insightLMU Research
Issue 02 · 2009
life sciences
S u sann e W e d l ich
evolutionary tinkering
New species can only arise if also new genes are formed. Duplication, which replicates
already existing genes, is one of the most important mechanisms for increasing the
number of genes. Evolutionary Biology Professor Wolfgang Stephan and his team were
able to show the importance of natural selection in such processes. Plant Biologist
­Professor Dario Leister, on the other hand, has demonstrated the existence of a ­hitherto
unknown path to the generation of new genes: in this process, foreign genetic material
that had been incorporated into cells millions of years ago is built into new gene
­arrangements.
No head, no organs, no muscle tissue: If you look at it like that, there is not a lot to Trichoplax adhaerens. If you don’t know what you’re looking for, you’ll probably not even notice
this creature in the sea of only a few millimeters in length – and miss the chance for an
­insight into the early evolution of multicellular life forms. This inconspicuous animal
­probably came into being more than 700 million years ago and therefore at an early stage
of the transition from unicellular to multicellular life forms. So, its anatomy is also ­extremely
simple: Two outer cell layers surround a body cavity within which cells move around
freely. Conversely, the genetic configuration of Trichoplax is all the more com­prehensive by
comparison: The animal has some 11,000 genes, which, on a purely quantitative basis,
­corresponds to approximately half the number of human genes.
At first, this is not particularly surprising. After all, a large number of genes is necessary in
any organism in order to maintain complex cell life. However, Trichoplax additionally has
gene arrangements that also exist, in a related form, in humans, where they mediate
­physiological functions that do not exist in simple unicellular life forms. Researchers con­
sider this as an example for what is probably the most significant evolutionary mechanism
for the creation of new genes: an already existing gene is duplicated, so that it is then
­present in an additional copy. Because the original gene continues to fulfill its tasks, the new
genetic arrangement is not necessary for the maintenance and function of the organism.
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Any changes in the newly created gene therefore do not have an immediate impact.
­Evolution therefore provides the option to experiment with new gene variants at practically
zero risk. Thus, as a rule the new gene changes faster than the original identical template,
until ultimately the duplicates will fundamentally differ in their structure and function. This
can lead to one of the genes retaining its original task, while the other one takes on a new
one. How exactly this happens had been substantially unclear until now – because there
seems to be a mechanism counteracting the divergence of gene copies: gene conversion.
In this process, individual segments of a gene copy are copied to the duplicate. Thus, for
example, a strongly mutating part of one of the gene copies could be replaced by an un­
changed counterpart from the other gene copy. In this way, over time, mutations should be
uniformly distributed over duplicated genes, and their evolution would therefore be tied
together by means of gene conversion.
ESCAPE MECHANISMS
How new genes are established after duplication in a population is the subject of the
­research undertaken by evolutionary biologists like Professor Wolfgang Stephan. “During
evolution increasingly complex life forms appear”, he reports. “This was possible only
­because genes with new functions were also created. And, one of the central tasks we evo­
lutionary geneticists are confronted with is to explain this process of functional differen­
tiation.” In this respect, Professor Stephan and his team were faced with the question of
how quickly duplicates must evolve differentially in order to escape the process of gene
conversion. The researchers also wanted to know the mechanism that made this escape
possible. “Ultimately, our results have shown that positive natural selection enables func­
tional differentiation of two gene copies and thus plays an unexpectedly significant role in
this process”, says Wolfgang Stephan. “However, this only works if it is very strong – and
also if, at the same time, gene conversion between the duplicates is suppressed.” Natural
selection is one of the most important mechanisms of evolution. It refers to the pressure
exerted by the environment on individuals. It is mediated over their genetic capability to
adapt and their reproductive success associated with it. Although the researcher in­vestigated two neighboring genes of the fruit fly Drosophila melanogaster that had been
generated by duplication: ph-d (distal) and ph-p (proximal), together they constitute the
locus ph (for “polyhomeotic”). “The duplication of ph took place at least 25 to 30 million
years ago”, he says. “Both copies are, however, still structurally and functionally very
­similar, which can probably be ascribed to gene conversion.” Nonetheless, differences
were noticed in the regulation of the two genes – which is a first sign of functional­
divergence. Therefore, Professor Stephan and his team analyzed the duplicate sequences
looking for unambiguous signs of strong positive selection.
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W H E R E T H E S ELE C T I O N P R E S S U R E H A S T H E S T R O N G E S T E F F E C T
A possible consequence of strong positive selection is a so-called selective sweep. Typical
for the occurrence of a selective sweep is a reduction of nucleotide variation in the genome
at a site where the beneficial mutation arose. And, indeed, the scientist found this signature
of a sweep in the area of the ph gene. Furthermore, he was even able to delimit the region
where the selection pressure is likely to have the strongest effect: This area is in the ph-p
gene. Then the question was: how was it possible to achieve neofunctionalization – i.e.
the evolution of a new function – although gene conversion occurred in this area in the
course of evolution? “We presume that this mechanism was inactivated in the relevant area
because too many changes took place in this non-coding part of the gene too quickly”, says
Wolfgang Stephan. “When the two gene duplicates are very different due to changes in a
copy, gene conversion becomes impossible.“ To date, natural selection as an aid to
­neofunctionalization has been considered implausible, because of its counterpart gene
© Source: LMU Munich
conversion.“
1 Reduction of nucleotide diversity in the polyhomeotic region of an African D. melanogaster population. The estimated target of
selection is indicated by an arrow. It is located within the large intron of ph-p. The lower part of the figure shows the sequenced 18
fragments, the structure of the genes in the region, and the physical distances (in kilobases).
“However, our result is surprising also from another point of view. After all, natural selec­
tion means that individuals producing more descendants than their fellow species members
successfully transmit their gene variants.” This fitness is transmitted via an individual’s
ability to adapt to the environment. Accordingly, scientists had expected to find selective
sweeps in particular in genes that directly participate in the adaptation of organisms. These
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could be genes that convey resistance to environmental toxins or have an effect on sensory
perception. “However, the gene we examined probably has no direct environmental
­connection, but, rather, regulates the activity of hundreds of other genes. It is thus possible
that natural selection plays an even greater role in neofunctionalization than had been
imagined to date.”
The team surrounding Professor Dario Leister and his collaborator Dr. Tatjana Kleine, on
the other hand, was confronted with a distinctly environment related gene: While they were
examining another mechanism of gene origination, the researchers came upon an arrange­
ment in Arabidopsis that may possibly generate a survival advantage for the plant when
subjected to salt stress. However, this gene does not originate from duplication, but, in fact,
contains at least partially foreign material. This friendly takeover is the fortunate result of
a process called endosymbiosis. Millions of years ago, free living proteobacteria and
­cyano­bacteria have been taken up by host cells. As they were originally able to live in­
dependently, they had their own genetic material.
Mitochondrium
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7 Overview of known types of inter-organelle DNA
transfer. 1, mitochondrion-to-nucleus; 2, plastid-to-
© Source: LMU Munich
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nucleus; 3, plastid-to-mitochondrion; 4, nucleus-tomitochondrion; 5, mitochondrion-to-plastid. Such
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complete gene transfers hardly ever take place any
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more, says Dario Leister. Nonetheless, there is still
always genetic material being transferred from the
Chloroplast
plastids and mitochondria into the cell nucleus.
Kern
However, the intruders gradually lost their autonomy and finally transformed into cell
­organelles, i.e. into components of the host cells having their own individual function. For
example, they constitute the energy generating mitochondria of higher organisms. In addi­
tion, algae and plants also harbour so-called plastids, which are themselves descendants of
the endosymbionts. A special form are chloroplasts. They carry out photosynthesis, there­
fore generating sugar, as an energy carrier, as well as oxygen, from sunlight, water, and
carbon dioxide. In the meantime, the organelles possess only a fraction of the genetic in­
formation of their precursors; the major part of their genotype has been transferred step by
step into the core of the host cell, the cell nucleus. This transfer took place mainly in the
early phase of the concerted evolution. In this process, complete genes, that means
DNA sequences encoding proteins, were also transferred and integrated into the genetic­
material of the host.
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“Such complete gene transfers hardly ever take place any more”, says Dario Leister. “None­
theless, there is still always genetic material being transferred from the plastids and mito­
chondria into the cell nucleus – and this in surprisingly large amounts. These DNA sections
are entirely randomly built in anywhere into the host’s genetic material, which as a rule
takes place without any problems. However, this can also cause potentially damaging
changes if for example an important gene is destroyed by the introduction of the foreign
material.“ The researchers then investigated whether the transfer can also have a positive
effect. Or, more accurately: can late evolutionary DNA transfers from the organelles to the
cell nucleus lead to generation of new functional genes? To date it was known that new
genes are indeed generated by means of similar mechanisms, among other things through
a redistribution of genetic material within the cell nucleus. Some examples could be the
fusion or splitting of already existing genetic arrangements. In addition there are also
­so-called mobile DNA elements that randomly introduce themselves into the genetic
­information.
“We wanted to know whether a fusion of organelle and cell nucleus DNA could cause a
positive effect, and have demonstrated that new genes continue to be formed in the cell
nucleus by means of adoption, adaptation and modification of the genetic material of the
organelles”, reports Dario Leister. In a first step, the scientists looked for former organelle
DNA sequences in the cell nucleus DNA sequences of humans, Arabidopsis, rice and yeast
that are now integrated in or next to the genes of their hosts. This can be done by using
computer software that can compare DNA sequences and search for similarities between
the sequences. The number of DNA sequences from organelles that integrated into the cell
nucleus DNA was surprisingly high, and in fact it is likely that many more of these sequenc­
es can no longer be identified as such because of their small size.
Genes carry the instructions for the construction of proteins, which are a cell’s most im­
portant function owners. The genes of higher organisms, however, consist of encoding
portions, the exons, and non-encoding portions, called introns. If a protein is to be synthe­
sized, first a copy of the gene that contains these instructions is made. If it cannot be
­copied, it is a “pseudogene”. The introns are then cut out of this molecule and the exons
are combined. Finally, these combined building blocks contain the instructions for the
­synthesis of a protein. The genetic contribution of the organelle DNA can consist of indi­
vidual exons, but is therefore essential for the structure of the entire gene as well as of the
corresponding protein.
In order to prove that by the still ongoing DNA transfer from the organelles into the cell
nucleus it is actually possible that functional genes can be newly formed, the scientists
examined an Arabidopsis gene whose sequence consists of cell nucleus and chloroplast
DNA. By comparing this sequence with other already known plant sequences, they were
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able to date the insertion event of the organelle DNA into the cell nucleus DNA to approxi­
mately 50 million years ago. “We were able to detect a transcript of the gene”, says Dario
Leister. “A lot of it was produced under salt stress conditions. This gene acts as a model for
the creation of a new gene by the combination of sequences of organelle and cell nucleus
DNA, and may help the plant acquire a selection advantage under salt stress. This is a gene
generation path that was hitherto unknown. It would seem that evolution works like a
­tinkerer: It gets genetic information from wherever it can and tinkers it together.”
Professor Dr. Dario Leister has held the Chair for Botany since 2005.
Previously he was a Heisenberg Fellow of the German Research Association.
[email protected]
www.botanik.bio.lmu.de/ueber_uns/professuren/leister/englisch/homepage_leister_en
Professor Dr. Wolfgang Stephan has held the Chair for Evolutionary Biology since 2000. He is a member
of the Senate Commission on Biodiversity Research of the German Research Foundation (DFG),
and the speaker of the DFG Research Unit on “Natural Selection in Structured Populations.”
[email protected]
www.zi.biologie.uni-muenchen.de/evol/EvoBio
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