Plasmopara viticola, is the main disease affecting vineyards in subtropical Brazil. Here, we collected 94 P. viticola isolates from four organic and conventional vineyards in the two main grape-growing states of Brazil to evaluate the sensitivity to the quinone outside inhibitor (QoI) azoxystrobin by pheno- and genotyping assays. The impact of location, production system and sensitivity to QoI fungicides on the population genetics and structure of P. viticola was determined using 10 microsatellite markers. Cytochrome b sequencing revealed that 28 and 100% of the isolates from vineyards under organic and conventional management carried the G143A mutation, respectively. The G143A mutation was associated with high levels of azoxystrobin resistance. Three out of the 94 isolates analyzed carried the M125I alteration, not previously described in P. viticola, which was associated with a five-fold reduction in azoxystrobin sensitivity compared to wild-type isolates. Haplotype network analysis based on cytochrome b gene sequences suggested that the Brazilian populations are more closely related to the European than the North American population. A total of six haplotypes were identified, with two of them carrying the G143A mutation. Microsatellite analysis revealed high allelic and genotypic variation among the four populations. Population differentiation analyses indicated that state of origin directly influences the population biology of P. viticola, while production system and QoI sensitivity have little effect. Great genetic diversity, sexual reproduction and high levels of admixture were observed in Rio Grande do Sul State. In contrast, populations in São Paulo State were dominated by a few clonal genotypes, and no admixed genotype was detected between the two genetic pools identified in the state. This study raises the hypothesis that winter weather conditions influence the overwinter survival strategy with profound effects in the population biology of P. viticola.Downy mildew, caused by  Plasmopara viticola, is considered one of the most destructive diseases of grapevine worldwide. Under favorable weather conditions and absence of control measures, the pathogen can infect all green parts of the grapevine causing losses up to 100%  in cytochrome b, which is associated with high resistance levels , South-eastern Brazil, and two in Rio Grande do Sul State (PPv3 and PPv4), Southern Brazil, in the 2018/19 growing season. Two locations consisted of vineyards under organic production system for at least six years (PPv1 and PPv4), where only copper and sulfur based products were used to control foliar diseases. The other two vineyards (PPv2 and PPv3) were cultivated under conventional production system, where disease control was based on application of multi- and single-site fungicides throughout the growing season. Distances between sampled vineyards ranged from 37 to 820 km . VineyarVitis vinifera cv. Cabernet Sauvignon grown in a greenhouse. Inoculated leaves were placed in Petri dishes (150 mm) containing 1% water-agar  amended with streptomycin sulfate (0.5 g L–1) and incubated in the dark at 22°C for 24 h. Then, Petri dishes were opened in the laminar-flow hood for 2 h to allow any remaining droplets to evaporate from the leaves, and then they were incubated at 22°C with a photoperiod of 16 h. Successive subcultures were performed at 7 to 10-day intervals during the whole study.In each vineyard, symptomatic leaves were collected randomly at a minimum distance of 10 m from each other and placed in individual plastic bags for transport. Upon arrival at the laboratory, each leaf was placed on wet filter paper in a moist plastic box (11 × 11 × 3.5 cm) and incubated at 22°C with a 16 h photoperiod for 24 h to stimulate downy mildew sporulation. Then, sporangia from a single-lesion were collected using a sterile needle, suspended in 150 μL sterile distilled water at 4°C and inoculated by depositing 15 droplets (10 μL each) onto the abaxial surface of a healthy leaf of For each isolate, three downy mildew lesions excised with a 10-mm cork borer were ground in liquid nitrogen, and DNA was extracted using a modified cetyl trimethyl ammonium bromide (CTAB) method . The genb gene of all isolates was amplified using primers CB 279F (–1), 8.5 μL of nuclease-free water, 1 μL of each primer (10 μM) and 12.5 μL of GoTaq Colorless Master Mix  in a Bio-Rad T100 thermal cycler . Amplification conditions were 94°C for 1 min; followed by 40 cycles at 94°C for 1 min, 55°C for 1 min and 72°C for 1 min; with a final DNA extension at 72°C for 8 min. PCR products were visualized on 2% agarose gels stained with SYBR Safe , and purified using the Wizard SV Gel and PCR Clean-Up System kit (Promega), following the manufacturer’s instructions. PCR products were sequenced on an ABI 3130xl Genetic Analyzer  using the PCR primers. Sequences were assembled, aligned and analyzed with Geneious v.10.0.2  was used at the following concentrations: 0, 0.001, 0.01, 0.1, 1, 10, and 100 μg mL–1. Leaves from the fourth through the sixth node distal from the shoot tip of potted plants of ‘Cabernet Sauvignon’ grown in a greenhouse were excised, superficially disinfected for 1 min in 0.5% sodium hypochlorite and triple rinsed in sterile distilled water. Subsequently, the leaves were dried at room temperature and then soaked in fungicide solution for 1 min. Leaves were placed with abaxial surface facing upward in 150-mm Petri dishes containing 1% WA with streptomycin sulfate (0.5 g L–1). Fresh sporangia were harvested from 7-day-old cultures into sterile distilled water at 4°C, and the suspension was adjusted to 5 × 104 sporangia mL–1. Each detached leaf was inoculated by placing 10 droplets (10 μL each), evenly distributed, on the abaxial surface and incubated at 22°C in the dark for 24 h. Thereafter, the Petri dish lids were removed for 2 h to allow the leaf surface to dry, and then they were incubated at the same temperature under a 16 h photoperiod. At 7 days after inoculation, the sporulation in each droplet was visually estimated using the following scale: 0 = no sporulation; 1 = light sporulation; 2 = the sporulation spot was smaller than the inoculum droplet; 3 = the sporulation spot was the same size as the inoculum droplet and; 4 = the sporulation spot was larger than the initial inoculum droplet (50) was determined by linear regression analysis of the sporulation inhibition percentage versus log10-transformed fungicide concentration using Microsoft Excel software. The minimum inhibitory concentration (MIC), the lowest concentration able to completely inhibit P. viticola growth, was also determined. The experiment was performed twice and the combined data demonstrated that variances were homogeneous according Levene’s test (P > 0.05). Levene’s test was performed using the package lawstat v.3.3 , 3.25 μL of nuclease-free water, 0.5 μL of each primer (10 μM) and 6.25 μL GoTaq Colorless Master Mix (Promega). PCR amplification was performed as follows: 94°C for 4 min followed by 38 cycles of 30 s at 94°C, 30 s at 54°C, and 35 s at 72°C; and a final extension at 72°C for 5 min. After amplification, the PCR products were multiplexed in two sets according to their size and fluorescent dye attached to the primer (TM 600 LIZ® (Applied Biosystems) as an internal size standard. Allele sizes were scored using GeneMarker v.3.0.1  vineyard population (PPv1-4); (ii) production system ; (iii) QoI sensitivity  and; (iv) state of origin (Rio Grande do Sul and São Paulo). We used GenAlEx v.6.503 software (NA), number of private alleles and unbiased Nei’s gene diversity  was estimated using the rarefaction method to account for variable population sizes in FSTAT v.2.9.4 (E5) and genotypic diversity including Stoddart and Taylor (G) and Simpson (λ) indices were calculated in poppr. G is a genotypic diversity index based on sample size  and the standardized index of association (rd) were calculated using clone-corrected data in poppr. The IA is a multilocus measure of linkage disequilibrium based on the distribution of the number of different alleles between all pairs of individuals , was assessed by 999 permutations. Loci were tested to determine whether they were under Hardy–Weinberg equilibrium in the R package pegas v.0.11  using the R package adegenet v.2.1.1 (K) in the dataset using the Bayesian Information Criterion (BIC) without prior information on group membership. The optimal number of principal components retained for the DAPC was determined by cross-validation using the ‘xvalDapc’ function, with a training set consisting of 90% of the data and then used to predict the groups of the 10% of remaining observations (poppr using Bruvo’s distance (The genetic population structure of  v.2.1.1 . DAPC is v.2.1.1 . At firsrvations . After rdistance .FST) was used to estimate the genetic differentiation between populations in Arlequin 3.5.2.2  and conventional (PPv2 and PPv3) production systems. A total of five polymorphic sites were identified in the dataset. However, only two of them were non-synonymous mutations resulting in the replacement of methionine [M (ATG)] by isoleucine [I (ATA)] at codon 125 (M125I) and glycine [G (GGT)] to alanine [A (GCT)] at codon 143 (G143A). The M125I alteration was found only in isolates from PPv4 (13%). All isolates from PPv2 and PPv3, 4% from PPv1 and 52% from PPv4 carried the G143A mutation .The haplotype network analysis revealed the existence of six haplotypes in Brazilian populations, three of them previously reported in the European population . The remaining three new haplotypes, never reported before, were identified in the population PPv4 (BRA and BRB) and in both PPv3 and PPv4 (BRC). Among all Brazilian sequences analyzed, the frequency of haplotypes IR, IS, IIS, BRB, BRC, and BRA was approximately 61, 28, 4, 3, 3, and 1%, respectively . Haploty50 and MIC values of 0.02 and 0.1 μg mL–1, respectively. Isolates carrying the M125I alteration had a five-fold increase in EC50 compared to wild-type isolates and a MIC of 1 μg mL–1. High resistance levels were observed in isolates carrying G143A (EC50 and MIC > 100 μg mL–1).Three azoxystrobin sensitivity levels were identified among the 14 Brazilian isolates tested . The firH and AR measures varied from 0.35 to 0.60 and 1.78 to 5.82, respectively. Overall, measures of genetic diversity showed small or no differences between populations within the production system and QoI sensitivity levels. When considering the state of origin, the Rio Grande do Sul cluster of populations had higher genotypic diversity  than that from São Paulo . Genotype abundance was also higher in Rio Grande do Sul (E5 = 0.95), if compared to São Paulo (E5 = 0.63), indicating that most genotypes occur at the same frequency.The genotype accumulation curve tended to a plateau from the 7th microsatellite, indicating that the 10 microsatellite loci had enough power to discriminate a significant number of MLGs . All 10 IA and rd) were not statistically different from zero for populations from Rio Grande do Sul (PPv3 and PPv4), which is expected under a scenario of random mating (P = 0.001), indicating that asexual reproduction is preponderant in this population from São Paulo. The number of loci under Hardy–Weinberg equilibrium was four, eight and nine for PPv1, PPv3, and PPv4, respectively. Those estimates were not calculate for PPv2 due to its small population size after clone correction (only three distinct MLGs), which could result in loss of power to reject the null hypothesis  and MLG7 (n = 3 from PPv1 and n = 20 from PPv2)  . MeanwhiFST analyses showed significant genetic differentiation between populations at all levels (FST = 0.04) and great to very great differentiation in other pairwise comparisons (FST = 0.20 to 0.44). Moderate differentiation was detected among isolates grouped by production system (FST = 0.05) and QoI sensitivity (FST = 0.08). Very great genetic difference was observed between the São Paulo and Rio Grande do Sul populations (FST = 0.27). The AMOVA results confirmed these findings and showed that 28.83% of the genetic variation could be explained by differences among populations at vineyard level and 31.82% between populations with respect to the state of origin  carried A143 cytochrome b alleles, while highly sensitive isolates (EC50 = 0.02 μg mL–1) carried G143 alleles (50 = 0.11 μg mL–1) was observed in a few isolates harboring the M125I alteration, which was never reported before, to the best of our knowledge. M125 is located within the heme b1 distal lobe of the QoI binding site formed by amino acid residues 120–150 close to the surface region of cytochrome b (Saccharomyces cerevisiae (A126T) and many plant pathogens, including P. viticola (F129L) (S. cerevisiae and is therefore not likely associated with a fitness cost (b gene of Pythium paroecandrum were associated with a 30-fold reduction (EC50 > 14.6 μg mL–1) in azoxystrobin sensitivity compared to wild-type isolates  . Interesess cost . Thus, fisolates . Howeverld doses .b sequencing revealed that all isolates analyzed from vineyards under conventional management had the G143A mutation , a maximum of three QoI applications preventively in mixture with effective partners from different cross-resistance groups should be used per vine cycle to avoid resistance development. However, the fungicide partner and its dose in the mixture significantly affect the success of QoI resistance management strategies. For instance, applications of the phthalimide fungicide folpet in mixture with QoIs delayed selection pressure in P. viticola compared to solo QoI applications, while the mixture with the dithiocarbamate fungicide mancozeb had low efficiency  were observed in Rio Grande do Sul compared to São Paulo . A recent study also revealed that P. viticola populations from São Paulo had low genetic diversity, where 19 of the 55 MLGs were clonal and represented 93% of the isolates sampled  and QoI sensitivity have little or no effect. It seems that the population genetic differences between Rio Grande do Sul and São Paulo are related to the distinct winter weather conditions, which have a profound influence on the reproductive mode and, consequently, in the allelic and genotypic diversity of P. viticola populations. As expected, populations from conventional vineyards had higher frequencies of G143A QoI-resistant isolates than those from organic vineyards. A new non-synonymous amino acid alteration at codon 125 in the cytochrome b, associated with a slight reduction in azoxystrobin sensitivity, was reported for the first time in P. viticola. Moreover, Brazilian P. viticola populations are more closely related to the European than the North American population based on cytochrome b analysis. Therefore, this study provides a solid support for further epidemiological studies and fungicide resistance management for downy mildew in subtropical regions.The results of the present study showed that the population genetics of https://www.ncbi.nlm.nih.gov/genbank/, MT329677–MT329682.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: RS, MC-G, LA, and BF designed the experiments. RS performed the experiments. RS and AO analyzed the data. All authors participated in writing and editing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a shared affiliation, though no other collaboration with several of the authors  at time of review.


D-loop of mitochondrial DNA as a maternally inherited genetic marker, we address the question of the origin and dispersal routes of domestic chicken of the Middle East (Iraq and Saudi Arabia), the northern part of the African continent (Algeria and Libya) and the Horn of Africa (Ethiopia).Indigenous domestic chicken represents a major source of protein for agricultural communities around the world. In the Middle East and Africa, they are adapted to hot dry and semi-dry areas, in contrast to their wild ancestor, the Red junglefowl, which lives in humid and sub-humid tropical areas. Indigenous populations are declining following increased demand for poultry meat and eggs, favouring the more productive exotic commercial breeds. In this paper, using the D-loop of 706 chicken samples from Iraq (n = 107), Saudi Arabia (n = 185), Algeria (n = 88), Libya (n = 23), Ethiopia (n = 211) and Pakistan (n = 92) show the presence of five haplogroups , suggesting more than one maternal origin for the studied populations. Haplogroup E, which occurred in 625 samples, was the most frequent in all countries. This haplogroup most likely originates from the Indian subcontinent and probably migrated following a terrestrial route to these different countries. Haplotypes belonging to haplogroup D were present in all countries except Algeria and Libya, it is likely a legacy of the Indian Ocean maritime trading network. Haplogroup A was present in all countries and may be of commercial origin. Haplogroup B was found only in Ethiopia. Haplogroup C was only detected in the South-Western region of Saudi Arabia and in Ethiopia.The analysis of the mtDNA The results support a major influence of the Indian subcontinent on the maternal diversity of the today’s chicken populations examined here. Most of the diversity occurs within rather than between populations. This lack of phylogeographic signal agrees with both ancient and more recent trading networks having shaped the modern-day diversity of indigenous chicken across populations and countries. Undoubtedly, village chickens are a valuable genetic resource for the countries around the world due to their adaptation to the local environment, including their higher resistance against endemic diseases. They supply high-quality protein and represent a major source of income to poor communities. Therefore, they contribute greatly to food security, poverty alleviation and management of natural resources . The maiThe Red junglefowl is the main ancestor of the domestic chicken . Its natMtDNA analysis has been used intensively to unravel the history of domestic chickens . AnalysiD-loop sequences and Asian haplotypes of reference from Liu et al. [This study aims to unravel the history and diversity of indigenous chicken from the Middle East, Northern and the Horn of Africa. It includes chicken from (i) Pakistan, a putative ancient centre of origin for domestic chicken in the northern part of the Indian subcontinent , (ii) Iru et al.  was analn = 54, 26%) and H_4 present only in Pakistani chicken .Eighty-eight haplotypes defined by 63 polymorphic sites were identified in the 706 sequences  and significant (P ≤ 0.05) levels of genetic diversity compared to the other regions (North-East and South), with haplotype diversities of 0.725 ± 0.053 (Baghdad) and 0.712 ± 0.105 , but 0.438 ± 0.121 for Misan in the South-East, and 0.182 ± 0.144 for Basra in the South (Table P ≤ 0.001) and significant (P ≤ 0.05) level of diversity in the Central region of the country  haplotypes diversity differences found between Tlemcen and Tiaret, Tlemcen and Mascara, Adrar and Tiaret and Oran and Tiaret  for nucleotide variation between Mascara and Tlemcen on the first level, and between Tiaret and Tlemcen on the second level  and nucleotides diversities  compared to the haplotypes (0.363 ± 0.131 and 0.182 ± 0.144) and nucleotides (0.001 ± 0.0004 and 0.0009 ± 0.0007) diversities for Mascara and Tiaret, respectively. Significant (P ≤ 0.001) and significant (P ≤ 0.05) haplotypes diversities for Adane, Arabo, Kumato, Loya, Meseret and Shubi  or significant (P ≤ 0.05) differences among populations , a (n = 31), C (n = 3) and B (n = 1). Haplogroup a is represented by 7 haplotypes, B by one, C by two and haplogroup D by 11 haplotypesReference haplotypes from Liu et al.  were useHaplogroup E is represented by different numbers of individuals and proportions in the six countries. It is present in 87 (99%), 169 (80%), 84 (79%), 21 (91%), 84 (91%) and 180 (97%) birds out of 625 for Algeria, Ethiopia, Iraq, Libya, Pakistan and Saudi Arabia, respectively. Haplogroup A is found in 31 samples . Haplogroup B and C were only found in Ethiopia and Saudi Arabia. One haplotype from Ethiopia was identical to the reference haplotype B, while C was identified as one Ethiopian haplotype and one Saudi haplotype (represented by two samples from the South-West region). Haplotypes belonging to haplogroup D were present in all countries except Algeria and Libya. This haplogroup was represented by eleven haplotypes beside the reference one. It includes 34 (16%), 8 (7%), 3 (7%) and 1 (0.5%) samples, out of 46, for Ethiopia, Iraq, Pakistan and Saudi Arabia respectively.Network analysis allows further visualisation of the diversity of the haplotypes and their relationships. The most frequent haplotypes , belong to haplogroup E  suggest a complex demographic history for our populations. We also performed mismatch distribution and Bayesian Skyline plot (BSP) analyses to gather more information regarding possible past population expansion.The demographic pattern for each population and each country was examined. The Mean Absolute Error (MAE) values were moderate to low Table . Resultsjima’s D . Its powThe mismatch distribution graphs show three patterns: uni-, bi- and multi-modal  have been reported in another studies [In this study, we analysed the mtDNA genetic diversity of 706 village chickens from Algeria, Ethiopia, Iraq, Libya, Pakistan and Saudi Arabia with the aim of unravelling their history and origin. To the best of our knowledge, it represents the first study on the  studies , 41, 42.D-loop mtDNA diversity. This is not the case when we consider all haplogroups together for each country , with E the commonest. This probably reflects more than one route of introduction and origin for the Saudi Arabian chicken. Haplogroup E likely originated from the Indian subcontinent following a terrestrial and maritime route while other haplogroups may have reached the country following maritime trading network on the Arabian Gulf and the Red Sea. This is further supported by the genetic diversity values observed for the different regions, where the eastern part on the Arabian Gulf shows higher diversity (0.856 ± 0.044) compared to the central (0.631 ± 0.084) and western (0.702 ± 0.041) regions of the country.Five haplogroups  have been identified in Ethiopian populations, with possibly different historical backgrounds of introduction. As mentioned before, the E haplogroup, the commonest, probably originated from the Indian subcontinent via either a terrestrial route along the Nile River Basin and/or arrival through the coastal areas of the Horn of Africa. The latter was the most likely route for the second most frequent haplogroup D. Within the country, the South region displays the highest haplotype (0.929 ± 0.021) and nucleotide (0.0150 ± 0.0006) variations compared to the other three Ethiopian regions. Then the Central - East, North and West regions with little differences among them.D-loop mtDNA diversity of Ethiopian, Iraqi, Libyan, Pakistani and Saudi chickens are higher than those previously reported for Iranian, Turkey and Egyptian chickens in the literature [Compared to other studies, the terature –46. Simiterature , 22, 47.A high frequency of one haplotype (H_3) was found in all countries examined here Fig. , which lD-loop mitochondrial DNA as a genetic marker for phylogeographic analysis within country.Overall, AMOVA showed similarity among countries, with most of the variation observed among individuals within populations Table . HoweverD-loop sequencing information. Combined with findings from other studies, the results presented here add further support for a main Indian subcontinent origin for chickens of the countries examined here, as well as for the importance of the Indian Ocean maritime trading network for the dispersal of the species. However, no or low phylogeographic structure was observed overall across the studied populations, showing the limitation of the D-loop chicken mitochondrial DNA diversity for this purpose.This paper presents for the first time the genetic diversity of Algerian, Iraqi and Libyan indigenous chickens using mtDNA n = 92), Iraq (n = 107), Libya (n = 23), Saudi Arabia (n = 185), Algeria (n = 88) and Ethiopia (n = 211). Iraqi samples were collected from five different areas  and Karbala (n = 12); and (iii) South with two regions in the South-East and southern part of the country, Misan (n = 24) and Basra (n = 11). In Saudi Arabia, samples were collected from 17 sites divided into three groups, East ), Central ) and West ). Algerian chicken samples were divided into two groups, North (Mascara (n = 20), Oran (n = 17), Tiaret (n = 11) and Tlemcen (n = 18)) and Central (Adrar (n = 22)). Due to the political situation, sampling in Libya was limited to one population representing the North-West part of the country along the Mediterranean Sea.This study was conducted on 706 samples. It includes chicken from Pakistan . The Ethiopian populations were divided into four groups: North (Meseret and Mihquan), Central-East , West  and South . All the blood samples were collected from free-range scavenging or semi-scavenging village chickens following standard veterinary practice approved by the relevant authority in each country and written or verbal consent from the farmers to sample their birds.In Ethiopia, chicken samples were collected from 19 different sites from both the highland and lowland parts of the country. Ten samples from each site were examined except for four populations  (Whatman Biosciences) using the MACHEREY-NAGEL DNA extraction kit or from full blood preserved in ethanol using the Qiagen kit following the manufacturers’ instructions.D-loop region were amplified using AV1F2 (5′-AGGACTACGGCTTGAAAAGC-3′) [Five hundred and forty-nine base pairs of the mtDNA AAGC-3′)  as the fAAGC-3′)  as the rAAGC-3′) –19. The D-loop [https://www.ncbi.nlm.nih.gov/genbank/), accession numbers MK920994-MK921699.An Applied Biosystems 3730 DNA Analyser was used for Sanger sequencing. For each sample, the primer sequences were trimmed to generate the 549-bp sequence fragment and correct possible base-calling errors using the proseq3 version 3.5 software . SequencD-loop . This dan = 22), the full mtDNA was retrieved from full-genome sequencing data using the Genome Analysis Toolkit (GATK V3.7) [D-loop selected for further analysis.For all the Ethiopian and some of the Iraqi samples (TK V3.7) , 24. Theπ) and the average number of nucleotide differences [DnaSP v5 was used to identify polymorphic sites, the number of haplotypes and to calculate haplotype diversity (Hd), nucleotide diversity , Algerian samples were divided into two groups, . Ethiopian samples were divided into four groups ((Meseret and Mihquan), , , and ), and Saudi Arabian were divided into three groups (East (n = 45), Central (n = 43) and West (n = 97)). All Libyan and Pakistani samples were grouped as a single Libyan or Pakistani country population.The analysis of molecular variance was implemented using Arlequin v 3.5.2 with 1000 permutations . It was Fs [The demographic profiles for each population were calculated from mismatch distribution patterns . The MeaFs  and TajiFs  were estFs .Ne to the likelihood of stationary distribution. The BSPs were standardised using the molecular mutation rate of evolution for chicken mtDNA, 3.13 × 10− 7 mutations/site/year (m/s/y) following Alexander et al. [Bayesian Skyline Plots (BSPs)  were invr et al. . This esAdditional file 1: Fig. S1. mtDNA D-loop variation of 88 haplotypes found in the 706 village chicken. Fig. S2. Median-Joining network for Algerian haplotypes (n = 13). Fig. S3. Median-Joining network for Ethiopian haplotypes (n = 36). Fig. S4. Median-Joining network for Iraqi haplotypes (n = 18). Fig. S5. Median-Joining network for Libyan haplotypes (n = 10). Fig. S6. Median-Joining network for Pakistani haplotypes (n = 19). Fig. S7. Median-Joining network for Saudi Arabia haplotypes (n = 26). Fig. S8. Maximum likelihood tree for the 136 haplotypes and references from Liu et al. [Fig. S9. Median-Joining network for 136 haplotypes of the collected and downloaded samples. Fig. S10. Mismatch distribution patterns of populations included in this study. Table S1. Downloaded sequences from GeneBank. Table S2a. Haplotype diversity significant differences among Iraqi populations. Table S2b. Nucleotide diversity significant differences among Iraqi populations. Table S3a. Haplotype diversity significant differences among Algerian populations. Table S3b. Nucleotide diversity significant differences among Algerian populations. Table S4a. Haplotype diversity significant differences among Ethiopian populations. Table S4b. Nucleotide diversity significant differences among Ethiopian populations. Table S5a. Haplotype diversity significant differences among Saudi regions. Table S5b. Nucleotide diversity significant differences among Saudi regions. Table S6. Location, sample size and genetic diversity of haplogroup E.u et al. . Fig. S9


The Griffon vulture was once a widespread species across the region of Southeast Europe, but it is now endangered and in some parts is completely extinct. In the Balkan Peninsula the largest Griffon vulture inland population inhabits the territory of Serbia. We present, for the first time, the genetic data of this valuable population that could be a source for future reintroduction programs planned in South-eastern Europe. To characterize the genetic structure of this population we used microsatellite markers from ten loci. Blood samples were collected from 57 chicks directly in the nests during the ongoing monitoring program. We performed a comparative analysis of the obtained data with the existing data from three native populations from French Pyrenees, Croatia, and Israel. We have assessed the genetic differentiation between different native populations and determined the existence of two genetic clusters that differentiate the populations from the Balkan and Iberian Peninsulas. Furthermore, we analysed whether the recent bottleneck events influenced the genetic structure of the populations studied, and we found that all native populations experienced a recent bottleneck event, and that the population of Israel was the least affected. Nevertheless, the parameters of genetic diversity suggest that all analysed populations have retained a similar level of genetic diversity and that the Griffon vulture population from Serbia exhibits the highest value for private alleles. The results of this study suggest that the Griffon vulture populations of the Balkan Peninsula are genetically differentiated from the populations of the Iberian Peninsula, which is an important information for future reintroduction strategies. Gyps fulvus fulvus Hablizl, 1783) (hereafter Griffon vulture) belongs to the scavenger guild composed of 8 species distributed in the Old World1. The IUCN Red List status of African-Eurasian vultures has witnessed a drastic population decline in recent years and the majority of species were listed as Critically Endangered2. Griffon vulture species is an object of several international conservation conventions and directives. It is currently listed as a priority species for protection by the European Union , Multi-species Action Plan for avian scavenger MsAP2, Convention on Biological Diversity, Bonn Convention, Bern Convention, CITES and BirdLife international. Thanks to the conservation efforts, Griffon vulture is today considered among the least concerns globally3. Europe alone is the home to more than 32,400–34,400  pairs of birds, but although this is a large number of individuals, only 10% of all European Griffon vultures live outside the Iberian Peninsula5.Eurasian Griffon vulture  and completely extinct in others . Today the only viable populations inhabit the territories of Serbia, Croatia, Crete and Bulgaria9. Although a Mediterranean species, it can be found in mainland enclaves that are located in gorges and canyons of the central Balkan Peninsula, which represent biodiversity hotspots11. Unfortunately, the range of this species has almost completely diminished and in Serbia they can be found only in two regions of its western part, in the gorges of the Drina river tributaries12.Although Griffon vulture populations have been recovered in Southwest Europe, the populations are still declining in many parts of Southeast Europe , Caucasus region, the Middle East and North Africa13. This negative demographic trend lasted until the ‘90s when Griffon vulture population decreased to only 10 breeding pairs12. Thanks to the successful conservation efforts, Griffon vulture population in Serbia is now the largest in the Balkan Peninsula, and constantly increasing. During the 201914 census 260 pairs of birds were detected, out of which 164 were breeding pairs. Although the conservation efforts of Griffon vulture in Serbia seem to represent a success story, no genetic analysis has been performed so far, and there are no data of its overall genetic variability.The Griffon vulture population in Serbia, similarly to many Balkan countries, experienced a rapid demographic decline starting from the mid-twentieth century, mainly due to the mass poisoning of the birds and the implementation of new veterinary measures that prohibited the deposition of dead animals in nature19. Because of this, IUCN has recognized genetic diversity as one of the three forms of biodiversity that deserve conservation, along with species and ecosystem diversity20. In territories where a species is extinct, like it is the case with the Griffon vulture in parts of the Balkan Peninsula, the programs of reintroduction sometimes are the only way of restoring the population21. These programs have to pay close attention to the genetic structure of the source population and founding individuals, in order to avoid post-reintroduction genetic drift that could have negative consequences24. The problem of genetic diversity and reintroduction of the Griffon vulture was addressed in the paper of Le Gouar25. They demonstrated that a proper choice of individuals used for the founding population can help to retain overall genetic diversity.Genetic diversity analysis is of great importance in modern-day conservation, and without knowing the genetic status of the population it is hard to implement proper conservation measures and secure long term survival of the total population in the nowadays fast-changing environment and habitat fragmentation26. The proposed genetic uniformity of Griffon vulture populations is contrary to the high levels of among-population differentiation that was observed for other vulture species like the Bearded vulture, Gypaetus barbatus27. It was also demonstrated that the geographical barriers can cause differentiation in the populations of Cooper’s Hawk, Accipiter cooperii28, as well as between the Bearded vulture populations of the Pyrenees and its reintroduced populations in the Alps29. Interestingly, isolation on islands was also identified as the cause of genetic differentiation for the populations of Egyptian vulture, Neophron percnopterus30 and Griffon vulture populations of Sardinia, Crete and Cyprus18. The strong phylogeographic pattern was detected within the populations of Cinereous vulture, Aegypius monachus32, which was explained by the philopatric behaviour that can cause a limited gene flow among populations32. Similarly, subtle population structuring detected in the Cape vulture, Gyps coprotheres, populations in South Africa could also be assigned to regional natal philopatry33. Given the fact that Griffon vulture, Cinereous vulture and Cape vulture share many behavioural similarities including the philopatric behaviour it could be possible that some level of genetic differentiation could be observed between the geographically distant Griffon vulture populations as well.Due to the high mobility of this species and known migrations undertaken by immature birds, it was believed that Griffon vulture populations represented a uniform population within its European range, with low genetic differentiation, as suggested by genetic analyses34 that are planned in Eastern Serbia and other parts of the Balkan Peninsula. We present a comparative analysis of the obtained data and those published previously on native populations of the Griffon vulture from Croatia, Israel and the Pyrenees in France25. Inclusion of the biggest inland Griffon vulture population from the Balkan Peninsula should improve the results of the comparative analysis which will allow us to determine if there is a detectable level of genetic differentiation between geographically distant populations, a pattern that was observed for the Cape vulture and the Cinereous vulture a species with similar behavioural traits as the Griffon vulture.In this paper we present for the first time genetic diversity data obtained from the largest and most viable Griffon vulture population that inhabits the part of the Balkan Peninsula with a continental climate. Depending on the genetic structure, it could potentially be the appropriate source population for future reintroduction programsO) and expected (HE) heterozygosity were found to be very close in all populations, ranging from 0.53 to 0.60 for HO and from 0.55 to 0.60 for HE. No deviation from Hardy–Weinberg equilibrium was detected in any of the analysed populations  and Spain, since there were no detectable genetic differences between these two populations. These results indicate that the population of the Pyrenees, together with the reintroduced populations, can be used to represent the population of the Iberian Peninsula.Analysis performed by the DAPC method Fig.  showed tK = 2 is the most likely scenario , and the Pyrenees in France, published in Le Gouar36. Presented data demonstrate that Griffon vulture population from Serbia exhibits similar values of genetic diversity as other native populations. Similar values of the parameters of genetic diversity were observed for the Griffon vulture populations of Cyprus and Spain26 although a slightly different set of microsatellite loci were used in that study. Compared to other endangered vulture species, the Griffon vulture population of Serbia exhibits higher values of genetic diversity than the ones observed in the populations of the Indian vulture (G. indicus) from Pakistan26, Cape vulture (G. coprotheres) from South Africa33, Cinereous vulture (A. monachus) from Spain, Georgia, Armenia and Kazakhstan32, and White-tailed eagles  from Norway, Estonia, and Germany37. Interestingly the population of Griffon vulture from Serbia exhibits similar values for parameters of genetic diversity when compared with the populations of the Bearded vulture (Gypateus barbatus) from the Pyrenees38, White-rumped vulture  from Pakistan26, and the Golden eagle (Aquila chrysaetos canadensis) from North America39, while it exhibits lower values in comparison to the White-backed vulture (G. africanus) from Namibia26. Although the population of Griffon vulture from Serbia experienced a serious bottleneck during the last decade of the twentieth century, it retained its rich genetic diversity which can be observed even when compared to the biggest and historically most stable population of Europe, the population from the Iberian Peninsula i.e. Pyrenees40. This finding corresponds with the observation that the similar long-lived species like White-tailed eagles retained relatively high levels of genetic diversity even after they had experienced a bottleneck event37. Some parameters, like the number of private alleles and the mean effective number of alleles, even suggest that the Balkan Peninsula, i.e. populations from Croatia and Serbia, exhibit higher genetic diversity than the ones observed in the Pyrenees. The highest number of private alleles based on a sample of 12 individuals was detected in the Griffon vulture population of Serbia, suggesting that this population could act as an important source of genetic variation34.The endangered Griffon vulture species of the Balkan Peninsula is the last inland population adapted to the continental climate. It could thus be an important source for the reintroduction programs in Southeast Europe. In the present paper we have assessed the genetic variability using the highly variable microsatellite loci described and developed for conservation and reintroduction programs43 and, further, that the detection of linkage disequilibrium for unlinked loci suggests a recent reduction in effective population size44. The effective population sizes estimated for the analysed populations showed that Israel has the biggest population, followed by Serbia and the Pyrenees.Considering the bottleneck in the Griffon vulture population of Serbia, the observed G–W index is the lowest observed among all the populations analysed, but is still relatively high and close to the observed values for other analysed native and introduced populations. According to the G–W index, the populations with the values closest to value 1 are considered to be the most stable populations, which is, according to our analysis, the population of Israel. Wilcoxon test, which evaluates the probability of recent bottleneck events based on heterozygosity excess, even suggests that the highest probability for this event is detected for the population of the Pyrenees, and not the population of Serbia. In addition, even the linkage disequilibrium between the pairs of loci suggests a higher probability of the recent bottleneck event for the population of the Pyrenees than the population of Serbia. It has been shown that the reduction in population size leads to a non-random association of alleles for unlinked loci46 forming a new colony in 1995 in the Mileševka Gorge46. These birds might have helped to maintain the high genetic diversity in the population of Serbia, thus reversing the negative effects of the previous rapid population decline.In addition, the discrepancy between the expected and observed bottleneck effects for the population of Serbia can be explained by possible immigration of the birds from nearby related colonies/populations. It is possible that after war conflicts in the region in the early ’90 s the four known Griffon vulture colonies in Eastern Herzegovina (Bosnia and Herzegovina) didn’t truly disappear, instead, they might have resettled in Serbia25. This confirms that, although all introduced populations exhibit high genetic variations, they form one continuous and uniform population with their source population  in Europe the European Griffon vulture populations retreated to refugia in North Africa and the Arabian Peninsula55. After the end of LGM the recolonization of Europe occurred in two directions: across Gibraltar into the Iberian Peninsula and across Bosporus into the Balkan Peninsula. This hypothesis is supported by the results which show that the population of Israel is the only population without a recent bottleneck. It is plausible that during the initial colonization of Europe from the Middle East the populations of the Iberian and the Balkan Peninsulas went through a founder effect and successive bottleneck, which led to the prevalence of two different genetic clusters, thus differentiating them.Another interesting result for the Griffon vulture population from Israel suggests that the Middle East could be recognized as a hybridization/admixture zoneEven though the two distinct groups can be recognized, AMOVA results suggest that the highest genetic variation is observed within populations and less between them. Since the within-population variance based on microsatellites is generally high, it can lead to an underestimation of variance among the populations. However, STRUCTURE analysis supports the finding that these populations highly overlap in their genetic composition, since they share two genetic clusters.57, although rare cases of extra-pair copulation within the same colony have been observed58 but never confirmed by genetic analyses59. Furthermore, it has a wide dispersion and migrates throughout the Mediterranean. In their migrations, the birds from Serbia can be found almost exclusively in the eastern Mediterranean towards the Arabian Peninsula, but when the nesting period approaches, these birds return to the region in which they have hatched14. This natal philopatric behaviour is important because it can act as a mechanism for increasing genetic differences between the populations that nest in distinct regions, especially in combination with somewhat different climatic conditions. In conservation biology, this effect of philopatry is well recognized as it can be one of the causes of phylogeographic structuring due to geographic isolation caused by this kind of behaviour. This long-term isolation can be used as a major criterion to identify population, i.e. evolutionary significant unit, that deserves separate management or priority for conservation due to its high distinctiveness60. The effect of natal philopatry on genetic differentiation of Cinereous vulture populations from the Iberian and Balkan peninsulas was used to explain the observed genetic differences, resulting in the fact that they are now recognized as evolutionary significant units worthy of protetction32. New data presented in our paper show that the Griffon vulture populations from the Balkan and Iberian Peninsulas are genetically differentiated as well, and that they may have been adapted to different climatic conditions. This raises the question whether the introduction of foreign birds to the Balkan Peninsula is justified. The morphological differences, as well as the observed genetic differences25, should be a rationale to pay closer attention to the local populations as the source for reintroduction programs. The fact that a small percentage of introduced birds from the Iberian Peninsula to Bulgaria survived suggests that the continental Balkan population deserves different conservation strategy and that the reintroduction should not be performed with foreign birds but the ones from the already existing and viable native populations of the Balkan Peninsula. The observed differences between the populations of the Balkan and Iberian Peninsulas could be the result of a non-adaptive processes, like a bottleneck and/or a founder effect, as well as the result of the natal philopatry. In the present paper, we used selectively neutral markers which limit the conclusions about adaptive differences, enabled from the loci under selection or the morphometric data. Nevertheless, the experience of reintroduction with the birds from the Iberian Peninsula into the Bulgaria region may suggest that these differences could be the result of local adaptations as well.Griffon vulture species is considered to be socially monogamous and once formed couples stay together61 at – 20 °C. Blood samples were collected via venal puncture using sterile equipment with respect to animal welfare, in accordance with relevant guidelines and regulations. During the sampling, thorough morphological analysis of chicks was performed  and they were tagged by the licensed personnel. For genetic analyses, we used samples from 57 different nests in order to avoid sampling of siblings. None of the birds was sacrificed or injured during the sampling process. All the procedures applied in this study were reviewed and approved by the Ministry of Nature Protection of the Republic of Serbia and the ethical committee of the Institute for Biological Research “Siniša Stanković”.In the present study we have analysed new samples from the Griffon vulture population that inhabits the region of South-western Serbia, the gorge of the river Uvac. Uvac belongs to the protected area of the “Special nature reserve Uvac” (43°24′N 19°56′E), founded in 1971 to protect the Griffon vultures. The reserve includes three artificial lakes at altitudes from 818 to 972 m a.s.l.: Uvačko (972 m a.s.l.), Zlatarsko (876 m a.s.l.) and Radoinjsko (818 m a.s.l.). The lakes are situated in a mountainous region with steep limestone cliffs. The birds used in the present study were sampled between the years 2013 and 2019, as part of the continuous monitoring program that includes marking the young birds directly in the nests. Samplings were performed from the end of May to the beginning of June before they leave the nests. The blood samples were collected from the chicks directly in the nests and stored in sodium citrate or QUEENS buffer25. The introduced populations were presented by the data from the populations of Baronies (N = 90), Causes (N = 86), Verdon (N = 41), Navaceles (N = 15) and Diois (N = 45)25. In the comparative analyses, we used the raw data (amplicon lengths) provided by Prof. Le Gouar . In order to be able to compare genetic data generated in two different laboratories, six samples from the Griffon vulture population of Croatia used in the study of Le Gouar25 were reanalysed. The feathers from the same birds used in Le Gouar25 were chosen for the DNA extraction in order to be processed in the same way as the new samples from Serbia. For all analyses performed in the present work, we used data for individual birds which had at least 5 genotyped loci, thus creating the differences in the sample sizes for the same populations used in our analysis and Le Gouar25. In a previous study, sample sizes for Baronies, Verdon, and Diois were smaller than the sizes used in our analysis, because the authors excluded the birds of known Spanish origin from the group of these samples, which they used to create a representative group for Spain. Considering that no genetic structuring between the introduced groups and between introduced groups and the native population in Ossau was observed in Le Gouar25 we included these birds in the samples for Baronies, Verdon, and Diois. Furthermore, due to the fact that in the same work the low level of genetic differentiation between the samples from Ossau and Spain was observed, we dubbed the Ossau as the population of the French Pyrenees derived from the Iberian Peninsula.For the comparative analysis we used genetic data of the native Griffon vulture populations of Croatia (N = 37), Israel (N = 33), and the Pyrenees in France  published in Le Gouar36 and the other five were chosen from Gautschi35 (Table 4)2SO4, 2.5 mM MgCl2, 0.8 mM dNTP mix, 1 U of reverse Taq polymerase  and 10 pmol of each forward and reverse primer. In order to verify the reliability of data, 10% of samples were reamplified for second time.The DNA extraction was performed using the GeneJET Genomic DNA Purification Kit (Thermo Fischer Scientific Cat.No. K0721). The DNA concentration and quality were checked both by a spectrophotometer  and agarose gel electrophoresis. We used ten microsatellite loci. Five were taken from Mira35 Table . Microsa35 Table . PCR was35 Table  using th35 Table  and 35 s35 Table . AmplifiFor fragment analysis, the first and second multiplex reactions were multipooled by the GF9C1 reaction, while the third multiplex reaction was multipooled by the GF3F3 reaction. All reactions were mixed in equal volumes and plated as one reaction in a volume of 0.5 µL. Each amplification mix contained five different loci. GeneScan 600 LIZ Size Standard was used to score alleles . Fragment analysis was performed on the 3130 Genetic Analyzer . Data were analysed using Gene Mapper Software .O) and expected (HE) heterozygosity, random match probability and mean number of pairwise differences. Random match probability represents the parameter that expresses the probability that two randomly picked individuals from a population have a matching genotype, and is calculated as the sum of square frequencies62. The mean number of pairwise differences represents the measure of differences between all pairs of haplotypes in the sample. Calculations were performed using Arlequin ver. 3.5.2.263 and HP-Rare 1.164. The effective number of alleles per locus was calculated using the following formula: pi is the frequency of an ith allele. Arlequin software was used for assessing genetic differentiation among populations through Analysis of molecular variances (AMOVA) and estimating pairwise population and overall FST values. Statistical significance of all performed tests was assessed with 10,000 permutations. The matrix of pairwise population FST values was visualized both by R functions and two-dimensional scaling (non-metric MDS) using PAST 3.2565. Hardy–Weinberg equilibrium was tested using Arlequin software with 1,000,000 number of steps in MC and 100,000 dememorization steps.In order to assess the genetic diversity of analysed populations, we determined the following parameters for each locus and population: number of alleles, number of alleles based on a minimal sample size (obtained by rarefaction), number of private alleles based on a minimal sample size (obtained by rarefaction), observed 67 was used and parameterized to 25% of stepwise mutation. The effect of the recent bottleneck was also evaluated based on the Garza–Williamson index (G–W) which represents the ratio between the number of alleles and allelic range and was calculated using Arlequin software. Linkage disequilibrium between the pairs of loci was estimated using the likelihood ratio test in Arlequin software with 10,000 numbers of steps in MC and 10,000 dememorization steps. Effective population sizes for the analysed populations were calculated with NeEstimator 2.168 using the linkage disequilibrium method and monogamy mating model. In order to correct the probabilities when multiple tests were done simultaneously, we performed a sequential Bonferroni test for all performed analyses69.In order to detect whether a significant number of targeted loci featured heterozygosity excess, we used the Wilcoxon signed-rank test implemented in the software Bottleneck v.1.2.0270 using the R package adegenet71. This method consists of performing linear discriminant analysis (LDA) on the principal components analyses (PCA) transformed matrix. The number of retained PCs was estimated using randomly repeated cross-validation (100 iterations) which consisted of performing DAPC on 90% of randomly sampled training set observations (stratified sampling was used so that the training set consisted of 90% of the observations from each population) after retaining 10–83 PCs and using the obtained models to predict the groups (populations) in the remaining 10% of samples (test set). Average prediction success per group was used as the metric to choose the optimal model.Clustering of the analysed populations and the observed distances among samples were presented using discriminant analysis of principal components (DAPC)75. We also performed the same analysis including the reintroduced populations. The analysis was performed using the admixture model with a burn length of 10,000 and a Markov Chain Monte Carlo (MCMC) of 100,000 randomizations. The range of the possible number of clusters (K) was from 1 to 10, with a series of 10 runs for each K. STRUCTURE harvester  was used to analyse the results obtained by STRUCTURE. Based on the results generated by STRUCTURE this software creates a plot of the mean likelihood value per K value and calculates the highest value of the second-order rate of change (Delta K) according to the method of Evanno77 in order to detect the number of K groups that best fits the data set. The model choice criterion, LnP(D), implemented in the STRUCTURE which detects the true K as an estimate of the posterior probability of the data for a given K was evaluated as well. The most likely scenario was chosen and used to graphically plot both the analysed individuals and populations.In order to estimate the number of genetic clusters represented in the native populations of the Griffon vulture we used the STRUCTURE v 2.3.4 softwareSupplementary Figures.Supplementary Tables.


Malus sieversii, the progenitor of the apple, is exclusively distributed along the Tien Shan mountains. Habitat fragmentation and hybridization between M. sieversii and the cultivated apples have been proposed to be the causal mechanism of the accelerated extinction risk. We examined the genetic diversity pattern of eleven wild and domesticated apple populations and assessed the gene flow between M. sieversii and the cultivated apples in Kazakhstan using thirteen nuclear microsatellite loci. On average, apple populations harbored fairly high within-population diversity, whereas population divergences were very low suggesting likely influence of human-mediated dispersal. Assignment results showed a split pattern between the cultivated and wild apples and frequent admixture among the apple populations. Coupled with the inflated contemporary migration rates, the admixture pattern might be the signature of increased human intervention within the recent past. Our study highlighted the prevalent crop to wild gene flow of apples occurring in Kazakhstan, proposing an accelerated risk of genetic swamping.There is growing attention given to gene flow between crops and the wild relatives as global landscapes have been rapidly converted into agricultural farm fields over the past century. Crop-to-wild introgression may advance the extinction risks of rare plants through demographic swamping and/or genetic swamping.  Prunus fruticosa, a rare shrub distributed in Eurasia, with the widely cultivated cherries, P. avium and P. cerasus is one of the compelling examples showing likely risk of genetic swamping [Inter-specific gene flow between rare species and the allied taxa that are commonly distributed may result in the increased extinction risk at least locally for the rare species ,2,3,4. Hswamping . A recenswamping . With thMalus Mill. (Rosaceae) comprises about 25 to 47 species including one of the most important fruit crops, M. domestica [Malus [M. domestica Borkh.) and the two main contributors, M. sieversii (Ledeb.) M. Roem. and M. sylvestris (L.) Mill. is a well-recognized example of such taxonomic challenges [M. sieversii [M. sieversii has primarily contributed to the genetic diversity of the apples along with a series of hybridizations and introgressions by secondary contributors  Borkh. and M. orientalis Uglitz.) [The genus omestica . The spea [Malus ,11. Hybrallenges ,14,15,16ieversii ,18,19. IUglitz.) ,21.M. sieversii  is an insect pollinated long-lived tree that can grow up to 10 m and flower in May [M. sieversii can also vegetatively regenerate by stem shoots, adventitious root formation particularly under unfavorable conditions where the plants are physically damaged and/or competing on resources with neighboring plants [M. sieversii is exclusively native to the Tien Shan mountain range residing mostly mid elevation (900–1600 m) [M. sieversii and the cultivated apple may further aggravate the extinction risk. As apple farming expands in the regions, the natural habitats likely adjoin the local orchards shaping secondary contact zones. M. sieversii is currently listed as vulnerable on the International Union for Conservation of Nature and Natural Resources (IUCN) red list. Despite the great importance of conserving the wild apple populations, only a handful of studies examined the extent and impact of hybridization in the wild M. sieversii populations by domesticated apples [M. sieversii populations showed a signature of hybridization with the apple cultivars [M. sieversii. The results suggest the likely influence of hybridization and introgression of cultivated apples on wild M. sieversii populations.r in May ,22. The r in May ,23. M. sg plants . The plag plants . M. siev–1600 m) ,20. With–1600 m) . Moreoved apples ,16,24,25ultivars  also fouM. sieversii). Although caution must be taken [M. sieversii populations and apple cultivars. The genetic diversity pattern of M. sieversii and how the wild populations spatially structured over heterogeneous landscapes were also investigated. Given the recent disturbances of natural habitats and the surge of agricultural activities, we hypothesized that there would be a high level of hybridization between the wild M. sieversii and cultivated apples, leading to a risk of genetic swamping. Genetic diversity is expected to be well maintained due to the potential hybrid events. Genetic studies often found an escalated infra-specific genetic diversity pattern in M. sieversii, possibly in association with closely related, M. kirghisorum and M. niedzwetzkyana [Malus niedzwetzkyana Diek, an endangered congener that is closely related, in our analysis to assess the influence of the related taxa (M. sieversii). In the present study, we aimed to determine the potential risk of genetic swamping resulting from gene flow between the cultivated apples and the wild progenitor  across two consecutive springs of 2018 and 2019 from Kazakhstan . All required permits were prepared and obtained by the local collaborator prior to the field sampling. We collected young leaves from 124 individuals of wild and domesticated apples . The quantity and quality of extracted DNAs were evaluated in a NanoDrop ND1000  and visualized in 1% agarose-gel electrophoresis.® PCR premix  according to the manufacturer’s protocol. For multiplexing, we fluorescently labeled  the PCR products on the 5′-end of forward primers for every locus. The detailed amplification conditions and the information on the 17 SSR loci are provided in the https://www.geneious.com) for the microsatellite profiling. The scoring results were then manually checked and corrected before finalizing the microsatellite profiles. We estimated the frequency of null alleles using INEST v. 2.2 [We amplified 17 SSR loci using the primer pairs described in three previous studies ,32,33. TT v. 2.2 . p values) were applied. As the 17 microsatellite markers were isolated from different chromosomes, we assumed that each marker is not in linkage. We filtered out four markers from all downstream analyses due to significant deviation from HWE (p < 0.01). Pairwise FST between all population pairs was calculated in Arlequin using 1000 permutations to ensure statistical robustness. We conducted a Mantel Test to examine Isolation by Distance using the pairwise genetic distances (Slatkin’s linearized FST = FST/(1 − FST)) and log-transformed Euclidean distances for all population pairs in GENALEX [To avoid clone sampling, we removed the redundant genets from the analyses. For the clonality test, we used GenoDive v. 2.0b23 , whereby GENALEX ,40. The We employed a Bayesian model-based clustering approach to examine population structure using the correlated allele frequencies model with admixture  implemen−4/allele/generation [Because we aimed to examine the level of crop to wild gene flow, both historical and contemporary migration rates among the wild apple populations and the cultivated apples were estimated as a proxy for the gene flow. To avoid the computational challenges and parameter calculation biases, we redefined groups based on the genetic clusters inferred from the STRUCTURE  implemented in Arlequin and BOTTLENECK v. 1.2.02 ,54, respM. sieversii and M. niedzwetzkyana), identical clones were not observed. However, over half of the domesticated apples turned out to be clones in both the two apple populations  with low to moderate frequencies . However, null alleles were only present in one population for two (loci 4 and 6) of the five loci, thus those loci remained in the downstream analyses. Among the wild apple samples (ST among 11 populations (56 population pairs) differed among population pairs, however, on average, the values were low  and the linearized geographic distances with a marginally significant statistical support  averaged over 13 SSR loci varied among the 11 populations . The Alawere low . On averST results  than across groups  and the smallest at TalEW (θ1 = 0.85). The θ values observed in the MIGRATE-N result are comparable to the ones observed in long-lived shrubs . p values for the nine populations failing to reject the null hypothesis i.e., the equilibrium state of population demography (The G-W indices (M-ratio) for all ten populations  were below the critical threshold, M = 0.73 estimated from the data using Critical_M  exhibited heterozygote deficiency  and were not in HWE. The reduced heterozygosity was likely derived from presence of null alleles. To avoid violating assumptions in coalescent-based approaches and biases in estimating F high He . It is w high He .Malus taxa is not well investigated, it can roughly be inferred from an estimate (~1.5 km) of an allied species  in Rosaceae [The lowered genetic variation among populations is pronounced in outcrossing trees ,65. LikeRosaceae . HoweverThe Bayesian- and distance-based assignment patterns (PCoA and STRUCTURE) were consistent with the population divergence pattern separating domesticated and wild apples . As expeThe high level of dispersal potential might also be the causal mechanism of hybridization between the wild and cultivated apples. Crop-to-wild gene flow is relatively common in trees as they are generally equipped for long distance dispersal . Recent M. niedzwetzkyana (red fleshed apple), was assigned primarily to the cluster of the cultivated apple genotypes, although the assignment pattern was more complex at K5 sharing its genetic affinity the highest with TalE and TalW (https://www.iucnredlist.org). Although the statistical power is low due to small sample size for the species, our study demonstrated that the genetic integrity of M. niedzwetzkyana might have been eroded by cultivated apples, at least in Kazakhstan. Thus, there is an urgent need to monitor the level of admixture between the crop apples and M. niedzwetzkyana throughout its distribution. Unexpectedly, the endangered wild apple, and TalW a,b. The It surely is worthwhile to examine the genetic structure and the population divergences as a proxy of gene flow, yet the genetic structure per se is not the direct measure of gene flow . Given tCoupled with the risk of genetic swamping, there is also concern for demographic effects such as population bottlenecks. Based on the G-W indices, most populations might have experienced a series of population declines in the past, yet no recent bottlenecks were observed for the wild apple populations . It alle


Cynodon dactylon (L.) Pers. (common Bermuda grass) has a limited capacity to grow at low temperatures, which limits its geographical range. Exploring its evolutionary relationship across different environmental gradients is necessary to understand the effects of temperature change on the genetics of common Bermuda grass. In this study, high-throughput transcriptome sequencing was performed on 137 samples of C. dactylon from 16 latitudinal gradients to explore the differential molecular markers and analyze genetic diversity and structure along latitudinal gradients at different temperatures. We primarily sampled more high-quality single nucleotide polymorphisms (SNPs) from populations at lower and middle latitudes. Greater intraspecific genetic variation at each level of temperature treatment could be due to factors such as wind pollination and asexual breeding. Populations of C. dactylon at high latitudes differed from populations at middle and low latitudes, which was supported by a principal component analysis (PCA) and genetic structure analysis, performed at different temperatures. We observed more genetic variation for low-latitude populations at 5 °C, according to an analysis of three phylogenetic trees at different temperature levels, suggesting that low temperatures affected samples with low cold resistance. Based on the results of phylogenetic analysis, we found that samples from high latitudes evolved earlier than most samples at low latitudes. The results provide a comprehensive understanding of the evolutionary phenomenon of landscape genetics, laying the groundwork for future structural and comparative genomic studies of C. dactylon. Festuca arundinacea Schreb and Lolium perenne L., have been studied [C. dactylon is a warm-season turfgrass primarily ranging from latitudes of 45° N to 45° S. It is widely used for lawns, parks, and sports fields [C. dactylon is an ideal species for studying genetic and genomic evolution because of its extraordinary morphological diversity and ecological adaptations. Additional studies are needed to better understand the ability of populations to adapt to global climatic changes that are altering local environmental conditions [C. dactylon is more sensitive to cold stress than heat stress [C. dactylon slows, while leaf senescence occurs at 10 °C [C. dactylon to low temperatures have been studied [C. dactylon tolerate various environmental stresses [Cynodon dactylon (L.) Pers. (common Bermuda grass) with a wide distribution at different temperatures.A major focus of evolutionary biology is understanding how genetic variation drives local adaptions across populations spanning wide geographical and environmental gradients. While genetic mutations and genetic drift tend to induce divergence in plant populations, natural selection produces similar adaptations to similar environments. It is important to characterize relative genotypic differences across different locations based on multiple environmental tests . Natural studied ,3. Adapt studied ,5. C. das fields . C. dactnditions . Temperanditions . Warm-senditions . C. dactt stress ,11. Whenat 10 °C . The res studied . There iOpisthopappus Shih populations by single nucleotide polymorphisms (SNP) was performed in order to obtain valuable information about population genetics [C. dactylon with different directions of stem growth was studied to better understand genetic diversification [Genetic divergence among individuals within a population can lead to population divergence through adaptive selection responding differently to environmental factors. Additionally, populations could converge if similar genetic variants in different populations are selected. Transcriptomics can help identify climatic factors affecting plant traits and performance. As more plant genomes and transcriptomes are sequenced, further exploration of the causes and effects of evolution becomes possible. Transcriptome sequencing has resulted in a better understanding of genetic and genomic diversification in non-model plants ,15,16,17genetics . The trafication . There ification . Studyinfication ,27. In tC. dactylon samples to further analyze their population genetics. Considerable genetic variation was estimated among populations  = 0.526, 0.521, 0.529 and polymorphism information content (PIC) = 0.280, 0.277, 0.282 at 5 °C, 20 °C and 35 °C, respectively. Observed heterozygosity (HO) estimates among all populations and expected heterozygosity (HE) were also calculated to indicate genetic diversity varied among populations. Diversity estimates for some populations at high latitude were a little lower than for the others.C. dactylon, which could be due to the gene introgression of different genetic populations, particularly those that shared similar geographic origins. Individuals at high latitudes evolved earlier than most individuals at low latitudes. No obvious linear trend of genetic similarity was observed among populations from low to high latitudes, suggesting a divergent pattern of evolution in C. dactylon along latitudinal boundaries. We also observed frequent introgressive hybridization in C. dactylon along latitudinal gradients.We analyzed the relationships between different populations along latitudinal gradients using SNP data at different temperature levels by constructing three trees for each temperature treatment . The phyAfter additional analysis of molecular variance (AMOVA) analyses, we found that more variation occurred at a temperature of 5 °C  and 35 °C , compared to 20 °C . High genetic differentiation was detected within populations at different temperature levels. Additionally, lower genetic differentiation was observed among populations from all latitudes when grown in low and high temperatures than when grown at 20 °C, whereas relatively low genetic differentiation was observed within populations at 20 °C .C. dactylon transcriptome sequence  were used to investigate genetic structure at different temperatures using principal component analysis (PCA)  were grouped in cluster three. The high-latitude populations grouped in cluster two were clearly separated from the others. The principal component analysis (PCA)  and cluster two (b). However, at axis two, cluster one (a) consists of low-latitude populations that could be separated from cluster one (b) based on the results of PCoA on C. dactylon populations at 5 °C.SNPs based on the is (PCA) . The PC1is (PCA)  revealedC. dactylon (K) from one to 10. The best K-value along latitudinal gradients for our analysis was estimated as K = 2, K = 3 and K = 3 at 5 °C, 20 °C, and 35 °C, respectively , could confer a competitive advantage in a changing environment. These results agree with the published results of our analysis of the genetic structure using expressed sequence tag-derived simple sequence repeats (EST-SSR) [C. dactylon at high latitudes differed from other populations at middle latitudes and lower latitudes based on an analysis of their genetic structure. The results of both the genetic structure analysis and the PCA analysis corresponded to growth habits [The relationship between local selection and adaptation with climate variables across different geographic locations could be associated with longstanding evolutionary mechanisms. Local selection and genetic drift could shape genetic diversity and differentiation patterns for EST-SSR) . Higher EST-SSR) ,31. Thish habits ,33,34. Mh habits . Proposeh habits . Some ofC. dactylon at low latitudes could emerge directly from most individuals at high latitudes, according to the results of our phylogenetic analysis at different temperatures. Most individuals at low latitudes are descendants of populations located at high latitudes, which could be due to the evolution of genetic diversity along latitudinal gradients. Biogeographic studies have demonstrated a southward migration towards warmer regions [Additionally,  regions ,37. Our  regions . PhylogeC. dactylon. Genetic resources of wild plant species are helpful for plant breeding [C. dactylon. Individuals at low latitudes have likely adapted to relatively higher temperatures, while low temperatures affected the genetic variation of populations at low latitudes. Across evolutionary time, individuals typically appeared at low latitudes subsequent to appearing at high latitudes, indicating that C. dactylon could have evolved from northern latitudes to southern latitudes. An analysis of genetic structure determined that populations of C. dactylon at high latitudes differed from populations at middle latitudes and low latitudes. Understanding genetic variation across geographic boundaries provides a deeper understanding of the study of evolutionary patterns. Our study suggests a valuable pattern of population genetics at different latitudes for the genomic evolution of C. dactylon. Additionally, some populations and individuals can be used to breed strains that are highly resistant to cold temperatures. This analysis will provide the foundation for future structural and comparative genomic studies of C. dactylon, and will also provide additional insight into the evolutionary phenomenon of landscape genetics.These results demonstrate the importance of identifying the geographical source of a sample during an experiment, as well as the need to study different temperature levels in order to fully understand how genetic variation affects the adaptive abilities of breeding ,40, so tA. millepora to adapt to gradual warming, while novel genetic mutations could induce further adaptations [C. dactylon for functional analysis associated with temperature (unpublished paper).Genetic variation among populations can occur as an adaptation to a wide range of climatic conditions, including temperature ,42. Inveptations . High teptations . Additioptations . Increasptations . To makeCynodon dactylon (L.) Pers. were collected from 16 geographic sites across China, ranging from 22°35′ N to 36°18′ N  according to the manufacturer’s instructions. The RNA concentration and quality were determined using a NanoDrop spectrophotometer  and an Agilent Bioanalyzer . High-quality mRNA obtained from the total RNA sample and oligo dT primer was then used for reverse transcription (RT) to generate cDNA, using a cDNA synthesis kit . All of the products of PCR reactions following RT were purified using AMPure PB beads , while the DNA fragments were selected using the BluePippin System. The products were then quantified using an Agilent 2100 Bioanalyzer , while the sequencing libraries were prepared using Illumina TruSeq kits. Paired-end sequencing with an insert size of ∼150 bp was performed using the Illumina HiSeq 2000 platform. Clean reads were obtained using a stringent filtering process and an adapter.SNPs for each individual were analyzed using the GATK  software . From trThe population structure at different latitudes was analyzed using the ADMIXTURE software, with a burn-in period of 10,000 generations and 50,000 iterations . We test


Onopordum alexandrinum is a near-endemic to Egypt and neighboring countries. Its habitats are designated as priority sites for conservation in the south and east Mediterranean regions. We hypothesize that variation in morphological, reproductive, and molecular traits could provide a survival strategy that allows the species to occupy landscapes including anthropogenic habitats (barley and abandoned fields) and natural habitats (sand dunes and desert plateau) with different soil resources along the western Mediterranean coast of Egypt. The results indicated that plant functional traits associated with high rates of resource acquisition and growth  occurred in populations located in abandoned fields with high soil resources. The genetic diversity analyses indicated similarity in genetic diversity of the present populations of O. alexandrinum in barley and abandoned fields with those of sand dunes. However, the genetic structures of these populations were different from those of natural desert plateau, which suggests reduced rates of gene flow. In this framework, it is essential to monitor and reduce the anthropogenic activity which will not only support the conservation of genetic diversity within populations but will also help ensure the resilience of O. alexandrinum in the face of environmental and climatic changes. Understanding the patterns and processes associated with changes in functional and genetic traits is one of the key goals in the preservation and conservation of rare and threatened species . In thisUnderstanding environmental forces  responsible for changes in functional traits and genetic diversity is important in species conservation because it reflects how a population survives under fluctuated conditions  ,10,11. SInvestigation of intraspecific variation in functional and genetic traits may further improve the accuracy and resolution of studies of conservation and community ecology ,18. ThisOnopordum alexandrinum Boiss. (Asteraceae) is a plant near-endemic to Egypt and neighboring countries [O. alexandrinum populations has been dramatically reduced and become highly fragmented by natural and anthropogenic factors such as climate change and habitat destruction. According to our survey, the populations exist in natural habitats (sand dunes and desert plateaus) and in anthropogenic habitats (barley crops and abandoned fields). Despite its threatened status due to reductions of population size and limited geographic distribution, no research has focused on the population structure and genetic diversity of this species. Therefore, resolving the intraspecific variation in both functional and molecular traits of O. alexandrinum is a priority for plant conservationists in the south and east Mediterranean regions. As the two approaches provide different but complementary information, an understanding of both aspects of trait variability and pattern is desirable for the development of appropriate conservation strategies. Accordingly, we hypothesize that variation in morphological, reproductive, and molecular traits could provide a survival strategy that allows the species to occupy landscapes including anthropogenic habitats (barley and abandoned fields) and natural habitats (sand dunes and desert plateau) with different soil resources along the western Mediterranean coast of Egypt. In this context, the following questions were addressed: (1) Are there intraspecific variations in morphological and reproductive traits among populations of O. alexandrinum in anthropogenic and natural habitats? (2) How large is the genetic diversity within populations and genetic differentiation among these populations? (3) Do local soil resources affect these variations? (4) Is it possible to draw general conclusions for long-term monitoring and the establishment of a conservation strategy for O. alexandrinum?ountries ,33,34, wountries . In Egyp−, SO4−−, and organic matter , as well as contents of total nitrogen (24.46 ppm) as well as Ca++ and Mg++ . The soil of the desert plateau was at the opposite end of resource availability, with the lowest percentages of Cl− and SO4−−  as well as Mg++ content (0.25 mg/100 g). It was obvious that the barley fields had the highest values of Na+ (3.21 mg/100 g) and total phosphorous (11.33 ppm). The desert plateau had the opposite trend, with the lowest content of Ca++ and Na+ .Most of the measured soil variables were significantly different in the four habitats . The soiO. alexandrinum showed significant differences for all the measured traits (2/mg) but the lowest leaf dry matter content (261.4 mg/g). Individuals from desert plateau had the shortest height (50.67 cm), highest leaf dry matter content (634.05 mg/g), thickest leaves (547.27 μm), highest seed production (213.33 seeds/capitulum), the lowest specific leaf area (3.35 mm2/mg), and the lowest seed mass (0.63 g/100 seeds). Individuals from barley fields had the lowest number of seeds (129 seeds/capitulum) but the heaviest seed mass (1.41 g/100 seeds).The populations of d traits . Individ−, SO4−−, Ca++, Mg++, total nitrogen, and total phosphorus (4−− (r = 0.47), Ca++ (r = 0.55), Mg++ (r = 0.61), total nitrogen (r = 0.65), and total phosphorus (r = 0.49). Leaf dry matter content was correlated with both soil Ca++ (r = 0.48) and total P (r = 0.51). Among the reproductive traits, seed number was clearly related to all measured variables except SO4−−, K+, and total P.The correlation results indicated that the significant drivers of soil-trait relationships were Closphorus . All of The six random amplified polymorphic DNA (RAPD) primers and six inter simple sequence repeat (ISSR) primers produced 32 and 45 unambiguous and reproducible bands, respectively . In the O. alexandrinum habitats as revealed by various RAPD and ISSR primers is shown in The total percentage of polymorphism of ISSR markers (53.33%) was higher than that of RAPD markers (31.25%) . In addiThe RAPD and ISSR data were combined for cluster analysis. The obtained cluster dendrogram is shown in O. alexandrinum is characterized by intraspecific phenotypic variation in morphological traits , which is consistent with previous studies  along with higher contents of organic matter and total nitrogen. Specific leaf area was found to be positively correlated with the availability of soil SO4−−, Ca++, Mg++, total N, and total P, as also demonstrated experimentally by Al Haj Khaled et al. [Our results indicated that sh flora ). The pod et al. . Wright d et al.  also docd et al. ,40,41.O. alexandrinum demonstrated the two strategies of phenotypic plasticity in the different habitats, with the highest specific leaf area and the lowest leaf dry matter content in the abandoned fields, representing the first strategy, while the opposite was in the case the natural population of the desert plateaus, which representing the second strategy. In addition, the leaves of desert plateau populations had high leaf dry matter content and were thicker than those of abandoned fields. Cornelissen et al. and Kleyer et al. [Wright et al. and Reich ,42 arguer et al. ,43 demonr et al. . Gross er et al.  reportedr et al. ,43,46.O. alexandrinum in desert plateaus had the highest seed production and the lowest seed mass, while those in barley fields had large seeds but low seed production. These results confirm intraspecific phenotypic variation in the seed traits of O. alexandrinum. The low seed production of O. alexandrinum in disturbed barley fields could be related to setting fewer seeds when facing increased environmental constrains or competition from crowded barley and other species, while larger seed mass will allow higher survival, establishment, and growth rates for the seedling [Seed production and seed mass normally display opposite trends in different habitats ,48,49. Tseedling . Seedlinseedling . Kitajimseedling  also conseedling ,51. TherO. alexandrinum indicated that the range and average polymorphic banding pattern of ISSR were higher than those of RAPD. This indicates that ISSR markers were superior to the RAPD marker in their capacity of providing more informative bands, as found in other studies [The molecular diversity analysis for the accessions of  studies ,54,55,56 studies , greater studies , higher  studies . However studies ,60,61,62O. alexandrinum are comparable with those reported for other rare and endemic Asteraceae [Onopordum species [O. alexandrinum in the study area were largely affected by anthropogenic disturbance and the geographical distribution of the accessions. The populations from anthropogenic habitats (barley and abandoned fields) with the adjacent sand dunes were clearly clustered together and separated from the natural population from desert plateau. This suggests that the populations of these anthropogenic fields could have originated from the surrounding sand dunes [In our study, the values of the polymorphic markers of ISSR and RAPD in teraceae ,64,65; h species ,67 and A species ,69. Gene species ,70. The  species ,72,73. Cnd dunes . The prend dunes ,76. On tO. alexandrinum along the western Mediterranean coast of Egypt. Therefore, the current study focused on the understanding of its ecology and genetic diversity, which influence a population’s ability for survival within different habitats, in order to direct its conservation programs. The debate about whether ecological factors contribute more than genetic factors in driving species to extinction has been recently quantitatively assessed for different endangered species [O. alexandrinum responded to the changes in soil resources modified by anthropogenic activity. Therefore, plant functional traits associated with high rates of resource acquisition and growth, such as high values of vegetative height and specific leaf area, and low values of leaf dry matter content and seed mass, were found in the populations of abandoned fields with high soil resources. Conversely, populations with low values of specific leaf area and vegetative height and high values of leaf dry matter content and seed mass were recorded in desert plateaus with low soil nutrient levels. Similarly, plant genetic traits are affected by the creation of rich habitat patches of barley and abandoned fields within regions that formerly supported only natural populations of O. alexandrinum. Therefore, natural populations of sand dunes exist and remain fragmented among the populations of barley and abandoned fields, as indicated by similarity in genetic diversity and the lack of genetic differentiation. In other words, the present populations of O. alexandrinum in barley and abandoned fields were likely to be derived from previously large populations of sand dunes. This suggests that increasing gene flow to the anthropogenic fields may lead to increasing mate availability and a broadening of the genetic base among these populations, which may increase the ability of O. alexandrinum to survive local selection pressures. On the other hand, the results indicated that the gene structures of these populations are different from those of populations from natural desert plateau, which suggests reducing rates of gene flow, which may lead to the deterioration of diversity in local populations of O. alexandrinum. The genetic distance between the sand dune and desert plateau populations could imply that intraspecific hybridization of both genotypes in restoration sites could result in increases in the number, size, and fitness of populations, and consequently aid in the conservation of O. alexandrinum. In this framework, the preservation of the genetic structure and maintenance of the unique genetic traits of O. alexandrinum populations may depend on maintaining a balance between isolation from and connectivity with neighboring populations [Anthropogenic activity has led to a dramatic reduction in the natural habitats of  species . Many th species ,79. Thes species . Our resulations . In thisThe study was carried out along the western sector of Mediterranean coastal land of Egypt that extends for about 550 km between Alexandria westward to Sallum at the Libyan border and with an average width of 20 km in a north–south direction . The lanO. alexandrinum along the western Mediterranean coast of Egypt. The barley and abandoned fields are the most fertile habitats, which are located on the sandy plains and depressions in the study area [O. alexandrinum. The field survey and measurements were carried out during March 2018.We selected two anthropogenic habitats (barley and abandoned fields) and two natural habitats (sand dunes and desert plateau), which represent the main distribution of udy area . The bar+, K+, Ca++ and Mg++) were determined according to Allen [Three composite soil samples were collected randomly from three different locations (0 to 50 cm depth) within each plot and subsequently pooled for each plot. Soil samples were brought to the laboratory, air dried at room temperature, and sieved through a 2-mm sieve. The estimation of organic carbon was carried out using air dried soil samples. Chlorides, sulfates, total N, total P, and extractable cations  was calculated in order to evaluate the type of relationship between the measured functional traits  and estimated soil variables.One-way analysis of variance (ANOVA) was used to test the significance of variations in the soil variables and the measured morphological and reproductive traits in the different studied habitats. Simple linear correlation coefficient  or absence (0) of bands of various molecular weight sizes. The DNA fingerprint patterns obtained from RAPD and ISSR analyses were converted into binary data matrices containing arrays of 0s and 1s. Only polymorphic and reproducible bands were considered for the analysis. Data were analyzed using cluster analysis to generate a dendrogram illustrating the relationships among the four populations of different habitats based on biochemical and molecular data. The percentage of polymorphic bands was determined according to the following equation:All data analyses were performed using SYSTAT version 7.0 .Populations of natural habitats such as sand dunes and desert plateaus should be considered a high priority for conservation in order to expand population size and genetic diversity. Such an action will not only be an effective manner of supporting the conservation of genetic diversity within populations but will also help to ensure resilience in the face of environmental and climatic changes.