The Problem with Gorilla Mitochondrial DNA Analysis
A mere glance at a map of the distribution of gorillas in Africa reveals
a striking pattern. In contrast to chimpanzees, which occur more or less
continuously across equatorial Africa, gorillas are limited to two discontinuous
areas in West and East Central Africa. While western gorillas are relatively
numerous, with an estimated total population of up to 110,000 individuals
distributed over about 709,000 km², gorillas in eastern Africa are
much more limited in number and found principally in scattered populations
(Sarmiento 2003). This pattern raises interesting questions, such as the
length of time western and eastern gorillas have been separate from one
another, and whether the populations have been very different in size
for a long time or only rather recently.
To address such questions, scientists have often turned to laboratory
analysis to estimate the relative amounts and geographical pattern of
genetic variation present in representatives of the populations of interest.
A commonly-used tool in such studies is analysis of the mitochondrial
DNA (mtDNA), a type of DNA found in all cells, but separate from the genomic
DNA that forms the chromosomes. Some peculiar properties of mtDNA, such
as a particularly high rate of evolution and maternal inheritance, make
it especially informative for studying the evolution of populations in
the last tens of thousands or few million years (Avise 2000).
Research published in the last decade on the pattern of mtDNA evolution
in gorillas estimated a date of about 2.2 million years ago for the split
between western and eastern gorilla mtDNA (Ruvolo 1996) and suggested
that the amount of variation within western gorillas was about ten times
greater than that found within eastern gorillas (Garner and Ryder 1996)
- a striking difference in comparison with results from studies of
other animal populations. However, while mountain gorillas were sampled
intensively from their small range in the wild, far fewer western gorillas
were analyzed and most of those were captive individuals whose ultimate
origins in Africa were unknown. This made it difficult to directly compare
levels of variation between western and eastern gorillas since the sampling
schemes were so different. Another remaining question of interest was
determining how the genetic variation was distributed across the range
of western gorillas.
We decided to conduct a study of gorilla mtDNA using samples from wild
gorillas. We relied upon noninvasive samples such as feces or hair that
could be collected from nests without disturbing the animals. Back in
the lab, we used standard polymerase chain reaction (PCR) techniques to
make copies of our target mtDNA segment of interest - the hypervariable
segment of the control region. This segment is frequently used in studies
examining variation within species as it contains the most variation in
the mitochondrial genome.
We almost immediately ran into difficulties because we often found more
than the expected one unique sequence per individual. The multiplicity
of sequences was suspected to be due to the inadvertent inclusion of pieces
of mtDNA that had become copied to the nuclear genome, so-called "nuclear
insertions of mtDNA" or "numts" (Lopez et al. 1994). These
numts occur in a variety of animal genomes, and if recently-integrated
numts very similar to the mtDNA segment of interest are present, it can
be very hard to reliably distinguish real mtDNA from numts (Bensasson
et al., 2001). Some suggestions for distinguishing authentic mtDNA sequences
from numts rely upon comparison of questionable sequences with those of
assumed authenticity, and a new study investigating mtDNA variation in
wild gorillas relies upon such comparisons (Clifford et al., 2004). Troubled
by the uncertainty inherent in such subjective comparisons, we decided
to investigate the matter directly.
We used a method ("long-range" PCR) that could produce only
the authentic mtDNA from two individuals, a western and an eastern gorilla,
and compared the results to the collection of sequences obtained from
those same individuals using conventional PCR methods (Thalmann et al.,
2004). We had hoped to see consistent differences between the authentic
mtDNAs and the imposter numt sequences, so that we would be able to use
these differences as criteria for determining authenticity of sequences.
Unfortunately, the numt sequences were so numerous and so similar to the
authentic sequences, no reliable criteria could be devised that would
allow distinguishing of the authentic sequences. This was especially disappointing
as the long-range PCR procedure required to produce authentic mtDNA sequences
requires the use of high-quality DNA samples derived from blood or tissue,
and so we have as yet no way to determine authentic mtDNA sequences from
our fecal and hair specimens.
Interestingly, we also conducted the same analysis using representatives
of the other great ape species, and had no difficulties in reliably generating
authentic mtDNA sequences from humans, chimpanzees, bonobos and orangutans.
The underlying reasons why numts are prevalent in some animal genomes,
and relatively infrequent in others, are currently not known (Bensasson
et al., 2001).
The take-home message of our study was that all conclusions based upon
analyses of gorilla mtDNA control region variation should be considered
suspect. A total of three sequences from captive gorillas have been authenticated.
Some of the rest of the data generated may also eventually be proven to
be authentic and hence usable, but an objective assessment is impossible
at present time since the means for direct validation - DNA from
blood or tissue samples - are not available from most individuals.
This suggests that insights into patterns of genetic variation in gorillas
will depend upon analysis of genetic segments occurring in the nuclear
DNA.
In a new study, researchers used analysis of 50 nuclear DNA segments sampled
from captive individuals to infer a level of nucleotide diversity in gorillas
twice as high as that in humans, but only slightly higher than in chimpanzees
(Yu et al., 2004). If the technical challenges of working with DNA from
noninvasive samples can be surmounted, application of a similar approach
of surveying multiple, independently-evolving nuclear DNA segments from
wild individuals may provide a reliable means towards obtaining more detailed
insights into the population history of gorillas.
Linda Vigilant, Olaf Thalmann and Brenda Bradley
|
The problem with numts
I) A segment of mtDNA becomes integrated in the nuclear DNA. II) Potential outcome of a PCR amplification targeting a DNA segment that is present in the mtDNA and also in the nuclear DNA. III) Tree analysis depicting close relationships among authen-tic mtDNA sequences and recently integrated nuclear copies. |
References
Bensasson, D. et al. (2001) Mitochondrial pseudogenes: evolution's misplaced
witnesses. Trends Ecol. Evol. 16:314-321.
Clifford, S. L. et al. (2004) Mitochondrial DNA phylogeography of western
lowland gorillas (Gorilla gorilla gorilla). Mol. Ecol., in press.
Garner, K. J. & Ryder, O. A. (1996) Mitochondrial DNA diversity in
gorillas. Mol. Phylogenet. Evol. 6:39-48.
Lopez, J. V. et al. (1994) Numt, a recent transfer and tandem amplification
of mitochondrial DNA to the nuclear genome of the domestic cat. J. Mol.
Evol. 39:174-190
Ruvolo, M. (1996) A new approach to studying modern human origins: hypothesis
testing with coalescence time distributions. Mol. Phylogenet. Evol. 5:202-219
Sarmiento, E. E. (2003) Distribution, taxonomy, genetics, ecology, and
causal links of gorilla survival: The need to develop practical knowledge
for gorilla conservation. In: Taylor, A. B. & Goldsmith, M. L. (eds.)
Gorilla Biology: A Multidisciplinary Perspective. Cambridge (Cambridge
University Press).
Thalmann, O. et al. (2004) Unreliable mtDNA data due to nuclear insertions:
a cautionary tale from analysis of humans and other great apes. Mol. Ecol.
13:321-335
Yu, N. et al. (2004) Nucleotide diversity in gorillas. Genetics, in press
Dr. Linda Vigilant works at the Max
Planck Institute for Evolutionary Anthropology, Leipzig, Germany, and
runs a research laboratory in which tools of genetic analysis are applied
to questions of the reproductive strategies, kinship, dispersal, population
histories of wild primates.
Olaf Thalmann is a graduate student at the MPI in Leipzig, using
genetic analysis of samples from wild gorillas to infer the population
structure and long-term demographic history of this species.
Dr. Brenda Bradley did her dissertation at Stony Brook University
on the molecular ecology of wild gorillas and is now a postdoc at the
MPI for Evolutionary Anthropology in Leipzig.