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Y chromosome haplotyping in Scandinavian wolves (Canis lupus) based on microsatellite markers

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The analysis of mitochondrial DNA sequences has for a long time been the most extensively used genetic tool for phylogenetic, phylogeographic and population genetic studies. Since this approach only considers female lineages, it tends to give a biased picture of the population history. The use of protein polymorphisms and microsatellites has helped to obtain a more unbiased view, but complementing population genetic studies with Y chromosome markers could clarify the role of each sex in natural processes. In this study we analysed genetic variability at four microsatellite loci on the canid Y chromosome. With these four microsatellites we constructed haplotypes and used them to study the genetic status of the Scandinavian wolf population, a population that now contains 60-70 animals but was thought to have been extinct in the 1970s. In a sample of 100 male wolves from northern Europe we found 17 different Y chromosome haplotypes. Only two of these were found in the current Scandinavian population. This indicates that there should have been at least two males involved in the founding of the Scandinavian wolf population after the bottleneck in the 1970s. The two Scandinavian Y chromosome haplotypes were not found elsewhere in northern Europe, which indicates low male gene flow between Scandinavia and the neighbouring countries.
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Molecular Ecology (2001)
10
, 1959–1966
© 2001 Blackwell Science Ltd
Blackwell Science, Ltd
Y chromosome haplotyping in Scandinavian wolves
(
Canis lupus
) based on microsatellite markers
A.-K. SUNDQVIST,* H. ELLEGREN,* M. OLIVIER† and C. VILÀ*
*
Department of Evolutionary Biology, Uppsala University, Norbyvägen 18 D, SE-752 36 Uppsala, Sweden,
Stanford
Human Genome Center, Department of Genetics, Stanford University School of Medicine, 975 California Avenue, Palo Alto,
CA 94304, USA
Abstract
The analysis of mitochondrial DNA sequences has for a long time been the most extensively
used genetic tool for phylogenetic, phylogeographic and population genetic studies. Since
this approach only considers female lineages, it tends to give a biased picture of the popu-
lation history. The use of protein polymorphisms and microsatellites has helped to obtain
a more unbiased view, but complementing population genetic studies with Y chromosome
markers could clarify the role of each sex in natural processes. In this study we analysed
genetic variability at four microsatellite loci on the canid Y chromosome. With these four
microsatellites we constructed haplotypes and used them to study the genetic status of the
Scandinavian wolf population, a population that now contains 60–70 animals but was
thought to have been extinct in the 1970s. In a sample of 100 male wolves from northern
Europe we found 17 different Y chromosome haplotypes. Only two of these were found in
the current Scandinavian population. This indicates that there should have been at least two
males involved in the founding of the Scandinavian wolf population after the bottleneck
in the 1970s. The two Scandinavian Y chromosome haplotypes were not found elsewhere
in northern Europe, which indicates low male gene flow between Scandinavia and the
neighbouring countries.
Keywords
: grey wolf, haplotype, isolation, microsatellites, migration, Y chromosome
Received 7 November 2000; revision received 22 March 2001; accepted 27 March 2001
Introduction
The grey wolf (
Canis lupus
) was once spread all over North
America, Europe and Asia (Mech 1970), but in the early
Middle Ages extermination of wolves began in central and
northern Europe (Carbyn
et al
. 1995). Today, wolves have
disappeared from most of Europe, USA, Mexico and
southern and eastern Asia (Mech 1970). In Scandinavia,
wolves were common until the mid-19th century (Wabakken
et al
. 1992). In 1966, legal protection of the wolf was
introduced in Sweden, but only a few years thereafter the
species was thought to be extinct from the Scandinavian
Peninsula (Naturvårdsverket 2000). However, in 1980, a
few wolves were seen in southern Sweden, in the county of
Värmland, and in southern Norway (Fig. 1; Wabakken
et al
. 1992). These animals formed a stable pack and since
1983 breeding has taken place almost every year in this
area, and the population size has increased. The wolf
population on the Scandinavian Peninsula was estimated to
be about 50–72 individuals in 1998 (Wabakken
et al
. 2001).
As there is no regular occurrence in northern Scandinavia,
the population may be isolated (Fig. 1). The management
situation is complicated since the population, though
small, causes large economic losses to sheep and reindeer
breeders (Naturvårdsverket 2000). Three alternatives have
tentatively been suggested to explain the origin of the
population (Ellegren
et al
. 1996; Wabakken
et al
. 2001):
immigrants from neighbouring populations (e.g. Finland
and Russia), release from captive populations, or survival
of a few individuals during the demographic bottleneck.
The current Scandinavian wolf population has low
levels of genetic variability. A study by Ellegren
et al
. (1996)
showed that all modern Scandinavian wolves analysed
(
n
= 12) shared the same mitochondrial DNA (mtDNA)
haplotype, and that the heterozygosity at 12 microsatellite
Correspondence: Carles Vilà. Fax: + 46–(0)18 4716310; E-mail:
carles.vila@ebc.uu.se
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© 2001 Blackwell Science Ltd,
Molecular Ecology
, 10, 1959–1966
loci had decreased over time after recolonization. Addi-
tional analyses, totalling over 30 individuals, have failed to
find additional mtDNA lineages (Vilà
et al
. in preparation).
All the current animals may be descendants of the indi-
viduals that founded the population in the early 1980s
and extensive inbreeding can be expected (Ellegren 1999).
Unless genetic contact can be established with more eastern
wolf populations, a continuous loss of genetic variability
may be inevitable. Inbreeding depression has been reported
in a study of captive wolves from Swedish zoos, e.g. reduction
in juvenile weight, as well as impaired reproduction and
longevity (Laikre & Ryman 1991). Moreover, a hereditary
form of blindness frequently appeared in the same captive
population as a result of inbreeding (Laikre
et al
. 1993). The
founders of the Swedish zoo population were two Swedish
and Finnish full-sib pairs captured in 195060 (Ellegren
et al
. 1996). The existence of deleterious alleles in the zoo
founders suggests that Scandinavian wolves have not been
purged of deleterious alleles and therefore that such alleles
might also be present in the contemporary wild popula-
tion. Furthermore, such alleles may currently exist at high
frequencies due to founding effects, potentially causing
inbreeding depression.
Population genetic studies have been greatly facilitated
during the last 10–15 years by the introduction of new
DNA-based approaches. Sequence analysis of mtDNA and
genotyping of highly variable nuclear microsatellites have
become the two standard tools in most genetic surveys in
animals, allowing inferences to be made on phylogenetic
relationships and maternal gene flow (mtDNA), phylogeo-
graphic patterns and levels of genetic variability (mtDNA
and microsatellites), and fine-scale population structure
(mainly microsatellites). Recent advances in human genome
analysis (Lahn & Page 1997) and in human population
genetics (Casalotti
et al
. 1999; Hurles
et al
. 1999; Pritchard
et al
. 1999; Su
et al
. 1999; Thomas
et al
. 2000) now suggest
that Y chromosome haplotyping may develop to become
the third main tool in the next generation of genetic studies
of natural populations. Y chromosome analysis has the
obvious advantage that it specifically follows paternal gene
flow, thus complementing studies of maternal gene flow
based on mtDNA. Several features make the Y chromo-
some a suitable target for population genetics analysis.
Although not having the high mutation rate characteristic
of mtDNA (Seielstad
et al
. 1999), the Y chromosome offers an
essentially inexhaustible source of polymorphisms given
that its size is several orders of magnitude larger than that
of mtDNA and that most of the chromosome, in contrast to
mtDNA, consists of noncoding DNA. Moreover, it repres-
ents a single segregating unit, i.e. being a nonrecombining
chromosome (with the exception of the very small pseudo-
autosomal region), and haplotypes composed of a very
large number of segregating sites can thus be constructed.
In the case of the Scandinavian wolf population, analysis
of Y chromosome haplotypes can, among other things,
provide information on the number of founding males.
There should have been at least as many male founders as
the number of Y chromosome haplotypes present in the
population, assuming no mutation. Additionally, the com-
parison of haplotypes in different populations can be used
to trace the origin of the Scandinavian wolf population. It
can also be used to reconstruct male migration patterns
between populations in northern Europe. Migration of
wolves from other populations (in Finland and Russia;
Fig. 1) might be the only natural way to restore the genetic
diversity in Scandinavian wolves and management
decisions are, and will be, taken depending on the exist-
ence of possible migration (Naturvårdsverket 2000). To
establish a system for Y chromosome haplotyping in wolf
populations we report here the development of locus-
specific microsatellite markers on the wolf Y chromo-
some, based on sequence data presented by Olivier &
Lust (1998) and Olivier
et al
. (1999). We also describe an
initial characterization of their variability in a haplotypic
context. Moreover, we apply these markers to the Scandin-
avian wolf population to address questions about founding
and migration pertinent to conservation issues.
Fig. 1 Present distribution of wolves in Scandinavia and sur-
rounding areas (shaded area). The location of the first appear-
ance of wolves in the early 1980s is indicated with an asterisk
(adapted from Wabakken et al. 2001).
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Molecular Ecology
, 10, 1959–1966
Materials and methods
Strategy for the development of locus-specific markers
Olivier
et al
. (1999) identified two different microsatellite
sequences (termed MS34 and MS41) from the canine Y
chromosome using a random amplified polymorphic
DNA (RAPD) approach (Olivier & Lust 1998). When
primers for polymerase chain reaction (PCR) amplification
flanking these microsatellite sequences were constructed,
however, none of the primer pairs revealed locus-specific
amplification in dogs. In both cases two fragments,
normally of different size, were obtained from amplification
of male dog DNA, while no amplification was obtained
from females (Olivier
et al
. 1999). We obtained a similar
pattern when we used the primers for amplification of
wolf DNA (data not shown). Since duplication of DNA
sequences is a common event on the Y chromosome
(Jobling
et al
. 1996; Lahn & Page 1997; Tilford
et al
. 2001),
this observation suggests that both these microsatellites
had been duplicated during the evolution of the canine
Y chromosome. To test for this possibility and, if correct,
to be able to develop locus-specific markers that sub-
sequently could be integrated to allow Y chromosome
haplotyping, we cloned amplification products obtained
from wolf DNA with the original primers reported by
Olivier
et al
. (1999) and screened the clones with single-
strand conformation polymorphism (SSCP).
DNA extraction and amplification
DNA was initially extracted from tissue samples from
two male wolves from different populations in Europe
(Latvia and Spain). A modified phenol/chloroform
protocol (Sambrook
et al
. 1989) was used, after digestion
with proteinase K. DNA was dissolved in water and the
concentration was measured with a fluorometer (Hoefer).
The specific canine Y chromosome primers MS34F plus
MS34R and MS41F plus MS41R, developed by Olivier
et al
.
(1999), were used to amplify the corresponding fragments
initially from the two male wolves. PCR reactions included
1
×
PCR buffer II (Perkin Elmer), 0.4 m
m
dNTPs, 0.5
µ
m
of
each primer, 5 mg/mL bovine serum albumin and 0.02 U
Ampli
Taq
Gold polymerase (Perkin Elmer). For MS34
3 m
m
MgCl
2
was used and for MS41 2 m
m
MgCl
2
. The
reactions were run in a total volume of 10
µ
L with 10–50 ng
DNA as template.
The PCR profile for MS34 included an initial denaturation
step at 95
°
C for 10 min, 14 touchdown cycles with 30 s at
95
°
C, annealing at 72
°
C for 30 s and decreasing 0.5
°
C each
cycle, and 1 min at 72
°
C, followed by 25 cycles of 95
°
C for
30 s, 65
°
C for 30 s and 72
°
C for 1 min. An extension step
of 10 min at 72
°
C was added after the last cycle. For MS41
the PCR profile was a similar touchdown reaction but
starting at a lower temperature, i.e. six cycles with
annealing at 68
°
C and decreasing 0.5
°
C each cycle,
followed by 32 cycles as above.
Cloning and sequencing
After PCR, the products were purified with QIAquick
PCR Purification Kit (Qiagen). Ligation of PCR products
into a pUC18 vector was performed at room temperature
for 2 h using the ‘New improved 2*Rapid ligation’ kit
(Promega). Plasmids were introduced into competent
cells by electroporation at 1800 V. Cells were incubated
at 37
°
C over night on agar plates containing X-gal and
IPTG. To be able to discriminate, in the ligations, between
the different Y chromosome fragments containing the
duplicated microsatellites, single strand conformation
polymorphism (SSCP; Hayashi 1991) analysis was used.
Cloned inserts were PCR amplified with the same primers
used previously and PCR products were denatured
and run in 12% acrylamide gels at 3 W for 16 h at 20
°
C.
Afterwards DNA was visualized using silver staining
(Bassam
et al
. 1991). Clones identified as containing
different inserts on the SSCP gel were selected for sequenc-
ing and template DNA was prepared with a QIAquick
PCR Purification Kit. Sequencing was performed using
BigDye terminator cycle sequencing chemistry on an ABI
377 instrument (Perkin Elmer). The corresponding protocols
recommended by the manufacturer were used.
Primer design and microsatellite amplification
New forward primers were designed from the obtained
sequences (see Results), in order to be able to amplify
independently individual copies of the duplicated frag-
ments. These primers were labelled with different fluor-
escence labels (6-FAM and TET) to allow them to be run
together in the same PCR reaction. The four microsatellite
loci identified by this procedure were named MS34A,
MS34B, MS41A and MS41B, and a corresponding nomen-
clature was used for the new forward primers.
The PCR mix for MS34A and MS34B included 1
×
PCR
buffer (Qiagen), 4 m
m
MgCl
2
, 0.8 m
m
dNTPs, 0.5
µ
m
forward primer A, 0.5
µ
m
forward primer B, 1.0
µ
m
reverse
primer and 0.02 U Hot Star
Taq
polymeras (Qiagen). The
PCR profile was 10 min at 95
°
C, followed by 35 two-step
cycles with 30 s at 95
°
C and 45 s at 72
°
C, and was com-
pleted with 10 min at 72
°
C.
The PCR mix for MS41A and MS41B included 1
×
PCR
buffer, 1.5 m
m
MgCl
2
, 0.8 m
m
dNTPs, 0.4
µ
m
forward primer
A, 0.5
µ
m
forward primer B, 0.9
µ
m
reverse primer and
0.02 U Ampli Taq Gold (Perkin Elmer). The PCR profile
included an initial denaturation step at 95
°
C for 10 min
and eight touchdown cycles, with 95
°
C for 30 s, 54
°
C for
30 s decreasing 0.5
°
C each cycle, and 72
°
C for 1 min,
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1962
A.-K. SUNDQVIST
ET AL.
© 2001 Blackwell Science Ltd,
Molecular Ecology
, 10, 1959–1966
followed by 35 cycles of 95
°
C for 30 s, 50
°
C for 30 s and
72
°
C for 1 min. An extension step of 72
°
C for 10 min was
added at the end.
Comparison of populations and data analyses
For population comparisons DNA was extracted from
male wolves originating from several populations in
northern Europe. Fourteen wild wolves from Scandinavia
and 13 wolves from the Swedish zoo population identified
as males by the collector and verified by us using ampli-
fication of the SRY gene (Meyers-Wallen
et al
. 1995) were
analysed. Additionally, 16 male wolves from Finland, 26
from Russia and 31 from the Baltic States were genotyped,
to be able to study the relationship between Scandinavian
wolves and animals from neighbouring populations. The
samples were genotyped on an ABI 377 instrument (Perkin
Elmer) and data were analysed using the software programs
genscan
and
genotyper
(Perkin Elmer).
Since the four markers analysed are located in the
nonrecombining region of the canine Y chromosome (see
Results), they can be combined into haplotypes. We used
the number of haplotypes in different populations as a
measure of genetic diversity. Because mutations in micro-
satellite loci are normally produced by the addition or
deletion of one or two repeat units (Ellegren 2000a), two
haplotypes that differ at multiple microsatellite loci or
by multiple repeat units are not likely to have recently
derived from each other.
Results
In order to isolate locus-specific markers from the Y
chromosome microsatellites amplified by two pairs of
primers reported by Olivier
et al
. (1999), we cloned PCR
products obtained in amplification with these primers on
male wolves (see Materials and methods). Two different
types of clones, within each individual, were identified for
MS34. Both types were about 330 base pairs (bp) in length,
and after sequencing they were found to differ by three
point mutations and one insertion/deletion of 2 bp in the
microsatellite flanking regions (Fig. 2a). They varied also
in the number of CA repetitive elements. Similarly, for
MS41 two different types of clones were identified within
each individual. These differed by two point mutations in
the c. 238 bp region amplified, one interrupting the repeat
array in the middle of the microsatellite, and one imme-
diately flanking it (Fig. 2b). Again, the number of CA
repeats was also different between the types. To allow
locus-specific amplification of the different microsatellites,
we used the deletion in MS34 and the point mutation
immediately adjacent to the microsatellite in MS41 to
design, for each marker, two new forward primers to be
used with original reverse primers (Fig. 2). These four new
primer pairs detecting the MS34A, MS34B, MS41A and
MS41B loci all yielded single fragments in amplification of
male wolf DNA. Since no amplification was still obtained
from female DNA (
n
= 138), we conclude that four locus-
specific microsatellite markers had been developed for
the nonrecombining region of the canid Y chromosome.
The four microsatellites were genotyped in 14 males
from the wild Scandinavian wolf population, in 13 males
from the Swedish zoo population and in 73 males from
other north European wolf populations. All markers were
polymorphic with two to nine alleles seen among the 100
male wolves analysed (Table 1). In a preliminary analysis
of haplotype diversity among north European (including
Scandinavian) wolves, 17 different haplotypes were found
Fig. 2 DNA sequences obtained for MS34
(a) and MS41 (b). Identical positions are
indicated with dots, and gaps in the
alignment are indicated with dashes.
Specific forward primers designed to
amplify independently each copy are
indicated by underlined sequences.
Microsatellite sequences are inside the
boxes.
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Y CHROMOSOME MICROSATELLITES IN SCANDINAVIAN WOLVES
1963
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Molecular Ecology
, 10, 1959–1966
(Table 2). Three of these haplotypes (A, B and C) were seen
in wild Scandinavian wolves, and a fourth one (D) was
found fixed in the Swedish zoo population (Table 3). A and
B differed by three repeat units at one locus (MS41B) but
were otherwise identical. C differed from A and B at three
loci (MS34B, MS41A, MS41B) by a total of nine and six repeat
units, respectively (Table 2). A total of four haplotypes were
observed in Finland, nine in Russia and 10 in the Baltic
States (Table 3). Haplotypes A, B and D were not found
outside Scandinavia, but haplotype C was also found in the
Baltic States. The haplotypic diversity is lower in Scandinavia
than in the other populations. The number of haplotypes
found per sample analysed was 0.21 in Scandinavia (0.15
excluding haplotype C, see below), and 0.25, 0.35 and 0.32
for Finland, Russia and Baltic States, respectively.
The temporal occurrence of haplotypes A, B and C in
Scandinavian male wolves is shown in Table 4. Haplotype
A was found in three animals collected during the period
1993–99, haplotype B was found in a total of 10 animals
collected during 1984–2000, while haplotype C was only
found in a single individual killed in 1977 in northern
Sweden.
Table 1 Characteristics of four wolf Y chromosome microsatellites
Microsatellite Primer sequence No. alleles Observed length (bp)
MS34A MS34AF 5-AGCCATTCCTGGCCGAGTGG-34 172–178
MS34R 5-GGTCCCCTTTTGCCATAGTGT-3
MS34B MS34BF 5-AGCCATTCCTGGCCGAGTCC-35 174–182
MS34R, as above
MS41A MS41AF 5-TCCTCTAATTTTCCCCTCTA-32 208–210
MS41R 5-CTGCTCGACCCTCTTCTCTG-3
MS41B MS41BF 5-TCCTCTAATTTTCCCCTCTC-39 212–228
MS41R, as above
Table 4 Time distribution of haplotypes A, B and C in the
Scandinavian wolf population. Each year corresponds to the date
when individual male wolves were killed
Haplotype A Haplotype B Haplotype C
1977
1984
1986
1986
1989
1992
1992
1993
1996
1997
1998
1999
2000
2000
Table 2 Y chromosome haplotypes found in the North European
wolves and allele sizes at four microsatellite loci (in base pairs)
Haplotype MS34A MS34B MS41A MS41B
A 172 182 208 214
B 172 182 208 220
C 174 178 208 226
D 172 180 208 216
E 178 176 208 216
F 176 176 208 218
G 176 178 208 222
H 174 178 208 228
I 172 180 208 212
J
172 180 208 214
K 178 176 208 218
L 174 178 208 222
M 176 178 210 224
O 172 182 208 216
P 178 174 208 216
Q 174 176 208 220
R 174 178 208 214
Table 3 Distribution of Y chromosome haplotypes across wolf
populations in northern Europe
Haplotype
Scand.
(14)
Zoo
(13)
Finland
(16)
Baltic states
(31)
Russia
(26)
A3
B10
C1 3
D13
E656
F53
G211
H332
I1
J
4
K510
L4
M41
O1
P1
Q1
R1
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ET AL.
© 2001 Blackwell Science Ltd,
Molecular Ecology
, 10, 1959–1966
Discussion
The isolation and characterization of polymorphic
microsatellite markers on the Y chromosome can help
explain the recovery of the Scandinavian wolf population
during the last decades. One important question is the
number of males that founded the population in the 1980s.
In this study we found three Y chromosome haplotypes
(A, B and C) among modern Scandinavian wolves.
Haplotype C, however, was only found in one individual
from northernmost Sweden killed in 1977. This was before
wolves re-appeared in southern Sweden and this particular
animal, and its relatives, were excluded as founders of
the extant population based on data from mtDNA and
autosomal microsatellite loci (Ellegren
et al
. 1996). Our
results therefore suggest that there could have been as few
as two males involved in the founding of the current
Scandinavian population, given that we only identified
two different Y chromosome haplotypes (A and B) in
samples from southern Scandinavia collected after 1983.
Together with the observation of a fixed mtDNA variant in
the population (Ellegren
et al
. 1996), a minimum of three
founders is suggested (two males and one female). This
agrees with the interpretation derived from the presence
of a maximum of five alleles at autosomal microsatellite
loci (Ellegren
et al
. 1996). Of course, the observation of
two paternal lineages only provides an absolute min-
imum number of male founders, given that multiple
males with identical Y chromosome haplotypes may have
been involved. Similarly, the presence of a fixed mtDNA
variant in Scandinavia represents a minimum number
of founding females because that variant is also very
common outside Scandinavia (Vilà
et al
. in preparation).
We cannot distinguish between the scenarios of two
males founding the population in the early 1980s and only
one early male founder and the other lineage being intro-
duced later. Haplotype A was first seen in a sample from
1993, which might be indicative of a relatively recent
recruitment. However, the limited sample size of males from
before 1993 precludes strong conclusions on this point.
Related to the question of the number of founders is the
origin of founders. If the current Scandinavian wolf popu-
lation descends from immigrants from northern Europe,
e.g. from Finland, Russia, or the Baltic States, we would
have expected to find haplotypes A and B also in these
populations. However, these haplotypes were not seen in any
of our 73 male samples from other north European coun-
tries. Assuming that these samples are from wolves of a
single panmictic population, for any haplotype present in
a frequency of 5% in this population the probability of
being missed in a sample of 73 is lower then 0.025. How-
ever, our preliminary data using mtDNA sequences and
microsatellites suggest that some fragmentation between
north European wolf populations exists (Vilà
et al
. in
preparation). In light of this, although our results are not
evidence of immigration from other North European
populations, it is probably premature to exclude other north
European wolf populations as the origin of the current
Scandinavian population.
The sudden appearance of wolves in southern Scandinavia
in the early 1980s, at least 1000 km from the closest regular
occurrence of Finnish or Russian wolves, led to speculation
about a possible origin from a release from the captive zoo
population (see Ellegren
et al
. 1996). According to the
pedigree for the Swedish zoo population only one sur-
viving paternal lineage should be expected. Indeed, all 13
zoo males analysed in this study shared the same haplo-
type (D), not found in the wild population. From this we
conclude that the paternal lines in the wild Scandinavian
wolf population cannot originate from the Swedish zoo
population.
The presence of Y chromosome lineages in the wild
population not observed in captive or in neighbouring
populations is compatible with the hypothesis that the
extant population could have been derived from some
Scandinavian male wolves surviving during the bottle-
neck. However, we cannot provide any direct support
for this idea. The analysis of these markers in ancient
wolf samples, collected before the population decline
in Scandinavia, could help to address the question.
The three haplotyes found in Scandinavia (A, B and C)
are not likely to have arisen from each other by mutation
in recent times. Assuming that mutations in microsatellite
sequences are normally produced by the addition or deletion
of one or, rarely, two repeat units (Ellegren 2000a), the dif-
ferences between these haplotypes correspond to several
mutational steps. The average mutation rate for human Y
chromosome microsatellites is in the order of 0.2% per
generation (Ellegren 2000b; Jobling & Tyler-Smith 2000).
If this figure is roughly applicable to other mammals and
considering that only a few generations have passed since
wolves reappeared in Scandinavia (mean generation time
of 3 years; Mech & Seal 1987), it indeed seems unlikely
that one of these haplotypes has recently derived from
another. A similar reasoning can be made for excluding a
recent mutational origin of the haplotype found in the
zoo population (D), which differed from the haplotypes
found in Scandinavia at two (A and B; totalling two and
three repeat units, respectively) or three (C; seven repeat
units) microsatellite loci.
The captive and wild Scandinavian wolf populations
show similarly low levels of genetic variability at auto-
somal markers (Ellegren 1999). Our results also indicate
relatively low diversity of Y chromosome haplotypes, con-
sistent with the low diversity of mtDNA variants (Ellegren
et al
. 1996; Vilà
et al
. in preparation). This reduced genetic
variability could be the result of the demographic bottle-
necks. Consequently, the establishment of gene flow with
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1965
© 2001 Blackwell Science Ltd,
Molecular Ecology
, 10, 1959–1966
other populations that could allow the recovery of some
variability is important for the long-term survival of the
Scandinavian wolf population. Our results indicate that
immigration of males might not be taking place and, until
more complete genetic evidence is available, effort should
be allocated to allow gene flow.
This study represents one of the first reports of Y chro-
mosome microsatellites in a wildlife species and their
application for fine-scaled Y chromosome haplotyping in a
natural population. We anticipate that more studies of this
kind will follow. Since dispersal patterns in many organ-
isms are characterized by pronounced differences between
sexes (Greenwood 1980, 1983), the ability to study specific-
ally gene flow in both sexes is highly warranted and will
add to the study of the effects of such differences on phylo-
geographic patterns. Moreover, since variation in male
reproductive success is often higher than that among
females (a consequence of the prevailing direction of sexual
selection, Andersson 1994), the ability to follow patrilines
can be particularly important in studies of social structures
and mating systems. Y chromosome haplotyping obviously
also represents a fast method to exclude possible fathers in
paternity studies and to determine multiple paternities in
single litters or clutches.
Admittedly, however, the use of Y chromosome haplo-
typing in population genetic studies of natural populations
is still often hindered by lack of sequence information
necessary for polymorphism screening. Identification of Y
chromosome microsatellites or single nucleotide poly-
morphisms (SNPs) should thus be given high priority in
population genetic studies. The combined use of SNPs and
microsatellites in Y chromosome haplotyping should allow
both deep-rooted events (slowly evolving SNPs) and more
recent population differentiation (fastly evolving microsat-
ellites; see de Knijff 2000; Forster
et al
. 2000) to be followed.
Acknowledgements
Samples for this study were kindly provided by Zanete Andersone,
Adriano Casulli, Inge Gade-Jørgensen, Ilpo Kojola, Rie Stagegaard,
Harri Valdmann and the Swedish National Veterinary Institute.
We wish to acknowledge Dr Jennifer Seddon, and two anonymous
reviewers for their helpful comments on the manuscript. This
research has been supported by Direktoratet for Naturforvaltning
(Norway), Swedish Environmental Protection Agency, the
Swedish Research Council for Agriculture and Forestry, the
Swedish Hunting Association, the Nordic Arctic Research Program,
and by the Olle Engkvist, Carl Trygger, Oscar and Lili Lamms,
and the Sven and Lilly Lawskis foundations. H.E. is a Royal
Academy of Sciences Research fellow supported by a grant from
the Knut and Alice Wallenberg foundation.
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... For 18 males (D = 3, W = 13, SH = 2) Y-STR was not determined. The distribution of Y-STR for the remaining 105 males is shown in Table 2, and compared with the findings of the same Y-STR haplotypes found by other studies in dogs, wolf-dog hybrids and in other wolf populations in Europe and Russia (SUNDQVIST et al., 2001;IACOLINA et al., 2010;RANDI et al., 2014). ...
... In IACOLINA et al. (2010) YH05 (named H3) was the most common in dogs but it was also found in five hybrid wolf-dog individuals. The only evidence of YH05 in wolves comes from SUNDQVIST et al. (2001) who found the same haplotype (named L) in four male wolves from Baltic States. In our study, two suspected hybrids carrying YH05 (WCRO32 and WCRO51) were confirmed as hybrids also by their low qw, while a third animal with YH05 (WG01) did not show any other (phenotypic, STR or mtDNA CR1) signs of hybridization. ...
... In our study, two suspected hybrids carrying YH05 (WCRO32 and WCRO51) were confirmed as hybrids also by their low qw, while a third animal with YH05 (WG01) did not show any other (phenotypic, STR or mtDNA CR1) signs of hybridization. These findings could suggest that (1) YH05 is a rare haplotype present in the Croatian wolf population, as in other wolf populations (SUNDQVIST et al., 2001), or (2) YH05 came from a dog and it is a sign of ancient introgression. The haplotype YH08 was found to be quite common in the Croatian wolf population, but the same haplotype was found in one male village dog which showed no apparent traces of admixture with wolves (q d = 0.995 CI 90% = 0,976-1,000). ...
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Wolf-dog hybridization is considered as one of the main threats for wolf conservation since the admixture and introgression of domestic genes may disrupt local adaptations and threaten the long term survival of wild wolf populations. We investigated the occurrence of wolf-dog hybridization in Croatia by analyzing a panel of 12 autosomal microsatellite markers using Bayesian admixture tests, and assessed its directionality by the use of maternally and paternally inherited markers in combination with morphometric data and morphological features. A systematic analysis of morphologic features and morphometric data was used to rank the studied individuals into either phenotypic wild-type wolves or suspected hybrids. By combining Bayesian assignment results with phenotypic features, we set three thresholds which differentiated wolves from hybrids with maximized hybrid detection and a minimized chance for false positive hybrid identification. On the basis of phenotype, out of 176 wild canids, 157 (89.2%) were categorized as wolves and 19 (10.8%) as suspected hybrids. On the basis of the Bayesian admixture tests and phenotype together, five (2.8 percent) animals were classified as wolf-dog hybrids, four of them as backcrosses with wolves, and one as a backcross with a dog. Mitochondrial DNA suggested that all hybrids originated from the mating of female wolves and male dogs. Two male hybrids had Y chromosome haplotypes common to both wolves and dogs, while the other two had wolf private Y chromosome haplotypes. One wolf had a dog Y-haplotype, indicating a past introgression of dog genes. All hybrids were found in Dalmatia, where wolves settled recently, and where they live close to humans, with a high rate of human-caused mortality. These conditions are considered as favorable for wolf-dog hybridization. However, we found a low hybridization prevalence in Croatia, which is nonetheless expected to persist as long as the conditions favoring its occurrence are met. The ecological, sociological, conservation and management implications of hybrid occurrence are yet to be determined. © 2018, University of Zagreb, Facultty of Veterinary Medicine. All rights reserved.
... Mivel ez a gonoszóma kizárólag apai ágon öröklődik, kiegészítésül szolgál a mtDNS-vizsgálatokhoz, pontosabb képet adva a leszármazási viszonyokról [36]. Ezen felül az Y-kromoszóma méretéből adódóan, szinte kifogyhatatlan forrása a polimorfizmusoknak, nagy részét nem kódoló régiók alkotják és nem rekombinálódik, emiatt szintén alkalmas populációgenetikai és hibridazonosítási vizsgálatokra [37]. ...
... Az eddigi módszerek fejlődésével és új technikák megjelenésével egyre megbízhatóbb eredményeket sikerül felmutatni a hibridizáció és a farkas-kutya elkülönítése terén [2,94]. Európában eddig Spanyolországban, Olaszországban, a Skandináv és a kelet európai térségben foglalkoztak a farkasok elterjedésével és kutyákkal történő kereszteződésükkel [16,37,51,61]. A farkasállományok genetikai vizsgálata Európa más térségeiben, így hazánkban is korán megkezdődött [6]. ...
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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.
... First, the genetic input at establishment in the 1980s and early 1990s was highly limited. Indeed, with just three founders, only a fraction of the genetic diversity of the source population in Finland, and possibly Russia, became represented in the Scandinavian population (Sundqvist et al. 2001;Vilà et al. 2003). Second, severe inbreeding and genetic drift further reduced the already limited diversity provided by the founders (no additional immigrant wolves reproduced within the Scandinavian population until 2008); only recently has there been some gene flow from the source population (Åkesson et al. 2016). ...
Article
Genetic drift can dramatically change allele frequencies in small populations and lead to reduced levels of genetic diversity, including loss of segregating variants. However, there is a shortage of quantitative studies of how genetic diversity changes over time in natural populations, especially on genome-wide scales. Here, we analyzed whole-genome sequences from 76 wolves of a highly inbred Scandinavian population, founded by only one female and two males, sampled over a period of 30 yr. We obtained chromosome-level haplotypes of all three founders and found that 10%–24% of their diploid genomes had become lost after about 20 yr of inbreeding (which approximately corresponds to five generations). Lost haplotypes spanned large genomic regions, as expected from the amount of recombination during this limited time period. Altogether, 160,000 SNP alleles became lost from the population, which may include adaptive variants as well as wild-type alleles masking recessively deleterious alleles. Although not sampled, we could indirectly infer that the two male founders had megabase-sized runs of homozygosity and that all three founders showed significant haplotype sharing, meaning that there were on average only 4.2 unique haplotypes in the six copies of each autosome that the founders brought into the population. This violates the assumption of unrelated founder haplotypes often made in conservation and management of endangered species. Our study provides a novel view of how whole-genome resequencing of temporally stratified samples can be used to visualize and directly quantify the consequences of genetic drift in a small inbred population.
... Based on a multiple-tube protocol (Taberlet et al. 1996) using procedures described in Fabbri et al. (2018), we genotyped fecal DNA samples amplifying them at 12 unlinked autosomal microsatellites (short tandem repeats [STRs]) selected for their polymorphism and reliable scorability for wolves and dogs ) and routinely used for genotyping low-content DNA samples in non-invasive genetic monitoring projects (Caniglia et al. 2013Fabbri et al. 2018), and a dominant 3-base pair (bp) deletion (named KB or CBD103DG23) of the b-defensin CBD103 gene (the K-locus; Anderson et al. 2009), which represents a reliable indicator of dog introgression in some Italian wolf subpopulations (Caniglia et al. 2013). Additionally, we sexed samples by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the ZFX/ZFY (zinc-finger protein) sequences and identified paternal haplotypes typing 4 STRs located on the Y chromosome (MS34A, MS34B, MS41A, and MS41B; Sundqvist et al. 2001) and maternal haplotypes analyzing 250 bp of the hypervariable domain of the mtDNA CR1 (Caniglia et al. 2013). We used the software Gimlet version 1.3.3 ...
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Introgressive hybridization between domestic dogs and wolves (Canis lupus) represents an em-blematic case of anthropogenic hybridization and is increasingly threatening the genomic integrity of wolf populations expanding into human-modified landscapes. But studies formally estimating prevalence and accounting for imperfect detectability and uncertainty in hybrid classification are lacking. Our goal was to present an approach to formally estimate the proportion of admixture by using a capture-recapture (CR) framework applied to individual multilocus genotypes detected from non-invasive samples collected from a protected wolf population in Italy. We scored individual multilocus genotypes using a panel of 12 microsatellites and assigned genotypes to reference wolf and dog populations through Bayesian clustering procedures. Based on 152 samples, our dataset comprised the capture histories of 39 individuals sampled in 7 wolf packs and was organized in bimonthly sampling occasions (Aug 2015−May 2016). We fitted CR models using a multievent formulation to explicitly handle uncertainty in individual classification, and accordingly examined 2 model scenarios: one reflecting a traditional approach to classifying individuals (i.e., minimizing the misclassification of wolves as hybrids; Type 1 error), and the other using a more stringent criterion aimed to balance Type 1 and Type 2 error rates (i.e., the misclassification of hybrids as wolves). Compared to the sample proportion of admixed individuals in the dataset (43.6%), formally estimated prevalence was 50% under the first and 70% under the second scenario, with 71.4% and 85.7% of admixed packs, respectively. At the individual level, the proportion of dog ancestry in the wolf population averaged 7.8% (95% CI = 4.4−11%). Balancing between Type 1 and 2 error rates in assignment tests, our second scenario produced an estimate of prevalence 40% higher compared to the alternative scenario, corresponding to a 65% decrease in Type 2 and no increase in Type 1 error rates. Providing a formal and innovative estimation approach to assess prevalence in admixed wild populations, our study confirms previous population modeling indicating that reproductive barriers between wolves and dogs, or dilution of dog genes through backcrossing, should not be expected per se to prevent the spread of introgression. As anthropogenic hybridization is increasingly affecting animal species globally, our approach is of interest to a broader audience of wildlife conservationists and practitioners.
... We genotyped our samples using eight autosomal loci (CXX.172, CXX.204, CXX.225, CXX.250, CXX.2, 468, 502, 622;Ostrander et al. 1993Ostrander et al. , 1995 and two Y-linked loci (99,035, MS34B; Bannasch et al. 2005;Sundqvist et al. 2001) originally developed in domestic dogs (Table S2), but for most of them extensively used in wolf-like canids population genetics and hybrid detection (e.g., Adams and Waits 2007;García-Moreno et al. 1996;Gottelli et al. 1994;Hindrikson et al. 2012;Iacolina et al. 2010;. ...
Article
Despite the known genetic permeability among wolf-like canids, there is currently no evidence of gene flow between the recently acknowledged African wolf (Canis lupaster) and domestic dogs (C. lupus familiaris). We genotyped African wolves across their range, together with African domestic dogs and ‘reference’ grey wolves (C. l. lupus; not occurring in Africa). Northwestern African wolves showed (1) the greatest genetic diversity as observed from microsatellite loci and mitochondrial + Y-chromosome markers, and (2) possible signs of past admixture with grey wolves. We detected two zones of hybridization between domestic dogs and African wolves, in northwestern Senegal and central Ethiopia. Hybrids were intermediary in the nuclear genetic space separating African wolves from domestic dogs (and grey wolves), and were in majority assigned to domestic dogs in STRUCTURE. Hybrids showed mitochondrial DNA haplotypes of African wolves, suggesting gene flow directionality between male African dogs and female African wolves. The roaming of feral and shepherds’ dogs in degraded habitats occupied by African wolves may have promoted hybridization. Our results provide evidence that, subsequent to the possible hybrid origin of C. lupaster, the genome of the African wolf is still subject to admixture with C. lupus descendants. This could lead to the genetic dilution of endemic African wolf lineages, such as in eastern Africa, but may also imply disease prevalence and competition for resources with domestic dogs. Our study also is the first to show a significant level of differentiation (ΦST and FST) between North African and West African wolves. Wider genetic screening of African wolves across their range should depict more accurately their population dynamics and the potential stakes related to gene flow with domestic dogs.
... Additionally, a molecular sexing test was conducted for NIS following Seddon (2005). Male samples (both tissue and scats) were genotyped for six Y-linked microsatellites (Sundqvist et al. 2001;Bannasch et al. 2005; Online Resource 2, Tables S2 and S3). Finally, following Caniglia et al. (2013), the 3-bp indel correlated to the black coat color (Candille et al. 2007;Galaverni et al. 2017) on the β-defensin K-locus was screened in all samples. ...
Article
Full-text available
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.
... For example, the addition of Y chromosome markers, even if relatively conserved, provided patrilineal histories (see e.g. Sundqvist et al. 2001, Brändli et al. 2005, Cruciani et al. 2007. Nuclear markers were added in the form of intron sequences (exons were often too conserved within species to yield a meaningful resolution) (Smit et al. 2008, Maswanganye et al. 2017 in an attempt to understand migration or address the gene tree vs species tree problem. ...
Article
Phylogeography examines the spatial genetic structure of species. Environmental niche modelling (or ecological niche modelling; ENM) examines the environmental limits of a species’ ecological niche. These two fields have great potential to be used together. ENM can shed light on how phylogeographical patterns develop and help identify possible drivers of spatial structure that need to be further investigated. Specifically, ENM can be used to test for niche differentiation among clades, identify factors limiting individual clades and identify barriers and contact zones. It can also be used to test hypotheses regarding the effects of historical and future climate change on spatial genetic patterns by projecting niches using palaeoclimate or future climate data. Conversely, phylogeographical information can populate ENM with within-species genetic diversity. Where adaptive variation exists among clades within a species, modelling their niches separately can improve predictions of historical distribution patterns and future responses to climate change. Awareness of patterns of genetic diversity in niche modelling can also alert conservationists to the potential loss of genetically diverse areas in a species’ range. Here, we provide a simplistic overview of both fields, and focus on their potential for integration, encouraging researchers on both sides to take advantage of the opportunities available.
... Several studies have also used a limited set of microsatellites, mainly four markers that we previously described (Sundqvist, Ellegren, Olivier, & Vilà, 2001), to define Y chromosome haplotypes in wolves and, mostly, dogs (Benson, Patterson, & Wheeldon, 2012;Brown et al., 2015;Fabbri et al., 2014;Randi et al., 2014;Sacks et al., 2013;Vilà, Walker et al., 2003;Wheeldon et al., 2013 ...
Article
Full-text available
Analyses of Y chromosome haplotypes uniquely provide a paternal picture of evolutionary histories and offer a very useful contrast to studies based on maternally inherited mitochondrial DNA. Here we used a bioinformatic approach based on comparison of male and female sequence coverage to identify 4.7 Mb from the grey wolf Y chromosome, likely representing most of the male‐specific, non‐ampliconic sequence from the euchromatic part of the chromosome. We characterized this sequence and then identified ≈1,500 Y‐linked SNPs in a sample of 145 re‐sequenced male wolves, including 75 Finnish wolf genomes newly sequenced in this study, and in 24 dogs and eight other canids. We found 53 Y chromosome haplotypes, of which 26 were seen in grey wolves, that clustered in four major haplogroups. All four haplogroups were represented in samples of Finnish wolves, showing that haplogroup lineages were not partitioned on a continental scale. However, regional population structure was indicated because individual haplotypes were never shared between geographically distant areas, and genetically similar haplotypes were only found within the same geographical region. The deepest split between grey wolf haplogroups was estimated to have occurred 125,000 years ago, which is considerably older than recent estimates of the time of divergence of wolf populations. The distribution of dogs in a phylogenetic tree of Y chromosome haplotypes supports multiple domestication events, or wolf paternal introgression, starting 29,000 years ago. We also addressed the disputed origin of a recently founded population of Scandinavian wolves and observed that founding as well as most recent immigrant haplotypes were present in the neighbouring Finnish population, but not in sequenced wolves from elsewhere in the world, or in dogs. This article is protected by copyright. All rights reserved.
... Each DNA sample was amplified by Polymerase Chain Reaction (PCR) and genotyped, through a multiple-tube approach (Taberlet, 1996;Caniglia et al., 2014), at 12 canine unlinked autosomal microsatellites (FH2004, FH2079, FH2088, FH2096, FH2137, CPH2, CPH4, CPH5, CPH8, CPH12, C09.250, C09.253), which have been already successfully used for individual identifications in longterm non-invasive monitoring projects of the Italian wolf population ; probability of identity computed in reference wolf population, among unrelated individuals PID=9.6 × 10 −9 and expected full-sibling PIDsibs=3.5 × 10 −4 ), for forensic applications and for the discrimination between wolves, dogs and their first two-three generation hybrids through Bayesian assignment procedures Randi et al., 2014). DNA Samples were also sexed by a PCR-RFLP assay of the zinc-finger protein gene ZFX/ZFY (Lucchini et al., 2002) and paternal haplotypes in male individuals were identified by the amplification of four Y-linked STRs (MS34A, MS34B, MSY41A, MSY41B; Sundqvist et al., 2001). Autosomal and Y-linked STRs were amplified in seven multiplexed reactions using the QIAGEN Multiplex PCR kit (Qiagen Inc., Hilden, Germany). ...
Article
Full-text available
The Italian wolf population, close to extinction in the mid-19th century, now counts about 1800 individuals. Its ongoing expansion raises social conflicts, especially in agricultural and semi-urbanized areas. Thus, monitoring wolf distribution, abundance and impact on the farming economy is a priority for conservation. We analysed canid DNA from 57 swabs from livestock kills, 13 faeces and 21 carcasses, to estimate the minimum number of individuals, their genetic variability and taxon (wolf, dog or hybrids), reconstruct the structure of local wolf packs, and describe the possible hunting patterns in a hitherto poorly investigated area of the Central Apennines. We genotyped, at the mitochondrial DNA control region and at 12 autosomal and four Y-linked microsatellites, 38 swab, three faecal and 19 muscular samples, corresponding to 42 individuals that Bayesian and Multivariate analyses assigned to 28 wolves, nine dogs and five admixed individuals. The minimum number of detected wolves ranged annually from three (2009) to 13 (2011), whereas parentage analyses identified at least three packs with a mean minimum home range of 60 ± 48 km2 and a mean pack size of 4.0 ± 0.9 individuals. The identification of the genetic profiles of the animals involved in the predations revealed that livestock were killed by at least 13 wolves and four dogs, identifying cases of single-individual attacks and cases of cooperation of individual pairs. Integrating information from multigenerational pedigrees with predation patterns we could hypothesize that i) one pack increased livestock attacks after its disruption; ii) one pack showed a mother-offspring collaboration; iii) another pack started livestock predations after two unrelated individuals established a breeding pair. Our analyses of livestock predation events provided useful information on wolf population dynamics, that can be incorporated into local wolf management actions in areas where a regular monitoring is lacking and the predation risk is high.
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Why have males in many species evolved more conspicuous ornaments and signals such as bright colours, enlarged fins, and feather plumes, as well as larger horns and other weapons than females? Darwin's explanation for such secondary sex traits, the theory of sexual selection, became his scientifically perhaps most controversial idea. It suggests that the traits are favoured by competition over mates. After a long period of relative quiescence, theoretical and empirical research on sexual selection has erupted during the last decades. This book describes the theory and its recent development, reviews models, methods, and empirical tests, and identifies many remaining open problems. Among the topics discussed are the selection and evolution of mating preferences; relations between sexual selection, species recognition, and speciation; constraints on sexual selection; the selection of secondary sex differences in body size, weapons, and in visual, acoustic, and chemical signals. The rapidly growing study of sexual selection in plants is also reviewed. Other chapters deal with alternative mating tactics, and with the relationships among sexual selection, parental roles, and mating systems. The present review of this very active research field will be of interest to students, teachers, and research workers in behavioural and evolutionary ecology, animal behaviour, plant reproductive ecology, and other areas of evolutionary biology where sexual selection is a potential selection factor. In spite of much exciting progress, some of the main questions in the theory of sexual selection yet remain to be answered.