Conserv Genet DOI 10.1007/s10592-009-9947-4 RESEARCH ARTICLE Species identification using a small nuclear gene fragment: application to sympatric wild carnivores from South-western Europe R. Oliveira Æ D. Castro Æ R. Godinho Æ G. Luikart Æ P. C. Alves Received: 29 October 2008 / Accepted: 25 May 2009 Ó Springer Science+Business Media B.V. 2009 Abstract Species identification is essential for non- field. Due to its low cost, simplicity, and wide range of invasive studies of elusive and rare animals, and for identifiable species, this test shows great promise to facil- detecting illegal harvest or trade of wildlife species. itate studies in molecular ecology, conservation genetics, However, most molecular tests identify only a limited and forensic analysis, as well as DNA bar-coding projects. number of species or require multiple laboratory steps to distinguish many taxa. Additionally, most protocols use Keywords Species ID Á Nuclear gene Á IRBP Á mitochondrial DNA being, therefore, especially prone to SSCP Á Carnivores Á Conservation genetics Á problems such as nuclear insert copies, high intraspecific Non-invasive genetics diversity or heteroplasmy. Here, we developed a molecular test based on the polymorphisms detected on a small nuclear gene fragment (221 bp of the IRBP -Interphoto- Introduction receptor Retinoid-Binding Protein- exon 1). This fragment revealed 51 variable sites (including 12 non-synonymous Reliable identification of species is fundamental in and 19 species-specific sites), which enabled the successful molecular ecology, conservation biology, forensic sciences distinction of all 16 carnivore species native to South- and wildlife management because many studies in these western Europe. A SSCP (Single-Strand Conformational disciplines crucially depend on species identification for a Polymorphism) gel electrophoresis technique was also wide range of applications, such as the definition of geo- optimized to allow the simple and inexpensive application graphic distributions, estimation of densities, and the of this molecular test. Sequences and SSCP profiles were analysis of biological and behavioural parameters (Long consistent in identifying a total of 387 samples, including et al. 2008). However, detection and identification of spe- faeces (172) and hairs (17) collected non-invasively in the cies is difficult for rare and threatened taxa, mainly due to their low densities, nocturnal and elusive behaviour, and to the logistical and ethical difficulties involved in their R. Oliveira Á D. Castro Á R. Godinho Á G. Luikart Á capture and handling during traditional capture-mark- & P. C. Alves ( ) recapture approaches. This has prompted the development CIBIO, Centro de Investigac¸a˜o em Biodiversidade e Recursos Gene´ticos, Universidade do Porto, Campus Agra´rio de Vaira˜o, of non-invasive tools applied to samples such as faeces, 4485-661 Vairao, Portugal hair, feathers, urine, saliva, scent marks, sloughed skin and e-mail: [email protected] animal products illegally trafficked (e.g., Taberlet et al. 1999; Dale`n et al. 2004, Arrendal et al. 2007; Hogan et al. R. Oliveira Á D. Castro Á P. C. Alves Departamento de Zoologia e Antropologia, Faculdade de 2008; Jones et al. 2008; Sundqvist et al. 2008; Sastre et al. Cieˆncias da Universidade do Porto, Rua Campo Alegre s/n, 2009). However, non-invasive samples seldom allow spe- 4169-007 Porto, Portugal cies identification based on morphology alone (Davison et al. 2002). When sympatric carnivores have similar body G. Luikart Division of Biological Sciences, University of Montana, features, behaviour and feeding habits, visual discrimina- Missoula, MT, USA tion of scats and hairs are subjective and error-prone 123 Conserv Genet (Riddle et al. 2003;Go´mez-Moliner et al. 2004; Kurose In South-western Europe, 16 wild carnivore species are et al. 2005; Nagata et al. 2005; Pilot et al. 2007). There- sympatrically distributed, including animals from six dif- fore, non-invasive studies of carnivore species should ferent families: Mustelidae (weasel Mustela nivalis, wes- include genetic identification. tern polecat Mustela putorius, European mink Mustela During the last decade, a number of molecular protocols lutreola, American mink Mustela vison, stoat Mustela have been designed to identify mammal carnivores. Direct erminea, stone marten Martes foina, pine marten Martes sequencing of a diagnostic DNA fragment (e.g., Farrell martes, European otter Lutra lutra, Eurasian badger Meles et al. 2000; Murakami 2002; Adams and Waits 2007; meles); Canidae (wolf Canis lupus, red fox Vulpes vulpes); Karlsson and Holmlund 2007; Kitano et al. 2007) is still Felidae (European wildcat Felis silvestris, Iberian lynx relatively expensive for large-scale wildlife surveys, and Lynx pardinus); Viverridae (common genet Genetta gen- DNA mixtures (e.g., contamination with prey DNA) are etta); Ursidae (brown bear Ursus arctos) and Herpestidae complicated to decipher. Therefore, alternative protocols (Egyptian mongoose Herpestes ichneumon). For most of have been developed, like mtDNA RFLP (Restriction these species, especially the ones with high conservation Fragment Length Polymorphism) assays (e.g., Hoss et al. status such as the Iberian lynx, the European wildcat, the 1992; Paxinos et al. 1997; Hansen and Jacobsen 1999; wolf and the European mink, it is urgent to clarify their Mills et al. 2000; Williams et al. 2003; Lucentini et al. precise distribution, to elucidate behavioural, ecological 2007; Ruiz-Gonza´lez et al. 2008) and tests based on posi- and genetic features, and to evaluate biological relation- tive versus negative PCR amplifications using species- ships among populations and subspecies. Here we present a specific primers (e.g., Palomares et al. 2002; Dale`n et al. simple, rapid and inexpensive technique to identify all 2004; Kurose et al. 2005; Fernandes et al. 2007; Tobe and carnivore species in South-western Europe based on the Linacre 2008). Most of the protocols target few taxa, fre- high discriminatory power of a short fragment of the quently endangered species and sympatric ones, or species nuclear gene Interphotoreceptor Retinoid-Binding Protein from which non-invasive signs are usually similar. In this (IRBP), which can also be successfully applied to the context, Fernandes et al. (2007) reported the broadest identification of non-invasive and forensic samples. Based DNA-based method specifically designed for carnivore on its simplicity, high efficiency and reliability, and low discrimination, using species-specific mtDNA primers to cost, we further discuss its usefulness for carnivore man- identify 15 species in the Iberian Peninsula. More recently, agement and biodiversity conservation. real-time PCR and melt-curve analysis protocols have also been developed to distinguish a few carnivore species (Berry and Sarre 2007; O’Reilly et al. 2008). Materials and methods Despite the recent advances in developing new molec- ular assays, important limitations still exist. For instance, This study was divided in three main steps, including the although real-time PCR presents relatively low cost and is development, laboratory testing and practical implemen- less time-consuming when compared with methods tation of a novel DNA-based technique. requiring post-PCR manipulations, the protocol developed by Berry and Sarre (2007) showed that intraspecific poly- (a) Developing the assay for species discrimination: morphism or melt temperature overlap may occur between carnivores IRBP sequences analysis closely related species. Additionally, DNA-based methods specifically designed for carnivore species discrimination A fragment of approximately 1,040 bp of the IRBP gene was (e.g., Fernandes et al. 2007) are based solely on mtDNA PCR amplified with carnivore universal primers, following polymorphism, presenting some limitations: mtDNA Flynn and Nedbal (1998). Between two to five individuals evolves rapidly, which may result in intraspecific diversity belonging to each of the 16 species representing all natural too high for species discrimination proposes (e.g., O’Reilly extant carnivores from South-western Europe, plus the dog et al. 2008); heteroplasmy, which has been documented for and the domestic cat (Table 1), were sequenced for both several mammalian mitochondrial genomes (Hsieh et al. strands on a 3130xl Genetic Analyzer Sequencer (Applied 2001; Paneto et al. 2007); nuclear copies (NUMTS), which Biosystems/HITACHI). Sequences were aligned together are known to occur in different species (Zhang and Hewitt with previously published ones using the software SEQ- 1996; Lopez et al. 1996; Kim et al. 2006; Antunes et al. SCAPE 2.0 (Applied Biosystems). All new sequences were 2007); and mtDNA introgression which also is common in submitted to NCBI GenBank (accession numbers from mammals (e.g., Ballard and Whitlock 2004; Alves et al. GQ214060 to GQ214077). After analysing the entire frag- 2006). Therefore, new methodologies based on nuclear ment variability in MEGA 3.1 (Kumar et al. 2004), we genes could be more informative or provide extra infor- selected a short fragment of the first exon, which revealed mation to complement mtDNA-based protocols. high interspecific diagnostic polymorphism, thus showing 123 Conserv Genet Table 1 Number of tissue (and Species Samples Geographic location non-invasive) samples genotyped from each carnivore Canis sp. 24 (11) Portugal (14); Spain (7); Romania (3) species and their geographic location. The number of Felis spp. 108 (85) Portugal (85); Spain (9); Italy (5); Germany (5); samples from