Subspecies
identification of Chimpanzees Pan troglodytes (Primates: Hominidae) from the National Zoo of the Metropolitan Park
of Santiago, Chile, using mitochondrial DNA sequences
Javier Andrés Vega 1,
José Suazo 2, Susan Valerie Smalley 3,
Luis Rodrigo Cataldo 4, Guillermo Cubillos 5 & José Luis Santos 6
1,3,4,6 Department of Nutrition,
Diabetes and Metabolism, School of Medicine, PontificiaUniversidad Católica de Chile, Santiago, Chile
2 Instituto de Investigaciónen Ciencias Odontológicas, Facultad de Odontología,
Universidad de Chile, Santiago, Chile.
5 Animal Welfare and Behavior
Section, Zoológico Nacionaldel Parque Metropolitano de
Santiago, Chile
1 jnvega@uc.cl, 2 jsuazo@odontologia.uchile.cl,3 svsmalleym@gmail.com, 4 rcataldo@ciq.uchile.cl, 5 gcubillos@parquemet.cl,6 jsantos@med.puc.cl (corresponding author)
Abstract: Natural populations of
Chimpanzees Pan troglodytes are declining because of hunting and illegal
live animal trafficking. Four
subspecies of Chimpanzee have been reported: Pan troglodytes troglodytes, P.t. schweinfurthii, P.t. verus and P.t. ellioti, which have remained geographically
separated by natural barriers such as the rivers Niger, Sanagaand Ubangi in central Africa. Sequence analysis of mitochondrial DNA (mtDNA)
has been used for the determination of these subspecies, which indirectly can
also suggest their geographic origin. It was decided to identify the subspecies and the geographic origin of
three captive chimpanzees of the National Zoo of the Metropolitan Park of
Santiago (Chile), by analyzing their mitochondrial DNA. DNA was extracted from the saliva of
three adult chimpanzees (two males and one female). After the analysis of sequences of the
mitochondrial hypervariable region (HVI), a
phylogenetic tree was constructed using mitochondrial sequences of known Pantroglodytes subspecies. Molecular phylogeny analysis revealed
that the chimpanzees are likely to belong to three different subspecies: P.t. schweinfurthii,P.t. verusand P.t. troglodytes. Identification of subspecies of the
three chimpanzees of the National Zoo of the Metropolitan Park of Santiago
(Chile) was possible due to mtDNA analysis. Future identification of chimpanzees
will allow the development of a studbook for the chimpanzee subspecies in other
Latin American zoos.
Keywords: Genetic distance, geographical
localization, Hypervariable region.
doi: http://dx.doi.org/10.11609/JoTT.o3611.5712-7 | ZooBank:urn:lsid:zoobank.org:pub:FBCECF51-1A84-4FBD-85AF-1E8F0811E511
Editor: Mandar Paingankar, University of Pune, Pune,
India. Date
of publication: 26 May 2014 (online & print)
Manuscript details: Ms #
o3611 | Received 03 May 2013 | Final received 04 April 2014 | Finally accepted
07 April 2014
Citation: Vega, J.A., J. Suazo, S.V. Smalley, L.R. Cataldo,G. Cubillos & J.L. Santos (2014). Subspecies
identification of Chimpanzees Pan troglodytes (Primates: Hominidae) from the National Zoo of the Metropolitan Park
of Santiago, Chile, using mitochondrial DNA sequences. Journal of
Threatened Taxa 6(5): 5712–5717; http://dx.doi.org/10.11609/JoTT.o3611.5712-7
Copyright: © Vega et al. 2014. Creative Commons
Attribution 4.0 International License. JoTTallows unrestricted use of this article in any medium, reproduction and
distribution by providing adequate credit to the authors and the source of
publication.
Funding: Supported by VRAID 02/2009 project of the PontificiaUniversidad Católica de Chile.
Competing Interest: The
authors declare no competing interests.
Acknowledgements: We
thank the staff of the Metropolitan Park of Santiago for their support in
obtaining saliva samples. Primer sequences for mtDNAanalysis were kindly provided by Prof. George Perry (Anthropological Genomics
Laboratory, Penn State University).
For
figures & images -- click here
Natural populations of
Chimpanzees Pan troglodytes are declining due to hunting and illegal
live animal trafficking (Oates 2006). The Chimpanzee is as an Endangered species according to the IUCN Redlist (Oates et al. 2013).
Latin American zoos host many
wild-animal colonies (http://www.alpza.com), however, identification upon
arrival of any new organism has been made only from the phenotypic characteristics
like color pattern (feathers, plates, fur, scales), body size or dental formula
(Klukkert et al. 2012). In addition, the exact geographical
origin of many animals in captivity is usually unknown and the case of Chimpanzees in Latin American zoos are no exception
to this rule. Currently,
Chimpanzees are known by four phenotipically recognized subspecies: P.t.troglodytes, P.t. schweinfurthii,P.t. verus and P.t. ellioti (formerly calledP.t. vellerosus)
(Groves 2001; Oates et al. 2009). Interestingly, these four subspecies have different geographic origins
that have remained spatially separated during the course of evolution due to
natural barriers of central Africa, such as the rivers Niger, Sanaga and Ubangi (Kortlandt1983) (Fig. 1).
Sequence analysis of
mitochondrial DNA (mtDNA) in the hypervariableregion HVI is informative for the determination of Chimpanzee subspecies and
also indirectly allows inference of their geographic origin (Goldberg & Ruvolo 1997; Excoffier et al.
1999; Ely et al. 2005; Stone et al. 2010). Following this approach, subspecies of chimpanzees can be divided into
Western (P.t. verusand P.t. ellioti),
Central (P.t. troglodytes) and Eastern
Chimpanzees (P. t. schweinfurthii) (Gagneux 2002).
The aim of this study was to
identify the subspecies and the geographic origin of three captive chimpanzees
of the National Zoo of the Metropolitan Park of Santiago (Chile), by analyzing
their mitochondrial DNA.
Material and Methods
Ethics statement: The study was approved by the National Zoo of the Metropolitan
Park of Santiago (Chile) following the ethical criteria of The Latin American
Association of Zoological Parks and Aquariums (ALPZA).
Biological samples and laboratory methods: Mitochondrial DNA was
extracted from saliva of three adult captive Chimpanzees (Pan troglodytes)
of the National Zoo of the Metropolitan Park of Santiago using the Oragene Animal Kit (DNA Genotek,
Canada) following the manufacturer’s instructions. Specimen 1 (died in 2010) and specimen 2
were males with an estimated ages of 30 and 12 years
respectively, while specimen 3 was a 35 years female. The study was conducted during the year
2010.
A total of 100ng of DNA was
amplified by Polymerase Chain Reaction (PCR) with primers obtained in
literature 5’-CTCTGTTCTTTCATGGGGAAGC-3’(forward) and
5’-CGGGATATTGATTTCACGGAGG-3’ (reverse) (Goldberg & Ruvolo1997; Perry et al. 2012). Initially, the DNA was heated to 940C for five minutes,
followed by 35 cycles of denaturing, annealing, and elongation (940C
for 30 seconds, 540C for 30 seconds, and 720C for 30
seconds). The final extension occurred at 720C for 10 minutes. PCR products (388 base pairs) were
visualized by 1.5% agarose gel electrophoresis. The purified PCR products were sequenced
with the big dye terminator sequencing kit v3.1 (Applied Biosystems,
USA), using forward and reverse primers in two independent reactions, and
analyzed in the ABI prism 3730XL sequencer (Applied Biosystems,
USA).
Data analysis: For
the genetic distance analysis of mtDNA sequences, we
compared 28 complete mitochondrial sequences of Pan troglodytescomprising the four chimpanzee subspecies retrieved from Genbank(http://www.ncbi.nih.gov/genbank/), one mitochondrial sequence of the
related-species Bonobo (Pan paniscus) as an
out-group sequence, and sequences of the three chimpanzees from the Santiago
Zoo. The sequences were submitted
to Genbank, with the following accession numbers:
KJ606391, KJ606392 and KJ606393, for specimens 1 (Images 1,2), 2 (Image 3) and
3 (Images 4,5), respectively.
The similarity of our
sequences with Genbank database entries was carried
out using BLAST (http://blast.ncbi.nlm.nih.gov). All 32 sequences were aligned and
compared using the program CLUSTALW (http://www.ebi.ac.uk/clustalw), with gaps
and missing data eliminated from the dataset (“complete deletion option”). There were a total of 359 positions
analyzed in the final dataset. Phylogenetic trees were constructed with the
Program MEGA6.05 (Tamura et al. 2013) using the Maximum Likelihood method with
the Hasegawa-Kishino-Yano (HKY) model (lowest BIC
score= 3846) and a gamma distribution (5 discrete gamma categories). (Posada & Crandall 2001). All positions containing gaps and
missing data were eliminated. The construction of the phylogenetic tree
allows the placement of mitochondrial sequences according to their genetic
similarity in comparison with Pan troglodytes mtDNA sequences of specimens of known subspecies. The
branch support for the correct location of branches was assessed through 1,000
bootstrap replicates.
Results
Specimen 1 showed 98%
similarity with P.t. schweinfurthii,
specimen 2 showed 95% similarity with P.t. verus and specimen 3 showed 98% similarity with P.t. troglodytes. Results of BLAST analysis suggest
that the specimen 1 from the Santiago Zoo was closely related to P.t. schweinfurthii,
specimen 2 was grouped with P.t. verus and specimen 3 was closely related to P.t. troglodytes. Nucleotide sequences of the three
chimpanzees of the Metropolitan National Zoo Park of Santiago and 29 reference
sequences of Chimpanzees from different geographical locations were used for
the construction of a maximum likelihood phylogenetic tree (Fig. 2). The bootstrap values,showed in the nodes of the branches, within the different clusters of
chimpanzees are relatively high. Therefore, it is likely that specimen 1 was of the subspecies P.t. schweinfurthii,
the specimen 2 is likely to be P.t. verus and the specimen 3 (female) is likely to be P.t. troglodytes.
Discussion
The correct subspecies
classification of Chimpanzees in captivity may contribute in the efforts to
reintroduce and establish the captive population’s management programs with
individuals of known origin, minimizing the likelihood of deleterious genetic
effects caused by inbreeding (Goldberg & Ruvolo1997). In this context, genetic marker
studies with nuclear and mitochondrial DNA sequences show that the nucleotide diversity
in chimpanzees is four times higher than in humans, which favors the use of mtDNA for identifying subspecies of chimpanzees (Stone et
al. 2002). It is also worth
mentioning that the extent of genetic differentiation among chimpanzee
subspecies is similar to the one found in human populations (Fischer et al.
2006). In this study, we have
successfully identified, with high probability, three chimpanzee specimens of
the National Zoo of Santiago of Chile, illustrating the utility of mtDNA in genetic identification (Ely et al. 2005).
Wild animal colonies
represent a valuable opportunity for the ex situ conservation of the species
and provide an important source of information for animal management
plans. Given the current situation
of the populations of chimpanzees held in captivity, it is desirable to develop
an identification plan involving Latin American zoological institutions. Zoos and primate rehabilitation centers
could play a major role in the reception and subsequent reintroduction of
animals, through development of studbooks for captive chimpanzees to minimize
inbreeding and to preserve maximum genetic diversity (Carlsen2009; Hvilsom et al. 2013). It should be noted that the release of
captive chimpanzees in wild areas could be carried out successfully,
representing a potential benefit to the conservation of this species (Goossens et al. 2005).
A recent research on the
genetic characterization of captive chimpanzees in Europe using 30 polymorphic
microsatellite markers revealed that 40% of genotyped individuals were of western
African origin, 18% of central Africa origin, 5% of eastern African origin and
23% hybrids (Hvilsom et al. 2013). A limitation of our study derives from
the use of a single mitochondrial marker instead of nuclear polymorphic
markers, which affects the accuracy of our subspecies classification.
In conclusion, identification
of subspecies of the three chimpanzee specimens of the National Zoo of the
Metropolitan Park of Santiago (Chile) was carried out by mtDNAanalysis. A more elaborate genomic
characterization with additional markers will facilitate the implementation of
cooperative management plans and may constitute the initial steps for the
creation of a studbook for chimpanzee subspecies of the region.
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