 5, Flying fox; 6, Tree ; 7, Gliding lemur; 8, Microbats.

. the enriched DNA fraction. In this way we hoped to achieve two aging for plant products and that stiff competition for the )

0 goals. First, we hoped to improve resolution at great distances phytophagous resources might make life difficult for the gliders 1

0 where the low temperature of the melting point of heteroduplexes in the presence of their actively flying descendant. This 'just so' 2 tends to obscure relationships. By using GC-rich DNA that melts evolutionary story fits the scanty evidence that is available d e t at a higher temperature we hoped to 'pull' these curves to higher for these groups in that the first definitive flying fox fossil, a

d temperature ranges and, if possible, better resolutions. Second, Archaeopteropus, appears near the -Oligocene boundary, (

r we aimed to see whether the use of GC-rich DNA gave the same around the time when the paromomyid gliders disappear from e h relationships between the bats that were seen with unfractionated the fossil record. The fact that the living colugo is a primary foli­ s i l DNA. If the AT bias is playing a role in artificially shortening vore and can therefore avoid competition with flower-, nectar,­ b u distances between bats because of a common AT bias, then one and fruit-eating flying foxes is also consistent with this view. P might expect that GC-rich DNA would give greater distances According to this scenario, the flying foxes are a relatively new e h group within the primate assemblage, having been preceded by a t than AT-rich DNA comparisons in the same taxa. This expecta­

y tion has been fulfilled for some flying fox-microbat comparisons, long history of gliders, such as the extinct paromomyids. This b early prior branch is often forgotten by those with the unrealistic d suggesting that AT bias plays a role in artificially shortening dis­ e t tances between taxa that share a strong AT bias. expectation that the hypothesis predicts short distances between n

a flying foxes and primates. The recency of flying foxes, in con­ r g trast to the stem branch of primate gliders, is supported by the

e The hypothesis

c fossil record, as shown in Fig. 2a. This shows a long fossil record n The flying primate hypothesis is illustrated in the literal-meta­ e of microbats that goes back towards the dawn of the Tertiary or c i l phorical drawing of Fig. 1. It is proposed that a very early branch perhaps even earlier, compared with the much more recent fossil r of the primates, perhaps the paromomyid branch, gave rise to a e record of flying foxes. The recency of mega bats is also supported d gliding assemblage, of which the living representative is the by the extreme morphological and biochemical similarity across n u

colugo, Cynocephalus. The living are gliders with close the flying fox assemblage and by DNA-DNA hybridization work y a neural, behavioural and biochemical affinities to primates. There that indicates that the living flying foxes diverged around 25 mil­ w IY e is some fossil evidence for an early primate-related assemblage lion ago. t a that were gliders. IS The 'flying primate' hypothesis proposes that If one wants to adhere to the idea that mammalian flight had a G

t these early primate gliders were succeeded by active powered common origin and that the flying foxes, along with the micro­ e

n flyers, the flying foxes, or megabats, Megachiroptera. All extant bats, are part of a monophyletic assemblage of bats, then some i b flying foxes and colugos are phytophagous, so perhaps this awkward corollaries must be considered. All of the just-men­ a S

whole gliding flying assemblage was phytophagous, with an ori­ tioned pieces of evidence for a recent origin of the flying foxes y

b gin in the Palaeotropics coinciding with the present distribution would tend to lead one to conclude that they have originated

d of flying foxes and colugos. One might expect that powered fly­ more recently than the microbats. The many highly specialized e c ers would be more successful than their gliding ancestors at for- features of microbats which are present in all living microbats, u d o r p e R South African Journal of Science Vol. 91 September 1995 JOHN T. ROBINSON FESTSCHRIFT 479

a MICROBATS MEGABATS Fig. 2. a, Fossil record of bats: Microbats have a fossil record that extends to the Late Palaeocene: even the oldest Myr .Chantwaria .Omo microbat have highly derived features (such as an • Notonycteris enlarged cochlea and modifications of the hand wing for Miocene • Yunnan echadronycteris advanced flight); the gondwanatherian of 10 o Foumas2 Bondesius is very similar to that of , in Lo Fournas 8 support of other zoogeographical evidence that microbats Sainte­ • Riversleigh megadennatids Catherine originated before the dawn of the Tertiary. In contrast, the 20 • fossil record is recent. The oldest unequivocal La Colomb. Propotto megabat fossil, Archaeopteropus, had cuspidate teeth and E. African Oligocene extra phalanges unlike the derived condition found in all 30 • Leuconoe extant megabats; the record is therefore consistent with a • Robiac • Cuvierimops Archaeo­ recent origin of megabats, as is the DNA-DNA hybridiza­ _~Wallia __._ Necr.omantis. p.!.Br~u=- tion evidence that suggests a divergence of megabats no Eocene more than 30 Myr ago. A greater relative age of the micro­ 40 Tabemacle Butte • Mathhes'a bat divergence could help explain both the biochemical • Vespertiliavus. • ~tehlinia • Chamblon' and familial diversity of microbats (17 living families, • Cecli.onyctens • D.zzya • Chambli • . many of which are presently difficult to relate to each 50 • Palaeochiropteryx -. Honrovits. Vielase other) compared with the uniformity of megabats in both • Ageina • • Archaeonycteris of these respects. b, Distances between the two kinds of -- .. Wyonycteris • AusrralOriyCferiS .- bats, dermopterans and primates, estimated from DNA Paleocene Beard 60 hybridization using GC-rich tracer DNA. TERTIARY

70 CRETACEOUS • ? Bondesius ferox

microbats which are all distinct from each other, and which have b MICROBATS deep, obscure relationships to each other. 2C1 The difficulties experienced in trying to relate them with molecular techniques MEGABATS suggest a long history of divergence, as does their Gondwanan biogeography. These considerations lead one to wonder whether A: Using 50% GC fractionation microbats were in fact present in the Cretaceous.7 The work of

. 20

) Pierson using immunological methods to study the different 0

1 microbat families supports the idea that the microbat divergence 0

2 MICROBATS took place in the Cretaceous, 80-100 million ago. d e t a Methods d (

DERMOPTERANS

r Analytical centrifugation. The relative proportions of AT-rich e

h and GC-rich DNA were estimated from buoyant densities as s i l b

u B: Extrapolation to more GC fractionation. MEGABATS Table I. Ordinal AT content (as a percent­ P

e age) of mammalian DNA: estimated from 14 12 10 8 6 4 2°C h

t buoyant-density centrifugation.

I I I I I I I y I I I I Megachiroptera b 100 80 40 20 10

d Pterupus polioceplwlus Myr 74 e t Pterupus alecto 73 n a

r and are also present in the oldest fossils, must therefore, if one M icrochiroptera g follows this line, have been lost in the flying fox assemblage. Of Hipposiderus galeritus e 70 c

n special interest here is the laryngeal sonar which characterizes Chiroderma salvini 70 e c microbats. We know laryngeal sonar must have been present in Rhinolophus creaghi 68 i l

r Earliest Eocene microbats, since Icaronyteris and Australonyc­ Primates e d teris both had greatly enlarged cochleas, even more elaborate Homo sapiens 61 n

u than some living microbat families, all of which have this spe­

Dermoptera y cialization unique to the Microchiroptera. It is a mystery why a Cynocephalus variegatus 61 w this sophisticated ability, so useful to a nocturnal flying , e Carnivora t would have been lost by the flying foxes only to be re-acquired in a Panthera uncina 61 G

a greatly inferior form (i.e. tongue clicking in one of flying t Felis domesticus 60 e fox, Rousettus). n i M icrochiroptera b There are many other unparsimonious difficulties with the a Myotis myotis 59 S notion that the flying foxes have emerged from an earlier flying

y 2 MyOlis lucifugus 57

b ancestor that is linked to the microbats (see Pettigrew' ) . In con­

d trast to the flying foxes whose origins seem to be recent, the Insectivora e c micro bat origins go far back. There are 17 living families of Erinaceus europea 56 u d o r p e R 480 JOHN T. ROBINSON FESTSCHRIFT South African Journal of Science Vol. 91 September 1995

0 . 14 Megabat Pleropus AT Results Base composition from buoyant density 0. 12 There was a wide variation of the modal buoyant density of DNA in the mammals we studied. This is to be contrasted with 0. 1 \ the situation in birds where the modal buoyant densities are very ~ c: \ ::1 0.08 tightly clustered over the whole avian kingdom. As reported pre­ 8 \ a \ viously, the 'lightest' DNA with the greatest shift towards high c: .2 0 .06 \ AT was found in the fiying fox, Pteropus. Next in order was Cro­ 13 £ \ cidura, a shrew whose DNA was also found to be greatly biased 0.0' \ towards high AT. In this respect it is interesting to note that Cro­ \ cidura's AT bias is not shared with other '' such as 0.02 \ \ the , which had levels of AT which were similar to those , found in other mammals. Next in line after Crocidura were the rhinolophoid and phyllostymoid microbats, including Rhino­ lophus, Hipposideros (Rhinolophoidea) and Chiroderma (Phyl­ Temperature (OC) lostomidae). In contrast, some microbats had no increase in AT Fig. 3. Normal behaviour of fractionated DNA: Dobsonia versus Pter­ content. These included Myotis from the family Vespertiliono­ opus. When GC-rich tracer DNA is used for the comparison, note that diae and Noctilio from the family Noctilionidae. Shifts to high the melting points for both the Ptempus-Ptempus homoduplex and for the Pteropus-Dob.wnia hybrid have shifted to higher temperatures. This AT were also observed in carnivores, primates and the colugo. At is to be expected since GC-rich DNA melts at a higher temperature than the other end of the extreme, monotremes showed a GC bias in AT-rich DNA . Note, however. that the distance between Pteropus and their DNA. Dobsonia is the same for both the GC-rich and AT-rich comparison. This is the behaviour of DNA that would be expected in the absence of any Normal behaviour of higher melting GC-rich DNA differential rate of change that is related to base composition. We call When we used the GC-rich DNA to make hybrids, we found, this normal behaviour of DNA. as expected, that the melting point of the hybrids had shifted to a higher temperature. In fact, the separation between the melting revealed in a cesium chloride gradient. 17 The taxa that were stud­ points of homoduplex and heteroduplex stayed approximately ied. along with modal buoyant densities and the percentage AT the same even though the melting point with the GC-rich DNA content of the DNA, are shown in Table I . had shifted to a higher temperature. This is shown in Fig. 3, where it can be seen that the heteroduplex formed from Pteropus DNA fractionation . We used the TED machine to fractionate and Dobsonia DNA melts at a higher temperature if the GC-rich . DNA according to its melting point. In initial experiments we ) tracer is used, but that the separation between the Pteropus 0

1 split the fractions around the modal melting point so that we had homoduplex and the Pteropus-Dobsonia heteroduplex is approx­ 0

2 a GC-rich fraction with all of the high melting points and an AT­ imately the same whether we used the GC-rich or the AT-rich d rich fraction with the fractions collected below the modal tem­ e tracer for comparison. This behaviour is what one would expect t a perature. In later experiments we used a super-GC-rich fraction if there is no peculiarity of mutation rate that is dependent upon d (

which took the fractions containing the 25%. This method will the base composition of the DNA. We call this 'normal' beha­ r e be described in more detail subsequently (Pettigrew and Kirsch. viour of the DNA and we observed it in many of our compari- h s i in prep.). sons. l b u

P t.TGC b I • • I e a Pteropus·Phinolophus AT/GC I h t

I t.T AT 0.16 0 . 16 • y I b 0.14 0 .14 I d e t J!l 0.12 0 . 12 n § E

a ::1 0 r 8 , g '0 0.1 0 . 1 , '0"

e c: 0 c .~ . 0.08 ·u 0 .08 , n £ e e u. ., c i 0.06 ,

l 0 .06 • r ,

e , 0.04 0.0' d n , u , 0.02 l:.':':':':;';'=';'''-''-- 0.02 ------.--- y " , " a w N '" ~ (I) 0 N '" \Q ., ~ N .. '" N V '" e ...... ,... (I) (I) co co ., 0> 0> 0> 0> 0> 0> 0> t

a Temperature (OC) Temperature (OC) G

t Fig. 4. Anomalous behaviour of DNA: Pteropus versus Rhino[ophus. In this case, the use of GC-rich DNA leads to a dramatic change of the distance e n between Ptempus and the comparison taxon, Rhino[ophus. While the GC-rich Pteropus-Ptempus homoduplex shifts to a higher melting point, the GC­ i b rich Pteropus-Rhino[ophus heteroduplex not only does not shift to higher temperatures, it shows a slight movement in the reverse direction. In other a

S words, the distance between Pteropus and Rhino[ophus is increased significantly by the use of GC-rich DNA as the tracer. We call this anomalous

y behaviour of DNA. In our study, such anomalies, where the distance increased with GC-rich DNA, were most marked in comparisons involving taxa b with a high AT content of their DNA. For example, rhinolophoid and phyllostomid microbats share the highest DNA content of mammals along with d e megabats; the greatest anomalies were seen in comparisons of these taxa and were much reduced in the case of comparisons with the other microbat c u families that have lower AT content. d o r p e R South African Journal of Science Vol. 91 September 1995 JOHN T. ROBINSON FESTSCHRIFT 481

Preropus vs. Microbats Fig. 5. Distance between Pleropus (AT solid. GC 0.16 scored) and a number of different micro bats to show Nocti/io the marked variation in microbat-megabat distance. 0 .14 There is a systematic relation between the known AT 0. 12 content of the microbat DNA and the distance meas­ E.. ured. The greatest distance obtained was from the 0 0" 0. 1 comparison with NOCliiio (30°C). a microbat with (; c: the smallest AT content. The closest distance (22°C) .2 13 0.08 was obtained in the comparison with Rhinoiophus ~ \ and Hipposideros. microbats with the most extreme 0 .06 ./ \ AT bias known. Other cases lie between these extremes. with the phyllostomid, Chimderma, hav­ 0 .0. '...... -,.- .... -_ \ ing a shortened distance like the rhinolophoids, that 0 .02 -" --- \ agrees with its moderate AT bias. and the vespertil­ --~:~, ionid. Scolophiius, lying with NOCliiio, fitting its absent AT bias. o .. .. o "'" '" '" "'" ..'" .." ...... '" '"" ..'" Temperalure (Oc)

Anomalous behaviour of DNA that AT bias in base composition has a dramatic effect on dis­ Anomalous behaviour of DNA was observed in some compar­ tances measured using DNA hybridization. Since the distances isons. By 'anomalous' we mean that the distance between taxa between some microbat families and flying foxes were in the was not maintained when GC-rich DNA was used as a tracer order of 28-30°C using our methods, and because such tempera­ instead of AT-rich tracer. In all cases, the anomaly involved an tures represent the base of the eutherian radiation, they are still increase in the separation of the melting curves when GC-rich compatible with the notion that microbats and flying foxes have DNA was used. This increase was most marked in comparisons arisen separately from the mammalian line. This is true only if between Pteropus. the flying fox. and the microbats Rhinolophus we exclude the Rhinolophoid microbats that show a consistent and Hipposideros (Rhinolophoidea) (Fig. 4). In these compari­ tendency to be close to flying foxes. Since the separation sons the distance between the microbat and megabat increased by between Rhinolophoids and flying foxes can be increased with as much as 10 degrees when the GC-rich tracer was used for GC enrichment of the DNA, perhaps even more enrichment will comparison. A similar anomaly was also observed for the Phyl­ lead to even further separation of these two groups. This expecta­ lostomid microbat Chiroderma. On the other hand, anomalies tion tends to be supported by the fact that other microbats which do not show such anomalous behaviour of their DNA, Noctilio,

. were less obvious with the microbats Noctilio (Noctilionidae) ) to take a good example. have long distances whether AT-rich or 0 and Myotis (Vespertilionidae). In the case of the microbat Noc­ 1 GC-rich DNA is used for comparison. On the other hand, if one 0 tilio, the distance observed with the AT-rich DNA was very large 2 and comparable to the distance that we observed with the GC­ wants to place more weight on the shorter distances (as does the d e Fitch Algorithm), then one is left with a phylogeny of the bats t rich DNAs in the comparisons where there was a marked anom­ a which is difficult to sustain on close examination. In this phylo­ d aly. In other words, there was a general pattern of decreased dis­ ( geny, the Rhinolophoid microbats are closer to flying foxes than r tances in the AT-rich comparisons that showed anomalies (Fig. e they are to the other families of microbats. Since all microbat h 5). To put this another way, those microbats which did not show s i l large anomalies in the GC-AT comparison also tended to have families are considered to form a monophyletic group on the b basis of their acoustic and laryngeal sonar specializations, it has u longer distances from the flying foxes than those microbats P which showed large anomalies. Putting all this together, it seems never been seriously suggested that they are not a natural group. e

h Paraphyletic assemblages of microbats and flying foxes have t likely that distances estimated using AT-rich DNA tend to under­

y estimate the true distances between the taxa under consideration appeared in the literature from time to time. For example, b Pierson2o found a strong affinity between Emballonurid micro­

d and this effect is greatest when the taxa share large AT biases in e t their DNA base composition. bats and flying foxes, and in recent molecular work both Mega­ n dermatid and Phyllostomid microbats have been aligned with a The new data give a tree of bats where flying foxes and Rhi­ r 21 g flying foxes to the exclusion of other microbats. While the het­ nolophoid microbats are combined to the exclusion of other e

c erodox result of the splitting of microbats by the flying foxes has microbat families. While this tree may provide some support for n

e appeared on a number of different occasions and might therefore

c the idea that bats are monophyletic, it is a new hypothesis about i

l have to be given serious consideration, the reservations already

the relationships of microbats which seems unlikely to be cor­ r expressed tend to be confirmed by the fact that megabats group e rect. There is presently no reason to question the monophyly of d with a different family of microbats on each of these occasions' n the microbats, so the fact that the present data tend to split the u

Is it a coincidence that each of the families of microbats (Rhi­

y microbats tends to cast doubt on this tree rather than provide

a nolophoid, Phyllostomid and Emballonurid), which has been much support for the monophyly of bats. An alternative view­ w

e aligned with flying foxes in these studies, is noted for its high AT

t point is that the flying fox-Rhinolophoid affinity can be a content? There are known to be cases of very high AT content explained by the fact that these two kinds of bats also share the G

(e.g. Plasmodium) where the AT content of DNA is so high that t greatest AT biases seen in mammals. Perhaps greater fractiona­ e codon usage is affected. For example, asparagines are substituted n of the DNA can provide further separation of Rhinolophoids i tion

b for glycines in the histone proteins of Plasmodium. In other

a from flying foxes.

S words, AT biases can be sufficiently extreme as to affect the

y expression of amino acids in proteins and therefore might affect b Conclusions and discussion d the epitopes that were measured in Pierson's study (Fig. 6). Sim­ e c The present study has not provided unequivocal support for ilarly, codon usage in proteins like haemoglobins might affect the u d the flying primate hypothesis. On the other hand, it has revealed assessment of the true affinities of those mammals that have high o r p e R 482 JOHN T. ROBINSON FESTSCHRIFT SOUlh African Journal of Science Vol. 91 September 1995

I. Linnaeus e. (1758). Systema naturae per regna tria naturae. secundum classes. ordines. genera, . cum characteribus. differentiis. synonymis. locis. Toms I. Editio Decima, Reformata. Holmiae. Impensis direct. Laurentii Sal vii. Stockholm. I-A{' 2. Leche W. (1886). Ueber der Savgethier gattung Guleopitiu:cus. K. Jvenska - dATP Vetensk. Akad. Hundl .• NS. 21(11).1-92. 3. Winge H. (1892). Jordfundne og nulevende flagemus (Chiroptera) fra logoa GC AT ~ santa, mina gereas. brasilien. Museo Lundi (Copenhagen) 2. 1-65. U ATP 4. Smith J.D. (1976). Chiropteran evolution. In Biology of Buts of tiu: New World Fumily PhyllostomatidiJe, part I. eds RJ. Baker. J.K. Jones Jr & D.e. Carter. pp. 49-69. Spec. Publ. Texas Tech. University, Lubbock. -CH,~ I 5. Smith J. D. (1977). Comments on flight and the evolution of bats. In Mujor Pullerns of Evolution. eds M.K. Hecht. P.e. Goody and B.M. ~ Hecht. pp. 427-437. Plenum. New York. 6. Sholey K.D. (1982). Developments in vertebrtJIe flight: Climbing und gliding of mlimflUlIJ lind reptiles. und tiu: flupping flight of birds. Ph.D. thesis. Uni­ 1. 0 radicals preferentially oxidise G. versity of Bristol. 2. Methyla~on of C increases at high temperatures. increasing T. 7. Pettigrew J.D" Jamieson B.G.M .. Robson S.K .. Hall L.S .. McAnally K.1. and 3. Precursor pools of dATP affected by cy1osolic ATP. 4. Reduced genome size and smaller nuclei exaggerate 1-4. Cooper H.M. (1989). Phylogenetic relations between microbats. megabats and primates (Mammalia: Chiroptera and Primates). Phil. Trans. R. Soc. B.325.489-559. Fig. 6. Mutational biases towards high AT. Aerobic metabolism may 8. Pettigrew J.D. (1986), Flying primates? Megabats have the advanced pathway have a number of consequences for the mutational biases that accom­ from eye to midbrain. Science 231. 1304-1306. pany DNA replication and repair. Note that all of these known effects 9, Rosa M.G.P, and Schmid L.M, (1994). Topography and extent of the visual point in the same direction, to increase AT content at the expense of GC field representation in the superior colliculus of the megachiropteran. Ptero­ content. pus, VisUlJI Neuroscience 11.1037-1057, 10. Rosa M.G.P., Pettigrew J,D. and Cooper H.M. (1995). Unusual pattern of reti­ AT content. We favour further exploration of this viewpoint, nogeniculate terminations in the controversial primate. Tursius. Brain Behuv. rather than suggesting that the microbats do not represent a natu­ Evol, (in press), ral assemblage. II. Buhl E.H.and Dann J.F. (1991). Cytoarchitecture. neuronal composition. and entorhinal afferents of the flying fox Hippocampus. Hippocumpus 1. The controversy surrounding the origin of mammalian flight 131-152. can be seen to cut across a number of major current areas in bio­ 12, Pettigrew J.D. (1995). Flying primates: Crashed. or crashed through? Symp, logy. The discovery of base compositional biases along the mam­ Zool. Soc. Lond. 67. (in press). malian genome, the isochore phenomenon,l7 is still unexplained 13, De Jong W.W .. Leunissen J.A.M. and Wistow GJ. (1993). Eye lens crystal­ in an evolutionary context. The present investigation supports the lins and the phylogeny of placental orders: evidence for a .

) macroscelid-paenungulate ? In Mummal Phylogeny: Plucentu/s. eds F.S .

0 view that a consideration of base composition is important in Szalay. MJ. Novacek and M.e. McKenna. pp. 5-12. Springer-Verlag, New 1

0 accessing phylogenetic relationships. Our discoveries that some York. 2 bats have extreme base compositional biases in the DNA com­ 14, Schreiber A .• Bauer D., and Bauer K. (1994). Mammalian evolution from d e serum protein epitopes. Bioi. 1. Linn. Soc. 51. 359-376. t pared to others may help in the understanding of the origin of a 15. Pettigrew J.D. (1994). Flying DNA. Curro Bioi. 4. 1-4. d these biases. While the DNA hybridization results have provided (

16. Arrighi F.E .. Udicker W.Z. Jr.. Mandel M. and Bergendahl J. (1972). Hetero­ r no strong support for the flying primate hypothesis, the fractio­ e geneity of CsCI buoyant densities of chiropteran DNA. Biaciu:m. Genet. 6, h nation has certainly pushed distances between microbats and 27-39. s i l megabats to values that are comparable with those between any 17. Bernardi G. (1993). The vertebrate genome: isochores and evolution. Malec. b u orders at the base of the mammalian radiation. Our findings are Bioi. Evol. 10. 189-204. P

18. Beard K,e. (1990), Gliding behaviour and palaeoecology of the alleged pri­

e not inconsistent with the flying primate hypothesis, provided that

h mate family Paromomyidae (Mammalia: Dermoptera). NtJlure 345. 340-341. t one accepts the possibility of a considerable underestimate of the 19. Kirsch J.A,W.. FlanneryT.. Springer M,S. and Lapointe F-J. (1995). Camping y distance between megabats, whose extreme AT bias is well-doc­ b in a different tree: Phylogeny of the Pteropodidae (Mammalia: Chiroptera)

d umented, and some microbats which also have biased AT con­ based on DNA hybridization. with evidence for bat monophyly. Aust. 1. Zoo/. e t tents in their DNA. (in press). n

a 20. Pierson E.D. (1986). Molecular systematics of tiu: Microchiroptera: Higiu:r r LD.P. thanks Ihe organizers for the opportunity to contribute to the g taxon relutionships und biogeography. Ph.D, thesis. University of California.

e Festschrift for J.T. Robinson. The work was carried out while J.D.P. was Berkeley. c on sabbatical in the laboratory of J.A.W.K. in Madison, supported by a n 21. Stanhope MJ .. Czelusniak J .. Si J.-S. Nickerson J. and Goodman M. (1992). e grant from the University of Queensland and by the external travel fund c A molecular perspective on mammalian evolution from the gene encoding i l

of the Vision. Touch and Hearing Research Centre, a Commonwealth interphotoreceptor retinoid binding protein. with convincing evidence for bat r e Special Research Centre of the Australian Research Council. monophyly. Molec. phylogenet. Evol. 1. 148-160, d n u y a w e t a G t e n i b a S y b d e c u d o r p e R