Genetic identification of endangered North African ungulates using noninvasive sampling TERESA LU  ISA SILVA,*†‡ RAQUEL GODINHO,*† DIANA CASTRO,* TERESA AB  AIGAR,‡ JOS  E CARLOS BRITO*† and PAULO C  ELIO ALVES*† § *CIBIO/InBIO, Centro de Investigac ß~ ao em Biodiversidade e Recursos Gen eticos, Universidade do Porto, Campus de Vair~ ao, Vair~ ao 4485-661, Portugal, †Departamento de Biologia, Faculdade de Ci ^ encias, Universidade do Porto, Rua do Campo Alegre s/n, Porto 4169-007, Portugal, ‡Estaci on Experimental de Zonas  Aridas (EEZA), CSIC, Carretera de Sacramento s/n, Almer  ıa, Spain, §Wildlife Biology Program, University of Montana, Missoula, MT 59812, USA Abstract North African ungulates include several threatened and emblematic species, yet are poorly studied mainly due to their remoteness and elusiveness. Noninvasive sampling provides a useful approach to obtain ecological and genetic information essential to guide conservation actions. The very first and most important step in conservation planning is to accurately identify species, and molecular genetics has been proved to be a useful tool. Several molecular genet- ics protocols are available for species identification, even for samples with poor quality DNA, such as faeces, hairs or bones. Most of these protocols use mitochondrial DNA for barcoding despite this marker being especially prone to problems, including mtDNA introgression, nuclear insert copies, high intraspecific diversity or heteroplasmy. In this work, we developed a molecular method based on polymorphisms in small fragments of the mitochondrial cyto- chrome b (cytb, mtDNA) and the nuclear kappa casein genes (KCAS, nDNA) for identifying endangered North African ungulates. These fragments revealed polymorphisms, including species-specific variation, which allowed species identification of nine ungulate species that co-occur in North Africa. The method was validated across more than 400 samples, including different types of noninvasive samples collected in the field. The simplicity, high reli- ability and relative low cost of the described method make it a promising tool to improve ecological studies of the North African ungulates and consequently, the implementation of more efficient management and conservation plans for these endangered ungulates. Keywords: conservation genetics, cytochrome b, deserts, gazelles, kappa casein, molecular method, species ID Received 8 March 2014; revision received 19 September 2014; accepted 22 September 2014 Introduction Accurate species identification is an essential tool in many disciplines, including conservation biology, ecol- ogy and forensics sciences (Long et al. 2008; Steele & Pires 2011). For elusive, rare and/or endangered species the use of noninvasive genetic sampling is particularly relevant, as it allows important data collection from the simple use of hairs, faeces, urine, skulls, egg shells, feath- ers, among others, avoiding the capture, handling or even the observation of individuals (Beja-Pereira et al. 2009; Lampa et al. 2013). The molecular identification of species through the use of noninvasive sampling is rela- tively recent (Waits & Paetkau 2005) and has contributed in the last decade to the study of elusive or rare species (Van Vliet et al. 2008; Beja-Pereira et al. 2009; Oliveira et al. 2009; Chaves et al. 2012; Godinho et al. 2012; Barbosa et al. 2013; Monterroso et al. 2013), mainly due to the powerful technical advances in areas such as DNA extraction, sequencing, microsatellite analysis and SNPs development, which expanded the use of molecular methods in conservation (DeSalle & Amato 2004; Beja- Pereira et al. 2009). In the last decade, the concept of DNA barcoding has profoundly increased the molecular identification of spe- cies (Floyd et al. 2002; Hebert et al. 2004) and barcoding initiatives highly accelerated biodiversity assessments (Smith et al. 2005; Hajibabaei et al. 2007). The idea behind the barcoding concept was to select a universally recog- nized gene for identification of most, if not all, organisms on Earth. This gene, or few genes, would show high interspecific but low intraspecific levels of variation thus establishing a barcoding gap (Hebert et al. 2003). The Correspondence: Teresa Lu  ısa Silva, Fax: 351 252661780; E-mail: tl_silva@cibio.up.pt © 2014 John Wiley & Sons Ltd Molecular Ecology Resources (2014) doi: 10.1111/1755-0998.12335