Molecular Ecology Resources (2009) 9, 754-758 doi:10.1111/j.l755-0998.2008.02390.x MOLECULAR DIAGNOSTICS AND DNA TAXONOMY Identification of the endangered small red brocket deer (Mazama bororo) using noninvasive genetic techniques (Mammalia; Cervidae) SUSANA GONZALEZ,* JESUS E. MALDONADO/r JORGE ORTEGA/tJ: ANGELA CRISTINA TALARICO,§LETICIABIDEGARAY-BATISTA,*,**JOSE EDUARDO GARCIA! and JOSE MAURICIO BARBANTI DUARTEg *Unidad de Genetica de la Conservation, Departamento de Genetica, IIBCE-Facultad de Ciencias/UdelaR, Montevideo, Uruguay, tCenterfor Conservation and Evolutionary Genetics, NZP/NMNH, Smithsonian Institution, 3001 Connecticut Ave. NW, Washington D.C. 20008, USA, %Laboratorio de Ictiologia y Limnologia, Posgrado en Ciencias Quimico-Biologicas, Escuela National de Ciencias Biologicas, lnstituto Politecnico National, Prolongation de Carpio y Plan de Ayala s/n, Col. Santo Tomds, 11340 Mexico, %Nucleo de Pesauisa e Conservacao de Cervideos (NUPECCE), Departamento de Zootecnia, FCAV/UNESP, Sao Paulo State University, Via de Acesso Paulo Donato Castellane, s/n, CEP 14884-900, Jaboticabal-SP, Brazil, fCentro Academico de Vitoria. Universidade Federal de Pernambuco, 55608-680 Vitoria de Santo Antao — PE, Brazil Abstract The small red brocket deer Mazama bororo is one of the most endangered deer in the Neotropics. The great morphological similarities with three other sympatric brocket deer species, coupled with the fact that they inhabit densely forested habitats complicate detection and prevent the use of traditional methodologies for accurate identification of species. The ability to determine the presence of this endangered species in an area is crucial for estimating its distribution range, and is critical for establishing conservation management strategies. Here we describe a fast and reliable noninvasive genetic method for species identification of Mazama species from faeces. We designed a primer set that amplifies a short 224-bp fragment of the cytochrome b and demonstrate its effectiveness in successful amplification of DNA isolated from both tissue and faecal samples. This fragment contains a BSTNI/ECORII digestion site that is unique to the endangered M. bororo. The digested polymerase chain reaction products yielded a 160-bp fragment that is clearly visible in a 2% agarose gel. Two other diagnostic sites were identified to differentiate the other three sympatric species, Sspl (M. gouazoubira) and Afllll (M. americana, and M. nana). Keywords: cryptic species, Mazama, noninvasive sampling, small red brocket deer, species identification Received 3 April 2008; revision accepted 9 ]une 2008 Brocket deer (Genus Mazama) are widely distributed in the detect and observe, especially in their densely vegetated Neotropics from southern Mexico to Argentina (Weber & habitats (Vogliotti & Duarte in press). Gonzalez 2003). The taxonomy, distribution, ecology, and The different species vary in size from small to medium status of every species in the genus Mazama remain unclear but they share morphological characters in common, such (Wemmer 1998). Brocket deer occupy a broad range of forest as the presence of spiked antlers in males, and the overall ecosystems but in general are found in closed environments skull morphology. However, pelage colouration varies such as forests and thickets, only occasionally venturing greatly from light brown, reddish brown to grey, and the into open areas. They are very secretive and difficult to body size ranges from less than 10 to 40 kg (Duarte & Jorge 1996; Duarte & Merino 1997). Correspondence: Susana Gonzalez, de Genetica de la Conservation, The genus Mazama is highly diverse and is composed of Departamento de Genetica, IIBCE-Facultad de Ciencias/UdelaR, Montevideo, Uruguay, E-mail: [email protected] 10 species with a wide geographical range from southern "Present address: Departament de Biologia Animal, Universitat Mexico to Argentina (Weber & Gonzalez 2003; Wilson & de Barcelona, Av. Diagonal 645,08028, Barcelona, Spain. Reeder 2005). The recent discovery (or rediscovery) of © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd MOLECULAR DIAGNOSTICS AND DNA TAXONOMY 755 three new species has raised considerable interest in the Smith et al. 2006). Another application involves the devel- taxonomic status and phylogenetic relationships of members opment of fast and reliable methods to identify species from of the genus Mazama (Duarte & Merino 1997; Medellin et al. shed integument and faeces (Kohn et al. 1999). Developing 1998; Duarte & Jorge 2003; Duarte el al. 2008). There are four a better method to distinguish brocket deer species will species of brocket deer known to occur in sympatry in the enhance the conservation management strategies that Atlantic rainforest: the red brocket deer M. americana, the target this threatened species. Genetic methods can be used brown brocket deer M. gouazoubira, the Brazilian dwarf to identify species more accurately when more conventional, brocket deer M. nana, and the recently described small red visual techniques of species identification prove inconclusive brocket deer M. bororo (Duarte & Jorge 2003). This species (Smith et al. 2006; Rudnick et al. 2007; van Vliet et al. 2007). is one of the most endangered Neotropical deer inhabiting We used the mitochondrial cytochrome b gene (cyt b) to small and highly isolated fragments of the remaining develop a quick, inexpensive and robust assay for discrim- Brazilian Atlantic forest (Duarte & Jorge 2003; Weber & inating among the three brocket deer species that can Gonzalez 2003). This area is one of the most important bio- potentially occur in the same areas with M. bororo (Duarte diversity hotspots (Myers et al. 2000) but it is quickly dis- & Jorge 2003). appearing due to intensive habitat fragmentation by We first used reference samples of known species origin human activity. Because of the very close morphological to characterize the patterns of variation in the mitochondrial similarity between the endangered small red brocket deer DNA (mtDNA) cyt b region, both within and among these M. bororo and the more common red brocket deer M. amer- brocket deer species. We then developed a new set of primers icana, information on their distribution and demography specifically designed from several species of Mazama and a was until recently inaccurate and very sparse. system of restriction enzyme digestion of polymerase chain The different species of brocket deer are morphologically reaction (PCR)-amplified mtDNA that can be easily and nearly indistinguishable but they have striking karyotypic reliably applied to distinguish the four species that can differences (Duarte & Jorge 1996,2003). It was only through potentially occur in the same geographical range of M. the karyotypical characterization that the small red brocket bororo. The restriction fragment length polymorphism (RFLP) deerM. bororo was discriminated and described as a distinct assay was then tested on mtDNA extracted from deer faeces species from the red brocket deer M. americana (Duarte & that were collected in the potential distribution range of Jorge 2003). Since then, karyotyping has been proposed as this species. one of the most reliable tools for species identification and distinguishing M. bororo from other sympatric species of Reference sample brocket deer (Duarte & Jorge 1996, 2003; Duarte & Merino 1997; Vogliotti & Duarte in press). However, this method is Blood samples used for marker development and for impractical, since it requires the capture of individuals screening were collected over a 14-year period (1990-2004) to collect fresh and adequate amounts of blood samples to and were stored at the cell bank of the Deer Research and conduct the cytogenetic technical procedures. This secretive Conservation Center at the Universidade Estadual Paulista. species is extremely difficult to capture and sampling Sample information regarding morphology, cytogenetics, procedures can be highly stressful to the animals. Efforts biochemical genetics and geographical location was taken to locate and capture animals can also be expensive and into account in the selection process. We attempted to time-consuming. For instance, it took 2 years of intensive maximize the use of samples that could only potentially fieldwork to locate and capture the first wild M. bororo occur in the same areas with M. bororo. A sample of 49 (Vogliotti & Duarte in press). This first record prompted the animals with known species designations that inhabit urgent need to explore alternative methods such as non- the potential areas of sympatry was selected. The sample invasive sampling in combination with molecular genetic consisted of seven M. americana, 27 M. gouazoubira, eight methods that would allow faster and more efficient strategies M. nana and seven M. bororo. for detection and determination of the geographical range of this very rare and elusive species. Extraction, amplification and mtDNA sequencing Molecular markers provide powerful tools with many potential applications for conservation biology and wildlife DNA extraction from tissue samples followed procedures management (Kohn et al. 1999). For example, noninvasive modified from Medrano et al. (1990). PCR amplifications faecal DNA sampling has been extremely useful in other of a 480-bp fragment of the 3' end of the cyt b gene were numerous studies for identifying species and individuals performed with primers L14724 and H15149 following in an area, evaluating distribution, sex ratio and estimating