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D I S C OV ER I ES
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MICHEL OHMER
DNA Barcoding as a Solution in the Speciation Debate?
The Cautionary Tale of the African Elephant
A relatively recent innovation in evolutionary biology has been
the use of mitochondrial DNA for creating phylogenetic trees of
divergence among closely related taxa. Combined with nuclear DNA
analysis, an especially important application for mitochondrial DNA
(mtDNA) involves resolving taxonomic debate about an evolutionary lineage; at the finest scale, mtDNA can provide insight into the
classification of a species. Recently, however, it has been suggested
that one or two reference mtDNA genes, if sequenced and standardized, may serve as a “barcode” of sorts for the correct identification
and classification of all life (Hebert). Such a statement promised to
resolve much of the debate surrounding species identification today,
especially in cases where morphology and genetics seemed to conflict,
or hybridization was apparent but ambiguous enough to cause dispute
among evolutionary biologists. When scientists realized that they had
overlooked an entire species of African elephant in 2001, the consequence of this application met its critical significance. Hardly of a size
to be missed, this giant relic of a once speciose order is endangered
and declining in numbers, making the classification of its species of
particular importance to conservation biologists and political advocates alike. However, this species classification mix-up led to claims
of even further divergence among African elephant lineages, and soon
all was returned to the age-old “what is a species?” debate. This debate
uncovered the dangers of relying too heavily on mtDNA, especially in
groups of organisms, such as elephants, where female philopatry and
matriarchal social structure exist. Therefore, there is a limit to which
mtDNA can serve as a “DNA barcode” for species identification, and
this limitation can lead to crucial mistakes with possibly long-reaching
consequences (Hebert).
The idea of DNA barcoding presented by Hebert et al. is by no
means new, as Craig Moritz and Carla Cicero pointed out in 2004,
because molecular markers have been used in the past. However, their
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proposal of a DNA barcoding technique for the classification of the
diversity of life is set apart by its stress on an increase in scale and standardization, in which only one or two reference mtDNA genes would
be selected to be sequenced in every taxon on earth, from poison dart
frogs to Giant Anteaters. Such an ambitious plan, however, could never
be supported fully unless a wide range of taxa were sequenced and
then tested for accuracy. Thus, the debate of elephant taxonomy was a
perfect opportunity to test this claim.
African elephants, at one time classified simply as Loxodonta
Africana, live within fragmented ranges throughout sub-Saharan
Africa, a shadow of their former majestic selves that once traversed
the entire continent. Their population numbers have declined by half
since 1979, with an estimated only 400,000 to 500,000 African individuals left (Barnes). This rapid population decline has largely been a
result of commercial poaching for ivory and the encroachment of everincreasing human settlements, pushing elephant populations into the
boundaries of protected areas and preventing them from the large-scale
migration they once performed in search of food and water resources.
This has lead to even greater problems, for as a keystone species and
an ecosystem engineer that often leaves destruction and disarray in its
path, its habitat restriction has thus put an even greater strain on the
ecosystems that it was once an integral part of. Not surprisingly, African elephants have received their fair share of research attention, but
until recently very little was known definitively about their population
differentiation throughout the continent and thus whether speciation
within L. africana had occurred. It has long been obvious to scientists,
however, that two groups of elephants, the widely distributed savannah elephant and the shy and morphologically distinct forest elephant,
did indeed exist, but until recently the extent of their divergence had
remained controversial. At best, these two groups had been classified
as sub-species within L. africana: L. a. africana and L. a. cyclotis,
respectively. Indeed, to the human eye the distinctiveness of the forest
elephant is difficult to miss: they are smaller, have longer, straighter
tusks, and round, instead of pointed, ears (Sikes). Nonetheless, these
differences were not seen as meaningful indicators of significant evolutionary divergence because it was assumed that these elephant subspecies intermixed at the edges of forests, creating a hybrid zone that
would prevent complete speciation. However, it has now been confirmed by key morphological and nuclear genetics studies that two
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African elephant species should indeed be recognized: L. africanas
and L. cyclotis.
The resolution of this speciation debate arose from what had been
needed for quite some time: a wide-ranging genetic phylogeny of
allelic divergence among African elephant populations from all over the
continent (including both assumed forest and savannah populations).
Morphological evidence provided by the analysis of 295 elephant
skulls by Grubb et al. offered only half the story, for morphological
evidence can serve as a launching board to understanding the speciation
of a lineage, but is rarely conclusive enough to define an extant species.
That is why a team of researchers led by Alfred L Roca and Nicholas
Georgiadis undertook the task of acquiring dart-biopsy samples from
195 elephants in 21 African elephant populations. These samples were
then tested for DNA sequence variation at four nuclear gene sites (1732
base pairs). DNA sequence data was taken both from short exon, or
coding, segments and long intron, or non-coding segments, but only
the intron segments could be used to determine phylogenetic diversity or relatedness as they evolve much more rapidly (Roca GE). Furthermore, by focusing on introns, researchers could exclude forces of
natural selection (as they do not act on introns), therefore making the
results more reliably dependent on acts of random genetic drift within
the elephant populations (Vogel).
Only 52 of the base pairs proved to be reliable in demonstrating
differentiation, but these conclusively supported a change in current
elephant taxonomy. Even after analyzing the phylogenies of these populations with three distinct methods (maximum evolution, maximum
parsimony, and maximum likelihood), a deep genetic division between
forest and savannah populations of African elephants was evident
(Roca GE). In fact, African forest and savannah elephants were found
to be over half as diverged from one another as they are diverged from
their distant relative the Asian elephant, Elaphas maximus, which has
long been known to be in a completely different genus (Vogel).
These newly defined species, according to Roca et al., were not
without their instance of hybridization. According to the Biological
Species Concept, reproductive isolation is of utmost importance for
defining a species (Mayr; O’Brien). But as has been shown by the
countless researchers who have attempted to define a species, this
species concept does not even come close to becoming a perfect rule
(Harrison). Therefore, based upon comparisons with the genetic integ67
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rity of the parent species, the hybridization present in only one of the
21 populations surveyed suggests little overall homogenizing power
(Roca GE).
When the news of two African elephant species hit the scientific
community, the idea for creating a “barcode of life” was already beginning to blossom. As previously mentioned, this revelation proposed
that mitochondrial DNA could serve as a core “global bioidentification system” for all animals (Hebert). Citing the inherent limitations
of morphological keys in the identification of species and taxa, Hebert et al. reported that the mitochondrial genome of animals, with its
limited recombination and haploid inheritance mode, not to mention
the greater ease with which mitochondrial DNA can be acquired, had
promise to be a microgenomic identification system with far reaching consequences (Hebert). This astounding statement would be put
to the test within many different species, and these newly introduced
elephant species were not without exception. A team of University of
California, San Diego researchers, led by Lori S. Eggert, decided to
reanalyze the organization of African elephant species using mitochondrial cytochrome b control regions, as well as nuclear microsatellites,
and their findings were yet even more astonishing.
Eggert et al. was quick to notice that Roca et al. had only sampled
21 African elephant populations, from only 11 of the 37 nations within
which the elephants reside. If different species of elephant could exist, supposedly diverging over 2.63 million years ago, why not utilize
mitochondrial DNA from elephant dung and greatly increase the range
of elephants sampled to be sure no hidden population structure had
been missed (Roca GE)? Surveying elephant dung, after all, was much
more feasible than using dart biopsies, as dart biopsies require actually
seeing, and then hitting, an elephant with a dart! Using feces allowed
Eggert et al. (Eggert) to survey an unprecedented amount of data, collecting anywhere from 20 to 50 samples at each of the 27 population
locations they analyzed. And what’s more, mitochondrial DNA could
be easily extracted from this dung, providing a wealth of data that other
researchers had only hoped to attain using only nuclear genetic information. Comparing the evolutionary patterns found within the sequenced
mitochondrial DNA with four microsatellite regions, this research team
was surprised to find a complicated web of genetic divergence among
African elephants. In fact, they did not find any deeply divergent lineages that corresponded to the clear-cut forest and savannah species
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Roca et al. had discovered using solely nuclear DNA. They had determined that there was evidence for not two species of African elephant,
but actually three broad groups: two groups of forest elephant, one in
Central and one in West Africa, and one group of savannah elephant.
Such a distinct difference in diversification demonstrated by mtDNA
and nuclear DNA presents a problem: which is painting a true picture
of speciation within the Loxodonta genus? Eggert et al. (Eggert), as
well as other researchers who used mtDNA to analyze African elephant
population differentiation, saw many possible explanations for why
nuclear DNA may have failed to detect other divergences that mtDNA
did. These included the fact that mtDNA is more variable and thus is
able to detect more subtle divergence, whereas nuclear DNA evolves
much more slowly (Eggert). Also, other researchers have speculated
that the maternal inheritance of mtDNA increases its “sensitivity,” thus
allowing it to better detect underlying population structure within taxa.
These researchers further suggest that the female philopatry of elephant
herds, which encourages gene flow through males, may result in lower
differentiation in nuclear genes (Nyakaana PS). Yet, couldn’t this same
explanation be used to support the use of nuclear loci? Which, then,
mtDNA control regions or nuclear DNA loci, paint an accurate picture
of the phylogeny of the largest mammal that walks this earth?
The answer to this question lies in the classification of this type
of evolutionary conundrum, termed cytonuclear dissociation. Cytonuclear dissociation occurs when nuclear and mitochondrial genomes
of a certain lineage represent two distinct evolutionary histories (Roca
CGD). When cytonuclear dissociation occurs, the natural history of a
lineage must be compared to the history of its genes in order to infer
the true identity of its diversification, both currently and in the past.
Roca et al. (CGD), realizing that he did not sufficiently address the use
of the mtDNA African elephant genome (at all), decided to put his previous research to the test by comparing it with his own mitochondrial
DNA analysis. He determined that, indeed, some savannah elephants
in locations distantly separated from forest elephants carried forest
elephant mitochondrial DNA, sometimes with a frequency of up to
90% among individuals (Roca CGD). Instead of determining outright
what might have caused this, Roca et al. (CGD) took the opposite approach to solving this mystery: by eliminating all other possible options. His team determined that such a high rate of forest mtDNA occurrence in some savannah populations could not have simply been
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the result of a few dispersing females, especially since elephant social
structure is built upon philopatry, in which females are non-dispersing
(Roca CGD, Nyakaana DNA). Random hybridization was further ruled
out again due to the two species’ very limited hybrid zone, and the fact
that a mix of forest and savanna nuclear alleles would have come with
hybridization, and that was not at all the case. With these two options
ruled out, a third, in which unidirectional hybridization was occurring,
seemed more promising. In this model, some forest females are lured
to mate with larger (and thus more dominant) savannah males. Over
many generations of such a backcrossing, forest nuclear alleles would
be diluted and eventually deleted, while forest mtDNA, passed on maternally, would be preserved. This explanation fits with elephant behavioral tendency for philopatry, where females remain with the natal
herd for life, and males disperse and thus allow for gene flow across
herds and theoretically populations (although most elephants no longer
have the freedom to travel out of their home ranges). The extent of
forest mtDNA in contrast to savannah suggests that at some time in
the distant past, savannah bull elephants were able to greatly out-compete forest elephant males in mating forest females, presumably when
the climate would have allowed the extension of forest elephants into
savannah elephant ranges simply because there was more forest at that
time (due to much higher levels of precipitation in the area in comparison to the relatively dry current conditions) (Roca CGD). Therefore, the story the mitochondrial DNA depicts in this lineage is overall
vastly misleading, and would consequently lead to the assumption of
more speciation than is actually present.
The African elephant speciation debate demonstrates that with
empirical observation, one theory can disprove another and provide
insight into future scientific advances. Even if mitochondrial DNA barcodes could correctly identify 100 bird species on the first try (Hebert),
they are simply not informative in species that experience male-biased
gene flow and almost no female gene flow. In other words, mtDNA can
lead a researcher astray when studying a species that has an overall matriarchal social structure, in which the passing on of mtDNA through
females from (sometimes ancient) unidirectional hybridization could
occur. The confusion that resulted from this case study of scientific
debate points to the utmost importance of such an exception, especially
if it was hoped that mitochondrial DNA barcoding could extend to the
identification of all eukaryotic life, most importantly—all species.
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The consequences, thus, of this population genetics study within
one of the most celebrated, and currently endangered taxa of the world,
are many-fold. Evolutionary biologists came away from this controversy with not only a more accurate taxonomy of African elephants,
but also having learned an important lesson in the ongoing saga of
species identification, increasing our appreciation for the complexity of the diversity of life. In fact, one researcher’s idea that mtDNA
could actually resolve elephant systematics proved to be quite incorrect (Blaxter). But what are the far-reaching impacts of being able to
define a species as a species? In the case of the African elephants, as is
the case in many other endangered lineages, two species are better than
one. As stated earlier, it is believed that only about one half million
African elephants now exist, reduced from their past population levels
of 1.3 million only twenty years earlier. But now, the situation will
seem, and is, much more dire: only a small percentage of that half
million are forest elephants, and savannah elephants make up the rest,
decreasing both of their population numbers dramatically. For conservation biologists attempting to sway the influence of the politician,
numbers don’t lie, and paradoxically, the poorer the current scene for
elephants, the harder governments will work to protect them against
poaching and encroachment. But knowing the evolutionary history of
lineage can be even more important: it can point biologists in the right
direction in understanding why a species might be in decline, and cite
these specific reasons for reform. The forest elephant, living only within thick forests, may be much more rare than the savannah elephant
simply because of the rapid deforestation occurring in many African
countries. As forests become more fragmented, the forest elephant’s
habitat is again opened for savannah bulls to encroach upon forest females, resulting in a reproductive competition that the male forest elephants are bound to lose. Thus, the study of population genetics and the
diversification among lineages can bring evolutionary biologists to not
only new levels of understanding in the realm of species identification,
but also in the process of classifying the life around us, we are better
able to understand its fragility and recognize its strengths.
The case of the African elephant demonstrates that it is an oversimplification of life’s diversity to confine species identification to a single
mitochondrial DNA sequence. Since the introduction of the DNA barcoding concept in 2003, it has not only been overwhelmingly praised,
but also staunchly criticized. While the large-scale standardization of
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reference gene sequencing in identifying taxa would indeed be beneficial, according to Craig Moritz et al. (Moritz), greatly increasing the
rate of discovering biological diversity, its use is limited and must be
noted as having clear caveats to application. Even as I write this, proponents of “The Barcode of Life” are compiling sequencing data for
the cytochrome c oxidase I gene (COI-5’) in animal, plant, and other
phyla. While such a “barcode” promises great ease in species identification and classification, the species debate of the African elephant
clearly demonstrates that one must exercise caution when attempting
to use a single gene sequence to resolve the taxonomy of the more than
10 million unique species on this earth.
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Works Cited
Barnes, R. F. W. et al. “African elephant database 1998.” International
Union for the Conservation of Nature (IUCN), Gland, Switzerland,
1999.
Blaxter, M. “Molecular systematics: Counting angels with DNA.” In
Nature 421 (2003): 122–24.
Eggert, L. S. et al. “The evolution and phylogeograhy of the African
elephant inferred from mitochondrial DNA sequence and nuclear
microsatellite markers.” Journal Proc. R. Soc. Lond. 269 (2002):
1993--–2006.
Harrison, Richard. “Linking Evolutionary Pattern and Process.”
Endless Forms: Species and Speciation. New York, Oxford:
Oxford UP, 1998.
Hebert, Paul et al. “Biological identifications through DNA barcodes.”
The Royal Society 270 (2003) 313–21.
Mayr, E. Principles of Systemic Zoology. New York: McGraw Hill,
1969.
Moritz, Craig and Carla Cicero. “DNA Barcoding: Promise and
Pitfalls.” PLoS Biology 10 (2004): 1529–31.
Nyakaana, Silvester et al. “DNA evidence for elephant social behavior
breakdown in Queen Elizabeth National Park, Uganda.” Animal
Conservation 4 (2001): 231–37.
Nyakaana, S., P. Arctander, and H. R. Siegismund. “Population structure
of the African savannah elephant inferred from mitochondrial
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control region sequences and nuclear microsatellite loci.” Heredity
89 (2002): 90–98.
O’Brien, S. J. and E. Mayr. “Bureaucratic Mischief: Recognizing
Endangered Species and Subspecies.” Science 251 (1991): 1187–
88.
Roca, Alfred L. et al. “Genetic Evidence for Two Species of African
Elephant in Africa.” Science 293 (2001): 1473–77.
Roca, Alfred L., Nicholas Geordiadis, and Stephen J. O’Brien.
“Cytonuclear genomic dissociation in African elephant species.”
Nature Genetics 37 (2005): 96–100.
Sikes, S. K. The Natural History of the African Elephant. London:
Weidenfeld and Nicholson, 1971.
Vogel, Gretchen. “ECOLOGY: African Elephant Species Splits in
Two.” Science 293 (2001) 1414a.
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