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Genetic assessment of the Iberian wolf Canis lupus signatus captive breeding program

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The main goal of ex situ conservation programs is to improve the chances of long term survival of natural populations by founding and managing captive colonies that can serve as a source of individuals for future reintroductions or to reinforce existing populations. The degree in which a captive breeding program has captured the genetic diversity existing in the source wild population has seldom been evaluated. In this study we evaluate the genetic diversity in wild and captive populations of the Iberian wolf, Canis lupus signatus, in order to assess how much genetic diversity is being preserved in the ongoing ex situ conservation program for this subspecies. A sample of domestic dogs was also included in the analysis for comparison. Seventy-four wolves and 135 dogs were genotyped at 13 unlinked microsatellite loci. The results show that genetic diversity in Iberian wolves is comparable in magnitude to that of other wild populations of gray wolf. Both the wild and the captive Iberian wolf populations have a similarly high genetic diversity indicating that no substantial loss of diversity has occurred in the captive-breeding program. The effective number of founders of the program was estimated as �16, suggesting that all founders in the studbook pedigree were genetically independent. Our results emphasize also the genetic divergence between wolves and domestic dogs and indicate that our set of 13 microsatellite loci provide a powerful diagnostic test to distinguish wolves, dogs and their hybrids.
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Genetic assessment of the Iberian wolf Canis lupus signatus captive breeding
program
Oscar Ramirez
1
, Laura Altet
1
, Conrad Ensen
˜at
2
, Carles Vila
`
3
, Armand Sanchez
1
&
Alfredo Ruiz
4,
*,
1
Departament de Cie
`ncia Animal i dels Aliments, Universitat Auto
`noma de Barcelona, Barcelona, Spain;
2
Parc Zoolo
`gic de Barcelona, Barcelona, Spain;
3
Department of Evolutionary Biology, Uppsala University,
Uppsala, Sweden;
4
Departament de Gene
`tica i Microbiologia, Facultat de Cie
`ncies – Edifici C, Universitat
Auto
`noma de Barcelona, 08193, Bellaterra, Barcelona, Spain (*Corresponding author: E-mail: Alfredo.
Ruiz@uab.es)
Received 20 June 2005; accepted 20 January 2006
Key words: captive breeding, dog, genetic diversity, microsatellites, wolf
Abstract
The main goal of ex situ conservation programs is to improve the chances of long term survival of natural
populations by founding and managing captive colonies that can serve as a source of individuals for future
reintroductions or to reinforce existing populations. The degree in which a captive breeding program has
captured the genetic diversity existing in the source wild population has seldom been evaluated. In this
study we evaluate the genetic diversity in wild and captive populations of the Iberian wolf, Canis lupus
signatus, in order to assess how much genetic diversity is being preserved in the ongoing ex situ conservation
program for this subspecies. A sample of domestic dogs was also included in the analysis for comparison.
Seventy-four wolves and 135 dogs were genotyped at 13 unlinked microsatellite loci. The results show that
genetic diversity in Iberian wolves is comparable in magnitude to that of other wild populations of gray
wolf. Both the wild and the captive Iberian wolf populations have a similarly high genetic diversity indi-
cating that no substantial loss of diversity has occurred in the captive-breeding program. The effective
number of founders of the program was estimated as 16, suggesting that all founders in the studbook
pedigree were genetically independent. Our results emphasize also the genetic divergence between wolves
and domestic dogs and indicate that our set of 13 microsatellite loci provide a powerful diagnostic test to
distinguish wolves, dogs and their hybrids.
Introduction
During the last centuries, more than 300 vertebrate
species have become extinct and the population
sizes of many other have been so reduced as to
consider them threatened species (Baillie et al.
2004). This has given rise to an increased aware-
ness for the need of developing in situ and ex situ
conservation programs. The aim of these pro-
grams is to improve the chances of long term
survival of the natural populations. Ex situ
programs require the founding and managing of
captive colonies that can serve as a source of
individuals for future reintroductions or to rein-
force existing populations and have already shown
their potential to rescue highly endangered species,
like for the California condor (Gymnogyps cali-
fornianus, Geyer et al. 1993) or the Przewalski’s
horse (Equus przewalskii, Bouman and Bouman
1994). In these cases only a handful of individuals
survived in the wild when the captive breeding
program started. However, in many cases the aim
Conservation Genetics (2006) 7:861–878 Springer 2006
DOI 10.1007/s10592-006-9123-z
of the ex situ conservation programs goes beyond
the survival of some individuals and their descen-
dants, but targets the conservation of the genetic
diversity over long periods. Genetic diversity is
essential to ensure the conservation of the evolu-
tionary potential that could allow the population
to adapt to changing environments (Frankham
et al. 2002).
Conservation of genetic diversity in a captive
population is a difficult task. These populations
are usually established by a very small number of
individuals, which can lead to dramatic founding
effects (Hedrick 2005). Additionally, the growth of
the population is usually very restricted because of
available infrastructures. This puts an upper
bound to the size of the captive population, which
translates into a small effective population size. In
a random mating population, the portion of the
heterozygosity that is lost every generation as a
result of the random genetic drift is 1/(2N
e
), where
N
e
is the effective population size (Hedrick 2005).
This means that a population with 10 contributing
founders will retain initially 95% of the original
heterozygosity and, if the population remains at
this size, it will keep losing 5% of the heterozy-
gosity every subsequent generation. On the other
hand, a minimum of 30 founders are needed for a
95% probability of capturing an allele with a fre-
quency of 0.05 in the source population (Frank-
ham et al. 2002). Different management strategies
can be implemented to minimize the loss of genetic
diversity in captive populations (Ballou and Lacy
1995; Ballou and Foose 1996; Frankham et al.
2002; Russello and Amato 2004).
In spite of the importance to ensure its success,
the degree in which a captive breeding program
has captured the genetic diversity existing in the
source wild population has seldom been evaluated
(Wyner et al. 1999; Storme et al. 2004). In this
study we evaluate the genetic diversity in wild and
captive populations of the Iberian wolf in order to
assess how much genetic diversity is being pre-
served in the ongoing ex situ conservation pro-
gram.
The gray wolf had an extensive distribution
covering Europe, Asia and North America, but as
a result of the human persecution its populations
have been fragmented and reduced across most of
its range and especially across Europe (Boitani
2003). The largest population in Western Europe
survived in the Iberian peninsula, where the total
wolf population was estimated to be around 2000–
2500 wolves in the early 1990s (Blanco et al. 1992).
The Iberian wolf was identified as a separate
subspecies, Canis lupus signatus, by Cabrera
(1907). Although this subspecies is not commonly
recognized (Nowak 2003; Sillero-Zubiri et al.
2004), a morphometric analysis (Vila
`1993) de-
scribed differences in skull shape separating Ibe-
rian wolves from wolves in Italy and other
populations in Eastern Europe. Additionally,
mitochondrial DNA data and microsatellite fre-
quencies showed a notable differentiation between
Iberian wolves and those found elsewhere in Eur-
asia (Vila
`et al. 1999; Lucchini et al. 2004). These
two lines of evidence indicate that Iberian wolves
have been separated from all other European
wolves for a long time and demand a separate
management, recognizing its evolutionary poten-
tial (Crandall et al. 2000).
As in the rest of Europe, the Iberian wolf was
intensely hunted because of livestock depredation
and the competition with humans for wild prey,
resulting in a population decline during the 19th
and 20th centuries (Ensen
˜at 1996; Figure 1). Al-
though in recent years the range of the Iberian
wolf may have been expanding, isolated nuclei
south of the Duero river may have disappeared
(Alonso et al. 1999; however, see also Blanco and
Corte
´s 2002) and have been declared of priority
concern (Habitats Directive of the European Un-
ion). In 1994, the European Breeding of Endan-
gered Species Programme (EEP) started a breeding
program for the Iberian wolf with 40 animals (23
males and 17 females) distributed in 13 institu-
tions. According to the studbook these animals
derived from 15 founders. Currently the EEP
population is composed of 51 wolves (27 males
and 24 females) distributed in nine lineages (a
lineage is a founder or pair of founders and all
their descendants) (see Figure 2). Three of the
lineages have a wild ancestry (the founders are
wild animals), the origin of five other is unknown
(the founders are captive Iberian wolves) and one
has a mixed origin (one founder is a wild wolf and
the other is a captive animal). Thus, although the
number of founders in the studbook is 15, some of
them could have been related and the actual
number of (genetically independent) founders for
the EEP populations is not known.
In this study we have analyzed the microsatel-
lite variability in the wild and captive population
862
of the Iberian wolf: (i) to provide an estimate of
the amount of genetic variability in this subspecies;
(ii) to compare the level of variability and differ-
entiation between the EPP and the wild population
in order to assess if the managed captive popula-
tion is representative of the diversity existing in the
wild and to assess its adequacy for a possible
reintroduction; (iii) to study the relationships
within the EEP population including animals with
unknown ancestry and estimate the effective
number of founders; (iv) to provide a system for
individual identification and reliable parentage
testing which allows us to optimize the genetic
management of the EEP population; and (v) to
assess the differentiation between wolves and dogs
and determine the likelihood of detecting wolf–dog
hybrids.
Materials and methods
Samples
Three samples, adding to a total of 74 Iberian
wolves, were used for the present study. The first
group (WILD) included 20 wild wolves captured
in different Spanish provinces: Asturias (seven),
Cantabria (four), Orense (one), Palencia (two),
Pontevedra (one), Valladolid (one) and Zamora
(four). The second group (EEP) was composed of
Figure 1. Historical reduction of the Iberian wolf geographical distribution in the Iberian Peninsula (Ensen
˜at 1996).
12 3 4 5 6
78
9
12 3 4
Figure 2. Pedigree of the EEP Iberian wolf population. The 15 founders are indicated by shaded (or gray) symbols. The pedigree
comprises nine lineages (1 Caba
´rceno, 2 Madrid-1, 3 Maiztegui, 4 Rotterdam, 5 Jerez, 6 Biorama, 7 Guadalajara, 8 Madrid-2,
9 Santillana) and includes the 29 individuals analyzed in this study (solid or gray symbols).
863
29 wolves from a total of 51 wolves included in the
breeding program of the EEP. The EEP popula-
tion comprises nine lineages (Figure 2). Three of
theses lineages (Cabarceno, Madrid-2 and San-
tillana) have a wild ancestry. One of them (Maiz-
tegui) has a mixed origin (one founder with wild
ancestry and another one of unknown origin). The
origin of the other five lineages is unknown. The 29
individuals of the EEP analyzed here belong to
nine different institutions affiliated with the pro-
gram (Barcelona, Caba
´rceno, Guadalajara, Jerez,
Lisbon, Madrid, Santillana, Vergel and Vizcaya)
and represent eight of the nine lineages present in
the EEP population, all except Rotterdam (Fig-
ure 2). The third group (CAPTIVE) was com-
posed of 25 captive wolves from 13 different
institutions. This third sample includes animals
that were already captive when the EEP program
started in 1994 but, for different reasons, were not
included in the captive breeding program. In
addition, a sample of 135 unrelated domestic dogs
provided by local breeders was also included in
our study for comparative purposes. These dogs
belong to 35 different breeds including the three
autochthonous breeds (Spanish Mastiff, Pyrenean
Mastiff and Pyrenean Mountain Dog) most used
by Spanish shepherds to protect livestock.
Microsatellite markers
Genomic DNA from blood samples was isolated
as described elsewhere (Francino et al. 1997). A
total of 13 microsatellites were analyzed: eight
dinucleotide markers CPH5 and CPH9 (Fredholm
and Wintero 1995) and CXX366, CXX403, CXX
410, CXX442, CXX459 and CXX474 (Ostrander
et al. 1995); and five tetranucleotide markers
CXX2001, CXX2010, CXX2130, CXX2054 and
CXX2158 (Francisco et al. 1996). The analyzed
microsatellites are autosomal and unlinked, pro-
viding independent markers (Mellersh et al. 1997,
2000). Genomic DNA was amplified using two
multiplex PCR reactions with seven markers in
multiplex-1 (CPH5, CXX366, CXX2158, CPH9,
CXX2130, CXX474 and CXX459) and six in
multiplex-2 (CXX2001, CXX2010, CXX2054, CXX
403, CXX410 and CXX442). The two multiplex
PCR reactions were carried out in 10 ll of final
reaction mixture containing PCR buffer (1),
1.5 mM MgCl
2
, 0.2 mM of each dNTP (PE Bio-
system), 1 U of Taq polymerase (Life Technolo-
gies Inc.), and 30–40 ng of genomic DNA. Primer
concentration was optimized for each marker: 0.2
lM for tetranucleotide markers, 0.3 lM for
CPH5, CPH9 and CXX366 and 0.4 lM for the
other dinucleotide markers. One primer from each
pair was fluorescently labeled with 6-FAM, TET
or HEX. Thermocycling profiles were 3 min at
94 C followed by 25 cycles of 94 C (30 s), 58 C
for multiplex-1 and 55 C for multiplex-2 (30 s)
and 72 C (30 s), followed by a final extension of
15 min at 72 C in an MJ Research Hot-Bonnet.
Labeled PCR products were analyzed by capillary
electrophoresis in an ABI 3100 Genetic Analyzer
(Applied Biosystems) and automatically sized rel-
ative to an internal standard (PRISM GENE-
SCAN-350
TM
TAMRA, Applied Biosystems) with
the GeneScan
TM
Analysis 3.5 software (Applied
Biosystems).
Mitochondrial DNA
Mitochondrial DNA (mtDNA) was analyzed in
eight wolves of the EEP population (representa-
tive of the eight lineages analyzed in this study) by
the amplification of a 313 bp fragment of the left
domain of the mitochondrial control region (Sac-
cone et al. 1987; Taberlet 1996). Primers used were
LoboMit-F 5¢-CTCCACCATCAGCACCCAAA-
G-3¢and LoboMit-R 5¢-GTAACCCCCACGT-
TAGTATG-3¢. PCR was carried out with the
PCR Core Kit Plus (Roche) in 50 ll of final
reaction mixture containing PCR buffer including
2.5 mM of MgCl
2
, 0.2 mM of each dNTP, 0.2
lM of each primer, 0.5 U of Uracil DNA gly-
cosylase and 2 U of Taq polymerase. Thermocy-
cling profiles were 3 min at 94 C followed by 35
cycles of 94 C (30 s), 60 C (30 s) and 72 C
(30 s), followed by a final extension of 3 min at
72 C in an MJ Research Hot-Bonnet thermocy-
cler. PCR products were purified with the Con-
cert
TM
Rapid PCR Purification System (GIBCO
BRL) and sequenced using the dideoxy method
with Big Dye
TM
Terminator Cycle Sequencing
Ready Reaction Kit, version 2.0 (Applied Bio-
system) and analyzed by capillary electrophoresis
in an automated DNA sequencer ABI PRISM
310 (Applied Biosystem). Mitochondrial DNA
sequences were aligned using Multalin software
(Corpet 1988).
864
Data analysis
Expected (H) and observed (H
o
) heterozygosity
within each population were calculated for each of
the 13 microsatellites analyzed in this study using
the BIOSYS-2 software package (Swofford and
Selander 1999). Deviations from Hardy–Weinberg
equilibrium were tested using the GENEPOP 3.1
program (Raymond and Rousset 1995) with a
Markov chain method to estimate the exact P value
(Guo and Thompson 1992). Mean observed and
expected heterozygosities, and mean number of
alleles (A) were compared between populations by
a two-way analysis of variance (Sokal and Rohlf
1995) to take into account the variation between
loci. Multilocus observed and expected heterozyg-
osities were compared within each population by a
t-test for paired comparisons (Sokal and Rohlf
1995). Genetic differentiation between the WILD,
EEP and CAPTIVE populations was analyzed by
the decomposition of gene diversity (Nei 1973).
The FSTAT program (Goudet 2000) was used to
estimate the gene diversity for the whole popula-
tion (H
T
), the mean gene diversity within popula-
tions (H
s
), the gene diversity among populations
(D
ST
) and the coefficient of gene differentiation
(GST ¼DST=HT). The same software was used to
estimate F
ST
statistics using the approximation
described by Weir and Cockerham (1984). Par-
entage Exclusion (PE) and Combined Parentage
Exclusion (CPE) probabilities were calculated on
the basis of the estimated allele frequencies (Ja-
mielson 1994). Polymorphism Information Con-
tent (PIC) and PE values were calculated assuming
that the genotypes of both parents were known
(Botstein et al. 1980; Jamielson 1994).
Genetic pairwise distances among the 74 Ibe-
rian wolves and 135 domestic dogs were calculated
using the measure (1 )Ps) where Ps is the pro-
portion of shared alleles averaged over loci (Bow-
cock et al. 1994). A tree was constructed from the
distance matrix using the Neighbor-Joining clus-
tering algorithm (Saitou and Nei 1987). The dis-
tance matrix was obtained with the program
MICROSAT (Minch et al. 1995) and the tree de-
rived using the MEGA 2.0 package (Kumar et al.
2001). In addition, all genotypes were screened
using a Bayesian admixture procedure imple-
mented in the STRUCTURE software (Pritchard
et al. 2000; http://www.pritch.bsd.uchicago.edu).
STRUCTURE was used with 10
6
iterations,
following a burn-in period of 10,000 iterations, to
estimate the number of clusters (K) using only ge-
netic information. The number of clusters was
estimated by computing the posterior probabilities
for values from K=1 to K=10. We first analyzed
only the wolf samples (WILD, EEP and CAP-
TIVE). In a second set of runs, both wolves and
dogs were included in the analyses. For the wolf
samples, this probability appeared to be bimodal
(or multimodal) with relatively small changes of ln
Pr(X/K) beyond K=1. When wolf samples were
analyzed together with dogs, ln Pr(X/K) increased
substantially from K=1 to K=2 with modest
increases beyond the latter Kvalue (the maximum
was not reached even with K=10).
The effective number of founders of the EEP
population was estimated by two different meth-
ods. In both cases, the OVERALL wolf sample
was considered as the best representation of the
source population. First, we solved for Nthe
equation for the expected number of alleles (k)
remaining after a founding event:
k¼mXð1piÞ2N
where mis the number of alleles and p
i
the fre-
quency of each allele in the source population
(Denniston 1978). Second, we used the following
maximum likelihood method. If the frequency of
allele iin the source population is p
i
, the proba-
bility that this allele is excluded from a founding
sample of 2Ngenes is ð1piÞ2Nand the proba-
bility that it is present in such a sample is
1ð1piÞ2N. Therefore, the probability that a
particular combination of kalleles is observed in
the founding sample (whereas the other m)kal-
leles are absent) is:
L¼Y
k
i¼1
11pi
ðÞ
2N
hi
Y
mk
j¼1
1pj

2N
The number of founders can be estimated as the N
value that maximizes the likelihood function L.
We found this estimate graphically by plotting the
ln Lfunction and looking for the maximum.
Results
We sequenced the mitochondrial DNA control
region of eight wolves of the EEP population, one
wolf from each lineage. We found three (lu1, lu2
865
and lu4) of the four haplotypes previously de-
scribed by Vila
`et al. (1999) in the Iberian wolf. A
fourth haplotype described for the wild population
(lu3) was not represented in any individual of the
EEP population. This haplotype, however, had
been previously observed in a single individual
collected in Portugal (Vila
`et al. 1999). The pres-
ence of these haplotypes in EPP confirms its origin
from Iberian wolves, without apparent introgres-
sion of wolves from other localities. Although the
mtDNA is maternally inherited and only offers
partial information, the relative uniformity of all
the sampled wolves, without individuals clearly
differentiated from the rest of the population (see
below), points in the same direction.
We analyzed the variation at 13 microsatellite
loci in 74 Iberian wolves divided in three samples:
WILD, EEP and CAPTIVE. All microsatellites
were highly polymorphic in the three populations
(Table 1; see Appendix A for allelic frequencies
per locus and population). Allelic diversity (A, the
number of alleles per locus) ranged from two to
seven with an average of 4.77 in WILD wolves,
from three to eight with an average of 4.92 in the
EEP sample and from two to eight with an average
of 4.61 in CAPTIVE. No significant differences
were observed between the three samples (ANO-
VA’s F=0.28, df=2/24; P=0.76). Overall, allelic
diversity ranged in the Iberian wolf from 3 to 10
with an average of 6.23.
Expected (H) and observed (H
o
) heterozygosi-
ties were calculated for each microsatellite locus in
each of the three samples and in the overall pop-
ulation (see Appendix A). In WILD wolves, H
varied between 0.44 and 0.76 with an average of
0.653 (Table 1). Similar values were observed in
the EEP and CAPTIVE samples which show an
average Hof 0.591 and 0.657, respectively (the
three values are not significantly different:
ANOVA’s F=2.84, df=2/24, P=0.078). Expected
heterozygosity in the overall sample was 0.651.
Observed heterozygosity was similarly high in the
three samples (Table 1) which are not significantly
different (ANOVA’s F=1.66, df=2/24, P=0.21).
Deviations from Hardy–Weinberg (H–W) were
tested for all loci in each population (see Appendix
A). In WILD animals, three loci showed a signif-
icant deviation from H–W. In the EEP and
CAPTIVE samples, nine and eight loci, respec-
tively, deviated from H–W (see Appendix A). All
significant deviations (but one) are due to a deficit
of heterozygotes and a corresponding excess of
homozygotes. The multilocus observed heterozy-
gosity was significantly different from the expected
heterozygosity in the EEP sample (t=)4.59,
df=12; P=0.0006) and the CAPTIVE sample
(t=)5.02; df=12; P=0.0003) but not in the
WILD sample (t=)2.14; df=12; P=0.053). The
apparent level of consanguinity F¼ðHHoÞ=H
is generally positive (Table 1, see also Appendix
A). Average Fin the WILD, EEP and CAPTIVE
samples was 0.153, 0.270 and 0.326, respectively.
The heterozygote deficiency is likely the result of
the fragmentation in the populations (Wahlund
effect). In fact, the differences are largest for the
CAPTIVE and EEP populations, where the entire
population is fragmented among different institu-
tions. This fragmentation is associated with a
small census and high inbreeding within each
institution (see Figure 2). For certain loci (e.g.
CXX403), allelic dropout (failure to amplify one
of an individual’s two alleles) might have con-
tributed to the heterozygote deficit because the
DNA samples of the WILD and CAPTIVE pop-
ulations were old (Pemberton et al. 1995).
Allelic diversity (A) and expected heterozygos-
ity (H) in domestic dogs were 10.38 and 0.79,
respectively (Table 1). Genotype frequencies at all
Table 1. Microsatellite variability estimates in three samples of Iberian wolf and a dog sample
Sample NAH
o
HF PIC CPE
WILD 17.1 4.77 0.534 0.653 0.153 0.582 0.999
EEP 28.1 4.92 0.429 0.591 0.270 0.523 0.997
CAPTIVE 20.9 4.61 0.460 0.657 0.326 0.587 0.999
OVERALL 66.1 6.23 0.469 0.651 0.279 0.593 0.999
DOG 126.5 10.38 0.579 0.792 0.280 0.763 0.999
N=sample size (mean number of individuals per locus); A=allelic diversity (mean number of alleles per locus); H
o
and H=observed
and expected heterozygosities; F=inbreeding coefficient, PIC=polymorphism information content; CPE=combined parentage
exclusion.
866
13 microsatellite loci deviated significantly from
Hardy–Weinberg expectations. Observed hetero-
zygosity (H
o
) was significantly lower than expected
heterozygosity (t=)7.58; df=12; P=0.000006)
resulting in an apparent inbreeding coefficient of
F=0.28. This value is consistent with the inclusion
in the study of dogs from different breeds, which
represent reproductively isolated units and pro-
duce a Wahlund effect.
The high allelic diversity and high heterozy-
gosity for most loci result in a high polymorphism
information content (PIC). For the Iberian wolf,
the most informative marker was generally the
CXX2001 locus whereas the locus CXX2010
showed the lowest PIC values (see Appendix A).
The average PIC value was 0.582 for the WILD
sample, 0.523 for the EEP sample, 0.587 for the
CAPTIVE sample and 0.593 for the OVERALL
sample (Table 1). The combined paternity exclu-
sion (CPE) values were also very high. These
observations indicate that this microsatellite set
can be used with high confidence for individual
identification and to ascertain the paternity in the
EEP or other Iberian wolf samples. For the dog
sample, the most informative locus was CXX2158
(PIC=0.972) and that with the lowest PIC (0.593)
was CPH5. The average PIC was 0.763 (Table 1).
To evaluate the genetic differentiation between
the three Iberian wolf samples, we used Nei’s
decomposition of gene diversity (Table 2). Values
of the coefficient of gene differentiation (G
ST
) were
generally low (from 0.001 to 0.073) with an aver-
age value of 0.024. Thus 97.6% of total gene
diversity was contributed by the diversity found
within the samples and only 2.4% was due to be-
tween sample diversity. Therefore, the degree of
differentiation was quite small. The multilocus F
ST
(Weir and Cockerham 1984) was 0.038, which al-
though significant (P<0.01) was relatively small
indicating again little differentiation between wolf
samples. Pairwise F
ST
values were 0.0598 for EEP
versus WILD, 0.0346 for EEP versus CAPTIVE,
and 0.0123 for WILD versus CAPTIVE.
The neighbor-joining tree based on the pro-
portion of shared alleles between individuals
showed two clearly separated groups correspond-
ing to Iberian wolves and dogs (Figure 3). The
three samples of wolves did not form separate
clusters but were interspersed, in good agreement
with the little differentiation between them. Cluster
analyses were performed using the STRUCTURE
software (Pritchard et al. 2000) with the wolf
samples alone and with wolves and dogs together.
For the wolf samples, the number of clusters var-
ied but each predefined sample (WILD, EEP,
CAPTIVE) was not assigned to any single cluster
but was split into two or more clusters. This
indicates that the three wolf populations can not
be easily separated from each other. When wolf
samples were analyzed together with dogs and two
clusters assumed the three predefined wolf samples
were assigned to the same cluster with proportion
of membership 0:985. Increasing the number of
clusters caused the splitting of the dog sample
while all wolves remained in one group. We con-
cluded that no consistent differentiation is revealed
by this clustering procedure between the three
Iberian wolf samples analyzed here.
To estimate the effective number of founders in
the EEP population, we considered the OVER-
ALL sample as the best representation of the
source wild population. The number of alleles
present in the OVERALL sample was 81 whereas
64 alleles were observed in the EEP sample. Thus,
17 alleles have been seemingly lost because of the
founder effect. When we solved for Nthe equation
which predicts the number of alleles remaining
after a founding event (see above) we get an esti-
mate for the number of founders of 17.8. The
alternative method, based in a maximum likeli-
hood approach, gave a similar value, 16.4.
Table 2. Microsatellite genetic differentiation among the
WILD, EEP and CAPTIVE samples of Iberian wolf
Microsatellite locus H
T
H
S
D
ST
G
ST
CXX2001 0.787 0.759 0.028 0.035
CXX2010 0.505 0.500 0.005 0.010
CXX2054 0.616 0.615 0.001 0.001
CXX403 0.635 0.607 0.028 0.044
CXX410 0.747 0.692 0.055 0.073
CXX442 0.487 0.484 0.003 0.006
CPH5 0.724 0.716 0.008 0.011
CXX366 0.476 0.480 0.004 0.008
CXX2158 0.648 0.652 0.004 0.006
CPH9 0.768 0.758 0.010 0.013
CXX2130 0.697 0.666 0.031 0.044
CXX474 0.651 0.611 0.041 0.063
CXX459 0.752 0.750 0.002 0.003
Total 0.653 0.638 0.016 0.024
H
T
=total gene diversity; H
S
=average gene diversity within
populations; D
ST
=gene diversity among populations;
G
ST
=coefficient of gene differentiation.
867
Discussion
Genetic variability in Iberian wolves and other
canids
Microsatellites have been used to estimate the level
of genetic variability in populations of several
Canis species (Table 3). In wild populations of
gray wolf (C. lupus), allelic diversity (A) and ex-
pected heterozygosity (H) are usually high reach-
ing the highest values (6.4–6.8 and 0.72–0.74) in
North America (particularly Canada), the Balkan
Peninsula (Greece and Bulgary) and Latvia and
Finland (Table 3). Genetic variability is also rela-
tively high in wild populations of coyote (C. la-
trans). On the other hand, the captive Mexican
wolf (C. lupus baileyi) population shows reduced
levels of microsatellite variability and the Ethio-
pian wolf (C. simensis), the most endangered
canid, shows the lowest variability values of all
studied Canis populations. Seemingly, Canis pop-
ulations show an inverse correlation between
microsatellite variability and degree of endanger-
ment, as has been found in other threatened taxa
(Spielman et al. 2004).
Our estimates of allele diversity, A, and ex-
pected heterozygosity, H, for the WILD sample of
Iberian wolves are relatively high, 4.76 and 0.653,
and comparable in magnitude to those of other
wild populations of gray wolf (Table 1). Until
now, only one study provided information on the
genetic diversity of Iberian wolves using micro-
satellite markers (Lucchini et al. 2004). In their
study, a mixed sample of 32 wild and captive
individuals was genotyped for 18 microsatellite
loci. Their estimates for Aand H(4.7 and 0.60) are
similar to those observed here. Both studies indi-
cate relatively high levels of genetic variability in
the Iberian wolf populations, comparable to that
in the large and continuous North American wolf
populations. This suggests that the population
changes suffered by the Iberian Wolf in the 20th
century (see Introduction, Figure 1) may have not
caused a severe reduction of genetic variability. It
0.1
Dog EEP
WILD CAPTIVE
Figure 3. Unrooted neighbor-joining tree of the individual microsatellite multilocus genotypes based on the distance (1-Ps) where Ps is
the proportion of shared alleles. Three samples of Iberian wolves (WILD, solid triangles; EEP, gray triangles; CAPTIVE, open
triangles) and a sample of dogs (solid circles) belonging to 35 different breeds were included in the analysis.
868
is worth noting however, that all the individuals
sampled here and in the previous study come from
the relatively large population north of the Duero
River. The Iberian wolf populations south of the
Duero River are smaller and the situation there is
considered critical. The level of genetic variability
in these populations is unknown.
Variability levels in the EEP population
The long term goal of ex situ conservation pro-
grams is to preserve enough genetic diversity as to
make viable a future reintroduction in natural
habitats. The EEP population of Iberian wolves
started in 1994 with wolves derived (without ge-
netic management) from 15 founders. It has been
managed since then using a minimum kinship
strategy (Ballou and Lacy 1995) but within the
limitations set by difficulties of individual ex-
change between institutions (Figure 2). If 15 were
the actual number of independent founders, we
could assume that a high proportion of the vari-
ability present in the wild population would be still
found in the EEP population. Several of the
Table 3. Microsatellite variability in populations of gray wolf and other wolf-like canid species
Taxon Population nN A H
o
HReferences
Canis lupus Vancouver 10 12.6 3.4 0.421 0.566 1
Kenai 10 18.9 4.1 0.536 0.581 1
Alberta 10 18.2 4.5 0.605 0.668 1
Minnesota 10 19.8 6.3 0.532 0.686 1
Southern Quebec 10 20.0 6.4 0.593 0.741 1
Northern Quebec 10 13.3 4.1 0.533 0.565 1
Northwest Territories 10 20.9 6.4 0.547 0.721 1
Scandinavian (Captive) 29 29.0 2.9 0.51 2
Scandinavian (Wild) 29 13.0 3.1 0.52 2
Alberta 10 32.0 4.4 0.553 0.581 3
Central Rocky Mountains 10 59.0 4.1 0.634 0.607 3
Italy 18 103 4.4 0.440 0.490 4
Croatia 18 24.0 5.4 0.630 0.690 4
Greece+Bulgaria 18 39.0 6.8 0.690 0.730 4
Turkey+Israel 18 7.0 3.7 0.660 0.670 4
Saudi Arabia 18 7.0 2.4 0.480 0.420 4
Latvia 18 38.0 6.8 0.710 0.730 4
Finland 18 13.0 5.5 0.690 0.730 4
Canis lupus signatus Spain 18 32.0 4.7 0.500 0.600 4
Canis lupus baileyi Certificada (Captive) 10/20 20.9/20.9 2.5/2.5 0.503/0.403 0.437/0.457 5/6
Ghost Ranch (Captive) 10/20 10.0/10.0 1.3/1.5 0.04/0.174 0.103/0.128 5/6
Aragon (Captive) 10/20 8.0/8.0 1.6/1.5 0.3/0.211 0.253/0.255 5/6
Canis rufus Captive 10 29.9 5.3 0.507 0.548 1
Canis simensis Web Valley 9 22.8 2.8 0.304 0.355 7
Sanetti 9 16.4 2 0.179 0.201 7
Canis latrans Washington 10 15.9 5.8 0.54 0.666 1
Kenai 10 12.8 4.9 0.554 0.627 1
Alberta 10 16.8 6.1 0.653 0.702 1
Minnesota 10 18.4 5.7 0.649 0.709 1
Maine 10 16.2 6.1 0.596 0.702 1
California 10 22.1 6.9 0.502 0.644 1
Canis familiaris 32 breeds 9 35.0 6.4 0.729 0.57 7
24 breeds 10 95.0 7 0.55 0.75 8
n=number of microsatellite loci; N=sample size (mean number of individuals per locus); A=allelic diversity (mean number of alleles
per locus); H
o
and H: observed and expected heterozygosities. References: (1) Roy et al. (1994); (2) Ellegren (1999); (3) Forbes and
Boyd (1996); (4) Lucchini et al. (2004); (5) Garcia-Moreno et al. (1996) (6) Hedrick et al. (1997); (7) Gottelli et al. (1994); (8) Altet
et al. (2001).
869
founding individuals, however, were of unknown
origin. Thus the effective number of founders was
doubtful and the actual level of genetic variability
in the EEP population unknown.
Our estimates of allelic diversity (A) and ex-
pected heterozygosity (H) in the EEP population,
4.92 and 0.591, are comparable to those of the
WILD sample (Table 1). This indicates that a
substantial proportion of the variability present in
the wild is preserved in the EEP population. As a
matter of fact, the two variability measures were
not significantly different when the three samples
included in this study (WILD, EEP and CAP-
TIVE) were compared. Thus, the pooled sample
(OVERALL) likely provides the best estimate of
variability levels in the Iberian wolf population.
The absence of a significant differentiation be-
tween the WILD, EEP and CAPTIVE samples is
corroborated by the decomposition of the gene
diversity. The proportion of the total gene diver-
sity which is contributed by differences between
the three samples is only 2.4%. Thus each of the
three samples contains almost all (97.6%) of the
total diversity. The absence of differentiation be-
tween the three Iberian wolf samples and the rep-
resentation of wild genetic diversity in the EEP
population are also evident when considering the
neighbor-joining tree (Figure 3). In this tree, as in
the Bayesian clustering analysis, members of the
three samples appear interspersed without a clear-
cut clustering of any groups.
The number of founders in the EEP population
was estimated by two different methods. The first
method, based on the number of alleles present in
the EEP population, gave an estimate of 17.8. The
second method, based on a maximum likelihood
approach, takes into account not only the number
of alleles but also which particular set of alleles is
present in the EEP population. This method re-
sulted in a value, 16.4, very close to the number of
founders in the studbook. These results suggest
that all the founders were in fact genetically inde-
pendent and all of them contributed effectively to
the genetic make-up of the EEP population. The
relatively high number of independent founders
and the subsequent genetic management explains
the high proportion of the original variability still
present in the EEP population. The EEP popula-
tion has a much higher genetic diversity than the
captive Scandinavian wolf population (Ellegren
1999), the three captive Mexican wolf populations
(Garcı
´a-Moreno et al. 1996; Hedrick et al. 1997)
or the captive C. rufus population (Roy et al.
1994). These captive populations were started with
a smaller number of founders than the EEP pop-
ulation. In fact, a significant correlation between
expected heterozygosity and number of founders is
observed when all six populations are analyzed
together (r=0.89, df=5; P=0.019). This correla-
tion explains 80% of the variance in heterozy-
gosity and suggests that the number of founders is
the chief determinant of the extant genetic diver-
sity in these captive breeding populations. The
captive Scandinavian and Mexican wolf popula-
tions show signs of different degrees of inbreeding
depression (Laikre and Ryman 1991; Laikre et al.
1993; Fredrickson and Hedrick 2002; however, see
also Kalinowski et al. 1999). Nevertheless, the
captive Mexican wolf population is used for rein-
troduction in the South West of the United States
(USFWS 1998). So far, no signs of inbreeding
depression have been observed in captive Iberian
wolves, although no systematic study has been
conducted. However, the observed heterozygosity
is significantly lower than expected under Hardy–
Weinberg equilibrium for the EEP sample. This is
the result of the fragmentation induced by the fact
that there are multiple captive breeding facilities,
which has led to high levels of inbreeding in most
lineages (Figure 2). This high inbreeding could
lead to fitness depression in the future as observed
in other captive wolf breeding programs. A man-
agement aimed at minimizing kinship (Ballou and
Lacy 1995) is in place, but its success will be
dependent on facilitating the exchange of individ-
uals between centers.
Microsatellite variation in Iberian wolf and
domestic dogs
Hybridization can occur between many species of
the canid family (Gray 1954; Lehman et al. 1991;
Mercure et al. 1993; Roy et al. 1996; Wayne and
Brown 2001). Wolves and dogs coexist across most
of the wolf’s range, and their close relationship,
caused by their recent divergence (Vila
`et al. 1997),
suggests that hybridization could be a problem for
the conservation of wolf populations (Nowak
1979; Wayne and Jenks 1991; Gottelli et al. 1994;
Roy et al. 1994). Hybridization has the potential
to produce morphological, physiological and
behavioral changes in domestic and wild canids
870
(Mengel 1971; Thurber and Peterson 1991; Lari-
viere and Crete 1993). For example, wolf–dog
hybrids tend to have synanthropic behavior and
are more difficult to control than wolves (Ander-
sone et al. 2002). In the Iberian Peninsula, wolves
can be found at low densities in many rural areas,
coexisting with agricultural and livestock activi-
ties, and occasional wolf–dog hybridization could
occur. Genetic analyses did not show any evidence
of introgression of dog mitochondrial DNA in
Spanish and Italian wolves (Vila
`and Wayne 1999;
Randi et al. 2000). More recent studies by micro-
satellite genotyping reported evidence of occa-
sional wolf–dog hybridization in Italy and
elsewhere in Europe (Andersone et al. 2002; Randi
and Lucchini 2002; Vila
`et al. 2003).
In our study, the sample of domestic dogs
shows high microsatellite variability (Table 1).
The allelic diversity (A) in particular is much
higher than for the Iberian wolf (10.5 versus 6.2).
Among the 136 alleles observed in domestic dogs,
63 are shared between dogs and Iberian wolves
whereas 72 are exclusive of dogs. On the other
hand, out of the 80 alleles observed in the Iberian
wolf, only 17 alleles are exclusive of wolves. The
differentiation between dogs and Iberian wolves is
clearly demonstrated in the phylogenetic tree built
with all individuals analyzed here (Figure 3). This
tree shows two neatly separated groups with a
single individual overlap and the cluster analysis
with the program STRUCTURE showed that
wolves and dogs are clearly separated. These re-
sults suggest that no individual wolf of hybrid
origin has been included in our Iberian wolf
sample and emphasize the genetic divergence be-
tween wolves and domestic dogs. They also
indicate that our set of 13 microsatellite loci
provide a powerful diagnostic test to distinguish
wolves, dogs and their hybrids.
Acknowledgements
This study would not have been possible without
the help of the institutions who collected and
provided wolf and dog samples: Zoo Aquarium de
la casa de Campo, Parc Zoolo
`gic de Barcelona,
Parque Ecolo
´gico Bizcaia, Jardim Zoolo
´gico de
Lisboa, Parque Zoolo
´gico de Jerez, Zoolo
´gico
Municipal de Guadalajara, Zoolo
´gico de Santill-
ana del Mar, Safari Park Vergel, and Servei Vet-
erinari de Gene
`tica Molecular (UAB). This work
was supported by grant BMC2002-01708 from the
Direccio
´n General de Investigacio
´n, Ministerio de
Ciencia y Tecnologı
´a (Spain).
Table A.1. Allele frequencies, expected and observed heterozygosities (Hand H
o
), inbreeding coefficient (F), polymorphism infor-
mation content (PIC) and parentage exclusion (PE) for 13 microsatellite loci analyzed in three samples of Iberian wolf and a dog
sample. N=sample size (number of individuals analyzed per locus)
LOCUS ALLELES WILD EEP CAPTIVE OVERALL DOG
CXX2001 120 0.004
124 0.026
128 0.163
132 0.053 0.328 0.071 0.174 0.096
136 0.105 0.034 0.143 0.087 0.011
140 0.421 0.138 0.429 0.304 0.252
144 0.237 0.345 0.190 0.268 0.285
148 0.079 0.138 0.095 0.109 0.133
150 0.105 0.017 0.048 0.051
154 0.026
156 0.024 0.007 0.004
N19 29 21 69 135
H
o
0.579 0.448 0.619 0.536 0.681
H0.755 0.747 0.761 0.789 0.803
F0.238 0.404*** 0.190 0.322*** 0.152***
PIC 0.700 0.689 0.713 0.751 0.772
Appendix A
871
Table A.1. Continued
LOCUS ALLELES WILD EEP CAPTIVE OVERALL DOG
PE 0.522 0.499 0.516 0.580 0.610
CXX2010 214 0.017 0.007
222 0.425 0.242 0.340 0.328 0.026
226 0.575 0.741 0.660 0.665 0.159
230 0.396
234 0.204
238 0.196
242 0.019
N20 29 25 73 135
H
o
0.650 0.172 0.208 0.315 0.585
H0.529 0.492 0.467 0.502 0.739
F)0.307 0.572** 0.559** 0.307** 0.209***
PIC 0.369 0.327 0.348 0.355 0.696
PE 0.185 0.171 0.174 0.181 0.508
CXX2054 144 0.025 0.007 0.048
148 0.075 0.196 0.070 0.026
150 0.325 0.293 0.239 0.289 0.144
154 0.450 0.638 0.522 0.557 0.278
158 0.050 0.014 0.141
162 0.050 0.069 0.056 0.089
166 0.025 0.043 0.007 0.137
170 0.089
174 0.044
178 0.004
N20 29 23 72 135
H
o
0.700 0.448 0.182 0.437 0.741
H0.697 0.511 0.627 0.603 0.845
F)0.004 0.125 0.715*** 0.277*** 0.125**
PIC 0.630 0.428 0.572 0.540 0.825
PE 0.441 0.243 0.370 0.348 0.691
CXX403 232 0.004
244 0.004
252 0.004
258 0.008
260 0.028
262 0.082
264 0.400 0.768 0.420 0.549 0.152
266 0.195
268 0.150 0.036 0.200 0.118 0.066
270 0.275 0.178 0.340 0.264 0.028
272 0.150 0.018 0.040 0.062 0.066
274 0.025 0.007 0.121
276 0.093
278 0.090
280 0.028
282 0.023
284 0.008
N20 28 24 72 128
H
o
0.100 0.357 0.250 0.250 0.703
H0.737 0.384 0.678 0.631 0.892
F0.867*** 0.071 0.636*** 0.596*** 0.212***
PIC 0.672 0.337 0.601 0.556 0.879
872
Table A.1. Continued
LOCUS ALLELES WILD EEP CAPTIVE OVERALL DOG
PE 0.477 0.189 0.392 0.360 0.777
CXX410 86 0.007
93 0.175 0.327 0.375 0.292 0.040
97 0.101
102 0.015
105 0.067
108 0.450 0.052 0.188 0.208 0.118
110 0.050 0.021 0.021 0.096
112 0.100 0.500 0.354 0.347 0.193
114 0.225 0.121 0.062 0.132 0.037
116 0.244
118 0.056
120 0.004
122 0.022
N20 29 24 73 135
H
o
0.6 0.655 0.609 0.625 0.755
H0.722 0.636 0.715 0.738 0.862
F0.172 )0.030 0.152 0.154** 0.124***
PIC 0.661 0.560 0.638 0.686 0.843
PE 0.469 0.358 0.435 0.489 0.721
CXX442 139 0.004
155 0.125 0.179 0.208 0.176
157 0.225 0.053 0.167 0.134 0.088
159 0.007
161 0.650 0.768 0.625 0.690 0.098
163 0.564
165 0.129
167 0.087
169 0.023
N20 28 24 72 132
H
o
0.600 0.393 0.391 0.451 0.401
H0.524 0.382 0.544 0.478 0.641
F)0.149 )0.028 0.285* 0.058 0.375***
PIC 0.453 0.334 0.480 0.427 0.612
PE 0.268 0.185 0.288 0.251 0.434
CPH5 105 0.019
107 0.067 0.022 0.023 0.288
109 0.017 0.015 0.115
111 0.500 0.310 0.364 0.371 0.500
113 0.100 0.052 0.136 0.091 0.078
119 0.067 0.017 0.023
121 0.266 0.345 0.364 0.333
123 0.259 0.114 0.144
N15 29 22 66 134
H
o
0.467 0.552 0.636 0.561 0.410
H0.683 0.727 0.724 0.726 0.650
F0.324** 0.245* 0.124 0.230*** 0.369***
PIC 0.612 0.661 0.650 0.675 0.593
PE 0.421 0.459 0.452 0.485 0.396
CXX366 156 0.050 0.009
160 0.067 0.025 0.150 0.091 0.345
164 0.700 0.750 0.675 0.701 0.388
873
Table A.1. Continued
LOCUS ALLELES WILD EEP CAPTIVE OVERALL DOG
166 0.220
170 0.043
172 0.133 0.036 0.004
174 0.050 0.018
176 0.100 0.125 0.175 0.136
N15 20 20 55 116
H
o
0.533 0.150 0.200 0.273 0.224
H0.494 0.427 0.504 0.473 0.683
F)0.082 0.655*** 0.609*** 0.426*** 0.673***
PIC 0.445 0.392 0.441 0.439 0.617
PE 0.277 0.239 0.260 0.275 0.407
CXX2158 252 0.006
264 0.012
268 0.042
272 0.077 0.053 0.050 0.048
276 0.077
282 0.077 0.125 0.111 0.110 0.053
286 0.5 0.607 0.611 0.580 0.053
290 0.154 0.054 0.056 0.080 0.137
294 0.030
296 0.059
298 0.107 0.222 0.100 0.113
302 0.060
306 0.018 0.010 0.047
310 0.018 0.010 0.024
314 0.115 0.018 0.040 0.036
316 0.077 0.020
320 0.060
324 0.053
328 0.036
332 0.036
340 0.018
N13 28 9 50 84
H
o
0.385 0.500 0.333 0.440 0.643
H0.723 0.608 0.595 0.637 0.937
F0.478** 0.181*** 0.455 0.312*** 0.315***
PIC 0.666 0.572 0.512 0.607 0.972
PE 0.489 0.398 0.324 0.435 0.861
CPH9 133 0.022
137 0.219 0.207 0.136 0.186 0.425
139 0.406 0.190 0.386 0.306
141 0.138 0.059 0.022
143 0.094 0.138 0.023 0.091 0.011
145 0.034 0.015 0.396
147 0.060
149 0.015
151 0.159 0.052 0.049
153 0.281 0.293 0.296 0.291
N16 29 22 67 134
H
o
0.688 0.621 0.682 0.657 0.425
H0.722 0.810 0.736 0.778 0.657
874
Table A.1. Continued
LOCUS ALLELES WILD EEP CAPTIVE OVERALL DOG
F0.049 0.237*** 0.075** 0.157*** 0.354***
PIC 0.645 0.766 0.671 0.738 0.594
PE 0.441 0.596 0.475 0.563 0.400
CXX2130 292 0.026
294 0.100 0.017 0.053 0.048 0.149
296 0.026
298 0.067 0.017 0.053 0.040 0.101
300 0.017
302 0.052 0.132 0.063 0.162
304 0.036
306 0.500 0.345 0.500 0.429 0.066
308 0.079
310 0.233 0.552 0.157 0.357 0.066
312 0.101
314 0.100 0.024 0.092
316 0.009
318 0.017 0.105 0.039 0.044
320 0.004
322 0.022
N15 29 19 63 114
H
o
0.600 0.414 0.526 0.492 0.693
H0.694 0.583 0.710 0.664 0.906
F0.140 0.294* 0.264* 0.282*** 0.236***
PIC 0.628 0.497 0.659 0.625 0.894
PE 0.439 0.304 0.480 0.435 0.803
CXX474 107 0.167 0.397 0.250 0.295 0.189
109 0.071
111 0.067 0.091 0.045 0.196
113 0.733 0.431 0.341 0.469 0.122
115 0.237
117 0.022
119 0.015
121 0.133
123 0.033 0.138 0.318 0.175 0.015
133 0.034 0.016
N15 29 22 66 135
H
o
0.4 0.276 0.773 0.469 0.526
H0.444 0.648 0.728 0.664 0.834
F0.102 0.579*** )0.062* 0.295*** 0.370***
PIC 0.393 0.564 0.656 0.601 0.809
PE 0.232 0.357 0.451 0.398 0.663
CXX459 137 0.286 0.190 0.357 0.266
139 0.017 0.047 0.023
141 0.017 0.008
145 0.072 0.023
147 0.107 0.047 0.039 0.008
149 0.393 0.431 0.357 0.398 0.074
151 0.107 0.190 0.024 0.117 0.219
153 0.036 0.155 0.072 0.102 0.125
155 0.024 0.008 0.090
157 0.109
159 0.071 0.016 0.184
875
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... A few studies implemented Bayesian clustering in STRUCTURE with no hybrids detected i.a.: (i) Ramirez et al. (2006), where the authors conducted genetic assessment of wild and captive Iberian wolves (Canis lupus signatus). In addition to Bayesian clustering, NJ algorithm in MEGA 2.0 (Kumar et al., 2001) was implemented, however, no hybrids were detected among 74 individuals (nevertheless, 54 of all wolves were included in the breeding program and not all of them can be defined as "purely wild" individuals) (ii) Fabbri et al. (2007) did not yield any hybrids among 435 distinct genotypes. ...
... To summarize the most important conclusions drawn by the authors of the above studies: (i) hybrid identification based on phenotype is not reliable, and even individual wolves with morphological traits suggesting hybridization should be classified on the basis of genetic markers (Galaverni et al., 2017;Kusak et al., 2018) (ii) because of the asymmetrical character of WDH, the wolf dog hybrid identification should be based on biparental markers (e.g., Vilà et al., 2003;Hindrikson et al., 2012) (iii) the great majority of studies suggested a low proportion or complete lack of admixed individuals suggesting no threat to the gray wolf 's genetic integrity in both, small and widely distributed populations (i.a., Randi and Lucchini, 2002;Ramirez et al., 2006;Verardi et al., 2006;Godinho et al., 2007Godinho et al., , 2011Iacolina et al., 2010;Caniglia et al., 2014;Lorenzini et al., 2014;Dufresnes et al., 2019;Korablev et al., 2021;Smeds et al., 2021). However, there were a few exceptions in Europe-small, regional populations where the number of admixed individuals was significantly higher (Bassi et al., 2017;Salvatori et al., 2019;Santostasi et al., 2021). ...
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Significant development of genetic tools during the last decades provided opportunities for more detailed analyses and deeper understanding of species hybridization. New genetic markers allowed for reliable identification of admixed individuals deriving from recent hybridization events (a few generations) and those originating from crossings up to 19 generations back. Implementation of microsatellites (STRs) together with Bayesian clustering provided abundant knowledge regarding presence of admixed individuals in numerous populations and helped understand the problematic nature of studying hybridization (i.a., defining a reliable thresholds for recognizing individuals as admixed or obtaining well-grounded results representing actual proportion of hybrids in a population). Nevertheless, their utilization is limited to recent crossbreeding events. Single Nucleotide Polymorphisms (SNPs) proved to be more sensible tools for admixture analyses furnishing more reliable knowledge, especially for older generation backcrosses. Small sets of Ancestry Informative Markers (AIMs) of both types of markers were effective enough to implement in monitoring programs, however, SNPs seem to be more appropriate because of their ability to identify admixed individuals up to 3rd generations. The main aim of this review is to summarize abundant knowledge regarding identification of wolf-dog hybrids in Europe and discuss the most relevant problems relating to the issue, together with advantages and disadvantages of implemented markers and approaches.
... Parameters of linear regression models for two-dimensional allele frequency spectra. References (67)(68)(69)(70)(71)(72)(73)(74)(75)(76) ...
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The observation that small isolated populations often suffer reduced fitness from inbreeding depression has guided conservation theory and practice for decades. However, investigating the genome-wide dynamics associated with inbreeding depression in natural populations is only now feasible with relatively inexpensive sequencing technology and annotated reference genomes. To characterize the genome-wide effects of intense inbreeding and isolation, we performed whole-genome sequencing and morphological analysis of an iconic inbred population, the gray wolves ( Canis lupus ) of Isle Royale. Through population genetic simulations and comparison with wolf genomes from a variety of demographic histories, we find evidence that severe inbreeding depression in this population is due to increased homozygosity of strongly deleterious recessive mutations. Our results have particular relevance in light of the recent translocation of wolves from the mainland to Isle Royale, as well as broader implications for management of genetic variation in the fragmented landscape of the modern world.
... Previous genetic analyses revealed that most of the diversity present in the wild wolf population was also present in the captive population due to the large number of founders used (Ramirez et al., 2006). The Sierra Morena wolf sample comes from an animal found road-killed in 2003 in Northeastern Andalusia (Southern Spain), with general wolf-like appearance that did not suggest dog admixture and preserved by the Andalusian Regional Government. ...
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Allee effects reduce the viability of small populations in many different ways, which act synergistically to lead populations towards extinction vortexes. The Sierra Morena wolf population, isolated in the south of the Iberian Peninsula and composed of just one or few packs for decades, represents a good example of how diverse threats act additively in very small populations. We sequenced the genome of one of the last wolves identified (and road‐killed) in Sierra Morena and that of another wolf in the Iberian Wolf Captive Breeding Program, and compared them with other wolf and dog genomes from around the world (including two previously published genome sequences from northern Iberian wolves). The results showed relatively low overall genetic diversity in Iberian wolves, but diverse population histories including past introgression of dog genes. The Sierra Morena wolf had an extraordinarily high level of inbreeding and long runs of homozygosity, resulting from the long isolation. In addition, about one third of the genome was of dog origin. Despite the introgression of dog genes, heterozygosity remained low because of continued inbreeding after several hybridization events. The results thus illustrate the case of a small and isolated wolf population where the low population density may have favored hybridization and introgression of dog alleles, but continued inbreeding may have resulted in large chromosomal fragments of wolf origin completely disappearing from the population, and being replaced by chromosomal fragments of dog origin. The latest population surveys suggest that this population may have gone extinct. This article is protected by copyright. All rights reserved.
... Assessment of genetic diversity in natural and captive populations is an important step to understand population structure, history, and hybrid status better when developing breeding programs for conservation management of threatened species [21][22][23]. Molecular genetic markers such as mitochondrial DNA (mtDNA) and biparentally inherited nuclear DNA microsatellites can facilitate the ability to characterize population diversity, assign possible origins of individuals, and identify hybrids and their parents [14,15,24,25]. Although the 12 captive crocodile populations in Thailand contain both C. siamensis and C. porosus, the C. siamensis captive-bred population is currently considered the most important source of individuals for a reintroduction program. ...
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The Siamese crocodile (Crocodylus siamensis) and Saltwater crocodile (C. porosus) are two of the most endangered animals in Thailand. Their numbers have been reduced severely by hunting and habitat fragmentation. A reintroduction plan involving captive-bred populations that are used commercially is important and necessary as a conservation strategy to aid in the recovery of wild populations. Here, the genetic diversity and population structure of 69 individual crocodiles, mostly members of captive populations, were analyzed using both mitochondrial D-loop DNA and microsatellite markers. The overall haplotype diversity was 0.924–0.971 and the mean expected heterozygosity across 22 microsatellite loci was 0.578–0.701 for the two species. This agreed with the star-like shaped topology of the haplotype network, which suggests a high level of genetic diversity. The mean ratio of the number of alleles to the allelic range (M ratio) for the populations of both species was considerably lower than the threshold of 0.68, which was interpreted as indicative of a historical genetic bottleneck. Microsatellite markers provided evidence of introgression for three individual crocodiles, which suggest that hybridization might have occurred between C. sia-mensis and C. porosus. D-loop sequence analysis detected bi-directional hybridization between male and female individuals of the parent species. Therefore, identification of genetically non-hybrid and hybrid individuals is important for long-term conservation management. Relatedness values were low within the captive populations, which supported PLOS ONE | https://doi.org/10.1371/journal.pone.
... Captive populations may also serve as a reservoir of genetic material that can be utilized for the re-establishment or reinforcement of wild populations and, thus, considered essential in the prevention of extinction of a species (Read and Harvey, 1986;Lacy, 1993). Animals in ex situ conservation are also expected to have an improved survival rate as genetic resource when they are reintroduced into the natural population (Ramirez et al., 2006). ...
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Information on the degree of inbreeding is very important in the effective management of captive populations of animals in zoos. For the North Persian leopard (Panthera pardus saxicolor), in particular, no report on this aspect is available. This study evaluated the effect of inbreeding on fitness traits and the possible occurrence of purging in a captive population of the leopard based on pedigree records of the Australasian Regional Association of Zoological Parks and Aquaria covering the period 1955–2008. The significance of individual, sire or dam inbreeding on individual and litter fitness traits and litter size was analyzed using linear mixed models. The study showed that individual inbreeding significantly decreased survival at days 30 and 90 (weaning age) after birth, while litter inbreeding significantly decreased litter survival at days 7, 30 and 90. There was also a corresponding decrease in litter size when the dam was inbred. Purging of genetic load is possible with increased survival of the individual and litter when the dam is inbred. However, enhanced zoo management has to be considered with increased survival of individuals. With the unpredictable response of traits to inbreeding, designing breeding programs for captive populations should be geared toward maximizing genetic diversity and minimizing the rate of inbreeding.
... These authors described the first wolf-dog hybrid in Portugal, on the more stable and apparently expanding subpopulation. Previous to Godinho et al. (2011a), several authors failed to detect hybrids in the Iberian Peninsula Ramirez et al., 2006). It is important to mention that despite this single case of hybridization, the Iberian wolves and free-ranging dogs in the study area are genetically distinct, showing high average posterior probability (99%) of assignment to their own clusters. ...
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Human population expansion has promoted contact between wildlife and domestic animals with severe ecological consequences, such as anthropogenic hybridization. In Portugal, Iberian wolf (Canis lupus signatus) populations are considered “Endangered” and co-habit with humans so the risks of hybridization with free-ranging dogs, and livestock depredation can be particularly high. Our aim was to report the occurrence of wolf-dog hybridization in an endangered Iberian wolf sub-population, located in the south of the Douro river, Portugal. We used mitochondrial DNA and microsatellite data to investigate putative hybrids between Iberian wolves and dogs. Here, we report for the first time a wolf-dog hybrid located in the south of the Douro river. This is the second hybrid found in Portugal, and even if hybridization cases are still considered rare, they can be particularly problematic in isolated, fragmented and endangered populations, such as the one studied here. Appropriate management and conservation measures are recommended.
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The observation that small, isolated populations often suffer reduced fitness as a result of inbreeding depression has guided conservation theory and practice for decades. However, investigating the genome-wide dynamics associated with inbreeding depression in natural populations is only now feasible with relatively inexpensive sequencing technology and annotated reference genomes. To characterize the genome-wide effects of intense inbreeding and isolation, we sequenced complete genomes from an iconic inbred population, the gray wolves (Canis lupus) of Isle Royale. Through comparison with other wolf genomes from a variety of demographic histories, we found that Isle Royale wolf genomes contain extensive runs of homozygosity, but neither the overall level of heterozygosity nor the number of deleterious variants per genome were reliable predictors of inbreeding depression. These findings are consistent with the hypothesis that severe inbreeding depression results from increased homozygosity of strongly deleterious recessive mutations, which are more prevalent in historically large source populations. Our results have particular relevance in light of the recently proposed reintroduction of wolves to Isle Royale, as well as broader implications for management of genetic variation in the fragmented landscape of the modern world.
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This study aimed to evaluate the genetic variation and examine the association of inbreeding level on mortality risk (at days 7, 30 and 180– weaning age) of Mhorr gazelle in captivity for the year 1969–2000 as recorded in the studbook record kept by Australasian Regional Association of Zoological Parks and Aquaria (ARAZPA). The effective number of founders, ancestors and founder genomes was found to be 3.42, 3, and 1.44 for the studied reference population. The reference population is composed of animals which are alive, with known parents and known sex. Animals that are less than 10 years old (based on birth dates up to 2008) with no remarks on its death are considered alive. The population may not have experienced a severe bottleneck, as the values on the effective number of founders and ancestors are almost equal. However, the effective number of founder genomes is low, which demonstrates gene loss due to genetic drift. The mean inbreeding coefficients of the individual, sire and dam were found to be 0.2971 +0.1043, 0.2300 +0.1141 0.2339 +0.1070, respectively. The maximum inbreeding level of the population is 0.5247 (52.47%). This means that parent–offspring or full-sib mating must have happened. The increase in inbreeding level of an individual was found to be significantly associated (p< 0.10) with an increase in mortality risk at day 180 or weaning age. Increasing inbreeding level of sires was found to be significantly associated with increasing risk in mortality at day 30, which indicates that inbred parents also can influence the survival of an offspring. Efficient breeding programs are as important for decreasing mortality in captive populations, as the provision of optimum zoo management practices.
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This study investigates the gene pool of Portuguese autochthonous dog breeds and their wild counterpart, the Iberian wolf subspecies (Canis lupus signatus), using standard molecular markers. A combination of paternal and maternal molecular markers was used to investigate the genetic composition, genetic differentiation and genetic relationship of native Portuguese dogs and the Iberian wolf. A total of 196 unrelated dogs, including breed and village dogs from Portugal, and other dogs from Spain and North Africa, and 56 Iberian wolves (wild and captive) were analyzed for nuclear markers, namely Y chromosome SNPs, Y chromosome STR loci, autosomal STR loci, and a mito-chondrial fragment of the control region I. Our data reveal new variants for the molecular markers and confirm significant genetic differentiation between Iberian wolf and native domestic dogs from Portugal. Based on our sampling, no signs of recent introgression between the two subspecies were detected. Y chromosome data do not reveal genetic differentiation among the analyzed dog breeds, suggesting they share the same patrilineal origin. Moreover, the genetic distinctiveness of the Iberian wolf from other wolf populations is further confirmed with the description of new mtDNA variants for this endemism. Our research also discloses new molecular markers for wolf and dog subspecies assignment, which might become particularly relevant in the case of forensic or noninvasive genetic studies. The Iberian wolf represents a relic of the once widespread wolf population in Europe and our study reveals that it is a reservoir of unique genetic diversity of the grey wolf, Canis lupus. These results stress the need for conservation plans that will guarantee the sustainability of this threatened top predator in Iberia.
Book
The biological diversity of our planet is being depleted due to the direct and indirect consequences of human activity. As the size of animal and plant populations decrease, loss of genetic diversity reduces their ability to adapt to changes in the environment, with inbreeding depression an inevitable consequence for many species. This textbook provides a clear and comprehensive introduction to the importance of genetic studies in conservation. The text is presented in an easy-to-follow format with main points and terms clearly highlighted. Each chapter concludes with a concise summary, which, together with worked examples and problems and answers, emphasise the key principles covered. Text boxes containing interesting case studies and other additional information enrich the content throughout, and over 100 beautiful pen and ink portraits of endangered species help bring the material to life.
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Gene flow can effectively suppress genetic divergence among widely separated populations in highly mobile species. However, the same may not be true of species that typically disperse over shorter distances. Using mtDNA restriction-site and sequence analyses, we evaluate the extent of divergence among populations of two small relatively sedentary North American canids, the kit and swift foxes (genus Vulpes). We determine the significance of genetic differentiation among populations separated by distance and those separated by discrete topographic barriers. Our results show the among-population component of genetic variation in kit and swift foxes is large and similar to that of small rodents with limited dispersal ability. In addition, we found two distinct groupings of genotypes, separated by the Rocky Mountains, corresponding to the traditional division between kit and swift fox populations. Previous workers have characterized these morphologically similar populations either as separate species or subspecies. Our mtDNA data also suggest that kit and swift fox populations hybridize over a limited geographic area. However, the sequence divergence between kit and swift foxes is similar to that between these taxa and the arctic fox (Alopex lagopus), a morphologically distinct species commonly placed in a separate genus. This result presents a dilemma for species concepts, and we conclude that kit and swift foxes should be recognized as separate species.
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Mitochondrial DNA (mtDNA) genotypes of gray wolves and coyotes from localities throughout North America were determined using restriction fragment length polymorphisms. Of the 13 genotypes found among the wolves, 7 are clearly of coyote origin, indicating that genetic transfer of coyote mtDNA into wolf populations has occurred through hybridization. The transfer of mtDNA appears unidirectional from coyotes into wolves because no coyotes sampled have a wolf-derived mtDNA genotype. Wolves possessing coyote-derived genotypes are confined to a contiguous geographic region in Minnesota, Ontario, and Quebec, and the frequency of coyote-type mtDNA in these wolf populations is high (>50%). The ecological history of the hybrid zone suggests that hybridization is taking place in regions where coyotes have only recently become abundant following conversion of forests to farmlands. Dispersing male wolves unable to find conspecific mates may be pairing with female coyotes in deforested areas bordering wolf territories. Our results demonstrate that closely related species of mobile terrestrial vertebrates have the potential for extensive genetic exchange when ecological conditions change suddenly.
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Genetic divergence and gene flow among closely related populations are difficult to measure because mutation rates of most nuclear loci are so low that new mutations have not had sufficient time to appear and become fixed. Microsatellite loci are repeat arrays of simple sequences that have high mutation rates and are abundant in the eukaryotic genome. Large population samples can be screened for variation by using the polymerase chain reaction and polyacrylamide gel electrophoresis to separate alleles. We analyzed 10 microsatellite loci to quantify genetic differentiation and hybridization in three species of North American wolflike canids. We expected to find a pattern of genetic differentiation by distance to exist among wolflike canid populations, because of the finite dispersal distances of individuals. Moreover, we predicted that, because wolflike canids are highly mobile, hybrid zones may be more extensive and show substantial changes in allele frequency, relative to nonhybridizing populations. We demonstrate that wolves and coyotes do not show a pattern of genetic differentiation by distance. Genetic subdivision in coyotes, as measured by theta and Gst, is not significantly different from zero, reflecting persistent gene flow among newly established populations. However, gray wolves show significant subdivision that may be either due to drift in past Ice Age refugia populations or a result of other causes. Finally, in areas where gray wolves and coyotes hybridize, allele frequencies of gray wolves are affected, but those of coyotes are not. Past hybridization between the two species in the south-central United States may account for the origin of the red wolf.
Article
We describe a model-based clustering method for using multilocus genotype data to infer population structure and assign individuals to populations. We assume a model in which there are K populations (where K may be unknown), each of which is characterized by a set of allele frequencies at each locus. Individuals in the sample are assigned (probabilistically) to populations, or jointly to two or more populations if their genotypes indicate that they are admixed. Our model does not assume a particular mutation process, and it can be applied to most of the commonly used genetic markers, provided that they are not closely linked. Applications of our method include demonstrating the presence of population structure, assigning individuals to populations, studying hybrid zones, and identifying migrants and admixed individuals. We show that the method can produce highly accurate assignments using modest numbers of loci—e.g., seven microsatellite loci in an example using genotype data from an endangered bird species. The software used for this article is available from http://www.stats.ox.ac.uk/~pritch/home.html.