Species Identification Using a Small Nuclear Gene Fragment: Application

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 Nuclear gene IRBP mitochondrial DNA being, therefore, especially prone to SSCP Carnivores Conservation genetics 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 D. Castro R. Godinho G. Luikart capture and handling during traditional capture-mark- & P. C. Alves ( ) recapture approaches. This has prompted the development CIBIO, Centro de Investigaçaõ em Biodiversidade e Recursos Gene´ticos, Universidade do Porto, Campus Agra´rio de Vairaõ, 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 D. Castro 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 molecular 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 each geographical Bulgaria (2); Romania (2) location is also indicated Genetta genetta 14 Portugal (10); Spain (4) Herpestes ichneumon 10 Portugal (9); Spain (1) Lutra lutra 13 Portugal (4); Austria (9) Lynx pardinus 4 Spain (4) Martes foina 31 (18) Portugal (19); Spain (11); Greece (1) Martes martes 37 (17) Portugal (1); Spain (27); Italy (7); Finland (2) Meles meles 21 (6) Portugal (6); Denmark (2); Spain (11); Italy (2) Mustela erminea 12 Portugal (2); Spain (1); Czech Republic (1); Denmark (2); Great Britain (1); Luxemburg (1); Ireland (1); Italy (2); Norway (1) Mustela lutreola 5 Spain (5) Mustela nivalis 18 Portugal (5); Spain (2); Austria (1); Denmark (1); Italy (6); Finland (3) Mustela putorius 19 Portugal (9); Spain (5); Italy (3); Denmark (2) Mustela vison 8 Portugal (2); Spain (4); Denmark (2) Vulpes vulpes 89 (80) Portugal (29); Spain (60) Ursus arctos 14 (12) Italy (12); Spain (2)

great potential to easily and reliably identify all species. This After optimization of the experimental conditions, the best region was also selected because lower intraspecific varia- discrimination of all SSCP patterns was achieved using a tion is likely to occur in exons, when compared to intronic 12% polyacrylamide gel (59:1 acrylamide: methylbis- regions or other non-coding fragments. acrylamide) ran with 19 TBE buffer at constant voltage of 450 V and temperature of 20°C, during 6h30. Samples (b) Optimizing the routine protocol: sequencing and an were genotyped as follows: 1 ll of amplified product was alternative PCR-SSCP technique mixed with 4 ll of denaturing loading buffer (95% deionized formamide, 10 mm NaOH, 0.01% bromophenol To amplify the small selected fragment, we designed PCR blue and 0.01% xylenecyanol) and, then, denatured for primers targeting conserved regions among all species, 5 min at 95°C. Mixtures were kept on ice until 4 ll were which resulted in a PCR product of 221 bp: the forward loaded on the gel. Visualization was made by silver primer, IRBPex1.Fw (50-GAGAAAGCMCTGGCCATCCT-30), staining (SILVER SEQUENCETM Staining Reagents, and the reverse primer, IRBPex1.Rw (50-ACCAGGAGCC Promega Corporation, USA). TGGGGTCCTC-30) have their 50 positions at nucleotides 940 and 1160, respectively, of the human IRBP gene (c) Validating the PCR-SSCP method for species (accession number X53044). The optimal PCR profile was discrimination: testing robustness and reliability identified by testing a range of hybridization temperatures (55–65°C) using the following conditions: 4 min at 94°C Total DNA from 198 tissue samples belonging to the 16 followed by 30 cycles at 94°C for 30 s, 60°C for 30 s and carnivore species was extracted using a standard salting- 72°C for 30 s, and a final extension at 72°C for 5 min. PCR out procedure (Sambrook et al. 1989). Sampling was dis- amplifications were performed for all the individuals tributed throughout different geographical locations in sequenced in a), in a total volume of 15 ll containing: 19 Europe, in order to span potential genetic variants within

PCR buffer (Ecotaq), 1.8 mM MgCl2, 0.3 lM of each species (Table 1). Additionally, ten Iberian samples from primer, 0.3 mM of each dNTP, 1 ll of DMSO (Dimeth- domestic cats (5) and dogs (5) were used to test the pos- ylsulfoxide), 0.5 U of Taq polymerase (Ecotaq) and sibility of discriminating between them and their wild approximately 10 ng of genomic DNA. In order to opti- counterparts. mize an alternative protocol to the direct sequencing of this To evaluate the applicability of the method in non- fragment, the resulting PCR products were analyzed based invasive studies, faecal DNA of known origin was on SSCP gel electrophoresis using a vertical electropho- extracted for some species (Table 1) using a guanidine resis system apparatus (BIO-RAD ProteanÒ II xiCell). thiocyanate protocol (adapted from Gerloff et al. 1995;

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Godoy, personal communication). Sampling was per- compromising amplification yield was selected to reduce formed in different field sites across Iberian Peninsula, unwanted PCR products and maximize specificity (60°C), under a variety of exposure times and environmental con- although lower temperatures can be used to facilitate ditions (e.g., different temperatures and humidity levels), amplification for poorer quality samples (57–60°C; e.g., given that both fresh (less than 3–4 day old) and medium scats). The analysis of the partial IRBP-exon1 showed a aged scats (maximum estimated age of approximately total of 51 variable sites from which 12 are non-synonymous 1 month) were collected in both Mediterranean and and at least 19 are species-specific, thus revealing poten- Atlantic habitats. Non-invasive samples comprised 212 tially interspecific diagnostic polymorphism (Table 2). scats from Felis spp. (80), Canis sp. (11), Vulpes vulpes A specific and unique SSCP band pattern was obtained (80), Meles meles (6), Martes martes (17) and Martes foina for each of the 16 species in the 198 tissue samples (Fig. 1) (18), from which 143 (67.5%) were fresh and 69 (32.5%) and all individuals from the same species showed consis- were medium aged samples. One individual in captivity tent SSCP patterns, even for samples from distant geo- was non-invasively sampled for each of the above-men- graphic origins. Intra-specific polymorphism was identified tioned species (except for pine marten), in order to addi- for Felis spp., Canis sp., Martes foina, Mustela erminea tionally analyze a fresh and less exposed scat sample as and M. nivalis (accession numbers from GQ214054 to positive control of the non-invasive experiments. DNA GQ214059; Table 2), although this variation never over- from remotely plucked hairs of brown bear (12) and lapped with the patterns observed for other species. This European wildcat (5) was also extracted, using the Qiagen IRBP locus did not distinguish between wolf and dog or QIamp DNA Micro Kit according to manufacturer’s between wild and domestic cats. instructions. Until DNA extraction, both scat and hair We also verified the usefulness of this protocol using samples were stored in absolute ethanol (5 parts of ethanol non-invasive DNA from some of the studied carnivores. per part of sample) at room temperature. Considering only the scats where it was possible to extract In order to reduce possible cross-amplification of prey DNA, we successfully amplified and identified 81,13% items, only the external surface and tip of each scat (where (172/212) (79.72% fresh and 84.06% medium aged scats), intestinal epithelium cells of the predator accumulate) were showing that the method is both reliable and robust when used for DNA extraction. Nevertheless, contamination with applied to samples with low DNA quantity and quality prey and human DNA was monitored by extracting, (Fig. 2). Amplification success proved also not to be spe- amplifying, and SSCP identifying DNA from the lab cies sensitive (data not shown). All of the 17 plucked hair technicians and from a comprehensive array of potential samples from bear and wildcat were successfully identified. preys (Appendix). Laboratory procedures for non-invasive Although PCR amplifications were obtained for some samples were conducted in a separate and autonomous non-target species that may be part of carnivores’ diet in facility, under sterile conditions. A maximum of seven South-western Europe (32.69% of the potential prey spe- samples and one negative control were handled in each cies analysed), their SSCP profiles were easily distin- extraction round. For both invasive and non-invasive guishable from all the carnivore species. For all other samples, pre and post-PCR manipulations were conducted investigated prey species, including several mammals, in physically separated rooms. birds, fishes, amphibians, reptiles and a few invertebrates, All samples were genotyped with the technique described no amplification was detected (Appendix). above in b). However, for non-invasive samples, minor modifications were made to the original PCR-SSCP protocol: two independent PCRs of 40 cycles were performed, the Discussion PCR reaction volume was increased to 25 ll (all reagents proportionally) and 4 ll of DNA extract were used. Positive Development of DNA-based technologies to identify the and negative controls (from both extraction and PCR) were species of origin of unknown samples has been the focus of loaded on each gel in order to compare the non-invasive many conservation, wildlife, and forensic biologists. Yet, results with the expected species-specific conformations and financial and technical limitations still hinder their wide for contamination monitoring, respectively. and frequent use in conservation research and management plans (Broquet et al. 2007). In non-invasive studies, repe- ated PCR analyses and continuous monitoring of data Results quality is essential to ensure accurate data (Taberlet et al. 1996; Beja-Pereira et al. 2009), therefore, it is crucial to Highly robust and specific PCR amplifications of the IRBP- decrease the time and cost involved in each step of data exon 1 fragment were obtained for all target carnivore production. In this context, our relatively inexpensive, easy species. The highest annealing temperature without and quick PCR-SSCP approach can be considerably useful, 123 osr Genet Conserv

Table 2 Interspeciﬁc polymorphic positions of a 221 bp fragment from IRBP exon 1 in wild carnivores from South-western Europe

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2

2 2 2 3 3 3 4 4 4 4 4 5 5 6 6 6 7 7 7 7 7 7 8 8 9 0 0 0 0 2 2 2 3 4 4 4 5 5 5 5 6 6 7 7 8 8 8 8 9 9 0

1 4 5 0 1 3 2 3 5 6 8 1 3 0 5 9 2 3 4 6 8 9 7 8 9 5 6 7 8 5 8 9 2 1 4 7 3 4 5 9 8 9 1 4 0 3 6 9 5 7 1

European wildcat Felis s. silvestris C T C C C G A G A G T C A G A G G C A A C T C C T C A G C M A C G C A A G G A G C A C A C T C A T T T Iberian lynx Lynx pardinus ...... G . . . A ...... C ......

Egyptian mongoose Herpestes ichneumon ...... A . . G G ...... C . C . . . . G C T . . . . . T ......

Common Genet Genetta genetta ...... G . . A . A G G . . . T . . . C . C . . . . G . T . . . . . T . T . . . . . C

Brown bear Ursus arctos ...... G A A G G . . C . . . G . . G . . . . C . C . . . T G G T C . . . . G G . . . . . C .

European otter Lutra lutra . . T . A . T . . . G G G . G . C . . . . . A . C . G C . C . T . T . G T . . . . G G G . A T G . C C

Pine marten Martes martes . . T . . . C . . . G T G . G . C . . G . C G . C . G C . C . . . T G G T . . . . G G G . A T . . C C

Stone marten Martes foina . . T . . . C . . . G . G . G . C . . G . C G . C . G C . C . . S T G G C . . . . G G G . A T G . C C

Eurasian badger Meles meles . . T . . . C . . . G G G . G . C . . G . . G . C . G C . C . . . T G G C . . . A G G G . A T G . C C

Polecat Mustela putorius . . T T . . T . . . G G G K G C C . . G . . A . . . G C T C . T . T G G T . . A . G G G . A T G . C C

Stoat Mustela erminea . . T . . . T . . . G G G . G C C . . G . . A . . A G C T C . T . T G G T . . . . G G G Y A T G . C .

Weasel Mustela nivalis . . T T . . T . . . G G G . G C C . . G . . A . . Y G C T C . T . T G G T . . . . G G G . A T G . C C

European mink Mustela lutreola . . T T . . T . . . G G G . G T C . . G . . A . . . G C T C . T . T G G T . . . T G G G . A T G . C C

American mink Mustela vison . . T T . . T . . . G G G . G . C . . G . . . T C . G C T C . T . T G G T . . . . G G G . A T G . C C

Wolf Canis lupus G C . . . C C A C A A G . . . C . . . G T . R . C . G C . C G T . . C G C A G . . . . G T . T G C . .

Red fox Vulpes vulpes G C . . . C C . C . G A G . . C . . . G . . G . C . G C . C G . . C G T A G . . . . G T . T G C . C

Position 1 corresponds to position 940 of the Human IRBP exon 1 (accession no. X53044). Boxes represent species-speciﬁc nucleotide variations and shaded positions correspond to non-synonymous variation 123 Conserv Genet

Fig. 1 Separation of variants of Fs Lp Hi Gg Ua Ll Mma Mf Mme Mp Me Mn Ml Mv Cl Vv the IRBP gene (221 bp) in Iberian wild carnivores by a ab b a ab b ab a b SSCP analysis on 12% polyacrylamide gels. Visualization was done by silver staining. Fs, Felis silvestris; Lp, Lynx pardinus; Hi, Herpestes ichneumon; Gg, Genetta genetta; Ua, Ursus arctos; Ll, Lutra lutra; Mma, Martes martes; Mf, Martes foina; Mme, Meles meles; Mp, Mustela putorius; Me, Mustela erminea; Mn, Mustela nivalis; Ml, Mustela lutreola; Mv, Mustela vison; Cl, Canis lupus; Vv, Vulpes vulpes (a, b and ab represent intraspeciﬁc polymorphism found in Felis silvestris, Mustela nivalis and Canis lupus)

Felis silvestris Vulpes vulpes Ursus arctos other potential advantages: (1) all samples under analysis C C C can be sequenced or subjected to a PCR-SSCP without any prior assumptions based on morphologic identifications, which are often applied to non-invasive samples before PCR in order to reduce cost and time in multiple-amplifications approaches; (2) it helps solving the problem of false negatives in species-specific PCRs without the need to co- amplify an additional fragment to control PCR success Fig. 2 SSCP patterns obtained for five European wildcat and red fox (e.g., Zaidi et al. 1999; Mukherjee et al. 2007); (3) while in scats, and five brown bear hairs samples. The first lane for each species-specific assays negative results for the target spe- species corresponds to a positive control (C) cies do not allow the immediate identification of the true predator and further experiments are needed (Palomares especially in studies of carnivores from South-western et al. 2002; Fernandes et al. 2007; O’Reilly et al. 2008), in Europe. With a simple PCR and SSCP gel analysis, a the described assay a single positive PCR-SSCP will minimum of 40 samples can be simultaneously compared, directly give the final species identification and (4) DNA depending on the electrophoresis apparatus used. This mixtures can be easily detected if multiple SSCP patterns methodological approach allows reducing 5 times the costs appear in a single sample, while species-specific protocols relatively to direct sequencing. Thus, for long-term and/or or sequencing will not detect or easily decipher possible large-scale monitoring programs, this method can be both contaminations, respectively. Although the presence of faster and cheaper than sequencing, while much more contaminating DNA does not influence species ID when straightforward than possible RFLP tests that require sev- specific primers are used, further amplifications using other eral steps (e.g., PCR, restriction endonuclease digestion molecular markers might be sensitive to the non-detected etc.) for distinguishing all species. Nevertheless, sequenc- contaminations and result in wrong genotyping. Accord- ing the small (221 bp) IRBP fragment might be preferred ingly, we consider that the capacity of this assay to indi- in lower scale projects or when this kind of electrophoresis viduate target species from prey DNA should be viewed apparatus is not available. also as a tool for screening DNA samples for following Although mitochondrial assays are often thought to be research. When compared to protocols based on mtDNA more efficient than nuclear ones for non-invasive molecu- polymorphism, another advantage of our method, that may lar studies (due to the higher copy number per cell), the significantly reduce the time and cost of carnivore moni- IRBP nuclear fragment showed high amplification rates toring programs, is the fact that it can also be used as a pre- using both faecal (average 81.13%) and plucked hair screening tool to identify samples with amplifiable nuclear (100%) samples, while overcoming limitations inherent to DNA. In particular, the amplification success of this frag- the use of mtDNA. Concomitantly, this method has several ment allows selecting samples with sufficient nuclear DNA 123 Conserv Genet for further genetic studies requiring, for example, the world. Both analyzed under direct sequencing or PCR- amplification of microsatellites or other nuclear SNPs. SSCP assays, this diagnostic tool may help to improve our Species identification using partially degraded DNA, as knowledge on carnivores’ distribution and population sta- the one extracted from non-invasive samples, should rely tus in areas such as the Iberian Peninsula, which represents on the amplification of short DNA targets (Taberlet et al. an important hotspot of biodiversity where many species 1999; Morin et al. 2001; Broquet et al. 2007). Unfortu- are still poorly studied. As an example, it has already been nately, most nuclear genes have low mutation rates successfully applied in the study of Portuguese populations and therefore long fragments (500–1000 bp) are usually of European wildcat and red fox, revealing high efficiency required to detect enough variable sites for several species in solving erroneous identifications based on scats mor- identification. Given the small size of this highly variable phology alone (Castro et al. unpublished data). In that nuclear fragment (\250 bp), analysis of DNA from non- study, a total of 95 non-invasive samples from both species invasive samples, such as faeces or hairs, was notably were submitted to IRBP identification, with an average efficient (81.13% of identification success) and in the same success of 76.52%. level to success rates reported for mtDNA fragments (e.g., Molecular species ID is also an important means to 72%, Fernandes et al. 2007). The amplification was suc- identify species protected by legislation such as the Con- cessful in samples subject to different degradation factors vention for International Trade of Endangered Species in the field (e.g., high vs. low temperature and humidity) (CITES), and for detecting and monitoring poaching, ille- since samples were collected across the entire Iberian gal harvest and trade of protected species. DNA-based Peninsula in distinct Mediterranean and Atlantic land- species identification has helped detect fraudulent trade of scapes. These findings demonstrate the applicability of this several species and illegal wild animal hunting (An et al. molecular method in a range of conditions that are common 2007; for more examples see Allendorf and Luikart 2007, in non-invasive studies. The small size of the fragment also Chap. 20). In this context, the IRBP fragment here ana- made it suitable for SSCP analysis, since the optimal lyzed can be easily applied for controlling and monitoring amplicon size for detection of point mutations is around illegal trade and hunting of the endangered carnivore spe- 200 bp (Orita et al. 1989; Ortı´ et al. 1997). Due to its cies from South-western Europe. Finally, its high level of relatively short length and high variability, this genomic polymorphism and discriminatory power to distinguish portion is also highly promising for routine forensic species from the same family (and also its capacity to applications. differentiate some prey species included in our study, data Among non-invasive samples, faeces are often the most not shown), suggest that IRBP constitute a good candidate widely used but contain the highest concentration of non- gene to further distinguish other species around the world, target DNA, namely from bacteria and prey items (Bradley in order to improve the description of biodiversity within and Vigilant 2002). At the same time, PCR inhibitors are the barcoding framework, which still presents the limita- present in scats in higher concentrations than other sample tion of being based solely on mtDNA (Moritz and Cicero types, e.g., hair samples. In faecal samples, inhibitors may 2004; Rubinoff 2006). also vary according to predator’s diet (Murphy et al. 2003). Nevertheless, our assay did not show variation related to Acknowledgments We thank N. Ferrand and R. Faria for com- species or samples types. No confusion was introduced in ments on earlier versions of this manuscript. We are grateful to Portuguese National Tissues Bank/National Institute for Nature and species identification in cases of prey items contamination, Biodiversity Conservation (BTVS/ICN-B), P. Monterroso, F. Alvares, which suggests that even if prey DNA is co-extracted with B. Gomez-Moliner, C. Gortazar, J. Searle, F. Suchentrunk, E. Randi, the predator DNA, we will still correctly identify the J. Godoy, P. Ferreras, A. Ruiz-Gonza´lez, C. Ferreira, J. Vicente, Z. predator. Boratynski, T. Perez and J. Paupe´rio for providing samples. This work was partially financed by the research project PTDC/CVT/71683/ The SSCP is usually considered a simple molecular 2006, from FCT (Fundaçaõ para a Cieˆncia e a Tecnologia). R. Oli- technique (e.g., Sunnucks et al. (2000). In fact, due to its veira and R. Godinho worked under PhD (SFRH/BD/24361/2005) straightforwardness, high efficiency and low price, this and Post-Doc (SFRH/BPD/36021/2007) grants, respectively, both PCR-SSCP test provides an excellent universal protocol for financed by FCT. D. Castro was supported by a CIBIO grant and G. Luikart was supported by the Portuguese-American Foundation for identifying all sympatric carnivores from South-western Development, CIBIO and UP. Europe, and likely other continental regions around the

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Appendix PCR-SSCP results obtained for the 52 potential carnivore preys analysed in this study Group Common name Species N PCR-SSCP

Mammals Wild boar Sus scrofa scrofa 4 Yes Domestic pig Sus scrofa domesticus 4 Yes Domestic cattle Bos taurus 4No Domestic sheep Ovis aries 5No European roe deer Capreolus capreolus 2 Yesa Wild rabbit Oryctolagus cuniculus 6 Yesa Iberian hare Lepus granatensis 4 Yesa Brown hare Lepus europaeus 4 Yesa Broom hare Lepus castroviejoi 4 Yesa Horse Equus caballus 5 Yes Donkey Equus asinus 2 Yes Wild goat Capra pyrenaica 2No Domestic goat Capra hircus 2No Mouse Mus sp. 2 Yes Wood mouse Apodemus sylvaticus 2 Yes Greater white-toothed shrew Crocidura russula 2 Yes Crowned shrew Sorex coronatus 1 Yes Pygmy shrew Sorex minutus 2 Yes Spanish shrew Sorex granarius 1 Yes Pygmy white-toothed shrew Suncus etruscus 1 Yes Water shrew Neomys sp. 1 Yes Amphibians and reptiles Lataste’s viper Vipera latastei 2No Asp viper Vipera aspis 2No Iberian adder Vipera seoanei 2No Schreiber’s green lizard Lacerta schreiberi 5No Ocellated lizard Lacerta lepida 1No Viperine water snake Natrix maura 1No Common toad Bufo bufo 1No Natterjack toad Bufo calamita 1No Sharp-ribbed newt Pleurodeles waltl 1No Large psammodromus Psammodromus algirus 1No Birds Red-legged partridge Alectoris rufa 2No Chukar partridge Alectoris chuckar 2No Rock partridge Alectoris graeca 2No Common quail Coturnix coturnix 2No Japanese quail Coturnix japonica 2No Eurasian scops owl Otus scops 2No Common barn Owl Tyto alba 5No Eurasian woodcock Scolopax rusticola 5No Robin Erithacus rubecula 5No Domestic chicken Gallus gallus 5No Common blackbird Turdus merula 1No Song thrush Turdus philomelos 1No Redwing Turdus iliacus 1No Rock pigeon Columba livia 2No Turtle dove Streptopelia turtur 2No

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Appendix continued Group Common name Species N PCR-SSCP

Fishes and crustaceous Rainbow trout Oncorhynchus mykiss 5No Pike Esox lucius 2No Gudgeon Gobio gobio 2No Carp Cyprinus carpio 2No Pumpkinseed Lepomis gibbosum 2No Louisiana crayﬁsh Procambarus clarkii 2No a Faint ampliﬁcation that did not produce a clear SSCP pattern

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