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Forensic Science International: Genetics Supplement Series 4 (2013) e230e231

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Forensic Science International: Genetics Supplement Series

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Molecular identication of a Buffy-tufted-ear marmoset (Callithrix

aurita) incorporated in a group of invasive marmosets in the Serra dos
Orgaos National Park, Rio de Janeiro Brazil
R.S. Carvalho a,b, D.A. Silva a, S. Loiola a, D.G. Pereira b, E.F. Carvalho a,*, H.G. Bergallo b

Laboratorio de Diagnosticos por DNA, Universidade do Estado do Rio de Janeiro, Brazil

Programa de Pos-graduacao em Ecologia e Evolucao, Universidade do Estado do Rio de Janeiro, Brazil



Article history:
Received 23 August 2013
Accepted 2 October 2013

For years, illegal pet trade resulted in the introduction of marmosets from the species Callithrix jacchus
and C. penicillata in the Atlantic Forest of southeastern Brazil. Among other problems these primates are
able to produce fertile hybrids in crosses between themselves and also with its counterpart, the
endangered Buffy-tufted-ear marmoset (C. aurita) natural of this region. Molecular genetic data has
increasingly been used as support for issues related to biodiversity with great contribution to
conservation programs, because of its power to elucidate gene ow, introgression, levels of kinship and
hybridization. In forensics, this information has the potential to be used for the control of exotic species
and curb trafcking of native species. By sequencing using mitochondrial markers Cyt b and CO II, this
work shows the molecular characterization of a mixed group of marmosets found between the forest and
a urbanized area close to the Serra dos Orgaos National Park in Rio de Janeiro state. The results showed
that the group of marmosets here studied is formed by a male individual with phenotype and genotype
matching that of the endangered species C. aurita, among others which belong to the invasive species C.
penicillata and C. jacchus. This reinforces the importance of studies aimed at elucidating the dynamics of
gene ow and the viability of possible hybrids facing the conservation of native species. From the
forensics standpoint, the characterization based on molecular markers, ensures a more comprehensive
tool for identifying those species ghting illegal trade and preserving the endangered C. aurita.
2013 Elsevier Ireland Ltd. All rights reserved.

Callithrix aurita
Molecular markers

1. Introduction
The Convention on International Trade in Endangered Species of
Wild Fauna and Flora (CITES), established in 1973, refers to an
international agreement to protect wildlife from the international
trade, and Brazil was among the rst to subscribe to CITES. Another
major threat to wildlife is the internal trade of pets and which
represents a serious menace to local biodiversity. Native marmosets from central (Callithrix penicillata) and northeast (Callithrix
jacchus) Brazil have been recurrently introduced to regions outside
their original range [1,2] where they assume the characteristics of
invasive species. In Brazilian southeastern regions, there are
reports of competition and hybridization of those invaders with a
local endangered marmoset species (Callithrix aurita) endemic of
Rio de Janeiros mountains [3].
The Red listed Buffy-tufted-ear marmosets (C. aurita) are better
adapted to the Brazilian southeast mountains (altitudes over 700
meters), a characteristically different habitat from the other two

* Corresponding author. Tel.: +55 21 9984 8269; fax: +55 21 2334 0594.
E-mail address: (E.F. Carvalho).
1875-1768/$ see front matter 2013 Elsevier Ireland Ltd. All rights reserved.

species, nevertheless the invader populations, which were already

spread on the low lands, are increasing rapidly (considering their
adaptability to urbanized areas) and are climbing toward the
native marmosets original home.

Fig. 1. A male individual of C. aurita in the middle others from different species.

R.S. Carvalho et al. / Forensic Science International: Genetics Supplement Series 4 (2013) e230e231


Fig. 2. (A) Neighbor-Joining (TN93) tree of three Callithrix species, based on COII gene. (B) Neighbor-Joining (TN93) tree of three Callithrix species, based on Cyt b gene.

Primates molecular identication using the sequencing of

mitochondrial regions of the cytochrome b gene (Cyt b) and of the
cytochrome C oxidase gene subunit II (COII) has been shown to be
an effective, quick and low cost identication tool [4] to be broadly
used in forensics and biodiversity conservation.
In this article we compare the mitochondrial molecular
identication (CO II and Cyt b) of a group of marmosets (at the
species level), to reveal the presence of a native male individual of
the threatened C. aurita living among his invasive congener C.
penicillata and C. jacchus.
2. Methodology
Specimens of one specic group of Callithrix sp. were collected
at Nogueira, Petropolis, Rio de Janeiro, Brazil, an urban area at the
foothills of Serra dos Orgaos National Park. Blood in FTA was
submitted to the DNA extraction and purication protocol with the
Qiagen QIAamp DNA FFPE Tissue kit. The DNA were amplied and
sequenced using the pair of primers for CO II CAA AAC GCC GCA
TCY CCA ATC (forward)/GGC CTG GTC GTA TGG AAG (reverse), and
AGA ATG ATA TTT GTC CTC A (heavy). Sequencing products were
processed in an ABI 3500 capillary system, being visually checked
and aligned using the software Geneious 6.1.6. Our nal dataset
consisted of a 301 bp of the Cyt b gene, and the 450 bp for the CO II
gene. The alignments were visually inspected to minimize missing
data. The Neighbor-Joining (NJ) tree was constructed using the
software Geneious 6.1.6 with the Tamura Nei Parameter (TN93)
distance model.

potential to spread allopatric alleles back into the native, rare and
endemic populations of C. aurita, which are already highly
threatened by habitat loss. This case alerts to the critical situation
for the C. aurita marmosets suffering the impact of the two major
menaces to biodiversity: habitat loss and biological invasions.
4. Conclusions
The mitochondrial DNA sequencing methodology, with local
group observations, were able to identify and discriminate an
individual, from the rare and threatened Callithrix aurita, living as
the dominant male in a group composed of genotypically and
phenotypically different Callithrix individuals. This result reinforces the relevance of this genetic tool to demonstrate the
ecological impacts of illegal commerce and introduction of
allopatric primates.
Role of funding
This study was supported by CAPES and FAPERJ.
Conict of interest
We would like to thank Dr. Antonio Amorim for his continuous
intellectual support.

3. Results and discussion

The Neighbor-Joining trees, presented in Fig. 2A and B, separate
the three species of the Callithrix genus (C. jacchus, C. penicillata and
C. aurita) observed in this study. It shows four individuals from the
Nogueira group (Nogueira group 25) joined together to the mainly
C. jacchus branch, detaching and clustering the individual Nogueira
1 among the others characterized as C. aurita. The molecular result
matches the phenotypical differentiation for the genus, gathering
the visually distinguishable Nogueira 1 (Fig. 1) together with others
molecularly and phenotypically dened as C. aurita. Local
observation revealed that Nogueira 1 was the dominant male in
the group and, consequently, the main responsible for mating.
These mating events, if successful, will generate hybrids with the

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