Genetic Evidence Confirms Presence of Andean Bears in Argentina

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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 Conservacioń-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 (Garcıá-Rangel 2012). The lack of Dpto. A, C. P. 4400, Salta, Argentina conclusive data on its southern range makes it 3 Fundacioń Andı´gena, Me´rida, Venezuela difficult to develop realistic and accurate management 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 (Garcıá-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, Dıáz 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 BarituŃational 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

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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.

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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 Estacioń de Fauna We isolated DNA from fecal (20–30 mg) and hair Silvestre Martıń Vucetich collection at Universidad (7–30 hair follicles) samples using the DNeasy Tissue Nacional de Jujuy (A. Canedi, Estacioń 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) 166 SHORT COMMUNICATIONS conducted 2 replicates for each PCR, and all experi- ments included negative controls. We compared sequences with those in GenBank using BLAST (Basic Local Alignment Search Tool) search algorithm (Altschul et al. 1990). Sequences

having .80% similarity with our cytochrome b Reference sequences were aligned and compared using Clustal accession no. Detailed are sample sequence GenBank

W in Mega 5.10 (Tamura et al. 2011); alignments for (A260/280). Reference gi|195182420|FM177764.1 gi|195182420|FM177764.1 gi|195182420|FM177764.1 gi|195182420|FM177764.1 gi|195182420|FM177764.1 all haplotypes were unambiguous. We performed new haplotype phylogenetic reconstruction using maximum likelihood in MEGA 5.10 (Tamura et al. 2011). For the ) with maximum-likelihood tree, we conducted bootstrap % analysis with 10,000 replicates as a measure of GenBank 96 100 100 100 100 support for the clades obtained. We treated gaps as 100 missing data using the method of complete deletion. We used a maximum-likelihood search level of 1, the reference sequence T. ornatus

Nearest-Neighbor Interchange on random trees as the BLAST identities ( tree search method, and the automatic initial tree with very strong branch swap filter (Tamura et al. 2011). We developed the tree using a condensed tree with a topology cut-off value for consensus equal to 50%.

Results Six (2 fecal and 4 hair samples) of 9 samples analyzed were successfully amplified (Table 1). The sampling sites were about 150 km south of the reported range for the species (Goldstein et al. 2008; Fig. 1). Two haplotypes for Andean bear cytochrome type ng/uL A260/280 Haplotype feces 32.2 0.91 H1 hairs 164.9 2.23 H1 hairs 86.9 2.51 H1 hairs 208.7 1.75 H1 b sequences were identified (Table 1). Five samples hairs 51.1 1.86 H2 Sample and Basic Local Alignment Search Tool (BLAST) search results. (ANI30-3, ANI31-3, ANI32, FM1, FM3) had 100% DNA indicated as the ratio of the absorbance at 260 and 280 nm 0 0 0 0 identity with T. ornatus sequences haplotype 1 (H1; 0 08 15 22 04 09 - feces 52.8 1.93 H1 9 9 9 9 9 Zhang and Ryder 1993, Yu et al. 2007, Krause et al. 9 43 41 10 41 40 10 u u u u u west 2008) by BLAST analysis. Sample FM 7 from Salta u : GI195182420 (Krause et al. 2008). 64 64 65 64 64 represented a new haplotype (H2) assigned to T. Longitude 0 0 0 0 ornatus with 96% similarity corresponding to 4 0 45 41 08 36 38 -65 9 9 9 9 9 unique changes, all transitions (Appendix 1). The 9 21 20 45 20 20 45 u u u u u occurrence of this haplotype was confirmed with 2 u south replicates in different PCR assays. Highest sequence Latitude similarity in non-Ursidae mammalian species in fragments amplified were those of white-lipped peccary (Tayassu pecari), with 88% Andean bear Tremarctos ornatus sequence identity. Phylogenetic analysis showed that haplotypes obtained from Jujuy and Salta’s samples constitute a clade with other Ursidae species, and the node had strong bootstrap support (85%; Fig. 3).

Discussion Whereas previous reports described Andean bear type, nanograms per microliter DNAsequences (ng/uL), from and purity GenBank of Table 1. Andean bear genetic samples used in this study ID Sample date Province ANI30 Jan 2006 SALTA 23 ANI31 Sep 2007 SALTA 23 ANI32 Jul 2008 JUJUY 23 FM1 Aug 2008 SALTA 23 FM3 Jun 1993 JUJUY 23 sign and reports from local people (which are subject FM7 Aug 2008 SALTA 23

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Fig. 3. Molecular Phylogenetic Analysis of Andean bear genetic samples (collected during 1993, and between 2006 and 2008, in Argentina) by maximum likelihood. The phylogenetic tree was constructed using aligned sequences of the 115–base-pair cytochrome b gene fragment from our 6 samples (this work) and 16 reference sequences from GenBank (representing 9 species). Gene Identity numbers correspond to accession numbers in GenBank: T. ornatus—GI: 195182420, GI: 159524410, GI: 121592344, GI: 418050, GI: 1122915 (Zhang and Ryder 1993, Talbot and Shields 1996, Yu et al. 2007, Krause et al. 2008); U. maritimus—GI: 533214488 and GI: 533214492 (Cronin et al. 2013); U. arctos—GI: 882164 and GI: 171853395 (Calvignac et al. 2008); A. simus—GI: 195182389 (Krause et al. 2008); U. americanus—GI: 397174839 (Miller et al. 2012); T. pecari—GI: 1655920 (Theimer and Keim 1998); P. tajacu—GI: 76781115 (Gongora et al. 2006); C. wagneri—GI: 1655916 (Theimer and Keim 1998); P. concolor—GI: 472834804 and GI: 472834806 (Caragiulo et al. 2013). Bootstrap values are indicated on the tree branches. to misidentification), our results provide compelling establish appropriate conservation units and better scientific documentation that Andean bears occur in understand genetic relationships between Argenti- Argentina and .150 km further south than previ- nean and Bolivian populations. Unfortunately, ously thought. The small cytochrome b fragment information on the origin from the wild of the 5 (115 bp) we described was suitable for identification reference sequences from GenBank included in our of Andean bears, even in samples containing phylogenetic analysis was not reported (Zhang and degraded DNA. Though the phylogenetic analysis Ryder 1993, Talbot and Shields 1996, Yu et al. 2007, highlights the identity of the Andean bear cyto- Krause et al. 2008). chrome b sequence, the tree obtained does not Mitochondrial DNA is inherited by maternal necessarily reflect the evolutionary relationship of lineage, and thus the probability of distinguishing species analyzed (Pamilo and Nei 1988). individuals with this marker is very low, such Phylogeographic analysis of the Andean bear that estimating number of animals sampled using using mtDNA markers could provide useful infor- mtDNA yields conservative estimates (DeYoung mation on the extent of genetic diversity in its and Honeycutt 2005). Analysis of our 6 samples geographic distribution (DeYoung and Honeycutt suggests the presence of 2–4 bears. The 2 samples 2005). Such information could be used to help from the southernmost site were collected 15 years

Ursus 25(2):163–171 (2014) 168 SHORT COMMUNICATIONS apart; therefore, they likely represent at most 2 Jayat et al. 2009, Del Moral and Lameda Camacaro bears. Among the 4 samples from the northern 2011, Rumiz et al. 2012). We recommend collection sites, 2 haplotypes were identified, representing .2 of additional samples to better determine the individuals. presence and distribution of Andean bears in A more accurate measure or estimate of individual Argentina. Meanwhile, the occurrence of this flag- numbers in Argentinean samples can be obtained ship species in Argentina’s Yungas ecoregion under- using microsatellite markers (Selkoe and Toonen lies the need to develop and implement conservation 2006, Da Fontoura-Rodrigues et al. 2008, Viteri and policies to preserve the biodiversity of this region. Waits 2009, Sharma et al. 2013). A number of population genetics studies that have included estimates of T. ornatus population size have been Acknowledgments reported. These studies made use of microsatellite We express our gratitude to Dr. A. Canedi loci originally cloned from domestic dogs (Canis (Argentina) for providing biological samples from lupus familiaris), brown bears (Ursus arctos), and his personal collection and for his invaluable support black bears (U. americanus; Ruiz-Garcia 2003, Ruiz- on the field work in Jujuy Province. We thank Dr. S. Garcia et al. 2005, Viteri and Waits 2009). Develop- Gonza´lez for her support and suggestions. Special ing the use of these markers for extending our study thanks to B. Angell and Dr. P. Gill for assistance will depend on bear non-invasive samples and their with the English style of the manuscript. We also amplification success. appreciate useful comments on an earlier version of The Andean bear moves along an altitudinal the manuscript by Dr. M. Fitz-Earle, Dr. D. gradient that includes several different habitat types, Garshelis, and anonymous referees. following seasonal patterns of food resources (Peyton 1980, Suarez 1988, Cuesta et al. 2003, Paisley and Garshelis 2006, Goldstein et al. 2008). Several anthro- Literature cited pogenic activities that have modified the landscape also ALTSCHUL, S.F., W. GISH,W.MILLER, E.W. MYERS, AND modify effective habitat availability for the bear as well D.J. LIPMAN. 1990. Basic local alignment search tool. as its dispersal behavior (Garcıá-Rangel 2012). The Journal of Molecular Biology 215:403–410. regions of Santa Cruz, Chuquisaca, and parts of Tarija BEJA-PEREIRA, A., R. OLIVEIRA, P.C. ALVES, M.K. in Bolivia and northern Argentina have undergone SCHWARTZ, AND G. LUIKART. 2009. Advancing ecolog- drastic changes in land use, including significant loss of ical understandings through technological transforma- forest (Killeen et al. 2007, Clark et al. 2012). Habitat tions in noninvasive genetics. Molecular Ecology Resources 9:1279–1301. loss or food availability could involve changes in BENSON, D.A., I. KARSCH-MIZRACHI, D.J. LIPMAN,J. geographic distribution of the Andean bear and may OSTELL, AND E.W. SAYERS. 2010. GenBank. Nucleic explain its presence in Argentina. Therefore, the Acids Research 38:D46–D51. presence of bears in northern Argentina would not BIANCHI, A.R., AND C.E. YAN˜ EZ. 1992. Las precipitaciones necessarily be due to population expansion. Conse- en el noroeste argentino. Publicacioń que contiene la quently, it will be important to conduct an intensive informacioń pluviome´trica mensual para 450 locali- sampling effort in potential distribution areas already dades del Noroeste Argentino. Second edition. Instituto identified in the Bolivian Yungas and Chaco Serrano Nacional de Tecnologıá Agropecuaria, Estacioń Exper- forest (Velez-Liendo et al. 2013) and also include imental Agropecuaria, Salta, Argentina. [In Spanish.] potential distribution areas in the same ecoregion BROWN, A., AND D. RUMIZ. 1989. Habitat and distribution system in Argentina. of the spectacled bear (Tremarctos ornatus) in the Our study provides further insights involving non- southern limit of its range. Pages 93–103 in M. invasive sample collection and molecular tools to Rosenthal, editor. First international symposium on the spectacled bear. Lincoln Park Zoological Gardens, detect and study Andean bears (see Garshelis 2006, Chicago, Illinois, USA. McKelvey et al. 2006). We used a short fragment CABRERA, A.L., AND A. WILLINK. 1980. Biogeografıáde within the mtDNA cytochrome b gene to confirm the Ame´rica Latina. Secretaria General de la Organizacioń occurrence of Andean bear in a debated area of its de los Estados Americanos, Washington, DC, USA. [In geographic range in northwest Argentina (Brown Spanish.] and Rumiz 1989, Mares et al. 1989, Ojeda and Mares CALVIGNAC, S., S. HUGHES,C.TOUGARD,J.MICHAUX,M. 1989, Dıáz et al. 2000, Del Moral and Bracho 2009, THEVENOT,M.PHILIPPE,W.HAMDINE, AND C. HANNI.

Ursus 25(2):163–171 (2014) SHORT COMMUNICATIONS 169

2008. Ancient DNA evidence for the loss of a highly DIÁZ, M.M., J.K. BRAUN, M.A. MARES, AND R.M. divergent brown bear clade during historical times. BARQUEZ. 2000. An update of the taxonomy, systemat- Molecular Ecology 17:1962–70. ics, and distribution of the mammals of Salta Province, CARAGIULO,A.,I.DIAS-FREEDMAN,J.A.CLARK,S.RABINO- Argentina. Occasional Papers Sam Noble Oklahoma WITZ, AND G. AMATO. 2013. Mitochondrial DNA sequence Museum of Natural History 10:1–52. variation and phylogeography of Neotropic pumas (Puma FARRELL, L.E., J. FOMAN, AND M.E. SUNQUIST. 2000. concolor). Mitochondrial DNA 25:304–312. Dietary separation of sympatric carnivores identified CASTELLANOS, A. 2011. Andean bear home ranges in the by molecular analysis of scats. Molecular Ecology Intag region, Ecuador. Ursus 22:65–73. 9:1583–1590. CAVELIER, J., D. LIZCANO,E.YERENA, AND C. DOWNER. FIGUEROA, J., AND M. STUCCHI. 2009. El Oso Andino: 2011. The mountain tapir (Tapirus pinchaque) and Alcances sobre su historia natural. Asociacioń para Andean bear (Tremarctos ornatus): Two charismatic, la Investigacioń y Conservacioń de la Biodiversidad- large mammals in South American tropical montane AICB, Lima, Peru. [In Spanish.] cloud forests. Pages 172–181 in L.A. Bruijnzeel, F. GARCIÁ-RANGEL, S. 2012. Andean bear Tremarctos ornatus Scatena, and L.S. Hamilton, editors. Tropical montane natural history and conservation. Mammal Review cloud forests: Science for conservation and management. 42:85–119. Cambridge Univ. Press, Cambridge, England, UK. GARSHELIS, D.L. 2006. On the allure of noninvasive genetic CHAVES, P.B., V.G. GRAEFF, M.B. LION, L.R. OLIVEIRA, sampling: Putting a face to the name. Ursus 17:109–123. AND E. EIZIRIK. 2012. DNA barcoding meets molecular GOLDSTEIN, I., S. PAISLEY,R.WALLACE, J.P. JORGENSON,F. scatology: Short mtDNA sequences for standardized CUESTA, AND A. CASTELLANOS. 2006. Andean bear– species assignment of carnivore noninvasive samples. livestock conflicts: A review. Ursus 17:8–15. Molecular Ecology Resources 12:18–35. ———, X. VELEZ-LIENDO,S.PAISLEY, AND D.L. GARSHELIS CLARK, M.L., T.M. AIDE, AND G. RINER. 2012. Land [IUCN SSC BEAR SPECIALIST GROUP]. 2008. Tremarctos change for all municipalities in Latin America and the ornatus. In International Union for Conservation of Caribbean assessed from 250-m MODIS imagery (2001– Nature. 2013. International Union for Conservation of 2010). Remote Sensing of Environment 126:84–103. Nature red list of threatened species. Version 2013.2. CRONIN, M.A., M.M. MCDONOUGH,H.HUYNH, AND R.J. http://www.iucnredlist.org/details/22066/0. Accessed 8 BAKER. 2013. Genetic relationships of North American Mar 2014. bears (Ursus) inferred from amplified fragment length GONGORA, J., S. MORALES, J.E. BERNAL, AND C. MORAN. polymorphisms and mitochondrial DNA sequences. 2006. Phylogenetic divisions among collared peccaries Canadian Journal of Zoology 91:626–634. (Pecari tajacu) detected using mitochondrial and CUESTA, F., M.F. PERALVO, AND F.T. VAN MANEN. 2003. nuclear sequences. Molecular Phylogenetics and Evo- Andean bear habitat use in the Oyacachi River Basin, lution 41:1–11. Ecuador. Ursus 14:198–209. IRWIN, D.E. 2002. Phylogeographic breaks without geo- DA FONTOURA-RODRIGUES, M.L., C.A.V. LIMA-ROSA,L. graphic barriers to gene flow. Evolution 56:2383–2394. TCHAICKA, F.P. VALDEZ, F.H.G. RODRIGUES, R.C. DE JANECˇ KA, J., R. JACKSON,Z.YUQUANG,L.DIQIANG,B. PAULA, M.P. GOUGH, W.E. JOHNSON, S.L. BONATTO, MUNKHTSOG,V.BUCKLEY-BEASON, AND W. MURPHY. AND E. EIZIRIK. 2008. Cross-amplification and charac- 2008. Population monitoring of snow leopards using terization of 13 tetranucleotide microsatellites in noninvasive collection of scat samples: A pilot study. multiple species of Neotropical canids. Molecular Animal Conservation 11:401–411. Ecology Resources 8:898–900. JAYAT, P., P.E. ORTIZ, AND M.D. MIOTTI. 2009. Mamı´feros DEL MORAL, J.F., AND A.E. BRACHO. 2009. Indicios de la selva pedemontana del noroeste argentino. indirectos de la presencia del oso andino (Tremarctos Pages 273–316 in A.D. Brown, P.G. Blendinger, T. ornatus Cuvier, 1825) en el noroeste de Argentina. Loma´scolo, and P. Garcıá Bes, editors. Selva pede- Revista del Museo Argentino de Ciencias Naturales montana de las Yungas: Historia natural, ecologıáy 11:69–76. [In Spanish.] manejo de un ecosistema en peligro. Ediciones del ———, AND F.I. LAMEDA CAMACARO. 2011. Registros de Subtro´pico, Yerba Buena, Tucumań, Argentina. [In ocurrencia del oso andino (Tremarctos ornatus Cuvier, Spanish.] 1825) en sus lı´mites de distribucioń nororiental y KILLEEN, T.J., V. CALDERON,L.SORIA,B.QUEZADA, M.K. austral. Revista del Museo Argentino de Ciencias STEININGER,G.HARPER, L.A. SOLO´ RZANO, AND C.J. Naturales Nueva Serie 13:7–19. [In Spanish.] TUCKER. 2007. Thirty years of land-cover change in DEYOUNG, R.W., AND R.L. HONEYCUTT. 2005. The Bolivia. AMBIO: A Journal of the Human Environ- molecular toolbox: Genetic techniques in wildlife ment 36:600–606. ecology and management. Journal of Wildlife Manage- KRAUSE, J., T. UNGER,A.NOÇON, A.S. MALASPINAS, S.O. ment 69:1362–1384. KOLOKOTRONIS,M.STILLER,L.SOIBELZON,H.SPRIGGS,

Ursus 25(2):163–171 (2014) 170 SHORT COMMUNICATIONS

P.H. DEAR, A.W. BRIGGS, S.C.E. BRAY, S.J. O’BRIEN, in felid conservation. European Journal of Wildlife G. RABEDER,P.MATHEUS,A.COOPER,M.SLATKIN,S. Research 59:1–16. PA¨ A¨ BO, AND M. HOFREITER. 2008. Mitochondrial RUIZ-GARCIA, M. 2003. Molecular population genetic genomes reveal an explosive radiation of extinct and analysis of the spectacled bear (Tremarctos ornatus)in extant bears near the Miocene–Pliocene boundary. the northern Andean area. Hereditas 138:81–93. BMC Evolutionary Biology 8:220. ———, P. OROZCO-TERWENGEL,A.CASTELLANOS, AND L. MARES,M.A.,R.A.OJEDA, AND R.M. BARQUEZ. 1989. Guide ARIAS. 2005. Microsatellite analysis of the spectacled to the mammals of Salta Province, Argentina. Illustrations bear (Tremarctos ornatus) across its range distribution. by E. Guanuco, P. Caplonch, and N. Giannini. University Genes and Genetics Systems 80:57–70. of Oklahoma Press, Norman, Oklahoma, USA. RUMIZ, D.I., A.D. BROWN, P.G. PEROVIC, S.C. CHALUKIAN, MCKELVEY, K.S., J.V. KIENAST, K.B. AUBRY, G.M. G.A.E. CUYCKENS,P.JAYAT,F.FALKE, AND D. KOEHLER, B.T. MALETZKE, J.R. SQUIRES, E.L. LIND- RAMADORI. 2012. El ucumar (Tremarctos ornatus), mito QUIST,S.LOCH, AND M.K. SCHWARTZ. 2006. DNA y realidad de su presencia en la Argentina. Mastozoo- analysis of hair and scat collected along snow tracks to logı´a Neotropical 19:359–366. document the presence of Canada lynx. Wildlife Society SELKOE, K.A., AND R.J. TOONEN. 2006. Microsatellites for Bulletin 34:451–455. ecologists: A practical guide to using and evaluating MILLER,W.,S.C.SCHUSTER,A.J.WELCH,A.RATAN,O.C. microsatellite markers. Ecology Letters 9:615–629. BEDOYA-REINA,F.ZHAO,H.L.KIM,R.C.BURHANS,D.I. SHARMA, S., T. DUTTA, J.E. MALDONADO, T.C. WOOD, H.S. DRAUTZ,N.E.WITTEKINDT,L.P.TOMSHO,E.IBARRA- PANWAR, AND J. SEIDENSTICKER. 2013. Spatial genetic LACLETTE,L.HERRERA-ESTRELLA,E.PEACOCK,S.FARLEY, analysis reveals high connectivity of tiger (Panthera G.K. SAGE,K.RODE,M.OBBARD,R.MONTIEL,L. tigris) populations in the Satpura–Maikal landscape of BACHMANN,O´ .INGO´ LFSSON,J.AARS,T.MAILUND,Ø.WIIG, Central India. Ecology and Evolution 3:48–60. S.L. TALBOT, AND C. LINDQVIST. 2012. Polar and brown SUAREZ, L. 1988. Seasonal distribution and food habits of bear genomes reveal ancient admixture and demographic spectacled bears Tremarctos ornatus in the highlands of footprints of past climate change. Proceedings of the Ecuador. Studies on Neotropical Fauna and Environ- National Academy of Sciences 109:E2382–E2390. ment 23:133–136. NSUBUGA, A., M. ROBBINS,A.ROEDER,P.MORIN,C. TALBOT, S.L., AND G.F. SHIELDS. 1996. A phylogeny of the BOESCH, AND L. VIGILANT. 2004. Factors affecting the bears (Ursidae) inferred from complete sequences of amount of genomic DNA extracted from ape faeces three mitochondrial genes. Molecular Phylogenetics and the identification of an improved sample storage and Evolution 5:567–575. method. Molecular Ecology 13:2089–2094. TAMURA, K., D. PETERSON,N.PETERSON,G.STECHER,M. OJEDA, R.A., AND M.A. MARES. 1989. A biogeographic NEI, AND S. KUMAR. 2011. MEGA5: Molecular analysis of the mammals of Salta Province, Argentina: evolutionary genetics analysis using maximum likeli- Patterns of species assemblage in the Neotropics. Texas hood, evolutionary distance, and maximum parsimony Tech University Press, Lubbock, Texas, USA. methods. Molecular Biology and Evolution 28:2731– PAGE` S, M., C. MAUDET,E.BELLEMAIN,P.TABERLET,S. 2739. HUGHES, AND C. HA¨ NNI. 2009. A system for sex TELETCHEA, F., J. BERNILLON,M.DUFFRAISSE,V.LAUDET, determination from degraded DNA: A useful tool for AND C. HA¨ NNI. 2008. Molecular identification of palaeogenetics and conservation genetics of ursids. vertebrate species by oligonucleotide microarray in Conservation Genetics 10:897–907. food and forensic samples. Journal of Applied Ecology PAISLEY, S., AND D. GARSHELIS. 2006. Activity patterns and 45:967–975. time budgets of Andean bears (Tremarctos ornatus)in THEIMER, T.C., AND P. KEIM. 1998. Phylogenetic relation- the Apolobamba Range of Bolivia. Journal of Zoology ships of peccaries based on mitochondrial cytochrome b 268:25–34. DNA sequences. Journal of Mammalogy 79:566–572. PAMILO, P., AND M. NEI. 1988. Relationships between gene UNTERGASSER, A., H. NIJVEEN,X.RAO,T.BISSELING,R. trees and species trees. Molecular Biology and Evolu- GEURTS, AND J.A. LEUNISSEN. 2007. Primer3Plus, an tion 5:568–583. enhanced web interface to Primer3. Nucleic Acids PEYTON, B. 1980. Ecology, distribution and food habits of Research 35:W71–W74. spectacled bears (Tremarctos ornatus) in Peru. Journal VELEZ-LIENDO, X., D. STRUBBE, AND E. MATTHYSEN. 2013. of Mammalogy 61:639–652. Effects of variable selection on modelling habitat and ———, E. YERENA, D.I. RUMIZ,J.JORGENSON, AND J. potential distribution of the Andean bear in Bolivia. OREJUELA. 1998. Status of wild Andean bears and Ursus 24:127–138. policies for their management. Ursus 10:87–100. VITERI, M.P., AND L.P. WAITS. 2009. Identifying polymor- RODGERS, T.W., AND J.E. JANECˇ KA. 2013. Applications and phic microsatellite loci for Andean bear research. Ursus techniques for non-invasive faecal genetics research 20:102–108.

Ursus 25(2):163–171 (2014) SHORT COMMUNICATIONS 171

WAITS, L.P., AND D. PAETKAU. 2005. Noninvasive genetic mammalian family that experienced rapid speciation. sampling tools for wildlife biologist: A review of BMC Evolutionary Biology 7:198. applications and recommendations for accurate data ZHANG, Y.P., AND O.A. RYDER. 1993. Mitochondrial DNA collection. Journal of Wildlife Management 69:1419– sequence evolution in the Arctoidea. Proceedings of the 1433. National Academy of Sciences 90:9557–9561. WAYNE, R.K., E. GEFFEN, D.J. GIRMAN, K.P. KOEPFLI, Received: 21 April 2014 L.M. LAU, AND C.R. MARSHALL. 1997. Molecular Accepted: 23 September 2014 systematics of the Canidae. Systematic Zoology Associate Editor: M. Fitz-Earle 46:622–653. YERENA, E. 1998. Protected areas for the Andean bear in South America. Ursus 10:101–106. Supplemental Material ———, AND D. TORRES. 1994. Spectacled bear conservation and dispersal corridors in Venezuela. Bears: Their Appendix 1. Andean bear (Tremarctos ornatus) Biology and Management 9:169–172. sequences obtained from Argentinean samples. The YU, L., Y.W. LI, O.A. RYDER, AND Y.P. ZHANG. 2007. region shown corresponds to the 115-bp cytochrome b Analysis of complete mitochondrial genome sequences fragment. The transition changes are indicated with increases phylogenetic resolution of bears (Ursidae), a gray background.

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