Genetic diversity and spatial genetic structure of Caucasian apple (Malus orientalis Uglitzk.) populations based on microsatellite markers for conservation strategy

Genetic diversity and spatial genetic structure of Caucasian apple (Malus orientalis Uglitzk.) populations based on microsatellite markers for conservation strategy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genetic diversity and spatial genetic structure of Caucasian apple (Malus orientalis Uglitzk.) populations based on microsatellite markers for conservation strategy Hamid Bina, Farrokh Ghahremaninejad, Shahin Zarre This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6546353/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Nov, 2025 Read the published version in BMC Ecology and Evolution → Version 1 posted 12 You are reading this latest preprint version Abstract In Eurasia, M. orientalis Uglitzk. (Caucases apple) is a tree with important ecological and economic (fruit) benefits. We measured eight quantitative morphological traits, none of which showed significant differences among the investigated Caucasian apple populations. In this research, we used 26 microsatellite (SSR) markers to investigate genetic diversity and define unit conservation in the Caucases apple. The mean values of genetic diversity,allelic richness (Ar), private allele (Ap), expected heterozygosity (H E ) and observed heterozygosity (H O ) were 1.74, 0.21, 0.65 and 0.76, respectively. In the regions studied, three major genetic clusters and two significant genetic barriers were discovered. Our gene flow findings revealed that there is little connection between M. orientalis population in the Caucasus indicating habitat fragmentation. Two regions in the Hyrcanian forest and one in the Zagros forest were identified as having the highest priority for conserving the genetic diversity of the Caucases apple. Microsatellite marker Conservation unit Genetic diversity M. orientalis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction As a Southwest Asian nation, Iran exhibits remarkable climatic, topographic, and soil diversity, fostering exceptional biodiversity. The country harbors approximately 8,000 flowering plant species (distributed across 167 families and 1,200 genera), with roughly 1,700 being endemic ( 1 ). These species thrive across four distinct ecological zones, Hyrcanian, Zagros, Irano-Turanian, and Khalij-Omanim, each characterized by unique physiographical and climatic conditions. Among these, Iran's primary forest ecosystems are concentrated in the Hyrcanian and Zagros regions ( 2 ). The Hyrcanian forests of Iran is a biodiversity hotspot in northern Iran, support a rich assemblage of temperate flora, including Fagus orientalis Lipsky (oriental beech), Carpinus betulus L. (hornbeam), and the relict Parrotia persica (DC.) C.A.Mey. (Persian ironwood). This region also hosts valuable fruit-bearing species such as Punica granatum L. (pomegranate), Betula pendula Roth (Birch tree) and wild members of the M. orientalis , which enhance genetic diversity and ecosystem resilience ( 2 – 4 ). The understory features shrubs like Berberis integerrima Franch. and Ruscus hyrcanus Woronow, while the endangered Taxus baccata L. (English yew) persists in shaded ravines. However, invasive species, overharvesting of Malus and other wild fruit trees, and land-use changes threaten these unique plant communities ( 2 , 3 , 5 , 6 ). The Zagros woodlands, Iran’s largest oak-dominated ecosystem, are characterized by drought-adapted species such as Quercus brantii Lindl (Persian oak) and Pistacia atlantica Desf. (wild pistachio). The Malus genus, particularly M. orientalis , thrives in these semi-arid highlands alongside wild pear ( Pyrus syriaca Boiss.) and almond ( Prunus dulcis (Mill.) D.A.Webb). Steppe vegetation, including Astragalus spp. and Salvia spp., dominates the understory, while remnants of Crataegus L. (hawthorn) and Celtis L. (nettle tree) mark degraded forest edges (Heshmati, 2007; Zarre et al., 2024 ). Despite their ecological adaptability, Zagros species face severe pressures from charcoal production, overgrazing, and climate-induced aridification, which disproportionately affect wild fruit trees like Malus and endemic oaks ( 2 , 7 , 8 ). The genus Malus Mill (Rosaceae) includes 25 to 47 species, which are among the most economically important crops worldwide and are widespread in North America, East Asia, Europe, and the Caucasus ( 9 ). Caucases wild apples play an important role in the local diet of rural and peri-urban communities ( 4 , 10 , 11 ). This species has been cultivated in the Caucasus region since ancient times primarily for the exceptional nutritional value of its fruits (more than 4000 years ago) ( 12 , 13 ). The Caucasian apple is one of the ancestors of the Mediterranean cultivar apple ( 10 , 14 , 15 ). Gene flow along the Silk Road facilitated their spread from Asia to Europe, with M. orientalis serving as a key ancestor of Caucasian cultivar. Wild Malus species are ecologically vital, providing food for wildlife and serving as genetic reservoirs for breeding stress-resistant apple varieties. Their conservation is critical to maintaining both ecosystem stability and agricultural resilience ( 8 ). The habitat of this species in Iran is located across the Trade Silk Route in two areas: Hyrcanian forest and Zagros forest ( 16 – 18 ). These mountainous regions differ in slope, elevation, vegetation, and ecological conditions; however, both are Middle Eastern hotspots of plant diversity ( 11 , 19 – 21 ). Habitat fragmentation of Caucasian apple populations in Iran poses significant challenges for biological conservation, evolutionary studies, and genetic diversity assessments. This fragmentation primarily leads to population isolation, restricting gene flow among wild Iranian apple populations. Such isolation can result in reduced genetic diversity, increased inbreeding, and potential loss of adaptive potential over time ( 10 , 22 , 23 ). The protection of habitats against the negative effects of fragmentation helps increase the ability of native plants to adapt to reduce the extinction rates of natural populations ( 24 ). Gene flow can enable the exchange of variability between distant populations and, by increasing dispersal, can reduce the rate of extinction of species ( 12 , 25 – 29 ). To select candidate populations with the highest priority for conservation, molecular markers are often used to understand spatial genetic structure, which allows the identification of natural populations for conservation ( 30 – 33 ). Knowledge of the spatial genetic structure of species can help in understanding the geographic and dispersal patterns of a species over time and space ( 34 ). In many studies focused on such aims, the genetic diversity pattern of a species is defined by analyzing its demographic structure and estimating the gene flow, genetic drift, Nei’s distance, and migration pathways of the species ( 35 – 38 ). The comparison of differentiation in particular populations is helpful for determining conservation units and inferring the nature of selection in geographic regions ( 39 – 42 ). Researchers have used various molecular markers like ISSR, RFLP and SSR to illustrate the spatial genetic structure and demographic history of species. These markers can provide accurate information about the evolution of natural stands and help select wild populations with high conservation priority ( 43 – 45 ). Microsatellites or simple sequence repeats (SSRs) are among the most powerful molecular markers that can be used for the identification of conservation units and the study of genetic diversity. Compared with other markers, microsatellites are characterized by high information content and versatility as molecular tools for germplasm characterization ( 46 – 49 ). In the present study, we applied SSR markers to characterize microsatellite loci in the Caucasus apple to 1) describe the spatial genetic structure of populations across the Silk Trade Route, with a particular focus on Iranian populations; 2) assess gene flow and genetic diversity; and 3) identify a conservation unit to conserve the genetic diversity of Caucasian apple. Material and Methods Sampling The data used in this study were obtained from our previously published research on the genetic diversity and domestication of apple ( M. orientalis ) in Iran and the Caucasus ( 8 ). The Caucasus apple samples we used were collected from two main regions of Iran: the Hyrcanian forest, including western Hyrcanian (Asalem region: 5 samples), central Hyrcanian (Tilak region: 14 samples, Sngdehsari region: 9 samples, Shit region: 4 samples, Vatna region: 8 samples, Gaznsara region: 16 samples, Abesk region: 9 samples), and eastern Hyrcanian (Vatna region: 14 samples, Arsam region: 4 samples, Gorgan region: 14 samples, Dorak region: 9 samples, Toskestan region: 10 samples, Siamarzkoh region: 9 samples, Sheshab region: 7 samples)—and the Zagros forest (Nozhyan: 8 samples, Droud: 4 samples, Sepdkoh: 10 samples, Marivan: 6 samples, Sagez: 2 samples, Bufloo: 5 samples), totaling 167 samples. All plant samples were morphologically identified by Dr. Farrokh Ghahremaninejad, a specialist in botany at the Department of Plant Sciences, Faculty of Biological Sciences, Kharazmi University. Voucher specimens have been deposited at the Kharazmi University Herbarium (herbarium code: T) under the deposition numbers 25760 to 25927. The habitats of Malus orientalis in Iran are not located in protected areas or national parks; therefore, no specific permission was required for sampling in these regions. DNA Extraction Genomic DNA was isolated from dried leaf samples of Caucasian apple using the NucleoSpin Plant II kit (Macherey-Nagel, Germany) according to the manufacturer's protocol. For microsatellite analysis, performed multiplex PCR amplification using a Qiagen multiplex PCR kit with 26 SSR markers, following established protocols ( 8 , 15 , 50 ). Control genotypes were cross-validated against the 2013 reference dataset. Only multilocus genotypes with < 20% missing data were included in subsequent analyses. The reliability of these markers for population genetics studies has been well documented in prior research ( 15 ). In this study, parameters of genetic diversity for each microsatellite locus were calculated via FSTAT v2.9.3 ( 51 ). ADZE software was employed to estimate the population genetic diversity, including allelic richness (Ar) and private allele (Ap), using standardized sample sizes of ADZE = 2 ( 52 ). The number of different alleles (Na), number of effective alleles (Ne), expected heterozygosity (He) and observed heterozygosity (Ho), within-population inbreeding coefficient FIS and Hardy–Weinberg equilibrium were calculated with GENEPOP 4.2 ( 53 , 54 ). The inverse distance weighted (IDW) interpolation function implemented in the Geographic Information System (GIS) software ArcGIS 9.3 ( 55 ) was used to estimate the geographic patterns of H O and H E for all 20 populations of the Caucasus apple. We explored the relationships among clusters via principal component analysis (PCA) with the adegenet package in the R environment ( 56 ). Analysis of molecular variance (AMOVA) was performed via GENALEX v6.5 ( 57 ) to infer the genetic differentiation between populations from Hyrcanian forest and Zagros forest. Barrier analysis, which is based on the ( 58 ) maximum difference algorithm, was conducted to define significant genetic breaks and spatial differences between populations. Genetic barriers were assessed via BARRIER 2.2 ( 59 ). Genetic Structure and Gene Flow Analysis We assessed the genetic structure of the Caucasus apple via STRUCTURE v2.3.4 software ( 60 ). This tool was used to examine range-wide genetic clustering among populations of Caucasian apple. Ten independent runs were performed for each K from 2–10 with 200,000 burn-in steps followed by 500,000 Markov chain Monte Carlo (MCMC) steps. The STRUCTURE output was analyzed and visualized by using StructureSelector ( 61 ). To infer historical gene flow (Nm) patterns, MIGRATE-N v3.6 ( 62 ) was used to estimate the effective population sizes (θ) and mutation-scaled immigration (M) among the groups identified by STRUCTURE. Additionally, we used the Bayesian method implemented in the software BayesAss (BA3) to estimate current gene flow between populations ( 63 ). Estimates via this method are based on migration over the last few generations. We selected the best run for allele frequencies and posterior probability densities of inbreeding coefficients with different random seeds among three independent runs. The Bayesian deviance was calculated to determine the best run via the script of ( 64 ), which was applied in R ( 65 ). To obtain a correct acceptance rate, the values of the parameters ranged between 20% and 40% ( 66 ). The inbreeding coefficient was set at 0.35, and the migration rate was set at 0.75 for the final analysis. For allele frequencies, the default setting of mixing parameters was used. The analysis was conducted with 10 6 burn-ins and 10 7 MCMC iterations; every 1,000th iteration was sampled. The results were visualized via QGIS 2.18 'Las Palmas' ( 67 ). CIRCUITSCAPE software (McRae & Beier, 2007) was used to evaluate the influence of topographic complexity as a resistance factor affecting gene flow among populations. This software models populations as nodes connected by resistors, based on the principles of electrical circuit theory and the connectivity-resistance approach. While altitude alone may not fully capture topographic complexity, we used a digital elevation model (DEM) to derive a resistance surface for the habitats of Malus orientalis in Iran. To statistically validate this resistance model, we compared the CIRCUITSCAPE-derived resistance distances with a null model of isolation-by-distance (IBD) using Mantel tests ( 68 ). Unit of conservation selection To determine the conservation priority of the Caucasus apple, we used reserve selection analysis via DIVA-GIS software ( www.diva-gis.org ). The reserve selection analysis algorithm, which is based on allele frequency in each population, determines the minimum number of geographic units necessary to maintain genetic diversity ( 69 ). The populations with the highest allelic richness are given first priority for conservation, and the priorities of other populations are selected on the basis of the first population. After the first priority of populations for conservation, new alleles were found in the next priority populations that were not observable in the previous populations ( 69 ). Morphological parameters studied Eight quantitative morphological traits were used in this study (leaf area, leaf angle with petiole, leaf length, petiole length, maximum leaf length, maximum leaf width at 0.1 of leaf length, maximum leaf width at 0.9 of leaf length, and stomatal morphology) ( 70 , 71 ). Statistical parameters and AMOVA were computed via SPSS software ( 72 ), and PCA for morphology was performed via the Clustvis website ( 73 ). Results Genetic diversity of Caucasian Apple populations This study identified 26 loci with 455 alleles for wild apples. All the loci were polymorphic for the population of Caucasian apples. The range of alleles per locus ranged from 7 (CH02c03b) to 29 (CH04e03), and the average number of alleles was 17.5 (Table S1). The average number of alleles per population was 5.22, with values ranging from 2.07 (Droud region) to 7.5 (Vatna region). In this study, the mean gene flow (Nm) was calculated to be 0.6 (Table S2). The Vaz and Sangdeh regions of the Hyrcanian forest have the highest H O and H E values, whereas the Nozhyan and Droud regions of the Zagros forest have the lowest H O and H E values. Overall, the observed heterozygosity in the Zagros forest populations was lower than that in the Hyrcanian forest populations (Table 1 ). The FIS parameter for Caucasian wild apple in Iran was estimated to be in the range of -0.83–0.11, but the mean for populations from Zagros forest was negative (Table 1 ). Overall, Caucasian apple populations in the Hyrcanian forest showed greater mean allelic richness compared to those in the Zagros forest. However, the mean number of private alleles was higher in Zagros forest populations (Fig. 2 ). Table 1 ) Estimated genetic diversity parameters for M. orientalis in Iran on the basis of genotyping samples Pop Province Latitude (X) Longitude (Y) N Na Ne I H E H O PPL (%) FIS Hyrcanian Forest Asalem Gilan 37.75 48.86 5 5.38 4.29 1.48 0.75 0.81 100 0.07 Abesk Mazandaran 36.29 52.62 9 6.50 4.49 1.60 0.81 0.8 100 0 Sangdeh Sari Mazandaran 36.1 53.28 9 6.38 4.34 1.56 0.73 0.83 100 0.11 Ganasara Mazandaran 36.33 52.09 16 7.42 4.69 1.61 0.76 0.76 100 0 Shit Mazandaran 36.54 53.37 4 3.57 2.86 1.09 0.75 0.78 100 0.03 Tilak Mazandaran 36.07 53.61 14 6.92 4.32 1.56 0.75 0.69 100 -0.08 Vaz Mazandaran 36.31 52.94 8 5.19 3.72 1.38 0.85 0.46 100 -0.83 Gorgan Gorgan 36.71 54.3 14 6.15 4.00 1.42 0.82 0.45 100 -0.81 Vatna Gorgan 36.66 53.99 14 7.50 4.67 1.64 0.67 0.7 100 0.03 Sheshab Gorgan 36.9 55.13 7 5.80 4.27 1.50 0.76 0.75 100 -0.01 Arsam Gorgan 36.67 54.66 4 2.80 2.34 0.87 0.73 0.71 100 -0.02 Tokestan Gorgan 36.76 54.53 10 5.80 3.68 1.43 0.7 0.74 100 0.05 Dorak Gorgan 36.75 54.38 9 5.96 4.50 1.54 0.7 0.76 100 0.081 Siamarzkoh Gorgan 36.79 55.1 9 6.23 4.18 1.55 0.7 0.74 100 0.05 Average 5.82 4.02 1.45 0.74 0.71 100 -0.09 Zagros Forest Marivan Kordestan 35.4 46.34 6 4.84 3.70 1.38 0.72 0.73 100 0 Saghez Kordestan 36.46 46.6 2 2.80 2.63 0.95 0.79 0.73 100 -0.08 Buflo Kordestan 36.25 46.23 5 4.57 3.69 1.34 0.7 0.58 100 -0.2 Sepedkoh Lorestan 33.95 48.49 10 4.88 3.34 1.29 0.72 0.74 100 0.02 Nozian Lorestan 33.23 48.58 8 3.69 2.89 1.11 0.65 0.69 96 0.06 Droud Lorestan 33.24 33.24 4 2.07 1.94 0.65 0.83 0.44 88 - Average 3.81 3.03 1.12 0.73 0.65 97.3 -0.04 Total 167 Mean 5.25 3.74 1.35 0.74 0.69 99.2 -0.08 N = numner of samples, Na = Number of Alleles, Ne = Effective Number of Alleles, I = Shannon's Information Index, H e = Expected Heterozygosity, H o = Observed Heterozygosity, PPL%= Polymorphic Loci Percentage, FIS = Inbreeding Coefficient According to the pairwise FST analysis, most apple populations from the Hyrcanian and Zagros forests showed very low genetic differentiation, with FST values ranging from 0.005 to 0.30 (Fig. 2 b, Table S2). Statistical significance of pairwise FST values was tested using 10,000 permutations in Arlequin. Although the FST between Mrivan and Vatna was very low genetic differentiation (FST = 0.005). Moderate levels of genetic differentiation was observed between some western and central populations of the Hyrcanian forest, with FST values above 0.25 (P < 0.001), indicating moderate genetic structure among these regions (Fig. 3 b, Table S2). On the basis of our findings from the AMOVA, the majority of variance (80%) was discovered within samples, with only 4% and 8% of variance found within groups and populations within groups, respectively (Table 2 ). Table 2 The results of the AMOVA test in two main regions of Caucus apples in this study Source of changes Degrees of freedom Sum of squares Components of variance Percentage of Variance Between Hyrcanian forest and Zagros forest 1 74.71 0.45 4 Between populations from Hyrcanian forest and Zagros forest 18 454059 0.87 8 Among samples 147 1573.85 0.88 8 within samples 167 1493 8.94 80 Total 333 3596.15 11.15 100 Population structure and genetic differentiation The STRUCTURE results revealed that the Caucasus apple samples exhibited a high level of heterozygosity and indicated five genetically distinct clusters corresponding to five main geographic regions, with the clearest separation observed at K = 5. This K value showed the best geographical pattern for the apple populations, and five distinct clusters were identified, each associated with a main geographic region, with no new groups appearing in the STRUCTURE analysis after K = 5 (Fig. 3 a). At K = 3, populations from Nozhyan, Droud, and Sepedkoh (purple group) were separated from all others, while apple populations from Marivan, Saghez, and Buflo clustered with the Hyrcanian populations (blue group). At K = 4, the Zagros populations split into two subgroups (red and purple), and most Hyrcanian populations remained in the blue group, except those from Asalem, Abesk, and Toskestan. K = 3 was supported as the optimal number of clusters based on the ΔK method (Fig. S4), although K = 5 showed the clearest geographic pattern. STRUCTURE bar plots for K = 3 to K = 6 are shown in Fig. 3 a. We used Monmonier's maximum difference algorithm to find potential genetic barriers between populations of Caucasian apples to complement our analysis and detect potential genetic discontinuity. Among Caucasians, there are two statistically significant genetic barriers (bootstrap support 90%). The first is in the forest of Hyrcanin, and the second is in the forest of Zagros. This caused apple populations from the Sheshab, Siamarzkoh, and Arsam regions in the Hyrcanina forest to be separated from other apple populations and caused apple populations from the Nozhyan region in the Zagros forest to be separated from other apple populations (Fig. 4 ). PCA Results of principal component analysis (PCA) for all populations in this study illustrated that PC2 and PC3 both explain 5.71 percent and 4.37 percent of the variance, respectively. The populations of the Zagros forest were divided into two groups via PCA; however, populations from northern Zagros overlapped with those from the central part of the Hyrcanian forest. The populations from the west of the Hyrcanian forest have mixed with the populations from the central area of the forest the most (Fig. 5 ). Gene flow between the habitats of M. orientalis To estimate gene flow, stands of the Caucasus apple were classified by geographical region. Our findings illustrate some gene flow between provinces. Gorgan populations were the predominant source of exogenous allelic variants, and strong gene flow was observed from Gorgan into Mazandaran, Gilan, and Kordestan populations (Ne respectively from Golestan into Mazandaran, Gilan and Kordestan provinces: 0.172, 0.185, 0.47). We observed minor gene flow from Gilan's population into Lorestan's population (Ne = 0.05) as well as minor gene flow from Kordestan's population into Mazandaran's population (Ne = 0.01). Little or no gene flow between Zagros region populations (Lorestan Province and Kordestan Province (Ne = 0.01) was detected, suggesting that M. orientalis in Zagros are isolated from each other (Fig. 3 c). This result is confirmed by CIRCUITSCAPE analysis, which reveals the existence of a strong topographic barrier between populations from the Zagros Mountains. In contrast, for Hyrcanian forest populations, some admixture in populations was apparent. Lowlands near the Caspian Sea allow undisturbed gene flow between Hyrcanian populations. Interestingly, the connection between Zagros and Gorgan is better than that between Zagros and the western part of the Hyrcanian region, which is confirmed both by MIGRATE-N and CIRCUITSCAPE (Fig. 4 ). Identification conservation units of Caucasus apple The minimum number of geographic units required to preserve the genetic diversity of the Caucasus apple in the two main regions—the Hyrcanian forest and the Zagros forest was analyzed. Our findings indicate that in the Hyrcanian forest, the Toskestan, Siamarzkoh, Abesk, and Asalem regions are designated as the first priority for conservation, while the Sangdehsari and Vaz regions are the second priority.In the Zagros forest, the Sepedkoh region is the first priority for conservation, whereas the Bufloo, Sgez, and Nozhyan regions are the second priority. Additional areas, marked in orange and green (Fig. 1 ), have lower conservation priority. Morphology of Caucasus apples A bar chart of the seven characteristics of the leaves of Iranian wild apple is shown in Figure S1. The leaf angle parameter widely used to describe the morphology of plants ( 74 ) was the most variable parameter in this study. The maximum variance of the analyzed statistics was observed for the population of Siamarzkoh for leaf length and leaf area. The measurement of the maximum leaf width at 0.1 of the leaf length and the maximum leaf width at 0.9 of the leaf length revealed that the Vatna region has the maximum variance. Analysis of variance (ANOVA) and PCA of morphological traits The analysis of variance of the length of the petiole revealed significant differences among the Buffalo region and other regions in this study. On the basis of leaf width, population of Buflo has been signed with all regions except thepopulation of SangdehSari. The population of Tuskestan and population of Sheshab region from Hyrcanian forest and the Grin region from Zagros forest have been clustered with other regions on the basis of the maximum length of the leaf. Analysis of variance of the maximum leaf width at 0.1 and maximum leaf width at 0.9 length revealed significant differences among the population of Sheshab, population of Tuskestan, and population of Vatna regions from Hyrcanian forest and other regions. The leaf areas of the Grin region and Vatna region were significantly different from those of the other Zagros forest regions (Fig. S2). Fig. S3B summarizes the principal component analysis results of the morphological data for the wild apple plants. We used seven principal components that explained the variation between Hyrcanian forest and Zagros forest. These principal components explained approximately 14.2% and 67.6% of the variation (PC1 and PC2), respectively, and, on the basis of the studied morphological traits, we did not observe any pattern of differences between the Hyrcanian forest and Zagros forest (Fig. S3A). Discussion Several analyses of the spatial genetic structure of the Caucasus apple were previously performed and described in the literature ( 10 , 11 , 75 , 76 ). However, the present study is the first large-scale genetic analysis of stands of this species from Iran, across the natural range from the Hyrcanian forest to the Zagros, using a large set of SSRs (26 SSR markers). Our findings confirmed that the gene pool of the Caucasus apple moved from East Asia to Europe across the Trade Silk Road. Genetic diversity within populations of M. orientalis According to our results, many populations of M. orientalis in the Hyrcanian forest (Asalem and Gorgan regions), as well as in the Zagros forest (Droud and Nozhyan regions), are genetically unique. High genetic diversity among Iranian populations across the natural range of species in the Zagros and Hyrcanian forests is confirmed by high dissimilarity between populations and limited dispersal between them ( 77 ). Previous research has suggested genetic similarities between stands in Iran and Central Asia. Using molecular markers, proven that discovered that Iranian wild apples in Hyrcanian forests are closely related to apple populations in Central Asia. These results could indicate that apples could migrate across the northern part of the Silk Road ( 11 ). In conservation genetics, allelic richness is the most important parameter, and it can be used to forecast historical bottleneck populations ( 78 – 80 ). The average allelic richness value in Iran (6.2 ± 0.04) is lower than that in Europe and China ( 75 ). A decrease in allelic richness resulted in a reduction in the effective population size, increasing the vulnerability of these populations to various unfavorable conditions ( 80 , 81 ). Another important parameter is expected heterozygosity, which is a good indicator of genetic diversity ( 82 ). The mean H E for Caucasus apple in Iran was 0.65, which was lower than the H E for M. orientalis in Europe and China (0.80), indicating that population growth in Iran is not uniform ( 83 , 84 ). However, it is easy to explain why the Caucasus apple is found in two main regions in Iran: the Hyrcanian forest and the Zagros forest, both of which have different climatic conditions and vegetation, which can lead to nonuniform growth ( 85 ). The FIS parameter predicts future generations' genetic diversity, and a negative value indicates that regeneration could fail ( 86 ). The lower value of this parameter for Iranian wild apple (-0.81 ± 0.02) than for European wild apple (0.04 ± 0.06) suggests that the regeneration of Iranian populations can be disrupted. The gene pool of various Malus species has been strongly influenced by human impact since ancient times because apple fruits were already important crops in the era of the Sumerians ( 87 ). Thus, it is difficult to recognize whether natural populations of M. orientalis were shaped more by natural processes or by long-term human influence. Gene flow Gene flow and migration have a significant impact on adaptation and may lead to population homogeneity ( 88 ). In the prevuous study about spatila genetic structure of M. orientalis concluded that the geographical location of a population has no effect on the genetic structure of wild apples ( 14 ), but we found a strong link between genetic structure and geographic region in this study. The results of the gene flow analysis revealed strong migration from the eastern part of the Hyrcanian forest to populations in the western part of this region and, with lower intensity, to the northern part of the Zagros forest. According to ( 76 ), Iran has been one of the main regions through which various taxa from the Malus genus moved from Central Asia to Europe across the Trade Silk Road. Our results support this theory and suggest that Iran served as a link between East Asia and Europe in the transportation of genetic variability in the Caucasus apple ( 76 , 89 ) The topography of the central part of Iran creates an enormous barrier for gene flow between the Hyrcanian and Zagros populations. Additionally, the northern and southern parts of the Zagros range are poorly connected; the Migrate-n results show that admixture in Lorestan is very low. The situation on the coast of the Caspian Sea is completely different gene flow along coastal lowlands is theoretically undisturbed, which allows for the exchange of genetic variability between Hyrcanian stands. Thus, northern populations are genetically similar, as indicated by the STRUCTURE and PCA results. The Caucasus apple is one of the minor ancestors of the European Caucasus apple ( 14 ). Long periods of cultivation and crossing between various taxa of Malus could have affected natural populations of ancestral species as well; however, it is difficult to quantify this impact. Spatial genetic structure of M. orientalis Gene affinity and exchange between populations can be determined by analyzing the spatial genetic structure of populations ( 24 ). The whole Iranian range of the species was divided into three clusters via STRUCTURE analysis, which differs from the results of previous studies ( 10 , 11 , 90 ). One of these variations may be the response of Iranian wild apples in the Hyrcanian and Zagros forests to population size and dispersal ( 91 ). In contrast to previous studies, our findings reveal a strong spatial genetic structure between wild apple populations in the Hyrcanian and Zagros forests ( 10 , 75 ). We hypothesize that the differences between Iranian wild apples are due to two gene pools with similar geographic origins. Interestingly, the population from Gilan is similar to Zagros stands; this particular population is quite far from other Hyrcanian stands; however, the topography of Iran hinders the natural flow of genes from Gilan to the Kordistan region. It is possible that the similarity of the gene pool in these two regions is connected with human impact. Stands from Lorestan differ strongly from both Hyrcanian and northern Zagros populations. The isolation of this area is supported by the results of migration analysis, as well as CIRCUITSCAPE and PCA. The distinctiveness of this area, which is difficult to assess due to topographic barriers, may indicate that a unique gene pool exists, which should be protected with high priority. Although STRUCTURE analysis revealed broad-scale clustering patterns, Monmonier’s algorithm identified localized genetic barriers in the Hyrcanian and Zagros forests. This difference is not unexpected, as STRUCTURE detects genome-wide patterns assuming Hardy Weinberg and linkage equilibrium, while Monmonier’s method identifies sharp spatial genetic discontinuities regardless of such assumptions (Manel et al., 2003; Chen et al., 2007; Prates et al., 2016). The genetic barrier around Nozhyan aligns with a distinct STRUCTURE cluster at K = 3, supporting its genetic uniqueness. Other barriers, particularly in the Hyrcanian forest, may represent incipient or incomplete divergence, gene flow reduction, or adaptation to local environments not yet strong enough to form fully distinct STRUCTURE clusters (Storfer et al., 2007; Blair et al., 2012). These results reflect the complementary nature of barrier and clustering analyses in revealing both historical genetic structure and ongoing differentiation processes (Sexton et al., 2014). Conservation of Caucasian apple Caucasian apple is one of the main ancestors of cultivated apples in the Caucasian region (Bina et al., 2022). These wild apple species play a crucial role in sustaining ecosystems, since they are an important source of food for wildlife. Additionally, they are valuable sources for apple breeding, helping to develop features like stress resistance against infections and dehydration. Therefore, it is crucial to protect this species' germplasm from serious threats like the negative effects of habitat fragmentation and futher challenges ( 90 ). Future projects such as regeneration and breeding are needed to conserve the genetic diversity of wild plants ( 92 ). Asia is the main center of genetic diversity in the genus Malus , implying that wild apple habitats in Asia have a large gene pool that needs to be preserved for future generations ( 93 ). However, no studies on the genetic diversity of this important species have been reported, and our study is the first in this field. To define a conservation unit for the Caucasian apple gene pool, we used reserve selection analysis. Our findings revealed three high-priority and medium-priority populations for conservation in two main regions. On a local scale, this illustrates the Caucasus region's lack of consideration of M. orientalis genetic conservation. Conclusion In this study, we discovered two high-priority regions in the Hyrcanin forest for preserving M. orientalis genetic diversity, indicating that M. orientalis management in the Hyrcanin forest would need additional attention. In the Zagros and Hyrcanin forests, we discovered ageographic barrier between the population with the highest priority and other populations. As a result, corridors between populations are suggested in this situation ( 94 ). Finally, on the basis of these results, we can conclude that Iran is one of the key sources of the M. orientalis gene pool that would be useful for cultivating apple breeding programs, but it does not have good habitat conditions and would require performance conservation programs. Declarations Ethics approval and consent to participate: Not applicable Consent for publication: All authors have read and approved the final manuscript and consent to its publication in the journal. Availability of data and materials: The data supporting the findings of this study are available at Zenodo under the DOI: 10.5281/zenodo.6981530. Competing interests: The authors declare that they have no competing interests. Funding: This work is based upon research funded by the Iran National Science Foundation (INSF) under project No. 4034387 Authors’ contributions: H.B., F.Gh. and Sh.Z. conceived and designed the experiments; F.Gh. and Sh.Z. obtained funding, H.B., F.Gh. and Sh.Z. analysed the data; H.B. wrote the original draft and preparation of the figures; H.B., F.Gh. and Sh.Z. gave critical inputs in final draft and revisions. Acknowledgements: This work is based upon research funded by the Iran National Science Foundation (INSF) under project No. 4034387 References Pushpangadan P, Nair K, Ahmad M. Biodiversity and medicinal plant wealth of South Asian countries: country reports of Bangladesh, Bhutan, India, Iran, Maldives, Nepal, Pakistan and Sri Lanka. 2004. Heshmati G. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6546353","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":452861430,"identity":"663f5c6f-4507-4494-a17f-8f04b380e815","order_by":0,"name":"Hamid Bina","email":"","orcid":"","institution":"Kharazmi University","correspondingAuthor":false,"prefix":"","firstName":"Hamid","middleName":"","lastName":"Bina","suffix":""},{"id":452861431,"identity":"f4000a50-a83e-4c17-ac6b-12ad7b2ec244","order_by":1,"name":"Farrokh Ghahremaninejad","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqElEQVRIiWNgGAWjYBAC9gYexoeNDRDOAaK08BzgYTYkWQubJEwLcYCHf+2xypk7DjPwtx9gPFxBlBaJd2k3N545zCBxJoHh4BlitNhLnDG7+bDtMAPDDQaGg0Q5kAeopRCkRZ54Lfw9ZowbgVoMSLCFx1hyZls6j+GZxAZibTlj+LG3zVpO7vjhwx+J0sIgkQDRysDASJwGBgb+A0QqHAWjYBSMgpELAH+RNezplbnIAAAAAElFTkSuQmCC","orcid":"","institution":"Kharazmi University","correspondingAuthor":true,"prefix":"","firstName":"Farrokh","middleName":"","lastName":"Ghahremaninejad","suffix":""},{"id":452861432,"identity":"e4497106-c819-4042-9c0f-c4d61ff9f96e","order_by":2,"name":"Shahin Zarre","email":"","orcid":"","institution":"University of Tehran","correspondingAuthor":false,"prefix":"","firstName":"Shahin","middleName":"","lastName":"Zarre","suffix":""}],"badges":[],"createdAt":"2025-04-28 09:38:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6546353/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6546353/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12862-025-02474-9","type":"published","date":"2025-11-10T15:57:46+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82563269,"identity":"20e80281-54f6-4a64-b9b0-c20a6573a12c","added_by":"auto","created_at":"2025-05-13 01:57:27","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1436013,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of \u003cem\u003eM. orientalis\u003c/em\u003e inIran and conservation priority areas based on DIVA-GIS analysis. The map highlights regions with high, medium, and low conservation priority, considering private alleles and allelic richness.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6546353/v1/219fe57457d5eda817a26dfb.png"},{"id":82561967,"identity":"0db4918f-af8a-4f89-9081-9f787b172519","added_by":"auto","created_at":"2025-05-13 01:41:27","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":49913,"visible":true,"origin":"","legend":"\u003cp\u003eIDW interpolation of allelic richness (Ar) and private allele richness (Ap) for the 20 populations of \u003cem\u003eM. orientalis \u003c/em\u003eon the basis of 26 SSRs (N=167)\u003c/p\u003e","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6546353/v1/ac7befc2a521a994cccfe666.png"},{"id":82561971,"identity":"18aebb1c-6c61-4a1e-a008-d4ec8830cceb","added_by":"auto","created_at":"2025-05-13 01:41:27","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":406822,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Population structure of \u003cem\u003eM. orientalis\u003c/em\u003e based on STRUCTURE analysis for K3 to K6 \u003cstrong\u003eb.\u003c/strong\u003e Matrix of pairwise F\u003csub\u003eST\u003c/sub\u003e values between sites of \u003cem\u003eM. orientalis\u003c/em\u003e. C: Gene flow diagrams of the Caucasus apple samples from 5 provinces. The colors correspond to provinces: Gorgan (Blue), Gilan (Green), Lorestan (Purple), Kordestan (Red) and Mazandaran (green). The direction of an arrow represents the direction of gene flow from one region to another. The width of the arrows denotes the relative amount of gene flow within the habitat of the species \u003cem\u003eM. orientalis\u003c/em\u003e (that is, the wider the arrow is, the greater the degree of gene flow). The ‘humps’ in the estimates of contemporary gene flow represent gene flow originating from sample sites within provinces. The patterns for each diagram are independent; that is, similar widths of arrows or humps do not represent the same amount of gene flow across each of the diagrams.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6546353/v1/b8b123fae52501154fe66b5f.jpeg"},{"id":82562711,"identity":"530af500-f2a3-42c0-9b05-741c9343c0fa","added_by":"auto","created_at":"2025-05-13 01:49:27","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":717199,"visible":true,"origin":"","legend":"\u003cp\u003eThe highest and lowest conductances are shown in yellow and dark blue, respectively, according to the CIRCUITSCAPE results and genetic discontinuities between Caucasian apple populations identified by BARRIER overlaid on a DEM. On the basis of Monmonier’s algorithm with 1000 bootstrap replicates, two significant genetic barriers were detected among the 20 populations of the Caucasu apple t. The dotted lines (white) represent the Voronoi tessellation\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6546353/v1/81b523d4d15800d58421da9d.jpeg"},{"id":82562708,"identity":"6105df03-e843-40f4-8a2b-03147ad77997","added_by":"auto","created_at":"2025-05-13 01:49:27","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":202802,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal component analysis (PCA) created on the basis of individual genotypes (N=167)\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6546353/v1/86a6752f4edb9b42fea91b3c.jpeg"},{"id":96105213,"identity":"93dbd32f-f03e-4499-b71c-d33ddeb05e0a","added_by":"auto","created_at":"2025-11-17 16:10:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3661413,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6546353/v1/6914c9f0-3ca0-4610-b7b1-f56bee3b5498.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genetic diversity and spatial genetic structure of Caucasian apple (Malus orientalis Uglitzk.) populations based on microsatellite markers for conservation strategy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAs a Southwest Asian nation, Iran exhibits remarkable climatic, topographic, and soil diversity, fostering exceptional biodiversity. The country harbors approximately 8,000 flowering plant species (distributed across 167 families and 1,200 genera), with roughly 1,700 being endemic (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). These species thrive across four distinct ecological zones, Hyrcanian, Zagros, Irano-Turanian, and Khalij-Omanim, each characterized by unique physiographical and climatic conditions. Among these, Iran's primary forest ecosystems are concentrated in the Hyrcanian and Zagros regions (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Hyrcanian forests of Iran is a biodiversity hotspot in northern Iran, support a rich assemblage of temperate flora, including \u003cem\u003eFagus orientalis\u003c/em\u003e Lipsky (oriental beech), \u003cem\u003eCarpinus betulus\u003c/em\u003e L. (hornbeam), and the relict \u003cem\u003eParrotia persica\u003c/em\u003e (DC.) C.A.Mey. (Persian ironwood). This region also hosts valuable fruit-bearing species such as \u003cem\u003ePunica granatum\u003c/em\u003e L. (pomegranate), \u003cem\u003eBetula pendula\u003c/em\u003e Roth (Birch tree) and wild members of the \u003cem\u003eM. orientalis\u003c/em\u003e, which enhance genetic diversity and ecosystem resilience (\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). The understory features shrubs like \u003cem\u003eBerberis integerrima\u003c/em\u003e Franch. and \u003cem\u003eRuscus hyrcanus\u003c/em\u003e Woronow, while the endangered \u003cem\u003eTaxus baccata\u003c/em\u003e L. (English yew) persists in shaded ravines. However, invasive species, overharvesting of \u003cem\u003eMalus\u003c/em\u003e and other wild fruit trees, and land-use changes threaten these unique plant communities (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe Zagros woodlands, Iran\u0026rsquo;s largest oak-dominated ecosystem, are characterized by drought-adapted species such as \u003cem\u003eQuercus brantii\u003c/em\u003e Lindl (Persian oak) and \u003cem\u003ePistacia atlantica\u003c/em\u003e Desf. (wild pistachio). The \u003cem\u003eMalus\u003c/em\u003e genus, particularly \u003cem\u003eM. orientalis\u003c/em\u003e, thrives in these semi-arid highlands alongside wild pear (\u003cem\u003ePyrus syriaca\u003c/em\u003e Boiss.) and almond (\u003cem\u003ePrunus dulcis\u003c/em\u003e (Mill.) D.A.Webb). Steppe vegetation, including \u003cem\u003eAstragalus\u003c/em\u003e spp. and \u003cem\u003eSalvia\u003c/em\u003e spp., dominates the understory, while remnants of \u003cem\u003eCrataegus\u003c/em\u003e L. (hawthorn) and \u003cem\u003eCeltis\u003c/em\u003e L. (nettle tree) mark degraded forest edges (Heshmati, 2007; Zarre et al., \u003cem\u003e2024\u003c/em\u003e). Despite their ecological adaptability, Zagros species face severe pressures from charcoal production, overgrazing, and climate-induced aridification, which disproportionately affect wild fruit trees like \u003cem\u003eMalus\u003c/em\u003e and endemic oaks (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe genus \u003cem\u003eMalus\u003c/em\u003e Mill (Rosaceae) includes 25 to 47 species, which are among the most economically important crops worldwide and are widespread in North America, East Asia, Europe, and the Caucasus (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Caucases wild apples play an important role in the local diet of rural and peri-urban communities (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). This species has been cultivated in the Caucasus region since ancient times primarily for the exceptional nutritional value of its fruits (more than 4000 years ago) (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). The Caucasian apple is one of the ancestors of the Mediterranean cultivar apple (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Gene flow along the Silk Road facilitated their spread from Asia to Europe, with \u003cem\u003eM. orientalis\u003c/em\u003e serving as a key ancestor of Caucasian cultivar. Wild \u003cem\u003eMalus\u003c/em\u003e species are ecologically vital, providing food for wildlife and serving as genetic reservoirs for breeding stress-resistant apple varieties. Their conservation is critical to maintaining both ecosystem stability and agricultural resilience (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). The habitat of this species in Iran is located across the Trade Silk Route in two areas: Hyrcanian forest and Zagros forest (\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). These mountainous regions differ in slope, elevation, vegetation, and ecological conditions; however, both are Middle Eastern hotspots of plant diversity (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eHabitat fragmentation of Caucasian apple populations in Iran poses significant challenges for biological conservation, evolutionary studies, and genetic diversity assessments. This fragmentation primarily leads to population isolation, restricting gene flow among wild Iranian apple populations. Such isolation can result in reduced genetic diversity, increased inbreeding, and potential loss of adaptive potential over time (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). The protection of habitats against the negative effects of fragmentation helps increase the ability of native plants to adapt to reduce the extinction rates of natural populations (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). Gene flow can enable the exchange of variability between distant populations and, by increasing dispersal, can reduce the rate of extinction of species (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR26 CR27 CR28\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). To select candidate populations with the highest priority for conservation, molecular markers are often used to understand spatial genetic structure, which allows the identification of natural populations for conservation (\u003cspan additionalcitationids=\"CR31 CR32\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Knowledge of the spatial genetic structure of species can help in understanding the geographic and dispersal patterns of a species over time and space (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). In many studies focused on such aims, the genetic diversity pattern of a species is defined by analyzing its demographic structure and estimating the gene flow, genetic drift, Nei\u0026rsquo;s distance, and migration pathways of the species (\u003cspan additionalcitationids=\"CR36 CR37\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). The comparison of differentiation in particular populations is helpful for determining conservation units and inferring the nature of selection in geographic regions (\u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Researchers have used various molecular markers like ISSR, RFLP and SSR to illustrate the spatial genetic structure and demographic history of species. These markers can provide accurate information about the evolution of natural stands and help select wild populations with high conservation priority (\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Microsatellites or simple sequence repeats (SSRs) are among the most powerful molecular markers that can be used for the identification of conservation units and the study of genetic diversity. Compared with other markers, microsatellites are characterized by high information content and versatility as molecular tools for germplasm characterization (\u003cspan additionalcitationids=\"CR47 CR48\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the present study, we applied SSR markers to characterize microsatellite loci in the Caucasus apple to 1) describe the spatial genetic structure of populations across the Silk Trade Route, with a particular focus on Iranian populations; 2) assess gene flow and genetic diversity; and 3) identify a conservation unit to conserve the genetic diversity of Caucasian apple.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSampling\u003c/h2\u003e \u003cp\u003eThe data used in this study were obtained from our previously published research on the genetic diversity and domestication of apple (\u003cem\u003eM. orientalis\u003c/em\u003e) in Iran and the Caucasus (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). The Caucasus apple samples we used were collected from two main regions of Iran: the Hyrcanian forest, including western Hyrcanian (Asalem region: 5 samples), central Hyrcanian (Tilak region: 14 samples, Sngdehsari region: 9 samples, Shit region: 4 samples, Vatna region: 8 samples, Gaznsara region: 16 samples, Abesk region: 9 samples), and eastern Hyrcanian (Vatna region: 14 samples, Arsam region: 4 samples, Gorgan region: 14 samples, Dorak region: 9 samples, Toskestan region: 10 samples, Siamarzkoh region: 9 samples, Sheshab region: 7 samples)\u0026mdash;and the Zagros forest (Nozhyan: 8 samples, Droud: 4 samples, Sepdkoh: 10 samples, Marivan: 6 samples, Sagez: 2 samples, Bufloo: 5 samples), totaling 167 samples. All plant samples were morphologically identified by Dr. Farrokh Ghahremaninejad, a specialist in botany at the Department of Plant Sciences, Faculty of Biological Sciences, Kharazmi University. Voucher specimens have been deposited at the Kharazmi University Herbarium (herbarium code: T) under the deposition numbers 25760 to 25927. The habitats of Malus orientalis in Iran are not located in protected areas or national parks; therefore, no specific permission was required for sampling in these regions.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eDNA Extraction\u003c/h3\u003e\n\u003cp\u003e Genomic DNA was isolated from dried leaf samples of Caucasian apple using the NucleoSpin Plant II kit (Macherey-Nagel, Germany) according to the manufacturer's protocol. For microsatellite analysis, performed multiplex PCR amplification using a Qiagen multiplex PCR kit with 26 SSR markers, following established protocols (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Control genotypes were cross-validated against the 2013 reference dataset. Only multilocus genotypes with \u0026lt;\u0026thinsp;20% missing data were included in subsequent analyses. The reliability of these markers for population genetics studies has been well documented in prior research (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, parameters of genetic diversity for each microsatellite locus were calculated via FSTAT v2.9.3 (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). ADZE software was employed to estimate the population genetic diversity, including allelic richness (Ar) and private allele (Ap), using standardized sample sizes of ADZE\u0026thinsp;=\u0026thinsp;2 (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e). The number of different alleles (Na), number of effective alleles (Ne), expected heterozygosity (He) and observed heterozygosity (Ho), within-population inbreeding coefficient FIS and Hardy\u0026ndash;Weinberg equilibrium were calculated with GENEPOP 4.2 (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e). The inverse distance weighted (IDW) interpolation function implemented in the Geographic Information System (GIS) software ArcGIS 9.3 (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e) was used to estimate the geographic patterns of H\u003csub\u003eO\u003c/sub\u003e and H\u003csub\u003eE\u003c/sub\u003e for all 20 populations of \u003cem\u003ethe\u003c/em\u003e Caucasus apple. We explored the relationships among clusters via principal component analysis (PCA) with the \u003cem\u003eadegenet\u003c/em\u003e package in the R environment (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e). Analysis of molecular variance (AMOVA) was performed via GENALEX v6.5 (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e) to infer the genetic differentiation between populations from Hyrcanian forest and Zagros forest. Barrier analysis, which is based on the (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e) maximum difference algorithm, was conducted to define significant genetic breaks and spatial differences between populations. Genetic barriers were assessed via BARRIER 2.2 (\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eGenetic Structure and Gene Flow Analysis\u003c/h3\u003e\n\u003cp\u003eWe assessed the genetic structure of the Caucasus apple via STRUCTURE v2.3.4 software (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e). This tool was used to examine range-wide genetic clustering among populations of Caucasian apple. Ten independent runs were performed for each K from 2\u0026ndash;10 with 200,000 burn-in steps followed by 500,000 Markov chain Monte Carlo (MCMC) steps. The STRUCTURE output was analyzed and visualized by using StructureSelector (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e). To infer historical gene flow (Nm) patterns, MIGRATE-N v3.6 (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e) was used to estimate the effective population sizes (θ) and mutation-scaled immigration (M) among the groups identified by STRUCTURE. Additionally, we used the Bayesian method implemented in the software BayesAss (BA3) to estimate current gene flow between populations (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e). Estimates via this method are based on migration over the last few generations. We selected the best run for allele frequencies and posterior probability densities of inbreeding coefficients with different random seeds among three independent runs. The Bayesian deviance was calculated to determine the best run via the script of (\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e), which was applied in R (\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e). To obtain a correct acceptance rate, the values of the parameters ranged between 20% and 40% (\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e). The inbreeding coefficient was set at 0.35, and the migration rate was set at 0.75 for the final analysis. For allele frequencies, the default setting of mixing parameters was used. The analysis was conducted with 10\u003csup\u003e6\u003c/sup\u003e burn-ins and 10\u003csup\u003e7\u003c/sup\u003e MCMC iterations; every 1,000th iteration was sampled. The results were visualized via QGIS 2.18 'Las Palmas' (\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e). CIRCUITSCAPE software (McRae \u0026amp; Beier, 2007) was used to evaluate the influence of topographic complexity as a resistance factor affecting gene flow among populations. This software models populations as nodes connected by resistors, based on the principles of electrical circuit theory and the connectivity-resistance approach. While altitude alone may not fully capture topographic complexity, we used a digital elevation model (DEM) to derive a resistance surface for the habitats of \u003cem\u003eMalus orientalis\u003c/em\u003e in Iran. To statistically validate this resistance model, we compared the CIRCUITSCAPE-derived resistance distances with a null model of isolation-by-distance (IBD) using Mantel tests (\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eUnit of conservation selection\u003c/h3\u003e\n\u003cp\u003eTo determine the conservation priority of the Caucasus apple, we used reserve selection analysis via DIVA-GIS software (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ewww.diva-gis.org\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.diva-gis.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). The reserve selection analysis algorithm, which is based on allele frequency in each population, determines the minimum number of geographic units necessary to maintain genetic diversity (\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e). The populations with the highest allelic richness are given first priority for conservation, and the priorities of other populations are selected on the basis of the first population. After the first priority of populations for conservation, new alleles were found in the next priority populations that were not observable in the previous populations (\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eMorphological parameters studied\u003c/h3\u003e\n\u003cp\u003eEight quantitative morphological traits were used in this study (leaf area, leaf angle with petiole, leaf length, petiole length, maximum leaf length, maximum leaf width at 0.1 of leaf length, maximum leaf width at 0.9 of leaf length, and stomatal morphology) (\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e). Statistical parameters and AMOVA were computed via SPSS software (\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e), and PCA for morphology was performed via the Clustvis website (\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e).\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eGenetic diversity of Caucasian Apple populations\u003c/h2\u003e \u003cp\u003eThis study identified 26 loci with 455 alleles for wild apples. All the loci were polymorphic for the population of Caucasian apples. The range of alleles per locus ranged from 7 (CH02c03b) to 29 (CH04e03), and the average number of alleles was 17.5 (Table S1). The average number of alleles per population was 5.22, with values ranging from 2.07 (Droud region) to 7.5 (Vatna region). In this study, the mean gene flow (Nm) was calculated to be 0.6 (Table S2).\u003c/p\u003e \u003cp\u003eThe Vaz and Sangdeh regions of the Hyrcanian forest have the highest H\u003csub\u003eO\u003c/sub\u003e and H\u003csub\u003eE\u003c/sub\u003e values, whereas the Nozhyan and Droud regions of the Zagros forest have the lowest H\u003csub\u003eO\u003c/sub\u003e and H\u003csub\u003eE\u003c/sub\u003e values. Overall, the observed heterozygosity in the Zagros forest populations was lower than that in the Hyrcanian forest populations (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The FIS parameter for Caucasian wild apple in Iran was estimated to be in the range of -0.83\u0026ndash;0.11, but the mean for populations from Zagros forest was negative (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Overall, Caucasian apple populations in the Hyrcanian forest showed greater mean allelic richness compared to those in the Zagros forest. However, the mean number of private alleles was higher in Zagros forest populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e) Estimated genetic diversity parameters for \u003cem\u003eM. orientalis\u003c/em\u003e in Iran on the basis of genotyping samples\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"13\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePop\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eProvince\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLatitude (X)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLongitude (Y)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eN\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eNa\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNe\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003eI\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eH\u003csub\u003eE\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eH\u003csub\u003eO\u003c/sub\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c12\"\u003e \u003cp\u003ePPL (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c13\"\u003e \u003cp\u003eFIS\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"13\" rowspan=\"14\"\u003e \u003cp\u003eHyrcanian Forest\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAsalem\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGilan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e37.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e48.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.07\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbesk\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMazandaran\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e52.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSangdeh Sari\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMazandaran\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e53.28\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGanasara\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMazandaran\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e52.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e7.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eShit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMazandaran\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e53.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTilak\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMazandaran\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e53.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6.92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-0.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVaz\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMazandaran\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e52.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5.19\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-0.83\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGorgan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGorgan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e54.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-0.81\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eVatna\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGorgan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e53.99\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e7.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSheshab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGorgan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e55.13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.27\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eArsam\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGorgan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36.67\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e54.66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTokestan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGorgan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e54.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.43\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDorak\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGorgan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e54.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5.96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.54\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.081\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSiamarzkoh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGorgan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e55.1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e6.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.55\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5.82\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e4.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-0.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003eZagros Forest\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMarivan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKordestan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e35.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e46.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4.84\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.38\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSaghez\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKordestan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e46.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.63\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.79\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-0.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBuflo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eKordestan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e36.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e46.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-0.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSepedkoh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLorestan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e33.95\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e48.49\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e4.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.34\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.29\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eNozian\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLorestan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e33.23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e48.58\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e2.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e96\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e0.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDroud\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLorestan\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e33.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e33.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e2.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e1.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.83\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAverage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e3.81\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e97.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-0.04\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e167\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e5.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c8\"\u003e \u003cp\u003e3.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c9\"\u003e \u003cp\u003e1.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c10\"\u003e \u003cp\u003e0.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c11\"\u003e \u003cp\u003e0.69\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c12\"\u003e \u003cp\u003e99.2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c13\"\u003e \u003cp\u003e-0.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"13\"\u003eN\u0026thinsp;=\u0026thinsp;numner of samples, Na\u0026thinsp;=\u0026thinsp;Number of Alleles, Ne\u0026thinsp;=\u0026thinsp;Effective Number of Alleles, I\u0026thinsp;=\u0026thinsp;Shannon's Information Index, H\u003csub\u003ee\u003c/sub\u003e= Expected Heterozygosity, H\u003csub\u003eo\u003c/sub\u003e= Observed Heterozygosity, PPL%= Polymorphic Loci Percentage, FIS\u0026thinsp;=\u0026thinsp;Inbreeding Coefficient\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAccording to the pairwise FST analysis, most apple populations from the Hyrcanian and Zagros forests showed very low genetic differentiation, with FST values ranging from 0.005 to 0.30 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, Table S2). Statistical significance of pairwise FST values was tested using 10,000 permutations in Arlequin. Although the FST between Mrivan and Vatna was very low genetic differentiation (FST\u0026thinsp;=\u0026thinsp;0.005). Moderate levels of genetic differentiation was observed between some western and central populations of the Hyrcanian forest, with FST values above 0.25 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001), indicating moderate genetic structure among these regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eb, Table S2).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOn the basis of our findings from the AMOVA, the majority of variance (80%) was discovered within samples, with only 4% and 8% of variance found within groups and populations within groups, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eThe results of the AMOVA test in two main regions of Caucus apples in this study\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSource of changes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDegrees of freedom\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSum of squares\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eComponents of variance\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003ePercentage of Variance\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBetween Hyrcanian forest and Zagros forest\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e74.71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBetween populations from Hyrcanian forest and Zagros forest\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e454059\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmong samples\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e147\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1573.85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ewithin samples\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e167\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1493\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.94\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e333\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3596.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003ePopulation structure and genetic differentiation\u003c/h3\u003e\n\u003cp\u003eThe STRUCTURE results revealed that the Caucasus apple samples exhibited a high level of heterozygosity and indicated five genetically distinct clusters corresponding to five main geographic regions, with the clearest separation observed at K\u0026thinsp;=\u0026thinsp;5. This K value showed the best geographical pattern for the apple populations, and five distinct clusters were identified, each associated with a main geographic region, with no new groups appearing in the STRUCTURE analysis after K\u0026thinsp;=\u0026thinsp;5 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). At K\u0026thinsp;=\u0026thinsp;3, populations from Nozhyan, Droud, and Sepedkoh (purple group) were separated from all others, while apple populations from Marivan, Saghez, and Buflo clustered with the Hyrcanian populations (blue group). At K\u0026thinsp;=\u0026thinsp;4, the Zagros populations split into two subgroups (red and purple), and most Hyrcanian populations remained in the blue group, except those from Asalem, Abesk, and Toskestan. K\u0026thinsp;=\u0026thinsp;3 was supported as the optimal number of clusters based on the ΔK method (Fig. S4), although K\u0026thinsp;=\u0026thinsp;5 showed the clearest geographic pattern. STRUCTURE bar plots for K\u0026thinsp;=\u0026thinsp;3 to K\u0026thinsp;=\u0026thinsp;6 are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ea.\u003c/p\u003e \u003cp\u003eWe used Monmonier's maximum difference algorithm to find potential genetic barriers between populations of Caucasian apples to complement our analysis and detect potential genetic discontinuity. Among Caucasians, there are two statistically significant genetic barriers (bootstrap support 90%). The first is in the forest of Hyrcanin, and the second is in the forest of Zagros. This caused apple populations from the Sheshab, Siamarzkoh, and Arsam regions in the Hyrcanina forest to be separated from other apple populations and caused apple populations from the Nozhyan region in the Zagros forest to be separated from other apple populations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePCA\u003c/h2\u003e \u003cp\u003eResults of principal component analysis (PCA) for all populations in this study illustrated that PC2 and PC3 both explain 5.71 percent and 4.37 percent of the variance, respectively. The populations of the Zagros forest were divided into two groups via PCA; however, populations from northern Zagros overlapped with those from the central part of the Hyrcanian forest. The populations from the west of the Hyrcanian forest have mixed with the populations from the central area of the forest the most (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eGene flow between the habitats of\u003c/b\u003e \u003cb\u003eM. orientalis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo estimate gene flow, stands of the Caucasus apple were classified by geographical region. Our findings illustrate some gene flow between provinces. Gorgan populations were the predominant source of exogenous allelic variants, and strong gene flow was observed from Gorgan into Mazandaran, Gilan, and Kordestan populations (Ne respectively from Golestan into Mazandaran, Gilan and Kordestan provinces: 0.172, 0.185, 0.47). We observed minor gene flow from Gilan's population into Lorestan's population (Ne\u0026thinsp;=\u0026thinsp;0.05) as well as minor gene flow from Kordestan's population into Mazandaran's population (Ne\u0026thinsp;=\u0026thinsp;0.01). Little or no gene flow between Zagros region populations (Lorestan Province and Kordestan Province (Ne\u0026thinsp;=\u0026thinsp;0.01) was detected, suggesting that \u003cem\u003eM. orientalis\u003c/em\u003e in Zagros are isolated from each other (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). This result is confirmed by CIRCUITSCAPE analysis, which reveals the existence of a strong topographic barrier between populations from the Zagros Mountains. In contrast, for Hyrcanian forest populations, some admixture in populations was apparent. Lowlands near the Caspian Sea allow undisturbed gene flow between Hyrcanian populations. Interestingly, the connection between Zagros and Gorgan is better than that between Zagros and the western part of the Hyrcanian region, which is confirmed both by MIGRATE-N and CIRCUITSCAPE (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eIdentification conservation units of Caucasus apple\u003c/h2\u003e \u003cp\u003eThe minimum number of geographic units required to preserve the genetic diversity of the Caucasus apple in the two main regions\u0026mdash;the Hyrcanian forest and the Zagros forest was analyzed. Our findings indicate that in the Hyrcanian forest, the Toskestan, Siamarzkoh, Abesk, and Asalem regions are designated as the first priority for conservation, while the Sangdehsari and Vaz regions are the second priority.In the Zagros forest, the Sepedkoh region is the first priority for conservation, whereas the Bufloo, Sgez, and Nozhyan regions are the second priority. Additional areas, marked in orange and green (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e1\u003c/span\u003e), have lower conservation priority.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMorphology of Caucasus apples\u003c/h2\u003e \u003cp\u003eA bar chart of the seven characteristics of the leaves of Iranian wild apple is shown in Figure S1. The leaf angle parameter widely used to describe the morphology of plants (\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e) was the most variable parameter in this study. The maximum variance of the analyzed statistics was observed for the population of Siamarzkoh for leaf length and leaf area. The measurement of the maximum leaf width at 0.1 of the leaf length and the maximum leaf width at 0.9 of the leaf length revealed that the Vatna region has the maximum variance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of variance (ANOVA) and PCA of morphological traits\u003c/h2\u003e \u003cp\u003eThe analysis of variance of the length of the petiole revealed significant differences among the Buffalo region and other regions in this study. On the basis of leaf width, population of Buflo has been signed with all regions except thepopulation of SangdehSari. The population of Tuskestan and population of Sheshab region from Hyrcanian forest and the Grin region from Zagros forest have been clustered with other regions on the basis of the maximum length of the leaf. Analysis of variance of the maximum leaf width at 0.1 and maximum leaf width at 0.9 length revealed significant differences among the population of Sheshab, population of Tuskestan, and population of Vatna regions from Hyrcanian forest and other regions. The leaf areas of the Grin region and Vatna region were significantly different from those of the other Zagros forest regions (Fig. S2). Fig. S3B summarizes the principal component analysis results of the morphological data for the wild apple plants. We used seven principal components that explained the variation between Hyrcanian forest and Zagros forest. These principal components explained approximately 14.2% and 67.6% of the variation (PC1 and PC2), respectively, and, on the basis of the studied morphological traits, we did not observe any pattern of differences between the Hyrcanian forest and Zagros forest (Fig. S3A).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eSeveral analyses of the spatial genetic structure of the Caucasus apple were previously performed and described in the literature (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e, \u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e). However, the present study is the first large-scale genetic analysis of stands of this species from Iran, across the natural range from the Hyrcanian forest to the Zagros, using a large set of SSRs (26 SSR markers). Our findings confirmed that the gene pool of the Caucasus apple moved from East Asia to Europe across the Trade Silk Road.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGenetic diversity within populations of\u003c/b\u003e \u003cb\u003eM. orientalis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAccording to our results, many populations of \u003cem\u003eM. orientalis\u003c/em\u003e in the Hyrcanian forest (Asalem and Gorgan regions), as well as in the Zagros forest (Droud and Nozhyan regions), are genetically unique. High genetic diversity among Iranian populations across the natural range of species in the Zagros and Hyrcanian forests is confirmed by high dissimilarity between populations and limited dispersal between them (\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e). Previous research has suggested genetic similarities between stands in Iran and Central Asia. Using molecular markers, proven that discovered that Iranian wild apples in Hyrcanian forests are closely related to apple populations in Central Asia. These results could indicate that apples could migrate across the northern part of the Silk Road (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn conservation genetics, allelic richness is the most important parameter, and it can be used to forecast historical bottleneck populations (\u003cspan additionalcitationids=\"CR79\" citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e). The average allelic richness value in Iran (6.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04) is lower than that in Europe and China (\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e). A decrease in allelic richness resulted in a reduction in the effective population size, increasing the vulnerability of these populations to various unfavorable conditions (\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e). Another important parameter is expected heterozygosity, which is a good indicator of genetic diversity (\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e). The mean H\u003csub\u003eE\u003c/sub\u003e for Caucasus apple in Iran was 0.65, which was lower than the H\u003csub\u003eE\u003c/sub\u003e for \u003cem\u003eM. orientalis\u003c/em\u003e in Europe and China (0.80), indicating that population growth in Iran is not uniform (\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e). However, it is easy to explain why the Caucasus apple is found in two main regions in Iran: the Hyrcanian forest and the Zagros forest, both of which have different climatic conditions and vegetation, which can lead to nonuniform growth (\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e). The FIS parameter predicts future generations' genetic diversity, and a negative value indicates that regeneration could fail (\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e). The lower value of this parameter for Iranian wild apple (-0.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02) than for European wild apple (0.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06) suggests that the regeneration of Iranian populations can be disrupted. The gene pool of various \u003cem\u003eMalus\u003c/em\u003e species has been strongly influenced by human impact since ancient times because apple fruits were already important crops in the era of the Sumerians (\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e). Thus, it is difficult to recognize whether natural populations of \u003cem\u003eM. orientalis\u003c/em\u003e were shaped more by natural processes or by long-term human influence.\u003c/p\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eGene flow\u003c/h2\u003e \u003cp\u003eGene flow and migration have a significant impact on adaptation and may lead to population homogeneity (\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e). In the prevuous study about spatila genetic structure of \u003cem\u003eM. orientalis\u003c/em\u003e concluded that the geographical location of a population has no effect on the genetic structure of wild apples (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), but we found a strong link between genetic structure and geographic region in this study. The results of the gene flow analysis revealed strong migration from the eastern part of the Hyrcanian forest to populations in the western part of this region and, with lower intensity, to the northern part of the Zagros forest. According to (\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e), Iran has been one of the main regions through which various taxa from the \u003cem\u003eMalus\u003c/em\u003e genus moved from Central Asia to Europe across the Trade Silk Road. Our results support this theory and suggest that Iran served as a link between East Asia and Europe in the transportation of genetic variability in the Caucasus apple (\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eThe topography of the central part of Iran creates an enormous barrier for gene flow between the Hyrcanian and Zagros populations. Additionally, the northern and southern parts of the Zagros range are poorly connected; the Migrate-n results show that admixture in Lorestan is very low. The situation on the coast of the Caspian Sea is completely different gene flow along coastal lowlands is theoretically undisturbed, which allows for the exchange of genetic variability between Hyrcanian stands. Thus, northern populations are genetically similar, as indicated by the STRUCTURE and PCA results. The Caucasus apple is one of the minor ancestors of the European Caucasus apple (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Long periods of cultivation and crossing between various taxa of \u003cem\u003eMalus\u003c/em\u003e could have affected natural populations of ancestral species as well; however, it is difficult to quantify this impact.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSpatial genetic structure of\u003c/b\u003e \u003cb\u003eM. orientalis\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGene affinity and exchange between populations can be determined by analyzing the spatial genetic structure of populations (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). The whole Iranian range of the species was divided into three clusters via STRUCTURE analysis, which differs from the results of previous studies (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e). One of these variations may be the response of Iranian wild apples in the Hyrcanian and Zagros forests to population size and dispersal (\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e). In contrast to previous studies, our findings reveal a strong spatial genetic structure between wild apple populations in the Hyrcanian and Zagros forests (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e). We hypothesize that the differences between Iranian wild apples are due to two gene pools with similar geographic origins. Interestingly, the population from Gilan is similar to Zagros stands; this particular population is quite far from other Hyrcanian stands; however, the topography of Iran hinders the natural flow of genes from Gilan to the Kordistan region. It is possible that the similarity of the gene pool in these two regions is connected with human impact. Stands from Lorestan differ strongly from both Hyrcanian and northern Zagros populations. The isolation of this area is supported by the results of migration analysis, as well as CIRCUITSCAPE and PCA. The distinctiveness of this area, which is difficult to assess due to topographic barriers, may indicate that a unique gene pool exists, which should be protected with high priority.\u003c/p\u003e \u003cp\u003eAlthough STRUCTURE analysis revealed broad-scale clustering patterns, Monmonier\u0026rsquo;s algorithm identified localized genetic barriers in the Hyrcanian and Zagros forests. This difference is not unexpected, as STRUCTURE detects genome-wide patterns assuming Hardy Weinberg and linkage equilibrium, while Monmonier\u0026rsquo;s method identifies sharp spatial genetic discontinuities regardless of such assumptions (Manel et al., 2003; Chen et al., 2007; Prates et al., 2016). The genetic barrier around Nozhyan aligns with a distinct STRUCTURE cluster at K\u0026thinsp;=\u0026thinsp;3, supporting its genetic uniqueness. Other barriers, particularly in the Hyrcanian forest, may represent incipient or incomplete divergence, gene flow reduction, or adaptation to local environments not yet strong enough to form fully distinct STRUCTURE clusters (Storfer et al., 2007; Blair et al., 2012). These results reflect the complementary nature of barrier and clustering analyses in revealing both historical genetic structure and ongoing differentiation processes (Sexton et al., 2014).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eConservation of Caucasian apple\u003c/h2\u003e \u003cp\u003eCaucasian apple is one of the main ancestors of cultivated apples in the Caucasian region (Bina et al., 2022). These wild apple species play a crucial role in sustaining ecosystems, since they are an important source of food for wildlife. Additionally, they are valuable sources for apple breeding, helping to develop features like stress resistance against infections and dehydration. Therefore, it is crucial to protect this species' germplasm from serious threats like the negative effects of habitat fragmentation and futher challenges (\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFuture projects such as regeneration and breeding are needed to conserve the genetic diversity of wild plants (\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e). Asia is the main center of genetic diversity in the genus \u003cem\u003eMalus\u003c/em\u003e, implying that wild apple habitats in Asia have a large gene pool that needs to be preserved for future generations (\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e). However, no studies on the genetic diversity of this important species have been reported, and our study is the first in this field. To define a conservation unit for the Caucasian apple gene pool, we used reserve selection analysis. Our findings revealed three high-priority and medium-priority populations for conservation in two main regions. On a local scale, this illustrates the Caucasus region's lack of consideration of \u003cem\u003eM. orientalis\u003c/em\u003e genetic conservation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn this study, we discovered two high-priority regions in the Hyrcanin forest for preserving \u003cem\u003eM. orientalis\u003c/em\u003e genetic diversity, indicating that \u003cem\u003eM. orientalis\u003c/em\u003e management in the Hyrcanin forest would need additional attention. In the Zagros and Hyrcanin forests, we discovered ageographic barrier between the population with the highest priority and other populations. As a result, corridors between populations are suggested in this situation (\u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e). Finally, on the basis of these results, we can conclude that Iran is one of the key sources of the \u003cem\u003eM. orientalis\u003c/em\u003e gene pool that would be useful for cultivating apple breeding programs, but it does not have good habitat conditions and would require performance conservation programs.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate:\u0026nbsp;\u003c/strong\u003eNot applicable\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eAll authors have read and approved the final manuscript and consent to its publication in the journal.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u0026nbsp;\u003c/strong\u003eThe data supporting the findings of this study are available at Zenodo under the DOI: 10.5281/zenodo.6981530.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis work is based upon research funded by the Iran National Science Foundation (INSF) under project No. 4034387\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions:\u0026nbsp;\u003c/strong\u003eH.B., F.Gh. and Sh.Z. conceived and designed the experiments; F.Gh. and Sh.Z. \u0026nbsp;obtained funding, H.B., F.Gh. and Sh.Z. analysed the data; H.B. wrote the original draft and preparation of the figures; H.B., F.Gh. and Sh.Z. gave critical inputs in final draft \u0026nbsp;and revisions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u0026nbsp;\u003c/strong\u003eThis work is based upon research funded by the Iran National Science Foundation (INSF) under project No. 4034387\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePushpangadan P, Nair K, Ahmad M. 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Effects of forest fragmentation on vertebrates in Douglas-fir forests. 1986.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-ecology-and-evolution","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"evob","sideBox":"Learn more about [BMC Ecology and Evolution](http://bmcevolbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/evob/default.aspx","title":"BMC Ecology and Evolution","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Microsatellite marker, Conservation unit, Genetic diversity, M. orientalis","lastPublishedDoi":"10.21203/rs.3.rs-6546353/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6546353/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn Eurasia, \u003cem\u003eM. orientalis\u003c/em\u003e Uglitzk. (Caucases apple) is a tree with important ecological and economic (fruit) benefits. We measured eight quantitative morphological traits, none of which showed significant differences among the investigated Caucasian apple populations. In this research, we used 26 microsatellite (SSR) markers to investigate genetic diversity and define unit conservation in the Caucases apple. The mean values of genetic diversity,allelic richness (Ar), private allele (Ap), expected heterozygosity (H\u003csub\u003eE\u003c/sub\u003e) and observed heterozygosity (H\u003csub\u003eO\u003c/sub\u003e) were 1.74, 0.21, 0.65 and 0.76, respectively. In the regions studied, three major genetic clusters and two significant genetic barriers were discovered. Our gene flow findings revealed that there is little connection between \u003cem\u003eM. orientalis\u003c/em\u003e population in the Caucasus indicating habitat fragmentation. Two regions in the Hyrcanian forest and one in the Zagros forest were identified as having the highest priority for conserving the genetic diversity of the Caucases apple.\u003c/p\u003e","manuscriptTitle":"Genetic diversity and spatial genetic structure of Caucasian apple (Malus orientalis Uglitzk.) populations based on microsatellite markers for conservation strategy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 01:41:23","doi":"10.21203/rs.3.rs-6546353/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-06-23T06:43:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-12T05:45:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-03T12:41:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"332175400952255451580983591481881327272","date":"2025-06-03T08:41:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"286429742626277417525589208687392313322","date":"2025-05-23T09:18:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"48805857874335234491767218939853081459","date":"2025-05-19T06:07:33+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"224613891498184998273650621682481512922","date":"2025-05-06T09:51:06+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-06T09:34:48+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-06T09:27:59+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-05-02T04:14:54+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-05-01T12:42:46+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Ecology and Evolution","date":"2025-05-01T12:41:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-ecology-and-evolution","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"evob","sideBox":"Learn more about [BMC Ecology and Evolution](http://bmcevolbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/evob/default.aspx","title":"BMC Ecology and Evolution","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f15d503f-0af0-481e-8b46-116dce96179d","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-11-17T16:05:05+00:00","versionOfRecord":{"articleIdentity":"rs-6546353","link":"https://doi.org/10.1186/s12862-025-02474-9","journal":{"identity":"bmc-ecology-and-evolution","isVorOnly":false,"title":"BMC Ecology and Evolution"},"publishedOn":"2025-11-10 15:57:46","publishedOnDateReadable":"November 10th, 2025"},"versionCreatedAt":"2025-05-13 01:41:23","video":"","vorDoi":"10.1186/s12862-025-02474-9","vorDoiUrl":"https://doi.org/10.1186/s12862-025-02474-9","workflowStages":[]},"version":"v1","identity":"rs-6546353","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6546353","identity":"rs-6546353","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}