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.