Molecular Identification of Primate Bushmeat Sold in Guinea-Bissau

Full text

DNA identification of primate bushmeat from urban markets in
Guinea-Bissau and its implications for conservation
Tânia Minhós
a,b,
⇑
,1
, Emily Wallace
a
, Maria J. Ferreira da Silva
a,c,1
, Rui M. Sá
a,d,e,1,2
, Marta Carmo
e
,
André Barata
f
, Michael W. Bruford
a
a
Organisms and Environment Division, School of Biosciences, Cardiff University, Biomedical Sciences Building, Room C/5.15, Museum Avenue, Cardiff CF10 3AX, Wales, UK
b
CESAM (Centre for Environmental and Marine Studies), DBA, Lisbon University, Campo Grande, Edifício C2, Piso 5, 1749-016 Lisboa, Portugal
c
CIBIO/InBio, Research Center in Biodiversity and Genetic Resources, Porto University, Campus Agrário de Vairão, 4485-661 Vairão, Portugal
d
Centre for Research in Anthropology, ISCTE, Avenida das Forças Armadas, 1649-026 Lisboa, Portugal
e
Department of Anthropology, Faculdade de Ciências Sociais e Humanas, Universidade Nova de Lisboa. Av. de Berna, 26-C, 1069-061 Lisbon, Portugal
f
Behaviour and Evolution Research Group, Psychology University of Stirling, Stirling FK9 4LA, Scotland, UK
article info
Article history:
Received 4 March 2013
Received in revised form 16 May 2013
Accepted 13 July 2013
Available online xxxx
Keywords:
Cercopithecus campbelli
Chlorocebus sabaeus
Colobus monkeys
Papio papio
Cytochrome c Oxidase subunit I (COI)
Hunting
Mitochondrial DNA
abstract
Hunting for bushmeat consumption is a major threat to wild populations. Assessing trade at markets pro-
vides a commonly used measure of its intensity and impact. However, most carcasses arrive at markets
already processed, which can pose serious challenges to its identification. We aimed to estimate the bias
induced by incorrect species identification on species-specific trade estimates. During a survey of primate
species traded in two urban markets in Guinea-Bissau, West Africa, we collected samples from 50 car-
casses, for which traders provided a priori species identifications. DNA barcoding was used to estimate
the bias posed by traders’ testimonies in this identification and to correct frequency estimates for each
traded species. Six of the ten extant primate species in Guinea-Bissau were found to be traded, with a
minimum estimate of 1550 individuals/dry season, based on the DNA barcoding. Molecular identification
showed that species with similar body size were frequently misidentified when relying on the informa-
tion provided by market traders only. Errors were particularly large in the case of the green monkey
(Chlorocebus sabaeus), identified only four times by market-holders but revealed to be the most traded
primate in Bissau after DNA barcoding. We highlight the importance using molecular tools to correctly
identify bushmeat species. Our study demonstrate that ignoring the possibility of a misidentification bias
can result in inadequate conservation policies by neglecting some of the most affected species.
!2013 Elsevier Ltd. All rights reserved.
1. Introduction
Unsustainable bushmeat consumption is considered a pan-
tropical conservation crisis (Milner-Gullanda et al., 2003; Macdon-
ald et al., 2012) and has been responsible for population declines
and local extinctions of large bodied mammals, including primates
(Alliance, 2006; Allebone-Webb et al., 2011).
A central component of research on the bushmeat trade is a
description of the trade chain and the traded species (e.g. Cowli-
shaw et al., 2005). This is usually attempted by analysing trade at
commercial markets, information that can be used to infer the
impact of hunting (Fa, 2007). Such surveys are considered quick-
er, cost-effective and more viable than the estimation of off-take
rates or of species abundances in hunted areas (Allebone-Webb
et al., 2011). Nevertheless, collecting reliable information in bush-
meat markets can be a complex task. As local laws protect some
traded species, vendors may be reluctant to discuss their involve-
ment in the trade and/or may deliberately misinform researchers
(Jenkins et al., 2011). Moreover, to aid meat preservation before
consumption, most carcasses arrive to West African markets in
a processed state: either smoked or cut into saleable portions
(Bowen-Jones and Pendry, 1999). In such cases, even experienced
observers may be unable to distinguish between species with
overlapping body size and shape, a factor that can be exacerbated
if juvenile individuals are also hunted. Incorrect identification of
carcasses can lead to inaccurate estimations of the frequency that
0006-3207/$ - see front matter !2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.biocon.2013.07.018
⇑
Corresponding author. Present address: IGC-Instituto Gulbenkian da Ciência,
Rua da Quinta Grande, 6, 2780-156 Oeiras, Portugal. Tel.: +351 916754288.
E-mail addresses: trodrigues@igc.gulbenkian.pt (T. Minhós), emilywallace44@
gmail.com (E. Wallace), FerreiraDaSilvaMJ@cardiff.ac.uk (M.J. Ferreira da Silva),
ruimoutinhosa@gmail.com (R.M. Sá), martacarmo@facebook.com (M. Carmo),
andrevlbarata@gmail.com (A. Barata), BrufordMW@cardiff.ac.uk (M.W. Bruford).
1
These authors contributed equally to this manuscript and are first co-authors of
the work.
2
Present address: Department of Pathological Morphology and Parasitology,
Faculty of Veterinary Medicine, University of Veterinary and Pharmaceutical Sciences,
Palackého 1-3, 612 42 Brno, Czech Republic.
Biological Conservation 167 (2013) 43–49
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a species is traded, which may have serious consequences to the
final interpretation of results and subsequent allocation of conser-
vation efforts (Fong et al., 2007; Rönn et al., 2009; Ntie et al.,
2010). Therefore, it is important to evaluate the extent of incor-
rect identifications.
Molecular identification has been proposed as a suitable meth-
od for the confirmation of carcasses at species level (e.g. Baker and
Palumbi, 1994). DNA barcoding derived from mitochondrial and
other DNA sequences (Hebert et al., 2004; Rönn et al., 2009; Kimw-
ele et al., 2012) has been shown to reliably identify some species
used in the bushmeat trade. However, molecular identification ap-
plied to market surveys still focuses on a limited number of pro-
tected species (e.g. Baker and Palumbi, 1994; Walsh et al., 2003;
Baker, 2008; Olayemi et al., 2011; Kimwele et al., 2012) and the
bias induced by incorrect species identification on trade estimates
has been only considered in few cases (e.g. Baker et al., 2006;
Clarke et al., 2006a, 2006b).
We analyzed the primate bushmeat trade at two markets in Bis-
sau, the capital city of Guinea-Bissau. Although previous reports
describe the hunting of large numbers of primates for sale in
important urban markets (Casanova and Sousa, 2007; Cá, 2008),
such trade has not been quantified to date. We used DNA barcod-
ing to determine the diversity and quantities of nonhuman prima-
tes sold at these markets and assess both the merits of applying
this method and the conservation implications of the results. This
study is the first, to our knowledge, where molecular species iden-
tification is integrated into an African urban market survey and
species commercial volume is assessed by vendor identification
and corrected using a molecular approach.
2. Methods
2.1. Bushmeat trade
We focused on two urban bushmeat markets (Fig. 1): (i) ‘‘Chap-
a’’, a permanent market in the center of Bissau where domestic
meat is also sold and (ii) ‘‘Rampa’’, closely located to a harbor,
where fish is sold along with domestic and wild meat. The markets
were visited in 2010 for 19 days during the dry season (March to
June). Visits took no more than 30 min to avoid hindering the nor-
mal function of the trade. Visits were conducted over three periods
(1st to 5th March, 30th March to 10th April and 19th May to 6th
June). We counted all primate carcasses existent at the markets
at the time of the visit, recorded the identification provided by
the traders (A.1, Appendix) and the price. The number of bushmeat
traders varied across the days (2–6 traders/day) and all were ap-
proached. Traders were always able to respond promptly to our
enquiries. Restaurants specializing in bushmeat dishes were also
visited to gather additional information on primate meat con-
sumption. Fifty tissue samples were collected from primate car-
casses for molecular identification (A.2, Appendix) and stored in
99% ethanol at room temperature. We targeted the freshest tissue
from inside the carcasses to assure better quality DNA and avoid
cross-contamination problems (including with human DNA).
2.2. Molecular identification
2.2.1. Laboratory procedures
Whole genomic DNA was extracted, at Cardiff School of Biosci-
ences (UK), using DNeasy Blood and Tissue kits (Qiagen, Hilden,
Germany), following the manufacturer’s protocol, with the excep-
tion of an overnight lysis step. A 623 base pair (bp) fragment from
the Cytochrome c Oxidase subunit I (COI) standard barcode region of
the mitochondrial DNA (mtDNA) was analyzed using three primer
sets (Lorenz et al., 2005,A.3, Appendix). Primer sets were used
sequentially when samples did not amplify a PCR product with
the previous set. ‘‘OWMCOI’’ was used first, followed if necessary
by ‘‘VERTCOI’’ and finally ‘‘FOLMER’’ (Folmer et al., 1994). There
was no COI voucher sequence for Campbell’s mona monkey (Cerco-
pithecus campbelli). Although we initially attempted COI marker,
this species was only confirmed using a 359 bp fragment of the
12S rRNA mtDNA gene (primers by Kessing et al. (1989) and mod-
ified by Kocher et al. (1989),A.3, Appendix), for which a voucher
sequence was available on Genbank. Along with all putative sam-
ples from C.campbelli, we sequenced one DNA sample from each
of the other species as controls.
PCRs comprised 10
l
l Qiagen Master Mix, 6
l
l RNase free water
and 2
l
l of 10!primer mix (containing each primer at 2
l
M). 2
l
l
of template DNA was added, resulting in 20
l
l final volume. The
thermal cycling program, performed on an Applied Biosystems
GeneAmp PCR System 9700, comprised 15 min at 95 "C, 40 cycles
of 30 s at 94 "C, 90 s at 50 "C and 90 s at 72 "C, followed by a final
extension step of 10 min at 72 "C. PCR products were visualized on
a 1% agarose gel and purified with 10 units of Exonuclease I and 5
units of Antarctic Phosphatase (New England Biolabs), using a cy-
cling program of 37 "C for 30 min, 80 "C for 20 min and 12 "C for
5 min. Samples were sequenced bidirectionally using Macrogen
Europe’s EZ-seq direct service (Macrogen Europe, Meibergdreef
31, 1105 AZ, Amsterdam, The Netherlands). Consensus sequences
were obtained in forward and reverse orientation by visual com-
parison using Bioedit Version 7.0.5.3 (Hall, 1999) and sequences
were manually aligned.
We checked for the presence of NUMTS (following Bensasson
et al., 2001; Anthony et al., 2007; Song et al., 2008) and found no
evidence for this type of contamination.
2.2.2. Species assignment
To assign query sequences to species level, we followed the
method described in Frézal and Leblois (2008). We searched the
NCBI (http://www.ncbi.nlm.nih.gov/), inPRIMAT (www.inpri-
mat.org) and BoLD databases (December 2010–March 2011) for
Fig. 1. Bushmeat trade in Guinea-Bissau. Top: Primate carcasses generally arrive at
the markets whole, charred and disemboweled. Bottom: Preparation of primate
meat stew at a specialized restaurant (locally called Abafatórios or Bafatórios). The
white circle indicates a baboon’s head.
44 T. Minhós et al. /Biological Conservation 167 (2013) 43–49

the most similar sequence – tentatively assigned this a ‘‘voucher’’
status – (see A.4, Appendix) and used genetic distances to cluster
the query sequences to the voucher. Mega 5.01 (Tamura et al.,
2011) was used to construct Neighbor Joining (NJ) trees with boot-
strap support (1000 replicates) of each node. All sequences pro-
duced (COI and 12SrRNA) were included along with the voucher
sequences.
2.3. Vendor identification error rate
Vendor identification error rate for each species was defined as
the percentage of mismatches between molecular and vendor
identification in the tissue samples collected for each species (i.e.
(TS–TL)/TS !100, where TS is the total number of tissue samples
collected per species using vendor identification, and TL is the
number of tissue samples per species where vendor identification
matched molecular identification) (Tables 1 and A.1). The 95% con-
fidence interval for a binomial probability was calculated using the
Clopper–Pearson exact method (Soper, 2013).
The number of individuals traded per species was defined as the
range between the minimum number of individuals and the poten-
tial maximum number of individuals present at the markets. We
defined the minimum number of individuals as the number of tis-
sue samples molecularly assigned to a given species and the poten-
tial maximum number of individuals per species as the sum of the
proportion of vendor’s correct identifications (A) and the propor-
tion of vendor’s incorrect identifications (B) for that species. We
calculated the proportion of correct identifications (A), using the
following formula: A = (NM !TL)/TS, in which NM represents the
number of vendor records per species. To estimate B, we first cal-
culated (1) the number of vendors’ records of all other species be-
sides the one being considered, IM (i.e. vendor records for all
species minus vendor records for each species); (2) the number
of vendor’s misidentifications, TNL (i.e. number of tissue samples
molecularly assigned to the species minus the number of correct
vendor identification, TL) and (3) the number of tissue samples
of all other species besides the one being considered, NSOS (i.e.
number of tissue samples collected for all species minus number
of tissue samples collected for each species). We used the formula:
B = IM !(TNL/NSOS).
We estimated the species-specific proportion in the trade be-
fore DNA barcoding (using vendors identifications only) and after
molecular specific assignment (using the potential maximum
number of individuals of each species present at the markets –
A + B). We extrapolated the trade for each species for the dry sea-
son period (November to May, 212 days). This was done before
molecular identification (using the number of carcasses observed
at the markets, as identified by the vendors) and after molecular
identification (using the potential maximum number of individuals
of each species).
3. Results
3.1. Trade at Bissau markets
We registered 150 primate carcasses from six different species:
western red colobus (Procolobus badius), western-black and white
colobus (Colobus polykomos), C.campbelli, guinea baboon (Papio pa-
pio), patas monkey (Erythrocebus patas) and green monkey (Chlor-
ocebus sabaeus). All carcasses were found whole, without tail,
smoked and disemboweled. We observed carcasses of both adult
and juvenile individuals. P.papio and C.polykomos carcasses were
the easiest to visually identify, whereas it was very difficult to dis-
tinguish between C.sabaeus and C.campbelli. The smallest car-
casses of P.badius and P.papio were also difficult to distinguish
from C.sabaeus and C.campbelli, due to similarities in terms of
body size and shape.
The traders based their prices in specimen body size rather than
weight. P.papio males were the largest and therefore most expen-
sive (10,000–15,000 CFA, approximately 22–33 US$). The price of
baboon females and carcasses of other species (regardless of the
sex) varied between 4000 and 8000 CFA (approximately 8–16
US$). Carcasses were mostly sold whole and to restaurants,
although we also observed purchases by private customers. At res-
taurants specializing in bushmeat (locally called Abafatórios or
Bafatórios), primate meat was regularly consumed as a snack
(stewed with bread, Fig. 1) whilst drinking alcohol. The meal (four
pieces of primate meat, not including bread) cost approximately 1
250 CFA (approximately 2.60 US$).
More primates were sold at Chapa (113) than Rampa (37) and
the average daily frequency across periods, measured as prima-
tes/day, was of 9.5 (SD = 7.4). However, this varied across the dry
season in both markets: increasing in the second period of the
study from 9.6 to 16.8 [(Chapa: 7.4 (SD = 6.8) to 11.6 (SD = 7.0)
and Rampa: 2.2 (SD = 2.2) to 5.2 (SD = 10.0)] and decreasing in
the third period, to 2 (SD = 6) [(Chapa: 2 (SD = 6) and Rampa: 0
(SD = 0)] prior to the rainy season.
3.2. Species assignment
We barcoded 46 samples using the OWMCOI primers, two sam-
ples (P.badius and C.campbelli) with VERTCOI and two samples (C.
polykomos) with FOLMER. In the neighbor-joining tree, all COI se-
quences grouped together in six highly supported clusters (100%
bootstrap support) (Fig. 2). The six clusters correspond to the six
species referred to by the traders (C.campbelli,P.badius C.polyko-
mos,P.papio,E.patas and C.sabaeus). The 12S rRNA tree (Fig. A.5,
Appendix) did not show such highly supported clades but all puta-
tive C.campbelli sequences grouped together along with the vou-
cher (Fig. 1S, Appendix). In the phenograms, samples are labeled
according to vendor identification but are grouped according to ge-
netic similarity with voucher sequences. We inferred that se-
quences grouping together with the respective voucher sequence
belonged to the same species.
3.3. Vendor identification error rate
The vendor identification error rate across species was 19.1%
(Tables 1 and A.1 for details on calculations). C.campbelli showed
the largest error (59.1%, 0.207 6p60.636), followed by P.badius
(40.0%, 0.262 6p60.878) and P.papio (15.4%, 0.545 6p60.981).
All tissue samples collected from carcasses identified by vendors
as E.patas,C.polykomos and C.sabaeus were molecularly assigned
to those species. The majority of samples morphologically misiden-
tified were molecularly assigned to C.sabaeus (11 samples labeled
as C.campbelli and three samples labeled as P.badius)(Table 1).
According to vendor identification, C.campbelli was the most
traded species (61%). After the correction using DNA-barcoding,
C.sabaeus and C.campbelli became traded at similar frequencies
(32.9% and 29.7%, respectively) (Table 1). C.campbelli and P.badius
decreased 31% and 2.9%, respectively, in their contribution to the
trade while all other species increased, on average by 8.4%
(SD = 14.6); the largest increase being C.sabaeus (30.3%) (Table 1).
We extrapolated a total of 1550 primates being traded in Bissau
over the dry season, predicting that over 450 specimens of each C.
sabaeus and C.campbelli are sold (Table 1). After applying the DNA-
barcoding, the extrapolated estimate of C.sabaeus traded increased
dramatically, from 47 to 510 individuals/dry season. On the other
hand, the number of traded C.campbelli decreased from 1015 to
462 individuals/dry season.
T. Minhós et al. /Biological Conservation 167 (2013) 43–49 45

4. Discussion
4.1. Molecular identification
This work constitutes the first evaluation of the bushmeat trade
in Guinea-Bissau, adding to the current knowledge of this major
conservation concern faced in Western and Central Africa. Our re-
sults highlight the bias that can occur when relying exclusively on
vendor identification. The most striking errors were for C.sabaeus
and C.campbelli. According to vendor identification, C.sabaeus
was identified only four times in 150 records whereas C.campbelli
was the most hunted species (91 records). After amending records
using DNA-barcoding, C.sabaeus became the most hunted species
and although C.campbelli remains one of the most hunted species,
its frequency was considerably reduced. Our findings emphasize
that the difficulties in species identification faced by observers
when conducting market surveys must not be neglected. If bush-
meat surveys aim to evaluate the diversity and quantity of species
traded, impact of hunting in wild populations or even consumer
preference should take the possibility of substantial misidentifi-
cation levels into account. In our case, carcasses of C.campbelli,C.
sabaeus and P.badius were frequently confounded probably be-
cause they are very similar in terms of body size and shape when
smoked. Many other studies based on market surveys, which
traded species (especially large-bodied mammals) reach the mar-
kets in an altered state (either smoked or butchered), may be fac-
ing similar species misidentification problems as was also
highlighted by Baker (2008). DNA-barcoding proved to be an effec-
tive tool to correctly estimate species-specific trade frequency,
which is crucial when planning adequate conservation policies.
4.2. Dimension of the primate bushmeat trade in Guinea-Bissau
Market surveys confirmed that six of the ten primate species
listed for Guinea-Bissau are hunted for bushmeat consumption
and sold in the country’s capital. Nevertheless, evidence exists
for trade of Western lesser spot-nosed guenon (Cercopithecus pet-
aurista buettikoferi) in other markets (Bijago Archipelago, R. Sá, pers
obser) and of primate body parts for medicinal and ritual practices
(Sá et al., 2012).
Our projection of approximately 1550 primate carcasses is rep-
resentative of the volume of bushmeat traded in Bissau, as Chapa
and Rampa are two of the few stable trading points. However,
these figures do not reflect the total mortality due to hunting, since
a substantial proportion of the hunted animals are not expected to
reach urban markets (e.g. 59% in southwestern Ghana, Cowlishaw
et al., 2005). We do not know this proportion for Guinea-Bissau but
like in other Western and Central African countries, bushmeat con-
sumption is a widespread practice in rural areas for subsistence
and commercial hunting as well as for bushmeat restaurants (Casa-
nova and Sousa, 2007; Cá, 2008).
Trade in Guinea-Bissau does not seem to be very different from
other West-African countries. Results presented by Fa et al. (2006)
suggest that from the 6892 carcasses recorded in Nigeria and
10,185 in Cameroon, only 10% and 17% were from primates. Con-
sidering these percentages and using their estimates of annual ur-
ban per capita consumption of all bushmeat (0.06 carcass/
inhabitant in Nigeria and 0.04 carcass/inhabitant in Cameroon), it
can be inferred that 0.006 primate carcasses are being consumed
per capita in urban areas in Nigeria and 0.007 in Cameroon each
year. Our findings point to a minimum of 0.0032 primate carcasses
consumed per capita in Bissau (1550 carcasses/488,581 urban
inhabitants, data on number of urban inhabitants in Bissau from
CIA, 2012). However, our projection covers only the dry season.
Market traders, restaurant owners and clients confirmed that
bushmeat is scarcer during the rainy season probably because for-
ested areas are more difficult to access and some become isolated.
The trade during this period needs to be investigated more deeply
as it could potentially increase the already large figures of bush-
meat trade in Bissau.
4.3. Conservation implications
Our study demonstrates that the bias in species misidentifi-
cation can be high when based exclusively on vendor identification
and, consequently, may lead to erroneous conclusions. However,
in many bushmeat market surveys, species identification is con-
ducted by trained observers, and for that reason, we would expect
a smaller misidentification bias. Although it was not under the
scope of this study, we believe that this bias should be evaluated
in the future. Should this bias be found to be significant, we would
recommend the use of DNA barcoding. Importantly, these errors
are likely to be study-specific and dependent on local conditions,
on the species being traded and how the carcasses/meat are pro-
cessed. Bias should therefore be evaluated in each case and taken
into consideration in species-specific trade frequencies and in the
Table 1
Molecular assignment of primate specimens found at Bissau bushmeat markets.
Species
a
Specimens at the
markets
b
Vendor error
rate
c
Specimens traded after
molecular assignment
d
Species-specific trade
e
Deviation
f
Extrapolation
g
Minimum Maximum Before
molecular ID
After
molecular ID
Before
molecular ID
After
molecular ID
Cercopithecus campbelli 91 59.1 11 41.4 60.7 29.7 "30.8 1015 462
Erythrocebus patas 4 0 2 7 2.7 5.0 +2.4 45 78
Procolobus badius 22 40 7 16.4 14.7 11.8 "2.9 246 183
Colobus polykomos 3 0 3 3 2.0 2.2 +0.2 34 34
Papio papio 26 15.4 12 25.4 17.3 17.9 +0.7 290 283
Chlorocebus sabaeus 4 0 15 45.7 2.7 32.9 +30.3 45 510
Average 19.1
Total 150 1674 1550
a
Species found at the bushmeat markets in Bissau.
b
Number of specimens per species as identified by the vendor.
c
Vendor identification error rate (%).
d
Range of number of individuals traded per species: minimum (i.e. number of tissue samples molecularly assigned to the species) and maximum (i.e. sum between
proportion of vendor’s correct identification (A) and the proportion of vendor’s incorrect identification (B)).
e
Species percentage of trade (%) before and after correction by molecular tools.
f
Deviation of species-specific trade (%) after correction by molecular tools ("denotes reduction and + denotes increase).
g
Extrapolation of trade for the entire dry season (November to May, 212 days) based on the maximum number of specimens traded after molecular assignment. Note that
the maximum number of specimens traded after molecular assignment is for 19 days (e.g. calculations for C.campbelli: 41.4 !212/19).
46 T. Minhós et al. /Biological Conservation 167 (2013) 43–49

conclusions drawn by the researchers. Identification of species
using DNA barcoding can also be an important tool for law enforce-
ment to detect illegal trade in protected species and/or to verify
information given by butchers and/or meat transporters regarding
the type of meat they handle. All of these actions will benefit the
conservation of regional biodiversity.
Fig. 2. COI neighbor-joining tree (1000 replicates) calculated using Kimura’s 2-parameter genetic distance. The six highly supported clusters (100% support), labeled in the
figure correspond to six species: (1) Chlorocebus sabaeus; (2) Erythrocebus patas; (3) Cercopithecus campbelli; (4) Papio papio;(5) Colobus polykomos and (6) Procolobus badius.
Each sample’s label reflects the trader’s identification. Capitalized labels refer to vouchers retrieved from databases. All voucher sequences are labeled with the original code
from the database except the ones that came from visually confirmed specimens in Guinea-Bissau (indicated with GB).
T. Minhós et al. /Biological Conservation 167 (2013) 43–49 47

Despite its apparent advantages, DNA-barcoding is still not fre-
quently applied to bushmeat market surveys. There are a range of
reasons for this, including (1) budgetary, time and expertise con-
straints; (2) degradation of DNA which hinders the amplification
of fragments longer than 500 bp required by COI universal primers;
(3) lack of voucher specimens, incorrectly labelled sequences or
those of low quality in repository databases and (4) limitations
of mtDNA including amplification of the same sequence in differ-
ent species due to introgression, co-amplification of nuclear DNA
(numts), heteroplasmy and low sequence divergence among taxa.
Additionally, cryptic species or those that display wide phenotypic
variation might represent a problem for the assignment of voucher
sequences (Valentini et al., 2009). A wider use of DNA barcoding
would benefit from the validation, variety, quality and availability
of voucher sequences in dedicated repository databases such as
BoLD, GenBank and inPrimat. This would increase the accurate
detection of the illegal trade of threatened species and would be
of great interest of national and intergovernmental agencies such
as bushmeat task force or the IUCN.
Our study constitutes the first survey to the Guinea-Bissau ur-
ban bushmeat markets. Through previous interviews with local
people and hunters, P.papio,C.sabaeus and colobus monkeys were
thought to be the most hunted species in Guinea-Bissau (Cá, 2008;
Casanova and Sousa, 2007). However, our molecular data showed
that C.campbelli is the second most affected species by the bush-
meat trade. This was never previously reported and may indicate
a decrease in the availability of species that were heavily targeted
in the past. For example, hunters described P.badius as a very eas-
ily hunted species and that large number of individuals from the
same social group could be killed during the same hunting session
(Ferreira da Silva, 2012). This may have cause a dramatic decrease
in their population and therefore, influence current bushmeat
trade dynamics. Future conservation policies should consider such
changes in the hunting dynamics.
Our extrapolation of 1550 primates traded per dry season also
suggests that commercial hunting might have an important impact
on harvested populations, which may compromise their survival in
the near future. Guinea-Bissau’s decades of civil war and political
instability has hampered law enforcement and the CITES conven-
tion has been signed but not ratified by Guinea-Bissau. The hunting
of primate species throughout the country became illegal only re-
cently (Anon., 2011), although it was already prohibited within
protected areas (IBAP, 2007). While chimpanzees, baboons and col-
obus monkeys have been studied in southern areas of Guinea-Bis-
sau (Hockings and Sousa, 2011; Sousa et al., 2011; Ferreira da Silva
et al., 2013; Minhós et al., 2013; Sá, 2013), information on the two
most traded primates is still lacking. Efforts need to be made in or-
der to: (1) decrease the bushmeat trade, (2) identify and monitor
the most hunted and important primate populations to be pro-
tected, (3) law enforcement and (4) develop alternative economic
activities to the bushmeat trade. The fact that in Guinea-Bissau,
populations of some hunted primate species have become extinct
in areas where they were recently known to occur (Casanova and
Sousa, 2007; Minhós, 2012) or now display hunting-related
changes in their dispersal strategies (Ferreira da Silva, 2012), stres-
ses the urgency of such actions. Guinea-Bissau is one of the poorest
countries in the world. Education and job opportunities are extre-
mely limited and the bushmeat trade is an appealing and lucrative
activity. This situation is likely to be more extreme in rural areas.
Any conservation policy should be combined with education
efforts as well as the provision of viable alternative long-term prof-
itable activities.
Our study demonstrates that DNA barcoding can provide a valu-
able tool to overcome misidentification bias and more accurately
estimate bushmeat species trade, enabling a more comprehensive
approach when designing conservation measures.
Acknowledgments
We would like to acknowledge the support of D. Starin (visits to
the markets and useful comments to the manuscript), vendors of
bushmeat markets (allowing visits and providing information), J.
Lello (statistical advices), C. Sousa and C. Casanova (research activ-
ities), IBAP- Institute of Biodiversity and Protected Areas (logistics).
We thank Direcção Geral de Florestas e Fauna and Instituto da Con-
servação da Natureza e Florestas (ICNF) for the CITES licenses (PT/
LI-0370/2010 to PT/LI-0376/2010 and 11-PT-LX0984/C to 11-PT-
LX0986/C). We acknowledge the comments by the three anony-
mous reviewers that strongly improved the manuscript. Portu-
guese Foundation for Science and Technology (FCT) funded this
work, under Ferreira da Silva, Minhós and Sá grants (SFRH/BPD/
88496/2012, SFRH/BPD/87396/2012, and SFRH/BD/35797/2007).
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.biocon.2013.
07.018.
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