Genetic structure in two northern muriqui populations (Brachyteles hypoxanthus, Primates, Atelidae) as inferred from fecal DNA

Genetics and Molecular Biology, 31, 1, 166-171 (2008)
Copyright by the Brazilian Society of Genetics. Printed in Brazil
www.sbg.org.br
Short Communication
Genetic structure in two northern muriqui populations
(Brachyteles hypoxanthus, Primates, Atelidae) as inferred from fecal DNA
Valéria Fagundes1, Marcela F. Paes1, Paulo B. Chaves1, Sérgio L. Mendes1, Carla de B. Possamai2,3,
Jean P. Boubli4* and Karen B. Strier5
1Departamento de Ciências Biológicas, Universidade Federal do Espírito Santo, Vitória, ES, Brazil.
2RPPN Feliciano Miguel Abdala, Caratinga, MG, Brazil.
3Pontifícia Universidade Católica de Minas Gerais, Belo Horizonte, MG, Brazil.
4Conservation and Research for Endangered Species, Zoological Society of San Diego, San Diego, CA, USA.
5Department of Anthropology, University of Wisconsin-Madison, Wisconsin, USA.
Abstract
We assessed the genetic diversity of two northern muriqui (Brachyteles hypoxanthus Primata, Atelidae) populations,
the Feliciano Miguel Abdala population (FMA, n = 108) in the Brazilian state of Minas Gerais (19°44’ S, 41°49’ W) and
the Santa Maria de Jetibá population (SMJ, n = 18) in the Brazilian state of Espírito Santo (20°01’ S, 40°44’ W). Fecal
DNA was isolated and PCR-RFLP analysis used to analyze 2160 bp of mitochondrial DNA, made up of an 820 bp
segment of the gene cytochrome c oxidase subunit 2 (cox2, EC 1.9.3.1), an 880 bp segment of the gene cytochrome
b (cytb, EC 1.10.2.2) and 460 bp of the hypervariable segment of the mtDNA control region (HVRI). The cox2 and
cytb sequences were monomorphic within and between populations whereas the HVRI revealed three different pop-
ulation exclusive haplotypes, one unique to the SMJ population and two, present at similar frequencies, in the FMA
population. Overall haplotype diversity (h = 0.609) and nucleotide diversity (π = 0.181) were high but reduced within
populations. The populations were genetically structured with a high fixation index (F = 0.725), possibly due to his-
ST
torical subdivision. These findings have conservation implications because they seem to indicate that the popula-
tions are distinct management units.
Key words: Brachyteles, conservation genetics, fecal DNA, mtDNA, PCR-RFLP.
Received: April 27, 2007; Accepted: September 25, 2007.
The muriqui or woolly spider monkey (Brachyteles
The IUCN Red List cites the northern muriqui as a
Spix 1823: Primates, Atelidae), endemic to Brazil, is the
critically endangered species because only about 900 indi-
largest Neotropical primate and was once widespread in the
viduals are known dispersed in 12 populations, five of
southeastern Atlantic Forest of Brazil. Aguirre (1971) esti-
which contain less than 20 members. Some of the popula-
mated a total population of 2,791-3,226 muriquis, contrast-
tions are restricted to small, unprotected and isolated forest
ing with a population of about 400,000 he reckoned would
fragments extending from the south of the state of Bahia
have existed in 1500. In the last decade this genus has been
throughout Minas Gerais and Espírito Santo states as well
split into two species (Groves, 2005), the northern muriqui
as along the Mantiqueira Mountains (Serra da Mantiqueira)
(Brachyteles hypoxanthus Kuhl 1820) and the southern
on the borders of the states of Minas Gerais, Rio de Janeiro
muriqui (Brachyteles arachnoides É. Geoffroy 1806). Cur-
and São Paulo (Rylands et al., 2003b; Mendes et al., 2005).
rent population estimates for the northern muriqui have
The southern muriqui is distributed along the Serra do Mar
indicated at least 864 individuals in the wild and data avail-
from the south of Paraná state to Rio de Janeiro state, is
able for the southern muriqui suggest a minimum popula-
listed as endangered in the IUCN Red List and no more than
tion of about 1,300 (Melo and Dias, 2005).
1300 are known to occur in relatively large and well pro-
tected areas (Melo and Dias, 2005).
Send correspondence to Valéria Fagundes. Laboratório de Gené-
The small size and fragmented distribution of north-
tica Animal, Departamento de Ciências Biológicas, Universidade
ern muriqui populations compromises the ecological via-
Federal do Espírito Santo, Av. Marechal Campos 1468, 29043-900
bility of this species (Brito and Grelle, 2006). Their
Maruípe, Vitória, ES, Brazil. E-mail: vfagunde@npd.ufes.br.
*Present address: Department of Anthropology, The University of
advanced age of 9 years at first reproduction and long
Auckland, Auckland, New Zealand.
inter-birth interval of three years, make the small persistent

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Fagundes et al.
167
populations more vulnerable to unfavorable demographic
conditions than primates with faster life histories or larger
populations (Strier et al., 2006). Habitat reduction and
hunting have probably forced surviving northern muriqui
populations into bottlenecks and reduced or eliminated op-
portunities for gene flow through populations, resulting in
changes in the frequency of alleles, loss of alleles, or both
(Young and Clarke, 2000) similar to those documented in
the golden lion tamarin (Grativol et al., 2001).
Population viability analysis (PVA) has suggested
that only muriqui populations of at least 700 monkeys
would be genetically viable (Strier, 1993/1994; Brito and
Grelle, 2006) but no empirical genetic data was included in
these analyses. Although a preliminary allozyme analysis
comparing one population of each species reported a high
fixation index (Pope, 1998), knowledge of genetic structure
of more populations is crucial for muriqui conservation
planning (Fagundes, 2005).
Standardization of a non-invasive method for DNA ex-
traction from muriqui feces is an important tool for assessing
the conservation status and behavioral ecology of these mon-
keys (Chaves et al., 2006). Furthermore, data on the levels of
genetic variability and differentiation in muriqui populations
as well as pedigree reconstruction and information regarding
Figure 1 - Map depicting the easternmost portion of Brazil. Sampling
the relatedness between individuals and the extent of in-
sites of the Brachyteles hypoxanthus populations are highlighted. The
breeding can contribute to the planning of effective conser-
FMA population in Minas Gerais (MG) state is roughly 150 km from the
vation strategies for these species.
municipality of Santa Maria de Jetibá (SMJ) in Espírito Santo (ES) state.
We used the Polymerase Chain Reaction and Restric-
tion Fragment Length Polymorphism analysis (PCR-
man presence. The other population, designated the SMJ
RFLP) to evaluate the genetic diversity of two northern
population, is in privately owned forest fragments in the
muriqui populations and characterize the distribution of ge-
municipality of Santa Maria de Jetibá (SMJ) in the state of
netic variability within and between populations. Fecal
Espírito Santo (20°0’ S, 40°44’ W). The forest patches con-
samples
from
126
free-ranging
northern
muriquis
taining the muriqui range from 60 ha to 350 ha and are
(Brachyteles hypoxanthus Kuhl 1820) were collected im-
highly fragmented due to agricultural activities and un-
mediately after defecation and stored at 4 °C or room tem-
evenly connected to each other by hilltop corridors. This
perature (~24-28 °C) in 50 mL polypropylene vials contai-
metapopulation has been estimated to be comprised of 115
ning a layer, about 1 mm to 4 mm deep, of desiccated silica
muriqui (Mendes et al., 2005), although only about 16%
beads to dehydrate the feces (Chaves et al. 2006). Each in-
(n = 18) could be sampled because monitoring of this popu-
dividual sampled was identified by its natural markings by
lation only started in 2001.
the experienced field researchers who collected the fecal
We extracted DNA from the feces using the QIAamp
samples within the ambit of a collecting license (number
DNA Stool Minikit (Qiagen) and assessed the quality and
363/2001) issued by the Brazilian National Environmental
amount using 1% (w/v) agarose gels stained with
Agency (Instituto Brasileiro do Meio Ambiente e dos Re-
0.5 μg/mL ethidium bromide. Appropriate primers were
cursos Naturais Renováveis – IBAMA). Two populations,
used for the PCR amplification of a total of 2160 base pairs
separated by 150 km, were studied (Figure 1). One popula-
(bp) from the following three mitochondrial DNA
tion is in a private conservation unit (Reserva Particular do
(mtDNA) segments: 820 bp of the gene cytochrome c
Patrimônio Natural Feliciano Miguel Abdala, RPPN-
oxidase subunit 2 (cox2, EC 1.9.3.1), amplified using the
FMA) of 957 hectares (ha) located in the state of Minas
L6955/H7766 primer pair (Ashley and Vaughn, 1995);
Gerais (19°44’ S, 41°49’ W) and is designated the FMA
880 bp of the gene cytochrome b (cytb, EC 1.10.2.2), ampli-
population. This population is made up of four groups
fied using the MVZ05/MVZ16 primer pair (Smith and
(three mixed-sex and one only-male group) totaling 226
Patton, 1993); and the 460 bp hypervariable segment of the
northern muriquis (Strier et al., 2006) of which nearly 50%
mtDNA control region (HVRI), for which we designed a
(n = 108) were sampled between 2001 and 2002, once this
novel specific primer pair consisting of a 5’-CTACTCCCT
population has been monitored over the last 25 years by K.
GAATAACCAAC-3’ forward primer (Mono1) and a 5’-
B. Strier and her coworkers and animals are used to the hu-
AGCGAGAAGAGCGGCAAATG-3’
reverse
primer

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168
Genetic structure of northern muriqui
(Mono2), which were based on the Brachyteles sequence
For the restriction fragment data analysis, one allele
(GenBank AF213966) with the 3’ annealing positions
was represented by one fragment obtained with an enzyme,
(L15463 for Mono1 and H15890 for Mono2) from the
and the set of all alleles of a specific digestion (i.e., the
Cebus albifrons mtDNA sequence (GenBank AJ309866).
cleavage pattern) received a capital letter (Table 1). The
The specificity of the HVRI primers was shown by their in-
composite haplotype panel for each monkey included the
ability to amplify human DNA, probably due to the 19 mis-
cleavage pattern of all the enzymes (Table 2, see Bates,
matches (12 forward and 7 reverse) between the primers,
2002 for details). Genetic diversity analyses were calcu-
which had been derived for monkey sequences, and human
lated using ARLEQUIN 3.01 (Excoffier et al., 2005) after
DNA. An in silico restriction simulation was performed
converting composite haplotypes into a binary matrix (Ta-
with Brachyteles HVRI sequence from Genbank using
ble 2) based on the presence or absence of alleles (restric-
BIOEDIT 7.0.5.3 (Hall, 1999) to identify restriction sites
tion fragments). The population parameters calculated
and restriction fragment sizes. Analysis of the human
were haplotype frequency, haplotype diversity (h), nucleo-
HVRI restriction sites excluded cross-contamination of
tide diversity (π), mean number of pairwise difference be-
muriqui PCRs with human DNA (Figure 2).
tween haplotypes, and Wright’s fixation index (FST,
The PCR was carried out in a final volume of 50 μL of
Wright, 1951).
1X Taq buffer containing 3 mM MgCl2, 0.4 mM of each
All 80 restriction assays for cox2 (n = 49) and cytb
dNTP, 0.4 μM of each primer, 2.5 units of Platinum Taq
(n = 31) resulted in monomorphic haplotypes for the seven
DNA polymerase (Invitrogen) and 50 ng of DNA (Chaves
endonucleases (Table 2). Therefore, we concentrated on
et al., 2006). Amplifications were conducted at 92 °C for
analyzing all 126 muriquis for the HVRI segment based on
5 min, followed by 35-37 cycles of 92 °C for 60 s, 47 °C to
the following assumptions. Firstly, amplification success
52 °C for 30 s to 60 s and 72 °C for 30 s to 60s, with a final
for HVRI was 100% (against 20%-30% for cox2 and cytb),
extension at 72 °C for 5 min. After amplification the am-
which is amongst the highest rate ever reported for fecal
plicons were electrophoretically sized on 1% (w/p) agarose
DNA (Broquet et al., 2007 and references therein). This is
gel using 1 kb and 100 bp Ladders (Invitrogen). Mock PCR
likely to be due to the specificity of the primers we used, as
blanks were included to check for contamination.
well as the short-length segment, which are better suited to
Estimates of the number of nucleotide substitutions
analyses of highly degraded fecal DNA. Secondly, HVRI
(genetic polymorphisms) took into account the previous in
seems to be one of the most variable segments within mam-
silico analysis shown in Figure 2. Three to seven restriction
malian mtDNA (Aquadro and Greenberg, 1983; Sbisà et
endonucleases (Table 1) digested roughly 20 ng of the PCR
al., 1997), and is thus highly suitable for revealing within-
amplicons following the manufacturer’s protocol. Restric-
population variation. Finally, mtDNA evolves as a single
tion fragment sizes were determined using polyacrylamide
locus (Avise, 2004), thus analyses of the most variable seg-
gel electrophoresis (5 to 10% w/v) and ethidium bromide
ment can provide a rough estimate of the diversity of the en-
staining, with the gels being photographed under ultraviolet
tire mtDNA genome.
light. Restriction fragment sizes were estimated using 1 kb
The HVRI assay revealed 35 restriction fragments
and 100 bp ladders as reference (detailed procedure is
(Table 2), of which 17 (48.6%) were polymorphic. The
available upon request). Table 1 summarizes the PCR-
MseI and RsaI restriction enzymes generated more frag-
RFLP results obtained after cutting PCR amplicons.
ments (n = 8) and BsaI generated fewer fragments (n = 2),
while HinfI generated the longest fragment (460 bp, with no
restriction site). Based on the restriction site map shown in
Figure 2 one polymorphic restriction site was present for
HinfI (position 240 or 220), two sites were present for RsaI
(positions 56 and 313), and one site each was present for
MseI (position 251) and Tsp509I (around position 400 to
420). The NlaIII and HpyCH4IV sites were invariable. Af-
ter assembling the single digestion patterns generated by
each endonuclease the HVRI, RFLP identified three com-
posite haplotypes, with haplotype SMJ1 being exclusively
Figure 2 - The in silico restriction analysis resulted in the haplotype
present in all the 18 monkeys from the SMJ group, while
A?AABAA in the muriqui sequence, which is highly similar to muriqui
the other two haplotypes (FMA1 and FMA2) were unique
haplotypes found in the SMJ and FMA populations. However, this
haplotype was not in our assessment, possibly because this sequence is
to the FMA population and were nearly equal in frequency
from an individual from a different population. The human haplotype is
at 57.4% for FMA1 and 42.6% for FMA2 (Table 2). Differ-
markedly different from all muriqui haplotypes, indicating that if human
ences in pairwise distances between haplotypes (SMJ1/
DNA were amplified due to inadvertent contamination it would be readily
FMA1 = 12, SMJ1/FMA2 = 13 and FMA1/FMA2 = 9)
identified. The BsaI and HpyCH4IV restriction enzymes cannot cut the
human segment (GenBank AC_000021/Homo sapiens), while the HinfI
demonstrated the closer relationship between FMA1 and
restriction enzyme cannot cut muriqui DNA.
FMA2 than between either of these haplotypes and the

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Fagundes et al.
169
Table 1 - Restriction enzyme cleavage patterns of the genes cytochrome c oxidase subunit 2 (cox2, 820 bp), cytochrome b (cyt b, 880 bp) and the
hypervariable segment of mtDNA control region (HVRI, 460 bp) of the Brachyteles hypoxanthus populations from Santa Maria de Jetibá in the Brazilian
state of Espírito Santo (SMJ population, n = 18) and Feliciano Miguel Abdala in the Brazilian state of Minas Gerais (FMA population, n = 108). The total
number (n) of muriquis analyzed for each mtDNA segment is shown in parentheses in the first column.
Segment, number of individuals analyzed
Fragment sizes
Cleavage pattern†
Frequency
Population
(n) and restriction enzyme*
(bp)
(%)
cox2 (n = 49)
AluI
230, 190, 170, 130, 100
A
100
SMJ and FMA
HinfI
390, 430
A
100
SMJ and FMA
MboI
280, 270, 140, 130
A
100
SMJ and FMA
MspI
620, 200
A
100
SMJ and FMA
RsaI
270, 220, 200, 130
A
100
SMJ and FMA
cyt b (n = 31)
HaeIII
410, 200, 150, 120
A
100
SMJ and FMA
HinfI
480, 290, 60, 50
A
100
SMJ and FMA
MspI
560, 270, 50
A
100
SMJ and FMA
HVRI (n = 126)
HinfI
460
A
100
SMJ
HinfI
460
A
42.6
FMA
HinfI
240, 220
B
57.4
FMA
RsaI
200, 120, 70, 55,15*
A
100
SMJ
RsaI
150, 120, 70, 55, 55, 15*
B
57.4
FMA
RsaI
150, 120, 70, 65, 55
C
42.6
FMA
MseI
150, 80, 75, 60, 40, 35, 25*
A
100
SMJ
MseI
150, 80, 75, 60, 40, 35, 25*
A
42.6
FMA
MseI
150, 95, 80, 60, 40, 35
B
57.4
FMA
Tsp509I
355, 105
A
100
FMA
Tsp509I
300, 105, 55
B
100
SMJ
HpyCH4IV
165, 90,75, 70, 60
A
42.6
FMA
HpyCH4IV
165, 160, 75, 60
B
57.4
FMA
HpyCH4IV
165, 160, 75, 60
B
100
SMJ
NlaIII
180, 125, 80, 75
A
100
SMJ and FMA
BsaI
430, 30
A
100
SMJ and FMA
*The restriction enzyme 5’→3’ sites were as follows: AluI = AG/CT; HinfI = G/ANTC; MboI = /GATC; MspI = C/CGG; RsaI = GT/AC;
HaeIII = GG/CC; HinfI = G/ANTC; MseI = T/TAA; Tsp509I = /AATT; HpyCH4IV = A/CGT; NlaIII = CATG/; and BsaI = GGTCTCN/N4.
†A, B and C are the cleavage patterns (set of restriction fragments) for each enzyme.
–These fragments (15 bp and 25 bp) were not visualized in the gels, probably because they were lost.
SMJ1 haplotype. This finding suggests that the SMJ and
We also found a high fixation index of FST = 0.725
FMA populations have been isolated long enough to pre-
(p < 0.001), which is strongly indicative of genetic distinc-
vent gene flow and haplotype sharing.
tiveness between the SMJ and FMA populations. This FST
is considerably higher than the FST = 0.413 for allozyme
Overall haplotype (h = 0.609 ± 0.022) and nucleotide
polymorphisms of 12 muriqui (two from a what is now con-
diversity (π = 0.181 ± 0.095) were relatively high. Within-
sidered a B. arachnoides population and ten from a
population diversity indices were slightly lower in the
B. hypoxanthus population different to that studied by us)
FMA population (h = 0.494 ± 0.016; π = 0.127 ± 0.070)
reported by Pope (1998), who used this data to recom-
than in the SMJ population, which were both zero. The low
mended the elevation of the southern and northern forms of
number of haplotypes homogeneously distributed is an un-
the muriqui into separate species (B. arachnoides and
favorable scenario for the genetic diversity of the FMA
Brachyteles hypoxanthus respectively).
population, but it is better than the single haplotype found
Populations containing mtDNA haplotypes at signifi-
in the SMJ population.
cantly different frequencies or, in more extreme cases, pre-

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170
Genetic structure of northern muriqui
Table 2 - Composite haplotypes obtained after PCR-RFLP analysis of HVRI in 126 northern muriqui monkeys from Santa Maria de Jetibá in the Brazil-
ian state of Espírito Santo (SMJ population, n = 18) and Feliciano Miguel Abdala in the Brazilian state of Minas Gerais (FMA population, n = 108).
FMA1 and FMA2 represent the two haplotypes from FMA population and SMJ1 the one from SMJ population. The binary code should be read from left
to right, a ‘1’ indicating the production of a restriction fragment of the specified size (in bp) and a ‘0’ indicating that the fragment was not detected (see
footnote for key).
Haplotype
Composite haplotype*
Composite haplotypes converted to binary code†
Frequency %
Population
SMJ1
AAABBAA
10010110101101111110111110101111111
100
SMJ
FMA1
BBBABAA
01101110111111011101010110101111111
57.4
FMA
FMA2
ACBAAAA
10001111100111011101010101111111111
42.6
FMA
*Letters represent the cleavage pattern resulted of digestion with HinfI, RsaI, MseI, Tsp509I, HpyCH4IV, NlaIII and BsaI, respectively. For details see
Table 1.
†Starting from left (1) to right (35) in the binary code, the 35 restriction enzyme fragments used were as follows: 1. Hinf I (460 bp); 2. Hinf I (240 bp); 3.
Hinf I (220 bp); 4. RsaI (200 bp); 5. RsaI (150 bp); 6. RsaI (120 bp); 7. RsaI (70 bp); 8. RsaI (65 bp); 9. RsaI (55 bp); 10. RsaI (55 bp); 11. RsaI (15 bp); 12.
MseI (150 bp); 13. MseI (95 bp); 14. MseI (80 bp); 15. MseI (75 bp); 16. MseI (60 bp); 17. MseI (40 bp); 18. MseI (35 bp); 19. MseI (25 bp); 20. Tsp509I
(355 bp); 21. Tsp509I (300 bp); 22. Tsp509I (105 bp); 23. Tsp509I (55 bp); 24. HpyCH4IV (165 bp); 25. HpyCH4IV (160 bp); 26. HpyCH4IV (90 bp); 27.
HpyCH4IV (75 bp); 28. HpyCH4IV (70 bp); 29. HpyCH4IV (60 bp); 30. NlaIII (180 bp); 31. NlaIII (125 bp); 32. NlaIII (80 bp); 33. NlaIII (75 bp); 34.
BsaI (430 bp); and 35. BsaI (30 bp).
senting
population-exclusive
haplotypes,
have
been
A. Grativol, L. Oliveira, Y. Leite and two anonymous re-
referred as management units and seem to have been histor-
viewers provided invaluable improvements on early
ically separated during evolutionary time (Moritz, 1994).
versions of the manuscript. We also thank everyone who
In our study, since the FMA and SMJ northern muriqui
helped with this project, especially A.S. Perrone, I.D. Lou-
populations did not share haplotypes, it is reasonable to as-
ro, R. Nunes, L. Cajaiba, R. Santos, H. Dazilio, A. Araújo,
sume that they should be given management unit status and
G. Picoretti, G. Schulz, A. Santos, L. Carmo, F. Paim, M.
actively managed. In addition, the FMA and SMJ popula-
Iurck, K. Tolentino, V. Souza, D. Guedes, J. Oliveira, M.
tions are connected by low levels of gene flow, are func-
Tokuda, F. Ferreira, W. Silva and L.G. Dias.
tionally independent and carriers of a portion of the species
evolutionary legacy. As a caveat, caution must be exercised
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Associate Editor: Fábio de Melo Sene

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