A worldwide perspective on large carnivore attacks on humans.

Large carnivores have long fascinated human societies and have profound influences on ecosystems. However, their conservation represents one of the greatest challenges of our time, particularly where attacks on humans occur. Where human recreational and/or livelihood activities overlap with large carnivore ranges, conflicts can become particularly serious. Two different scenarios are responsible for such overlap: In some regions of the world, increasing human populations lead to extended encroachment into large carnivore ranges, which are subject to increasing contraction, fragmentation, and degradation. In other regions, human and large carnivore populations are expanding, thus exacerbating conflicts, especially in those areas where these species were extirpated and are now returning. We thus face the problem of learning how to live with species that can pose serious threats to humans. We collected a total of 5,440 large carnivore (Felidae, Canidae, and Ursidae; 12 species) attacks worldwide between 1950 and 2019. The number of reported attacks increased over time, especially in lower-income countries. Most attacks (68%) resulted in human injuries, whereas 32% were fatal. Although attack scenarios varied greatly within and among species, as well as in different areas of the world, factors triggering large carnivore attacks on humans largely depend on the socioeconomic context, with people being at risk mainly during recreational activities in high-income countries and during livelihood activities in low-income countries. The specific combination of local socioeconomic and ecological factors is thus a risky mix triggering large carnivore attacks on humans, whose circumstances and frequencies cannot only be ascribed to the animal species. This also implies that effective measures to reduce large carnivore attacks must also consider the diverse local ecological and social contexts.

Evolutionary applications of phylogenetically-informed ecological niche modelling (ENM) to explore cryptic diversification over cryptic refugia.

We used the fat dormouse (Glis glis), a species from a monotypic genus of family Gliridae, as a model to promote the understanding of patterns of cryptic diversification along the ancient Hyrcanian Forests, one of the old-growth relicts of the temperate deciduous forests worldwide. Mitochondrial Cytb data was used to investigate the phylogenetic status of two geographically-different populations of G. glis along the Hyrcanian Forests among all the worldwide known lineages of the species. Regarding phylogenetically informed partitioning of occurrence data, we then used two analytically different ENMs (i.e. environmental-space and geographic-space) to address whether niche divergence conforms G. glis diversification over the study area. Phylogenetic reconstruction showed significant heterogeneity between other fat dormouse lineages and those belonging to the Hyrcanian Forests as well as within the two hypothesized cryptic groups in the study area. Quantifying niche differences using the two ENM frameworks additionally confirmed divergence between the two cryptic lineages by indicating niche conservatism. The integration of phylogeny and ENM in this study confirms the development of distinct cryptic species and suggests that the Hyrcanian Forests, a well-known Pleistocene refugium, might contain multiple cryptic refugia for small forest-dwelling species during paleontological oscillations.

Conservation Below the Species Level: Suitable Evolutionarily Significant Units among Mountain Vipers (the Montivipera raddei complex) in Iran.

Northern and western mountains of Iran are among the most important biodiversity and endemism hot spots for reptiles in the Middle East. Among herpetofauna, the montivipers represent an emblematic and fragmented endemic group for which estimating their level of genetic differentiation and defining conservation priorities is urgently needed. Here, we present the most comprehensive phylogenetic study on the Montivipera raddei species group comprising all 5 known taxa, among which 3 are endemic to Iran. Based on 2 mitochondrial genes, phylogenetic and phylogeographic analyses revealed 3 major lineages each presenting very contrasting distribution areas. The Iranian montivipers are highly structured in clades showing low genetic diversity and corresponding to high altitude summits. Molecular dating revealed the role of Quaternary paleo-climatic oscillations and altitudinal movements of montivipers in shaping genetic diversity and differentiation of these sky-island taxa. In addition, the best scenario of historical biogeography allowed identifying 3 possible refugial areas in Iran most likely arising by vicariance. Based on our mitochondrial results and pending additional data, we recognize 3 candidate species among the M. raddei complex: M. raddei, Montivipera latifii, and Montivipera kuhrangica that are coherent with their geographical distribution. We propose that the most appropriate evolutionary significant units for conservation of the montivipers are represented by 13 units among which 6 are recognized as high priority. Finally, we suggest some recommendations to the IUCN as well as to the Iranian conservation policies with respect to conservation prioritization.

Integrating phylogenetic, phylogeographic, and morphometric analyses to reveal cryptic lineages within the genus Asaccus (Reptilia: Squamata: Phyllodactylidae) in Iran.

The Middle Eastern endemic genus Asaccus comprises Southwest Asian leaf-toed geckos. To date, this genus includes 19 species of leaf-toed geckos (seven in Arabia and 12 in the Zagros Mountains). Despite a recent study on the taxonomy and phylogeny of Asaccus species in Iran, controversies still remain surrounding the phylogeny and phylogeography of the genus. Here, we used an integrative approach to determine the phylogeny and phylogeography of Asaccus species using two mitochondrial genes (12 S and Cyt b), and one nuclear gene (c-mos). Our results uncovered 22 distinct lineages, demonstrating a significant cryptic diversity that challenges the current morphological classifications of these species. Phylogenetic analyses reinforce the monophyly of the Asaccus group, positioning A. montanus as a basal lineage, which supports a deep evolutionary divergence dating back to the Late Oligocene, approximately 27.94 million years ago. This genetic diversity also highlights the impact of historical climatic and geographical changes on species diversification. The findings advocate for an integrative approach combining both molecular and morphological data to resolve species identities accurately, thereby enhancing conservation strategies to protect these genetically distinct lineages.

Evaluation of venom diversity and antivenom quality from the venom of long-term captive vs recently wild captured Pseudocerastes persicus snake: An In vitro and In vivo study.

Snakebite envenomation is a life-threatening condition and antivenoms are used as the most effective treatment. Venom obtained from snakes in long-term captivity showed some variations in comparison to the venom of the wild snakes. The objective of this study is to compare the venom of the Pseudocerastes persicus under long-term captivity and wild conditions as well as the antivenom obtained from these venoms. We have analyzed venom samples and produced trivalent antivenoms using the venom of long-term captive (LTC) or recently wild-captured (RWC) Pseudocerastes persicus, and RWC Macrovipera lebetina, and Echis carinatus. The HPLC analysis revealed that the RWC snakes' venom had three peaks that were not present in the LTC snake's venom. Further analysis using MALDI-TOF and MS/MS showed that the fraction with a retention time (RT) of 14 min contained a toxin from the Kunitz-type serine protease inhibitor (KUT) class, while the fraction with RT 21 a peptide identified within the snake venom metalloproteinase (SVMP) class. The third peak was identified as a sphingolipid. Interestingly, the in vivo preclinical tests showed no significant differences in the effectiveness of the antivenoms. which could be due to the cross-immunogenicity or cross-reactivity between different toxins in the venom. According to our results, small variations in the venom composition of a species do not lead to a decrease in the efficacy of the polyvalent antivenom.

Detecting hybridization between Iranian wild wolf (Canis lupus pallipes) and free-ranging domestic dog (Canis familiaris) by analysis of microsatellite markers.

The genetic threat due to hybridization with free-ranging dogs is one major concern in wolf conservation. The identification of hybrids and extent of hybridization is important in the conservation and management of wolf populations. Genetic variation was analyzed at 15 unlinked loci in 28 dogs, 28 wolves, four known hybrids, two black wolves, and one dog with abnormal traits in Iran. Pritchard's model, multivariate ordination by principal component analysis and neighbor joining clustering were used for population clustering and individual assignment. Analysis of genetic variation showed that genetic variability is high in both wolf and dog populations in Iran. Values of H(E) in dog and wolf samples ranged from 0.75-0.92 and 0.77-0.92, respectively. The results of AMOVA showed that the two groups of dog and wolf were significantly different (F(ST) = 0.05 and R(ST) = 0.36; P < 0.001). In each of the three methods, wolf and dog samples were separated into two distinct clusters. Two dark wolves were assigned to the wolf cluster. Also these models detected D32 (dog with abnormal traits) and some other samples, which were assigned to more than one cluster and could be a hybrid. This study is the beginning of a genetic study in wolf populations in Iran, and our results reveal that as in other countries, hybridization between wolves and dogs is sporadic in Iran and can be a threat to wolf populations if human perturbations increase.

Effects of logged and unlogged forest patches on avifaunal diversity.

In the Hyrcanian forests of northern Iran, reduced-impact silviculture systems, (single-tree and group-tree selection) were applied over a large area, which generated different local habitat structures. The aim of this study was to assess the differences between treated and untreated areas of forest and their effect on avian richness, abundance and diversity (R.A.D). Birds were surveyed during the breeding season in 2009 by 100-point counts, equally distributed in the treated and untreated area. Avian R.A.D was significantly different and higher in the untreated area. Generally, forestry practices cause noticeable changes in canopy percentage, tree composition, snags and shrub number. Treated forest habitats in the area of study had a much more developed understory, fewer snags and fewer large diameter trees. The results highlighted the importance of forest maturity and showed that preventing silvicultural disturbances may not be the best solution for conserving and enhancing biodiversity. Rather, methods such as selective cutting seem an appropriate and sustainable way of forest management. It is suggested that forests should be managed to conserve structural elements which create favorable habitat for bird species, preventing future species losses due to logging practices.

MaxEnt brings comparable results when the input data are being completed; Model parameterization of four species distribution models.

Species distribution models (SDMs) are practical tools to assess the habitat suitability of species with numerous applications in environmental management and conservation planning. The manipulation of the input data to deal with their spatial bias is one of the advantageous methods to enhance the performance of SDMs. However, the development of a model parameterization approach covering different SDMs to achieve well-performing models has rarely been implemented. We integrated input data manipulation and model tuning for four commonly-used SDMs: generalized linear model (GLM), gradient boosted model (GBM), random forest (RF), and maximum entropy (MaxEnt), and compared their predictive performance to model geographically imbalanced-biased data of a rare species complex of mountain vipers. Models were tuned up based on a range of model-specific parameters considering two background selection methods: random and background weighting schemes. The performance of the fine-tuned models was assessed based on recently identified localities of the species. The results indicated that although the fine-tuned version of all models shows great performance in predicting training data (AUC > 0.9 and TSS > 0.5), they produce different results in classifying out-of-bag data. The GBM and RF with higher sensitivity of training data showed more different performances. The GLM, despite having high predictive performance for test data, showed lower specificity. It was only the MaxEnt model that showed high predictive performance and comparable results for identifying test data in both random and background weighting procedures. Our results highlight that while GBM and RF are prone to overfitting training data and GLM over-predict nonsampled areas MaxEnt is capable of producing results that are both predictable (extrapolative) and complex (interpolative). We discuss the assumptions of each model and conclude that MaxEnt could be considered as a practical method to cope with imbalanced-biased data in species distribution modeling approaches.

Convergent evolution of morphological and ecological traits in the open-habitat chat complex (Aves, Muscicapidae: Saxicolinae).

Open-habitat chats (genera Myrmecocichla, Cercomela, Oenanthe and relative) are a morphologically and ecologically cohesive group of genera with unclear phylogenetic relationships. They are distributed mostly in open, arid and/or rocky habitats of Africa and Eurasia. Here, we present the most comprehensive molecular phylogenetic analysis of this group to date, with a complete taxon sampling at the species level. The analysis, based on a multilocus dataset including three mitochondrial and three nuclear loci, allows us to elucidate the phylogenetic relationships and test the traditional generic limits. All genera are non-monophyletic, suggesting extensive convergence on similar plumage patterns in unrelated species. While the colour pattern appear to be a poor predictor of the phylogenetic relationships, some of the ecological and behavioural traits agree relatively well with the major clades. Following our results, we also propose a revised generic classification for the whole group.

Effect of habitat complexity on richness, abundance and distributional pattern of forest birds.

Structurally complex forests provide more diverse conditions in comparison to homogenous forests because of greater variety of microhabitats and trees. This study assesses the association of bird species richness, abundance, and distributional pattern with habitat complexity (HC) in Kheyrud Forest in the north of Iran. Birds were surveyed during spring 2009 by 100 point counts. In each point count six habitat features related to the index of HC were computed and scored from 0 to 3. Then the scores were summed and divided into two groups of low and high complexity, HC ≤ 6 and HC > 6, respectively. To compare bird richness and abundance in different HCs, a two sample t-test was used. Presence and absence of bird species at each plot as a dependent variable were compared with the vegetation characteristics as an independent variable by means of the Canonical Correspondence Analysis. The results revealed bird species richness and abundance were significantly higher in more complex habitats. Bird species can be divided into two groups, the first group including species which associated with late successional stages and the second group, species belonging to early successional stages. Numbers of birds belonging to the first group declined in less complex forests, whereas the numbers of birds belonging to the second group increased. At the stand scale, our results reveal that bird abundance and richness are strongly associated with the complexity of vegetation structure in the study area.

The phylogeny, phylogeography, and diversification history of the westernmost Asian cobra (Serpentes: Elapidae: Naja oxiana) in the Trans-Caspian region.

We conducted a comprehensive analysis of the phylogenetic, phylogeographic, and demographic relationships of Caspian cobra (Naja oxiana; Eichwald, 1831) populations based on a concatenated dataset of two mtDNA genes (cyt b and ND4) across the species' range in Iran, Afghanistan, and Turkmenistan, along with other members of Asian cobras (i.e., subgenus Naja Laurenti, 1768). Our results robustly supported that the Asiatic Naja are monophyletic, as previously suggested by other studies. Furthermore, N. kaouthia and N. sagittifera were recovered as sister taxa to each other, and in turn sister clades to N. oxiana. Our results also highlighted the existence of a single major evolutionary lineage for populations of N. oxiana in the Trans-Caspian region, suggesting a rapid expansion of this cobra from eastern to western Asia, coupled with a rapid range expansion from east of Iran toward the northeast. However, across the Iranian range of N. oxiana, subdivision of populations was not supported, and thus, a single evolutionary significant unit is proposed for inclusion in future conservation plans in this region.

The legacy of Eastern Mediterranean mountain uplifts: rapid disparity of phylogenetic niche conservatism and divergence in mountain vipers.

BACKGROUND: The orogeny of the eastern Mediterranean region has substantially affected ecological speciation patterns, particularly of mountain-dwelling species. Mountain vipers of the genus Montivipera are among the paramount examples of Mediterranean neo-endemism, with restricted ranges in the mountains of Anatolia, the Levant, Caucasus, Alborz, and Zagros. Here we explore the phylogenetic and ecological diversification of Montivipera to reconstruct its ecological niche evolution and biogeographic history. Using 177 sequences of three mitochondrial genes, a dated molecular phylogeny of mountain vipers was reconstructed. Based on 320 occurrence points within the entire range of the genus and six climatic variables, ecological niches were modelled and used to infer ancestral niche occupancy. In addition, the biogeographic history and ancestral states of the species were reconstructed across climate gradients. RESULTS: Dated phylogenetic reconstruction revealed that the ancestor of mountain vipers split into two major clades at around 12.18 Mya followed by multiple vicariance events due to rapid orogeny. Montivipera colonised coastal regions from a mountain-dwelling ancestor. We detected a highly complex ecological niche evolution of mountain vipers to temperature seasonality, a variable that also showed a strong phylogenetic signal and high contribution in niche occupation. CONCLUSION: Raising mountain belts in the Eastern Mediterranean region and subsequent remarkable changes in temperature seasonality have led to the formation of important centres of diversification and endemism in this biodiversity hotspot. High rates of niche conservatism, low genetic diversity, and segregation of ranges into the endemic distribution negatively influenced the adaptive capacity of mountain vipers. We suggest that these species should be considered as evolutionary significant units and priority species for conservation in Mediterranean mountain ecosystems.

Evolution and taxonomy of the wild species of the genus Ovis (Mammalia, Artiodactyla, Bovidae).

New insights for the systematic and evolution of the wild sheep are provided by molecular phylogenies inferred from Maximum parsimony, Bayesian, Maximum likelihood, and Neighbor-Joining methods. The phylogeny of the wild sheep was based on cytochrome b sequences of 290 samples representative of most of the sub-species described in the genus Ovis. The result was confirmed by a combined tree based on cytochrome b and nuclear sequences for 79 Ovis samples representative of the robust clades established with mitochondrial data. Urial and mouflon, which are either considered as a single or two separate species, form two monophyletic groups (O. orientalis and O. vignei). Their hybrids appear in one or the other group, independently from their geographic origin. The European mouflon O. musimon is clearly in the O. orientalis clade. The others species, O. dalli, O. canadensis, O. nivicola, and O. ammon are monophyletic. The results support an Asiatic origin of the genus Ovis, followed by a migration to North America through North-Eastern Asia and the Bering Strait and a diversification of the genus in Eurasia less than 3 million years ago. Our results show that the evolution of the genus Ovis is a striking example of successive speciation events occurring along the migration routes propagating from the ancestral area.

Comparison of venom from wild and long-term captive Gloydius caucasicus and the neutralization capacity of antivenom produced in rabbits immunized with captive venom.

Gloydius caucasicus (NIKOLSKY, 1916) is a member of the Viperidae family in Iran. Comprehensive understanding of the toxigenic characteristics of snake venom is important for clinical monitoring of snakebite patients and effective therapy. We compared the toxic activities of venoms and the neutralization capacity of antivenoms produced with venoms from wild adult (WA) with long-term captive adult (LCA) of G. caucasicus in order to obtain more effective antivenom from LCA in therapy, and subsequently protect G. caucasicus from overharvesting for its venom, which poses a real threat of extinction for the species. Our results showed that LD50 of WA and LCA were 16.8 µg/dose and 17.7 µg/dose, respectively. Lower hemorrhagic and necrotic (p ≥ 0.05), and higher coagulative and edematogenic activities (p ≤ 0.05) were observed in WA compared with LCA venom. Also, captive-born neonates exhibited weaker toxic activities compared with captive adult snakes, which could be an age-related difference. Study data illustrated that effective capacity of LCA antivenom to neutralize the toxic activities of WA viper venom. According to the results, about 0.4-4 µl of LCA antivenom is required to neutralize the toxic activities of 1 µg of WA venom, indicating its efficacy in treatment of snakebites in humans. On this basis, it is recommended that capture of wild snakes for their venom be discontinued to reduce their future extinction risk.

Anthropogenic food resources sustain wolves in conflict scenarios of Western Iran.

The feeding ecology of gray wolves has been investigated extensively worldwide. Despite previous studies on food habits of wolves in Asia and Iran, none has focused on the diet of the species in a scenario of depleted of wild prey and with recent records of attacks on humans. Here, we combined telemetry methods and scat analysis to study the diet of wolves in areas of Hamadan province, Iran, where medium to large wild prey is almost absent. Between October 2015 and March 2017, we studied the feeding behavior (by identifying feeding sites through clusters of GPS locations) of three wolves fitted with GPS collars, belonging to different wolf packs. We also collected and analyzed 110 wolf scats during the same period within the same areas. Overall, we investigated 850 clusters of GPS locations in the field, and identified 312 feeding sites. Most feeding clusters were linked to dumpsites and poultry farms around villages. We found 142 and 170 events of predatory (kill sites) and scavenging behavior, respectively. Prey composition based on kill sites was comprised of 74.6% livestock, 19.7% lagomorphs, 3.5% dogs, 1.4% red fox, and 0.7% golden jackal. Similarly, prey composition based on scavenging clusters was comprised of 79.9% livestock, 10.6% red fox, and 9.4% golden jackal. Scat analysis, however, indicated that livestock (34.3%), garbage (23.7%), poultry (16.0%), and European hare (15.4%) were the most frequent food items. We discuss the role of anthropogenic food sources in a context where agonistic wolf-human encounters occur recurrently, and suggest management guidelines regarding illegal dumping of animal carcasses and garbage dumpsites, in order to minimize wolf-human negative interactions.

Extinction risks of a Mediterranean neo-endemism complex of mountain vipers triggered by climate change.

Climate change is among the most important drivers of biodiversity decline through shift or shrinkage in suitable habitat of species. Mountain vipers of the genus Montivipera are under extreme risk from climate changes given their evolutionary history and geographic distribution. In this study, we divided all Montivipera species into three phylogenetic-geographic Montivipera clades (PGMC; Bornmuelleri, Raddei and Xanthina) and applied an ensemble ecological niche modelling (ENM) approach under different climatic scenarios to assess changes in projected suitable habitats of these species. Based on the predicted range losses, we assessed the projected extinction risk of the species relative to IUCN Red List Criteria. Our result revealed a strong decline in suitable habitats for all PGMCs (63.8%, 79.3% and 96.8% for Xanthina, Raddei and Bornmuelleri, respectively, by 2070 and under 8.5 RCP scenario) with patterns of altitudinal range shifts in response to projected climate change. We found that the mountains close to the Mediterranean Sea are exposed to the highest threats in the future (84.6 ± 9.1 percent range loss). We also revealed that disjunct populations of Montivipera will be additionally highly isolated and fragmented in the future. We argue that leveraging climate niche projections into the risk assessment provides the opportunity to implement IUCN criteria and better assess forthcoming extinction risks of species.

Evolutionary history and postglacial colonization of an Asian pit viper (Gloydius halys caucasicus) into Transcaucasia revealed by phylogenetic and phylogeographic analyses.

It has been generally acknowledged that glacial climates at the time of the Pleistocene altered the patterns of species distributions, prompting latitudinal and altitudinal distribution shifts in several species, including poikilothermic species commonly known for their thermal sensitivity. However, the historical phylogeographic patterns of such species have remained largely unknown. Here, we present the historical biogeographic, phylogenetic, and phylogeographic relationships of the Caucasian pit viper, G. h. caucasicus, based on two mtDNA (cyt b and ND4) and one nDNA (c-mos) genes. This pit viper represents the westernmost member of the Crotalinae subfamily in the Palearctic and occurs in a variety of habitats, from 30 m to 3,000 m above sea level. In Iran, it is distributed on the northern and southern slopes of the Alborz Mountains, rendering it a target for phylogenetic and phylogeographic studies of a terrestrial poikilothermic animal. Our study identified four Iranian lineages of G. h. caucasicus along the northeastern to northwestern slopes of the Alborz Mountains and southern Azerbaijan (Talysh Mountains). Diversification of the Iranian lineages highlights population expansion and subsequent isolation into four plausible refugial areas during the Quaternary paleo-climatic oscillations, confirmed by our molecular dating and historical biogeographic analyses. The results of coalescence-based simulations support the incursion of the species from northeastern Iran to the western end of the Alborz, and then toward Transcaucasia via two directions: northern and southern slopes of the Alborz Mountains. Furthermore, our results clearly implied that G. h. caucasicus should be elevated to species rank and further referred to as G. caucasicus (Nikolsky, 1916).

Upward Altitudinal Shifts in Habitat Suitability of Mountain Vipers since the Last Glacial Maximum.

We determined the effects of past and future climate changes on the distribution of the Montivipera raddei species complex (MRC) that contains rare and endangered viper species limited to Iran, Turkey and Armenia. We also investigated the current distribution of MRC to locate unidentified isolated populations as well as to evaluate the effectiveness of the current network of protected areas for their conservation. Present distribution of MRC was modeled based on ecological variables and model performance was evaluated by field visits. Some individuals at the newly identified populations showed uncommon morphological characteristics. The distribution map of MRC derived through modeling was then compared with the distribution of protected areas in the region. We estimated the effectiveness of the current protected area network to be 10%, which would be sufficient for conserving this group of species, provided adequate management policies and practices are employed. We further modeled the distribution of MRC in the past (21,000 years ago) and under two scenarios in the future (to 2070). These models indicated that climatic changes probably have been responsible for an upward shift in suitable habitats of MRC since the Last Glacial Maximum, leading to isolation of allopatric populations. Distribution will probably become much more restricted in the future as a result of the current rate of global warming. We conclude that climate change most likely played a major role in determining the distribution pattern of MRC, restricting allopatric populations to mountaintops due to habitat alterations. This long-term isolation has facilitated unique local adaptations among MRC populations, which requires further investigation. The suitable habitat patches identified through modeling constitute optimized solutions for inclusion in the network of protected areas in the region.

Is black coat color in wolves of Iran an evidence of admixed ancestry with dogs?

Melanism is not considered a typical characteristic in wolves of Iran and dark wolves are believed to have originated from crossbreeding with dogs. Such hybrid individuals can be identified with the combined use of genetic and morphological markers. We analyzed two black wolves using a 544 base pairs (bp) fragment of the mtDNA control region and 15 microsatellite loci in comparison with 28 dogs, 28 wolves, and four known hybrids. The artificial neural networks (ANNs) method was applied to microsatellite data to separate genetically differentiated samples of wolves, dogs, and hybrids, and to determine the correct class for the black specimens. Individual assignments based on ANNs showed that black samples were genetically closer to wolves. Also, in the neighbor-joining network of mtDNA haplotypes, wolves and dogs were separated, with the dark specimens located in the wolf branch as two separate haplotypes. Furthermore, we compared 20 craniometrical characters of the two black individuals with 14 other wolves. The results showed that craniometrical measures of the two black wolves fall within the range of wolf skulls. We found no trace of recent hybridization with free-ranging dogs in the two black wolves. Dark coat color might be the result of a natural combination of alleles in the coat-color-determining gene, mutation in the K locus due to past hybridization with free-ranging dogs, or the effect of ecological factors and adaption to habitat conditions.