Using DNA to track the origin of the largest ivoryseizure since the 1989 trade ban

Samuel K. Wasser*†, Celia Mailand*, Rebecca Booth*, Benezeth Mutayoba‡, Emily Kisamo§, Bill Clark¶,
and Matthew Stephens ***Center for Conservation Biology, Department of Biology, and Department of Statistics, University of Washington, Seattle, WA 98195; ‡Faculty of Veterinary Medicine, Department of Veterinary Physiology, Biochemistry, Pharmacology, and Toxicology, Sokoine University of Agriculture, Morogoro,
Tanzania; §Lusaka Agreement Task Force, Nairobi, Kenya; and ¶Interpol Working Group on Wildlife Crime and Department of Law Enforcement, Israel Nature and Parks Authority, Jerusalem 95463, Israel

Author contributions: S.K.W. and M.S. contributed equally to this work; S.K.W., E.K., and
B.C. designed research; S.K.W. and B.M. performed research; C.M., R.B., and M.S. analyzed
data; and S.K.W., B.C., and M.S. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS direct submission.
Freely available online through the PNAS open access option.
Abbreviations: CITES, Convention on International Trade in Endangered Species of Wild
Fauna and Flora; MCMC, Markov Chain Monte Carlo.
†To whom correspondence should be addressed. E-mail: wassers@u.washington.edu.
**Present address: Departments of Human Genetics and Statistics, University of Chicago,
Chicago, IL 60637.
© 2007 by The National Academy of Sciences of the USA
4228–4233 PNAS March 6, 2007 vol. 104 no. 10 www.pnas.org cgi doi 10.1073 pnas.0609714104

Edited by John C. Avise, University of California, Irvine, CA, and approved December 26, 2006 (received for review November 2, 2006)
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The illegal ivory trade recently intensified to the highest levels ever
reported. Policing this trafficking has been hampered by the
inability to reliably determine geographic origin of contraband
ivory. Ivory can be smuggled across multiple international borders
and along numerous trade routes, making poaching hotspots and
potential trade routes difficult to identify. This fluidity also makes
it difficult to refute a country’s denial of poaching problems. We
extend an innovative DNA assignment method to determine the
geographic origin(s) of large elephant ivory seizures. A Voronoi
tessellation method is used that utilizes genetic similarities across
tusks to simultaneously infer the origin of multiple samples that
could have one or more common origin(s). We show that this joint
analysis performs better than sample-by-sample methods in assigning
sample clusters of known origin. The joint method is then
used to infer the geographic origin of the largest ivory seizure since
the 1989 ivory trade ban. Wildlife authorities initially suspected
that this ivory came from multiple locations across forest and
savanna Africa. However, we show that the ivory was entirely
from savanna elephants, most probably originating from a narrow
east-to-west band of southern Africa, centered on Zambia. These
findings enabled law enforcement to focus their investigation to a
smaller area and fewer trade routes and led to changes within the
Zambian government to improve antipoaching efforts. Such outcomes
demonstrate the potential of genetic analyses to help
combat the expanding wildlife trade by identifying origin(s) of
large seizures of contraband ivory. Broader applications to wildlife
trade are discussed.

The illegal trade in elephant ivory has once again escalated to
the devastating levels that occurred before the 1989 Convention
on International Trade in Endangered Species of Wild
Fauna and Flora (CITES) ivory trade ban (1–5). Between
August 2005 and August 2006, there have been 12 major seizures
of African elephant ivory being shipped to the Far East, totaling
23,461 kg, plus 91 unweighed tusks. Most of this ivory was
deemed to be from freshly killed elephants (B.C., unpublished
observation). It is commonly assumed that customs intercepts
10% of all contraband (e.g., drugs, weapons, pirated compact
discs). We conservatively assume that this percentage is also the
case for ivory; most enforcement agencies do not ‘‘target’’ ivory
as they do drugs or weapons, and technological advances (such
as drug scanners and detection dogs) do not help with interception
of contraband ivory. Thus, the above 23,461 kg should
correspond to 234,610 kg of smuggled ivory from 23,000
elephants killed this past year. Knowing the origin of ivory in
such large seizures enhances understanding of where elephants
are being slaughtered and routes by which the contraband ivory
is smuggled. Law-enforcement efforts could be fruitfully focused
with such information. It also creates accountability that compels
nations to be more responsive to poaching in their country.

We previously described a method to infer the geographic origin
of individual samples of African elephant DNA (6). Here, we
extend the approach to multiple samples and apply this method
to infer the origin of the largest seizure of contraband ivory since
the 1989 ivory trade ban (the second largest seizure in the entire
history of the trade).

In late June 2002, an investigative team consisting of officers
from the Zambia Wildlife Authority, the Lusaka Agreement
Task Force, and the Anti-Corruption Bureau of Malawi uncovered
vital information concerning the shipment of a 20-ft
container packed with 6.5 tons of contraband elephant ivory in
Malawi, destined for the Far East. (Based on the above assumptions,
this would have resulted from poaching of between 3,000
and 6,500 elephants.) The container had been shipped via South
Africa to Singapore, where it was seized later that month. The
seizure contained 532 tusks of widely diverse sizes and weights.
The average weight of tusks was 11 kg, substantially larger than
the average tusk in the current ivory trade. The seizure also
contained 42,120 ‘‘hankos,’’ believed to have been manufactured
in Malawi. Hankos are round ivory cylinders, 6.5 cm in length
and 1.5–2 cm in diameter, cut from the solid portion of the tusk.
Some Asian communities carve their personal seal on the end of
these cylinders to be used as a prestigious stamp (7). The hankos
alone in this shipment were worth an estimated $8.4 million
(U.S.), and represented 20% of Japan’s annual hanko trade
(B.C., unpublished observation). The enormous size of this
consignment indicates the existence of an elaborate network in
the Far East that is capable, with a single delivery, to receive and
launder tens of thousands of hankos and hundreds of tusks into
existing legal markets.

Investigative work revealed that the ivory had been carried
from Zambia into Malawi in small lots, before shipping, but it
was unknown whether the ivory came from Zambian elephants.
Our analysis tested two broad competing hypotheses for the
origin of the seized ivory:

Hypothesis 1. The ivory originated from within, or in close
proximity to, Zambia and/or Malawi, the original shipping
locale. This hypothesis would require minimal preshipment
transport (smuggling), but the size of the seizure would suggest
that poaching intensity in this region was substantially greater
than previously believed or acknowledged.

Hypothesis 2. The ivory originated from numerous locations
across forest and savanna Africa, with stockpiles smuggled into
Malawi before shipping. This hypothesis, which suggests the
existence of a relatively sophisticated and widespread organizational
network, was supported by several factors, including the
large volume of the shipment, the considerable mean and
variation in tusk size, and extensive poaching in the nearby
Democratic Republic of Congo†† and Selous Game Reserve in
Tanzania.

Results
We selected 67 of the 532 tusks for DNA analysis, using a
stratified sampling scheme aimed at maximizing the chances of
acquiring tusks from multiple locations (see Materials and Methods).
Amplification success varied greatly across samples; a total
of 13 tusks had all 16 loci amplify successfully, and 23 tusks had
at least 14 loci amplify successfully, whereas 18 tusks had no loci
amplify successfully. In total, 37 tusks (55%) amplified at seven
or more loci and were included in the subsequent assignment
analysis [the cutoff of seven loci being chosen for consistency
with the way the reference database was assembled (6)]. Among
these 37 samples, the average number of successful loci was 13.5.
Hankos were excluded from these analyses because initial attempts
to amplify DNA from hankos were unsuccessful. Hanko
samples are derived from the core of the tusk and were subsequently
found to require a decalcification step before their
extraction; analyses of the hankos are ongoing.

DNA obtained from the tusks was compared with a reference
database of DNA samples of known geographic origin. The
reference data were from Wasser et al. (6), augmented with 165
samples from Zambia, Malawi, and Southern Tanzania. The
combined samples provided an updated reference database of
525 samples (see Materials and Methods). Initial comparison of
alleles obtained from each tusk against reference allele frequency
distributions for forest vs. savanna elephants suggested
that all of the tusks were most likely derived from savanna
elephants [likelihood ratios in favor of savanna origin, computed
as in Wasser et al. (6), ranged from 2.5 104 to 9.1 1010].
We developed a statistical assignment method to infer the
most likely savanna locations of the sampled tusks. Existing
assignment methods estimate the likely source of each tusk
independently, assuming the tusks were independently and
uniformly sampled from some set of possible sources. This
assumption is problematic here because it implies that the tusks
likely originated from a wide range of locations, essentially
ignoring the possibility that they came from a restricted region
(hypothesis 1). Our approach (see Materials and Methods) extends
the smoothed continuous assignment method for individual
DNA samples from Wasser et al. (6) to analyze multiple tusks
simultaneously, allowing that they may have arisen either from
a wide range of locations or from one (or a few) narrow
geographic region(s).

Fig. 1 A–D illustrates the improved performance that can be
achieved by analyzing multiple samples simultaneously rather
than one sample at a time. Specifically, the figure compares
results from our approach, which jointly analyzes multiple
samples, with results from sample-by-sample analysis using the
method described in ref. 6. We applied both methods to groups
of samples known to originate from Malawi (n 18) (Fig. 1A),
Zambia (n 29) (Fig. 1B), and the Selous Game Reserve in
Southern Tanzania (n 12) (Fig. 1C), with each analysis
constituting a random sample of half of the samples available
from its respective origin, and to a group of samples from
__________________________________________________________________________________________
††Mubalama, L. (2005) Rapport sur L’Enquete du Marche D’Ivoire la ville de Kinshasa, March
9–19, 2005, Wildlife Conservation Society and Monitoring of Illegal Killing of Elephants,
Kinshasa, Democratic Republic of Congo.
A
B
C
D
Footnote: Fig. 1. Comparison of results from the new assignment method for jointly
analyzing multiple samples (Left) with those obtained by independently analyzing
each sample by using the assignment method from Wasser et al. (6) (Right).
Results obtained for a batch of samples ofknownorigin from Malawi (A), Zambia
(B), and Selous Game Reserve in Tanzania (C) and for dung and tissue samples
originating from across savanna Africa (D) are shown. Circles show the estimated
location of origin of each sample, whereas crosses indicate locations of reference
samplesfromsavanna habitats used tomakethe assignments. In A–C, s are used
to indicate the actual locations of the samples of known origin.
Wasser et al. PNAS March 6, 2007 vol. 104 no. 10 4229 POPULATION
BIOLOGY
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numerous locations scattered throughout savanna Africa
(n 37, chosen to match the number of tusks analyzed from the
Singapore seizure) (Fig. 1D). The remaining halves of samples
from Zambia, Malawi, and Selous contributed to the reference
samples for these analyses. When applied to a batch of samples
originating from a limited geographical region (Fig. 1 A–C), our
joint analysis method is able to recognize this fact and produces
estimated locations of origin that are both accurate and compact.
In contrast, estimates from independent analysis of each sample,
although centered on approximately the correct location, are
considerably more diffuse and tend to (wrongly) suggest that the
samples came from a relatively wide geographic region. Conversely,
when applied to samples that actually originated from a
wide geographic region (Fig. 1D), our joint analysis method is
also able to deduce this from the data and produces estimated
locations of origin that are very similar to those from the
sample-by-sample analysis.

We applied the new joint analysis method to 37 tusks acquired
from the Singapore seizure. The results (Fig. 2 Left) suggest that
the tusks originated from a relatively restricted part of southern
Africa, concentrated near Zambia, lending support to hypothesis
1. The tusks were genotyped on the same platform and at the
same time as the reference samples from Malawi, Selous, and
Zambia and at a different time and platform than the majority
of the other reference samples from East and Savanna Africa.
We therefore checked to determine whether our results were not
unduly affected by unidentified systematic differences between
the way different reference samples were treated, by reanalyzing
the tusk DNA without the additional reference samples from
Zambia, Malawi, and Selous. The results (Fig. 2 Right) were
similar to those obtained with the additional reference samples,
with estimated tusk origins being slightly more diffuse and
centered slightly farther south.

Discussion
Using DNA, it is possible to determine, with near 100% accuracy,
whether an individual sample originated from a savanna or
forest elephant (6). The DNA from all of the tusks that we
examined from the Singapore seizure pointed to a savanna origin
for these samples. This simple inference alone immediately rules
out many countries that are habitat for forest elephants (Loxodonta
cyclotis); it also lends some support to the hypothesis that
the tusks may have originated from a restricted geographic
region rather than from a pool of many stockpiles from across the
continent. More sophisticated analytic methods, able to accurately
determine the likely geographic origin of DNA samples on
a finer scale, point to a relatively narrow band of Southern
Africa, centered on Zambia, as the likely source of tusks in this
seizure. The estimated locations of origin for the tusks spread
east and west from Zambia and may include regions of Mozambique
and savanna Angola from which no reference samples are
yet available. Reference samples from these locations could
increase the precision of these estimates and help to confirm or
rule out these countries as possible contributors to the seizure.
The 37 tusks analyzed here represent a subset of tusks that
produced the most complete genotype data. Although visual
inspection revealed no obvious systematic differences between
these tusks and others that failed to yield such complete data (see
Materials and Methods), it is difficult to entirely rule out the
possibility that the seizure could contain some tusks of different
origin that failed to produce good genotype data.
These caveats notwithstanding, the analysis of available DNA
data from these samples has greatly facilitated law-enforcement
efforts. As described in hypotheses 1 and 2, authorities strongly
suspected that this ivory had multiple origins, including forest
habitat. Our results caused law enforcement to substantially
narrow the area of origin and the trade routes being investigated.
These results also had a number of consequences for Zambia.
The seizure immediately followed Zambia’s application to
CITES for a one-off sale of their ivory stockpiles at COP12
(Conference of the Parties). That application maintained that
only 135 elephants were known to have been illegally killed in
Zambia during the previous 10 years, woefully shy of the
3,000–6,500 elephants we estimate to have been killed in Zambia
surrounding the seizure, let alone during that entire 10-year
period. Subsequent to being informed of our findings, the
Zambian government replaced its director of wildlife and began
imposing significantly harsher sentences for convicted ivory
traffickers in its courts. However, one still has to wonder whether
this will be enough.

Virtually no one has been prosecuted for this case. Moreover,
just 3 years after the Singapore seizure, when we were in the thick
of our DNA analyses, another 6 tons of ivory was seized in the
Philippines en route from Zambia. (That ivory was subsequently
stolen from the warehouse that Philippine customs had contracted
to hold the contraband.) This begs the question: How can
a poor country like Zambia, with only token international
assistance, have the physical capacity to act effectively against
________________________________________________________________________________________
Tusks from Seizure
with additional reference samples

Fig. 2. Assignment results for 37 tusks from the Singapore seizure. The estimated locations of origin (circles) of the 37 tusks analyzed are shown. (Left) Results
using the additional reference samples from Zambia, Malawi, and Selous. (Right) Results without these additional reference samples. Crosses are the same as in Fig. 1.
4230 www.pnas.org cgi doi 10.1073 pnas.0609714104 Wasser et al.
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criminals exploiting the dynamic market demands of the financially
robust Far East?

Wildlife trade represents a serious and growing area of
organized crime that can irreparably damage a country’s ecosystems
and economy and has demonstrable links to other
serious crime. The illegal ivory market exemplifies this. Elephants
are a keystone species, whose loss significantly alters
natural habitat. The ivory trade has corresponded with massive
declines in elephant numbers ( 50% continent-wide and up to
90% in some areas), including areas where habitat loss (the other
most likely cause of decline) has remained unchanged (8–11).
Moreover, the illegal trade this year has seen its largest increases
ever, based on marked increases in seizures without any commensurate
increases in the capacities of the seizing agencies
(B.C., unpublished observation). Much of this increase in trade
is being driven by wholesale prices of high-quality ivory in China
and Japan (12), which have risen from $100 per kilogram in the
late 1990s to $200 per kilogram by 2004 to a now staggering $750
per kilogram (B.C., unpublished observation). This disproportionately
large 3.5-fold rise in the past 2 years has raised concern
that commodity speculators may be buying up much of the ivory.
Certainly, these trends suggest that the market is being heavily
stimulated, adding to current fears that China’s growing demand
for illegal ivory could jeopardize elephants throughout Africa
and Asia (5, ††).

Given that syndicated ivory crime has reached such international
scale, we suggest that the most effective way to combat this
trade is to prevent the ivory from ever entering the international
market. Genetically tracking the origin of large ivory seizures can
help by identifying poaching hotspots, focusing urgently needed
policing of elephant poaching and associated trafficking in
contraband ivory. This approach places emphasis on saving
elephants before they are killed. By identifying common patterns
among large seizures, such as homogeneity of origin and proximity
to original shipping locale, our methods could also highlight
likely smuggling routes (e.g., major roads, train routes, or
nearby ports) and suggest how illegal ivory is being moved to
global markets outside Africa. These effects should also increase
tusk seizure rates, further helping to stop the trade before it
leaves Africa. Strategic changes in these smuggling patterns over
time could also be detected, as could changes in the quantity and
distribution of ivory from specific locales in the world’s major
ivory markets. Monitoring such changes, coincident with CITES
trade decisions, could provide critically needed tools to determine
whether sanctioned sales influence poaching rates across
the continent.

Although our methods can enhance the effectiveness of law
enforcement in wildlife trade, what is really needed is to combine
this with a major reinfusion of law-enforcement aid at the scale that
coincided with the 1989 ivory ban. For this reinfusion to occur,
industrialized nations need to be reeducated about the seriousness
of the poaching problem to encourage their governments to once
again provide this needed law-enforcement support. The United
Nations has declared many of Africa’s natural resources to be
‘‘World Heritage,’’ and the rest of the world needs to help protect
this shared heritage. To ensure that such aid is not endless,
law-enforcement aid needs to be coupled with education aimed at
reducing demand in the Far East and at engendering respect for
natural resources in Africa. Improved management is also needed
in Africa to restore the historical abilities of elephants to selfregulate
their population sizes and reduce elephant/human conflict.
Ironically, stopping poaching may help reduce such conflict, if
elephants can once again be made to feel safe enough to remain in
protected areas (13). Stopping poaching will also prevent loss of
tourism in wildlife-rich countries, along with the disproportionately
large amounts of foreign currency it generates.

The international community virtually stopped ivory poaching
once (14), and it can stop it again. The enhanced lawenforcement
effort that coincided with the 1989 ban dramatically
suppressed the illegal ivory trade. However, believing that the
problem was solved, western aid was largely withdrawn by 1993.
Law enforcement rapidly declined in poor African countries, and
poaching began to steadily increase all over again (14). A more
comprehensive approach is needed this time, one that combines
law enforcement with DNA analyses, education, and improved
management. We have to act now, before it is too late. We hope
that the results of this study will encourage such timely conservation
efforts, thereby helping to curb a criminal trade that is
once again imperiling elephants.

We also believe that these techniques can prove useful for
other species that are substantially represented in the wildlife
trade. The ability to acquire DNA from feces, coupled with new
methods that markedly enhance fecal sampling rates over large
remote areas (15), makes this approach highly feasible for a
diverse array of at-risk species.

Materials and Methods
(not included here)