Luis Llaneza

Genetic evidence for multiple events of hybridization between wolves and domestic dogs in the Iberian Peninsula.

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.

New techniques for an old disease: Sarcoptic mange in the Iberian wolf

Sarcoptic mange, a parasitic skin infection caused by the burrowing mite Sarcoptes scabiei, has been reported in over 100 mammals, including humans. In endangered species, mange causes conservation concerns because it may decimate isolated populations and contribute to extinction. The Iberian Peninsula still maintains one of the largest wolf (Canis lupus) populations in Europe. In Iberia, sarcoptic mange is endemic in red foxes (Vulpes vulpes) and the first confirmed wolf mange cases were recently reported. However, knowledge on S. scabiei in wolves is scarce because of the sampling difficulties inherent to research on scarce species. In order to describe wolf mange epidemiology and to infer conservation implications, this study combined traditional laboratory techniques with the revision of wolf carcass pictures taken by field biologists and original information obtained by camera trapping. A total of 125 necropsies and 8783 camera-trap days allowed insights into wolf mange epidemiology between 2003 and 2010. Living Sarcoptes mites were detected in 19% of the fresh carcasses. Alopecic (delayed) type IV hypersensitive response reactions were observed, while parakeratotic lesions were infrequent. The number of mites isolated per wolf ranged from 1 to 78, and had a negative correlation with the percentage of alopecic skin. No effect by sex on mange prevalence was found. Yearlings showed a lower probability to present mange-compatible lesions than pups or adults. Wolves with mange-compatible lesions had a lower kidney fat index than apparently healthy ones. ELISA testing of 88 sera yielded an antibody prevalence of 20%. Photo-trapping recorded mange-compatible lesions since 2003 with a peak in 2008. The percentage of wolves with mange-compatible lesions registered in camera-traps during 1 year correlated with the percentage of red foxes with lesions in the previous year. This is the first large survey on sarcoptic mange in the Iberian wolf. Necropsy data, with alopecia as the main feature and a slight effect on body condition, and trends derived from camera trapping coincided in showing a rather low prevalence and an apparently stable situation of the disease and its host, suggesting that this parasite is currently not a major threat for this wolf population. However, more information is needed in order to assess the effect of mange on aspects such as pup survival.

Toothless wildlife protection laws

Granting legal protection to an endangered species has long been considered a major milestone for its conservation and recovery. A multitude of examples such as wolves in the contiguous USA (Boitani 2003) or many large carnivore populations in Europe (Chapron et al. 2014) have revealed how instrumental wildlife protection laws can be for species recovery. However, legal obligations to conserve endangered species may be useless if the rule of law is not properly enforced. Such situation is not exclusive to countries with political instability or weak institutional capacities but can also be relevant, for instance, to member states of the European Union and therefore bound to European legislation on nature conservation.

Real-time assessment of hybridization between wolves and dogs: combining noninvasive samples with ancestry informative markers.

Wolves and dogs provide a paradigmatic example of the ecological and conservation implications of hybridization events between wild and domesticated forms. However, our understanding of such implications has been traditionally hampered by both high genetic similarity and the difficulties in obtaining tissue samples (TS), which limit our ability to assess ongoing hybridization events. To assess the occurrence and extension of hybridization in a pack of wolf–dog hybrids in northwestern Iberia, we compared the power of 52 nuclear markers implemented on TS with a subset of 13 ancestry informative markers (AIMs) typed in noninvasive samples (NIS). We demonstrate that the 13 AIMs are as accurate as the 52 markers that were chosen without regard to the power to differentiate between wolves and dogs, also having the advantage of being rapidly screened on NIS. The efficiency of AIMs significantly outperformed ten random sets of similar size and an additional commercial set of 18 markers. Bayesian clustering analysis implemented on AIMs and NIS identified nine hybrids, two wolves and two dogs. Four hybrids were unambiguously assigned to F1xWolf backcrosses. Our approach (AIMs + NIS) overcomes previous difficulties related to sample availability and informative power of markers, allowing a quick identification of wolf–dog hybrids in the first phases of hybridization episodes. This provides managers with a reliable tool to evaluate hybridization and estimate the success of their actions. This approach may be easily adapted for other pairs of wild/domesticated species, thus improving our understanding of the introgression of domestication genes into natural populations.

Wolf population genetics in Europe: a systematic review, meta-analysis and suggestions for conservation and management

The grey wolf (Canis lupus) is an iconic large carnivore that has increasingly been recognized as an apex predator with intrinsic value and a keystone species. However, wolves have also long represented a primary source of human–carnivore conflict, which has led to long‐term persecution of wolves, resulting in a significant decrease in their numbers, genetic diversity and gene flow between populations. For more effective protection and management of wolf populations in Europe, robust scientific evidence is crucial. This review serves as an analytical summary of the main findings from wolf population genetic studies in Europe, covering major studies from the ‘pre‐genomic era’ and the first insights of the ‘genomics era’. We analyse, summarize and discuss findings derived from analyses of three compartments of the mammalian genome with different inheritance modes: maternal (mitochondrial DNA), paternal (Y chromosome) and biparental [autosomal microsatellites and single nucleotide polymorphisms (SNPs)]. To describe large‐scale trends and patterns of genetic variation in European wolf populations, we conducted a meta‐analysis based on the results of previous microsatellite studies and also included new data, covering all 19 European countries for which wolf genetic information is available: Norway, Sweden, Finland, Estonia, Latvia, Lithuania, Poland, Czech Republic, Slovakia, Germany, Belarus, Russia, Italy, Croatia, Bulgaria, Bosnia and Herzegovina, Greece, Spain and Portugal. We compared different indices of genetic diversity in wolf populations and found a significant spatial trend in heterozygosity across Europe from south‐west (lowest genetic diversity) to north‐east (highest). The range of spatial autocorrelation calculated on the basis of three characteristics of genetic diversity was 650−850 km, suggesting that the genetic diversity of a given wolf population can be influenced by populations up to 850 km away. As an important outcome of this synthesis, we discuss the most pressing issues threatening wolf populations in Europe, highlight important gaps in current knowledge, suggest solutions to overcome these limitations, and provide recommendations for science‐based wolf conservation and management at regional and Europe‐wide scales.

Patterns of Exposure of Iberian Wolves (Canis lupus) to Canine Viruses in Human-Dominated Landscapes.

Wildlife inhabiting human-dominated landscapes is at risk of pathogen spill-over from domestic species. With the aim of gaining knowledge in the dynamics of viral infections in Iberian wolves (Canis lupus) living in anthropized landscapes of northern Spain, we analysed between 2010 and 2013 the samples of 54 wolves by serology and polymerase chain reaction (PCR) for exposure to four pathogenic canine viruses: canine distemper virus (CDV), canine parvovirus-2 (CPV), canine adenovirus 1 and 2 (CAV-1 and CAV-2) and canine herpesvirus. Overall, 76% of the studied wolves presented evidence of exposure to CPV (96% by HI, 66% by PCR) and 75% to CAV (75% by virus neutralization (VN), 76% by PCR, of which 70% CAV-1 and 6% CAV-2). This represents the first detection of CAV-2 infection in a wild carnivore. CPV/CAV-1 co-infection occurred in 51% of the wolves. The probability of wolf exposure to CPV was positively and significantly correlated with farm density in a buffer zone around the place where the wolf was found, indicating that rural dogs might be the origin of CPV infecting wolves. CPV and CAV-1 appear to be enzootic in the Iberian wolf population, which is supported by the absence of seasonal and inter-annual variations in the proportion of positive samples detected. However, while CPV may depend on periodical introductions by dogs, CAV-1 may be maintained within the wolf population. All wolves were negative for exposure to CDV (by VN and PCR) and CHV (by PCR). The absence of acquired immunity against CDV in this population may predispose it to an elevated rate of mortality in the event of a distemper spill-over via dogs.

Decoding Group Vocalizations: The Acoustic Energy Distribution of Chorus Howls Is Useful to Determine Wolf Reproduction

Population monitoring is crucial for wildlife management and conservation. In the last few decades, wildlife researchers have increasingly applied bioacoustics tools to obtain information on several essential ecological parameters, such as distribution and abundance. One such application involves wolves (Canis lupus). These canids respond to simulated howls by emitting group vocalizations known as chorus howls. These responses to simulated howls reveal the presence of wolf litters during the breeding period and are therefore often used to determine the status of wolf populations. However, the acoustic structure of chorus howls is complex and discriminating the presence of pups in a chorus is sometimes difficult, even for experienced observers. In this study, we evaluate the usefulness of analyses of the acoustic energy distribution in chorus howls to identify the presence of pups in a chorus. We analysed 110 Iberian wolf chorus howls with known pack composition and found that the acoustic energy distribution is concentrated at higher frequencies when there are pups vocalizing. We built predictive models using acoustic energy distribution features to determine the presence of pups in a chorus, concluding that the acoustic energy distribution in chorus howls can be used to determine the presence of wolf pups in a pack. The method we outline here is objective, accurate, easily implemented, and independent of the observers experience. These advantages are especially relevant in the case of broad scale surveys or when many observers are involved. Furthermore, the analysis of the acoustic energy distribution can be implemented for monitoring other social canids that emit chorus howls such as jackals or coyotes, provides an easy way to obtain information on ecological parameters such as reproductive success, and could be useful to study other group vocalizations.

Multimethod, multistate Bayesian hierarchical modeling approach for use in regional monitoring of wolves

In many cases, the first step in large-carnivore management is to obtain objective, reliable, and cost-effective estimates of population parameters through procedures that are reproducible over time. However, monitoring predators over large areas is difficult, and the data have a high level of uncertainty. We devised a practical multimethod and multistate modeling approach based on Bayesian hierarchical-site-occupancy models that combined multiple survey methods to estimate different population states for use in monitoring large predators at a regional scale. We used wolves (Canis lupus) as our model species and generated reliable estimates of the number of sites with wolf reproduction (presence of pups). We used 2 wolf data sets from Spain (Western Galicia in 2013 and Asturias in 2004) to test the approach. Based on howling surveys, the naïve estimation (i.e., estimate based only on observations) of the number of sites with reproduction was 9 and 25 sites in Western Galicia and Asturias, respectively. Our model showed 33.4 (SD 9.6) and 34.4 (3.9) sites with wolf reproduction, respectively. The number of occupied sites with wolf reproduction was 0.67 (SD 0.19) and 0.76 (0.11), respectively. This approach can be used to design more cost-effective monitoring programs (i.e., to define the sampling effort needed per site). Our approach should inspire well-coordinated surveys across multiple administrative borders and populations and lead to improved decision making for management of large carnivores on a landscape level. The use of this Bayesian framework provides a simple way to visualize the degree of uncertainty around population-parameter estimates and thus provides managers and stakeholders an intuitive approach to interpreting monitoring results. Our approach can be widely applied to large spatial scales in wildlife monitoring where detection probabilities differ between population states and where several methods are being used to estimate different population parameters.

Spatial assessment of wolf-dog hybridization in a single breeding period.

Understanding the dynamics of wolf-dog hybridization and delineating evidence-based conservation strategies requires information on the spatial extent of wolf-dog hybridization in real-time, which remains largely unknown. We collected 332 wolf-like scats over ca. 5,000km2 in the NW Iberian Peninsula to evaluate wolf-dog hybridization at population level in a single breeding/pup-rearing season. Mitochondrial DNA (MtDNA) and 18 ancestry informative markers were used for species and individual identification, and to detect wolf-dog hybrids. Genetic relatedness was assessed between hybrids and wolves. We identified 130 genotypes, including 67 wolves and 7 hybrids. Three of the hybrids were backcrosses to dog whereas the others were backcrosses to wolf, the latter accounting for a 5.6% rate of introgression into the wolf population. Our results show a previously undocumented scenario of multiple and widespread wolf-dog hybridization events at the population level. However, there is a clear maintenance of wolf genetic identity, as evidenced by the sharp genetic identification of pure individuals, suggesting the resilience of wolf populations to a small amount of hybridization. We consider that real-time population level assessments of hybridization provide a new perspective into the debate on wolf conservation, with particular focus on current management guidelines applied in wolf-dog hybridization events.

Toward reliable population estimates of wolves by combining spatial capture-recapture models and non-invasive DNA monitoring

Decision-makers in wildlife policy require reliable population size estimates to justify interventions, to build acceptance and support in their decisions and, ultimately, to build trust in managing authorities. Traditional capture-recapture approaches present two main shortcomings, namely, the uncertainty in defining the effective sampling area, and the spatially-induced heterogeneity in encounter probabilities. These limitations are overcome using spatially explicit capture-recapture approaches (SCR). Using wolves as case study, and non-invasive DNA monitoring (faeces), we implemented a SCR with a Poisson observation model in a single survey to estimate wolf density and population size, and identify the locations of individual activity centres, in NW Iberia over 4,378 km2. During the breeding period, posterior mean wolf density was 2.55 wolves/100 km2 (95%BCI = 1.87–3.51), and the posterior mean population size was 111.6 ± 18.8 wolves (95%BCI = 81.8–153.6). From simulation studies, addressing different scenarios of non-independence and spatial aggregation of individuals, we only found a slight underestimation in population size estimates, supporting the reliability of SCR for social species. The strategy used here (DNA monitoring combined with SCR) may be a cost-effective way to generate reliable population estimates for large carnivores at regional scales, especially for endangered species or populations under game management.