Ecological genetics and conservation genomics of wolf (Canis lupus)

In the present work, we apply both traditional and Next Generation Sequencing (NGS) tools to investigate some of the most important adaptive traits of wolves (Canis lupus). In the first part, we analyze the variability of three Major Histocompatibility Complex (MHC) class II genes in the Italian wolf population, also studying their possible role in mating choice and their influence on fitness traits. In the second section, as part of a larger canid genome project, we will exploit NGS data to investigate the transcript-level differences between the wolf and the dog genome that can be correlated to domestication

Wolf outside, dog inside? The genomic make-up of the Czechoslovakian Wolfdog

Background Genomic methods can provide extraordinary tools to explore the genetic background of wild species and domestic breeds, optimize breeding practices, monitor and limit the spread of recessive diseases, and discourage illegal crossings. In this study we analysed a panel of 170k Single Nucleotide Polymorphisms with a combination of multivariate, Bayesian and outlier gene approaches to examine the genome-wide diversity and inbreeding levels in a recent wolf x dog cross-breed, the Czechoslovakian Wolfdog, which is becoming increasingly popular across Europe. Results Pairwise FST values, multivariate and assignment procedures indicated that the Czechoslovakian Wolfdog was significantly differentiated from all the other analysed breeds and also well-distinguished from both parental populations (Carpathian wolves and German Shepherds). Coherently with the low number of founders involved in the breed selection, the individual inbreeding levels calculated from homozygosity regions were relatively high and comparable with those derived from the pedigree data. In contrast, the coefficient of relatedness between individuals estimated from the pedigrees often underestimated the identity-by-descent scores determined using genetic profiles. The timing of the admixture and the effective population size trends estimated from the LD patterns reflected the documented history of the breed. Ancestry reconstruction methods identified more than 300 genes with excess of wolf ancestry compared to random expectations, mainly related to key morphological features, and more than 2000 genes with excess of dog ancestry, playing important roles in lipid metabolism, in the regulation of circadian rhythms, in learning and memory processes, and in sociability, such as the COMT gene, which has been described as a candidate gene for the latter trait in dogs. Conclusions In this study we successfully applied genome-wide procedures to reconstruct the history of the Czechoslovakian Wolfdog, assess individual wolf ancestry proportions and, thanks to the availability of a well-annotated reference genome, identify possible candidate genes for wolf-like and dog-like phenotypic traits typical of this breed, including commonly inherited disorders. Moreover, through the identification of ancestry-informative markers, these genomic approaches could provide tools for forensic applications to unmask illegal crossings with wolves and uncontrolled trades of recent and undeclared wolfdog hybrids

Križanje vukova i pasa u Hrvatskoj

Wolf-dog hybridization is considered as one of the main threats for wolf conservation since the admixture and introgression of domestic genes may disrupt local adaptations and threaten the long term survival of wild wolf populations. We investigated the occurrence of wolf-dog hybridization in Croatia by analyzing a panel of 12 autosomal microsatellite markers using Bayesian admixture tests, and assessed its directionality by the use of maternally and paternally inherited markers in combination with morphometric data and morphological features. A systematic analysis of morphologic features and morphometric data was used to rank the studied individuals into either phenotypic wild-type wolves or suspected hybrids. By combining Bayesian assignment results with phenotypic features, we set three thresholds which differentiated wolves from hybrids with maximized hybrid detection and a minimized chance for false positive hybrid identification. On the basis of phenotype, out of 176 wild canids, 157 (89.2%) were categorized as wolves and 19 (10.8%) as suspected hybrids. On the basis of the Bayesian admixture tests and phenotype together, five (2.8 percent) animals were classified as wolf-dog hybrids, four of them as backcrosses with wolves, and one as a backcross with a dog. Mitochondrial DNA suggested that all hybrids originated from the mating of female wolves and male dogs. Two male hybrids had Y chromosome haplotypes common to both wolves and dogs, while the other two had wolf private Y chromosome haplotypes. One wolf had a dog Y-haplotype, indicating a past introgression of dog genes. All hybrids were found in Dalmatia, where wolves settled recently, and where they live close to humans, with a high rate of human-caused mortality. These conditions are considered as favorable for wolf-dog hybridization. However, we found a low hybridization prevalence in Croatia, which is nonetheless expected to persist as long as the conditions favoring its occurrence are met. The ecological, sociological, conservation and management implications of hybrid occurrence are yet to be determined.Križanje vukova i pasa jedna je od glavnih prijetnji očuvanju vukova jer se miješanjem i introgresijom gena domaćih životinja mogu narušiti lokalne prilagodbe te tako ugroziti dugoročni opstanak populacija vukova u prirodi. Istražili smo pojavu križanja vukova i pasa u Hrvatskoj analizom 12 autosomskih mikrosatelitskih markera Bayesian testom primjese te utvrdili smjer hibridizacije određivanjem markera nasljeđivanih i po majčinskoj i po očinskoj liniji, u kombinaciji s određivanjem morfometrijskih i morfoloških svojstava. Sustavna analiza morfoloških svojstava i morfometrijskih podataka korištena je za rangiranje istraživanih jedinki, bilo kao fenotipske divlje tipove vukova bilo kao sumnjive na križanost. Kombiniranjem rezultata Bayesian statistike s fenotipskim obilježjima postavili smo prag za razlikovanje vukova od križanaca, koji povećava vjerojatnost određivanja križanaca uz smanjivanje vjerojatnosti za pogrešno pozitivno određivanje. Temeljem fenotipa, od 176 divljih kanida 157 (89,2 %) bilo je kategorizirano kao vukovi, dok je 19 (10,8 %) bilo svrstano u sumnjive križance. Temeljem Bayesian testa primjese, pet (2,8 %) životinja bilo je svrstano u križance vuka i psa, četiri od njih određena su kao unatražno križani s vukovima, dok je jedan bio unatražno križan sa psom. Mitohondrijska DNK pokazala je da su svi križanci bili rezultat parenja vučice sa psom. Dva su muška križanca na Y-kromosomu imala haplotipove i od vukova i od pasa, dok su druga dva imala Y-kromosomske haplotipove dosad utvrđene samo kod vukova. Jedan je vuk imao pseći Y-kromosomski haplotip što je uputilo na introgresiju psećih gena. Svi su križanci pronađeni u Dalmaciji, gdje su se vukovi nedavno proširili te gdje žive blizu ljudi, s visokom stopom smrtnosti uzrokovane od ljudi. Te se okolnosti inače smatraju pogodujućima za nastanak križanaca vukova i pasa. Ipak, pronađena učestalost pojave križanaca bila je niska, ali i za očekivati je da će se zadržati sve dok postoje uvjeti koji pogoduju nastanku križanaca. Ekološke, sociološke te značajke hibridizacije za zaštitu i upravljanje populacijom vukova tek treba utvrditi

CONSERVATION AT THE INDIVIDUAL LEVEL: SUCCESSFUL REHABILITATION AND POST-RELEASE MONITORING OF AN ITALIAN WOLF (Canis lupus italicus) INJURED IN A CAR ACCIDENT.

This case report describes the rescue of an eight-month-old male Italian wolf (Canis lupus italicus), the victim of a car accident that caused it a pulmonary contusion, a fracture of the shaft of right femur, and a metaphyseal fracture of the left stifle. A lateral surgical approach was performed to treat the animal\u2019s multiple contusions and fractures. Afterwards the wolf was transferred to a wild animal recovery center for its rehabilitation, where it fully recovered. After 35 days in captivity the wolf was thus released into the supposed home-range of its original pack, and its movements were monitored by a GPS satellite collar. The collar worked correctly for 479 days. During that period the collar acquired a total of 1202 locations, indicating that the wolf had traveled at least 1590 km, with an average monthly distance (\ub1 SD) of 102 \ub1 40 km, exploring an overall area of about 270 km2. During the first 10 days after its release, the wolf remained in the area of its supposed native pack, whereas at about the age of 10 months the wolf began to make wide extraterritorial movements. The wolf\u2019s last localization was acquired on 13th May 2018, about 17 months after its release, at a linear distance of about 65 km from the release site. This preliminary data showed that the wolf was alive and travelled long distances after its release, and demonstrates how a multidisciplinary management approach can support the recovery and successful release into nature of a rescued wild animal belonging to a flagship species with a notable ecological role, such as the Italian wolf

Disentangling the taxonomic status and phylogeographic structure of Marmora\u2019s (Curruca sarda) and Balearic Warbler (Curruca balearica): a genetic multi-marker approach

Marmora\u2019s Warbler (Curruca sarda) and Balearic Warbler (C. balearica) are allopatric sibling species and were recently split mostly based on morphological and ethological characteristics. Here we provide the first phylogenetic and phylogeographic analyses of this species complex to support the taxonomic status of C. sarda and C. balearica in light of integrative taxonomy. We sampled the two taxa in most of their breeding ranges and we sequenced three mitochondrial and one nuclear gene region. All C. balearica individuals had private haplotypes for the four markers and formed monophyletic clades. Genetic distances between the two taxa were comparable with those found between other species belonging to the Curruca genus. Furthermore, most of the genetic variance was expressed at the interspecific level, rather than between different populations within taxa or between individuals within populations. Our results strongly support the current taxonomic status of these two warblers as distinct species

Očuvanje na individualnoj razini: uspješna rehabilitacija i praćenje talijanskog vuka (Canis lupus italicus) nastradalog na prometnici nakon puštanja na slobodu

This case report describes the rescue of an eight-month-old male Italian wolf (Canis lupus italicus), the victim of a car accident that caused it a pulmonary contusion, a fracture of the shaft of right femur, and a metaphyseal fracture of the left stifle. A lateral surgical approach was performed to treat the animal’s multiple contusions and fractures. Afterwards the wolf was transferred to a wild animal recovery center for its rehabilitation, where it fully recovered. After 35 days in captivity the wolf was thus released into the supposed home-range of its original pack, and its movements were monitored by a GPS satellite collar. The collar worked correctly for 479 days. During that period the collar acquired a total of 1202 locations, indicating that the wolf had traveled at least 1590 km, with an average monthly distance (± SD) of 102 ± 40 km, exploring an overall area of about 270 km2. During the first 10 days after its release, the wolf remained in the area of its supposed native pack, whereas at about the age of 10 months the wolf began to make wide extraterritorial movements. The wolf’s last localization was acquired on 13th May 2018, about 17 months after its release, at a linear distance of about 65 km from the release site. These preliminary data showed that the wolf was alive and travelled long distances after its release, and demonstrates how a multidisciplinary management approach can support the recovery and successful release into nature of a rescued wild animal belonging to a flagship species with a notable ecological role, such as the Italian wolf.Ovaj prikaz slučaja opisuje spašavanje osmomjesečnog mužjaka talijanskog vuka (Canis lupus italicus) koji je bio žrtva naleta vozila s posljedičnim nagnječenjem pluća, prijelomom vrata desne bedrene kosti prijelomom u području metafize lijevoga koljena. Primijenjen je lateralni kirurški pristup kako bi se sanirala višestruka nagnječenja i prijelomi u životinje. Nakon toga je vuk prevezen u centar za oporavak divljih životinja gdje se potpuno oporavio. Nakon 35 dana u zatočeništvu vuk je pušten u okoliš nalik na njegov prirodni te je praćen preko GPS ogrlice. Ogrlica je ispravno radila 479 dana. Za to je vrijeme pokazala 1202 lokacije, upućujući na to da je vuk prešao 1590 km, u prosjeku mjesečno 102 ± 40 km (± SD), istražujući područje od 270 km2. Prvih 10 dana nakon oslobađanja vuk je ostao u području nalik na njegov prirodni okoliš, no u dobi od 10 mjeseci počeo je odlaziti na šire područje. Posljednja je njegova lokacija zabilježena 13. svibnja 2018., oko 17 mjeseci nakon puštanja, na zračnoj udaljenosti od oko 65 km od mjesta ispuštanja. Ovaj preliminarni podatak pokazuje da je vuk bio živ i prelazio velike udaljenosti nakon oslobađanja. Također pokazuje da multidisciplinarni pristup spašavanju može pospješiti oporavak i uspješno vraćanje u prirodu divljih životinja koje pripadaju vodećim vrstama s velikom ekološkom važnošću, kao što je talijanski vuk

Evaluating the quality of patient-specific deformable image registration in adaptive radiotherapy using a digitally enhanced head and neck phantom

This paper presents a deformable image registration-based method for the quality assurance of head and neck adaptive radiotherapy using digitally post-processed anthropomorphic phantom image datasets. One of the main findings of this work is that spatial and dose errors are a function of the magnitude of the deformation and of the gradient of the dose distribution. This emphasizes the importance of performing patient-specific deformable image registration verification and, consequently, the need to develop and make available tools that are for this purpose

A reduced SNP panel to trace gene flow across southern European wolf populations and detect hybridization with other Canis taxa

[EN] Intra- and inter-specific gene flow are natural evolutionary processes. However, human-induced hybridization is a global conservation concern across taxa, and the development of discriminant genetic markers to differentiate among gene flow processes is essential. Wolves (Canis lupus) are affected by hybridization, particularly in southern Europe, where ongoing recolonization of historic ranges is augmenting gene flow among divergent populations. Our aim was to provide diagnostic canid markers focused on the long-divergent Iberian, Italian and Dinaric wolf populations, based on existing genomic resources. We used 158 canid samples to select a panel of highly informative single nucleotide polymorphisms (SNPs) to (i) distinguish wolves in the three regions from domestic dogs (C. l. familiaris) and golden jackals (C. aureus), and (ii) identify their first two hybrid generations. The resulting 192 SNPs correctly identified the five canid groups, all simulated first-generation (F1) hybrids (0.482≤Qi≤0.512 between their respective parental groups) and all first backcross (BC1) individuals (0.723≤Qi≤0.827 to parental groups). An assay design and test with invasive and non-invasive canid samples performed successfully for 178 SNPs. By separating natural population admixture from inter-specific hybridization, our reduced panel can help advance evolutionary research, monitoring, and timely conservation management.We thank S. Czarnomska, A. Galov, J. Harmoinen, E. Velli, D. Battilani, P. Aragno, P. Genovesi, and the Mam- mal Research Institute, Polish Academy of Sciences, for their assistance. We are also grateful to two anonymous reviewers for their constructive feedback that greatly improved our manuscript. Funding was provided to ISPRA by the Italian Ministry of Environment (MATTM; Direzione Tutela della Natura) and Regione Emilia Romagna (Assessorato Agricoltura) within a multi-year collaborative project to genotype and monitor the Italian wolf population. AVS was supported by a senior postdoctoral fellowship from Insubria University, Italy. RG was sup- ported by a research contract from the Portuguese Foundation for Science and Technolog

Men and wolves: Anthropogenic causes are an important driver of wolf mortality in human-dominated landscapes in Italy

Over the last 40 years the gray wolf (Canis lupus) re-colonized its historical range in Italy increasing human-predator interactions. However, temporal and spatial trends in wolf mortality, including direct and indirect persecution, were never summarized. This study aims to fill this gap by focusing on the situation of Tuscany and Emilia-Romagna regions, hosting a significant proportion of the Italian wolf population, by: (i) identifying the prevalent causes of wolf mortality, (ii) summarizing their temporal and spatial patterns and (iii) applying spatially-explicit Generalized Linear Models to predict wolf persecution. Between October 2005 and February 2021, 212 wolf carcasses were collected and subjected to necropsy, being involved in collisions with vehicles (n = 104), poisoned (n = 45), wounded with gunshot (n = 24) or blunt objects (n = 4) and being hanged (n = 2). The proportion of illegally killed wolves did not increase through time. Most persecution events occurred between October and February. None of our candidate models outperformed a null model and covariates such as the density of sheep farms, number of predations on livestock, or human density were never associated to the probability of having illegally killed wolves, at the municipal scale. Our findings show that conventional correlates of wolf persecution, combined with a supposedly high proportion of non-retrieved carcasses, fail to predict illegal wolf killings in areas where the species have become ubiquitous. The widespread spatial distribution of illegal killings indicates that persecution probably arises from multiple kinds of conflicts with humans, beyond those with husbandry. Wolf conservation in Italy should thus address cryptic wolf killings with multi-disciplinary approaches, such as shared national protocols, socioecological studies, the support of experts’ experience and effective sampling schemes for the detection of carcasses