SHORT COMMUNICATIONS Genetic evidence confirms presence of Andean bears in Argentina Mariana Cosse1,4, J. Fernando Del Moral population numbers (Goldstein et al. 2008). This Sachetti2,3, Natalia Mannise1,andMiguelAcosta2 bear is endemic to the tropical Andes and is adapted to most biomes present in this subregion (Cavelier et 1Gene´tica de la Conservacio´n-Departamento de Biodi- al. 2011). It inhabits areas of Venezuela, Colombia, versidad y Gene´tica, Instituto de Investigaciones Biolo´- Ecuador, Peru, and Bolivia (Yerena and Torres gicas Clemente Estable, Av. Italia 3318 C. P. 11600, 1994, Peyton et al. 1998, Yerena 1998, Figueroa and Montevideo, Uruguay Stucchi 2009), but the extent of its southern range is 2 a Proyecto Juco, Eduardo Wilde Nu 450, V Soledad, controversial (Garcı´a-Rangel 2012). The lack of Dpto. A, C. P. 4400, Salta, Argentina conclusive data on its southern range makes it 3 Fundacio´n Andı´gena, Me´rida, Venezuela difficult to develop realistic and accurate manage- ment plans for the species’ conservation, or to Abstract: The Andean bear (Tremarctos ornatus)is monitor changes in populations or their distribution found throughout the Andes Mountains from (Goldstein et al. 2006). The long-debated presence of Venezuela to Bolivia. However, little is known about this species in northwestern Argentina provides a its distribution and range in southern areas, includ- good example of how lack of conclusive data ing Argentina. Our objective was to develop a compromises our understanding of its distribution genetic marker to identify this species by analysis and range (Garcı´a-Rangel 2012). of non-invasive samples (i.e., hair or feces). We Residents of Bolivia near the Argentina border designed a primer pair to amplify a 115–base-pair have reported seeing Andean bear, which has led to fragment within cytochrome b of mitochondrial several studies suggesting that its presence in DNA. We successfully amplified the expected northern Argentina is likely (Mares et al. 1989, fragment in samples from Argentina (collected Ojeda and Mares 1989, Dı´az et al. 2000). For during 2 periods [1993 and between 2006 and example, Brown and Rumiz (1989) found signs of 2008]) having sequences highly similar to Andean Andean bear activities close to Baritu´National Park bear reference sequences in GenBank; and we (Argentina). More recently, Del Moral and Bracho identified 2 haplotypes in samples from northwest (2009) and Del Moral and Lameda Camacaro (2011) Argentina. We confirmed the presence of Andean provided similar indirect evidence of presence of the bears in Argentina, which extends the known species in Argentina, including descriptions of southern distributional limit about 150 km. Further tracks, food remains, and scat. These records, studies employing our approach will allow for a however, were not definitive, suggesting need for comprehensive assessment of the potential distribu- additional supportive data. tion and southernmost range of this species. Rumiz et al. (2012) further suggested that the evidence for the Andean bear’s presence in Argentina is Key words: Andean bear, Argentina, cytochrome b, inconclusive. These authors reviewed diverse publica- geographic distribution, mitochondrial DNA, tions with data from .800 survey sites in the same non-invasive sampling, Tremarctos ornatus, Yun- region, which used camera traps to monitor species gas Forest including felids, primates, and tapir, as well as species DOI: 10.2192/URSUS-D-14-00020.1 richness. The extensive surveying done in these studies, Ursus 25(2):163–171 (2014) however, did not show evidence of Andean bear presence (Rumiz et al. 2012). These authors suggested use of non-invasive DNA analysis to confirm species presence in surveyed areas. Non-invasive genetic The Andean bear (Tremarctos ornatus) is the only sampling (i.e., hair and feces samples) can provide extant species within subfamily Tremarctinae. It has conclusive evidence on species presence and may been categorized as vulnerable by the International provide important information for conservation of Union for Conservation of Nature Red List, and is rare and elusive species (Beja-Pereira et al. 2009). threatened with extinction because of decreasing DNA retrieved from non-invasive samples is often altered and degraded in a way similar to ancient 4email: [email protected] DNA, making its analysis more complex than with 163 164 SHORT COMMUNICATIONS Fig. 1. Andean bear sampling area in Argentina. The sites from which haplotype 1 (triangle) and haplotype 2 (square) samples were taken are indicated in the map. Signs of bear activity described in the Del Moral and Bracho (2009) and Del Moral and Lameda Camacaro (2011) are represented as stars. Gray areas show a section of the geographic range of the Andean bear in Bolivia (Goldstein et al. 2008). The rectangle on the inset highlights location of the sampling area in South America. DNA from tissue samples (Page`s et al. 2009). These of mtDNA, allowing for amplification of short samples, however, contain both nuclear and mito- fragments using universal vertebrate primers. There chondrial DNA (mtDNA), the latter of which is are also regions with interspecies sequence diversity, present at a higher copy number per cell (Waits but with little or no intraspecific variation such as that and Paetkau 2005). Consequently, success rates in in cytochrome b. Furthermore, cytochrome b sequenc- detecting sequences in mtDNA are greater than in es are readily available in GenBank for comparison studies analyzing nuclear sequences. In addition, and taxonomic identification of DNA samples analysis using non-invasive sampling methods has (Wayne et al. 1997, Farrell et al. 2000, Irwin 2002, been refined for analysis of short mtDNA fragments Janecˇka et al. 2008, Teletchea et al. 2008, Chaves et al. (200 base-pairs [bp] or shorter), further improving 2012, Rodgers and Janecˇka 2013). success (Teletchea et al. 2008). The cytochrome b We developed a cytochrome b–based assay to gene in the mitochondrial genome is a useful marker determine the taxonomic identity of environmental for vertebrate species identification, particularly in DNA present in feces and hair thought to be of the degraded samples (Teletchea et al. 2008, Chaves Andean bear. Our goal was to determine whether the et al. 2012). Conserved sequences are found in regions Andean bear was present in northwestern Argentina. Ursus 25(2):163–171 (2014) SHORT COMMUNICATIONS 165 Fig. 2. Andean bear sample collection, which occurred during 1993, and between 2006 and 2008, in Argentina. Hairs (A, B) and feces collected using sterile clamps (C). Study area range (Figueroa and Stucchi 2009, Castellanos We conducted the study in the Yungas Forest 2011). We collected samples that appeared to be ecosystem (Cabrera and Willink 1980), which is part Andean bear feces or hair (Fig. 2) and stored them in of the tropical Andes ecoregion. We selected areas by 15-mL or 50-mL tubes containing 10-g or 20-g silica taking into account their protection status, reported gel beads, respectively (Nsubuga et al. 2004). Nine of sightings of Andean bears by local people, and evidence the samples collected were selected for analysis based of bear activities from previous reports. The areas were on collection date, sample quantity, and avoiding within the Yungas Biosphere Reserve (13,287 km2; samples that appeared degraded. Fig. 1). One sampling area was in the Santa Maria We developed a method for Andean bear species mountains (23u209–23u219S, 64u419–64u439W), a 126- identification using DNA isolated from non-invasive km2 area at 700–1,000-m elevation; and the second area samples. We designed polymerase chain reaction (PCR) sampled was Ramada Barrosa (23u459–23u479S, 65u079– primers to specifically amplify cytochrome b sequences 65u109W), a 50-km2 area at 900–1,910-m elevation. in T. ornatus mtDNA using sequence data from Annual rainfall ranges from 1,000 to 1,800 mm, with GenBank (Zhang and Ryder 1993, Talbot and Shields 95% occurring in summer (Bianchi and Yan˜ez 1992). 1996, Yu et al. 2007, Krause et al. 2008, Benson et al. 2010). We designed a primer pair using Primer3Plus (Untergasser et al. 2007) to amplify a fragment within Methods the cytochrome b gene (TOCYTB FW 59-GACCTCC- We collected feces and hair samples during 2 CAACACCATCAAA-39 and TOCYTB RV 59-CTC- periods using different sampling methods and GACAGATATGGGCGACT-39). The predicted am- storage conditions. In 1993, 3 samples (2 fecal and plicon size was 155 bp, and because primers span 40 bp, 1 hair sample) were collected by Dr. A. Canedi. informative sequences are the central 115 bp. These samples were stored in the Estacio´n de Fauna We isolated DNA from fecal (20–30 mg) and hair Silvestre Martı´n Vucetich collection at Universidad (7–30 hair follicles) samples using the DNeasy Tissue Nacional de Jujuy (A. Canedi, Estacio´n de Fauna Extraction Kit (Qiagen, Hilden, Germany) or ZR Silvestre, Universidad Nacional de Jujuy, personal Soil Microbe DNA MicroPrepTM (Zymo Research, communication). Remaining samples were collected Orange, California, USA) following manufacturer’s by our team between 2006 and 2008 using systematic instructions. We conducted PCRs in 20 mL with 1x surveys of the Yungas Biosphere Reserve, an area of Immomix PCR Buffer (Bioline Reagents, London, approximately 180 km2. We surveyed about 100 km England, United Kingdom), 0.5 pM of each primer, with transects of 5–20 km. We searched for bear scat and 80–100 ng DNA extract. We used thermal and hair on foot or by horseback, and occasionally cycling with an initial denaturation at 95uC for used tracking dogs. We established transects along 10 minutes, followed by 40 cycles of 30 seconds mountain peaks, because Andean bear activity trails at 95uC, 1 minute at 56uC and 1 minute at 72uC, are commonly found in these areas elsewhere in its and a final extension for 15 minutes at 72uC. We Ursus 25(2):163–171 (2014) ornatus represented a new2008) by haplotype BLAST (H2) analysis.Zhang Sample assigned and FM Ryder 7 to 1993, from Yuidentity Salta et with al.