Mitochondrial DNA sequence variability in red-legged partridge, <Emphasis Type="Italic">Alectoris rufa</Emphasis>, Spanish populations and the origins of genetic contamination from <Emphasis Type="Italic">A. chukar</Emphasis>

The analysis of 135 mitochondrial D-loop sequences of the Iberian autochthonous red-legged partridge (Alectoris rufa) from wild population hunting bags from various locations and fowl runs in Spain yielded 37 different haplotypes. Among these, three haplotypes correspond to chukar partridges (Alectoris chukar), indicating genetic introgression from birds illegally introduced for restocking: three individuals carrying such haplotypes where found in natural populations, one appeared among those sampled on a mass reproduction farm and the remaining 10 in another fowl-run. The geographical origin of the contaminating chukar haplotypes could be assigned to the most easterly area of the chukar partridge geographical distribution in China. Molecular diversity parameters in the A. rufa samples indicate a considerable amount of genetic variation. Φ_ST showed significant differences among populations that are not explained by geographical distance alone. Particularly, one northern population (Palencia) shows a certain degree of genetic differentiation that could reflect a previously suggested subspecies division. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Detecting introgressive hybridisation in rock partridge populations (<Emphasis Type="Italic">Alectoris graeca</Emphasis>) in Greece through Bayesian admixture analyses of multilocus genotypes

The nominal subspecies of rock partridge (Alectoris graeca graeca) is widely distributed in Greece, where populations are declining due to over-hunting and habitat changes. Captive-reared chukars (A. chukar) have been massively released throughout the country, raising fear that introgressive hybridisation might have disrupted local adaptations leading to further population declines. In this study we used mtDNA control-region sequences and Bayesian admixture analyses of multilocus genotypes determined at eight microsatellite loci, to assess the extent of introgressive hybridisation in 319 wild rock partridges collected in Greece. A neighbour-joining tree split the mtDNA haplotypes into three strongly supported clades, corresponding to rock, red-legged (A.␣rufa) and chukar partridges. We did not detect any case of maternal introgression. In contrast, admixture analyses of microsatellite genotypes identified from four to 28 putative hybrids (according to different assignment criteria), corresponding to 1.2–8.8% of the samples, which were widespread throughout all the country. Power and limits of admixture analyses were assessed using simulated hybrid genotypes, which revealed that a small number of markers can detect all first and second generation hybrids (F ₁ and F ₂), and up to 90% of the first generation backrossess. Thus, the true proportion of recently introgressed rock partridges in Greece might be ca. 20%. These findings indicate that introgressive hybridisation is widespread, suggesting that released captive-bred partridges have reproduced and hybridised in nature polluting the gene pool of wild rock partridge populations in Greece.     

Genetic insight into Mediterranean chukar (Alectoris chukar, Galliformes) populations inferred from mitochondrial DNA and RAPD markers

The chukar (Alectoris chukar, Galliformes) is one of the most important game birds as it is widely distributed and hunted over the whole of its range. The aim of this work was to assess the genetic differentiation as well as the possible presence of hybrid specimens in A. chukar populations from Italy, Greece and Cyprus. To provide phylogenetic context, conspecific, allopatric specimens from Israel, Georgia, Armenia, Kazakhstan, Afghanistan, Pakistan, Mongolia, China and USA were compared. Sequencing of the mitochondrial DNA (mtDNA) Control Region supplied information on the ancestry of A. chukar populations, whereas Random Amplified Polymorphic DNA (RAPD) fingerprinting was used to assess whether hybridization had occurred. The Italian population was found to be an inter-specific mixture of A. chukar and A. rufa (i.e., the red-legged partridge) mtDNA lineages, whereas the representatives from Greece and Cyprus showed only the A. chukar maternal line. RAPD markers revealed introgression with A. rufa genes in the Italian population, whereas no A. chukar × A. rufa hybrid specimens were detected in the eastern Mediterranean populations. The genetic data obtained from the Italian A. chukar population as well as from a few Greek specimens pointed against their Mediterranean kinship, suggesting relationships with A. chukar subspecies from the easternmost part of the Asian continent.     

Mitochondrial DNA and microsatellite markers evidence a different pattern of hybridization in red-legged partridge (Alectoris rufa) populations from NW Italy

Genetic introgression with exotic genomes represents a major conservation concern for the red-legged partridge (Alectoris rufa, Phasianidae). In particular, massive releases of chukar partridges (Alectoris chukar) and/or red-legged × chukar partridge hybrids for hunting purposes have cast serious doubts on the Italian present-day occurrence of wild A. rufa populations not affected by introgressive hybridization. This study investigates the genetic structure of red-legged partridges populations in two ecologically different areas in Northern Italy. Analysis of maternal mitochondrial DNA and biparental microsatellite markers excluded the presence of hybridization in a typical agricultural habitat where hunting and release of reared birds are strictly banned. By contrast, signs of chukar introgression were detected in a perifluvial habitat unusual for the red-legged partridge in Italy. The present study documents the first red-legged partridge population with no genetic evidences of recent chukar introgression presently living in Italy. We recommend that urgent conservation actions are taken to preserve the genetic integrity of this population from the risk of hybridization with farm-reared birds and to support its long-term conservation.     

Noninvasive Genotyping of the Red-Legged Partridge (Alectoris rufa, Phasianidae): Semi-Nested PCR of Mitochondrial DNA from Feces

DNA-based studies using avian feces are scarce and deal only with large-sized species. The red-legged partridge, Alectoris rufa, is a medium-sized member of the order Galliformes. Our goal was to set up a fast, noninvasive procedure for mitochondrial DNA (mtDNA) genotyping of A. rufa fecal samples. We focused on the protected population from Elba Island (Tuscan Archipelago National Park, Italy). Dry A. rufa fecal samples (n = 30) were collected in winter. Both the cytochrome b gene (1,092 bp) and the control region (ca. 1,155 bp) were amplified by means of semi-nested PCRs. Twenty-five samples were successfully sequenced for both genes: 8 showed A. rufa mtDNA lineage and 17 chukar partridge (A. chukar), an exotic species. Mixed maternal ancestry suggests A. rufa × A. chukar hybridization. Our protocol allows noninvasive mtDNA genotyping of any Alectoris species and appears suitable to investigate protected populations as well as those existing either at very low density or inhabiting poorly accessible regions.     

Fitness consequences of anthropogenic hybridization in wild red-legged partridge (Alectoris rufa, Phasianidae) populations

Hybridization is a widespread phenomenon, which plays crucial roles in the speciation of living beings. However, unnatural mixing of historically isolated taxa due to human-related activities has increased in recent decades, favouring levels of hybridization and introgression that can have important implications for conservation. The wild red-legged partridge (Alectoris rufa, Phasianidae) populations have recently declined and the releases of farm-reared partridges have become a widespread management strategy. The native range of the red-legged is limited to the south-west of Europe (from Italy to Portugal). This species does not breed in sympatry with the chukar partridge (A. chukar), whose range is Eurasian (from Turkey to China). However, red-legged partridges have often been hybridized with chukar partridges to increase the productivity of farmed birds, and game releases may have spread hybrid birds into the wild. In this study, we investigated the fitness (survival and breeding) differences between hybrid and “pure” red-legged partridges in a wild population located in central Spain. Incubation probability was similar in hybrids and “pure” partridges. Hybrid females laid larger clutches than “pure” ones, but hatching success did not differ between hybrid and “pure” partridges. Hybrid birds had lower survival rate than “pure” ones, mainly because of higher predation rates. Our results show that, despite lower survival, hybrid partridges breed in natural populations, so this could increase extinction risk of wild pure partridge populations, through releases of farmed hybrid birds. The consequences of continued releases could be of vital importance for the long term conservation of wild red-legged partridges.     

<Emphasis Type="Italic">Mycobacterium avium</Emphasis> disease in wild red-legged partridges (<Emphasis Type="Italic">Alectoris rufa</Emphasis>)

Three wild red-legged partridges (Alectoris rufa) from intensively managed hunting areas in Spain were received for necropsy. They showed granulomatous lesions in different parts of the body, mainly in liver and spleen. Microscope examination of the granulomas showed central caseous necrosis and large amounts of acid-fast bacilli, surrounded by epitheloid cells, giant cells, and lymphocytes. Attempts to isolate and culture the bacillus in Colestsos medium were unsuccessful, but the polymerase chain reaction technique revealed the presence of microorganisms belonging to the Mycobacterium avium complex in one of the partridges. This is the first report of avian tuberculosis in free-living red-legged partridges.     

Genetic structure of chukar partridge ( Alectoris chukar) populations in the Longdong Loess Plateau, China

This study used polymerase chain reaction (PCR) and dideoxy direct sequencing methods to analyze the genetic structure of the chukar partridge (Alectoris chukar) in the Longdong Loess Plateau, northwestern China. A total of 491 nucleotides of mitochondrial DNA from the control region gene were sequenced for 78 chukar partridges from eight different populations. The 24 variable positions defined 25 haplotypes. The high gene flow (Nm=3.75) implied little genetic differentiation between the eight populations. All the populations shared the haplotype C_1, which suggested that they all came from a common ancestor. The 25 haplotypes were spread through the populations, but could be clustered into two groups. The haplotype similar index between the two groups was only 0.15, with a genetic distance of 0.43%. The genetic variation was significantly different between the two groups, which had been isolated for all of recorded history. The genetic structure of chukar partridge populations in the Plateau appears to be the result of the synergistic effect of a glacier, along with debris flow and human activities since the middle of the Pleistocene.Communicated by F. Bairlein     

The role of history vs. demography in shaping genetic population structure across an ecotone: chukar partridges (Alectoris chukar) as a case study

Recent advances in molecular ecology allow better assessment of the role of ecological and evolutionary processes in determining the dynamics of genetic diversity in natural populations. The chukar partridge ( Alectoris chukar ) is abundant and continuously distributed in the mesic Mediterranean regions of Israel, becoming scarce and patchily distributed in the arid Negev Desert. This demographic transition occurs across a main environmental transition, the Northern Negev ecotone. Populations of chukars along this gradient have been shown to differ from other populations in morphology, bilateral asymmetry, and in allozyme variation. The aim of this study is to infer whether historical (i.e. secondary contact of formerly vicariant populations) or recurrent demographic events (i.e. restriction to gene flow across the ecotone) have shaped the observed patterns of genetic diversity in chukar populations in Israel. We analysed mtDNA control-region sequences in 216 chukars collected from 28 localities (five distinct geographical regions). Results show that (1) average haplotype diversity is high ( h  = 0.93) while haplotype divergence is low (π= 0.8%), suggesting that extant populations diversified recently; (2) mtDNA gene diversity does not significantly differ across the distribution range; and (3) haplotype distribution differs among the sampling locations ( amova ; P  < 0.00031). Mantel test and autocorrelation analyses indicate that isolation-by-distance may explain the geographical partitioning of genetic diversity. These patterns have likely been produced by complex dynamics between past fragmentation of northern vs. intermediate and southern populations. Late Pleistocene climatic changes, fostering cycles of range contraction and expansion, may have led to genetic differentiation in allopatry, followed by restricted gene flow across secondary contact zones.     

Introgression of Alectoris chukar genes into a Spanish wild Alectoris rufa population

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.     

Sanitary risks of red-legged partridge releases: introduction of parasites

We studied the helminth community and body condition of 99 hunter-harvested red-legged partridges (Alectoris rufa) from Ciudad Real (Central Spain). Forty-six were sampled in two game estates where an important number of farm-reared red-legged partridges are released yearly. The remaining 53 were obtained from natural wild populations adjacent to one of the estates with releases. Four nematode species (Heterakis gallinarum, Aonchoteca caudinflata, Eucoleus contortus and Cheilospirura gruveli) and two cestode species (Raillietina (R.) tetragona and Skryabinia bolivari) were identified. The managed areas showed higher parasite diversity, with higher prevalences and intensities for all helminths found. Three of these species were typical of farm-bred partridges and two of these, A. caundinflata and S. bolivari, were found parasitizing adult partridges. This suggests introduction of these helminths into the breeding population of managed states. The birds sampled in the nonmanaged estates showed a better body condition, but no relation with parasite infection was found. Our results suggest that the release of farm-reared red-legged partridges, a strategy that is becoming a common practice in Spanish hunting areas, poses risk to wild populations because of introducing parasites. However, these results also suggest that simply stopping releases may be a good way to remove locally those parasites from populations, as the establishment of the introduced parasites seems limited.     

陇东黄土高原石鸡的分子系统地理结构

本文运用聚合链式反应和直接测序的方法测定陇东黄土高原 8个石鸡 (Alectorischukar)地理种群 78个样本的mtDNA控制区 4 91bp ,建立其分子系统地理结构。 78个样本共发现 2 4个变异位点 ,2 5种单倍型。 8个地理种群共享单倍型C1 ,6个种群共享单倍型C2 ,种群间有一定的基因交流。 8个地理种群的 2 5种单倍型在NJ树中相互混杂 ,没有形成独立的地理结构 ,但聚成两个分支。单倍型网络图显示 2 5种单倍型聚成两个星状的集群 ,分别以单倍型C1和C2为中心。这两个群间的遗传差异显著。陇东黄土高原石鸡的系统地理结构属于“系统发生连续 ,具有部分空间隔离”的地理格局。这种地理格局是更新世冰川、泥石流和人类活动共同作用的结果     

Introgression of chukar genes into a reintroduced red-legged partridge (Alectoris rufa) population in central Italy

Insight regarding the genetic origin and composition of the studied population of the red-legged partridge (Alectoris rufa) is likely to provide general and critical information for the appropriate management and possible conservation of the species. The reintroduced population of red-legged partridges living in Pianosa Island (National Park Tuscany Archipelago) has proven to be sustainable: captive-bred individuals, morphologically assigned to the taxon A. rufa, were released to the island approximately 20 years ago, establishing an apparently well-adapted population. We have investigated this population by means of 10 microsatellite loci in order to shed light on its genetic structure. Considering that A. rufa is known to crossbreed with A. chukar, the Pianosa Island population was compared at the molecular level with a red-legged partridge breeding stock (Aulla, MS) as well as with a population of pure A. chukar. Our results indicate that the red-legged partridge population from Pianosa, morphologically identified as A. rufa, is actually partly introgressed with A. chukar, questioning its genetic purity and the possible use of this population as a starting stock for future reintroductions elsewhere.     

Vocal signatures of the rally call of red-legged and rock partridges and of their hybrids

Red-legged (Alectoris rufa) and rock (A. graeca) partridges are two species belonging to the same genus, which hybridise at a contact zone located in the southern Alps. Hybrids are viable and fertile. Male rally calls (implicated in species isolation mechanisms) of the two species and their F1 hybrids were analysed and compared. Three temporal and frequency parameters were studied. Of all parameters studied, principal components analysis revealed that male rock partridge signals present a reduced stereotypy compared to those of the male red-legged partridge. It also indicated that the frequency of the maximum amplitude and the frequency band containing 80 % of the energy are the factors that differ the most between the two species. F1 hybrid males present an intermediate profile. These results are discussed in terms of species barrier problems within the Alectoris lineage.     

基于线粒体DNA控制区的石鸡华北亚种的种群历史动态研究

隶属于鸟纲鸡形目雉科的石鸡是一个多型种,在我国已有7个亚种被报道,其中石鸡华北亚种是我国的特有鸟。用石鸡华北亚种(Alectoris chukar pubescens)12个地理种群112个样本的线粒体DNA控制区(mt DNA CR)1154 bp序列的信息研究了石鸡华北亚种的种群历史动态。112个样本中共发现28个变异位点,定义了29种单倍型,其中12个地理种群的50个样本共享单倍型H1,8个地理种群的16个样本共享单倍型H4。地理种群间存在较大的基因流,且种群间没有由于地理距离产生隔离的证据。负的Tajima(D=-1.336,P0.05)和Fu(Fs=-1.720,P0.05)统计检验值及错配分布的单峰模式都支持石鸡华北亚种经历了种群扩张。石鸡华北亚种大部分种群的错配分布与种群过去的扩张相一致,扩张发生在晚更新世中期的第五寒冷期(0.027—0.06 Ma)。推测其扩张的原因可能为:1)更新世期间我国北方地区没有发生大规模的冰川,2)青藏高原的隆升使我国北方干旱化和荒漠化加剧利于石鸡种群的扩散。武都(WD)位于东洋界,而石鸡是典型的古北界的鸟种,表明WD种群可能是石鸡在东洋界的一个建群种,因此需要给予充分的关注。     

Prevalence of Escherichia coli, Salmonella sp. and Campylobacter sp. in the intestinal flora of farm-reared, restocked and wild red-legged partridges (Alectoris rufa): is restocking using farm-reared birds a risk?

For hunting purposes, several millions of red-legged partridges (Alectoris rufa) are released each year in Spain, and these releases have the potential to introduce new parasites and disease into wild populations. We studied the prevalence of Escherichia coli, Campylobacter sp. and Salmonella sp. in the intestinal flora of red-legged partridges from three different husbandry groups: farm-reared, restocked and natural populations. Prevalence of E. coli was significantly higher in farm-reared (45%, p = 0.01) and restocked partridges (60%, p < 0.001) than in wild ones (6%, p > 0.05). The prevalence of Campylobacter sp. (23%, 100 out of 444) did not differ significantly between these three husbandry groups, and Salmonella sp. was only detected in a group of partridge chicks on one of the farms studied (0.9%, 5 out of 544). These results suggest that farm-reared and restocked partridges can act as carriers of these three enteropathogens and highlight a potential risk of transmission to natural populations via the releases of farm-reared partridges. However, future investigations are needed regarding the relation of the isolated bacteria with zoonotic strains and dissemination of antibiotic resistant microorganisms, especially E. coli, and to better evaluate the effect that these three enteropathogens have on partridge health and on the success of restocking with farm-reared birds.     

A comparative genetic study of two groups of chukar partridges (<Emphasis Type="Italic">Alectoris chukar</Emphasis>) from Cyprus and Argentina,using microsatellite analysis

The aim of the present work is to estimate the usefulness of microsatellite genetic markers analysis to characterize and analyze the possible differences between a captive reared population and a wild one from the same species. The first sample consists of 27 chukar partridges (Alectoris chukar) bred in one farm in Argentina. The second one is composed of 31 chukar partridges coming from a wild Cyprus population (A. chukar cypriotes). We analyzed seven microsatellite loci: MCW135, MCW225, MCW276, MCW280, MCW295, LEI31, and ADL0142. The Argentina group showed higher genetic variation than the Cyprus did. Significant global F _IS value was found in the Argentina sample. Significant genetic differentiation exists between both groups (F _ST=0.394; p<0.01). The Argentina group did not show any signs of bottleneck. Results from Factorial Correspondence Analysis (FCA) suggest that the 58 partridges could be split into two distinct genetic clusters (Cyprus and Argentina). Nevertheless, in the light of PARTITION results, three Argentina individuals might be related to Cyprus. STRUCTURE is unable to assign these three animals to any of the two groups. This could be due to a single or repeated introduction of external individuals into the original Argentina group, so that these results would point to more than one origin for this population. This admixture of individuals could explain the high genetic variation observed in the Argentina farm. Global F _IS value would probably be higher without these immigrations; on the other hand, these admixtures could have prevented bottlenecks.     

Analysis of Genetic Diversity of Chukar Partridge (Alectoris chukar) Populations in Khorasan-e-Razavi Province of Iran by RAPD-PCR

RAPD markers were used to investigate population genetic parameters of an endangered partridge, Alectoris chukar, in four areas of Iran, as a part of a genetic conservation program. The aim of this study was to analyze the genetic similarity among these populations. Blood samples from 75 birds were used for DNA extraction and RAPD-PCR analysis of 67 loci, with 28 polymorphic bands (41.79%). The populations of Kalat-e-Nader and Mashhad were found to be closely related, as were the Torbat-e-Jaam and the Quchan populations. Mean heterozygosity for all populations was 0.4405 ± 0.0755. The results indicate that chukar partridge genetic diversity in Khorasan-e-Razavi province is sufficient and the amount of gene flow among populations is acceptable.     

陇东黄土高原石鸡的遗传多样性与保护

石鸡 (Alectorischukar)是我国北方重要的猎鸟 ,由于栖息地片断化和人类狩猎 ,陇东黄土高原上的石鸡数量正日益减少。本文用PCR直接测序的方法 ,测定了陇东黄土高原 8个石鸡种群mtDNA控制区I区和部分II区的4 91个碱基 ,探讨其遗传多样性。 78个样本共发现 2 4个变异位点 (占所测序列的 4 .89% )和 2 5种单倍型 (占所测样本的 32 .0 5 % )。 8个种群中 ,铜川种群的序列变异率、单倍型多样性和核苷酸多样性都最高 ,分别是 0 .4 7、0 .82和 0 .0 0 2 9;而红回种群的最低 ,仅分别为 0 .10、0 .2 8和 0 .0 0 0 8,这与红回种群受奠基者效应、遗传隔离和自然选择的作用有关。 8个种群共享 1种单倍型C1,说明它们来自共同祖先 ,是 1个单系群 ,属于 1个进化显著单元 ,但它们聚成两个集群。两集群间单倍型相似性指数仅 0 .15 ,遗传距离达 0 .4 3% ,单因素方差分析显示遗传变异差异显著 (F =5 .0 2 >F0 .0 5(14 ,1) =4 .0 6 ) ,分别有 13种和 10种单倍型为两个集群所特有。基于遗传差异性 ,陇东黄土高原的石鸡应分为两个管理单元进行保护 ,尤其对遗传变异和遗传多样性最高的铜川种群应进行重点保护。     

Daily variations of blood biochemical parameters in the red-legged partridge (Alectoris rufa)

The study of blood chemistry provides important information about many aspects of the biology and physiology of animals. It is known that many factors can influence the results of such analyses. A proper knowledge of these effects is necessary to correctly interpret the results. In this paper, we analyzed the daily variations of blood biochemical parameters of 32 captive red-legged partridges (Alectoris rufa) of both sexes to test the effect of the daily cycle on these parameters. We found daily variation in glucose, cholesterol, uric acid, triglycerides, and calcium, but not in total protein and creatinine. A sex effect was only significant in uric acid. The most reliable data were those obtained from blood samples collected in midday hours. Hence, this time is suggested for blood-sampling in future studies.