Human-mediated introgression of exotic chukar (Alectoris chukar, Galliformes) genes from East Asia into native Mediterranean partridges

Mediterranean red-legged (Alectoris rufa) and rock (Alectoris graeca) partridge populations are affected by genetic pollution. The chukar partridge (Alectoris chukar), a species only partly native to Europe, is the most frequently introgressive taxon detected in the genome of hybrid partridges. Both theoretical (evolutionary) and practical (resources management) matters spur to get insight into the geographic origin of the A. chukar hybridizing swarm. The phenotypic A. rufa populations colonizing the easternmost part of the distribution range of this species, the islands of Elba (Italy) and Corsica (France), were investigated. The analysis of both mitochondrial (mtDNA: Cytochrome-b gene plus Control Region: 2,250 characters) and nuclear (Short Tandem Repeats, STR; Random Amplified Polymorphic DNA, RAPD) genomes of 25 wild (Elba) and 20 captive (Corsica) partridges, disclosed spread introgression of chukar origin also in these populations. All mtDNA haplotypes of Elba and Corsica partridges along with those we obtained from other A. rufa (total, n = 111: Italy, Spain, France) and A. graeca (n = 6, Italy), were compared with the mtDNA haplotypes of chukars (n = 205) sampled in 20 countries. It was found that the A. chukar genes detected in red-legged (n = 43) and rock partridges (n = 4) of Spain, France and Italy as well as in either introduced (Italy) or native (Greece, Turkey) chukars (n = 35) were all from East Asia. Hence, a well-defined geographic origin of the exotic chukar genes polluting the genome of native Mediterranean A. rufa and A. graeca (inter-specific level) as well as A. chukar (intra-specific level), was demonstrated.     

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.     

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.     

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.     

The genetic structure of the loggerhead sea turtle (Caretta caretta) in the Mediterranean as revealed by nuclear and mitochondrial DNA and its conservation implications

The population genetic structure of the loggerhead sea turtle (Caretta caretta) nesting in the eastern Mediterranean was assessed by sequencing a fragment of the control region of the mitochondrial DNA (n = 190) and seven microsatellites (n = 112). The two types of markers revealed genetic structuring (mtDNA: γ_st = 0.212, P < 0.001; nDNA F _st = 0.006, P < 0.001), thus indicating that both females and males are philopatric and that gene flow between populations is restricted. Mitochondrial DNA data indicate that the female populations nesting on the islands of Crete and Cyprus have suffered a recent bottleneck or colonization event. However, no bottleneck or founder effect was revealed by nuclear markers, thus indicating male-mediated gene flow from other populations that would increase nuclear genetic variability. Crete, and to a lower extent Cyprus, are thought to play a central role in such male-mediated gene flow that may reduce the negative effect of genetic drift or inbreeding on the small populations of Lebanon and Israel. This population structure indicates that assessing population relevance only on the basis of genetic variability and size would be misleading, as some populations not fulfilling those requirements may play a relevant role in genetic exchange and hence contribute to the overall genetic variability.     

Allozyme differentiation in the Mediterranean firs (Abies, Pinaceae). A first comparative study with phylogenetic implications

Nineteen natural Mediterranean fir populations, belonging to eight species and to one natural hybrid (A. ×borisii-regis), were investigated by starch and polyacrylamide gel electrophoresis. A total of 31 alleles was scored at eight loci (IDH-B, ACP-A, PER-B, 6PGD-A, 6PGD-B, MNR-B, PGI-B, PGM-A. Great variation was observed in the heterozygosity among the population studied and ranged from 0.010 (A. pinsapo) to 0.328 (A. cephalonica). The interpopulation genetic diversity was about 26% of the total genetic diversity. From the dendrogram, new phylogenetic relationships were revealed. High affinity was observed between the Calabrian fir population and the one from north-west Greece as well as betweenA. equi-trojani grown in Asia Minor and the southern Greek populations. Species specific alleles were found inA. cilicica. From the findings of the present work, a new hypothesis concerning the taxonomy, distribution and evolution ofAbies species in the Balkan Peninsula is supported.     

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.     

Genetic diversity of Garra rufa Heckel, 1843 (Teleostei: Cyprinidae) in Anatolia

In the present study, mitochondrial DNA polymerase chain reaction-restriction-fragment length polymorphism (PCR-RFLP) and nuclear DNA inter-simple sequence repeat (ISSR) assays were used to assess the phylogenetic and phylogeographic relationships among Garra rufa samples from Anatolia. The complete mtDNA NADH 3/4 dehydrogenase (ND-3/4) gene amplified by PCR was digested with eight restriction enzymes. These enzymes produced 20 composite haplotypes for G. rufa populations. All the mtDNA haplotypes detected were highly diverged from each other and each lineage had a unique genetic profile. The evaluation of mtDNA PCR-RFLP data coupled with geological history of Anatolia indicated a deep genetic divergence among the mtDNA haplotypes of G. rufa populations from drainages of the Mediterranean and Persian Gulf, suggesting an early isolation of Tigris-Euphrates with Orontes river and other rivers draining into the Mediterranean Sea. In general, data from both mtDNA and nDNA were congruent.     

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。     

Genetic Structure of Przewalski’s Rock Partridge (Alectoris magna) Populations in the Longzhong Loess Plateau, China

In this paper, we have analyzed the nuclear DNA of Alectoris magna samples, collected from the Longzhong Loess Plateau. We used allelic variation at eight microsatellite markers to describe the genetic structure of A. magna populations. The primary goal of this study was to examine the population genetic structure and determine the extent of population differentiation among populations of A. magna. The average value of H _E (0.455) was smaller than H _O (0.477), and there was a heterozygote deficit at the MCW135 locus in the Lanzhou population and the Beidao population. The AB063167 locus in each population (except that of Jingyuan) was not in equilibrium (P < 0.05). The Lixian edge population had a lower proportion of genetic diversity than the central geographic populations; the Haiyuan and Jingyuan populations had higher genetic diversity than the central geographic populations. Analysis of population structure revealed clear differentiation among the eight populations of A. magna, suggesting strong isolation of these populations and correspondingly low levels of migration or gene flow. The A. magna populations of Longzhong Loess Plateau are separated into eastern and western populations by a clustering chart. Genetic data indeed suggest that patterns of speciation and population diversification of A. magna in the Longzhong Plateau have been affected by the stability of the climate, natural selection, and human intervention.     

Gene variation and gene flow inOrchis morio (Orchidaceae) from Italy

Data are presented on genetic variation at 27 enzyme loci of the Green-Winged orchid,Orchis morio, in 18 population samples from Italy. The existence in Italy of two subspecies, i.e. subspp.morio andpicta, is not supported by allozyme data. No genetic heterogeneity was found betweenmorio-like andpicta-like samples and specimens. Moreover, morphological transition between the two forms was observed in different Italian populations. The parameters of genetic variability estimated forO. morio populations are consistent with those found among monocotyledon plants, and among those outcrossing, animal-pollinated and with wind-dispersed seeds. Genetic diversity of ItalianO. morio is mostly within populations. Correspondingly, low values of interpopulational genetic distance were found. This appears to be due to high levels of gene flow, which were estimated with different methods. The lack ofO. longicornu from Italian samples, as well as of any hybrid withO. morio (F₁, backcrossed or recombinant individuals) is demonstrated on the basis of genetic data. It is concluded that recurrent reports ofO. longicornu from Italy are due to confusion withO. morio or with otherOrchis species.     

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.     

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.     

Consistency of population genetics parameters estimated from isozyme and RAPDs dataset in species of genus <Emphasis Type="Italic">Prosopis</Emphasis> (Leguminosae,Mimosoideae)

Genetic variability, population structure and differentiation among 17 populations of 5 species and 2 natural interspecific hybrids of section Algarobia of genus Prosopis were analyzed from data of 23 isozyme and 28 RAPD loci. Both markers indicated that the studied populations are highly variable. P. alba populations in average showed lower values of genetic variability estimates from isozyme data, but this trend was not observed for RAPD markers. The hierarchical analyses of the distribution of genetic variability showed that the highest proportion of variation occurred within populations, the differentiation among species was intermediate and the lowest component was observed among populations within species. The consistency between results from both dataset implies that they are not biased and reflect the actual genetic structure of the populations analyzed. The matrices of Euclidean distances obtained from the two sets of markers were highly correlated according to Mantel test. In both cases the corresponding phenogram and MDS plot tended to cluster conspecific populations while hybrid populations were not intermediate between putative parents. Some disagreements between isozyme and RAPD phenograms were observed mainly in the affinities of hybrid populations. Such inconsistencies might result from reticular rather than dichotomic evolutionary relationships. The phenetic associations retrieved gave no support to the division of the section Algarobia into series.     

Genetic characterization of common carp (Cyprinus carpio) populations from Greece using mitochondrial DNA sequences

Wild common carp from two lakes and two rivers in Greece were genetically characterized with sequencing analysis of two mitochondrial DNA segments: cytochrome b (1119 bp) and D-loop (646 bp). A total of 9 variable singleton sites and 7 unique haplotypes were detected. A common haplotype was found in three out of the four populations examined, which seems to be the ancestral one and represents the European origin of common carp from Greece. This haplotype could be also justified by the introductions reported with individuals belonging to the Central European race, into many natural habitats in Greece. Limited genetic variation — in Evros and Aliakmonas populations — could be due to bottleneck effects and small effective population sizes, whereas the different haplotypes found in Lake Volvi could represent different common carp stocks. Values of sequence divergence among Greek haplotypes ranged from 0.0006 to 0.0023. The Neighbour-Joining (NJ) phylogenetic tree constructed based on the combined sequences, reveals that the populations of common carp from Greece belong to the European group of populations — which is highly divergent from the South East-Asia cluster — and to the subspecies Cyprinus carpio carpio.     

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.     

Patterns of Genetic Variation in Swertia przewalskii, an Endangered Endemic Species of the Qinghai-Tibet Plateau

Swertia przewalskii Pissjauk. (Gentianaceae) is a critically endangered and endemic plant of the Qinghai-Tibet Plateau in China. RAPD and ISSR analyses were carried out on a total of 63 individuals to assess the extent of genetic variation in the remaining three populations. Percentage of polymorphic bands was 94% (156 bands) for RAPD and 96% (222 bands) for ISSR. A pairwise distance measure calculated from the RAPD and ISSR data was used as input for analysis of molecular variance (AMOVA). AMOVA indicated that a high proportion of the total genetic variation (52% for RAPD and 56% for ISSR) was found among populations; pairwise Φ _ST comparisons showed that the three populations examined were significantly different (p < 0.001). Significant genetic differentiation was found based on different measures (AMOVA and Hickory θ_B) in S. przewalskii (0.52 on RAPD and 0.56 on ISSR; 0.46 on RAPD and 0.45 on ISSR). The differentiation of the populations corresponded to low average gene flow (0.28 based on RAPD and 0.31 based on ISSR), whereas genetic distance-based clustering and coalescent-based assignment analyses revealed significant genetic isolation among populations. Our results indicate that genetic diversity is independent of population size. We conclude that although sexual reproduction and gene flow between populations of S. przewalskii are very limited, they have preserved high levels of genetic diversity. The main factors responsible for the high level of difference among populations are the isolation and recent fragmentation under human disturbance.     

Climatic and geographic effects on the spatial genetic pattern of a landbird species (Alectoris rufa) on the Iberian Peninsula

Understanding the spatial pattern of genetic diversity may be pivotal to adaptive conservation management of a given taxon. The red-legged partridge (Alectorisrufa, Linnaeus 1758) is naturally widely distributed from the Mediterranean to humid temperate zones. According to a recent study, the genetic structure of this species comprises five clusters, three of which are in the Iberian Peninsula (glacial refugia). Partridge demographic expansion events and climatic shifts during Pleistocene glaciations have been used to test the hypotheses concerning Iberian red-legged partridge distribution. We tested the existence of climatic and geographic relationships on genetic diversity/distances. We employed markers from two different genetic systems, such as part of the mitochondrial DNA control region (n = 113) and 20 species-specific microsatellite DNA loci (n = 377), including climatic and geographic factors from the 14 Iberian localities where A. rufa populations were sampled. Our results showed a mitochondrial genetic diversity pattern associated with a thermic gradient, and a decrease of genetic diversity in peripheral populations that concurred with the ‘abundant centre’ hypothesis. Overall, current climatic variables reliably described genetic variation and differentiation in the red-legged partridge, which may be a result of local species adaptation.     

RAPD variations among pure and hybrid populations ofPicea mariana, P. rubens, andP. glauca (Pinaceae), and cytogenetic stability ofPicea hybrids: Identification of species-specific RAPD markers

Random amplified polymorphic DNA (RAPD) analysis was used to determine genetic relationships amongP. mariana (black spruce),P. rubens (red spruce), andP. glauca (white spruce) and to assess the degree of polymorphism within populations from different provenances and among spruce hybrids. Eleven arbitrary decamer primers were used to amplify genomic DNAs extracted from embryogenic cultures and seedlings. Species-specific RAPD markers were identified.Picea mariana andP. rubens showed similar RAPD profiles confirming their close genetic relationship. Species-specific RAPD markers were identified and were useful in distinguishing white spruce from black and red spruces. RAPD differentiation between populations within each species was small. The level of polymorphism was much higher in spruce hybrid populations than in the pure species. Cytological analysis ofP. mariana ×P. rubens hybrids showed normal mitotic behaviour at prophase, metaphase, anaphase, and telophase. All the hybrids analyzed from different cross combinations were euploids.