Journal of Heredity Advance Access originally published online on March 29, 2007
Journal of Heredity 2007 98(2):179-182; doi:10.1093/jhered/esm001
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Brief Communications |
Introgression of Alectoris chukar Genes into a Spanish Wild Alectoris rufa Population
From the Laboratory of Cytogenetics and Molecular Genetics, Veterinary Faculty, C/Miguel Servet, 177, 50.013-Zaragoza, Spain (Tejedor, Monteagudo, and Arruga); the Department of Biology, Carleton University, 1125 Colonel By Drive, Ottawa, ON, K2M 2N3, Canada (Mautner); and the Ministry of the Interior, Nicosia 1453, Cyprus (Hadjisterkotis)
Address correspondence to Dr M. T. Tejedor at the address above, or e-mail: ttejedor{at}unizar.es.
In order to detect introgression of other Alectoris genus species into wild populations of Spanish Alectoris rufa, we studied a sample of 93 red-legged partridges (supposed to be A. rufa) captured in the island of Majorca. A set of 31 chukar partridges (Alectoris chukar) from Cyprus and 33 red-legged partridges (A. rufa) from one Spanish farm were also studied to provide suitable populations for comparison. Factorial correspondence analysis on microsatellite genotypes supported a clear distinction of birds from Cyprus, whereas partridges from Majorca and the Spanish farm overlapped in a wide area. The existence of A. chukar mitochondrial DNA in 16 individuals from Majorca indicated introgression into their maternal lineage even if their phenotypes were not different from A. rufa. Bayesian inference based on microsatellite analysis indicated a noticeable degree of genetic proximity to A. chukar only for one of these hybrids.
Red-legged partridge (Alectoris rufa) is a native species of the northern and western Mediterranean areas (Iberian Peninsula, Southern and Central France, Northwest Italy, and Corsica). High hunting pressure and fast transformation of rural areas have led to a decline in partridge populations throughout Spain (Lucio and Purroy 1992). Intensive repeated restocking from captive-reared individuals is usually practiced. Uncontrolled restocking may initiate genetic pollution problems by introducing interspecific hybrids with foreign species of the genus Alectoris (Baratti et al. 2005; Barbanera et al. 2005).
Microsatellites are often used for the characterization and genetic differentiation of partridge populations (Randi et al. 2003). Factorial correspondence analysis (FCA) shows patterns of differentiation based on microsatellite data (individual multilocus scores). Bayesian inference based on microsatellites identifies the population structure and evaluates the relative contribution of each inferred population into the genotypes of each individual and into the original groups. Both methodologies have been successfully applied to the detection of F1 interspecific hybrids (Baratti et al. 2005). On the other hand, mitochondrial DNA sequences analysis can detect the introgression of maternal lineages.
We have chosen the Island of Majorca as a study model to study the possible introgression of other Alectoris genera into wild populations of Spanish A. rufa. Majorca (3640 km2) is the largest of the Balearic Islands, located in the Mediterranean off the east coast of the Spanish mainland. Insularity prevents partridge migration; restocking from captive-reared individuals is the only means of contact with external populations and the only possible origin for interspecific hybrids. The captive-reared birds released for restocking come from Spanish continental farms, but we have no records of the exact number and location of the implicated farms. Partridges from Cyprus and from one Spanish farm are used as comparison points.
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Materials and Methods |
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Animals and DNA Extraction
We analyzed liver samples from 93 partridges captured throughout the Majorcan countryside, whose external phenotype was identical to that of Alectoris rufa (Linneo 1758). Feathers from 31 chukar partridges (Alectoris chukar) were collected from 4 geographical regions of Cyprus; this population was described elsewhere (Tejedor et al. 2005). There are not any A. rufa populations in Cyprus that could be naturally crossed to chukar partridges. Sterile blood samples were collected from 33 randomly chosen A. rufa reproductive individuals, bred in captivity in one Spanish farm (located in Catalonia), selected from a set of 28 collaborating continental farms as a representative farm involved in restocking A. rufa in Spain. We isolated DNA using the commercially available GENECLEAN kit (BioRad, Hercules, CA).
Microsatellite Analysis
We chose a set of 10 domestic chicken microsatellite loci for previous evaluation in partridges, but only 7 of these loci were polymorphic (MCW135, MCW225, MCW276, MCW280, MCW295, LEI31, and ADL0142). Data on the MCW series are available at the Web page of the Department of Animal Sciences of the Wageningen University (http://137.224.73.223/abg-org/hs/research/molecular/tables.html, cited 2006 June 1). Hanotte et al. (1997) and Pang et al. (1999) reported details on LEI31 and ADL0142, respectively. We identified microsatellite alleles on 8% denaturing polyacrylamide gels containing 8 M urea and stained with AgNO3 (Heukeshoven and Dernick 1985; Budowle 1991).
Data Analysis
GENETIX 4.05.2 (Belkhir et al. 1996–1998) computed several parameters of population genetics: number of unique alleles found in a population when it is compared with all the other populations and observed and unbiased expected heterozygosities (HO, HE). GENETIX also performed FCA (Benzecri 1973) on microsatellite data. Polymorphism information content (PIC) was calculated by means of the allele frequency module of CERVUS (version 2.0, Marshall et al. 1998). The Carlson method (Carlson et al. 2004), as implemented on HelixTree® Genetic Analysis Software version 5.1.2. (Golden Helix, Inc. http://www.goldenhelix.com/index.jsp, cited 2007 March 10), was used to search for eventual tight correlations between pairs of microsatellite loci. The Bayesian clustering procedure, implemented in STRUCTURE (Pritchard et al. 2000), identified the K (unknown) clusters of origin of the sampled individuals and assigned the individuals to these clusters. We used 100 000 iterations, following a burn period of 10 000 iterations. The proportion of membership (qi) estimates, for each individual, the fraction of its genome drawn from each of the K clusters. Individuals were assigned (on the basis of probabilities) to one cluster if their proportion of membership (qi) to that cluster was equal to or larger than an arbitrary threshold of 0.800 (Randi et al. 2003). Unassigned individuals are presumed to be admixed from several clusters.
Mitochondrial DNA Analysis
We designed a consensus polymerase chain reaction (PCR) primer pair (forward primer: 5'-cactacaccgcagacaccac-3'; reverse primer: 5'-tgggtgaaatgggattttgt-3') showing a 100% homology to a partial region of the mitochondrial Cytochrome b sequences available in GenBank for A. rufa and Alectoris graeca (Randi 1996) and for A. chukar (Kornegay et al. 1993) (accession numbers Z48775, Z48772, and L08378, respectively). It specifically amplifies a 580-bp fragment in the 3 species. PCR products were sequenced by using an ABI 3730XL automatic sequencer. The sequences obtained were used in BLASTn alignment searches in GenBank (Altschul et al. 1997).
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Results |
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Table 1 shows an overview of the microsatellite loci used in this study. Majorca birds showed 7 unique alleles, whose frequencies were low (0.0054–0.0860). The farm showed 2 unique alleles with low frequencies (0.0152). Cyprus did not show any unique alleles. PIC values ranged from 0.086 (MCW225, Farm) to 0.724 (MCW135, Farm). Expected heterozygosity values (HE) averaged over all loci were 0.535 (Majorca), 0.282 (Cyprus), and 0.516 (Farm). R2 values for every pair of polymorphic loci were always less than 0.8. The FCA plot of individual microsatellite genotypes (Figure 1) supported a clear distinction of birds from Cyprus, which grouped separately from all the other individuals on the first factorial component FC-I (90.39% of the total genetic variation). Partridges from the Majorcan and the Farm groups overlapped in a wide area. Second and third factorial components FC-II and FC-III only explained 6.89% and 2.71% of the total genetic variation, respectively. STRUCTURE suggested that the partridges studied split into 2 distinct genetic clusters. The proportions of membership of each sampled group in the 2 inferred clusters are shown in Table 2. STRUCTURE assigned all the partridges from Cyprus to cluster I. Cluster II included all individuals from Farm and 92 of the Majorcan partridges. The only unassigned Majorcan individual showed qi values supporting a possible mixing between clusters I and II (qI = 0.241, qII = 0.759).
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All the individuals provided a single PCR product of 580 bp when submitted to partial mitochondrial cytochrome b gene amplification. Table 3 shows the results of BLASTn sequences analysis. In the Majorcan group, 16 partridges (17.2%) presented a 100% sequence homology with the sequence for A. chukar (L08378 [GenBank] ), whereas the rest of the individuals showed a 100% homology with A. rufa (Z48775 [GenBank] ). All the Cyprus birds carried the A. chukar mitochondrial sequence. Every individual from the studied Farm showed the A. rufa mitochondrial sequence. Red-legged individuals carrying the A. chukar mitochondrial sequence are thus considered to be hybrids. Distribution of hybrid partridges did not follow any geographical pattern in Majorca. No mitochondrial sequences from any other species were found.
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Discussion |
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Birds are considered to be a taxon poor in microsatellites, and their transference between species is usually difficult (Caizergues 2005). However, chicken primers can be successfully amplified in the Alectoris genus. Randi et al. (2003) applied a set of 8 chicken microsatellite loci to the study of variability and population structure of rock partridge (A. graeca). Five of them (MCW135, MCW225, MCW276, MCW280, and MCW295) are used in this report. Baratti et al. (2005) used 10 completely different microsatellite loci to detect introgression of chukar genes into one Italian A. rufa population. We previously used our complete set of 10 microsatellite loci in A. chukar (Tejedor et al. 2005), but this is the first time this set has been exploited in A. rufa. Average HE values for Majorca and Farm groups were higher than values obtained by Baratti et al. (2005) in A. rufa (0.504 and 0.432) and by Randi et al. (2003) in A. graeca (0.22–0.48).
By BLAST analysis of the microsatellite sequences we have used to the latest chicken genome sequence, and they are located on the following chromosomes in chicken—ADL0142: GGA6; LEI31: GGA2; MCW135: GGA9; MCW225: GGA14; MCW276: GGA4; MCW280: GGA4; MCW295: GGA4. MCW295 (16.08 Mb) is distant from MCW276 (60.5 Mb) and MCW280 (65.8 Mb) on chromosome 4 and is genetically unlinked to them, but MCW276 and MCW280 are linked in chicken, although that may not be true in partridge.
Based on R2, the Carlson method (Carlson et al. 2004) determines groupings of markers that are in tight correlation. A minimum value of R2 = 0.8 is usually required to consider that 2 markers are tightly linked. Because the observed R2 for the studied 7 microsatellite loci is always less than 0.8, we have no evidence of close linkage among the loci that could result in redundant information in partridges. The heterozygosity and the PIC values indicate the suitability of these markers to analyze genetic variability. In summary, they could provide a reliable base to infer significant patterns of population genetic diversity, structuring, and clustering.
The existence of A. chukar mitochondrial DNA in red-legged partridges indicates A. chukar introgression into the maternal lineage of the affected individuals. Alectoris rufa and A. chukar do not share habitats, and natural hybridization areas do not exist (Randi 1996). Therefore, the detected hybridization has, most probably, a domestic origin. Alectoris chukar partridges offer easier management, larger body size, and better practical prolificacy in captivity; therefore, several companies are breeding them for meat production in Spanish farms. Hybrid individuals are difficult to distinguish from the pure A. rufa at the phenotypic level already at the first backcross generation (Negro et al. 2001). Such hybrids would have been later released during restocking. We consider that the percentage obtained (17.2%) is a first approximation (probably underestimated) of the current degree of A. chukar introgression into the local red-legged partridge species. Male-based introgression cannot be detected by mitochondrial analyses. In France, chukar mitochondrial DNA is present in 30% of the individuals in farms devoted to A. rufa breeding and 6% of the animals in wild populations (Queney G, personal communication, cited by Caizergues 2005). Chukar mitochondrial DNA has also been detected in partridge populations in Central Italy previously assumed to be A. rufa (Barbanera et al. 2005). Only in one Majorcan individual bearing A. chukar mitochondrial DNA did the inferred ancestry based on microsatellites point to possible contact with cluster I (qI = 0.241). If mitochondrial DNA analysis had not been available, no clues on the existence of chukar ancestors would have appeared in the rest of the hybrid birds from Majorca.
If A. chukar x A. rufa hybrids are introduced only once, the hybridization rate tends to remain stable in the population (Vallance M, cited by Caizergues 2005). If the original hybridization rate was high enough, microsatellite analysis could detect A. chukar introgression into the population (Baratti et al. 2005). Uncontrolled restocking repeatedly introduces such hybrids into Majorca. As deduced from their external phenotypes (completely similar to A. rufa), they are not F1 hybrids. In every backcross with A. rufa, the proportion of the A. chukar genome is decreased in the hybrids; after several generations, the estimated probability of belonging to cluster I is very low for these hybrid individuals.
Releasing foreign species and hybrid animals is strictly forbidden by Spanish Law 4/1989 for the Conservation of Natural Landscapes and Wild Fauna and Flora. To date, control of genetic purity of restocking individuals does not exist. This is the first evidence of the existence of such hybrids A. rufa x A. chukar in Majorca Island, in spite of the external phenotypes of the individuals. This evidence enhances the need for a control plan for genetic purity of red-legged partridges to be established all over the Spanish countryside.
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Acknowledgments |
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This work was funded by grant number RZ-2004-00011-00-00 from Spanish CICYT-INIA and by the European LEADER Plus Communitary Initiative "Rural Majorca." We thank "Fundació Sa Perdiu Roja," Santany municipal government, the Hunting Societies, and the local hunters for providing specimens and research funding. We are also grateful to Dr D. Savva and Mr B. Fowler (University of Reading, United Kingdom) for help in polishing the English original.
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Footnotes |
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Corresponding Editor: Jerry Dodgson
Received July 17, 2006
Accepted November 27, 2006
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