Batman's place in evolution

Some of Jack Pettigrew's peers regard the former director of the Vision, Touch and Hearing Centre at the University of Queensland as an eccentric, and some consider his hypothesis that bats are our distant cousins as somewhat, well, batty.

In 1992, for example, a news article in the international journal Science caricatured his ideas with an image of the flying monkeys from the Wizard of Oz.

But the neuroanatomist Wizard of Oz was not the first to conclude that the Megachiroptera - fruit bats and flying foxes - are flying primates.

Nearly 250 years ago the great Swedish naturalist Carl von Linne, inventor of the modern system of classifying living species, grouped bats and south-east Asia's colugo or flying lemur (Cynocephalus volans) with primates, because they shared key characteristics common to all primates.

By an accident of history, Linnaeus chose the world's only echo-locating fruit bat, the Egyptian rousette (Rousettus aegyptiacus), as his holotype, when he could have used any one of Europe's 40-plus insectivorous microbats.

The Egyptian rousette roosts in caves, using the echoes of its rapid tongue clicks - not ultrasonic squeaks - to find its way around in the dark. On a number of key characters, Linnaeus was unable to exclude it as a primate.

In a paper published in Philosophical Transactions of the Royal Society 1989, Pettigrew drew attention to more than 50 characters that he believes separate megabats from microbats.

His opponents, however, invoked evolution's parsimony principle: what chance that nature would invent flapping flight twice in mammals?

Their knockout punch was DNA sequencing experiments, which in the early 1990s grouped megabats with microbats. For Pettigrew to be right, DNA - the ultimate arbiter on taxonomic issues - had to be wrong. Morphology may mislead, but DNA tells no lies.

But if the DNA evidence is right, fruit bats must have made some highly improbable changes to the design specifications for the common ancestor of all bats - assuming that insect-eating, echo-locating bats, the second most diverse group of mammals on the planet, appeared first on the evolutionary stage.

Indeed, on any number of physical, physiological and behavioural characteristics, they've made a sport of vaulting over Richard Dawkins' lofty evolutionary peak, Mt Improbable, from their ancestral home in the Vale of Microbats, to reach Primate Plain.

Pettigrew's critics invited him to explain what fruit bats had evolved from, if not microbats. (The question can be reversed, however: the paucity of fossils from both groups has led a few to argue for a scheme in which microbats evolved from a megabat ancestor like R. aegypticus).

Pettigrew's answer, 20 years ago, paid homage to Linnaeus: fruit bats evolved from south-east Asia's flying lemur, the colugo - or, more accurately, megabats and colugos diverged from a colugo-like gliding primate.

---PB--- Flyers and gliders

If you can't fly, gliding is the second most efficient way of moving around a rainforest, foraging for fruit - the preferred food of both primates and fruit bats. And more than one group of animals adapted to clambering around on the trunks of tall trees has made the evolutionary leap to gliding flight.

Several groups of Australian marsupials have independently evolved gliding flight. So did small, feathered cousins of the warm-blooded Cretaceous predator, Velociraptor, which took to the trees and evolved into birds, sharing the skies with flying reptiles like Pterodactyl and Pteranodon.

In modern times, several rainforest frogs, lizards and even a snake have variously co-opted webbed feet or flared rib cages for short, gliding flights.

Until quite recently, zoologists puzzled over the colugo's place in the grand scheme of mammalian evolution - the two extant species, Cynocephalus volans (Philippines) and (Malaysia), are the last representatives of a unique mammalian order, the Dermoptera.

The strange, bug-eyed, cat-sized gliders forage nocturnally, like fruit bats, but eat only leaves, buds and flowers, not fruit. Uniquely among gliding mammals, the leathery gliding membrane or patagium encloses the wrists and ankles.

In microbats, the patagium stretches between greatly elongated metacarpals, the long bones that end in the knuckles. Fruit bats have their patagium stretched across elongated phalanges. The modified second finger ends in a hook on the leading edge of the wing. With wings folded, fruit bats clamber around a tree with almost lemur-like agility, says Pettigrew.

This striking divergence in the structure of the wing is more likely the result of convergent evolution in the descendants of unrelated gliding ancestors, he says. And this is just one at least 50 anatomical, physiological and behavioural traits separating megabats and microbas.

In the case of megabats and their colugo cousins, the fossil record offers a likely common ancestor: the paramomyids, whose fossils occur in rocks of early Paleocene (60 million years ago) to early Oligocene (35 million years ago) age. Their unusually long limb bones hint that they were gliders.

The earliest megachiropteran fossils are also of early Oligocene age. The coincidence suggests paramomyid gliders exited stage left, just as fruit bats entered from stage right, according to Pettigrew.

A rainforest frugivore that evolved flapping flight would be far more efficient at foraging than one limited to gliding - so efficient, says Pettigrew, that the fruit-eating paramomyid ancestors of fruit bats went rapidly extinct in the unequal contest for their preferred fruit diet. But one paramomyid lineage survived, because it had taken to herbivory and avoided competition with the new-wave fruit bats: the colugos.

---PB--- Begetting megabats

Taxonomists now regard the paramomyids as a sister group to the primate-like plesiadapoids, grouping them in the extinct order, the plesiadapiforms, which flourished in Europe and America from the Paleocene to early Eocene (~64-55 million years ago).

Around this time, all the modern mammalian orders were diversifying into a host of ecological niches vacated by the dinosaurs in the wake of the Cretaceous-Tertiary (K-T) Boundary mass extinction event 64.8 million years ago.

The plesiadapiforms are now regarded as proto-primates. Some resembled lemurs.

Pettigrew now invites us to join the dots: the paramomyids took to the air and evolved into colugos, which in turn, begat megabats. Meanwhile, their sister group, the plesiadapoids, evolved into our own ancestors, the primates.

And Pettigrew's scheme has recently found strong support, from a surprising quarter.

In the November 2 issue of Science, US and German molecular geneticists, led by Dr William Murphy of Texas A & M University, reported the results of their comparative DNA study of the three mammalian orders in the super-order Euarchonta: Primates (lemurs, marmosets, monkeys, and apes, including Homo), Dermoptera (colugos) and Scandentia (tree shrews).

They concluded that colugos are the long-elusive sister order to the primates and placed the divergence of primates and Dermoptera around 24 million years before dinosaur extinction - at 88.8 million years ago. The Scandentia were previous candidates as the sister group to primates, but they came much later - in the early Paleocene, around 63 million years ago.

But the latest analysis did not include fruit bats. The authors had no reason to include them in the quest for the sister group of primates - they were just bats.

What made the new analysis different was that it compared patterns of indels - INsertions and DELetions in the introns that divide genes into protein-encoding exons.

Where a point mutation simply swaps one base for another, indels physically change the length of a DNA sequence, by inserting two or more DNA bases at a specific point in the intron, or deleting two or more bases.

Pettigrew says that, with a point mutation, there is no telling in which of the two species the mutation occurred, without referring to other, related species. Over tens of millions of years, informative mutations become increasingly indistinct amid accumulating background "noise".

The US-German analysis did not employ DNA hybridisation - a technique developed in the late 1980s, where DNA from each of the species being compared is heated to the boiling point of water, separating the tightly bound strands of the double helix.

As the DNA mix cools, complementary sequences from the two species align and bind to each other; mismatched sequences remain separated. As they are reheated, the hybrid DNA separates at a temperature proportional to the time since the species diverged from a common ancestor. The lower the temperature, the longer the divergence time.

Indels are much rarer events. The genetic code is "read" in triplets, so any error that adds or deletes one or two bases creates a frameshift mutation that changes every amino acid downstream of that site, creating a nonsense protein. Non-lethal indels slowly accumulate, forming distinctive patterns across the genome. The more closely related the species, the more similar the patterns.

---PB--- DNA hybridisation

So why did early DNA sequencing studies group megabats and microbats, if, as Pettigrew contends, fruit bats are flying primates, descended from colugos?

Pettigrew doubts that molecular geneticists who have relied on DNA sequencing and other molecular evidence will take kindly to his explanation, first advanced in 1992, and since updated in the light of new findings about the nature of chromosomal DNA.

In the late 1970s, Italian molecular geneticist Professor Giacomo Bernardi discovered that the genomes of mammals are studded with long uninterrupted tracts of DNA consisting uniformly of a particular ratio of A-T to G-C base pairs, in which A-T base pairs dominate.

Pettigrew says the exceptionally high energy demands of flapping flight require very high levels of synthesis of adenosine triphosphate (ATP). Adenosine is a partner in the A-T base pair, and in species that employ flapping flight, some quirk of adenosine synthesis seems to bias mutations towards A-T.

A possible explanation for the A-T bias is that guanine is much more sensitive to oxidation than the cytosine, adenosine or thymine. DNA-repair enzymes misread oxidised G as an A, so there is a pernicious ratchet toward A-T that is exaggerated in organisms with the highest levels of oxidative metabolism - those that fly.

Birds, bees and bats share this surfeit of A-T in their DNA. In fact, says Pettigrew, fruit bat DNA consists of around 75 per cent A-T residues, the highest of any known vertebrate species. Eventually the A-T bias threatens orderly genome function, inducing a compensatory mechanism that loads up DNA with G-C isochores. So, not only does the fruit bat genome contain an excess of A-T sequences, it is expanded by extra G-C isochores.

G-C isochores are very "sticky", says Pettigrew, so when fruit bat DNA is heated to 100 degrees, the strands remain bound instead of separating and base-pairing with the DNA of the other species.

The genomes of microbats, the only other mammals possessing true, flapping flight, are also A-T rich. So, when fruit bat and microbat DNA is hybridised, the combination of the two mechanisms exaggerate the closeness of their relationship and may grossly underestimate when they diverged from a common ancestor.

In 1995 Pettigrew took sabbatical leave to work in the Wisconsin laboratory of eminent vertebrate taxonomist Professor John Kirsch. In a series of experiments, Pettigrew hybridised megabat DNA with DNA from four microbat lineages, including three families of rhinolopids, supposedly the closest relatives of megabats.

Each time, he added fractionated, G-C enriched DNA to the mix, to compensate for the A-T bias. He also hybridised megabat DNA with tree shrew, colugo and marsupial DNA, the latter as an out-group.

Now, instead of megabats and microbats being each other's closest relatives, the data indicated only a very distant relationship. And the supposed ancestors of megabats, the rhinolophids, came out as being least related to microbats.

Previously, DNA, protein and enzyme comparisons had indicated that megabats were closely related to rhinolophid microbats, - but not to the other microbat orders - yet this bizarre result sounded no alarm bells.

Pettigrew's experiments showed the close relationship between megabats and rhinolophid microbats was almost certainly an artefact of the extreme A-T bias in both taxa.

And Pettigrew observes that the isochore problem is also likely to have led to substantial underestimates of when major lineages within the Microchiroptera arose. He suspects the major microbat lineages diverged much earlier than most researchers believe.

---PB--- Megabat divergence

While the isochore problem does not invalidate DNA hybridisation as a tool for phylogeny, it may have caused molecular geneticists to underestimate divergence times within groups like birds, bats and insects.

Unless there is reliable fossil evidence, molecular geneticists rely on the youngest DNA-based estimate of when species diverged.

"When you're building evolutionary trees, if you begin with the assumption of a short distance between two species, it distorts all your other branch lengths," Pettigrew says. "Even a five per cent underestimate of the distance between two taxa distorts the tree, and in the case the megabat-microbat divergence, we're probably looking a 50 per cent underestimate.

"So I discarded the shortest distance, and relied on independent data from other researchers, including morphological comparisons."

Pettigrew decided to represent the relationships between four taxa as a three-dimensional tetrad, placing megabat, microbat and colugo at three vertices, and placing the tarsier (the famously big-eyed nocturnal primates) at the fourth vertex, representing the primate out-group.

Assuming the short megabat-microbat distance to be an artefact, he discarded it, and used the remaining five pair-wise distances to make a four-way comparison. "The results were very consistent," he says. "They all showed an underestimate of the megabat-microbat distance."

Tellingly, that underestimate was greatest for rhinolophid microbats, which have the second highest A-T content of any mammal, after megabats. The results support a close relationship between colugos and fruit bats, with humans next, and microbats being least related to the other three.

Pettigrew believes he is close to confirming that colugos and fruit bats are related, which would make them a sister group to the primates, by descent from a shared, 88 million-year plesiadapiform ancestor.

That would leave microbats out on a limb. When did they evolve, and from what ancestral glider? The fossil record offers no obvious candidate. A jaw, earbones and wingbones of one of the world's earliest fossil microbats, from Eocene (55mya) sedimentary rocks at Murgon in Queensland, are indistinguishable from today's species.

The fossilised egg of a noctuid moth from Martha's Vineyard, in Massachusetts, offers circumstantial evidence that echolocating bats were around 75 million years ago. (Noctuid moths have a unique 'ear' on the thorax that detects bat sonar, triggering a reflex that causes them to fold their wings and crash dive out of the bat's path.)

Mantises, which evolved in the early Cretaceous, (~125mya) also have a thoracic ear that triggers evasive action on detecting bat sonar, suggesting bats were among the earliest placental mammals.

The discovery of a primitive gliding mammal in inner Mongolia's Daguo fossil beds in 2005 extended the record for mammalian aerialists at least to the Jurassic-Triassic boundary, 140 million years ago. Not closely related to any modern or extinct mammal group, Volacotherium antiquum had teeth highly specialised for an insect diet - an unusual trait for a glider, says Pettigrew.

"Nobody is suggesting it is a microbat precursor, but it adds to the circumstantial evidence that bats could have evolved very early," he says.

---PB--- The eyes have it

It was the structure of the fruit bat eye, and the neural circuitry of the brain's visual regions, that originally alerted Jack Pettigrew to the remarkable possibility that fruit bats share an ancestor with primates, not microbats, and that mammals must thus have evolved flapping flight twice.

Both colugos and fruit bats share with primates a distinctive eye structure in which the nerves from the light-sensing retina at the rear of the eye project forward before turning back through a central gap in the retina to connect to the brain.

As a result, the primate eye has a small blind spot at the centre of its visual field. Behind the eyes, the optic nerves connect to the brain via a structure called the lateral geniculate nucleus (LGN). In the primate, fruit bat and colugo, the LGN has six interleaved layers of nerves - three from each eye - that integrate the separate images from each eye.

Megabats devote more of their neocortex - the outer "modern" layer of the brain where sensory input is processed - to vision than any other mammal. The corresponding region in microbats is relatively tiny, and the auditory region correspondingly large, as might be expected for a mammal that lives in a world dominated by sound.

Microbats leave their young at home, in communal nurseries, while foraging. Megabats, like primates, carry their young with them until they become too heavy. Newborn microbats do not open their eyes for several weeks; baby megabats open their eyes at birth.

Chromosome counts in the 17 different families of microbats vary widely, suggesting the lineages are ancient. All megabats have the same chromosome count, indicating a more recent origin.

Microbat and megabat sperm are very different, and sperm structure is a basic indicator of relationships between species.

Megabats and microbats carry external parasites, including fleas and flies, but they represent different families.

Blood serum proteins, including haemoglobin, clearly link megabats with primates, and indicate a distant relationship with microbats. Alpha-crystallin, the clear, crystalline protein that forms the lens of the eye, tells a similar tale.

Pettigrew says when fruit bats and colugos defecate, they reverse position and dangle head-up by their hooked wing "thumbs". The colugo shares this demure posture, hanging by its thumbs during defecation. In contrast, microbats dangle by their back legs and arch their backs to avoid soiling their fur.

Microbats take their prey on the wing, while other species hover on the wing to sip nectar from flowers. Fruit bats land in trees to forage for fruit, and despite their wings, are agile climbers, moving like lemurs, according to Pettigrew.

Fruit bats have mammary glands on the chest, like primates, not on the abdomen, like most mammals.

Male megabats have a soft, pendulous penis with a prominent glans, like primates. Microbats have a bony penis, sans glans.

And last August, Chinese researchers reported an almost definitive clue linking fruit bats to primates: fruit bats have a menstrual cycle - a universal trait in primates, but very rare among the world's other, 4000-odd mammals.

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