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Dewclaws in wolves as evidence of admixed ancestry with dogs



Vestigial first toes (dewclaws) on the hind legs are common in large dog (Canis lupus familiaris) breeds but are absent in wild canids, including wolves (Canis lupus). Based on observational criteria, dewclaws in wolves have been generally regarded as a clue of hybridization with dogs, although this was not substantiated by molecular evidence. By means of population assignment and genetic admixture analysis, we investigated individual genotypes of three dewclawed wolves from Tuscany (central Italy, 1993–2001). Based on 18 microsatellite markers, dewclawed wolves were not uniquely assigned to the Italian wolf population but appeared to be second or later generation backcrosses of wolf–dog hybrids. Alleles uniquely shared with dogs, and mitochondrial DNA and Y haplotypes identical to those of Italian wolves, further supported their admixed ancestry. Although patterns of dewclaw inheritance in wolf–dog hybrids and backcrosses have not been ascertained, we conclude that dewclaws in wolves, when present, are a clue of admixed ancestry, probably originating in areas where large dog breeds are involved in cross-matings. Other "atypical" morphological traits (e.g., white nails, atypical color patterns or body proportions, dental anomalies) as well might be reliable clues of admixed ancestry, and they deserve careful monitoring and molecular investigation.
Dewclaws in wolves as evidence of admixed
ancestry with dogs
Paolo Ciucci, Vittorio Lucchini, Luigi Boitani, and Ettore Randi
Abstract: Vestigial first toes (dewclaws) on the hind legs are common in large dog (Canis lupus familiaris) breeds but
are absent in wild canids, including wolves (Canis lupus). Based on observational criteria, dewclaws in wolves have
been generally regarded as a clue of hybridization with dogs, although this was not substantiated by molecular evi-
dence. By means of population assignment and genetic admixture analysis, we investigated individual genotypes of
three dewclawed wolves from Tuscany (central Italy, 1993–2001). Based on 18 microsatellite markers, dewclawed
wolves were not uniquely assigned to the Italian wolf population but appeared to be second or later generation back-
crosses of wolf–dog hybrids. Alleles uniquely shared with dogs, and mitochondrial DNA and Y haplotypes identical to
those of Italian wolves, further supported their admixed ancestry. Although patterns of dewclaw inheritance in wolf–
dog hybrids and backcrosses have not been ascertained, we conclude that dewclaws in wolves, when present, are a
clue of admixed ancestry, probably originating in areas where large dog breeds are involved in cross-matings. Other
“atypical” morphological traits (e.g., white nails, atypical color patterns or body proportions, dental anomalies) as well
might be reliable clues of admixed ancestry, and they deserve careful monitoring and molecular investigation.
Résumé : Des premiers orteils vestigiaux (ergots) existent fréquemment sur les pattes postérieures des races de chiens
(Canis lupus familiaris) de grande taille, mais sont absents chez les canidés sauvages, y compris les loups (Canis lu-
pus). En se basant sur des critères visuels, on a généralement considéré la présence d’ergots chez les loups comme un
indice d’une hybridation avec les chiens, bien que les données moléculaires n’aient pas appuyé cette conclusion. Nous
avons étudié les génotypes individuels de trois loups de Toscane (Italie centrale, 1993–2001) qui possèdent des ergots
au moyen d’une méthode d’assignation de population et d’une analyse de mélange génétique. D’après 18 marqueurs
microsatellites, les loups à ergots ne se regroupent pas exclusivement avec la population italienne de loups, mais ils
semblent être des hybrides loups–chiens issus de rétrocroisements de seconde génération ou de génération précédente.
Des allèles communs avec seulement les chiens, d’une part, et des haplotypes ADN mitochondrial et Y identiques à
ceux des loups italiens, d’autre part, viennent confirmer leur ascendance mixte. Bien que les patterns de transmission
des ergots chez les hybrides loups–chiens et chez les animaux issus de rétrocroisements n’aient pas été déterminés,
nous croyons que la présence d’ergots chez les loups est un indice d’une ascendance mixte, provenant de régions où
des chiens de races de grande taille sont impliqués dans les accouplements mixtes. D’autres caractères morphologiques
« particuliers » (e.g., tels que les ongles blancs, les patterns de couleur ou les proportions corporelles inusités et les
anomalies dentaires) peuvent aussi s’avérer être des indices fiables d’ascendance mixte; ils méritent un suivi attentif et
devraient donner lieu à une analyse moléculaire.
[Traduit par la Rédaction] Ciucci et al. 2081
Hybridization between wolves (Canis lupus) and domestic
dogs (Canis lupus familiaris) has often been feared in Eur-
asia in areas where human-induced low wolf densities, habi-
tat fragmentation, and large number of free-ranging dogs
increase the chances of nonpredatory wolf–dog encounters
(Boitani 1983, 1984; Bibikov 1985; Blanco et al. 1992;
Andersone et al. 2002). However, until recently wolf–dog
hybrids have been diagnosed solely on the basis of atypical
or anomalous morphological characters (i.e., size, propor-
tions, color patterns, etc.; Kronit 1971 cited in Andersone et
al. 2002; Boitani 1983; Bibikov 1985), a method whose reli-
ability is limited because it is essentially observational and
Can. J. Zool. 81: 2077–2081 (2003) doi: 10.1139/Z03-183 © 2003 NRC Canada
Received 3 October 2002. Accepted 7 October 2003. Published on the NRC Research Press Web site at on
23 January 2004.
P. Ciucci1and L. Boitani. Università di Roma “La Sapienza”, Dipartimento di Biologia Animale e dell’Uomo, Viale
dell’Università, 32-00185 Roma, Italy.
V. Lucchini and E. Randi. Istituto Nazionale per la Fauna Selvatica, Via Cà Fornacetta, 9-40064 Ozzano Emilia, Italy.
1Corresponding author (e-mail:
January 15, 2004 7:46:32 AM
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subjective. In addition, not all morphological variation has a
genetic basis, and hybrids are not always morphologically
distinguishable from the parental taxon (Allendorf et al.
Although recent DNA population-level studies failed to
reveal large-scale introgression of dog genes in European
wolf populations (Vilà and Wayne 1999; Randi et al. 2000;
Randi and Lucchini 2002), molecular genetic markers did al-
low detection of some wolf–dog hybrids in Europe (Vilà et
al. 1997, 2003; Randi et al. 2000; Andersone at al. 2002;
Randi and Lucchini 2002; Lucchini et al. 2004). In addition,
rare backcrosses of wolf–dog hybrids into the parental wolf
population were already reported in Italy by Randi and
Lucchini (2002) who called for the development of a suite of
morphological, behavioral, and molecular traits to aid in the
distinction of pure wolf populations and areas of potential
introgression. However, limited molecular evidence is cur-
rently available on the admixed nature of anomalous
morphological characters, which are generally considered
“atypical” in wolves.
The dewclaw, a vestigial first toe on the hind leg, is never
found in wolves but can be common in large dog breeds.
Dewclaws in wolves were never observed by several North
American and Eurasian wolf biologists, and were not found
in the large wolf-pelt collections from Spain, Portugal, and
Italy (J.C. Blanco, L.D. Mech, F. Petrucci–Fonseca, S. Reig,
C. Vilà, personal communications). A case was reported from
Extremadura (Spain), in an area where wolves, large mastiff
dogs, and putative wolf–dog hybrids were known to occur
(Teruelo and Valverde 1992: 261). Similarly, from a large
collection of dingo (Canis lupus dingo) skins in the Natural
History Museum of London, only one specimen displayed
dewclaws; however, its skin did not conform to the standard
of purebred dingoes (J. Clutton-Brock, personal communica-
tion). In coyotes (Canis latrans), dewclaws are commonly
believed by some researchers to be a sign of hybridization
with dogs (L.D. Mech, personal communication), and the
same anecdotal evidence appears to hold for wolves. A com-
pilation of observational material reviewed by wolf special-
ists from the Great Lakes area in North America suggested
that dewclaws in wolves indicated hybridization with dogs
(Duman 2001), although no molecular investigation sup-
ported this conclusion. More recently, Andersone et al.
(2002) found dewcalws in a litter of seven mongrel pups
from northern Latvia whose individual genotypes (16 micro-
satellites) showed that most of the alleles were common with
dogs, whereas their mitochondrial DNA (mtDNA) haplotype
was typical of other Latvian wolves.
In contrast to wolves, dewclaws can be quite common in
large domestic dogs. Although in some breeds the character
is maintained through artificial selection (e.g., Great Pyre-
nees, St. Bernard), in others (Bernese, Newfoundland) it keeps
reappearing in the population despite being consistently se-
lected against (Alberch 1985). Both developmental (Alberch
1985) and genetic (Galis et al. 2001) arguments have been
used to explain the occurrence of dewclaws in dogs (see Dis-
cussion). Based on these theoretical grounds, we hypothe-
sized that dewclaws in wolves represent a trait inherited
through cross-mating with dogs rather than an expression of
phenotypic variation in wolves. To test this hypothesis we
analyzed 18 canine microsatellite loci, supported by addi-
tional maternal and paternal genetic markers, to assess the
individual genotypes of dewclawed wolves and compared
them with Italian wolves and dogs.
In 1993–2001 we closely examined six wolves from the
Tuscany region (central Italy), four of which were illegally
or accidentally killed and two were livetrapped as part of a
radiotelemetry study. Three of these wolves displayed the
dewclaw on both hind legs, representing the first and only
wolves observed in Italy with this character out of 206 car-
casses examined from 1986 to 2001 (V. Guberti, personal
communication), and 19 wolves livetrapped since the 1970s
(Ciucci and Boitani 1998). Age of dead or livetrapped
wolves was estimated from patterns of dentition consump-
tion (Gipson et al. 2000). All dewclawed wolves (W520,
W535, W587) were from an area in south-central Tuscany,
which composes a secondary branch of the main wolf distri-
bution in Italy. The area consists of semiagricultural land-
scapes at about 300 m a.s.l. with extensive sheep production
and large numbers of maremma-type guard dogs, most of
which display dewclaws.
Wolf W535 was a 32-kg 6-month-old male livetrapped
and radio-collared in November 1993 in an area of the Prov-
ince of Siena where a wolf pack was being monitored by ra-
diotelemetry. W535 showed a high degree of association
with W390, the radio-collared alpha male of the pack and
potential father of W535, and displayed ecological and be-
havioral attitudes typical of wolves (P. Ciucci, L. Boitani,
E. Tedesco, and L. Artoni, unpublished data). Although
W390 did not display dewclaws and matched the phenotypic
standard of wolves, we never closely observed W535’s
mother or siblings. W535 died, apparently in good health,
from a vehicle collision in February 1994 at 9 months,
whereas W390 was illegally shot 1 month later. W520 was a
28-kg 9-month-old male poisoned in February 1999 also in
the Province of Siena, and W587 was an unaged female ille-
gally shot in November 2001 in the Province of Grosseto (N.
Cini, personal communication). Distances between the local-
ities where the three wolves were found ranged 20–60 km.
Phenotypically, the three dewclawed wolves were judged by
experienced observers to fall within the standard variability
of Italian wolves, with the exception of W520’s skull, which
at later inspection was, on average, smaller and displayed a
pronounced prognathism. W587 was found decapitated.
All dewclawed wolves were tissue-sampled for genetic
analyses, and DNA extraction and microsatellite genotyping
followed protocols by Randi and Lucchini (2002). We also
included in the analysis a set of 100 Italian wolves and 95
domestic and feral dogs as reference populations. Individual
genotypes of the dewclawed wolves were determined at 18
loci assigned to 16 different chromosomes (Neff et al. 1999)
as described in Randi and Lucchini (2002). Wolves and dogs
have distinct multilocus microsatellite genotypes, and hy-
brids can be identified by assignment procedures (Randi and
Lucchini 2002; Vilà et al. 2003). Multilocus genotypes of
dewclawed wolves were assigned to the Italian wolf or dog
populations using two procedures: a factorial correspondence
analysis (FCA; Benzécri 1973) of individual multilocus
scores computed using GENETIX version 4.02 (Belkhir et
© 2003 NRC Canada
2078 Can. J. Zool. Vol. 81, 2003
January 15, 2004 7:46:32 AM
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al. 1996–2001) and a Bayesian admixture analyses imple-
mented in STRUCTURE version 1.0 (Pritchard et al. 2000).
We used STRUCTURE with 100 000 iterations following a
burn-in period of 10 000 iterations to identify the number of
genetically distinct clusters that maximize the likelihood of
the data and to assign the individuals to the clusters using
only genetic information (Randi and Lucchini 2002; Luc-
chini et al. 2004). The amount of dog ancestry of the three
dewclawed wolves up to the second generation in the past
was estimated using STRUCTURE (options Usepopinfo = 1
and Genesback = 2), whereas their relatedness (r) was calcu-
lated using the program KINSHIP version 1.3.1 (Goodnight
and Queller 1999). To check for male wolf ancestry in
W535 and W520, we also investigated Y markers using four
microsatellites that map on Y chromosomes (Sundqvist et al.
2001). Further methodological details are provided in Randi
and Lucchini (2002) and Lucchini et al. (2004).
The FCA split the dog and Italian wolf populations into
two clearly separated clusters, and the three dewclawed
wolves were marginal to the Italian wolf genotype distribu-
tion (Fig. 1). In addition, the Bayesian procedure assigned
each wolf and dog to the correct populations with high q
values (>0.99) and small 90% confidence intervals (dog:
0.97–1; wolf: 0.99–1), where the qvalue represents the pro-
portion of the genome that originate from that particular
population. In contrast, dewclawed wolves were assigned to
the Italian wolf population with lower qvalues (W535: q=
0.95; W520: q= 0.90; W587: q= 0.76) and larger 90% con-
fidence intervals (W520: 0.58–1; W535: 0.75–1; W587:
0.60–0.92), providing further evidence of their admixed an-
cestry with dogs. Males W535 and W520 shared a unique Y
haplotype that is very common in the Italian wolf population
but absent in the dogs that we analyzed (V. Lucchini and E.
Randi, unpublished data). W520’s microsatellite alleles were
also found in Italian wolves, but W535 and W587 shared
with dogs alleles that were absent from the Italian wolf pop-
ulation (Table 1). All three dewclawed wolves shared the
unique mtDNA haplotype of the Italian wolf population
(Randi et al. 2000).
Estimates of the amount of dog ancestry up to the second
generation in the past revealed that W587 had a high compo-
nent of dog ancestry (q= 0.99) at the second generation,
whereas W535 and W520 had lower values (q= 0.014 and
q= 0.122, respectively), suggesting that their hybridization
origin could only be older than two generations. Kinship
analysis suggested that W535 and W520 could be first-
degree relatives (r= 0.31; cf. Lucchini et al. 2002), which is
further supported (p= 0.95) by a simulation using 1000 rep-
licates (Goodnight and Queller 1999). W390 was assigned to
Italian wolves by Bayesian admixture analyses.
Individual heterozygosities (H; Nei 1987) of W535,
W520, and W587 ranged from 0.32 to 0.52, and only W535
(H= 0.32) was lower than the average heterozygosity for all
Italian wolves (H= 0.44; Randi and Lucchini 2002;
Lucchini et al. 2004).
It has long been recognized that the occurrence of
dewclaws in dogs is correlated with body size and is more
frequent in large breeds (Darwin 1868: 35; Alberch 1985).
© 2003 NRC Canada
Ciucci et al. 2079
Fig. 1. Scores of individual wolf (Canis lupus) and dog (Canis lupus familiaris) microsatellite genotypes plotted on the first two axes
of a factorial correspondence analysis (FCA) performed using GENETIX.
CPH2 CPH6 CPH7 CPH12 C20.253 FH2132
W520 —
W535 94 (0.34)
W587 122 (0.15) 170 (0.32) 196 (0.09) 106 (0.76) 327 (0.05)
Note: Values in parentheses are the frequency of the allele in the dog population.
Table 1. Alleles (size given as base pairs, including primers) of six microsatellite loci that were observed in
the dewclawed wolves (Canis lupus), which are shared with dogs (Canis lupus familiaris) but are absent in
the Italian wolf population (cf. Randi and Lucchini 2002).
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From a developmental point of view, the phenotypic varia-
tion necessary for the development of extra toes appears to
be linked with the size (number of cells) of the embryonic
limb bud, which is larger in large dog breeds (Alberch
1985). By exceeding a minimum threshold value in the num-
ber of limb-bud cells necessary to specify for extra toes
during morphogenesis, dog breeders cannot eliminate the ap-
pearance of the trait by continuously selecting for large size
(Alberch 1985). On the other hand, although mutations for
polydactyly are quite common in amniotes, they are not
evolutionarily stable owing to the strong negative pleiotropic
effects present during the phylotypic stage (Lande 1978;
Galis et al. 2001). In line with this theory, there are no pub-
lished reports of dewclaws in wild canids except for a red
fox (Vulpes vulpes) shot in 1913 on the island of Biskops-
Arnö in Sweden (Lönnberg 1916). Being evolutionarily con-
strained in amniotes, polydactyly in large dog breeds could
have been fostered by intense artificial selection, whereas
the expected negative pleiotropic effects might be responsi-
ble for the shorter life, higher incidence of locomotory dis-
eases, and higher mortality rates reported for polydactylous
dogs (Galis et al. 2001: 639).
Although possible in principle, it is therefore quite un-
likely that polydactyly could stochastically appear and be
maintained in wolves, even at the level of the local, inbred
populations. Accordingly, the heterozygosity observed in the
three dewclawed wolves we collected did not reveal high
levels of inbreeding compared with the Italian wolf popula-
tion. Alternatively, the extreme rarity of dewclawed wolves
and their exclusive occurrence in an area with high densities
of large dog breeds suggest that the trait represents a charac-
ter possibly displayed by wolf–dog hybrids and backcrosses.
Although we did not investigate patterns of genetic inheri-
tance of the dewclaws either in dogs or in wolf–dog hybrids,
our results confirmed that the dewclawed wolves we col-
lected in Tuscany were wolves of admixed ancestry, which
supported our original hypothesis. Since they shared the
mtDNA and Y haplotypes of the Italian wolf population, but
their Bayesian assignment probabilities were not intermedi-
ate between dogs and wolves as expected for F1 hybrids
(Randi and Lucchini 2002), they were most likely back-
crosses of wolf–dog hybrids into the Italian wolf population.
The amount of dog ancestry estimated up to the second gen-
eration in the past revealed that the dewclawed wolves we
sampled might represent second (W587) or later (W520,
W535) generation backcrosses, with the number of alleles
uniquely shared with the dog population increasingly diluted
by the number of generations since the origin of the hybrid-
It is noteworthy that according to observational criteria
alone, and with the notable exception of dewclaws, experi-
enced observers would not have distinguished the three
wolves from pure wolves. Unfortunately, in all three cases
we could not examine parents or siblings for the presence of
dewclaws. The case reported by Andersone et al. (2002)
supported our findings that dewclaws in wolves originated
from cross-mating with dogs, even though the dewclawed
pups they analyzed were obviously different from the
phenotypic standard of Latvian wolves. As successively con-
firmed by molecular markers, both presumptive parents of
these pups shared some dog-like traits (color patterns, skull
traits), which indicated hybridization, but there was no men-
tion of dewclaws (Andersone et al. 2002).
Relatedness values among the three dewclawed wolves
suggest that at least two different hybridization events oc-
curred in this restricted area of Tuscany, reflecting local-
ly predisposing conditions (i.e., widespread, large-livestock
guard dogs; low density and social disruption of local wolf
packs; highly fragmented landscape). In addition, they share
the unique mtDNA haplotype of Italian wolves (Randi et al.
2000), confirming the previously described directionality of
wolf–dog (and wolf–coyote) hybridization (i.e., female wolves
mating with male dogs or coyotes; Boitani 1983; Lehman et
al. 1991; Gottelli et al. 1994; Roy et al. 1994; Vilà and
Wayne 1999; Randi et al. 2000; Randi and Lucchini 2002;
Vilà et al. 2003).
Although introgression of dog genes into the Italian wolf
population is apparently very limited and wolves and dogs
are genetically differentiated (Randi and Lucchini 2002), our
results provide additional evidence that wolves and dogs do
hybridize in the wild and that, at least occasionally, hybrids
can be successfully reintegrated into the wolf population
(Andersone et al. 2002; Randi and Lucchini 2002; Vilà et al.
2003; Lucchini et al. 2004). Following Allendorf et al.’s
(2001) categorization, these findings suggest that the Italian
wolf population might represent an intermediate case be-
tween type 4 and type 5 anthropogenic hybridization, with a
widespread pure wolf parental population interspersed with
few, localized areas of very limited introgression. In this re-
spect, as quantitative assessment of anthropogenic hybridiza-
tion is crucial to evaluate conservation scenarios (Allendorf
et al. 2001), intensive genetic sampling and monitoring are
urgently needed in areas of suspected hybridization. Map-
ping these areas on a regional scale would benefit from
screening diagnostic morphological traits that are reliable
clues of admixed ancestry (Randi and Lucchini 2002).
Among these, dewclaws on wolves are expected to be lim-
ited to cross-matings involving some large dog breeds. Other
presumptive morphological traits might be reliable as well
(e.g., white nails, atypical color patterns or body propor-
tions, dental anomalies; cf. Duman 2001), provided that their
molecular correlates are further investigated.
This study was funded by the Region of Tuscany and by
the Italian National Institute of Wildlife. We thank L. Artoni
and E. Tedesco for field assistance, and an anonymous ref-
eree for comments on an earlier draft of the manuscript.
C.D. Soulsbury kindly reviewed the English style of the text.
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... Aleandri" for diagnostic or forensic purposes. Individuals were classified as pure wolves by expert veterinarians and zoologists based on morphology and body mass [16,17]. ...
... Ten known admixed wolf x dog individuals (first-generation offspring of a female wolf and a male dog, as well as individuals showing clear dog-like morphological traits, cf. [5,16,17]), were identified by qualified wildlife scientists and genotyped with the validated protocol to check for signals of genetic admixture. Muscle tissues were stored at −20 • C or in five volumes of 95% ethanol at room temperature (or at +4 • C) until processed. ...
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The conservation of pure wolf populations in Europe is currently threatened by anthropogenic hybridization with dogs, through potential extinction of local gene pools due to replacement with domestic gene variants. Distinction of wolf–dog hybrids from wild ancestors is essential for development and implementation of management and conservation plans. Morphological traits are rarely helpful, and a genetic approach is the most effective to distinguish admixed individuals back to old backcrosses. To provide a molecular tool to address the issue, we optimized and validated a cost-effective protocol in multiplexed PCR format based on 22 STR canine loci, which allows us to distinguish genetically pure wolves from dogs, and, when used in association with a Bayesian assignment approach, is capable of statistically assigning admixed individuals to classes of hybrids with different levels of dog ancestry. Our method demonstrated high reliability, showing full repeatability and reproducibility of data with as little as 0.125 ng of genomic DNA, and was therefore suitable for the analysis of non-invasively collected samples and degraded DNA. The application of our STR panel to the appropriate assignment procedure unambiguously defined two genetically separated clusters for wolves and dogs, and successfully identified known hybrids as admixed individuals, which eventually were classified as recent hybrids and older backcrosses. The protocol, which is described here in detail, can be adopted by various laboratories that need this kind of diagnosis; furthermore, it would be capable of producing concordant results through inter-laboratory comparisons with wolf and dog control DNAs.
... foltos), és a szőr formája (pl. göndör), (ii) testi arányok (végtagok hossza, koponya méretei), (iii) farkaskarom megléte és (iv) fehér karomszín [27][28][29]. A kameracsapdás módszer is egy lehetséges megoldás a farkasok és kóborló kutyák, ill. ...
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ÖSSZEFOGLALÁS A szerzők a kutyák és farkasok különböző genetikai markerekkel történő elkülönítésének lehetőségeit vizsgálják és bemutatják ennek fontosságát az igazságügyi alkalmazás területén. A rendelkezésre álló farkas-és kutyaeredetű mintákból meghatározott mitokondriális kontrollrégió haplotípusok és a 14 vizsgált mikro-szatellita-alléleloszlás adatai alapján különbség látható a farkas-és kutyaminták között. Az eddigi hazai eredmények is alátámasztják annak lehetőségét, hogy különböző genetikai markerek párhuzamos vizsgálatával − amelyek megfelelnek az igazságügyi célú alkalmazás kritériumainak −, kellő valószínűséggel alátámasztható egy kérdéses eredetű minta alfajszintű besorolása. SUMMARY Background: After several decades of absence, the grey wolf (Canis lupus) has started recolonizing its former territories in Hungary at the beginning of the 21 st century. Due to the intense presence of mankind, wolves are forced to share great areas of land with humans, which potentially leads to several conflicts. From the wolves' perspective, it means the decimation of domestic livestock. As far as humans are concerned, these conflicts may manifest in the illegal hunting of wolves and trading with their products. When facing such case, it should be examined whether the perpetrator/victim is a wolf or a dog. Objective: The aim of our study was to test genetic methods which can be used for forensic application as well to distinguish between wolves, dogs, or their hybrids. Materials and Methods: Altogether 22 samples (hair, skin, faeces, saliva, and purified DNA) from wolves and wolf-dog hybrids were collected. For the comparative canine database DNA samples from Hungarian dog populations were used. After DNA isolation, the mitochondrial hypervariable region I (HVI) and 14 autoso-mal microsatellite markers were amplified by PCR (Polymerase Chain Reaction). Mitochondrial haplotypes determined by sequencing were grouped using PopART. Genetic profiles based on the detected microsatellite alleles were analysed using Structure 2.3.4 and were grouped based on a Bayesian approach. Results and Discussion: The mitochondrial control region (HVI) haplotypes were successfully determined from the examined samples; these sequences were uploaded to the GenBank database. We did not find similar point mutation patterns between wolves and dogs. However, difference between wolf and dog groups was shown based on the detected microsatellite allele distribution, to make the results even more reliable further markers and more wolf samples should be involved. Overall, our preliminary results support that simultaneous application of large number of genetic markers meeting the standards of forensic application criteria-, could be adequate to determine the precise taxonomic origin of questionable samples.
... It was possible to distinguish a phenotypically wild-type female (WD2) and two other individuals, a female (WD3) and a male (WD1), with signs of hybridization (Figs. 1, 2). The anomalous characters of each individual (Table 1) refer to the phenotypic markers of hybridization reported in the literature (Ciucci et al., 2003;Anderson, 2009;Ciucci, 2012;Caniglia et al., 2013;Galaverni et al., 2017). From behavioral observations, it was possible to define the hierarchy of the pack by identifying the dominant couple as WD1 and WD2. ...
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Some anomalies in the breeding cycle of a pack of wolf-dog hybrids in a semi-anthropized area in the central-western part of Calabria are described. The data were collected between October 2019 and March 2021 by direct observations and video-camera trapping. In addition to recording anomalous morphological and phenotypic traits present in varying degrees in some individuals of the pack, we documented for two consecutive years the breeding of a subordinate female that was about three months early compared with the normal wolf breeding cycle. Moreover , in spring 2020, it was possible to observe double breeding within the same pack, due to the regular reproduction of the dominant female.
... Reference wild parentals were selected from found-dead wolves collected across the Italian peninsular distribution range that showed the typical wild coat colour pattern and no other apparent dog-like traits such as white claws or spurs on the hind legs 26,31,33,91 . ...
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Anthropogenic hybridization is recognized as a major threat to the long-term survival of natural populations. While identifying F1 hybrids might be simple, the detection of older admixed individuals is far from trivial and it is still debated whether they should be targets of management. Examples of anthropogenic hybridization have been described between wolves and domestic dogs, with numerous cases detected in the Italian wolf population. After selecting appropriate wild and domestic reference populations, we used empirical and simulated 39-autosomal microsatellite genotypes, Bayesian assignment and performance analyses to develop a workflow to detect different levels of wolf x dog admixture. Membership proportions to the wild cluster (qiw) and performance indexes identified two qthresholds which allowed to efficiently classify the analysed genotypes into three assignment classes: pure (with no or negligible domestic ancestry), older admixed (with a marginal domestic ancestry) and recent admixed (with a clearly detectable domestic ancestry) animals. Based on their potential to spread domestic variants, such classes were used to define three corresponding management categories: operational pure, introgressed and operational hybrid individuals. Our multiple-criteria approach can help wildlife managers and decision makers in more efficiently targeting the available resources for the long-term conservation of species threatened by anthropogenic hybridization.
... Just as was often the practice with coyote-dog hybrids, wolf-dog hybrids were documented to segregate dog-like phenotypes (e.g., coat and eye coloration, markings, tail) and individuals carrying such traits were then often inferred to be of admixed ancestry (Iljin 1941). These traits are often relied upon for phenotype-based detection of dog-wolf interbreeding in Europe (e.g., Andersone et al. 2002;Randi et al. 2002;Ciucci et al. 2003;Vilà et al. 2003;Ardalan et al. 2011;Godinho et al. 2011;Lorenzini et al. 2014;Godinho et al. 2015;vonHoldt et al. 2017a). Such assumptions may be erroneous, as melanistic wolves of North America retain a dog-evolved genetic variant that is likely under natural selection but is not at all informative for the ancestry composition of a genome (Anderson et al. 2009;Schweizer et al. 2018). ...
This bibliography provides a collection of references that documents the evolution of studies evidencing interbreeding among Canis species in North America. Over the past several decades, advances in biology and genomic technology greatly improved our ability to detect and characterize species interbreeding, which has significance for understanding species in a changing landscape as well as for endangered species management. This bibliography includes a discussion within each category of interbreeding, the timeline of developing evidence, and includes a review of past research conducted on experimental crosses. Research conducted in the early 20th century is rich with detailed records and photographs of hybrid offspring development and behavior. With the progression of molecular methods, studies can estimate historical demographic parameters and detect chromosomal patterns of ancestry. As these methods continue to increase in accessibility, the field will gain a deeper and richer understanding of the evolutionary history of North American Canis.
... Experts did not agree on how to consider individuals bearing putative morphological traits indicating hybridization (e.g., Ciucci et al., 2003), but which are genetically identified as nonadmixed (Figure 4). Amongst the 13 experts who provided an explanation for their response, five suggested that anomalous phenotypic traits may not always be a reliable sign of admixture, and that diagnoses should always be based on genetic evidence. ...
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Anthropogenic hybridization is widely perceived as a threat to the conservation of biodiversity. Nevertheless, to date, relevant policy and management interventions are unresolved and highly convoluted. While this is due to the inherent complexity of the issue, we hereby hypothesize that a lack of agreement concerning management goals and approaches, within the scientific community, may explain the lack of social awareness on this phenomenon, and the absence of effective pressure on decision-makers. By focusing on wolf x dog hybridization in Europe, we hereby (a) assess the state of the art of issues on wolf x dog hybridization within the scientific community, (b) assess the conceptual bases for different viewpoints, and (c) provide a conceptual framework aiming at reducing the disagreements. We adopted the Delphi technique, involving a three-round iterative survey addressed to a selected sample of experts who published at Web of Science listed journals, in the last 10 years on wolf x dog hybridization and related topics. Consensus was reached that admixed individuals should always be defined according to their genetic profile, and that a reference threshold for admixture (i.e., q-value in assignment tests) should be formally adopted for their identification. To mitigate hybridization, experts agreed on adopting preventive, proactive and, when concerning small and recovering wolf populations, reactive interventions. Overall, experts' consensus waned as the issues addressed became increasingly practical, including the adoption of lethal removal. We suggest three non-mutually exclusive explanations for this trend: (i) value-laden viewpoints increasingly emerge when addressing practical issues, and are particularly diverging between experts with different disciplinary backgrounds (e.g., ecologists, geneticists); (ii) some experts prefer avoiding the risk of potentially giving carte blanche to wolf opponents to (illegally) remove wolves, based on the wolf x dog hybridization issue; (iii) room for subjective interpretation and opinions result from the paucity of data on the effectiveness of different management interventions. These results have management implications and reveal gaps in the knowledge on a wide spectrum of issues related not only to the management of anthropogenic hybridization, but also to the role of ethical values and real-world management concerns in the scientific debate.
... Recent mitochondrial DNA analyses, for example, suggests that dogs diverged from wolves as early as 134,000 years ago but were morphologically indistinguishable from their wild progenitors until 15,000-10,000 years ago when human populations became less mobile (Vila et al. 2002). Two possible routes for dog domestication are implied by molecular analyses: either a single event involving one wolf population Sundqvist et al. 2006) or multiple events in different localities with continued interbreeding between wolves and dogs in some areas (Ciucci et al. 2003;Tchernov and Valla 1997;Verardi et al. 2006;Verginelli et al. 2005). Molecular and archaeological evidence are not in precise agreement regarding the timing of dog domestication. ...
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Dogs live in 45% of households, integrated into various human groups in various societies. This is certainly not true for wolves. We suggest that dogs' increased tractability (meant as individual dogs being easier to control, handle and direct by humans, in contrast to trainability defined as performance increase due to training) makes a crucial contribution to this fundamental difference. In this study, we assessed the development of tractability in hand-raised wolves and similarly raised dogs. We combined a variety of behavioural tests: fetching, calling, obeying a sit signal, hair brushing and walking in a muzzle. Wolf (N = 16) and dog (N = 11) pups were tested repeatedly, between the ages of 3-24 weeks. In addition to hand-raised wolves and dogs, we also tested mother-raised family dogs (N = 12) for fetching and calling. Our results show that despite intensive socialization, wolves remained less tractable than dogs, especially in contexts involving access to a resource. Dogs' tractability appeared to be less context dependent, as they followed human initiation of action in more contexts than wolves. We found no evidence that different rearing conditions (i.e. intensive socialization vs. mother rearing) would affect tractability in dogs. This suggests that during domestication dogs might have been selected for increased tractability, although based on the current data we cannot exclude that the differential speed of development of dogs and wolves or the earlier relocation of wolves to live as a group explains some of the differences we found.
Despite the generally positive trend of European populations, the wolf (Canis lupus) is still today a challenging species to conserve, particularly in the most anthropogenic southern European countries, because of its conflict with humans. In this chapter we summarize the dynamics of wolf distribution in Italy, one of the most densely populated European countries, over the last 50 years. We track changes in the wolf’s diet by comparing its change in Italy with other countries, with the aim of understanding how these changes may have affected the evolution of the human-predator conflict in Italy. In particular, we summarize the results of studies both in Italy and in other European countries to clarify the true impact of wolf predation on both livestock and wild ungulates, which represent the two main causes of predator-human conflict. In order to provide specific insight about the past and the current distribution and feeding habits of the wolf in Italy, and to take stock of the conflict between wolves and humans, we present three case studies. All were carried out over recent decades in northern Italy, i.e. in the area where wolf packs, and particularly their ability to produce dispersing individuals, could affect the future of the entire Italian population. Finally, we consider how to mitigate wolf-human conflict and suggest effective management of wolf populations.
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Representing a form of anthropogenic hybridization, wolf–dog interbreeding may potentially compromise the ecological and evolutionary traits of local wolf populations and corrode social tolerance towards wolves. However, estimates of the extent of wolf–dog hybridization in wolf populations are scarce, especially at a multi-pack scale and in human-dominated landscapes. Using non-invasive (n = 215) and invasive (n = 25) samples of wolf-like canids collected in the Province of Grosseto (central Italy, 2012–2014), we assessed the extent of wolf–dog hybridization based on multi-locus genotypes (16 and 49 loci for non-invasive and invasive samples, respectively) and Bayesian clustering techniques. Based on a total of 72 genotypes, the minimum proportion of admixed individuals in our sample was 30.6%, comprising 8 out of the 13 surveyed packs; however, by correcting for the proportion of admixed individuals undetected using the 16-loci compared with the 49-loci marker set (26.7%), we suspect the rate of recent admixture could be closer to 50%. While we did not detect any F1 hybrid, four admixed individuals had a non-negligible probability of being first-generation backcrosses, one of which likely derived from a backcross of a F1 hybrid into the dog population. Complementary genetic markers (i.e., Y-haplotype and K-locus) or anomalous morphological traits further indicated widespread occurrence of admixed individuals of older generations of backcross. This high level of admixture raises serious wolf conservation concerns and exemplifies the expected dynamics of wolf–dog hybridization if left unmanaged in human-dominated landscapes. The implications of our findings need to be urgently upscaled for the implementation of management interventions that cannot be procrastinated any longer at the regional and national scale.
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The distribution, status and conservation problems of the wolf Canis lupus in Spain were studied in 1987 and 1988. Wolves regularly occurred over 100,000 km2, mainly in the northwest quarter of the country, with an estimated 294 breeding pairs, i.e. 1,500 and 2,000 wolves in early spring and autumn, respectively, For 1987, reports were received of 309 wolves killed in Spain (excluding Asturias) but the actual number could range from 550 to 750 every year, mostly illegal killings. Annual damage to livestock was estimated at nearly one million US $, losses being disproportionately high in mountain areas due to the lack of protection of free-ranging livestock. The number of stray dogs is higher where wolves are absent and no extensive wolf-dog hybridization has been recorded. Problems related to wolf management are discussed.
Mitochondrial DNA (mtDNA) genotypes of gray wolves and coyotes from localities throughout North America were determined using restriction fragment length polymorphisms. Of the 13 genotypes found among the wolves, 7 are clearly of coyote origin, indicating that genetic transfer of coyote mtDNA into wolf populations has occurred through hybridization. The transfer of mtDNA appears unidirectional from coyotes into wolves because no coyotes sampled have a wolf-derived mtDNA genotype. Wolves possessing coyote-derived genotypes are confined to a contiguous geographic region in Minnesota, Ontario, and Quebec, and the frequency of coyote-type mtDNA in these wolf populations is high (>50%). The ecological history of the hybrid zone suggests that hybridization is taking place in regions where coyotes have only recently become abundant following conversion of forests to farmlands. Dispersing male wolves unable to find conspecific mates may be pairing with female coyotes in deforested areas bordering wolf territories. Our results demonstrate that closely related species of mobile terrestrial vertebrates have the potential for extensive genetic exchange when ecological conditions change suddenly.
Genetic divergence and gene flow among closely related populations are difficult to measure because mutation rates of most nuclear loci are so low that new mutations have not had sufficient time to appear and become fixed. Microsatellite loci are repeat arrays of simple sequences that have high mutation rates and are abundant in the eukaryotic genome. Large population samples can be screened for variation by using the polymerase chain reaction and polyacrylamide gel electrophoresis to separate alleles. We analyzed 10 microsatellite loci to quantify genetic differentiation and hybridization in three species of North American wolflike canids. We expected to find a pattern of genetic differentiation by distance to exist among wolflike canid populations, because of the finite dispersal distances of individuals. Moreover, we predicted that, because wolflike canids are highly mobile, hybrid zones may be more extensive and show substantial changes in allele frequency, relative to nonhybridizing populations. We demonstrate that wolves and coyotes do not show a pattern of genetic differentiation by distance. Genetic subdivision in coyotes, as measured by theta and Gst, is not significantly different from zero, reflecting persistent gene flow among newly established populations. However, gray wolves show significant subdivision that may be either due to drift in past Ice Age refugia populations or a result of other causes. Finally, in areas where gray wolves and coyotes hybridize, allele frequencies of gray wolves are affected, but those of coyotes are not. Past hybridization between the two species in the south-central United States may account for the origin of the red wolf.
We describe a model-based clustering method for using multilocus genotype data to infer population structure and assign individuals to populations. We assume a model in which there are K populations (where K may be unknown), each of which is characterized by a set of allele frequencies at each locus. Individuals in the sample are assigned (probabilistically) to populations, or jointly to two or more populations if their genotypes indicate that they are admixed. Our model does not assume a particular mutation process, and it can be applied to most of the commonly used genetic markers, provided that they are not closely linked. Applications of our method include demonstrating the presence of population structure, assigning individuals to populations, studying hybrid zones, and identifying migrants and admixed individuals. We show that the method can produce highly accurate assignments using modest numbers of loci—e.g., seven microsatellite loci in an example using genotype data from an endangered bird species. The software used for this article is available from
Out of the 3.5 million domestic dogs Canis familiaris living in Italy, c850 000 are free to move in and out of villages. These are an immense reservoir to the 80 000 feral dogs whose biology is very much like that of wolves C. lupus. In wolf areas, with a mean density of c1 wolf/100km2, there are 150-310 free-ranging dogs/100km2 and 24-82 feral dogs/100km2, densities increasing from central to S Italy. Direct and indirect competition of different sorts result: competition for food, as both wolves and dogs feed mainly at the open dumps outside villages and to a less extent on livestock; competition for range, as movements of loners and young wolves in search of new territories are limited by the presence of packs of up to 20-25 dogs; 'genetic' competition, as loners and isolated female wolves may interbreed with dogs and their offspring be more fitted to the Italian environment than wolves, due to their dog-like look. Consequences for wolf conservation are discussed.-Author
A review of fossil evidence on the rates of limb loss in tetrapods indicates that millions of years are required for complete loss of external traces of limbs. Morphological series of intermediate stages of limb loss within genera in the lizard families Scincidae and Teiidae show that there are evolutionary pathways of body elongation and reduction of limb size relative to skull length, accompanied by loss of limb elements beginning distally. Evolutionary elongation of the lizard body occurs through an increase in the coefficient of allometric growth of the body with respect to the skull, which precedes structural reduction of the limbs. A review of embryological studies of limbed and limbless forms suggests that in amniotes the distal to proximal sequence of structural reduction evolves by the progressively earlier onset of cell death which usually occurs to form the spaces between the digits, in conjunction with the normal proximo-distal sequence of determination of mesodermal elements in limb development. Following a presentation of data on digital variation in lizards, genetic studies of digit loss and polydactyly are summarized which indicate a polygenic influence on structural variation. Using these data, mathematical models are constructed which show that weak selection pressures can produce geologically rapid structural changes. The mechanism of reexpression of long lost structures of the limb (such as cetacean pelvic limbs and atavistic digits in the horse, dog, and guinea pig) is considered in a view of information on appendicular mutations in the mouse.
We evaluated the accuracy and precision of tooth wear for aging gray wolves (Canis lupus) from Alaska, Minnesota, and Ontario based on 47 known-age or known-minimum-age skulls. Estimates of age using tooth wear and a commercial cementum annuli-aging service were useful for wolves up to 14 years old. The precision of estimates from cementum annuli was greater than estimates from tooth wear, but tooth wear estimates are more applicable in the field. We tended to overestimate age by 1-2 years and occasionally by 3 or 4 years. The commercial service aged young wolves with cementum annuli to within ±1 year of actual age, but under estimated ages of wolves ≥9 years old by 1-3 years. No differences were detected in tooth wear patterns for wild wolves from Alaska, Minnesota, and Ontario, nor between captive and wild wolves. Tooth wear was not appropriate for aging wolves with an underbite that prevented normal wear or severely broken and missing teeth.