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IMAGINE that for years you’ve been using that old clock in the village
square to tell the time, making sure you get to the pub soon after it opens.
Then one day you decide to find out how this ancient timepiece works. So
you climb the steps of the clock tower, throw open the doors of the clock’s
housing, peer inside in search of the mechanism that all these years has
kept you more or less punctual, only to discover that it’s full of pigeon
feathers, dead rats and other unspeakable debris – nothing that could possibly
turn the hands of the clock in anything like a regular manner. Naturally,
you con clude that the clock cannot work after all. Except, of course, you
know it does.

That, roughly speaking, is how some biologists are coming to feel about
the molecular evolutionary clock. ‘It works, so long as you don’t look too
closely to try to understand what’s driving it,’ says Hampton Carson, a
geneticist at the University of Hawaii. This is bad news for the molecular
clock, because it is in danger of being investigated out of existence.

The idea of the molecular clock goes back 25 years, and is seductively
simple. As two related species diverge through evolutionary time they accumulate
genetic differences, or fixed mutations, at a roughly steady rate. This
means that if you measure the genetic difference between two such species,
and you calculate the rate of accumulation of mutation – the rate at which
the clock ticks – then you can work out when the lineages split from each
other. ‘There are lots of examples where this kind of thing has been done,’
says Vincent Sarich, a biochemist at the University of California at Berkeley
and a champion of the molecular clock. ‘There is a clock, no doubt about
¾±³Ù.’

Sarich and his colleague Allan Wilson have used the theory of the molecular
clock on creatures small, feathered and furred. This body of work includes
the timing of the evolutionary split between humans and apes, and, more
recently the announcement by Wilson and his colleagues that Mitochondrial
Eve – the mother of us all – lived in Africa some 200 000 years ago (see
‘All about Eve’, ¿ìè¶ÌÊÓƵ, 14 May 1987). Not surprisingly, Sarich and
Wilson now find it more than a little irritating to have people tell them
their prized tool does not exist after all. According to Sarich, ‘They’re
just theoreticians who don’t have real world concerns . . . There’s a refusal
to recognise that biologists don’t require precise answers. You don’t need
99 per cent accuracy; 80 per cent will do just fine, thank you.’

So, is it a question of how well the molecular clock keeps time – 80
per cent accuracy as against 99 per cent? Or is it a matter of whether the
clock exists at all? Biologists pondered this at a recent meeting at the
Banbury Conference Center of the Cold Spring Harbor Laboratory, New York.
For Sarich the experience was frustrating: ‘It felt like being in a time
warp. I might as well have been back in the 1960s, with the recognition
that we were given.’ Philip Gingerich, a palaeontologist at the University
of Michigan, was equally frustrated, but for different reasons. ‘I came
expecting to be shown lots of clocks, but I haven’t seen any,’ he said.
Francisco Ayala, a geneticist at the University of California, Irvine, took
a more optimistic stance. ‘I am confident we will find a good clock,’ he
said. ‘But first we must learn to recognise ¾±³Ù.’

The molecular clock concept has always attracted controversy, with various
groups dismissing the idea, often despite evidence in support of it. ‘Twenty-five
years ago the situation was much simpler,’ commented Emile Zuckerkandl,
who with Linus Pauling developed the concept in the early 1960s and coined
the term ‘molecular clock’ in 1965. ‘For most people, the answer to the
question of whether there is a clock was, ‘No’. Very few biologists would
take the suggestion seriously, because they ‘knew’ that nothing in evolution
moved at anything like a regular pace.’

This certainty that a molecular clock could not exist was a reaction
to a particularly bizarre piece of theoretical nonsense, orthogenesis, that
had entranced evolutionary biologists for several decades early in the century.
The notion was that evolution was driven internally and inexor ably in particular
directions and at a steady rate. According to this theory, sabre-toothed
tigers were the agents of their own extinction, because, having begun to
elongate in dramatic fashion, their long, curved canines were destined to
grow longer still, eventually leaving the animals incapable of closing their
jaws. Extinct, with their mouths agape. Similarly, certain oysters, Gryphea,
alive in the Mesozoic era evolved themselves out of existence, because,
following an internal trajectory, their coiled shells coiled once too often,
thus clamming themselves shut in a prison of their own making. ‘Embarrassing,
embarrassing,’ is Gingerich’s observation on the business of orthogenesis.

Passion in the Ivory Tower

‘Having disposed of orthogenesis, biologists came to recognise what
is in fact blindingly obvious, that morphological evolution runs at all
kinds of rates,’ said Zuckerkandl. ‘There is nothing constant about it at
all. And the very reasonable inference was that there was unlikely to be
anything regular about molecular evolution either. So, molecular clocks
could not exist. Period.’

Reasonable inference or not, there was without doubt a measure of pure
emotional reaction against the suggestion of a regularity in evolution,
even in the molecular realm. ‘I know the aftermath of the orthogenesis episode
prejudiced me against the idea of a molecular clock for a long time,’ admits
Gingerich. The palaeontologist from Michigan had plenty of company, and
that band swelled even further when Sarich and Wilson’s foray into palaeoanthropologists’
territory produce figures for the ape/human split that were dismissed as
nonsense by the scholars who were supposed to know about these things.

Zuckerkandl and Pauling built the molecular clock concept by comparisons
of amino acid sequences in haemoglobin and cytochrome c. They garnered support
from Walter Fitch and Emanuel Margoliash of the University of Wisconsin,
who in 1967 used cytochrome c sequences to reconstruct a family tree of
a wide range of mammalian species, which more or less coincided with what
palaeontologists believed on the basis of anatomy. Sarich and Wilson used
other proteins – serum albumins – for their comparison of apes and humans,
and instead of marking off differences in amino acid sequences among the
different species, they determined genetic divergence by an quantitative
immunological technique. It is a different, less direct, method of counting
the ticks of the clock.

Using this technique, they worked out that apes and humans diverged
about 5 million years ago, practically yesterday compared with the 15 to
30 million years that palaeo anthropologists believed in the late 1960s.
‘We were variously ignored, abused and scorned,’ remembered Sarich. ‘But
look at the figure they talk about now – about 7 million years. We were
more or less right.’

In the two and a half decades since the clock concept was advanced,
the application of the theory to the relationship between apes and humans
has been the one most thoroughly scrutinised. This has involved many different
methods of listening to the clock ticking, including protein electrophoresis,
amino acid sequencing, restriction mapping of mitochondrial DNA, and sequencing
of mitochondrial and genomic DNA. Virtually all answers fall between the
5 to 10 million year ago period, with 7 to 8 million usually given as a
good average. ‘If you need better evidence that there’s a molecular clock
working in there somewhere, I don’t know what it is,’ says Sarich.

In 1967, however, when Sarich and Wilson published their paper in Science,
the data were seen to be the result of an assumption of a clock, not proof
of it. Even Fitch and Margoliash’s mammalian family tree was far from perfect.
What was needed was some fundamental argument in support of the clock concept,
a real theoretical base. The neutral theory of molecular evolution appeared
to provide this sound base. The idea of neutrality was much in the air at
the time, but unformed as a theory. That step was taken by the brilliant
Japanese geneticist, Motoo Kimura, who published a mathematical description
of the theory in 1968.

‘The theory does not deny the role of Darwinian positive selection in
determining the course of adaptive evolution,’ Kimura said recently, ‘but
it assumes that only a small fraction of DNA (or protein) changes are adaptive.’
So, if the great majority of genetic change proceeds unmolested by positive
or negative selection, genetic mutation would indeed accumulate at a more
or less steady rate – the molecular clock would therefore tick with metronomic
regularity. Here, apparently, was the mechanism that kept the cogs turning
in clocklike order. The neutral theory provided for many observers the rationale
for the existence of a global clock, one that, with appropriate calibration,
might be applied to any conceivable biological comparison.

But it was not to be. ‘Yes, the clock concept gave birth to the neutral
theory, but perhaps not legitimately,’ says Zuckerkandl. ‘The theory might
be right in many important respects, but it does not give us the basis of
a molecular clock.’ In the mid-1970s Fitch worked with Chuck Langley of
the National Institute of Environmental Health Sciences, North Carolina,
to produce a series of protein sequence comparisons among more than a dozen
mammalian species, measured against a timescale of evolutionary divergence.
‘This showed that there is a rough match between amino acid substitutions
and time passed,’ explained Fitch. ‘But it also showed that the rate of
change is by no means constant. The neutral theory in the strict sense cannot
explain the rough clock we see.’

So in recent years there has been increasing emphasis on selection and
how it might operate to give a degree of constancy in the accumulation of
mutations over long periods of time. While he is no champion of the neutral
theory, Zuckerkandl nevertheless is concerned that sentiments might be swinging
too far in the selectionist direction. ‘The neutralists had a big appetite
in their day, and wanted to consume the entire genome,’ he said at the Banbury
meeting. ‘The selectionists should not make the same mistake.’ In other
words, selection may well be an important element in genetic change, but
other processes are at work too in various parts of the genome, and these
must be taken into account in building a complete pattern of genetic change.

Obviously, if selection on a particular gene continued unrelentingly
at a constant pressure, you might expect to see a constant rate of substitution
in the DNA over long periods of time. But selection is likely to shift from
time to time, in response to ecological change, for instance, thus producing
the local inconstancies that Fitch and Langley saw.

You can see the effects of different levels of selection pressure, by
looking at proteins that are subject to different degrees of functional
constraint – that is, the structure of some proteins can be altered slightly
without impairing their function, while others can tolerate little or no
change at all. Some proteins are fundamental to life, and any modification
of their structure utterly alters their function. Histone, for instance,
is very tightly constrained, and accumulates mutations very slowly, whereas
globin is less constrained and has a higher rate of accumulation – the histone
clock ticks very slowly, the globin clock fast. Differences of this sort
would not necessarily undermine the theory of a global molecular clock:
you could have a histone clock or a globin clock. But, as Fitch noted, ‘wherever
you look for it, you also see variation in rates through time’. Exit the
cherished notion of a global molecular clock.

The most striking variation in rate is a slowing down. This is not just
the fact that as time passes there is an ever increasing chance of a double
hit – one base of DNA being substituted for another at a site that had already
been substituted earlier in the gene’s history. But this does not account
for all the observed cases of ‘slowdown’. It could be the result of increased
functional constraint, increased efficiency of DNA replication and repair
mechanisms, or a combination of the two. Whatever the cause, the consequence
is that ‘you are stuck with local clocks at best’, said Morris Goodman,
of Wayne State University, and a pioneer in studies of molecular evolution.

By local clock Goodman means the ability to establish a reasonable degree
of regularity of genetic change, probably using one or just a few genes,
focusing on a limited number of species, and looking through a narrow time
window. ‘For most practical purposes this is fine,’ says Goodman. ‘But you
have to demonstrate the clock works each time you set one up, and you cannot
extrapolate outside the range you’ve defined.’

It was Goodman’s experimental footsteps that Sarich and Wilson were
stepping in when they produced the first molecular clock numbers for the
ape/human split. Although he did not date the divergence, Goodman earned
the Berkeley duo’s approbrium by suggesting that he could see a slowdown
in rates of protein evolution among the primates. It turned out that Goodman
was being misled to some extent by fallacious dates then championed by the
palaeoanthropologists. But, even with better calibration dates and much
more molecular data, he still identified a slowdown, which differs according
to the lineage. ‘It’s a real phenomenon,’ said Goodman, ‘no doubt about
it’ .

The differences in mutation rates that Goodman sees among relatively
closely related primate species may be greatly magnified when you look further
afield. According to Roy Britten, a molecular geneticist at the California
Institute of Technology, ‘Rates of DNA change can differ by as much as a
factor of five.’ For instance, DNA changes rapidly in sea urchins and Drosophila,
somewhat slower in rodents, and slowest of all in birds and primates, particularly
the higher primates. The differences, speculates Britten are ‘probably due
to evolutionary variation and selection of biochemical mechanisms such as
DNA replication or repair’.

Inconstant clocks

The one constant that is beginning to emerge concerning the ticking
of the molecular clock appears to be variation – variation at all conceivable
levels, that was the message of the recent Banbury meeting. Different rates
of evolution between different lineages. Different rates within a lineage
over different periods of time. And, in one spectacular example at least,
dramatically different rates of change within the genome of a single species.

Both Britten and Jeff Powell, a molecular biologist at Yale University,
described two species of Drosophila – melanogaster and simulans – that only
recently diverged from each other and are sufficiently genetically compatible
to be able to produce viable but infertile hybrids. While most of the DNA
in the genomes of these two species has not diverged much, as you would
expect given the small amount of time for accumulation of mutation, about
a third has started to diverge at a remarkable rate. The degree of genetic
difference is so great that, mixed together in the standard DNA-DNA hybridisation
test, they simply fail to recognise one another physically. This implies
a divergence of DNA sequence in this component of the genomes from these
two species of at least 30 per cent, 10 times greater than in the rest of
the genome.

If you look at single stretches of DNA within a genome, not at different
genomic territories, again you can see differences in mutation rate. For
instance, Britten showed a slide of the DNA sequence near to but outside
the coding region of a gene in Drosophila, and pointed to what amounts to
a mutation watershed: on one side a high rate of substitution, on the other
a low rate, the difference being 10-fold. ‘The interesting thing is that
this is a non-coding region of the DNA, with no obvious difference in function
on either side of the boundary,’ noted Britten. He wondered whether the
boundary marked the point of origin of two sets of replication machinery,
or replicons, with one prone to little error, the other prone to a lot.
Whatever the explanation, the observation further upsets the workings of
an already shaky molecular clock.

The one area of potential stability in all this uncertainty has always
been the so-called ‘silent site’, the third position in the three-letter
nucleotide code in genes that specifies amino acids. Substitutions at the
third position only occasionally cause a change in the amino acid specified
by the codon, which means the site is relatively unconstrained by selection
and may drift relatively freely. As a result, researchers frequently compare
silent-site substitutions among different species, with the assumption that
they are looking at mutation uninfluenced by selection – a pretty fair molecular
clock. But Margaret Riley, a population geneticist at the University of
Massachusetts, warns that ‘there’s no such thing as a clock with silent
sites, because we know about codon bias’. This phenomenon means that third
positions may under certain circumstances be less free to drift than biologists
once imagined. Silent sites in the same gene may drift at different rates
in different species, thus scuttling the perfect clock. ‘We don’t fully
understand codon bias,’ says Riley, ‘but everywhere we look, we find ¾±³Ù.’

So, will the theory of the silent-site clock have to be scrapped too:
‘If you were to look at substitution in silent sites in one gene among several
species, I would expect to see considerable variation in rate,’ says Riley.
‘But if you were to take a dozen genes, maybe the variation would average
out and you would have a reasonable clock.’ The same may apply to other
forms of the clock. Take a collection of proteins or genes, stand back and
you see that the rate of substitution is roughly constant over time. But,
hold up a magnifying glass to the innards of the clock to try to discover
where the constancy comes from, and the whole thing looks a mess, with rates
going in all directions. As Riley says: ‘It all depends on what you’re interested
¾±²Ô.’

At the centre of the current debate about the molecular clock is the
choice between the fundamental mechanics of the clock and the question of
utility. If you are interested in knowing how DNA evolves, how its various
components behave in the face of myriad subtle influences from selection
and factors in the genome’s microenvironment, then you come away from the
clock tower with images of pigeon feathers, dead rats and other debris.
There is no molecular clock, no metronomic ticking. But if you wish simply
to obtain reasonably accurate dates for when specific species diverged,
then you’ll be satisfied with limited, local clocks. (You probably won’t
even make the trip up the clock tower, and you’ll get to the pub on time,
as you always have.)

* * *

Are molecular clocks slowing down?

OF ALL the taxonomic groups that have come under the scrutiny of molecular
clocks, the primates have received most publicity. And it is among the proponents
of these molecular clocks that the idea of slowdown has been debated most
intensively. ‘You see slowdown wherever you look,’ said Morris Goodman of
Wayne State University, ‘but it looks really interesting in the higher primates.’

In the face of counterarguments from his critics at the University of
California at Berkeley, Goodman has long argued that a reduction in the
rate of the accumulation of mutations over time was inevitable. ‘In essence,
the slowdown hypothesis proposed that over eons of time natural selection
increased the internal complexity of life and, in safeguarding the new and
complex functions that had evolved, slowed the rate of molecular evolution,’
explained Goodman. From time to time, however, the pressure of natural selection
shifts as organisms experience new environments, and the rate of mutation
may increase dramatically for a while. But, overall, there will be a general
drift towards lower rates through time.

Here, then, Goodman is arguing for a gradually reduced rate of mutation
in the protein structure of the organism through evolutionary time. But,
he says, the phenomenon goes deeper, to the level of the DNA sequence, even
to sequences that do not directly code for amino acids. ‘The key premise
is that a majority of genomic DNA sequence changes are neutral changes having
little or no effect on the phenotype,’ explains Goodman. ‘Thus, decreases
in de novo mutation rates should decrease rates of DNA sequence change.’

But why should rates of new mutation decrease through evolutionary time?
The main reason is a fine-tuning of biochemical repair mechanisms that guard
the fidelity of DNA replication. But another factor is difference in generation
time in large and small species. For instance, for every human generation,
mice run through 100 generations. And, as the production of each generation
is an opportunity for mistakes to occur in DNA replication, you expect to
see a much higher rate of mutation in mice. There is, but it is only a five-fold
difference, not 100-fold.

The reason that the disparity is smaller than predicted is that it is
in the turnover of germline cells that replication errors are accumulated,
not simply at each new generation. ‘There is a correlation between generation
time and germline turnover, but it is clearly not a direct relationship,’
said Goodman. From this, you would expect the small-bodied (short generation
time) primates to have a higher rate of mutation than the large-bodied primates
(long generation time). As primates have tended to grow larger through evolutionary
time, the lower mutation rate associated with these species would also be
reflected in the group’s history.

By now Goodman and his colleagues have accumulated considerable sequence
data about the primates, particularly data from various globin genes. In
primates as a whole, the data show a drop in the rate of non-coding changes
through time. In addition, says Goodman, the studies ‘clearly demonstrate
that marked nonuniformities in the accumulation of mutations . . . have
occurred in different primate lineages,’ said Goodman. Rates among the small
prosimian primates such as tarsiers and galagos are highest, with New and
Old World monkeys clocking in about half this rate, and apes and humans
about half the monkey rate. Completing the trend, the greatest slowdown
seems to be among the human ancestors, the hominids.