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Hybridization between wolf and domestic dog: First evidence from an endangered population in central Portugal
Accepted Manuscript Title: Hybridization between wolf and domestic dog: first evidence from an endangered population in central Portugal Authors: Rita Tinoco Torres, Eduardo Ferreira, Rita Gomes Rocha, Carlos Fonseca PII: DOI: Reference:
S1616-5047(16)30221-X http://dx.doi.org/doi:10.1016/j.mambio.2017.05.001 MAMBIO 40908
To appear in: Received date: Accepted date:
28-12-2016 9-5-2017
Please cite this article as: Torres, Rita Tinoco, Ferreira, Eduardo, Rocha, Rita Gomes, Fonseca, Carlos, Hybridization between wolf and domestic dog: first evidence from an endangered population in central Portugal.Mammalian Biology http://dx.doi.org/10.1016/j.mambio.2017.05.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Hybridization between wolf and domestic dog: first evidence from an endangered population in central Portugal Rita Tinoco Torres1*, Eduardo Ferreira 1*, Rita Gomes Rocha1,2 and Carlos Fonseca1 *both authors contributed equally 1
Department of Biology & CESAM, University of Aveiro, 3810-193 Aveiro, Portugal 2
Departamento de Ciências Biológicas, Centro de Ciências Humanas e Naturais,
Universidade Federal do Espírito Santo, Av. Fernando Ferrari 514, Goiabeiras, 29075-910, Vitória, ES, Brazil Corresponding author: Rita T. Torres CESAM & Department of Biology, University of Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal E-mail: [email protected] Abstract Human population expansion has promoted contact between wildlife and domestic animals with severe ecological consequences, such as anthropogenic hybridization. In Portugal, Iberian wolf (Canis lupus signatus) populations are considered “Endangered” and co-habit with humans so the risks of hybridization with free-ranging dogs, and livestock depredation can be particularly high. Our aim was to report the occurrence of wolf-dog hybridization in an endangered Iberian wolf sub-population, located in the south of the Douro river, Portugal. We used mitochondrial DNA and microsatellite data to investigate putative hybrids between Iberian wolves and dogs. Here, we report for the first time a wolf-dog hybrid located in the south of the Douro river. This is the second hybrid found in Portugal, and even if hybridization cases are still considered rare, they can be particularly problematic in isolated, fragmented and endangered populations, such as the one studied here. Appropriate management and conservation measures are recommended. Keywords: Iberian Peninsula; ; ; ; , Canis lupus signatus, hybridization, conservation genetics, molecular tools.
As human population increases in numbers and expand, also into wildlife suitable habitats, the contact between wildlife and domestic animals is likely to intensify, with several ecological consequences, such as anthropogenic hybridization (Allendorf et al., 2001). Anthropogenic hybridization, triggered by human-induced changes (e.g., habitat modification, fragmentation, species (re)introductions) (Allendorf et al., 2001), has prompted intense debate
(Vilà and Wayne, 1999). It has been suggested that anthropogenic hybridization has several undesirable consequences, jeopardizing the genetic integrity of populations, eventually causing their extinction (Lescureux and Linnell, 2014). One of the classic examples of the potential deleterious contact between wildlife and domestic animals is the hybridization between wolf (Canis lupus) populations and free-ranging dogs (Canis lupus familiaris). Wolf-dog hybridization has long been acknowledged in Europe (Randi, 2011), though occurring with different frequencies: low frequency in the western regions (e.g., Italy: Randi and Lucchini, 2002; Iberian Peninsula: Godinho et al., 2011a) and high in the eastern part of the continent (e.g., Estonia, Latvia, Bulgaria: Randi et al. 2000; Andersone et al., 2002; Hindrikson et al., 2012; Caniglia et al., 2014). Contrary to the remarkable wolf recovery, which occurred in several European countries (Chapron et al., 2014), the distribution of the Iberian wolf (Canis lupus signatus), an endemic subspecies of the Iberian Peninsula, has been declining throughout the 20th century in Portugal (Torres and Fonseca, 2016), where it is considered “Endangered”. Several studies report that hybridization is particularly problematic in small and fragmented wolf populations, inhabiting humanized landscapes, where free-ranging dogs are common (Vilà and Wayne, 1999; Godinho et al., 2011a) and where their feeding ecology is largely based on livestock (Torres et al., 2015; Torres and Fonseca, 2016). Consequently, it is vital to identify the degree of hybridization between wolf and free-ranging dogs in endangered and small populations, such as the subpopulation presented in this study. Here, we investigate the occurrence of wolf-dog hybrids in an Iberian wolf subpopulation in central Portugal (south of the Douro river), at the south-western edge of its distribution, using mitochondrial DNA and microsatellite data. The study was conducted in central-west Portugal, within an area of 750 km2, which encompasses the range of 3 wolf packs (Arada, Montemuro and Cinfães packs, Figure 1) (for more details of the study area please see Torres et al., 2015). In Portugal, genetic studies have demonstrated the existence of two apparently isolated wolf subpopulations, separated by the river Douro (Godinho et al., 2011a): the packs north of this river are considered stable and appear to be locally expanding; while the packs south of this river (only 6 confirmed packs) are isolated from the remaining populations, showing high levels of fragmentation and low genetic diversity (Godinho et al., 2011a; Hindrikson et al., 2016). A total of 47 transects were distributed throughout the study area, and were monthly inspected, which corresponds to 130.4 km per month (smallest transect: 0.6 km; largest transect: 7.4 km), from 2011 to 2014. We also collected scats opportunistically while travelling to and between transects. During this period, a total of 93 scat samples were carefully collected (to avoid contamination) in the territory of the three packs and stored immediately in 96% ethanol, and after a few hours at -20ºC. During the same time period, saliva and hair samples were randomly collected from 26 domestic dogs (mainly shepherd, stray and hunting dogs) from the same area.
Hair samples were also collected from a juvenile animal with a phenotypic appearance of a wolf (J0A) found dead in the territory of the Arada pack, in September 2014 (unknown cause of death). The DNA was isolated from scats, saliva or hair samples, one to two weeks after collection using the QIAGEN® QiAamp DNAStool kit, and from tissue using a standard salt-out extraction procedure (Bruford et al., 1992). All laboratory procedures were held in dedicated facilities, and with all due care in order to avoid contamination of samples. Scats, saliva and hair samples usually have low amount of DNA, with low average quality. A 442bp-length fragment of the D-loop (mtDNA) was sequenced for all samples, using the primers Thr-L 15926 and DL-H 16340 (Vilà et al., 1999). However, the comparison of mtDNA haplotypes was based on a shorter sequence (261bp), following Vilà et al. (1999), since no polymorphism was detected outside this shorter fragment. This fragment is frequently used in the molecular distinction between wolf and dog (e.g., Godinho et al., 2011a). All samples were also genotyped for a panel of 24 microsatellite markers, based on at least three replicate genotypes for each marker. This panel included the 18 markers included in the Canine Genotypes™ 1.1 (Finnzymes®) and six additional markers, that were amplified in two multiplex reactions: the first including the markers C04.140, C20.253 (Ostrander et al 1993), FH2001 and FH2161 (Francisco et al, 1996); the second including the markers CPH14 (Fredholm and Wintero, 2009) and DBAr (Kerns et al 2004). Mitochondrial haplotypes were compared with those from wolves and domestic dogs identified by Vilà et al. (1997), that we retrieved from Genbank (Accession numbers: AF005280.1–314.1; AF008135.182.1; https://www.ncbi.nlm.nih.gov/genbank/). Comparisons of mtDNA haplotypes were performed using a neighbour-joining phylogenetic tree, generated with the algorithm implemented in MEGA 6 (Tamura et al., 2013). General diversity indices, assignment tests and principal coordinate analysis (PCoA) were performed using GenAlEx 6.501 (Peakall and Smouse, 2012). For the PCoA, and subsequent NEWHYBRIDS and STRUCTURE analysis, our dataset of wolf and dog genotypes was complemented with a dataset of genotypes of Iberian wolf and domestic dog (Godinho et al., 2011a), freely available on Dryad (Godinho et al., 2011b). This dataset included 408 genotypes based on 42 autosomal microsatellite markers. After discarding the most incomplete genotypes from the dataset, we included in our analysis: 191 domestic dog genotypes, 197 wolf genotypes and 8 wolf-dog hybrids, identified in Godinho et al (2011a). We only incorporated in our analyses the data pertaining to the 24 microsatellite markers used for genotyping our samples. In order to allow the calibration and merging of the two datasets, a reference sample (SMLM88, from the Portuguese National Tissue Collection ICNF) was used. This sample, that has been previously genotyped at Godinho’s laboratory, was the first sample to be genotyped in our lab. We used this sample to calibrate the allele calling procedure and genotyped all samples using the calibrated allele calling. Additional checking was performed by carefully inspecting the allele frequency distributions on both datasets. Posterior probabilities of inferred ancestry of individuals to parental and hybrid classes were estimated using
NEWHYBRIDS 1.0 (Anderson and Thompson, 2002), after five replicate runs (each with 106 iterations with a 105 iterations period of burnin), using the merged datasets. The most likely number K of different genetic clusters and posterior probability of assignment of individual’s genotype to inferred genetic clusters were estimated using STRUCTURE (Falush et al., 2003), after five replicate runs (each with 106 iterations with a 105 iterations period of burnin; K=2; with admixture model with correlated allele frequencies and no prior of individual identification). From the 93 sampled scats, 51 (55%) were positively identified as belonging to Iberian wolf, based on their mtDNA haplotype. The remaining belonged to dog (15) and fox (1) or did not successfully match any species (26). All wolves sampled in the study area shared a single haplotype, matching the W1 wolf haplotype characterized by Vilà et al. (1997). None of the sampled dogs had this haplotype, and all had haplotypes already identified in Iberian populations of domestic dogs. The collected animal (J0A) presented the same W1 haplotype. Ten individual genotypes (based on a sample of 31 wolf scats, with three genotypes sampled three or more times) were identified in the Arada pack territory (including the collected animal – J0A), six in the Cinfães pack (18 wolf scats, one genotype sampled three times) five in the Montemuro pack (16 wolf scats, al genotypes sampled only once). Despite sharing the same mtDNA haplotype (W1) with wolves from the region, individual J0A presented a particularly high number of alleles (12) that were not shared with other wolves from the study area (Supplementary info, Table S1). On average, other sampled wolf genotypes only presented one allele that was not shared with other wolves from the study area. Ten out of these 12 alleles were however shared with the reference population of domestic dogs from the study area. Moreover, five out of these 12 alleles had been previously found only on domestic dogs (or wolf-dog hybrids) sampled by Godinho and collaborators (2011b) in the Iberian Peninsula. Therefore, it was not surprising that J0A was not clearly assigned to an Iberian wolf parental class (Figure 2A), being assigned with high posterior probability to one of two hybrid classes (84.5 % F1 hybrid; 13.3 % wolf backcross). Other results corroborated this finding: i) J0A genotype occupied an intermediate position between wolf and dog genotypes in the PCoA plot (Figure 2B); ii) J0A was assigned with similar likelihood values to both parental populations, using GenAlEx (Figure 2C); iii) J0A presented intermediate q proportions of genotype attributed to dog (67 %) and wolf (33 %) genetic clusters, estimated with STRUCTURE, while genotypes of pure Iberian wolves and dogs were always attributed to the corresponding cluster with an average posterior probability of 99 %; and iv) in all analyses, the assignment probabilities, inferred ancestries and relative positions of the individual J0A were similar to those of the hybrids previously detected by Godinho and collaborators. To the best of our knowledge, this study is the first to report a wolf-dog hybrid in the subpopulation located in the south of the Douro river, and the second report in Portugal (Godinho et al. (2011a). Godinho et al. (2011a) found 8 putative hybrids in the Iberian Peninsula, out of the
204 Iberian samples analysed, but the only hybrid collected in Portugal was from the northwestern region (Minho region), north of the Douro river. These authors described the first wolfdog hybrid in Portugal, on the more stable and apparently expanding subpopulation. Previous to Godinho et al. (2011a), several authors failed to detect hybrids in the Iberian Peninsula (Vilà and Wayne, 1999; Ramiréz et al., 2006). It is important to mention that despite this single case of hybridization, the Iberian wolves and free-ranging dogs in the study area are genetically distinct, showing high average posterior probability (99%) of assignment to their own clusters. Our case report is consistent with the results from Godinho et al. (2011a), which reported hybridization between female wolves and male dogs. Nevertheless, there are recent evidences of hybridization between male wolves and female dogs (Hindrikson et al., 2012). In fact, the same way female wolves are expected to give birth to their litters in the wild, it is possible that domestic or feral female dogs will give birth closer to human settlements. Consequently, monitoring of domestic dog populations, in regions of confirmed hybridization between wolf and dog, should be prioritized, otherwise genetic sampling will be biased and hybridization can be underestimate (Godinho et al. 2011a). Across Europe, hybridization has been reported in several countries, such as Bulgaria (Randi et al. 2000), Latvia (Andersone et al. 2012), Italy (Randi & Lucchini 2002; Verardi et al. 2006), Scandinavia (Vilà et al., 2003), and in the Iberian Peninsula (Godinho et al. 2011a), even tough with different frequencies. In Portugal, even if hybridization reports are still scarce, the risks and conservation consequences of hybridization are critical (Lescureux and Linnell, 2014). This is particularly problematic in the packs inhabiting our study area, as they are small and fragmented populations, meaning that hybridization can lead to the reduction or loss of specific adaptations, ultimately resulting in admixed population (Hindrikson et al., 2012). Interestingly, the hybrid reported here had the appearance of a wolf and it was morphologically described as a juvenile wolf. However, as we also sampled free-ranging dogs, we believe that we have minimized the potential bias of not collecting samples from possible hybrids that look like pure dogs (Godinho et al., 2011a). Regrettably, there are several factors in our study area that can favour hybridization: first, free ranging dogs are ubiquitous around villages since there is a widespread practice in rural Portugal for dog-owners to allow their dogs to freely roam, obviously increasing the chances of encounters between dogs and wolves; second, livestock constitutes most part of wolves’ diet (Torres et al., 2015), as wild prey diversity and density is low, which may force wolves to feed close to villages. Our study area is, like most of southern Europe, characterized by highly humanized habitats. Efforts to minimize and manage anthropogenic hybridization are challenging (Allendorf et al., 2001). We suggest the i) implementation of a long-term genetic (and population) monitoring plan of both wolf and free-ranging dog populations. These efforts should be focused not only on
wolf packs, but also on the dog populations in areas of confirmed wolf presence and; ii) development of projects aiming to understand the ecology and population dynamics of the hybrids (Lescureux and Linnell, 2014). The genetic monitoring throughout time is vital to understand the real degree of hybridization but also to understand how it evolves through time. Obviously, the presence of free ranging dogs in overlapping areas with wolves, is ultimately the main cause of hybridization. So, even if difficult to implement, conservationists and managers ought to develop effective programs to somehow control the populations of free ranging dogs. Some authors have proposed the eradication of hybrids from the populations (Vilà et al., 2003; Godinho et al., 2011a) but as hybrids do not have any legal status in Europe (Lescureux and Linnell, 2014), such actions raise ethical concerns (Lorenzini et al., 2014). Acknowledgements We are grateful to all the people and institutions that provided valuable assistance, particularly Associação de Conservação do Habitat do Lobo Ibérico (ACHLI). R.T. Torres and E. Ferreira were supported by a post-doctoral grant from FCT (SFRH/BPD/112482/2015 and SFRH/BPD/72895/2010, respectively). R.G. Rocha was supported by a research grant from Fundação de Amparo a Pesquisa e Inovação do Espírito Santo, Brazil (FAPES, ref: 0650/2015). We would like to thank University of Aveiro (Department of Biology) and FCT/MEC for the financial support to CESAM RU (UID/AMB/50017) through national funds and co-financed by the FEDER, within the PT2020 Partnership Agreement. We also thank three anonymous referees for constructive comments of this article. References Allendorf, F. W., Leary, R. F., Spruell, P., & Wenburg, J. K. (2001). The problems with hybrids: setting conservation guidelines. Trends Ecol Evol, 16(11): 613-622. Anderson, E. C., & Thompson, E. A. (2002). A model-based method for identifying species hybrids using multilocus genetic data. Genetics, 160, 1217–1229. Andersone, Z., Lucchini, V., Randi, E., & Ozolins, J. (2002). Hybridisation between wolves and dogs in Latvia as documented using mitochondrial and microsatellite DNA markers. Mamm. Biol., 67: 79–90. Bruford, M. W., Hanotte, O., Brookfield, J. F. Y., & Burke, T. (1992). Single-locus and DNA fingerprinting. In A. R. Hoelzel (Ed.), Molecular genetic analyses of populations. A Pratical Approach. (pp. 225–269). Oxford: IRL Press, Oxford. Caniglia, R., Fabbri, E., Galaverni, M., Milanesi, P., & Randi, E. (2014). Noninvasive sampling and genetic variability, pack structure, and dynamics in an expanding wolf population. J. Mammal. 95(1): 41-59.
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Figure 2 – Genetic evidences supporting the putative hybrid origin for individual J0A (identified with a grey arrow). A) Posterior probability of assignment of each individual to each of the two parental (wolf -white; dog - black) or four hybrid (F1, F2 or backcross – grey shades) classes, estimated using NEWHYBRIDS, for the dataset including: 221 domestic dogs (30 from the study area), 220 Iberian wolves (14 from the study area), eight known hybrids and the hybrid J0A. Each individual is represented by a vertical bar. B) Two-dimension plot of the first two variation axes of the principal coordinates analysis, based on the same set of 450 individual genotypes. C) Assignment probabilities, estimated with GenAlEx, for Iberian wolves, dogs and their hybrids. Black circles stand for dogs, white circles for wolves and grey circles for hybrids. SA – study area; Hb – other previously detected wolf-dog hybrids.
Figr-1
Figr-2
S1616-5047(16)30221-X http://dx.doi.org/doi:10.1016/j.mambio.2017.05.001 MAMBIO 40908
To appear in: Received date: Accepted date:
28-12-2016 9-5-2017
Please cite this article as: Torres, Rita Tinoco, Ferreira, Eduardo, Rocha, Rita Gomes, Fonseca, Carlos, Hybridization between wolf and domestic dog: first evidence from an endangered population in central Portugal.Mammalian Biology http://dx.doi.org/10.1016/j.mambio.2017.05.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Hybridization between wolf and domestic dog: first evidence from an endangered population in central Portugal Rita Tinoco Torres1*, Eduardo Ferreira 1*, Rita Gomes Rocha1,2 and Carlos Fonseca1 *both authors contributed equally 1
Department of Biology & CESAM, University of Aveiro, 3810-193 Aveiro, Portugal 2
Departamento de Ciências Biológicas, Centro de Ciências Humanas e Naturais,
Universidade Federal do Espírito Santo, Av. Fernando Ferrari 514, Goiabeiras, 29075-910, Vitória, ES, Brazil Corresponding author: Rita T. Torres CESAM & Department of Biology, University of Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal E-mail: [email protected] Abstract Human population expansion has promoted contact between wildlife and domestic animals with severe ecological consequences, such as anthropogenic hybridization. In Portugal, Iberian wolf (Canis lupus signatus) populations are considered “Endangered” and co-habit with humans so the risks of hybridization with free-ranging dogs, and livestock depredation can be particularly high. Our aim was to report the occurrence of wolf-dog hybridization in an endangered Iberian wolf sub-population, located in the south of the Douro river, Portugal. We used mitochondrial DNA and microsatellite data to investigate putative hybrids between Iberian wolves and dogs. Here, we report for the first time a wolf-dog hybrid located in the south of the Douro river. This is the second hybrid found in Portugal, and even if hybridization cases are still considered rare, they can be particularly problematic in isolated, fragmented and endangered populations, such as the one studied here. Appropriate management and conservation measures are recommended. Keywords: Iberian Peninsula; ; ; ; , Canis lupus signatus, hybridization, conservation genetics, molecular tools.
As human population increases in numbers and expand, also into wildlife suitable habitats, the contact between wildlife and domestic animals is likely to intensify, with several ecological consequences, such as anthropogenic hybridization (Allendorf et al., 2001). Anthropogenic hybridization, triggered by human-induced changes (e.g., habitat modification, fragmentation, species (re)introductions) (Allendorf et al., 2001), has prompted intense debate
(Vilà and Wayne, 1999). It has been suggested that anthropogenic hybridization has several undesirable consequences, jeopardizing the genetic integrity of populations, eventually causing their extinction (Lescureux and Linnell, 2014). One of the classic examples of the potential deleterious contact between wildlife and domestic animals is the hybridization between wolf (Canis lupus) populations and free-ranging dogs (Canis lupus familiaris). Wolf-dog hybridization has long been acknowledged in Europe (Randi, 2011), though occurring with different frequencies: low frequency in the western regions (e.g., Italy: Randi and Lucchini, 2002; Iberian Peninsula: Godinho et al., 2011a) and high in the eastern part of the continent (e.g., Estonia, Latvia, Bulgaria: Randi et al. 2000; Andersone et al., 2002; Hindrikson et al., 2012; Caniglia et al., 2014). Contrary to the remarkable wolf recovery, which occurred in several European countries (Chapron et al., 2014), the distribution of the Iberian wolf (Canis lupus signatus), an endemic subspecies of the Iberian Peninsula, has been declining throughout the 20th century in Portugal (Torres and Fonseca, 2016), where it is considered “Endangered”. Several studies report that hybridization is particularly problematic in small and fragmented wolf populations, inhabiting humanized landscapes, where free-ranging dogs are common (Vilà and Wayne, 1999; Godinho et al., 2011a) and where their feeding ecology is largely based on livestock (Torres et al., 2015; Torres and Fonseca, 2016). Consequently, it is vital to identify the degree of hybridization between wolf and free-ranging dogs in endangered and small populations, such as the subpopulation presented in this study. Here, we investigate the occurrence of wolf-dog hybrids in an Iberian wolf subpopulation in central Portugal (south of the Douro river), at the south-western edge of its distribution, using mitochondrial DNA and microsatellite data. The study was conducted in central-west Portugal, within an area of 750 km2, which encompasses the range of 3 wolf packs (Arada, Montemuro and Cinfães packs, Figure 1) (for more details of the study area please see Torres et al., 2015). In Portugal, genetic studies have demonstrated the existence of two apparently isolated wolf subpopulations, separated by the river Douro (Godinho et al., 2011a): the packs north of this river are considered stable and appear to be locally expanding; while the packs south of this river (only 6 confirmed packs) are isolated from the remaining populations, showing high levels of fragmentation and low genetic diversity (Godinho et al., 2011a; Hindrikson et al., 2016). A total of 47 transects were distributed throughout the study area, and were monthly inspected, which corresponds to 130.4 km per month (smallest transect: 0.6 km; largest transect: 7.4 km), from 2011 to 2014. We also collected scats opportunistically while travelling to and between transects. During this period, a total of 93 scat samples were carefully collected (to avoid contamination) in the territory of the three packs and stored immediately in 96% ethanol, and after a few hours at -20ºC. During the same time period, saliva and hair samples were randomly collected from 26 domestic dogs (mainly shepherd, stray and hunting dogs) from the same area.
Hair samples were also collected from a juvenile animal with a phenotypic appearance of a wolf (J0A) found dead in the territory of the Arada pack, in September 2014 (unknown cause of death). The DNA was isolated from scats, saliva or hair samples, one to two weeks after collection using the QIAGEN® QiAamp DNAStool kit, and from tissue using a standard salt-out extraction procedure (Bruford et al., 1992). All laboratory procedures were held in dedicated facilities, and with all due care in order to avoid contamination of samples. Scats, saliva and hair samples usually have low amount of DNA, with low average quality. A 442bp-length fragment of the D-loop (mtDNA) was sequenced for all samples, using the primers Thr-L 15926 and DL-H 16340 (Vilà et al., 1999). However, the comparison of mtDNA haplotypes was based on a shorter sequence (261bp), following Vilà et al. (1999), since no polymorphism was detected outside this shorter fragment. This fragment is frequently used in the molecular distinction between wolf and dog (e.g., Godinho et al., 2011a). All samples were also genotyped for a panel of 24 microsatellite markers, based on at least three replicate genotypes for each marker. This panel included the 18 markers included in the Canine Genotypes™ 1.1 (Finnzymes®) and six additional markers, that were amplified in two multiplex reactions: the first including the markers C04.140, C20.253 (Ostrander et al 1993), FH2001 and FH2161 (Francisco et al, 1996); the second including the markers CPH14 (Fredholm and Wintero, 2009) and DBAr (Kerns et al 2004). Mitochondrial haplotypes were compared with those from wolves and domestic dogs identified by Vilà et al. (1997), that we retrieved from Genbank (Accession numbers: AF005280.1–314.1; AF008135.182.1; https://www.ncbi.nlm.nih.gov/genbank/). Comparisons of mtDNA haplotypes were performed using a neighbour-joining phylogenetic tree, generated with the algorithm implemented in MEGA 6 (Tamura et al., 2013). General diversity indices, assignment tests and principal coordinate analysis (PCoA) were performed using GenAlEx 6.501 (Peakall and Smouse, 2012). For the PCoA, and subsequent NEWHYBRIDS and STRUCTURE analysis, our dataset of wolf and dog genotypes was complemented with a dataset of genotypes of Iberian wolf and domestic dog (Godinho et al., 2011a), freely available on Dryad (Godinho et al., 2011b). This dataset included 408 genotypes based on 42 autosomal microsatellite markers. After discarding the most incomplete genotypes from the dataset, we included in our analysis: 191 domestic dog genotypes, 197 wolf genotypes and 8 wolf-dog hybrids, identified in Godinho et al (2011a). We only incorporated in our analyses the data pertaining to the 24 microsatellite markers used for genotyping our samples. In order to allow the calibration and merging of the two datasets, a reference sample (SMLM88, from the Portuguese National Tissue Collection ICNF) was used. This sample, that has been previously genotyped at Godinho’s laboratory, was the first sample to be genotyped in our lab. We used this sample to calibrate the allele calling procedure and genotyped all samples using the calibrated allele calling. Additional checking was performed by carefully inspecting the allele frequency distributions on both datasets. Posterior probabilities of inferred ancestry of individuals to parental and hybrid classes were estimated using
NEWHYBRIDS 1.0 (Anderson and Thompson, 2002), after five replicate runs (each with 106 iterations with a 105 iterations period of burnin), using the merged datasets. The most likely number K of different genetic clusters and posterior probability of assignment of individual’s genotype to inferred genetic clusters were estimated using STRUCTURE (Falush et al., 2003), after five replicate runs (each with 106 iterations with a 105 iterations period of burnin; K=2; with admixture model with correlated allele frequencies and no prior of individual identification). From the 93 sampled scats, 51 (55%) were positively identified as belonging to Iberian wolf, based on their mtDNA haplotype. The remaining belonged to dog (15) and fox (1) or did not successfully match any species (26). All wolves sampled in the study area shared a single haplotype, matching the W1 wolf haplotype characterized by Vilà et al. (1997). None of the sampled dogs had this haplotype, and all had haplotypes already identified in Iberian populations of domestic dogs. The collected animal (J0A) presented the same W1 haplotype. Ten individual genotypes (based on a sample of 31 wolf scats, with three genotypes sampled three or more times) were identified in the Arada pack territory (including the collected animal – J0A), six in the Cinfães pack (18 wolf scats, one genotype sampled three times) five in the Montemuro pack (16 wolf scats, al genotypes sampled only once). Despite sharing the same mtDNA haplotype (W1) with wolves from the region, individual J0A presented a particularly high number of alleles (12) that were not shared with other wolves from the study area (Supplementary info, Table S1). On average, other sampled wolf genotypes only presented one allele that was not shared with other wolves from the study area. Ten out of these 12 alleles were however shared with the reference population of domestic dogs from the study area. Moreover, five out of these 12 alleles had been previously found only on domestic dogs (or wolf-dog hybrids) sampled by Godinho and collaborators (2011b) in the Iberian Peninsula. Therefore, it was not surprising that J0A was not clearly assigned to an Iberian wolf parental class (Figure 2A), being assigned with high posterior probability to one of two hybrid classes (84.5 % F1 hybrid; 13.3 % wolf backcross). Other results corroborated this finding: i) J0A genotype occupied an intermediate position between wolf and dog genotypes in the PCoA plot (Figure 2B); ii) J0A was assigned with similar likelihood values to both parental populations, using GenAlEx (Figure 2C); iii) J0A presented intermediate q proportions of genotype attributed to dog (67 %) and wolf (33 %) genetic clusters, estimated with STRUCTURE, while genotypes of pure Iberian wolves and dogs were always attributed to the corresponding cluster with an average posterior probability of 99 %; and iv) in all analyses, the assignment probabilities, inferred ancestries and relative positions of the individual J0A were similar to those of the hybrids previously detected by Godinho and collaborators. To the best of our knowledge, this study is the first to report a wolf-dog hybrid in the subpopulation located in the south of the Douro river, and the second report in Portugal (Godinho et al. (2011a). Godinho et al. (2011a) found 8 putative hybrids in the Iberian Peninsula, out of the
204 Iberian samples analysed, but the only hybrid collected in Portugal was from the northwestern region (Minho region), north of the Douro river. These authors described the first wolfdog hybrid in Portugal, on the more stable and apparently expanding subpopulation. Previous to Godinho et al. (2011a), several authors failed to detect hybrids in the Iberian Peninsula (Vilà and Wayne, 1999; Ramiréz et al., 2006). It is important to mention that despite this single case of hybridization, the Iberian wolves and free-ranging dogs in the study area are genetically distinct, showing high average posterior probability (99%) of assignment to their own clusters. Our case report is consistent with the results from Godinho et al. (2011a), which reported hybridization between female wolves and male dogs. Nevertheless, there are recent evidences of hybridization between male wolves and female dogs (Hindrikson et al., 2012). In fact, the same way female wolves are expected to give birth to their litters in the wild, it is possible that domestic or feral female dogs will give birth closer to human settlements. Consequently, monitoring of domestic dog populations, in regions of confirmed hybridization between wolf and dog, should be prioritized, otherwise genetic sampling will be biased and hybridization can be underestimate (Godinho et al. 2011a). Across Europe, hybridization has been reported in several countries, such as Bulgaria (Randi et al. 2000), Latvia (Andersone et al. 2012), Italy (Randi & Lucchini 2002; Verardi et al. 2006), Scandinavia (Vilà et al., 2003), and in the Iberian Peninsula (Godinho et al. 2011a), even tough with different frequencies. In Portugal, even if hybridization reports are still scarce, the risks and conservation consequences of hybridization are critical (Lescureux and Linnell, 2014). This is particularly problematic in the packs inhabiting our study area, as they are small and fragmented populations, meaning that hybridization can lead to the reduction or loss of specific adaptations, ultimately resulting in admixed population (Hindrikson et al., 2012). Interestingly, the hybrid reported here had the appearance of a wolf and it was morphologically described as a juvenile wolf. However, as we also sampled free-ranging dogs, we believe that we have minimized the potential bias of not collecting samples from possible hybrids that look like pure dogs (Godinho et al., 2011a). Regrettably, there are several factors in our study area that can favour hybridization: first, free ranging dogs are ubiquitous around villages since there is a widespread practice in rural Portugal for dog-owners to allow their dogs to freely roam, obviously increasing the chances of encounters between dogs and wolves; second, livestock constitutes most part of wolves’ diet (Torres et al., 2015), as wild prey diversity and density is low, which may force wolves to feed close to villages. Our study area is, like most of southern Europe, characterized by highly humanized habitats. Efforts to minimize and manage anthropogenic hybridization are challenging (Allendorf et al., 2001). We suggest the i) implementation of a long-term genetic (and population) monitoring plan of both wolf and free-ranging dog populations. These efforts should be focused not only on
wolf packs, but also on the dog populations in areas of confirmed wolf presence and; ii) development of projects aiming to understand the ecology and population dynamics of the hybrids (Lescureux and Linnell, 2014). The genetic monitoring throughout time is vital to understand the real degree of hybridization but also to understand how it evolves through time. Obviously, the presence of free ranging dogs in overlapping areas with wolves, is ultimately the main cause of hybridization. So, even if difficult to implement, conservationists and managers ought to develop effective programs to somehow control the populations of free ranging dogs. Some authors have proposed the eradication of hybrids from the populations (Vilà et al., 2003; Godinho et al., 2011a) but as hybrids do not have any legal status in Europe (Lescureux and Linnell, 2014), such actions raise ethical concerns (Lorenzini et al., 2014). Acknowledgements We are grateful to all the people and institutions that provided valuable assistance, particularly Associação de Conservação do Habitat do Lobo Ibérico (ACHLI). R.T. Torres and E. Ferreira were supported by a post-doctoral grant from FCT (SFRH/BPD/112482/2015 and SFRH/BPD/72895/2010, respectively). R.G. Rocha was supported by a research grant from Fundação de Amparo a Pesquisa e Inovação do Espírito Santo, Brazil (FAPES, ref: 0650/2015). We would like to thank University of Aveiro (Department of Biology) and FCT/MEC for the financial support to CESAM RU (UID/AMB/50017) through national funds and co-financed by the FEDER, within the PT2020 Partnership Agreement. We also thank three anonymous referees for constructive comments of this article. References Allendorf, F. W., Leary, R. F., Spruell, P., & Wenburg, J. K. (2001). The problems with hybrids: setting conservation guidelines. Trends Ecol Evol, 16(11): 613-622. Anderson, E. C., & Thompson, E. A. (2002). A model-based method for identifying species hybrids using multilocus genetic data. Genetics, 160, 1217–1229. Andersone, Z., Lucchini, V., Randi, E., & Ozolins, J. (2002). Hybridisation between wolves and dogs in Latvia as documented using mitochondrial and microsatellite DNA markers. Mamm. Biol., 67: 79–90. Bruford, M. W., Hanotte, O., Brookfield, J. F. Y., & Burke, T. (1992). Single-locus and DNA fingerprinting. In A. R. Hoelzel (Ed.), Molecular genetic analyses of populations. A Pratical Approach. (pp. 225–269). Oxford: IRL Press, Oxford. Caniglia, R., Fabbri, E., Galaverni, M., Milanesi, P., & Randi, E. (2014). Noninvasive sampling and genetic variability, pack structure, and dynamics in an expanding wolf population. J. Mammal. 95(1): 41-59.
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Figure 2 – Genetic evidences supporting the putative hybrid origin for individual J0A (identified with a grey arrow). A) Posterior probability of assignment of each individual to each of the two parental (wolf -white; dog - black) or four hybrid (F1, F2 or backcross – grey shades) classes, estimated using NEWHYBRIDS, for the dataset including: 221 domestic dogs (30 from the study area), 220 Iberian wolves (14 from the study area), eight known hybrids and the hybrid J0A. Each individual is represented by a vertical bar. B) Two-dimension plot of the first two variation axes of the principal coordinates analysis, based on the same set of 450 individual genotypes. C) Assignment probabilities, estimated with GenAlEx, for Iberian wolves, dogs and their hybrids. Black circles stand for dogs, white circles for wolves and grey circles for hybrids. SA – study area; Hb – other previously detected wolf-dog hybrids.
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