Dewclaws in wolves as evidence of admixed ancestry with dogs

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 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.

Full text

NOTE / NOTE
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
Introduction
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
2077
Received 3 October 2002. Accepted 7 October 2003. Published on the NRC Research Press Web site at http://cjz.nrc.ca 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: paolo.ciucci@uniroma1.it).
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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.
2001).
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.
Methods
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
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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).
Results
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).
Discussion
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-
ization.
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.
Acknowledgments
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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