Quaternary radiation and genetic structure of the red fox
Vulpes vulpes in the Mediterranean Basin, as revealed by
allozymes and mitochondrial DNA
F. Frati1, G. B. Hartl2, S. Lovari1*, M. Delibes3 and G. Markov4
1
Department of Evolutionary Biology, Ethology and Behavioural Ecology Group, University of Siena, via P. A. Mattioli 4, 53100, Siena, Italy
Institut furr Haustierkunde der Christian-Albrechts-Universitart zu Kiel, Biologiezentrum, Olshausenstra3e 40, D-24118 Kiel, Germany
3
Consejo Superior de Investigaciones Cientificas, Estacion Biologica de Don ana, Avda. de Maria Luisa s/n. Pabello’ n del Peru’ , 41013 Sevilla, Spain
4
Institute of Zoology, Bulgarian Academy of Science, 1, Tsar Osvoboditel bul., BG-100, Sofia, Bulgaria
2
(
Abstract
The Quaternary dispersal of the red fox Vulpes vulpes in the Mediterranean area was evaluated through the
study of allelic variation at 45 enzyme loci in 120 individuals from 10 sampling sites. A 375 bp fragment of
the mitochondrial cytochrome b gene was also sequenced in a total of 41 specimens from the same sampling
locations. Nine allozyme loci were polymorphic. The proportion of polymorphic loci per population (P)
ranged from 0 to 15.6%, and expected average heterozygosity (H) from 0 to 4.4%. A total of 18 different
Cyt b haplotypes were detected. Most of them were confined to only one population. Both allozyme and
mtDNA data implied that our fox populations were genetically fairly isolated from one another, suggesting
low gene flow between them. This isolation should be of comparatively recent origin according to the slight
differentiation among Cyt b haplotypes. Fox populations appeared to belong to two genetically distinct
groups. With a mean value of Nei’s D = 0.024, genetic distance between these groups was similar to that
detected at subspecies level in taxa of large mammals. This pattern may have originated from different
colonization waves during Quaternary glaciations and deglaciations. Red foxes from Sardinia were more
closely related to the Bulgarian foxes than to the Iberian ones. However, repeated introductions to Sardinia
probably also occurred from Central Italy and Spain, as suggested by the presence of haplotype A and a
typical Central Italian allele, Ck-290.
Key words: Vulpes vulpes, allozymes, mitochondrial DNA, haplotypes, population genetics
INTRODUCTION
The red fox Vulpes vulpes (L., 1758) is the living wild
mammal with the widest “natural distribution“ (Nowak,
1991: 1050). Its range extends from most of North
America to the whole of Europe, through nearly all of
Asia, North Africa, and most of Australia, where it was
introduced in the 19th century. Such an unusually wide
distribution is probably the result of the great biological
plasticity of this species. In the Mediterranean area, the
red fox is not only present all around the basin rim, but
also on several islands, e.g. Sicily, Sardinia and Corsica.
Its arrival in Sicily probably occurred during the
Middle/Upper Pleistocene, through temporary land
bridges during the highest peak of the last glacial
episode (Tagliacozzo, 1993). The origin of the Sardinian
red fox Vulpes vulpes ichnusae Miller, 1907, a recognized
*All correspondence to: Dr S. Lovari, Department of Evolutionary
Biology, Ethology and Behavioural Ecology Group, University of
Siena, via P. A. Mattioli 4, 53100, Siena, Italy; email: lovari@unisi.it
subspecies (Toschi, 1965: 290), is dubious. It might be
the only autochthonous living mammal of Sardinia and
Corsica (Malatesta, 1970; Esu & Kotsakis, 1983) or it
may have been introduced by humans in the Early
Neolithic, about 7000 years ago (Vigne, 1992; Masseti,
1993). In this paper, we investigate the genetic identity
and origin of the Sardinian subspecies of red fox by
comparing it with other populations from Southern
Europe and the Near East.
Furthermore, the red fox has been widely distributed
in the Mediterranean region throughout the Middle
Pleistocene and Early Holocene (Kurte’ n, 1968; Bonifay,
1971; Capasso Barbato & Minieri, 1978; Ballesio, 1979).
It may thus have undergone intense population movements during glaciations and deglaciations (cf. Sage &
Wolff, 1986). Another aim of this paper has been to
compare genetically a sample of populations of red
foxes in an attempt to reconstruct indirectly the Quaternary dispersal of this species in the Mediterranean
area.
Genetic markers, such as allozymes and mitochondrial
SP2
IT1
SP1
IT3
AU1
IT2
BU2
IT4
BU1
IS1
Fig. 1. Collecting sites of red foxes (see Table 1 for explanation of acronyms).
Table 1. Collecting sites and acronyms of the 10 populations,
and sample sizes for the allozyme and the DNA screenings
Country
Sampling areas
Sample size
Allozymes DNA
Spain
SP1
SP2
Italy
IT1
IT2
IT3
IT4
Donana Natl. Park; Sevilla
prov.
Valladolid prov.
10
5
9
3
Siena prov.
Sardinia region
Maremma Reg. Park;
Grosseto prov.
Palermo prov.
42
19
3
9
5
3
2
2
8
6
17
6
2
3
4
3
120
41
Austria
AU1
Tullner Feld, Lower Austria
Bulgaria
BU1
BU2
Vitoscha
Rila
Israel
IS1
Grofit
Total
DNA sequences, have been widely used to assess genetic
variability in natural populations and to establish intraspecific evolutionary relationships. Allozymes are a
powerful tool for assessing levels of genetic variability in
mammals (Hartl, Willing & Nadlinger, 1994), and
electrophoretic variation in red foxes from Denmark was
investigated by Simonsen (1982). Mitochondrial genes
provide useful information on variation and differentiation at the population level in both vertebrates and
invertebrates (Avise et al., 1987; Simon et al., 1994), and
sequence data on the cytochrome b gene have been
already gathered in mammals (Irwin, Kocher & Wilson,
1991) and fox-like canids in particular (Geffen et al.,
1992).
MATERIALS AND METHODS
Collection of samples
Standard allozyme and DNA analyses have been carried
out on samples of liver tissue removed from foxes freshly
killed by hunters in the course of control operations (i.e.
rabies monitoring and livestock protection) and regular
hunting (Table 1) from selected sites in the Mediterranean range (Fig. 1). Several sampling areas were protected, which exerted some ethical constraints on the
collection of red fox specimens. Therefore, several
sample sizes were small and the relevant information has
to be taken with caution. Tissue samples were preserved
at -80 °C prior to analyses.
Electrophoretic study
A total of 33 isozyme systems representing 45 presumptive structural loci (Table 2) were examined by
horizontal starch gel electrophoresis according to
routine methods (Hartl & Hor ger, 1986; Grillitsch et al.,
Haplotype A
50
100
150
200
250
300
350
375
Fig. 2. Sequence of Cyt b haplotype A. The 26 variable sites are indicated with an asterisk (*) while underlined codons show
amino acid replacements in at least one of the other haplotypes.
Table 2. Enzyme systems studied and presumptive loci scored
in the red fox
Enzyme (abbreviation, E.C. number)
Loci scored
Glycerophosphate dehydrogenase (GDC, 1.1.1.8) Gdc
Sorbitol dehydrogenase (SDH, 1.1.1.14)
Sdh
Lactate dehydrogenase (LDH, 1.1.1.27)
Ldh-1, Ldh-2
Malate dehydrogenase (MDH, 1.1.1.37)
Mdh-1, Mdh-2
Malic enzyme (ME, 1.1.1.40)
Me-1, Me-2
Isocitrate dehydrogenase (IDH, 1.1.1.42)
Idh-1, Idh-2
6-Phosphogluconate dehydrogenase (PGD,
Pgd
1.1.1.44)
Glucose dehydrogenase (GDH, 1.1.1.47)
Gdh
Glucose-6-phosphate dehydrogenase (GPD,
Gpd
1.1.1.49)
Glutamate dehydrogenase (GLUD, 1.4.1.3)
Glud
NADH diaphorase (DIA, 1.6.2.2)
Dia-1, Dia-2
Catalase (CAT, 1.11.1.6)
Cat
Superoxide dismutase (SOD, 1.15.1.1)
Sod-1, Sod-2
Purine nucleoside phosphorylase (NP, 2.4.2.1)
Np
Aspartate aminotransferase (AAT, 2.6.1.1)
Aat-1, Aat-2
Glutamate pyruvate transaminase (GPT, 2.6.1.2) Gpt
Hexokinase (HK, 2.7.1.1)
Hk
Creatine kinase (CK, 2.7.3.2)
Ck-2
Adenylate kinase (AK, 2.7.4.3)
Ak
Phosphoglucomutase (PGM, 2.7.5.1)
Pgm-1, Pgm-2
Esterases (ES, 3.1.1.1)
Es-1, Es-2
Acid phosphatase (ACP, 3.1.3.2)
Acp-1, Acp-2
Fructose-1,6-diphosphatase (FDP, 3.1.3.11)
Fdp
3-Galactosidase (3-GAL, 3.2.1.23)
ft-Gal
3-Glucuronidase (3-GUS, 3.2.1.31)
ft-Gus
Peptidases (PEP, 3.4.11)
Pep-1, Pep-2
Aminoacylase-1 (ACY-1, 3.5.1.14)
Acy-1
Adenosine deaminase (ADA, 3.5.4.4)
Ada
Carbonic anhydrase (CA, 4.2.1.1)
Ca
Fumarate hydratase (FH, 4.2.1.2)
Fh
Aconitase (ACO, 4.2.1.3)
Aco-1, Aco-2
Mannosephosphate isomerase (MPI, 5.3.1.8)
Mpi
Glucosephosphate isomerase (GPI, 5.3.1.9)
Gpi
1992). The interpretation of band-patterns was carried
out as outlined by Harris & Hopkinson (1976) and
Harris (1980). The most common allele in red foxes
from Sardinia was designated arbitrarily ‘100’. Variant
alleles were designated according to the relative mobility
of the corresponding allozymes. Allelic frequencies,
indices of genetic variation within and among popula-
tions, genetic distances, and dendrograms were
calculated using the BIOSYS-1 (release 1.7) program of
Swofford & Selander (1989) and the PHYLIP-package
of Felsenstein (1993). In populations with sample sizes
>10 we tested for an agreement of observed and expected genotypic frequencies using Fisher’s exact test
with pooling of genotypes for rare alleles (Swofford &
Selander, 1989).
Mitochondrial (mt) DNA study
Owing to the longer procedure and the technical difficulty, DNA sequence data were gathered in a subset of
only 41 specimens, randomly chosen at similar
numbers from the 10 populations (Table 1). Total
DNA was extracted from about 1 cm3 of liver tissue of
each specimen according to the protocol outlined in
Simon, Franke & Martin (1991). Briefly, the procedure
included grinding the tissue in homogenizing buffer,
differential centrifugation to enrich the mitochondrial
fraction, incubation with proteinase k and SDS to
digest proteins and disrupt membranes, phenol/
chloroform extraction, and ethanol precipitation. A
portion of the mitochondrially encoded gene for cytochrome b (Cyt b) was amplified by the Polymerase
Chain Reaction (PCR — Saiki et al., 1985) using the
primers 5’-CAGAATGATATTTGTCCTCA-3’ and
5’-GATATGAAAAACCATCGTTG-3’ (modified, respectively, from H15149 and L14724 of Irwin et al.
[1991]). The Cyt b gene was used because of previous
usage in studies on fox-like canids (Geffen et al., 1992).
PCR amplification was performed for 35 cycles with a
denaturation step of 1 min at 94 °C, an annealing step
of 1 min at 45 °C and an extension step of 1 min 10 s
at 72 °C. Double-stranded PCR products were run on a
1% Low Melting Point Agarose gel, the band excised
from the gel and the DNA purified by phenol-chloroform extraction and ethanol precipitation. Purified
DNA was sequenced by the double-stranded protocol
of Hsiao (1993) with both amplification primers, generating large regions of sequence overlap. The
sequenced fragment encompasses the 5’-end 375 base
Table 3. Allelic frequencies and genetic variation in 10 red fox populations (allozymes). All. = allele, P = proportion of
polymorphic loci (99% criterion), A = mean number of alleles per locus, Ho = observed heterozygosity, H = expected heterozygosity (Nei, 1978). P, A, and H are calculated over 45 presumptive loci
Population
Locus
All.
SP1
SP2
Me-1
100
117
125
100
125
100
57
107
100
120
130
100
90
100
80
100
78
100
110
-100
-68
1.000
0.950
0.050
0.100
0.900
1.000
1.000
1.000
1.000
1.000
1.000
4.4
1.0
0.7
0.6
0.944
0.056
0.944
0.056
0.056
0.944
0.944
0.056
1.000
1.000
1.000
1.000
1.000
8.9
1.1
1.0
1.0
Idh-2
Gdh
Dia-2
Ck-2
Pgm-2
Acy-1
Mpi
Gpi
P(%)
A
Ho(%)
H(%)
IT1
0.929
0.071
1.000
0.619
0.381
0.905
0.095
0.143
0.857
0.940
0.060
0.940
0.060
1.000
0.976
0.024
15.6
1.2
2.5
2.9
IT2
IT3
IT4
AU1
BU1
BU2
IS1
0.763
0.237
1.000
0.711
0.289
0.684
0.316
0.763
0.237
1.000
0.763
0.237
1.000
1.000
11.1
1.1
2.6
4.4
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
0.0
1.0
0.0
0.0
1.000
1.000
0.750
0.250
1.000
1.000
0.500
0.500
1.000
1.000
1.000
4.4
1.0
1.1
2.6
1.000
1.000
0.375
0.375
0.250
0.750
0.250
0.063
0.938
0.938
0.063
0.938
0.063
1.000
1.000
11.1
1.0
2.5
3.3
0.941
0.059
0.971
0.029
0.750
0.250
0.735
0.265
1.000
1.000
0.765
0.235
0.824
0.176
1.000
13.3
1.1
2.1
3.6
1.000
1.000
0.700
0.300
0.833
0.167
1.000
1.000
0.833
0.167
0.833
0.167
1.000
8.9
1.1
2.8
3.1
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
1.000
0.0
1.0
0.0
0.0
pairs of the gene, from the ATG initiation codon to
position 375 (Fig. 2). It codes for 125 amino acids.
Sequences were aligned by using the multiple alignment
program CLUSTAL V (Higgins, Bleasby & Fuchs,
1992). Sequence analysis was performed by using
MEGA (Kumar, Tamura & Nei, 1993) to estimate
several parameters of genetic variation including the
number of variable sites and values of genetic distance.
Evolutionay trees based on sequence data were inferred
using the same program. The program REAP
(McElroy et al., 1992) was used to derive estimates of
allelic and nucleotide diversity within populations.
Genetic heterogeneity based on haplotype sequences
(1st) was evaluated using the AMOVA treatment
(Excoffier, Smouse & Quattro, 1992). Heterogeneity of
genotype distribution among populations was also
tested with the Monte-Carlo y2 test of Roff & Bentzen
(1989) as implemented in REAP (1000 replicates).
The nucleotide sequences reported in this paper have
been deposited in the EMBL, GenBank and DDBJ
Nucleotide Sequence Databases under accession
numbers Z80957-Z80997.
Mpi, and Gpi. For each population, allelic frequencies
and indices of total electrophoretic variation are given
in Table 3. Deviations of genotypic frequencies from
Hardy-Weinberg equilibrium (P<0.05) were found at
Me-1 in IT1 and at Dia-2, Acy-1, and Mpi in BU1. In all
cases, there was an excess of homozygotes for the
respective rare allele.
Twenty-six nucleotide positions were variable (6.9%),
with only one substitution at each position (Fig. 2). The
variable sites were divided into 20 transitions and six
transversions; six substitutions were in 1st codon positions (four of them causing amino acid replacement),
four in 2nd codon positions (all causing amino acid
replacement) and 16 in 3rd codon positions (two of
them causing amino acid replacement). The variable
positions caused a total of 10 amino acid replacements.
Seven of these amino acid replacements fell among the
hypervariable sites (Irwin et al., 1991), three of them fell
among the slow evolving sites. By virtue of the combination of substitutions at the 26 variable sites, 18 different
haplotypes were detected among the 41 specimens
screened (Table 4). SP1, IT4 and IS1 were the most
uniform populations with only one haplotype observed
in each one. On the other hand, the most diverse
populations appeared to be BU1, AU1, IT2 and IT1
RESULTS
(Table 4), with the first one showing the highest value of
allelic diversity. Within-population nucleotide diversity
Genetic diversity within populations
was highest in AU1 and also high in BU1, BU2 and
Electrophoretic polymorphism was detected at nine out IT3. Amino acid replacements were found among inof 45 loci: Me-1, Idh-2, Gdh, Dia-2, Ck-2, Pgm-2, Acy-1, dividuals of the AU1, IT2, IT3 and BU2 populations.
Table 4. Distribution of Cyt b haplotypes among the populations studied
Population
Haplotypes
Sample
size
A B
C D E F
G H I
J
K L M N O P
Q R
SP1
SP2
IT1
IT2
IT3
IT4
AU1
BU1
BU2
IS1
1
5
2
2
-
2
-
1
-
1
1
-
1
-
2
-
1
-
2
-
1
-
1
-
1
-
1
-
2
-
2
1
-
1
-
5
-
2
-
3
5
3
9
5
3
2
6
2
3
3
Total
10
2
1
2
1
2
1
2
1
1
1
1
2
3
1
5
2
3
41
Table 5. Summary of F-statistics at all loci (over 10 fox
populations)
Locus
FIS
FIT
Me-1
Idh-2
Gdh
Dia-2
Ck-2
Pgm-2
Acy-1
Mpi
Gpi
0.240
-0.050
0.005
0.524
0.200
0.665
0.220
0.207
-0.024
0.337
0.014
0.417
0.769
0.863
0.791
0.318
0.320
-0.002
0.128
0.035
0.414
0.514
0.829
0.375
0.126
0.142
0.021
Mean
0.234
0.601
0.479
FST
Genetic diversity between populations
Except for Idh-2 and Gpi, allelic frequencies were
significantly different between populations (P < 0.01,
contingency y2 analysis, Swofford & Selander, 1989).
According to a hierarchical analysis of gene diversity
(F-statistics, Table 5), 48% of the total diversity could
be attributed to differentiation among populations.
This result remained stable (FST = 0.445) when the
island population of Sardinia (IT2) and the somewhat
remote population from Grofit (IS1) were excluded
from the calculations.
Haplotype A (Fig. 2) was the commonest. It was
found in 10 specimens of four different populations.
Only two other haplotypes (D and N) were shared by
specimens from different populations. All other Cyt b
haplotypes were diagnostic for particular populations.
Sequence divergence among haplotypes ranged from
0 to 2.67% and, interestingly, it was maximum between
two specimens from the AU1 population (haplotypes I
and K). Confirming the observation derived from
allozyme data, overall among-population differentiation was quite high (1st = 0.459) and the Monte-Carlo
test demonstrated a significant heterogeneity of
genotype distribution among samples (y2 = 277.30,
P<0.001).
Allelic
diversity
Nucleotide
diversity (%)
0.000
0.667 ± 0.314
0.694 ± 0.147
0.800 ± 0.164
0.667 ± 0.314
0.000
0.933 ± 0.122
1.000 ± 0.500
0.667 ± 0.314
0.000
0.000
0.533
0.236
0.380
0.733
0.000
1.427
0.800
0.733
0.000
Genetic relationships among populations
Pairwise genetic distances among populations based on
electrophoretic data are given in Table 6. Overall
genetic relationships between populations are displayed
in a Neighbor-joining tree (Fig. 3). The topology of the
tree remained stable when the populations with very
small sample sizes (IT3, IT4) were excluded.
Haplotypes A, B, C, F, N and R were quite similar to
one another, showing one to two nucleotide substitutions only. Other pairs of similar haplotypes were D and
P, L and O, and H and K (each pair being differentiated
by only one nucleotide substitution). Interestingly, the
haplotypes of the SP2 foxes (A and Q) differed from the
SP1 foxes by four substitutions. Because of the low
degree of variation, evolutionary trees inferred from
sequence data were not statistically significant and they
are not set out here.
DISCUSSION
Information
derived from allozyme and mtDNA
analyses appear to be congruent for certain aspects,
but they also show elements of discrepancy for others.
Both data sets suggest quite a remarkable degree of
interpopulation differentiation among European
populations of red foxes. On the other hand, there
appears to be little or no correlation in the estimates of
the degree of differentiation within populations and of
the evolutionary divergence among them. The same
phenomenon was also observed in Russian and westernAsian trout populations (Bernatchez & Osinov, 1995).
In our case, one likely explanation for its occurrence
could be the small size of the mtDNA data set where the
effect of stochastic factors may play an important role.
The electrophoretic variation detected in our study
was considerably higher than that reported in a previous
investigation (Simonsen, 1982), with a total proportion
of polymorphic loci (Pt) of 20%, a weighted mean P of
11.4%, and a mean H of 2.15%. Simonsen (1982) found
21 enzyme loci completely monomorphic in a sample
of 282 red foxes from Denmark. This result is most
Table 6. Matrix of pairwise unbiased genetic distances according to Nei (1978) — above the diagonal — and of modified Rogers
distances (Wright, 1978) — below the diagonal — among fox populations
SP1
SP2
IT1
IT2
IT3
IT4
AU1
BU1
BU2
IS1
SP1
SP2
IT1
IT2
IT3
IT4
AU1
BU1
BU2
IS1
0.013
0.151
0.120
0.150
0.122
0.160
0.114
0.100
0.150
0.000
0.154
0.121
0.150
0.128
0.162
0.117
0.104
0.146
0.023
0.024
0.106
0.097
0.146
0.046
0.137
0.133
0.213
0.014
0.014
0.011
0.170
0.107
0.123
0.052
0.061
0.180
0.023
0.023
0.009
0.029
0.201
0.091
0.196
0.187
0.211
0.012
0.013
0.019
0.008
0.038
0.166
0.096
0.086
0.201
0.025
0.026
0.001
0.014
0.007
0.024
0.153
0.150
0.211
0.012
0.013
0.019
0.002
0.039
0.006
0.023
0.022
0.180
0.009
0.009
0.017
0.002
0.035
0.003
0.021
0.000
0.177
0.023
0.021
0.047
0.033
0.045
0.038
0.045
0.033
0.031
-
IS1
SP2
IT1
SP1
AU1
IT3
BU2
BU1
IT2
IT4
Fig. 3. Genetic relationships among the fox populations
studied (allozyme data, modified Rogers distance [Wright,
1978], Neighbor-joining tree).
probably due to the different sets of enzyme screened, as
it also happened in some mustelid species, which were
completely monomorphic in Simonsen (1982) but partially highly polymorphic in Hartl et al. (1988). Only
two out of nine polymorphic loci in our study were
examined by Simonsen (1982). Except for the foxes
from Spain, differences in P and A between populations
may be determined by differences in sample sizes
(rs = 0.99, P<0.001 and rs = 0.84, P = 0.017, respectively).
Average heterozygosity in Sardinia was high in relation
to P and A. This may occur in bottlenecked and/or
isolated populations (cf. Hartl & Pucek, 1994), as many
rare alleles are lost in such situations, but frequencies of
some of them may increase dramatically. Thus, high
levels of H are generated even if P and A are low (Nei,
Maruyama & Chakraborty, 1975).
Electrophoretic estimates of genetic variation within
populations were paralleled by those obtained from the
mtDNA sequence only in the Iberian and Israeli populations, which were identified as the genetically most
homogeneous ones. However, in the other populations,
mtDNA sequence analysis revealed a somewhat lower
diversity in IT1 and a considerably higher diversity in
AU1, IT3 and SP2 than allozyme data. The most
evident inconsistency between allozyme and mtDNA
data occurred in the comparison of genetic divergence
between the two populations from Spain, very similar in
terms of allozyme frequencies (Fig. 3), but relatively
different in terms of Cyt b sequences.
Both allozyme and mtDNA data suggest that presently these fox populations are genetically fairly
isolated from one another. Almost 50% of the total
allozymic diversity (FST = 0.479) and of the total sequence variation (1st = 0.459) was due to divergence
between populations. When Ck-2 and Dia-2 with partially fixed differences in allelic frequencies among
populations were excluded, FST remained still as high
as 31%. Such a value is much higher than that observed
among subspecies of the red deer Cervus elaphus (22%;
Gyllensten et al., 1983). As a possible consequence of
isolation, the Israeli population was fixed for a private
allele at the Dia-2 locus, while in AU1 a private allele at
the locus Gdh had a comparatively high frequency
(0.250). Differentiation between populations was even
more pronounced in mtDNA, where most haplotypes
were restricted to only one population (Table 4). All
haplotypes were only slightly differentiated from one
another and, especially in the AU1 population, divergence between haplotypes was higher within
populations than between them. The reduction of gene
flow between populations may be of comparatively
recent origin. Smaller canid species are killed and/or
outcompeted by larger ones (Macdonald, 1992: 90).
Until recent times, the red fox must have survived in
sympatry with the wolf Canis lupus and jackals Canis
spp., in Europe and the Mediterranean Basin. Its
population density must have been kept low also by
other larger predators (striped Hyaena hyaena and
spotted Crocuta crocuta hyenas, leopard Panthera
pardus, lion Panthera leo and, perhaps, lynx Lynx lynx),
thus enhancing the turnover of breeders, lowering the
mean age of fox populations and favouring a high gene
flow. Presently, to some extent, hunting by humans
might locally mimic the action of natural predators,
but, in the long run, it may generate anti-Darwinian
effects. Failure of intensive hunting in consistently
reducing population size has been repeatedly demonstrated (Macdonald, 1980; Lade et al., 1996), and its
likely effect on genetic variability of red foxes has been
discussed elsewhere (Frati, F., Lovari, S. & Hartl, G.,
In prep.).
Because of the above situation, mtDNA sequences
did not help much to evaluate evolutionary relationships
among fox samples. According to allozyme genetic
distances (Table 6, Fig. 3) and the distribution of rare
allozyme alleles, the fox populations of the Mediterranean Basin could be divided into two genetically rather
distinct groups: one comprising Spain, the large Italian
islands (Sardinia and Sicily), and Bulgaria; the other
including peninsular Italy and Austria. The fixation of
the private allele Dia-2130 makes the Israeli population
appear to be the most differentiated one, but mean
distance values make it more similar to the first group
(D = 0.030 ± 0.006) than to the second one (D = 0.046 ±
0.001). With a mean value of D = 0.024, Nei’s (1978)
unbiased genetic distance between these groups was
similar to that detected at subspecies level in taxa of
large mammals (cf. Hartl et al., 1990). Within the first
main cluster of populations, IT2 was considerably more
closely related to the Bulgarian foxes than the Iberian
populations, whereby the distance between IT2 and the
Iberian foxes was of a magnitude similar to that
between the two major groups (Table 6, Fig. 3).
Two alternative hypotheses may be put forward to
explain our data. Red foxes from the East might have
been transported by early immigrants, since the Neolithic, to the Mediterranean islands and to Spain. If this
hypothesis is true, two aspects are hard to explain. The
red fox has been well presented all over the Iberian
peninsula since the Middle Pleistocene. It is most unlikely that the autochthonous Iberian foxes were
supplanted so totally by eastern immigrant foxes that
the presently quite high genetic distance (D = 0.023—
0.025) from the Austrian and the Italian populations
could develop. Furthermore, contemporary Iberian
foxes are genetically very homogeneous (H = 0.006—
0.01), which rules out the possibility of past interbreeding with a different gene pool. As to Sardinian red
foxes, as well as having common traits with balkanic
populations, their gene pool may well be a mixture of
genes from different contributing stocks (H = 0.026). In
fact, they share genetic markers with populations from
Central Italy (haplotype A and the allele Ck-290) and
from Northern Spain (haplotype A). There is little
argument against the likelihood of the red fox having
been repeatedly introduced by humans to Sardinia (cf.
Schur le, 1993, for wild ungulates) from both Italy and
Spain, given the intermediate geographic position of the
island.
More convincingly, the Quaternary radiation pattern
of the red fox in the Western Palaearctic may also
explain our results. In Europe, this canid has been
commonly found since the Middle Pleistocene (Kurte’ n,
1968; Bonifay, 1971; Capasso Barbato & Minieri, 1978;
Ballesio, 1979), but most likely it underwent extensive
population fluctuations during glacial and interglacial
changes. The red fox does not perform well in very
cold climates, e.g. in the Arctic, where it is replaced by
the cold-adapted Arctic fox Alopex alopex (cf. Nowak,
1991). It may be assumed that its distribution should
have shrunk and population densities should have
decreased during glaciations, while red foxes retreated
south in warmer pockets. Fragmentation of its distribution probably ensued. At the end of the Wurrm, red
fox populations should have spread northward to
occupy their previous range. One could speculate that
fox populations from northern ‘pockets’ and those
from southern ones reached some degree of genetic
differentiation during the Wur rm. Because of the relatively short time of separation, successful interbreeding
between these populations could still occur. A combination of effective physical barriers (e.g. the Pyrenees)
and geographic distance may have reduced extensive
panmixia. While the low altitude eastern passes of the
Alpine Arch have allowed migration of many mammalian species of Central Europe to Italy, the Pyrenees
may be a comparatively more effective barrier than the
Alps. This could explain why Spanish foxes have
maintained the previous genetic identity, whereas those
from Central Italy have not. The above explanation
may also account for the great genetic separation of
Israeli foxes. Quaternary glaciations and deglaciations
must have repeatedly stirred population movements of
mammals (cf. also moles Talpa spp. [Filippucci et al.,
1987; Loy, Di Marino & Capolongo, 1996]; Meridiopitymys [Chaline & Mein, 1979]; snow voles Chionomys
nivalis [Janeau & Aulagnier, 1997]; hares Lepus spp.
[Palacios, 1996]; southern chamois Rupicapra pyrenaica
[Masini & Lovari, 1988]; and, perhaps, wildcats Felis
silvestris [Ragni et al., 1993]). Alternation between
panmixia and isolation may have occurred a number
of times. The whole story must be quite complicated,
but we think that the second hypothesis is more
convincing than the first one, although further genetic
data on foxes from populations of North Africa, the
Near East and the Balkans may be necessary to bear
it out.
Acknowledgements
We are greatly indebted to Lucia Burrini, Lidia Fleba,
B. Massa, R. Mazzoni della Stella, Giorgia Romeo and
Y. Yom-Tov who kindly provided samples of red foxes.
G. Ficcarelli, R. Fondi, Rita Lorenzini, F. Masini, an
anonymous referee and, in particular, M. Masseti read
earlier versions of our manuscript critically, improving
it with many useful comments. FF and SL were partly
supported by grants from the Amministrazione Provinciale di Siena, M.U.R.S.T. 40% and M.U.R.S.T.
60%.
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