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Hybridisation with introduced chukars (Alectoris
chukar) threatens the gene pool integrity of native rock
(A. graeca) and red-legged (A. rufa) partridge populations
Marina Barilani
a
, Ariane Bernard-Laurent
b
, Nadia Mucci
a
, Cristiano Tabarroni
c
,
Salit Kark
d
, Jose
`Antonio Perez Garrido
e
, Ettore Randi
a,
*
a
Istituto Nazionale per la Fauna Selvatica (INFS), Via Ca
`Fornacetta 9, 40064 Ozzano Emilia (BO), Italy
b
Office National de la Chasse et de la Faune Sauvage (ONCFS), CNERA Faune de montagne, CADAM,
Pre
´fecture Est, F-06286 Nice cedex 3, France
c
Via Pertini 3, Casalecchio di Reno, Bologna, Italy
d
The Biodiversity Research Group, Department of Evolution, Systematics and Ecology, The Institute of Life Sciences,
The Hebrew University of Jerusalem, Jerusalem 91904, Israel
e
Departamento de Produccio
´n Animal II, Facultad de Veterinaria, Universidad de Leo
´n, Campus de Vegazana, 24071 Leo
´n, Spain
ARTICLE INFO
Article history:
Received 30 August 2006
Received in revised form
2 January 2007
Accepted 5 January 2007
Available online 6 March 2007
Keywords:
Alectoris
Introgression
Outbreeding depression
Admixture analysis
mtDNA control-region
Microsatellites
Partridge conservation genetics
ABSTRACT
The decline of over-hunted red-legged (Alectoris rufa) and rock (A. graeca) partridge popula-
tions has been contrasted with massive releases of captive-reared birds, often hybrids with
non-indigenous A. chukar. Released interspecific hybrids raise the risks of introgressive
hybridisation, and can contribute to further depress the fitness of native populations. Aim-
ing to assess the extent of hybridisation, we genotyped the mtDNA control-region and eight
nuclear microsatellites in 671 red-legged, rock and chukar partridges and hybrids, identi-
fied by phenotypic traits. Results reveal a diffuse occurrence of hybridisation: (1) 39 sam-
ples (6.2%) show mtDNA haplotypes discordant with their phenotypes, indicating
red-legged and chukar mtDNA introgression in native rock partridges; (2) admixture anal-
yses of the microsatellite genotypes identified 32 additional rock partridges (5.1%) hybri-
dised mainly with chukars. We analysed also 39 samples collected from a presumed
natural red-legged xrock partridge hybrid zone in the French Alps. Surprisingly, 28% birds
showed typical chukar mtDNAs, indicating hybridisation with introduced chukars or
hybrids. This hybrid zone led to an introgression cline of chukar alleles into neighbouring
Alpine rock partridges detectable up to 100 km, which was shorter than expected by neutral
genetic theory, and that suggested natural selection against hybrids. These findings indi-
cate that introgressive hybridisation may disrupt local adaptations in natural red-legged
partridge and rock partridge populations, and call for strict control of farming and restock-
ing operations.
2007 Elsevier Ltd. All rights reserved.
0006-3207/$ - see front matter 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biocon.2007.01.014
*Corresponding author: Tel.: +39 051 6512252; fax: +39 051 796628.
E-mail addresses: marinab11@libero.it (M. Barilani), a.bernard@oncfs.gouv.fr (A. Bernard-Laurent), nadia.mucci@infs.it (N. Mucci),
cristiano.tabarroni@iperbole.bo.it (C. Tabarroni), salit@cc.huji.ac.il (S. Kark), dp2jpg@unileon.es (J.A. Perez Garrido), met0217@iperbole.
bo.it (E. Randi).
BIOLOGICAL CONSERVATION 137 (2007) 57–69
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/biocon
1. Introduction
The role of hybridisation in generating biodiversity patterns,
as well as the conservation value and the legal status of hy-
brids in populations that are protected by law are controver-
sial (Allendorf et al., 2001; Mallet, 2005). Various models
have been developed to capture the complexities of hybrid
zones (Barton and Hewitt, 1985; Moore, 1987; Harrison,
1986), and to evaluate the evolutionary consequences of
hybridisation (Barton, 2001). Often, but not always, hybrids
found in stable hybrid zones have low fitness, and their tem-
poral persistence and evolutionary fate are unclear (Barton,
2001). Consequently, hybrid zones are sometimes regarded
to as evolutionary dead ends, or, in contrast, as genetic melt-
ing pots that can potentially foster the emergence of evolu-
tionary novelties (Seehausen, 2004).
Anthropogenic habitat changes, invasion of alien species
and translocation of captive-reared stocks or artificial hybrids
raise risks of genetic pollution and extinction of natural pop-
ulations via hybridisation (Rhymer and Simberloff, 1996). Ge-
netic admixture and introgression of ‘‘alien’’ alleles can
disrupt local adaptations, and can eventually lead to fitness
and population declines (i.e., outbreeding depression; Tem-
pleton, 1986). Risks of genetic pollution by introgressive
hybridisation are threatening both endangered and common
species (Rhymer and Simberloff, 1996; Allendorf et al., 2001).
The occurrence of natural hybridisation is relatively frequent
in birds (Grant and Grant, 1992), and anthropogenic hybridisa-
tion has been documented in game bird species, particularly
in galliforms and waterfowl (Rhymer and Simberloff, 1996;
Mank et al., 2004; Barilani et al., 2007).
The Alectoris partridges include seven closely related inter-
fertile species that are distributed in Eurasia, China and
southern Arabia (Johnsgard, 1988). Their distributions are lar-
gely allopatric, with the exception of two partially sympatric
species in Arabia. Natural hybridisation in parapatric Alectoris
contact zones was described in the southern French Alps (Ber-
nard-Laurent, 1984), in Thrace (Greece; Dragoev, 1974); and in
central China (Chen et al., 1999). A red-legged (Alectoris ru-
fa) x rock partridge (A. graeca) hybrid population, distributed
over an area ca. 15 km wide along the southern edge of the
French Alps, showed shorter than expected introgression of
red-legged allozyme and mtDNA markers in Alpine rock par-
tridge populations distributed up to ca. 150 km from the con-
tact zone. This suggests that the diffusion of hybrid
partridges in nature could be contrasted by outbreeding
depression (Randi and Bernard-Laurent, 1998, 1999).
Rock partridges have declined in the second part of the last
century in most of their native range, due to habitat changes
and over-hunting, which have led to the extinction of local
populations in parts of the Alps, Italian Apennine and Greece
(Bernard-Laurent and De Franceschi, 1994; Handrinos and
Akriotis, 1997). Population decline has been contrasted by
massive releasing of captive-reproduced partridges, often
using chukars (A. chukar) or hybrids with chukars (Randi
et al., 2003; Barilani et al., 2007). Red-legged partridges are
massively hunted and restocked throughout the entire spe-
cies range in Iberia, France and Italy (Aebischer and Potts,
1994). Earlier genetic analyses indicate that chukar mtDNA
haplotypes are widespread in most of the red-legged par-
tridge populations studied (Negro et al., 2001; Barbanera
et al., 2005; Baratti et al., 2004). Thus, the genetic integrity of
red-legged and rock partridges might be at risk of widespread
introgressive hybridisation (Potts, 1989; Bernard-Laurent
et al., 2001; Barilani et al., 2007). The concomitant decline of
natural populations and the risk of genetic pollution with
captive-reared hybrids are raising concerns about the conser-
vation of the declining partridges in Europe (Tucker and
Heath, 1994).
Detecting the presence of hybrids or admixed populations
can be problematic, particularly if the parental taxa are mor-
phologically similar (as some species of the Alectoris par-
tridges; Johnsgard, 1988), or if a limited number of
diagnostic markers is used in genetic screening (Boecklen
and Howard, 1997; Va
¨ha
¨and Primmer, 2006). However, nowa-
days, mitochondrial and abundant nuclear hypervariable
DNA markers (e.g., microsatellites) and new Bayesian statisti-
cal methods have dramatically improved the assessment of
cryptic population structure, population admixture analyses
and individual assignment testing (Beaumont and Rannala,
2004). In this study we aimed to investigate the extent of
hybridisation in red-legged and rock partridge populations
sampled in Iberia, France and Italy using Bayesian admixture
analyses of multilocus individual genotypes. We sequenced
the hypervariable part of the mtDNA control-region (Randi
and Lucchini, 1998) and genotyped eight microsatellites
(Barilani et al., 2007). Specifically, we aimed to: (1) probabilis-
tically assign individual partridges to one of the sampled
parental species or to hybrid groups of natural or artificial ori-
gin, using genetic data; (2) assess the extent of natural or
anthropogenic hybridisation in Alpine, Apennine and Sicilian
rock partridge populations; and (3) propose conservation
guidelines based on the assessment of hybridisation risk in
European partridges.
2. Materials and methods
2.1. Sample collection and morphological identification
We collected 671 tissue samples from nine geographic regions
across the native distribution range of the red-legged, rock
and chukar partridges (Tab le 1;Fig. 1). Red-legged partridges
(n= 123) were collected in Portugal, Spain and France between
1990 and 2002. Rock partridges (n= 416) were collected from
the French and Italian side of the Alps, the Italian Apennines
and Sicily between 1989 and 1999. We collected also 39 par-
tridges from the natural hybrid zone in the southern French
Alps (Alpes Maritimes; Figs. 1 and 2). Artificial hybrids have
been produced in captivity mainly by crossing red-legged
and rock partridges with chukars. Therefore, we also analysed
93 chukars sampled in 1990–2002 from native populations in
Greece, Israel and China.
Samples were assigned to one of the three Alectoris spe-
cies, or were identified as hybrids, based on diagnostic
morphological traits and geographic distributions, indepen-
dently of the genetic data. Red-legged partridges are easily
identified by the discontinuous border of the black gorget
with black spotting, and flank feathers with only one black
band. Rock partridges and chukars can be distinguished
mainly by lore color (black vs. white), ear-covert colors (black
58 BIOLOGICAL CONSERVATION 137 (2007) 57–69
with yellow extremities vs. chestnut) and by the narrower
width of the two black bands on the flank feathers in rock par-
tridge (Johnsgard, 1988). Partridges in the natural hybrid zone
showed variable combinations of red-legged and rock par-
tridge feather patterns (described by Bernard-Laurent, 1984;
sampling location 2 in Fig. 2). Partridges collected outside
the hybrid zone did not show morphologic traces of hybrid-
isation and are assumed to be genetically ‘‘pure’’. In the
French Alps (but not in Italy), commercial farming and
restocking of rock partridges has always been forbidden.
However, red-legged partridges close to the rock partridge
range were restocked until the beginning of the 1990s, when
tissues for this study were collected in the French Alps. Since
the mid 1990s, restocking of red-legged partridges close to the
distribution range of the French rock partridge is also forbid-
den (Bernard-Laurent et al., 2001). Red-legged partridges col-
lected in Portugal and Spain and chukars did not show any
morphological or genetic sign of hybridisation (see Section 3).
2.2. Laboratory methods
Tissue samples were individually stored at 20 C in 95%
ethanol. Total DNA was extracted using guanidine thiocyanate
(Gerloff et al., 1995). The 50half of the mitochondrial DNA
Table 1 – List of the species, population of origin and geographic location of the studied Alectoris samples
Species Population Region Samples provided by
A. rufa Portugal Alentejo D. Dias (Lisboa, Portugal)
Spain Andalucia J. A. Perez Garrido (Leo
´n, Spain) J. J.
Negro (Sevilla, Spain) E. Randi (Bologna,
Italy)
Hybrid zone France Alpes-Maritimes (Cipie
`res, Coursegoules) A. Bernard-Laurent (Nice, France)
France Alpes-Maritimes (mid Valley of Tine
´e and Valley of Cians) A. Bernard-Laurent
A. graeca France Alpes-Maritimes (high Valley of Tine
´e) A. Bernard-Laurent
France Hautes-Alpes (Champsaur, Queyras) A. Bernard-Laurent
France Ise
`re A. Bernard-Laurent
France Savoie A. Bernard-Laurent
Italy, Alps Cuneo Province E. Randi
Italy, Alps Valle d’Aosta Region E. Randi
Italy, Alps Novara, Vercelli provinces E. Randi
Italy, Alps Como, Lecco, Sondrio, Brescia provinces E. Randi
Italy, Alps Trento Province E. Randi, M. Paganin
Italy, Apennines Abruzzo Region E. Randi
Italy, Sicily Sicilia Region E. Randi
A. chukar North Israel Galilee P. Alkon (Las Cruces, USA)
S. Kark (Jerusalem, Israel)
E. Randi
Central China Gansu Zhou Tianlin, L. Naifa (Lanzhou, China)
Greece Kos and Karpathos Islands A. Sfougaris (Volos, Greece)
A. Giannakopoulos (Volos, Greece)
Fig. 1 – Distribution ranges, sampling areas and sample sizes of red-legged (Alectoris rufa; circles), rock (A.graeca; ellipses) and
chukar (A.chukar) partridges in Europe, and the A.rufa ·A. graeca hybrid zone (square). Details on populations sampled in the
framed region are shown in Fig. 2.
BIOLOGICAL CONSERVATION 137 (2007) 57–69 59
control-region (mtDNA CR) was PCR-amplified using the
external primer PHDL (tRNA
Glu
;5
0-aggactacggcttgaaaagc-30)
and the internal primer PH-H521 (50-ttatgtgcttga-ccgaggaac-
cag-30), and sequenced using primer PHDL (Randi and Luc-
chini, 1998). All samples were also genotyped at eight
microsatellites (Barilani et al., 2007) originally isolated in
Wageningen University (http://www.zod.wau.nl/abg/index.html)
from the chicken (Gallus gallus) genome: MCW118 (PCR
annealing temperature T
a
=55C), MCW135 (T
a
=55C),
MCW152 (T
a
=50C), MCW225 (T
a
=45C), MCW276 (T
a
=
60C), MCW280 (T
a
=55C), MCW295 (T
a
=50C), MCW323
(T
a
=50C). PCRs were done using the following thermal
cycle: (94·20)+[(94·3000)+(T
a
·3000) + (72·3000)] ·40 cycles +
(72·20). The amplicons were analysed using an ABI 3100
automated sequencer and programs GENESCAN 3.7 and GENOTYPER
2.1. Details of laboratory protocols are available upon request.
2.3. Analyses of sequence and microsatellite variability
Phylogenetic trees were obtained using MEGA 2.1 (Kumar
et al., 2001), with the neighbor-joining procedure (NJ; Saitou
and Nei, 1987) and Tamura-Nei’s (TN93; 1993) genetic dis-
tance model, which is appropriate to describe the evolution
of CR sequences. Support for the internodes in NJ tree was
assessed by bootstrap percentages (BP; Felsenstein, 1985)
after 10,000 resampling steps. We used also other distance
methods (i.e., genetic distances computed using b-distribu-
tions of variable sites), and a Bayesian procedure (MRBAYES
3.04; Huelsenbeck and Ronquist, 2001), which produced re-
sults very similar to the NJ tree. Commonly used summary
population genetic statistics (allelic frequencies, heterozy-
gosity and deviations from Hardy–Weinberg equilibrium)
were computed for each locus and population, and patterns
of differentiation were visualized by a factorial correspon-
dence analysis (FCA) of individual multilocus scores using
GENETIX 4.03 (Belkhir et al., 2001).
2.4. Admixture and hybridisation analyses
Maternal hybridisation was directly detected by discordant
mtDNA and morphologic traits, while biparental multilocus
genotypes were analyzed using a Bayesian clustering proce-
dure implemented in STRUCTURE2 (Pritchard et al., 2000; Falush
et al., 2003), which was designed to identify the K(unknown)
populations of origin of the sampled individuals, and assign
the individuals to the populations. Population clusters are
constructed by minimizing the departures from Hardy–Wein-
berg equilibrium (HWE) and linkage equilibrium (LE), which
could result from recent admixtures, migration or hybridisa-
tion. The samples are subdivided into a number of different
sub-populations (clusters) and, simultaneously, individuals
are assigned probabilistically to one (the population of origin)
or more than one cluster (the parental populations) if their
genotypes are admixed. STRUCTURE does not need perfect genet-
Fig. 2 – Distribution of red-legged (dark grey areas) and rock partridges (light grey areas) in south-eastern France and northern
Italy. Sampled populations: (1) red-legged partridges, Cipie
`res, Coursegoules (Alpes-Maritimes, France), 20–24 km from the
hybrid zone; (2) partridges collected within the hybrid zone, mid Valley of Tine
´e (Alpes-Maritimes). Rock partridges sampled
in: (3) high Valley of Tine
´e (Alpes-Maritimes, 14–28 km); (4) Cuneo, (Italy, 30–40 km); (5) Champsaur and Queyras (Hautes
Alpes, France 75–85 km); (6) Ise
`re (France, 80–90 km); (7) Savoie (170–190 km); (8) Valle d’Aosta (Italy, 130–150 km); (9) Novara
and Vercelli (Italy, 250 km); (10) Como, Lecco, Sondrio, Brescia (Italy, 350 km); (11) Trento (Italy, 550 km).
60 BIOLOGICAL CONSERVATION 137 (2007) 57–69
ic equilibrium to cluster individuals, but attempts to mini-
mise departures from HWE and LE within the inferred
clusters.
In this study we applied STRUCTURE with the admixture
model and allele frequencies uncorrelated. All simulations
were run with 10
5
iterations, following a burn-in period of
10
4
iterations, and were replicated four times. The optimal K
values were selected using the formula: D
ln P(D)
= [ln P(D)
k
lnP(D)
k1
](Garnier et al., 2004), where lnP(D) is the estimated
posterior probability of the data conditional to K. We per-
formed explorative analyses with K= 1–15, using all the sam-
ples that were reliably genotyped at eight microsatellites
(n= 668), or excluding the known hybrids (i.e., 39 partridges
with discordant mtDNA and 39 samples from the hybrid zone;
n= 590). Results showed that the probability of the data, and
the value of D
ln P(D)
, increased sharply from K=1toK= 4, then
very weakly up to asymptotic values that were obtained with
K= 9–10 (not shown). Therefore, we used STRUCTURE with the
following search strategy:
1. Assessment of the global genetic subdivision and identifi-
cation of the cryptic hybrids. We analysed the 590 pre-
sumptive non-hybrid samples, both using and not using
their mtDNA haplotypes as additional characters (the
haplotypes were coded as 1, rufa;2,graeca;3,chukar, based
on result of mtDNA phylogenetic analyses), without prior
population information (option USEPOPINFO = 0), and
K= 1–9. For the selected Kvalue, we assessed the average
coefficient of membership (Q) of each sampled population
(chukar, red-legged and rock partridges) to the inferred
clusters. Then, we assigned each genotype to the inferred
clusters, based on threshold values of the individual pro-
portion of membership (q
i
). Predictably, the threshold val-
ues will affect the accuracy of hybrid identifications
(Va
¨ha
¨and Primmer, 2006). Following empirical and simula-
tion results (Barilani et al., 2007) we selected an identifica-
tion threshold q
i
= 0.90, assigning each individual to one
cluster (species) if q
i
P0.90 (parental individuals), or
jointly to two clusters if the proportion of membership to
each one was q
i
< 0.90 (admixed individuals). In this way
we used STRUCTURE to estimate the posterior probability for
each individual to belong to one parental species, or to
have fractions q
i
of its genome originating from two paren-
tal species.
2. Identification of the admixed partridges. STRUCTURE was
run with K= 4, and option USEPOPINFO active, that is
indicating the reference population from where each
individual was sampled (POPFLAG = 1), except for all the
individuals that showed a mtDNA haplotype discordant
with prior species identification, or that showed admixed
genotypes (POPFLAG = 0). In this way we evaluated the
probability to assign, with individual proportion of mem-
bership q
i
P0.90, each putative hybrid to its sampled
populations, or to the sampled (0), first (1) or second (2)
past generation in another one or in more than one
population.
3. Genetic composition of the Alpine hybrid zone. STRUCTURE
was run with all the samples (n= 668), with K= 4 and
option USEPOPINFO active. The genetic composition of
individual genotypes was assessed using the identification
threshold q
i
= 0.90, as described above. Moreover, the soft-
ware NEWHYBRIDS (Anderson and Thompson, 2002) was used
for computing the posterior probability for each individual
to belong to each of six genotypic classes that originate
after two generations of hybridisation, that is: two paren-
tals (P
0
,P
1
), first generation hybrids (F
1
), second generation
hybrids (F
2
), backcrosses of F
1
with the first parental (B
0
);
backcrosses of F
1
with the second parental (B
1
). Posterior
distributions were evaluated after 10
5
Monte Carlo Markov
Chains, without using any individual or allele frequency
prior information. STRUCTURE can identify admixtures
among any number Kof parental populations, while
NEWHYBRIDS assumes that hybrid classes originated after
admixture of two parental species. Therefore, we assessed
hybridisation in the rock partridges using either the red-
legged or the chukar partridges as parentals, and exclud-
ing rock partridges from Sicily, as they were divergent from
all the other populations and were identified as a distinct
group by the software.
3. Results
3.1. Mitochondrial DNA sequence diversity, species
distinction and identification of maternal introgression
The mtDNA CR sequence alignment (431 nucleotide long)
showed 92 distinct haplotypes, including 36 haplotypes in
red-legged partridges, 21 in rock partridges and 35 in chukar
partridges, defined by 63 polymorphic sites (62 nucleotide
substitutions and one insertion/deletion). The NJ clustering
grouped these haplotypes into three monophyletic clades
supported by BP P89%, and largely corresponding with mor-
phologic red-legged, rock and chukar partridges (Fig. 3). The
average interspecific TN93 sequence divergence was
d= 0.042 (SD = 0.007). The species clades included also haplo-
types (indicated by arrows in Fig. 3) of birds with phenotypes
not corresponding with the mitochondrial identification. Six
red-legged haplotypes (R15, R17, R22, R24, R26 and R27) were
found in 27 rock partridges sampled in the French (n= 21)
and Italian Alps (n= 2), Apennines (n= 2) and Sicily (n= 2).
Six chukar haplotypes (C1, C2, C7, C14, C26 and C35) were
found in French red-legged (n= 6) and rock partridges (n= 3),
as well as in rock partridges from the Italian Alps (n= 2) and
Sicily (n= 1).
Partridges sampled within the Alpine hybrid zone (indi-
cated with HYZ in Fig. 3) showed mtDNA haplotypes both
from red-legged (11) and rock partridge (16) parentals, as ex-
pected. However, there were also 11 hybrid birds that unex-
pectedly showed chukar mtDNA haplotypes, which could
not originate from natural introgression, but from hybridisa-
tion with released captive-bred female chukars or hybrids.
The proportions of chukar, red-legged and rock partridge
mtDNAs that were found in the Alpine populations are shown
in Fig. 4a. This plotting indicates that the frequency of red-
legged mtDNA haplotypes introgressed in the Alpine rock par-
tridges sharply falls below 10% within 100 km distance from
the Alpine hybrid zone.
BIOLOGICAL CONSERVATION 137 (2007) 57–69 61
R20 (1) (rufa France)
R19 (1) (HYZ France)
R19 (13) (rufa France)
R18 (1) (rufa France)
R17 (20) (rufa France)
R11 (2) (rufa France)
R17 (1) (rufa Spain)
R11 (1) (rufa Portugal)
R17 (12) (graeca France)
R17 (1) (graeca Italian Alps)
R17 (2) (graeca Italian Apps)
R17 (5) (HYZ France)
R16 (1) (rufa France)
R15 (10) (rufa France)
R15 (1) (graeca France)
R13 (1) (rufa Spain)
R14 (1) (rufa France)
R14 (1) (HYZ France)
R12 (6) (rufa France)
R27 (1) (graeca France)
R25 (1) (rufa France)
R26 (1) (graeca France)
R29 (1) (rufa Spain)
R30 (3) (rufa Spain)
R28 (4) (rufa France)
R21 (2) (rufa France)
R22 (5) (graeca France)
R24 (1) (graeca France)
R22 (1) (graeca Italian Alps)
R22 (3) (HYZ France)
R23 (1) (HYZ France)
R9 (1) (rufa Spain)
R8 (4) (rufa Portugal)
R10 (1) (rufa Spain)
R31 (1) (rufa Spain)
R32 (1) (rufa Spain)
R35 (2) (rufa Portugal)
R36 (1) (rufa Portugal)
R34 (2) (rufa Spain)
R33 (1) (rufa Portugal)
R1 (2) (rufa Portugal)
R2 (4) (rufa Portugal)
R2 (13) (rufa Spain)
R7 (3) (rufa Spain)
R4 (2) (rufa Spain)
R3 (7) (rufa Spain)
R5 (1) (rufa Portugal)
R6 (1) (rufa Portugal)
C15 (2) (chukar Israel)
C16 (1) (chukar Israel)
C17 (4) (chukar Israel)
C21 (7) (chukar Israel)
C23 (1) (chukar Israel)
C18 (2) (chukar Israel)
C24 (1) (chukar Israel)
C19 (2) (chukar Israel)
C20 (1) (chukar Israel)
C22 (1) (chukar Israel)
C25 (3) (chukar Israel)
C32 (3) (chukar Israel)
C33 (1) (chukar Israel)
C30 (9) (chukar Israel)
C31 (1) (chukar Greece)
C34 (1) (chukar Israel)
C28 (1) (chukar Israel)
C29 (2) (chukar Israel)
C26 (1) (rufa France)
C26 (3) (chukar Israel)
C27 (2) (chukar Israel)
C35 (1) (graeca France)
C14 (1) (rufa France)
C14 (9) (HYZ France)
C8 (2) (chukar China)
C12 (1) (chukar China)
C13 (6) (chukar China)
C11 (2) (chukar China)
C9 (1) (chukar China)
C10 (1) (chukar China)
C1 (3) (rufa France)
C2 (1) (rufa France)
C1 (22) (chukar China)
C5 (1) (chukar China)
C1 (3) (chukar Greece)
C1 (2) (graeca France)
C1 (1) (graeca Italian Alps)
C1 (2) (HYZ France)
C3 (2) (chukar China)
C4 (2) (chukar China)
C6 (1) (chukar China)
C7 (1) (chukar Greece)
C7 (1) (graeca Italian Alps)
G20 (1) (graeca Sicily)
G21 (3) (graeca Sicily)
G19 (29) (graeca Sicily)
G22 (7) (graeca Sicily)
G8 (2) (graeca France)
G7 (1) (graeca Italian Alps)
G1 (35) (graeca France)
G3 (1) (graeca France)
G1 (53) (graeca Italian Alps)
G5 (1) (graeca Italian Alps)
G6 (1) (graeca Italian Alps)
G2 (3) (graeca France)
G2 (13) (graeca Italian Alps)
G17 (1) (graeca Italian Apps)
G18 (1) (graeca Italian Apps)
G16 (1) (graeca Italian Apps)
G15 (1) (graeca Italian Apps)
G9 (90) (graeca France)
G13 (15) (graeca France)
G14 (13) (graeca France)
G4 (2) (graeca Italian Alps)
G9 (25) (graeca Italian Alps)
G13 (5) (graeca Italian Alps)
G9 (16) (graeca Italian Apps)
G14 (19) (graeca Italian Apps)
G9 (7) (HYZ France)
G14 (2) (HYZ France)
G10 (2) (graeca Italian Alps)
G11 (35) (graeca France)
G11 (5) (graeca Italian Alps)
G11 (7) (HYZ France)
0.005
A. rufa
A. chukar
A. graeca
HYZ
graeca
HYZ
graeca
graeca
graeca
graeca
graeca
graeca
graeca
graeca
HYZ
HYZ
HYZ
HYZ
HYZ
graeca
graeca
graeca
rufa
rufa
rufa, graeca
rufa
HYZ
HYZ
HYZ
98
96
100
86
89
(1) (graeca Sicily)
(2) (graeca Sicily)
graeca
Fig. 3 – Mid-point rooted neighbor-joining tree computed using MEGA and Tamura-Nei genetic distances between mtDNA
control-region haplotypes of rock, chukar and red-legged partridges. Bootstrap values of the main clades are indicated. The
arrows indicate all discordant haplotypes that were found in partridges phenotypically identified as A.rufa,A.chukar and A.
graeca, or that were sampled in the hybrid zone (HYZ; location no. 2 in Fig. 2). Haplotype numbers and sampling locations are
indicated.
62 BIOLOGICAL CONSERVATION 137 (2007) 57–69
3.2. Genetic diversity and species distinction at
microsatellite loci
The eight microsatellite loci showed 62% (in Chinese chukars)
to 100% (in the hybrid zone) polymorphic loci (at the 95% le-
vel), and from 2.7 (in Chinese chukars) to 4.9 (in the hybrid
zone) alleles per locus, on average. Expected heterozygosity
was lowest in Sicilian rock partridges (H
E
= 0.26), and highest
in partridges of the Alpine hybrid zone (H
E
= 0.55), which
showed the highest levels of variability, an expected outcome
of genetic admixture. Observed and expected heterozygosi-
ties were similar in the samples, except in Sicily, where H
O
was lower than expected, and deviations from HWE were sig-
nificant (P< 0.05; estimated by permutations using GENETIX).
Microsatellite diversity was significantly partitioned among
the 11 sampled populations (Weir and Cockerham’s multilo-
cus F
ST
= 0.49, P< 0.001). The FCA plotting of individual geno-
types showed that chukars grouped separately from the other
partridges on the first factorial component CA-I, while the
red-legged and rock partridges were separated on CA-II
(Fig. 5). Partridges sampled in the hybrid zone plotted inter-
mediately, partially overlapping with both the red-legged
Km
0 100 200 300 400 500
mtDNA proportions
0
20
40
60
80
100
Km
0 100 200 300 400 500
STR hybrid proportions
0
20
40
60
80
100
Fig. 4 – (a) Proportions of A.graeca (empty triangles), A.chukar (filled squares) and A. rufa (filled circles) mtDNA haplotypes
found in Alpine partridges, plotted against distances (measured in km across the Alpine ridge) starting from the
red-legged population in Cipie
`res (population 1 in Fig. 2). Note that the proportions of A. chukar and A. rufa mtDNA
haplotypes in population 2, the hybrid zone, are identical. (b) Proportion of admixed microsatellite (STR) genotypes in the
same populations. Admixture proportions were computed as the percent of individuals that had admixed ancestry after the
assignment procedure (STRUCTURE,K= 4; see Section 3).
BIOLOGICAL CONSERVATION 137 (2007) 57–69 63
and rock partridge distributions. Some rock partridges (la-
belled in Fig. 5) and hybrids plotted towards the chukar
distribution.
3.3. Bayesian assessment of genetic subdivisions and
identification of cryptic hybrids
STRUCTURE was run with 590 presumptive non-hybrid samples,
both with and without their mtDNA haplotypes, USE-
POPINFO = 0, and K= 1–9. Optimal genetic subdivisions were
obtained with K=3or4(Fig. 6a). Almost all the samples were
univocally assigned to single clusters (with individual
q
i
P0.90), supporting the morphological species identifica-
tions, and some admixed genotypes were identified (Fig. 6b
and c). With K= 3, chukars were assigned to cluster 1 with
an average proportion of membership Q
1
> 0.99; red-legged
partridges were assigned to cluster 2 with Q
2
= 0.97 (samples
from France), or 99% (Portugal and Spain).The rock partridges
were assigned to cluster 3 with Q
3
> 0.98 (Fig. 6b). Very similar
results were obtained with K= 4, with the rock partridges
from Sicily being assigned to their own cluster 4(Q
4
= 97%),
whereas rock partridges from the Apennines were assigned
to both clusters 3 and 4 (cumulative Q
3
+Q
4
= 95%; Fig. 6c).
In these analyses we identified 28 putative admixed samples
(mainly in French rock and red-legged partridges; Fig. 6b and
c), which were jointly assigned to two clusters with individual
proportion of membership to each one q
i
< 0.90. All the other
samples were assigned to their specific cluster with individ-
ual q
i
P0.90.
STRUCTURE was then run with all the samples, including
those with discordant mtDNA and the 28 putative admixed
samples, except the partridges collected in the hybrid zone
(n= 629; using or not their mtDNA haplotypes, USE-
POPINFO = 1, and K= 4). Results confirmed the occurrence of
32 admixed individuals, which are listed in Table 2. There
were no discordant mtDNAs or admixed genotypes among
the red-legged partridges sampled in Iberia. In contrast, nine
presumed red-legged partridges from France showed chukar
or red-legged mtDNAs. Five red-legged partridges with chukar
mtDNAs were not identified as hybrids by STRUCTURE. One rock
partridge showing a red-legged mtDNA haplotype was sam-
pled within the red-legged partridge population (number 1
in Fig. 2), and could be a hybrid migrating out of the hybrid
zone. Sixteen rock partridges sampled close to the hybrid
zone (population 3 in Fig. 2) showed red-legged mtDNAs,
and seven of them were identified as red-legged xrock par-
tridge hybrids also by STRUCTURE. Moreover, in population 3
there were seven birds with rock partridge mtDNAs that were
identified as admixed by STRUCTURE. Admixed partridges were
identified also in populations 5–7, located far from the hybrid
zone. Three of them showed chukar mtDNAs. Ten additional
birds with rock partridge mtDNAs were identified as red-leg-
ged xrock partridge hybrids. Two birds were identified as hy-
brids with chukar by STRUCTURE. Eight of the 12 hybrids found
among the Italian Alpine, Apennine and Sicilian rock par-
tridges were also hybrids with chukars. Genetic analyses dis-
covered three possibly mislabelled individuals that were
labelled as rock partridges, but that showed red-legged
mtDNA and microsatellite genotypes. Most of the admixed
rock partridges had ancestry in the red-legged partridge or
in the chukar second past generation. Two samples from
the central Italian Alps, two from southern Apennines, and
three admixed samples from Sicily can result from released
captive-bred hybrids with chukars. Using the mtDNA as an
additional locus did not change these results.
3.4. Genetic composition of the Alpine hybrid zone
STRUCTURE with K= 4 split the 39 partridges sampled in the hy-
brid zone between the rock partridge cluster 1 (with Q
1
= 0.58),
and the red-legged partridge cluster 4 (with Q
4
= 0.37). Individ-
ual assignments further revealed that 77% of the samples
CA-II (8%)
498
398
612
600
570
566
602
548
417
Fig. 5 – Factorial correspondence analysis of individual microsatellite genotypes. Dark squares indicate partridges sampled in
the hybrid zone. Some outlier A. graeca are marked.
64 BIOLOGICAL CONSERVATION 137 (2007) 57–69
show detectable signals of admixture (Fig. 7): only three birds
(34, 38 and 39; 7.7%) could be assigned to the parental red-
legged partridges; six birds could be parental rock partridges
(1, 2, 4, 5, 6 and 7; 15.4%). All the other samples showed discor-
dant nuclear/mtDNA assignments (i.e., sample 3, which could
be identified as a rock partridge with red-legged partridge
mtDNA haplotype R17), or they were strongly admixed, show-
ing individual q
i
< 0.90. Among the admixed birds, only six
partridges (22–27; 15.4%) showed the intermediate q
i
values
(0.40 <q
i
< 0.60), which are expected in first generation hybrid
(F
1
). All the other partridges (24/39 = 61.5%) showed prevalent
rock or red-legged partridge genotypes, suggesting that they
are backcrosses (Fig. 7). Results obtained with NEWHYBRIDS
also suggests that partridges in the hybrid zone included
mainly F
2
and backcrosses: No hybrid partridge was assigned
to the F
1
genotypic class, but there were four F
2
(sample 1,
2, 14 and 31), and three backcrosses with rock partridges
(7, 8 and 22). Fourteen samples showing hybrid ancestry with
STRUCTURE were classified as parental rock partridges by NEWHY-
BRIDS. However, five of them had chukar (17, 37) or red-legged
partridge (20, 32, 39) mtDNAs. Three samples (12, 16, 18) were
concordantly identified as parental red-legged partridges. The
other samples were partially identified as F
2
or backcrosses.
This hybrid zone likely led to introgression of red-legged
partridge mtDNA haplotypes and microsatellite alleles into
the adjacent Alpine rock partridge populations (Fig. 2). The
proportion of admixed genotypes, computed as the percent of
individuals that had admixed ancestry after the assignment
K
2
LnP(D)
-14000
-13000
-12000
-11000
-10000
-9000
-8000
-7000
-6000
Δ
Δ
Δ
468
Fig. 6 – Results of STRUCTURE analyses. (a) Plot of ln P(D) and D
ln P(D)
= [lnP(D)
k
-lnP(D)
k1
] (computed following Garnier et al., 2004)
values for K= 1–9. (b) Assignment of individuals to population clusters for K= 3 and 4. Rock partridges (A. graeca) sampled
from the French Alps and the Italian Apennines are boxed.
Table 2 – Number of admixed red-legged (A. rufa) and rock (A. graeca) partridges, which showed discordant mtDNA
haplotypes, or that did not join their specific clusters with q
i
P
0.90
Admixed samples nmtDNA STRUCTURE Mislabelled
chukar graeca rufa rufa ·graeca chukar ·graeca
A. rufa France 9 6 0 3 3 0 0
A. graeca France 41 3 17 21 20 2 2 rufa
A. graeca Alps 5 2 1 2 0 1 1 rufa
A. graeca Apennines 4 0 2 2 1 2 0
A. graeca Sicily 3 1 0 2 0 3 0
Assignments were obtained using STRUCTURE with POPFLAG option not active, with eight microsatellites and the mtDNA as an additional locus and
K= 4. Mislabelled individuals are three samples which were labelled as A. graeca, but that showed A. rufa mtDNA and microsatellite genotypes.
BIOLOGICAL CONSERVATION 137 (2007) 57–69 65
procedure (STRUCTURE,K=4, PF = 1, mtDNA) in the sampled Al-
pine populations is plotted in Fig. 4b, which shows that the
peak of hybrids in population 2 (partridges sampled within
the hybrid zone) quickly drops down close to zero in rock par-
tridge populations less than 150 km away from the contact
zone.
4. Discussion
Invasive species and translocated populations are threatening
native populations by hybridisation, raising risks of genetic
extinction, loss of local adaptations or outbreeding depres-
sion (Templeton, 1986; Rhymer and Simberloff, 1996; Allen-
dorf et al., 2001). Worldwide translocations of non-
indigenous populations are threatening a number of species
of anseriforms (such as the New Zealand grey duck, Anas
superciliosa superciliosa; Rhymer et al., 1994; the white-
headed duck, Oxyura leucocephala;Munoz-Fuentes et al.,
2007) and galliforms (such as the Italian subspecies of grey
partridge, Perdix perdix italica;Liukkonen-Attila et al., 2002;
and wild populations of the common quail, Coturnix c. cotur-
nix, threatened by restocking with domesticated Japanese
quail, Coturnix c. japonica;Barilani et al., 2005).
Genetic data in this and other studies indicate that hybrid-
isation with chukars partridges is widespread across the en-
tire distribution range of the red-legged partridge (Tejedor
et al., 1994; Negro et al., 2001), as well as in introduced red-leg-
ged partridges in Britain (Potts, 1989) and central Italy (Baratti
et al., 2004; Barbanera et al., 2005). Between the 1960s and the
1980s chukars, or hybrids with chukars, were massively re-
leased to restock hunted red-legged populations in Iberia
(Dias, 1992; Arruga et al., 1996), France (Goodwin, 1986), and
Italy (Priolo, 1970), leading to widespread genetic pollution of
native populations. Hybrids, mainly with chukars, were found
also across the entire native distribution of the rock partridge.
4.1. Widespread hybridisation in rock partridges
Alectoris partridges speciated in allopatry during the Pleisto-
cene period, and did not evolve strong intrinsic mechanisms
LnP (D)
0.0 0.2 0.4 0.6 0.8 1.0
Samples
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
Fig. 7 – Genetic composition of partridges sampled in the Frenc hybrid zone (Alpes-Maritimes), estimated using STRUCTURE with
K= 4 genetic clusters. ln P(D) is the probability of each individual genotype (samples) to be assigned to one of four genetic
clusters. Light grey bars indicate the proportion of red-legged genes; dark grey bars indicate the proportion of rock partridge
genes, intermediate grey bars indicate the proportion of chukar genes. Individual mtDNA haplotypes (labelled as R=A. rufa;
C=A. chukar;G=A. graeca) are indicated.
66 BIOLOGICAL CONSERVATION 137 (2007) 57–69
of reproductive isolation since then (Randi and Bernard-Lau-
rent, 1998). This is examplified by the naturally hybridising
populations of red-legged and rock partridges in the French
contact zone (Bernard-Laurent, 1984; Randi and Bernard-Lau-
rent, 1999), and by the occurrence of hybrids in other natural
(Barilani et al., 2007) or introduced populations (Baratti et al.,
2004; Barbanera et al., 2005). While natural hybridisation is
strictly limited to three small contact zones (Randi and Ber-
nard-Laurent, 1999; Chen et al., 1999), the anthropogenic dif-
fusion of artificial hybrids seems to be much more
widespread, and can occur in most of the restocked popula-
tions throughout the distribution range of red-legged and
rock partridges (Dias, 1992; Bernard-Laurent et al., 2001; Negro
et al., 2001; Barilani et al., 2007). Field observations showed
that released red-legged partridges can mate in nature (Potts,
1989; Duarte and Vargas, 2004), and that interspecific hybrids
are fertile (Bernard-Laurent, 1990). However, genetic data sug-
gest that survival and diffusion of natural hybrids in Alpine
habitats might be constrained by natural selection (e.g., as
consequences of differential selective and/or competitive
pressures between hybrids and their parental populations;
Randi and Bernard-Laurent, 1999).
In this study, we identified the presence of chukar mtDNAs
and admixed microsatellite genotypes in red-legged and rock
partridges that were sampled close to the hybrid zone in the
southern French Alps. This hybrid zone contains a high pro-
portion (28%) of chukar mtDNA haplotypes, indicating gene
introgression from chukars, or from hybrids with chukars, re-
leased to restock the red-legged partridge populations close to
the hybrid zone. Rock partridges in this study were sampled
between 1989 and 1994, that is before wildlife conservation
laws prohibited the introduction of non-indigenous animal
species since 1995 in France, and 1997 in Italy. It would be
interesting to analyse again partridges originating from the
same study areas of the French Alps to assess if chukar genes
are still present in these populations today.
Chukar mtDNA and admixed genotypes were found also in
5.1% of rock partridges sampled in the Italian Alps, Apennines
and in Sicily. In principle, the presence of chukar alleles in the
Italian Alps might be due to occasional long distance dis-
persal or gene flow from the French hybrid zone. However,
the limited introgression of mtDNA haplotypes and admixed
microsatellite genotypes across the Alpine rock partridge
populations (Fig. 4a and b), suggests strong constraints to
gene flow. Thus, the admixed rock partridge genotypes most
probably originated as a result of releases of captive-reared
chukars or hybrids. The diffuse presence of chukar genes in
other parts of the rock partridge range (such as in Greece;
Barilani et al., 2007), and in translocated red-legged partridge
populations in central Italy (Baratti et al., 2004; Barbanera
et al., 2005), calls for stricter control of captive-breeding of
Alectoris partridges in Europe.
The Bayesian models (STRUCTURE and NEWHYBRIDS) used in this
study assume that individuals are genotyped at neutral un-
linked molecular markers, which should be in Hardy–Wein-
berg and linkage equilibrium in the reference populations.
The small number of genetic markers used in this study
would limit the power of the assignment analyses to the first
two-three generations of hybridisation and backcrossing
(Pritchard et al., 2000; Anderson and Thompson, 2002). These
simulations suggest that the proportion of admixed rock par-
tridges detected in this study in the Alps and Apennines
(about 5.0–6.0%) has been underestimated. These limitations
can, in principle, be overcome by increasing the number of
microsatellite (Gonzalez et al., 2005) or other markers (i.e.,
SNP; Morin et al., 2004). Fast and cheap RAPD markers can
be used in genetic screenings (Barbanera et al., 2005),
although dominant markers are less useful for detecting hy-
brids, and the unknown nature of RAPD variation makes it
difficult any genetic interpretation of the results.
4.2. Genetic admixture and introgression in hybridising
partridges
The partridges sampled in the French hybrid zone included
77% admixed genotypes, most of which (61.5%) were not F
1
,
but F
2
or some kind of backcross. The hybrid zone seems to
be predominantly composed by hybrids originated within
the hybrid zone, and not by migrants flowing from parental
red-legged or rock partridges. A sporadic occurrence of
hybridisation events, and a low frequency of F
1
are typical
of most natural hybrid zones where extensive hybridisation
and gene introgression are prevented by various behavioural
or genetic mechanisms, as predicted by the ‘‘tension zone’’
model (Barton and Hewitt, 1985).
The genetic structure of the hybrid zone is in agreement
with plumage trait variation, showing that 86% of partridges
have admixed color patterns, while only 10% have typical rock
partridge plumage, and 4% have pure red-legged partridge
phenotypes (Bernard-Laurent unpublished). In this study, we
morphologically identified eight putative rock partridges
among the 39 samples collected within the hybrid zone. How-
ever, two of them showed chukar mtDNA haplotypes, and one
of these two originated in the red-legged population. There-
fore, these two birds are probably hybrids that retained intro-
gressed chukar mtDNA. The other six rock partridges, which
had graeca mtDNA and were identified as graeca by STRUCTURE
or NEWHYBRIDS, could be tentatively identified as parental graeca.
Three apparently pure red-legged partridges were assigned to
rufa by the assignment procedures, and could be considered as
parentals migrating into the hybrid zone. The high frequency
of chukar mtDNA haplotypes in the hybrid zone suggests that
hybridising red-legged partridges were admixed with released
captive-bred chukars or hybrids. Thus, the consequences of
restocking with captive-reared partridges can be detected also
in the hybrid zone, which, from this point of view, should not
be considered as a natural one.
The composition of the hybrid zone as inferred from micro-
satellite data is in agreement with previously published allo-
zyme data (Randi and Bernard-Laurent, 1999). Introgression
of red-legged mtDNA CR haplotypes or microsatellite alleles
into the rock partridge Alpine populations is detectable up to
ca. 100 km away from the hybrid zone (Fig. 4), in agreement
with allozyme (see Randi and Bernard-Laurent, 1999; their Figs.
1 and 4), and mtDNA findings (Randi and Bernard-Laurent,
1998). The geographic diffusion of hybrid genomes is lower
than expected by neutral genetic cline models. Randi and Ber-
nard-Laurent (1999) estimated that neutral clines generated
after post-glacial secondary contact between red-legged and
rock partridges in the Alps should be about 1120–2750 km wide,
BIOLOGICAL CONSERVATION 137 (2007) 57–69 67
much wider than the observed clines of 100–150 km. Introgres-
sion could be constrained by natural selection against hybrid
partridges. If hybrids are unfit and their survival and dispersal
are constrained by natural selection, we expect negative im-
pacts from the restocking of natural populations.
4.3. Management and conservation implications and
suggestions
Red-legged and rock partridge populations are declining in
parts of their range. Uncontrolled restockings with captive-
reared red-legged and rock partridge, or their hybrids with
chukars, is leading to massive introgressive hybridisation, in
Iberia and France, and in parts of Italy and Greece. Urgent
conservation actions should include the implementation of
an officially accepted analytical-based protocol, aimed to
identify genetically pure or hybrid populations (both in nature
and in captivity), using DNA markers and admixture analyses.
Despite their morphologic similarity, the Alectoris partridges
are genetically well differentiated and can be identified by
molecular methods (Randi et al., 1998, 2003; Barilani et al.,
2007), by diagnostic plumage and vocalization traits (Ceugniet
et al., 1999). The mitochondrial and microsatellite markers
used in this paper and in Barilani et al. (2007), analysed with
the appropriate statistical procedures, can be applied to as-
sess the extent of hybridisation and gene introgression in par-
tridge populations. Population genetic analyses should be
used: (1) to map the distribution of pure natural populations
of partridges, and support their conservation in the wild; (2)
to enforce strict controls of the genetic status of partridge
stocks reproduced in breeding farms and used for restocking
or any other hunting activity. These actions, coupled with
strict limitations to restocking operations, which should be
allowed only under control, and in presence of technically
sound releasing and monitoring programs, should help in
preserving the gene pools of Alectoris species in Europe.
Acknowledgement
We deeply appreciate the collaboration of many people who
helped in sampling collection: D. Dias (for sampling in Portu-
gal), P. Alkon and N. Liu (for help during sampling in Israel and
China), I. Artuso, P. De Franceschi, A. Gentile, M. Paganin, M.
Pandolfi, M. Pellegrini (for sampling in Italy). A. Bernard-Lau-
rent was supported by the Office National de la Chasse et de la
Faune Sauvage. Funding for laboratory analyses were pro-
vided to E. Randi by the Ministero per le Politiche Agricole e
Forestali, and the Ministero per l’Ambiente e la Tutela del Ter-
ritorio, Direzione Conservazione della Natura.
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