New insights into the genetic composition and phylogenetic relationship of wolves and dogs in the Iberian Peninsula

Pires, A. E., Amorim, I. R., Borges, C., Simões, F., Teixeira, T., Quaresma, A., ... & Matos, J. (2017). New insights into the genetic composition and phylogenetic relationship of wolves and dogs in the Iberian Peninsula. Ecology and Evolution.

This study investigates the gene pool of Portuguese autochthonous dog breeds and their wild counterpart, the Iberian wolf subspecies (Canis lupus signatus), using standard molecular markers. A combination of paternal and maternal molecular markers was used to investigate the genetic composition, genetic differentiation and genetic relationship of native Portuguese dogs and the Iberian wolf. A total of 196 unrelated dogs, including breed and village dogs from Portugal, and other dogs from Spain and North Africa, and 56 Iberian wolves (wild and captive) were analyzed for nuclear markers, namely Y chromosome SNPs, Y chromosome STR loci, autosomal STR loci, and a mitochondrial fragment of the control region I. Our data reveal new variants for the molecular markers and confirm significant genetic differentiation between Iberian wolf and native domestic dogs from Portugal. Based on our sampling, no signs of recent introgression between the two subspecies were detected. Y chromosome data do not reveal genetic differentiation among the analyzed dog breeds, suggesting they share the same patrilineal origin. Moreover, the genetic distinctiveness of the Iberian wolf from other wolf populations is further confirmed with the description of new mtDNA variants for this endemism. Our research also discloses new molecular markers for wolf and dog subspecies assignment, which might become particularly relevant in the case of forensic or noninvasive genetic studies. The Iberian wolf represents a relic of the once widespread wolf population in Europe and our study reveals that it is a reservoir of unique genetic diversity of the grey wolf, Canis lupus. These results stress the need for conservation plans that will guarantee the sustainability of this threatened top predator in Iberia.

Domestic cat introgression in wildcat in Iberia

Oliveira, R., Godinho, R., Randi, E., Ferrand, N., & Alves, P. C. (2008). Molecular analysis of hybridisation between wild and domestic cats (Felis silvestris) in Portugal: implications for conservation. Conservation Genetics, 9(1): 1-11.

The endangered European wildcat (Felis silvestris silvestris) is represented, today, by fragmented and declining populations whose genetic integrity is considered to be seriously threatened by crossbreeding with widespread free-ranging domestic cats. Extensive and recent hybridisation has been described in Hungary and Scotland, in contrast with rare introgression of domestic alleles in Italy and Germany. In Portugal, the wildcat is now listed as VULNERABLE in the Red Book of Portuguese Vertebrates. Nevertheless, genetic diversity of populations and the eventual interbreeding with domestic cats remain poorly studied. We surveyed genetic variation at 12 autosomal microsatellites for 34 wild and 64 domestic cats collected across Portugal. Wild and domestic cats were significantly differentiated both at allele frequencies and sizes (F _ST=0.11, R _ST = 0.18, P < 0.001). Population structure and admixture analyses performed using Bayesian approaches also showed evidence of two discrete groups clustering wild and domestic populations. Results did not show significant genetic divergence among Northern, Central and Southern wildcats. Six morphologically identified wildcats were significantly assigned to the domestic cluster, revealing some discrepancy between phenotypic and genetic identifications. We detected four hybrids (approximately 14%) using a consensus analysis of different Bayesian model-based software. These hybrids were identified throughout all sampled areas, suggesting that hybridisation is of major concern for the appropriate implementation of wildcat conservation strategies in Portugal.

Oliveira, R., Godinho, R., Randi, E., & Alves, P. C. (2008). Hybridization versus conservation: are domestic cats threatening the genetic integrity of wildcats (Felis silvestris silvestris) in Iberian Peninsula?  Philosophical Transactions of the Royal Society B: Biological Sciences, 363(1505), 2953-2961.

Cross-breeding between wild and free-ranging domestic species is one of the main conservation problems for some threatened species. The situation of wildcats (Felis silvestris silvestris) in Europe is a good example of this critical phenomenon. Extensive hybridization was described in Hungary and Scotland, contrasting with occasional interbreeding in Italy and Germany. First analyses in Portugal revealed a clear genetic differentiation between wild and domestic cats; however, four hybrids were detected. Here, we extended the approach to Iberian Peninsula using multivariate and Bayesian analyses of multilocus genotypes for 44 Portuguese wildcats, 31 Spanish wildcats and 109 domestic cats. Globally, wild and domestic cats were significantly differentiated (F _ST=0.20, p<0.001) and clustered into two discrete groups. Diverse clustering methods and assignment criteria identified an additional hybrid in Portugal, performing a total of five admixed individuals. The power of admixture analyses was assessed by simulating hybrid genotypes, which revealed that used microsatellites were able to detect 100, 91 and 85% of first-generation hybrids, second-generation genotypes and backcrosses, respectively. These findings suggest that the true proportion of admixture can be higher than the value estimated in this study and that the improvement of genetic tools for hybrids detection is crucial for wildcat conservation.


See more on domestic cat introgression in wildcat

Dog attacks to livestock in Spain

Echegaray, J. & Vilà, C. (2010). Noninvasive monitoring of wolves at the edge of their distribution and the cost of their conservation. Animal Conservation, 13(2), 157-161.

Large predators are recolonizing areas in industrialized countries, where they have been absent for decades or centuries. As they reach these areas, the predators often encounter unwary livestock and unprepared keepers, which translates into large economic costs. The cost per individual may have important repercussions on the conservation and management of large predators. During the years 2003–2004, we collected 136 feces preliminarily identified as belonging to gray wolves Canis lupus along the north-eastern limit of the wolf range in the Iberia peninsula (Basque Country, Spain). Genetic analyses allowed us to identify the species of origin in 86 cases: 31 corresponded to wolves, two to red foxes Vulpes vulpes and 53 to dogs Canis familiaris. Among the wolves, we identified 16 different individuals. We estimated the cost of conserving wolves to be >€3000 per wolf per year, based on the cost of damage compensation and prevention during the 2003–2004 period. However, most of the wolf feces contained wild prey whereas dog feces contained mostly remains of domestic animals. This finding suggests that uncontrolled dogs could be responsible for some of the attacks on livestock, contributing to negative public attitudes toward wolf conservation and increasing its cost.

Dog-wolf hybrids in Iberia

GODINHO, R., LLANEZA, L., BLANCO, J. C., LOPES, S., ÁLVARES, F., GARCÍA, E. J., PALACIOS,V. CORTÉS, Y., TALEGÓN, J. & FERRAND, N. (2011). Genetic evidence for multiple events of hybridization between wolves and domestic dogs in the Iberian Peninsula.Molecular Ecology, 20(24), 5154-5166.

Hybridization between wild species and their domestic counterparts may represent a major threat to natural populations. However, high genetic similarity between the hybridizing taxa makes the detection of hybrids a difficult task and may hinder attempts to assess the impact of hybridization in conservation biology. In this work, we used a combination of 42 autosomal microsatellites together with Y-chromosome microsatellite-defined haplotypes and mtDNA sequences to investigate the occurrence and dynamics of wolf–dog hybridization in the Iberian Peninsula. To do this, we applied a variety of Bayesian analyses and a parallel set of simulation studies to evaluate (i) the differences between Iberian wolves and dogs, (ii) the frequency and geographical distribution of hybridization and (iii) the directionality of hybridization. First, we show that Iberian wolves and dogs form two well-differentiated genetic entities, suggesting that introgressive hybridization is not a widespread phenomenon shaping both gene pools. Second, we found evidence for the existence of hybridization that is apparently restricted to more peripheral and recently expanded wolf populations. Third, we describe compelling evidence suggesting that the dynamics of hybridization in wolf populations is mediated by crosses between male dogs and female wolves. More importantly, the observation of a population showing the occurrence of a continuum of hybrid classes forming mixed packs may indicate that we have underestimated hybridization. If future studies confirm this pattern, then an intriguing avenue of research is to investigate how introgression from free-ranging domestic dogs is enabling wolf populations to adapt to the highly humanized habitats of southern Europe while still maintaining their genetic differentiation.

See more about wild canid hybridisation with dogs

Iberian lynx naïf to domestic carnivores pathogens

Roelke ME, Johnson WE, Millán J, Palomares F, Revilla E, Rodríguez A, Calzada J, Ferreras P, León-Vizcaíno, Delibes M & O’Brien SJ. 2008. Exposure to disease agents in endangered Iberian lynx (Lynx pardinus). European Journal of Wildlife Research, 54: 171-178

The Iberian lynx (Lynx pardinus) is the most endangered felid species in the world. Lynx populations have decreased dramatically in size and distribution in the last four decades, thus becoming increasingly vulnerable to catastrophic events such as epizooties. From 1989 to 2000, serum samples were obtained from 48 free-ranging lynx captured in the Doñana National Park (DNP, n = 31) and mountains of Sierra Morena (SM, n = 17) in southern Spain. Samples were tested for antibodies against Toxoplasma gondii, feline herpesvirus 1 (FHV-1), feline calicivirus (FCV), feline/canine parvovirus (FPV/CPV), feline coronavirus, feline immunodeficiency virus (FIV), feline leukaemia virus and canine distemper virus (CDV) and for FeLV p27 antigen, to document baseline exposure levels. Antibodies against T. gondii were detected in 44% of lynx, with a significantly greater prevalence in DNP (61%) than in SM (12%). In DNP, prevalence was significantly higher in adult (81%) than in juvenile and sub-adult (41%) lynx, but no such difference was observed in SM. Low prevalences (≤11%) of minimally positive titres were found for FHV-1, FCV and FPV/CPV. This, combined with the lack of evidence for exposure to CDV, FIV and FeLV, suggests that these lynx populations are naïve and might be vulnerable to a disease outbreak in the future. Because of the reduced size of lynx populations, the documented low level of genetic variation (particularly in the DNP population) coupled with the recently documented state of immune depletion in a majority of necropsied lynx, it is important to better understand the threat and potential impact that disease agents might pose for the conservation of this endangered species. Future surveillance programs must include possible disease reservoir hosts such as domestic cats and dogs and other wild carnivores.

Pathogens on Iberian lynx

Millán J, Candela MG, Palomares F, Cubero MJ, Rodríguez A, Barral M, de la Fuente J, Almería S & Vizcaíno LL. 2009. Disease threats to the endangered Iberian lynx (Lynx pardinus). The Veterinary Journal, 182: 114-124

The Iberian lynx, (Lynx pardinus), is the most endangered felid in the world. To determine whether sympatric carnivores are reservoirs of pathogens posing a disease risk for the lynx, evidence of exposure to 17 viral, bacterial and protozoan agents was investigated in 176 carnivores comprising 26 free-living lynx, 53 domestic cats, 28 dogs, 33 red foxes (Vulpes vulpes), 24 Egyptian mongooses (Herpestes ichneumon), 10 common genets (Genetta genetta) and 2 Eurasian badgers (Meles meles) in the areas inhabited by the last two populations of Iberian lynx, both in Andalusia (South-Western Spain).

The results indicated that the lynx had low rates of contact with viral pathogens, with one seropositive finding each for feline leukemia virus, parvovirus and canine adenovirus-1, whereas contact with bacteria and protozoa appeared more frequent. Active infections with parvovirus, Ehrlichia spp., Mycobacterium bovis,Leptospira interrogans and Cytauxzoon spp. were confirmed. In contrast, 53% of the domestic cats were exposed to some infectious agent (prevalence range 4.5–11.4%). Antibodies to canine distemper virus and parvovirus were frequently found in dogs (32% and 42%, respectively) and foxes (30% and 12%). Past or present infections with parvovirus, Ehrlichia spp., Chlamydophila spp., M. bovis, Salmonella enterica, L. interrogans, Toxoplasma gondii, and Neospora caninum were also detected in these and other species surveyed.

Questionnaires to owners revealed that 14% of the dogs but none of the cats had been vaccinated, and no cat had been neutered. Based on the apparent absence of acquired immunity of the lynx against infectious agents, the frequent detection of agents among sympatric carnivores, and the reported lack of immunocompetence of the Iberian lynx, a disease outbreak among the local abundant carnivores may pose a serious disease risk for lynx conservation.

FeLV on Iberian lynx

Meli M.L., Cattori V., Martínez F., López G., Vargas A., Palomares F., López-Bao J.V., Hofmann-Lehmann R. & Lutz H. 2010. Feline Leukemia virus infection: A threat for the survival of the critically endangered Iberian lynx (Lynx pardinus): Veterinary Immunology and Immunopathology, 134: 61-67

The Iberian lynx (Lynx pardinus) is considered the most endangered felid species in the world. To date, less than 200 animals remain in the wild. Low numbers and genetic uniformity may contribute to render this species particularly susceptible to infectious diseases. Different pathogens have been identified in Iberian lynxes; including several feline bacterial and viral agents. Within a 6-month period starting in December 2006, 12 lynxes living in the northern part of the Doñana area were found to be infected with feline leukemia virus (FeLV). Eleven of these animals were antigenemic, and four of them died in the wild in less than 6 months since the first infected animal had been discovered. The remaining viremic lynxes were captured and allocated to a quarantine center to stop the spread of the infection. An additional three animals died shortly in the quarantine center due to acute anemic disease. Sequencing of the envelope surface unit gene revealed a common origin for the FeLV found in all lynxes. The sequences were closely related to FeLV-A/61E, originally isolated from cats in the USA. Our data demonstrate that, similarly to FeLV, the introduction of a new or particularly pathogenic infection brought into the small population of Iberian lynxes by other wild carnivores or feral cats and dogs roaming in the same habitats have severe consequences. It could result in epidemics that have the potential to eradicate the entire lynx population

Pathogens from domestic cat on Iberian lynx

Meli M., Cattori V., Martínez F., López G., Vargas A., Simón M.A., Zorrilla I., Muñoz A., Palomares F., López-Bao J.V., Pastor J., Tandon R., Willi B., Hofmann-Lehmann R. & Lutz H. 2009. Feline Leukemia Virus and Other Pathogens as Important Threats to the Survival of the Critically Endangered Iberian Lynx (Lynx pardinus). PLoS ONE, 4(3): e4744

Background

The Iberian lynx (Lynx pardinus) is considered the most endangered felid species in the world. In order to save this species, the Spanish authorities implemented a captive breeding program recruiting lynxes from the wild. In this context, a retrospective survey on prevalence of selected feline pathogens in free-ranging lynxes was initiated.

Methodology/ Principal Findings

We systematically analyzed the prevalence and importance of seven viral, one protozoan (Cytauxzoon felis), and several bacterial (e.g., hemotropic mycoplasma) infections in 77 of approximately 200 remaining free-ranging Iberian lynxes of the Doñana and Sierra Morena areas, in Southern Spain, between 2003 and 2007. With the exception of feline immunodeficiency virus (FIV), evidence of infection by all tested feline pathogens was found in Iberian lynxes. Fourteen lynxes were feline leukemia virus (FeLV) provirus-positive; eleven of these were antigenemic (FeLV p27 positive). All 14 animals tested negative for other viral infections. During a six-month period in 2007, six of the provirus-positive antigenemic lynxes died. Infection with FeLV but not with other infectious agents was associated with mortality (p<0.001). Sequencing of the FeLV surface glycoprotein gene revealed a common origin for ten of the eleven samples. The ten sequences were closely related to FeLV-A/61E, originally isolated from cats in the USA. Endogenous FeLV sequences were not detected.

Conclusions/Significance

It was concluded that the FeLV infection most likely originated from domestic cats invading the lynx's habitats. Data available regarding the time frame, co-infections, and outcome of FeLV-infections suggest that, in contrast to the domestic cat, the FeLV strain affecting the lynxes in 2007 is highly virulent to this species. Our data argue strongly for vaccination of lynxes and domestic cats in and around lynx's habitats in order to prevent further spread of the virus as well as reduction the domestic cat population if the lynx population is to be maintained.

First detection of FeLV on Iberian lynx

Luaces I, Doménech A, García-Montijano M, Collado VM, Sánchez C, Tejerizo JG, Glaka M, Fernández P & Gómez-Lucía E. 2008. Detection of Feline Leukemia virus in the endangered Iberian lynx (Lynx pardinus). Journal of Veterinary Diagnostic Investigation, 20: 381-385

Feline retroviruses are rarely reported in lynx species. Twenty-one Iberian lynx (Lynx pardinus) blood and tissue samples collected from Doñana National Park and Los Villares (Sierra Morena) in southern Spain during 1993–2003 were analyzed by polymerase chain reaction to amplify nucleic acids from feline retroviruses. Six samples were positive for Feline leukemia virus (FeLV), but no samples tested positive for Feline immunodeficiency virus. The BLAST analysis indicated that 5 of the 6 sequences were closely related to FeLV strain Rickard subgroup A, whereas 1 sequence was identical to FeLV. To the authors' knowledge, this is the first report of FeLV in the endangered Iberian lynx.

FeLV and Iberian lynx

López G, López-Parra M, Fernández L, Martínez-Granados C, Martínez F, Meli ML, Gil-Sánchez JM, Viqueira N, Díaz-Portero MA, Cadenas R, Lutz H, Vargas A & Simón MA. 2009. Management measure to control a feline leukemia virus outbreak in the endangered Iberian lynx. Animal Conservation, 12: 173-182

The feline leukemia virus (FeLV) is a retrovirus that affects domestic cats all over the world. Its pathogenic effects generally include anemia, immunosuppression or tumors. Dissemination over populations is linked to cat sociality, because the virus is transmitted by direct contact. Although the domestic cat is its common host, FeLV infection has also been described in some wild felids. In the Iberian lynx Lynx pardinus, some sporadic FeLV infection cases have been reported since 1994, but an outbreak with the involvement of several animals has never been described until now. During spring 2007, an FeLV outbreak hit the Doñana (SW Spain) population. The infection rapidly spread throughout the densest subpopulation throughout Doñana. Infected animals showed very acute anemic disease, most of them dying in o6 months. To avoid FeLV dissemination, a control program was carried out that included removal of viremic lynxes, vaccination of negative individuals and reduction of the feral cat population. The program was implemented both in Donana and in Sierra Morena populations. In Doñana, around 80% of the total lynx population and 90% of the outbreak focus subpopulation were evaluated. Seven out of the 12 infected individuals found died and two reverted to latency; the remaining viremic animals have been kept in captivity. The outbreak appears to have been successfully confined to the subpopulation where the virus appeared and no more cases have been found since August 2007. In the larger Sierra Morena population, 8% of the lynx population was surveyed. Thirtyfour uninfected Iberian lynxes were vaccinated at least once. The FeLV prevalence was found to be 27% in the Doñana population and 0% in the Sierra Morena population.