Nothofagus pumilio is one of their most ecologically important tree species . It presents great adaptability and a wide distribution range, making it a suitable model for predicting the performance of trees facing global climate change. N. pumilio forests are increasingly threatened by extreme climatic events and anthropogenic activities. This study aims to identify priority conservation areas and Genetic Zones (GZs) for N. pumilio, promoting the implementation of specific practices to ensure its management and long-term preservation. Thirty-five populations (965 trees) sampled across its distribution  were genotyped with SSRs, and geographical patterns of genetic variation were identified using Bayesian approaches. The phylogeographic patterns of the species and geomorphological history of the region were also considered. Six priority conservation areas were identified, which hold high allelic richness and/or exclusive allelic variants. Eighteen GZs were delineated based on the genetic structure of this species, and maps showing their distributional range were drawn up. Overall, this study defines management units based on genetic data for N. pumilio for the first time, which will facilitate the establishment of sustainable practices and highlight priorities for investment of conservation funding.Patagonian forests are the southernmost temperate forests in the world, and  The biological value of these forests lies partly in their high level of endemism and frequency of taxonomically isolated genera, despite low species richness in comparison with other South American forests1. Together with some conifer species, southern beeches of the Nothofagus genus constitute the main components of Andean Forests, which are recognized as providing a wide range of ecological, economic, and social benefits and services. Therefore, the management of these native forests is required in order to maintain the ecological dynamics of this unique ecosystem. Several studies have documented the role of climatic variability during the Quaternary in shaping the current distribution of Patagonian forests4. In addition, extreme climatic events have recently been suggested as one of the causal agents of current forest decline in Northern Patagonia5, e.g. severe droughts occurring after a wet period triggered N. pumilio decline6. In this regard, an increase in mean maximum summer temperature, overall warming, and a marked decrease in annual precipitation were detected in this region throughout the twentieth century8, threatening the persistence of these forests. Besides, anthropogenic and natural causes such as logging and more frequent fires10 also endanger these forests.Patagonian forests represent the most southern temperate forests in the world, and were isolated from other continental forests approximately 40–2.5 million years before presentNothofagus pumilio, commonly called lenga, is one of the most important species of Patagonian forests, since it has the largest distribution area of all native trees in the region, covering about 1.636 million hectares and occupying a range of about 20° latitude (from 36°S to 55°S) in Argentina. This species has a similar distributional range to the west of the Andes mountain range, in Chile11. Moreover, it occurs in heterogeneous environments along altitudinal, latitudinal, and longitudinal gradients that imply diverse and contrasting thermal, photoperiodic, and precipitation conditions, respectively. N. pumilio shows great adaptation or plasticity to extreme and contrasting environments, since it grows close to the timberline (cold-tolerant species) and thrives in a wide range of precipitation levels 12. In addition, several phenotypic variations have been described for this species across environmental clines; e.g., bud phenology variation and seedling growth associated with altitude14, and wood density related to soil moisture gradients15. Since N. pumilio exhibits great adaptability, it serves as a suitable model for prediction of the performance and fitness of trees to cope with global climate change in the southern hemisphere. Consequently, a research is currently being undertaken to determine whether local adaptation of this Southern beech evolved in a convergent way with respect to European tree species that are also distributed along broad environmental gradients . Natural hybridization between N. pumilio and N. antarctica has been observed in sympatric populations17 and it has been suggested that the latter acts predominantly as pollen receptor18. The introgression of the chloroplast genome from N. pumilio to N. antarctica would have occurred as a consequence of repeated backcrosses between hybrids and parents, and the former is clearly distinguished at the nuclear level19.Austrocedrus chilensis21, Nothofagus nervosa and Nothofagus obliqua23. According to the National Law N° 26331 on Minimum Requirements for Environmental Protection of Native Forests, most of the Argentinean N. pumilio forests (up to 97%) were included in areas of high (38%) or moderate (59.1%) conservation value24. Therefore, forest management is highly encouraged for this species, particularly the identification of priority conservation areas, in order to ensure compliance with this law and their long-term preservation. The identification of Genetic Zones (GZs) is one alternative of OGMU delineation. GZs are genetically homogeneous regions within which seeds can be transferred with low risk of causing change in the original genetic structure26. A requirement for the identification of GZs is the characterization of the population structure of a species, resulting from genetic signatures of evolutionary history, which can be assessed by neutral molecular markers. A second, well-known alternative for OGMU delineation is the definition of Provenance Regions (PRs), which aims to ensure the safe transference of propagation material without risk of maladaptation since it is based on variation in adaptive characters27. In addition, assessment of species’ genetic diversity with molecular markers could facilitate the identification of priority conservation areas29. Several authors have suggested that areas containing high allelic richness and/or exclusive allelic variants are meaningful for conservation purposes32, particularly because they play a key role as genetic diversity reservoirs or sources of locally-adapted genotypes.Government directives regulating the criteria and indicators for defining management units, such as Conservation Units (CU), became important worldwide during the last decades of the twentieth century. Appropriate management of native resources depends on understanding the nature and distribution of genetic diversity among and within species, and highlighting how this genetic variation is structured across their distribution range. Since most native forests are widely distributed and composed of hundreds of natural populations, management of their genetic resources cannot be based on single-population units. Thus, the establishment of operational genetic management units (OGMUs) is necessary for implementation of specific practices arising from conservation or restoration programs. In Argentina, OGMUs were delineated in three native species based on genetic criteria: N. pumilio forests. Genetically homogeneous regions  were first delineated by assessing the neutral variation of the target species across its natural distribution range in Argentina. Based on this, geographical patterns of N. pumilio genetic variation were recognized. In addition, areas holding high allelic richness and/or exclusive allelic variants were identified. Previous information from N. pumilio phylogeographic patterns33 was considered and new information from SSR markers was added.The aim of this work is to identify priority conservation areas in 34, due to its usefulness in determining the persistence and adaptive potential of a species. Therefore, indicators of genetic diversity should be considered when developing criteria and guidelines for forest management37. A set of genetic parameters was used here in order to characterize the N. pumilio populations . In particular, HE Nei for Tr population (0.757) was significantly higher than the HE Nei obtained for PG (0.581), Pi (0.573), BL (0.537), and HP (0.398) populations. In addition, both BL and HP exhibited the lowest heterozygosity values, suggesting that they hold lower genetic diversity than the other populations analyzed.Significant differences in unbiased heterozygosity were detected among populations using Wilcoxon’s test . Since this population is isolated from the main forest masses (western side of the species’ distribution) and located in an area that was covered by an ice field at the Last Glacial Maximum (LGM), we hypothesize that few individuals contributed to the recolonization process. In addition, a population bottleneck was identified in Sierra Colorada (SC), since it showed significant heterozygosis excess under both Infinite Allele Model -IAM and Two Phase Model -TPM mutation models .Inbreeding and bottlenecks are demographic events that shape population genetic variation, specifically, reducing allelic variants. Signs of inbreeding were detected in 12 populations  method . The indexes did not differ substantially, indicating that null alleles did not strongly bias the FST value. At the same time, the standardized Hedrick’s G′STH  was 0.201. These differentiation coefficients (FST and GST) were similar to those previously reported in other Nothofagus species such as N. nervosa (0.061) and N. obliqua (0.049)22.The differentiation coefficient for N. pumilio populations19. Based on these results, an estimation of population structure was carried out, implementing spatial clustering methods as recommended by Perez et al.38. In contrast, the genetic and geographic distances do correlate with each other when using maternally inherited cpDNA markers33. In this regard, it was suggested that the pollen dispersal capacity of N. pumilio greatly exceeds that of seed dispersal (75–100 times greater)39, as in other wind-pollinated trees41, which could partly explain the differences found between cpDNA and SSR results from IBD analyses.An isolation-by-distance (IBD) pattern was not supported by the Mantel test and the Spatial Genetic Structure (SGS) test  and Huemules (42° 50′S) in N. pumilio forests, since all individuals analyzed from these two sites exhibited either northern or southern haplotypes, respectively. The evolutionary significance of this lineage segregation prompted us to consider these findings as the first division criterion for exploration of the genetic structure of Argentinean N. pumilio forests located to the north and south of these boundaries  was reported as a putative relictual zone33 since it exhibits high genetic diversity in plants  and animals .From the genetic structure analysis (BAPS) of the ‘north’ subset, four main clusters were identified which grouped the populations with an adjustment probability of 0.941  were analyzed separately , other division criteria were also considered in defining the optimum number of clusters. A particular topographic configuration occurs at 44°S where there are changes in the orientation of the watersheds (north–south oriented mountain chains between 42°S and 44°S shift afterward to a west–east orientation). This feature contributed to different ice mass development during the glacial periodely Fig.  IIa. In 49. In addition, palynological records suggest a different paleoclimatic history for the area south of 50°S, which shaped the forest expansion in a different way  in southern Patagonia and Tierra del Fuego during the postglacial period3. Furthermore, a particular recolonization process was suggested for Tierra del Fuego Island, mainly led by trees from local periglacial refugia50. Currently, the N. pumilio Argentinean forest south of 44°S is highly fragmented along a narrow range on the Andes hillsides. Nonetheless, an exception to this configuration occurs on Tierra del Fuego Island, where N. pumilio forest probably retained or recovered its continuity. Moreover, this forest mass is present at low altitudes (less than 600 m a. s. l.) and is highly influenced by seasonal storm track patterns. In this study the five populations located in Tierra del Fuego were analyzed separately   was found only in GZ14 , and GZ18 BL   Fig. . N. pumiPa Table . AdditioN. pumilio was confirmed by assessing the hierarchical analysis of variance (AMOVA), where the proportion of variance due to both the ‘among- and within-’ group components were used as elements of comparison. Our proposal of eighteen GZs had a greater among-group than within-group component of variance, which is evidence of correct grouping (Table ’ GZs (Table The optimum number of genetic management units (in this case GZs) in ng Table . The phyZs Table  are consN. pumilio was assessed here by neutral molecular markers (SSRs) and was taken into account when delineating N. pumilio GZs in Argentina. This approach has been successfully used to define management units for several species , since microsatellite diversity was correlated with genomic diversity at population level, although not at individual level53. In addition, considering that microsatellite diversity may not reflect adaptive diversity54, when defining Conservation Units (CUs) and Provenance Regions (PRs) it would be appropriate to determine the genetic variation data based on adaptive traits55. Even though Chilean N. pumilio populations were not sampled, the current status of these forests could reflect similar variation to the Argentinean side, since they are probably subject to comparable threats. However, it may be difficult to achieve common management criteria on both sides of the mountain range (Andes Cordillera), since each country has specific regulations.The delineated GZs could facilitate the implementation of specific practices related to conservation programs by determining priority areas based on the genetic diversity of each OGMU. The genetic structure of AR) is commonly chosen as a genetic criterion in order to define priority areas for conservation32. This parameter is independent of sample size56 and suitable for determining historical processes such as bottleneck or admixture56, and inferring putative relictual zones31. Accordingly, GZ2, GZ3, and GZ6 require special attention due to their high genetic diversity , which could be evidence of genotypes adapted to specific environments60. Furthermore, a high LCA value could be indicative of genetic isolation30 and thus help to identify the putative location of species–specific refugia62. Based on the high LCA and/or Pa detected in this study , particular consideration should be given to this area, which is included within GZ9. This demographic event leads to a reduction in genetic diversity, which could threaten the adaptive potential of this population and, in an extreme case, lead to local extinction63. For this reason, strict in situ conservation management is highly recommended for this area, in order to prevent the loss of these genetic variants.In addition, since a relatively recent demographic bottleneck was inferred in the Finally, GZ7 and GZ13 displayed specific genetic composition, based on their genetic structure and admixture patterns. In particular, these zones combine an almost balanced mixture of two surrounding gene pools, and thus, special consideration is suggested for their operational forest management. Local sources must be chosen if reproductive materials are required for active restoration in these GZs, in order to preserve their characteristic genetic structures. In the case of severe loss of the GZ7 and GZ13 local forests , a balanced seed pool from the surrounding GZs is suggested for future tree-planting activities.N. pumilio management plan based on genetic knowledge and considering its entire distribution range (more than 2200 km). Six priority areas  were identified that could lead conservation action, specifically targeting in situ conservation. Furthermore, this study enabled the delineation of 18 N. pumilio GZs in Argentina, thus providing preliminary operational genetic management units that best fit the genetic structure of the target species. Maps showing the distributional range of each GZ in Argentina were drawn up. Reproductive material can thus be transferred within these ad hoc GZs with reduced risk of altering the original genetic structure. This possibility is important in light of the numerous active ecological restoration projects that have been developing in the N. pumilio forests on both sides of the Andes Range in the last decade. In addition, the recommendations suggested here are highly important for regulatory institutions that have to enforce compliance with national Law 26331, on the protection of native forests. Moreover, the 18 GZs constitute a first step towards the definition of both PRs and CUs, thus promoting preservation of the southernmost forest ecosystem of the world.This study presents the first scientific evidence for the definition of a N. pumilio populations (965 adult trees) included in this study  and from 71° 56′ to 66° 37′ in the longitudinal range. A minimum of 20 trees were sampled in each population, with the exception of Cholila and El Chalten sites where 18 and 19 samples were obtained (respectively) due to limited access. In every site, a minimum distance of 50 m between sampled trees was maintained in order to preclude the collection of closely-related individuals. In addition, only N. pumilio phenotypes were considered at the time of population sampling .Of the 35 dy Table , Fig. 1,64. Samples were stored at − 20 °C until PCR amplifications were carried out. Seven polymorphic loci  were analyzed using a specific set of microsatellite markers (nuSSRs) developed for this species by Soliani et al.65. SSR amplification conditions and PCR thermal profiles were set following the recommendations given by the authors65. Amplification reactions were performed with 20 ng of template DNA in a final volume of 11 µl, using 1X Green Buffer and GoTaq DNA Polymerase  for all primers, with the exception of Npum10 that was amplified with Platinum Taq . The M13 protocol66 was applied and PCR products were visualized on 1% agarose gel stained with Sybr Safe (Invitrogen). Individual genotyping was done using a 3130xl Genetic Analyzer sequencer  and the allele assignment with GeneMarker V2.7.4 .Total DNA was extracted from bud tissue following the procedure described by Dumolin et al.Na), the effective number of alleles (Ne), and the expected heterozygosity that considers the null allele effect (HE null), which were obtained with GenAlEx 6.5 software67. Unbiased heterozygosity described by Nei68 was also estimated with Info-Gen software69. The Wilcoxon test was implemented as an a posteriori multiple-comparison analysis, to identify the significant differences among populations. The presence of null alleles at both locus and population levels was tested with MICRO-CHECKER70 and their frequencies were estimated using FreeNA71. The presence of null alleles was considered in the estimation of inbreeding coefficients (FIS), which was calculated with INEST 2.272. Two approaches were tested for obtaining FIS: 1. an Individual Inbreeding Model (IIM) using a Bayesian approach (FIS′), and 2. a Maximum Likelihood approach (FIS”). In both cases, three different models were tested  at population level, and the model with the lowest Deviance Information Criterion (DIC) was considered as best fitting the data. Once the inbreeding model had been selected, the mean inbreeding coefficients (FIS′) were calculated. In addition, a jackknife procedure was performed to estimate the standard error (SE) of the FIS” values, which were then used to assess the significance of this coefficient using the Z-test based on the normal distribution theory.A set of genetic parameters was first obtained in order to characterize the populations under study Table . These p73. This approach computes the distribution of the expected heterozygosity from the observed number of alleles at population level, assuming the mutation-drift equilibrium model. A significant heterozygosity excess or deficiency is confirmed by performing a Wilcoxon sign-rank test for every mutation model . In this study, a past population bottleneck was inferred when heterozygosity excess was significant (p < 0.05) in at least two of the above-mentioned mutational models.In order to identify populations experiencing a relatively recent reduction in effective population size, a demographic bottleneck test was carried out using BOTTLENECK softwareFST74 and G′ST75 indexes. FST was then recalculated by implementing the ‘exclusion of null alleles’ (ENA) method in order to avoid a possible bias introduced by the presence of null alleles71. Confidence intervals (95% level) were obtained through a bootstrap re-sampling procedure. In addition, to evaluate the occurrence of an isolation-by-distance (IBD) pattern, a correlation between the genetic and geographic distances between populations was assessed using two approaches: a. The Mantel test, performed with Info-Gen and using the correlation between FST estimates and the logarithm of the geographic distances between population pairs; b. The Spatial Genetic Structure (SGC) test by GenAlEx 6.4, with which spatial autocorrelation coefficients  were obtained for ten distance classes (each one covering 200 km). In both approaches, 1000 permutations were considered when calculating statistical significance. In the SGC test, the upper and lower error limits for the 95% confidence interval were determined by carrying out 10,000 bootstrap samples.Genetic differentiation among populations was determined by estimating both 33, see Fig. 77. This analysis showed weak grouping, since the adjusted probability of the inferred number of clusters was 0.58632. This reinforced the decision of considering alternative division criteria before performing the final clustering.Due to the large latitudinal span of the area under study (more than 2200 km long) and the diverse evolutionary history of the region, multiple division criteria were considered  and Cholila, and 2. ‘South’ including the remaining 23 populations that exhibited southern cpDNA haplotypes and are located between Huemules and Bahía Lapataia (54° 50′S). The genetic structure of N. pumilio populations from each north and south group was then determined by a spatial clustering method using BAPS software. This approach has the advantage of considering the geographic locations of pre-assigned groups of individuals when defining the best number of clusters (K)77. In addition to this analysis, an admixture model within each inferred K was tested with STRUCTURE 2.2 software79 by running 500,000 Markov chain Monte Carlo (MCMC) steps, a burn-in of 100,000 iterations and five replicates for each inferred K in the entire analysis. Complementarily, UPGMA (Unweighted Pair Group Method using Arithmetic averages) dendrograms based on Nei’s distance (averaged over loci) were constructed to explore the relationships among the identified clusters.A phylogeographic break around 42°S latitude was previously reported for some species of 32 and Thomas et al.80. In this case, individual admixture coefficients for each inferred cluster (obtained by STRUCTURE) larger than 0.7 were considered. Each cell was assigned with the mean membership value of the individuals, enclosed by a circular neighborhood of 20 min diameter constructed around its center to graphically represent the inferred clusters. Raster package 2.14 was used to generate these maps in R software environment (R Core Team) and they were edited in ArcGIS 9.3 .A grid-based spatial analysis was carried out with a resolution of 2 min to visualize the geographical distribution and admixture pattern of inferred clusters. Separate raster maps were produced, considering a circular neighborhood cell of 20 min diameter around every sampled location, following van Zonneveld et al.Delineation of GZs was performed based on the previously-mentioned analyses and summarized in Fig. 81, in order to evaluate the number of clusters that best grouped the studied populations. The optimum number of genetic zones was confirmed based on the significant differences among and within the obtained clusters, as proposed by Pastorino and Gallo20; i.e., genetic variance should be maximum between populations of different groups and minimum within the groups.A hierarchical analysis of molecular variance (AMOVA) was performed using Arlequin 3.5Pa), the expected heterozygosity considering the null allele effect (HE null), and locally common alleles (LCA). The LCA average for each GZ was calculated considering alleles with frequencies higher than 5% and, at the same time, an occurrence probability of less than 25% of the defined GZs. In addition, genetic diversity was estimated by calculating allelic richness (AR) after rarefaction to a common sample size82 using FSTAT software83.To obtain valuable information for the definition of management plans, some genetic parameters for each defined GZ were calculated with GenAlEx and Info-Gen: private alleles (Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.


Echinococcus spp. is a very severe zoonotic helminth infection. Echinococcus shiquicus is a newly discovered species that has only been reported in the Qinghai and Sichuan provinces of the Qinghai-Tibet plateau, China where, to date, it has only been confirmed in Tibetan foxes and wild small mammal populations of the Tibetan plateau. Information on its genetic and evolutionary diversity is scanty. The aim of this study was to investigate the prevalence of E. shiquicus in plateau pikas (Ochotona curzoniae), a known intermediate host, and to determine the genetic variation and phylogenetic relationship of the E. shiquicus population in the Tibet region of China based on mitochondrial DNA.Cystic or alveolar echinococcosis caused by the larval stages of Echinococcus shiquicus samples were collected from Damxung and Nyêmo counties . The mitochondrial cox1 and nad1 gene sequences were analyzed, and the genetic diversity and epidemiology of E. shiquicus in the region were discussed based on the results.E. shiquicus in pikas in Damxung and Nyêmo counties was 3.95% (6/152) and 6.98% (9/129), respectively. In combination with previous public sequence data, the haplotype analysis revealed 12 haplotypes (H) characterized by two distinct clusters (I and II), and a sequence distance of 99.1–99.9% from the reference haplotype (H1). The diversity and neutrality indices for the entire E. shiquicus populations were: haplotype diversity (Hd) ± standard deviation (SD) 0.862 ± 0.035; nucleotide diversity (Hd ± SD) 0.0056 ± 0.0003; Tajima's D 0.876 (P > 0.05); and Fu’s F 6.000 (P > 0.05).The prevalence of E. shiquicus in plateau pikas in the Tibet Autonomous Region of China. The neutrality indices suggest a deficiency of alleles, indicative of a recent population bottleneck.This was the first analysis of the newly discovered  Echinococcus shiquicus, a newly described species of the genus Echinococcus, is currently believed to be limited and endemic to the Qinghai-Tibet plateau region of China  and 20 complete cox1-nad1 sequences from our previous investigation in Darlag county, Qinghai province [To investigate the genetic diversity, we analysed a total of 35 [Damxung  and Nyêmcox1 and nad1 were amplified and sequenced using E. shiquicus-specific primers (Table cox1 and nad1 genes (2505 bp), the E. shiquicus isolates formed 11 different haplotypes (H2–H12) (Table E. shiquicus reference sequence (H1) (GenBank accession no. AB208064 or NC_009460). Of these 11 haplotypes, haplotypes H2–H7 represent haplotypes from this study while haplotypes H6 and H8–H12 represent haplotypes from samples collected in Darlag County. The relationships between these haplotypes and their genetic diversities are shown in Table E. shiquicus population  were calculated using the concatenated cox1-nad1 sequences  and Fu’s F  were positive and non-significant, suggesting allele deficiency and a recent population bottleneck event of the E. shiquicus population in the Tibetan region.Diversity and neutrality indices for es Table . The totes Table . HaplotyE. shiquicus remains poorly understood [Echinococcus infection in the Qinghai-Tibet plateau cannot be overstated in terms of providing a near-accurate picture of the prevalence of the species in the region. For example, the challenge posed by misidentification was recently discussed by Boufana et al. who investigated the possible presence of E. shiquicus in hosts other than Tibetan foxes and pikas and found for the first time since the discovery of this helminth that dogs could potentially serve as a definitive host for this species [E. shiquicus in a previously identified E. granulosus sample and identified cross-reaction of primers as a major reason for missing the E. shiquicus in the first instance [The geographical distribution, epidemiology and zoonotic potential of derstood . Althougderstood , therebyderstood , 11 in t species . These ainstance .E. shiquicus infection in pikas in Damxung and Nyêmo counties of 3.95 and 6.98%, respectively. With the question of the zoonotic potential of E. shiquicus still unanswered [E. shiquicus infection observed in the study area and the prevalence of 23.75–37.5% previously reported in Darlag and Shiqu counties of Qinghai and Sichuan provinces, respectively [E. shiquicus infection for both humans and livestock in the region.The results of this study showed a prevalence of answered , the preE. shiquicus has evolved without bottleneck effect [E. shiquicus population could have possibly experienced a bottleneck effect in the course of evolution.Earlier studies have raised the controversial point of whether or not k effect , 24. HowAmong the 11 haplotypes identified in sequences reported previously from Qinghai province , H2 and E. shiquicus population from Tibet in comparison to previous isolates from Qinghai province. Our results demonstrate allele deficiency among the E. shiquicus population indicating a population bottleneck event in the Tibet Autonomous Region. However, we suggest that future studies on population genetics consider using a larger panel of isolates and examining other genetic markers in order to further elucidate the evolutionary status of E. shiquicus in the Qinghai-Tibet Plateau.In this study, we report the genetic diversity and the phylogenetic status of E. shiquicus in plateau pikas in the Tibet Autonomous Region of China, and the neutrality indices suggest a deficiency of alleles as expected from a recent population bottleneck.This study demonstrates the existence of Additional file 1: Fig. S1. Multiplex PCR results image of Echinococcus shiquicus samples based on three specific primers of E. multilocularis, E. shiquicus and E. granulosus.


Bactericera cockerelli (Šulc), a generalist herbivore that vectors a bacterial pathogen  of potato (Solanum tuberosum L.). Genome‐wide SNP data revealed two major genetic clusters—psyllids collected from potato crops were genetically similar to psyllids found on a common weed, Lycium spp., but dissimilar from those found on another common non‐crop host, Solanum dulcamara L. Most psyllids found on Lycium spp. and potato represented a single mitochondrial cytochrome oxidase I (COI) haplotype that has been suggested to not be native to the region, and whose arrival may have been concurrent with zebra chip disease first emerging. The putatively introduced COI haplotype usually co‐occurred with endosymbiotic Wolbachia, while the putatively resident COI haplotype generally did not. Genetic intermediates between the two genetic populations of insects were rare, consistent with recent sympatry or reproductive isolation, although admixture patterns of apparent hybrids were consistent with introgression of genes from introduced into resident populations. Our results suggest that both host–plant associations and endosymbionts are shaping the population genetic structure of sympatric psyllid populations associated with different non‐crop hosts. It is of future interest to explicitly examine vectorial capacity of the two populations and their potential hybrids, as population structure and hybridization might alter regional vector capacity and disease outbreaks.Changing climate and land‐use practices have the potential to bring previously isolated populations of pest insects into new sympatry. This heightens the need to better understand how differing patterns of host–plant association, and unique endosymbionts, serve to promote genetic isolation or integration. We addressed these factors in populations of potato psyllid,  In its western North American range, B. cockerelli comprises several unique COI haplotypes that differ in host use and endosymbionts , which causes zebra chip disease in potato plants (Solanum tuberosum L.)  and matrimony vine (Lycium spp.). While potato production is focused within two areas, one in southcentral Washington/northcentral Oregon and another in southern Idaho, these perennial hosts are distributed broadly over the entire region  and putatively introduced (western) psyllids differ in their host–plant associations or carry distinct endosymbionts. Third, we evaluate whether hybridization between genetically distinct psyllids occurs frequently. Our ultimate goal is to inform how the arrival of new psyllid genetic types might have influenced the sudden emergence of zebra chip disease in potato. Because our work was conducted at a broad geographic scale Figure , we hope22.1Solanum dulcamara L), and 11 matrimony vine (Lycium spp.) sites across three northwestern US states in 2016 . We used the DNeasy Blood & Tissue kit to extract psyllid DNA  by placing single insects in a microcentrifuge tube with 180 µl ATL buffer. We ground the insect tissue submerged in buffer for 1 min using a pestle driven by a MicroTube Homogenizer ; otherwise, the extraction was conducted following the kit protocol. One hundred µl AE buffer from the kit was used to elute DNA. The quantity of DNA was measured using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific).From each collection site and date, four to ten adult psyllids were haphazardly selected for DNA extraction  for NextRAD library preparation and sequencing as described in Fu et al. . DNA wasWe characterized the COI haplotype of each sample following the high‐resolution melting curve method  that trimmed reads using bbduk , which represents the number of putative ancestral populations. ADMIXTURE estimates the proportion of putative ancestry for each individual based on its genotype. We conducted ADMIXTURE runs in “unsupervised” mode because psyllid gene flow could occur among host plants and study sites. Ancestry coefficients from 50 runs were aligned and averaged using CLUMPAK  is admixed between two source populations A and B . In our instance, we were interested in examining individual psyllids that were most likely to be hybrids that were derived from the putatively introduced and resident populations. We picked two source populations based on the membership coefficients generated in ADMIXTURE (K = 2). We assumed that psyllids with 100% assignment to the lineage corresponding to the W haplotype  between any pair of populations from a non‐crop site (with pre‐ and post‐season samples) and a potato site in the same region. This allowed for a comparison of genetic differentiation between pre‐ and post‐season populations in relation to potato. FST was estimated using the package StAMPP in R  determined by majority rule. Sampling sites were nested within host plant species and nested within genetic groups (see Population genomic patterns). The function poppr.amova in R package poppr was used to conduct AMOVA  and other independent variables, for example, host plant, COI haplotypes, and geographic distance, we conducted MMRR , 0.25 µl Taq DNA polymerase , 20 µl of psyllid DNA, and 16.65 µl molecular grade water to reach 50 µl. The PCR was denaturation at 95°C for 3 min, followed by 25 cycles of 95°C for 30 s, 51°C for 30 s and 72°C for 1 min; a final extension was at 72°C for 10 min. PCR products were visualized on 1.5% agarose gels. Samples with a clear band of ~460 bp were processed in the second PCR, which included 4 µl 10 × PCR buffer, 7.2 µl 25 mM MgCl2, 1.2 µl BSA, 0.8 µl 10 mM dNTPs, 0.4 µl Taq DNA polymerase, 3 µl barcode primers, 10 µl of five time diluted first PCR products, and 13.4 µl molecular grade water. Dual barcode primers were ordered from IBEST , and each psyllid sample processed here received a unique barcode. The second PCR was denaturing at 95°C for 3 min, followed by 10 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min; the extension was at 72°C for 10 min. Samples showed a single band at the expected size of ~580 bp during gel electrophoresis. Equal molar of PCR amplicons from the second PCR were pooled and sequenced on Illumina MiSeq with a 2 × 300 bp configuration.To characterize the bacterial microbiota of individual psyllids, 77 insects that had sufficient DNA remaining after NextRAD sequencing were processed for 16S rRNA gene sequencing. Primers flanking the V3‐V4 hypervariable regions of the 16S rRNA gene  , where 76.2% of psyllids on bittersweet nightshade were of the resident northwestern COI haplotype, and 99.5% of psyllids on potato were of the putatively introduced western COI‐haplotype type. Of the psyllids collected from matrimony vine, 65% were western COI haplotype and 31.7% were northwestern COI haplotype. The frequency of COI haplotypes did not vary between the two potato growing areas in our study region .Among the 566 psyllids we examined, 359 (63.4%) and 191 (33.7%) were of the dominant western and northwestern COI haplotypes, respectively, and 16 (2.8%) were other COI haplotypes . Different COI haplotypes generally were associated with different host plant species . However, individuals were highly admixed, and the results were not easily interpretable. Thus, we focus on the results at lower K values. Most bittersweet nightshade‐ and potato‐collected psyllids were assigned to different groups at K = 2, while psyllids collected from matrimony vine were assigned to both groups . To avoid confusion, we will henceforth refer to genetic groups assigned by majority rule in ADMIXTURE while K = 2 as W and NW ADMIXTURE genetic groups.We found a similar pattern in ADMIXTURE as with the neighbor‐net clustering analysis. The cross‐validation error was minimized at eight clusters  showed that 20 insects  assigned to the NW ADMIXTURE genetic group were of the western COI haplotype  was the response. Geographic distance alone explained very little genetic distance (r2 ~ 0.02). COI haplotype and host plant explained more   geographic distance, (b) host plant identity, and (c) COI haplotype; genetic distance (y) Table .3.5FST changes of the pre‐ and post‐season populations . Population turnover at matrimony vine sites was also reflected in genetic group composition change. Sites with high FST between pre‐season and post‐season populations often exhibited a shift from the NW ADMIXTURE genetic group‐dominant to W ADMIXTURE genetic group‐dominant . The less abundant W. pipientis , C. ruddii‐1, was 100% identical to a known C. ruddii gene (KR045612.1). The strain associated with the W ADMIXTURE group, C. ruddii‐2, differed from C. ruddii‐1 by three nucleotides. The remaining three strains were unclassified and could only be identified to the Enterobacteriaceae family . We were unable to evaluate variation in psyllid endosymbiont communities among host plant species because of uneven sample sizes leading to heterogeneity of variance .Over 6 million paired‐end reads  were generated from the 16S rRNA sequencing of 77 samples. We focused on the taxa that were most prevalent in the dataset instead of describing taxa that occurred in a few samples with low reads. After filtering , seven bacterial strains were retained in the final dataset  harbored Wolbachia. The two Wolbachia‐infected psyllids in the NW ADMIXTURE genetic group were of the western COI haplotype, suggesting they were the product of successful matings between infected western COI‐haplotype females and uninfected northwestern COI‐haplotype males. Interestingly, differences in endosymbiont communities between genetic groups were not driven solely by Wolbachia; endosymbiont community structure remained distinct between the ADMIXTURE genetic groups when Wolbachia was removed from analyses . Two highly abundant C. ruddii strains were present in all psyllids except two, with ADMIXTURE genetic groups containing distinct strains . Psyllid genetic types also varied in their host use; potato fields harbored primarily W ADMIXTURE genetic type psyllids, and these psyllids used mainly matrimony vine, not bittersweet nightshade, as non‐crop hosts. While the results of hybridization between genetically distinct psyllids was not observed frequently, the genetic makeup of hybrids is consistent with the presence of We found that the W and NW ADMIXTURE genetic groups are distinct populations with limited gene flow, which could be consistent with the hypothesis that western psyllids have dispersed into the region recently  such a genetic sweep is indeed occurring (as noted above), (b) western psyllids are superior vectors of the zebra chip pathogen, (c) this enhanced transmission is linked to particular maternally inherited genes or particular endosymbionts, and (d) if hybrids also exhibit enhanced transmission. More generally, we recommend that future work should examine any impacts that endosymbionts other than Wolbachia have on potato psyllids’ ability to feed on different host plants, and if this in turn impacts pathogen transmission. Indeed, there is preliminary evidence from several other psyllid species that these endosymbionts might play an important role (Subandiyah et al., 2000; Fromont, Riegler, & Cook, Our study did not examine transmission of the zebra chip pathogen by the two genetic groups or their hybrids. However, this could be a fruitful area for future inquiry as hybridization has the potential to alter disease dynamics. Psyllids of the western COI haplotype are the predominant vector of the zebra chip pathogen in the region, while the LSO bacterium is less frequently found in northwestern COI haplotypes (Swisher et al., Wolbachia are shaping the genetic integration of putatively introduced and resident populations of a plant pathogen vector, the potato psyllid. In doing so, these factors might have the potential to alter regional vector capacity and zebra chip outbreaks. While future work is necessary to elucidate mechanisms and to confirm that such a trend is indeed taking place in this system, our work highlights the potential importance of incorporating vector genetic structure, host–plant associations, and endosymbionts to understand and predict disease dynamics in other systems. Understanding interactions among these factors and their impact on disease dynamics is increasingly important, as changing climate and land‐use practices will continue to bring allopatric insect vector populations together, with potentially dire consequences for disease outbreaks.Overall, our results suggest that both host–plant associations and Supplementary MaterialClick here for additional data file.


Saccharomycesbayanus scored the highest in citric and floral notes, and this was preferred over all the other wines that were evaluated. The wines aged in oak barrels were preferred to both, wines aged in the presence of oak chips as well as those aged without any wood contact. The use of climate chambers to dry the grapes that are going to be used for the elaboration of sweet wines appears to be an advantageous alternative to the traditional method, since it allows a more precise control of the process and highly valued sweet wines from a sensory point of view are obtained thereby.In this study, a climate chamber, as an alternative method, has been used to dry raisins and the sensory profiles of the sweet sherry wines obtained have been evaluated. Other important factors, namely grape variety, vintage, vinification conditions, as well as the ageing method and its length of time, have also been considered. When heavy rainfall had been registered, the musts extracted from grapes dried under controlled conditions in a climate chamber showed a lower intensity of the musty off-odor compared to those elaborated with sun-dried grapes. The wine fermented at low temperature with  The production of sweet wines is commonly carried out with dehydrated grapes and there are three different processes to perform the water loss of the grapes: dehydration at a controlled temperature, humidity and ventilation; drying with non-controlled conditions and withering with eventual control of temperature and humidity and natural ventilation . The sweWhen this traditional procedure is applied, grapes may suffer undesired alterations that would have an impact on the quality of the final product; rainfalls particularly may contribute to the growth of certain fungi, which would lead to the loss of a considerable proportion of the treated grapes. In a worse scenario, highly toxic metabolites might appear in the wines that have been elaborated using such grapes ,6,7,8. FThere are several alternatives for a controlled dehydration of the grape, which would bring about a number of considerable advantages. On the one hand, withering methods with natural ventilation have been widely employed for the production of sweet wines in Italy, where the use of artificially heated air is not allowed ,12,13,14Research studies on Pedro Ximénez and Muscat sweet musts and wines made from grapes dried both under sunlight and within climate chambers with forced ventilation can be found in the literature ,24,25,26With a view to proposing the use of climate chambers as an alternative to the traditional sun-drying method, we have evaluated the sensory profiles of sweet sherry wines elaborated from grapes dried in climatic chambers. Some important factors, such as grape variety, vintage, fermentation conditions, as well as the ageing method and time, have been considered in this study.Two grape varieties  from three consecutive vintages  were used for this research. The grapes were subjected to two different drying procedures: the traditional natural sun-drying method  and controlled drying in a climate chamber (C).For chamber drying, about 2000 kg of ripe grape bunches of each grape variety and vintage were collected from a local winery in the Jerez-Xérès-Sherry D.O. (Denomination of Origin) region. The grapes were dried in a climate chamber  for about 5 days at 40 °C and 10% relative humidity. The bunches were uniformly distributed in a single layer inside the chamber. For the sun drying method, the grapes were spread out also in a single layer on “esparto” grass mats to dry under sunlight for about 10–15 days. They were turned over every day and covered at night. Grape weight loss was periodically monitored for both methods and the drying process was considered as completed when such weight loss reached about 35% of the original weight, at 20–21° Baume.After this, grapes were separately destemmed, crushed and pressed by means of a vertical press (300 bars maximum). The initial pH of the must ranged between 3.6 and 3.8 and it was different for each experience, so it was adjusted to 3.5 by adding tartaric acid . The concentration of total sulphur dioxide was also set at 120 mg/L by adding potassium metabisulfite (Agrovin).Saccharomyces cerevisiae yeast inoculum  was employed. The fermentation was carried out at room temperature (less than 30 °C). In order to evaluate the effect of the addition of nitrogen, the conditions for experiment E2 were the same as for experiment E1 with the addition of some yeast nutrients . For the evaluation of the employment of pellicular maceration, in experiment E3 the conditions were the same as for experiment E1 but the grapes were crushed without previous destemming and submitted to prefermentative pellicular maceration for 24 h at 4 °C using 3 g/hL of pectolytic enzymes , and then pressed. In order to check the influence of the type of yeast in the fermentation, the must from experiment E5 was fermented under the same conditions as for experiment E3 but employing Saccharomyces bayanus . Finally, to investigate the effect of the temperature in the fermentation, in experiment E4, the must was fermented at low temperature (less than 10 °C) with S. bayanus. Taking into account the recommendations from the supplier, this yeast was more suitable than S. cerevisiae to carry out the fermentation at lower temperatures.Five different conditions were tested for the partial fermentation of the musts obtained from vintage 01 Muscat grapes dried in the climate chamber  with After that, the selected Muscat wine from vintage 01 was subjected to ageing for 1 year using the following 3 parallel methods: For the first method, the wine was poured into 30 L medium toast American oak barrels (samples B); for the second method, the wine was put in contact with oak chips  at a dose of 4 g/L (samples identified by Ch) and for the third method, ageing took place without any contact with oak, in stainless steel tanks (samples S).The sweet wines (Muscat and Pedro Ximénez) produced from vintages 02 and 03 were all aged in 30 L medium toasted American oak barrels (B). During this phase, all the wines were maintained in the same room at about 20 °C.During each ageing phase, the samples were taken once every month over a whole year (S0 to S12). All of the samples were stored at 4 °C until their analysis.The exclusively orthonasal sensory evaluation of the musts and wines was carried out in duplicate by a panel of between 13 and 17 members depending on the test. The samples were not tasted with the mouth, so the retronasal sensory evaluation was not performed. All the panel members had a medium level of experience in sensory analysis of sweet wines . In addiQuantitative descriptive analysis , and in Twenty-milliliter samples were presented in blue glasses , so thaAfter performing the triangle test, the judges were also asked to provide a quantitative assessment of the detected differences. The following 5-point scale was used to rate such differences: not present (0), low (1), medium (2), strong (3) and very strong (4).In respect to descriptive profile tests, as a preliminary stage to determine the appropriate descriptors that would better define the samples, a representative number of must and wine samples from the whole set were presented to the judges for them to provide qualitative descriptions for each one. The descriptors with a mention frequency over 5 were selected; i.e., fruity, citric, ripe fruit, raisin, floral, honey, herbaceous, vinous, lactic, musty, chemical character and oak. The final worksheet also included aromatic intensity and olfactory quality as descriptors. Each descriptor was scored according to a nine-point scale , and also the olfactory quality of each sample was evaluated according to a structured nine-point scale  . Table 2The statistical analyses were carried out using Statistica 8.0 , and Microsoft Office Excel 2010 applications.The treatment of the data from the triangle test was based on the tabulated statistic data  with resAn analysis of variance (ANOVA) was performed on the quantitative sensory data from the descriptive assessments. However, an acceptable dispersion of sensory data could complicate the discrimination between groups of samples for a particular descriptor . Therefore, it is proposed the subsequent application of multivariate statistical techniques that take into account the whole group of differences and similarities for all variables.A Principal Component Analysis (PCA) was performed in order to highlighting the similarity of the samples, and to determine the main contributors to any of the differences found between them. Missing data were replaced by the average value of that variable in the group. The minimum eigenvalue to select a principal component was set at 1.0, and a factor rotation according to varimax normalized method was applied to confirm a correlation with the PCA. Loadings greater than 0.7 identified those variables well correlated to PCs.In order to identify those descriptors that better differentiate between clusters of samples, a Linear Discriminant Analysis (LDA) was performed according to Wilks’ lambda statistic, and the so-called forward stepwise method was employed. According to this methodology, the discrimination model is built step-by-step by reviewing all the variables at each step and evaluating which one contributes the most to the discrimination between clusters. The F-to-enter and F-to-remove were set at 1 and 0.N = 13 judges, α = 0.05). In the case of Muscat, 10 out of the 13 judges identified the different sample, while in the discrimination of Pedro Ximénez musts, 8 judges detected the different sample. Given that according to the standard, 8 is the minimum number of coincidental judgments required to confirm a significant difference between the samples, we could conclude that the Muscat musts and also those of Pedro Ximénez presented a significantly different aromatic profile if the grapes had been raisined in the sun or in a climate chamber. The differences were valued by the judges as being of a moderate intensity .The musts obtained after traditional drying of the grapes from the first year (V01) were compared with those dried in a climatic chamber by means of a triangle test (p < 0.05) the musts from Muscat grapes treated by the 2 drying methods tested, being higher the scores for the musts obtained under controlled conditions. From Pedro Ximénez musts, the results were similar, although the differences were not significant. Having said that, the most interesting result is that those musts obtained from grapes dried in chambers were perceived as having a lower intensity in the olfactory fungal defect (musty). The detection of such a defect in the musts from either a drying method, seems to indicate that the grapes had been previously contaminated, probably due to the rainfalls during the days before that year’s harvesting period. As a result, the musts of grapes dried in climate chambers were better evaluated .An ANOVA was then applied to the scores obtained from the descriptive test on these V01 musts with the objective of identifying the aromatic notes that were responsible for the differences that had previously been confirmed by the triangle test. The resulting data are shown in When the data from the second vintage (V02) musts were analyzed, it was found that non-significant differences between the average scores for some of the defects in the Muscat musts could justify a better olfactory rating when their grapes had been dried in a climate chamber. However, this preference did not reach statistical significance. On the other hand, no differences could be confirmed for V03 musts that had been dried either in a climate chamber or by means of the traditional sun-drying method.It should be taken into account that the grapes from these vintages  arrived at the pilot plant in a better sanitary condition. This could explain their similar evolution regardless of the raisining method employed, as opposed to what happened with V01 grapes, which was characterized by intense rainfalls on the days prior to their harvest. Other authors neither found significant sensory differences between musts obtained by grapes dried traditionally or in a controlled way .Due to the intrinsic variance of sensory data, it is often difficult to confirm differences between samples based on the results obtained from an analysis of variance. Therefore, in order to evaluate the minor differences between each of these descriptors that may come into sight when comparing “asoleo” and climate chamber musts, a PCA was applied to the sensory scores of all the must samples from V01, V02 and V03. The PCA allowed determining four principal components that account for 86.7% of the total variance. The loads (data not shown) confirmed a relationship between PC1 (40.1% of the variance explained) and the drying method, given its correlation with olfactory descriptors such as ripe fruit and raisins; and PC2 (22.6% of the variance explained) that represents a greater aromatic complexity, as it correlates with aromatic intensity and citric and floral notes, which contribute to better aromatic quality. On the other hand, PC3 and PC4  are related to aromatic defects .When the sensory profiles were transferred to this new representation, it can be seen that for PC1 a, the raOn the other hand, PC3 and PC4 allowed us to differentiate the musts according to their harvest b. As canIn any case, the most interesting result from our research is that the Muscat grape musts from the 02 vintage (V02.M.C), whose grapes were treated in a climate chamber, were also located in this defect-free area. It is therefore confirmed that the use of a climate chamber for the raisining phase of the grapes avoided some of the risks associated to adverse weather conditions, by allowing the hygienic drying of the grapes and preventing the appearance of fungi and its associated aromatic defects, while food safety was also preserved. In addition, in some cases the musts would exhibit a greater aromatic complexity. It should also be added that the drying time was reduced by 50–66%. At this point, it could be concluded that the capacity of the sensory analysis as a methodology to control the hygienic and sanitary state of musts was demonstrated.1 (63) > E11 (56) > E31,2 (55) > E52 (43) > E22 (36), where the different superscripts indicate that these wines were perceived differently with regards to their olfactory qualities. The wine that had been fermented at low temperature with S. bayanus (E4) was the best rated, although the difference with the wine fermented with S. cerevisiae at room temperature (E1) was not significant. The nitrogen-added wine was granted the lowest score (E2), which agreed with the conclusions by other authors [S. cerevisiae (E3) was slightly better scored.The following step was the evaluation of some fermenting conditions in the production of sweet wine such as the type of yeast, the addition of nitrogen, the employment of pellicular maceration or the fermentation temperature . The win authors . Finallyp-values) between the wines produced. These are attributed the highest score in the ranking test for wine E4 (fermented with S. bayanus at low temperature), which was characterized by high scores in Muscat typical citric and floral notes , while is yeast ,42. CompSaccharomyces bayanus yeast, at a controlled temperature not greater than 10 °C, no nitrogen added and non-pellicular maceration in the presence of pectolytic enzymes. The above fermenting conditions were used for the rest of the research.Consequently, the E4 test was selected as the preferred fermenting conditions for the production of sweet wines from grapes dried in a climate chamber, namely with p < 0.05): oak, olfactory quality, vinous, raisin, citric, musty, honey, fruity and ripe fruit. The Muscat wine from V01 fermented under the preferred conditions (E4) was subjected to ageing for 1 year using oak chips (Ch), oak barrels (B) and stainless steel containers (S). A LDA where the ageing method was taken as the cluster variable was applied to the sensory profiles of the sampled wines. This would allow the identification of the descriptors that best differentiate them. Since a high percentage of the wines (86.9%) were correctly classified, a PCA was performed on such most significant descriptors  presented the lowest values for PC2, that is to say, they exhibited high intensity levels of citric notes, a typical character of Muscat wines when kept under these ageing conditions. Other authors  already On the other hand, wines aged in oak barrels (B) were found on the right area of the graph , which implies high intensity in oak notes and a very clearly perceived raisin note, both contributing to a highly valued olfactory quality.With regard to wines aged in contact with oak chips (Ch), their position on the plane indicates low aromatic intensities of positive character notes and therefore it did not appear to be the best ageing system in principle.This clear differentiation regarding ageing time corresponded to the discrimination observed for the same samples regarding the volatile composition . Thus, tThe previously selected vinification (E4) and ageing (B) methods were applied to the Muscat and Pedro Ximénez musts obtained from the V02 and V03 grapes dried in climate chambers, and the ageing process was monitored for 1 year. In the case of V01 grapes, only the wines from the Muscat variety were aged, since those from the Pedro Ximénez variety were accidentally contaminated by external causes at the beginning of the process and became unsuitable for this study.In order to identify the variables that best discriminate wines according to their ageing time length, a LDA was performed on the initial (S0), intermediate (S6) and final (S12) samples, where the ageing time would be the cluster variable. The descriptors with the highest discrimination capacity were: aromatic intensity, fruity, citric, ripe fruit, floral, herbaceous, oak, vinous and together with olfactory quality (data not shown). The exclusive use of these sensory descriptors minimizes the amount of unnecessary data and is expected to improve the estimate and interpretation of the new components found by the PCA analysis, which resulted in three PCs (explaining 74% of the variance). Since standardized coefficients greater than 0.7 were considered as significant, it could be confirmed that the descriptors aromatic intensity, oak and olfactory quality correlated well with PC1 (which explained 28.7% of the variance), as well as herbaceous, although this latter correlated inversely, since it was a negative coefficient. With the same criterion, PC2 (explains 25.9% of the variance) correlated well with the citric and floral notes; while PC3 (19.4% of the variance explained) did so with the fruity and ripe fruit notes. It can be seen from the graph that for each vintage and variety the wines were sorted according to their ageing time along PC1 . According to the loads of this component, the arrangement of the samples implies greater intensities of oak and aromatic intensity as the ageing process progresses, and what is more interesting, the perceived olfactory quality improved. The scores increased significantly over the first month, a behavior that has already been described by other authors  in relatIt could be concluded that with regards to wood ageing and from the sensory point of view, ageing in oak barrels improved the olfactory perception of all the sweet wines made from grapes dried in a climate chamber, whether it be Muscat or Pedro Ximénez. Nevertheless, the effect became more noteworthy when the grapes were from less optimal vintages from the aromatic point of view.Saccharomyces bayanus scored the highest in citric and floral notes, and this was preferred over all the other wines that were evaluated. Regarding the ageing stage, the wines aged in oak barrels were preferred to both, wines aged in the presence of oak chips as well as those aged without any wood contact. In addition, the use of climate chambers is a valuable alternative technique for the dehydration of grapes, compared to the traditional sun-drying method, because they allow a more precise control on the process and facilitate the solution to some production problems such as the musty off-odors associated to fungus contamination.In this study, sensory analysis was proven to be a reliable methodology to evaluate different process variables in the production of sweet wines, in which climate chambers to dehydrate the grapes were employed. The wine fermented at low temperature with


Large migrating insects, flying at high altitude, often exhibit complex behaviour. They frequently elect to fly on winds with directions quite different from the prevailing direction, and they show a degree of common orientation, both of which facilitate transport in seasonally beneficial directions. Much less is known about the migration behaviour of smaller (10–70 mg) insects. To address this issue, we used radar to examine the high-altitude flight of hoverflies (Diptera: Syrphidae), a group of day-active, medium-sized insects commonly migrating over the UK. We found that autumn migrants, which must move south, did indeed show migration timings and orientation responses that would take them in this direction, despite the unfavourability of the prevailing winds. Evidently, these hoverfly migrants must have a compass (probably a time-compensated solar mechanism), and a means of sensing the wind direction . By contrast, hoverflies arriving in the UK in spring showed weaker orientation tendencies, and did not correct for wind drift away from their seasonally adaptive direction (northwards). However, the spring migrants necessarily come from the south (on warm southerly winds), so we surmise that complex orientation behaviour may not be so crucial for the spring movements. This maWhile it is true that the smallest members of the daytime aerial fauna (micro-insects with body masses less than 10 mg) have no control over their movement direction once airborne , the proOwing to the enormous scale of these migrations, and the diversity of roles that the immigrants play, such seasonal movements are of great significance to ecosystem functioning . In the Episyrphus balteatus and Eupeodes corollae, hereafter collectively referred to as ‘migrant hoverflies’. These species make annual migrations into and out of higher latitude regions in copious numbers [figure 1a.One of the most abundant and important components of the day-flying meso-insects are hoverflies (Diptera: Syrphidae), particularly smaller species such as  numbers ,19,20 an numbers ,5. Migra numbers ,22. Cruc numbers , seeming numbers ,24. A re−1 [The self-powered airspeeds of migrant hoverflies (approx. 2–3 m s−1 ) will ty−1 . In the −1 ), which 2.(a)We studied the flight behaviour of migrant hoverflies greater than 150 m above ground level (a.g.l.) during their spring and autumn migrations of the 10 year period from 2000 until 2009 inclusive. The ‘spring migration period’ was defined as May and June, and the ‘autumn migration period’ as August and September , but we (b)Ep. balteatus  and Eu. corollae, two aphidophagous species which are known to be migratory in northern Europe [Ep. balteatus caught in the UK [Ep. balteatus in body mass. As the body mass range of Eu. corollae overlaps with that of Ep. balteatus, but all other migratory hoverflies have different body masses [xxσ and yyσ) of wild-caught Ep. balteatus were previously measured by us [xxσ/yyσ produced a value of 8.8 ± 4.0 (mean ± 1 s.d.) [xxσ/yyσ lying between 5 and 10, as these would all fall within the range of the laboratory measurements for our main study species. If migrant hoverflies fly with a body axis that has a substantial pitch angle relative to the horizontal, this would potentially effect the estimation of their xxσ/yyσ ratio and thus lead to mis-classification of targets. We confirmed that hoverflies migrate with a horizontal body axis by examining frames from a video of a mass immigration of Eristalis tenax hoverflies arriving at the northeastern tip of Cyprus in spring 2019: mean difference between body pitch angle and a perfectly horizontal reference line was 0.3° . Our radar filtering procedure led to a total of 367 817 individual targets categorized as migrant hoverflies during 2628 separate ‘migration occasions’ (any location/date combination when at least one migrant hoverfly was detected). Our previous analyses [The criteria and justification for identifying migrant hoverflies among the plethora of VLR-detected insects in our database  is explained in detail elsewhere , but we n Europe . Radar sn Europe . Firstlyn the UK  to charay masses , we can y masses  to seleced by us  using aned by us . Laborat 1 s.d.) , and, th(c)To analyse directional patterns, we used a subset of the data which we termed hoverfly ‘mass migrations’. These were selected by: (i) ordering all ‘migration occasions’ (when one or more migrant hoverflies had been detected), in the spring and autumn separately, from the highest number of hoverflies detected to the lowest; (ii) categorizing all ‘migration occasions’ as ‘mass migrations’ until cumulatively 75% of the total number of individual hoverflies in that season was reached; and (iii) removing all remaining migration occasions from the directional analyses. This ensures that only mass migrations are used to identify seasonal migration patterns. This process resulted in 434 of the hoverfly migration occasions  being defined as mass migrations accounting for 75% (155 538) of the individual hoverflies, with 186 in spring and 248 in autumn . For every individual hoverfly that passed through the beam, the VLR automatically recorded the migratory track vector (i.e. the displacement speed and direction relative to the ground); both of these parameters are largely determined by the wind vector but are also somewhat influenced by the insect's flight vector . The VLRr’  for each distribution; and (iii) the probability that the distributions of tracks and headings differed from a uniform distribution . All hoverfly migration events had significant common alignment of their track directions , but not all of them showed a significant degree of common orientation.The mean migration track and the mean flight heading, plus associated circular statistics, were calculated for each of the 434 mass migrations of hoverflies, using the Rayleigh test of uniformity . The folr-values  and from individual mass migration events.We then calculated the overall mean track and heading directions of the hoverfly mass migrations in the spring and autumn, by analysing the mean tracks and headings of the individual mass migrations with the Rayleigh test once again . We did α  or clockwise of the track  or further away from the seasonal PDM than the track , and then an analysis was carried out to identify the degree of correction for lateral wind drift in each season , or if there was a significant positive (which would indicate significant correction for wind drift) or negative bias [α and the 95% confidence interval (CI) [Φ  , and if val (CI) ,27. Thist angle’ , see thehttps://www.metoffice.gov.uk/), for the spring and autumn periods of 2000–2009 at the same altitude (24 records d−1) as our VLR sampling regime (150–1200 m a.g.l.). Seasonal downwind directions were also calculated using Rayleigh tests, and Watson–Wheeler tests were used to test for significant differences between the mean directions of insect and wind datasets [Modelled estimates of downwind directions and speeds above our radar locations were acquired from the UK Meteorological Office website (datasets . Becausedatasets ) to test3.a; directions were also very similar when calculated using all individual hoverflies rather than daily means, see the electronic supplementary material, figure S5). To test whether these seasonal directions could simply arise from a random selection of days for migration, we compared the distribution of downwind directions on all days: prevailing daytime winds averaged across the radar locations blew towards 61° in spring  but in a more eastwards direction, towards 84°, in the autumn , indicating active selection of favourably directed winds during spring mass migrations. In the autumn, the pattern was not so clear, as hoverfly mass migrations occurred on a considerably wider range of wind directions. However, there was a more southward component and a significant tendency towards the southeast on mass migration days . Thus, in the autumn, there is evidence for a certain degree of selectivity of winds (downwind directions towards the southeast versus east-northeast); however, the wind selectivity was not as pronounced as it was in the spring , nor was the mean downwind direction as close to the seasonal PDM in the autumn as the corresponding values in spring. Given the apparently reduced level of wind selectivity in the autumn, and the general unsuitability of the winds in this season, how then do autumn migrants achieve migration in the seasonally beneficial direction?Our previous study of migrant hoverflies showed that they exhibit seasonally beneficial migration directions, towards 342° in the spring and due south in the autumn  and south-southwest in the autumn , indicating a greater population-level preference (or capability) to orient in an advantageous direction in autumn. Thirdly, the clustering of individual headings around the mean during individual mass migrations was also significantly higher during the autumn . Fourthly, the mean heading direction in autumn (198°) was identical to the seasonal PDM (see below), whereas the corresponding values in spring  were rather different. Taken together, these results demonstrate that orientation performance was considerably higher in the autumn, with migrants seemingly more motivated to (and/or capable of) stronger orientation towards the seasonal PDM than during the spring.The migrant hoverflies exhibited a significant degree of common orientation of their flight headings during a high proportion of mass migrations . Similar to the migration tracks, the days with significant common orientation had mean headings in broadly seasonally beneficial directions: towards the northwest in the spring  during spring and autumn in two related analyses. Firstly, using the method of Green & Alerstam [α . There was no clear relationship of track versus α in the spring, with a very wide scatter of points , indicating that spring hoverflies make no attempt to correct for drift. By contrast, the pattern of track against α in the autumn was very different, with a strong linear relationship and much less scatter . The slope of 1.18  was significantly greater than 1 (p < 0.05), which indicates that the orientation strategy employed is the one we have previously termed CBDO [α = 0, and thus the direction in which there is no correction) gave a value of 197.7°  for the estimate of the seasonal PDM. We therefore used a value of 198° for the autumn PDM in the subsequent analyses. In order to look for evidence of time-compensation in the compass mechanism, we calculated the values of the mean track and mean heading of hoverfly mass migrations for each hour of the day and compared the hourly values with the overall mean value across the whole day . Time of day did not affect either directional measure ; in both cases, hourly mean values remained very close to the overall daily mean value and there was no systematic pattern in the variation . This result indicates that hoverflies fully compensate for the Sun's apparent motion over the course of the day [Next, we investigated the patterns of the angle Alerstam , we plotmed CBDO . The int the day .α was assigned a positive value) with mass migrations where the heading was further away . During the spring mass migrations, the mean value of α was not significantly different from 0°, and there were approximately equal numbers of positive and negative values, for both the total dataset, and the two subsets of data with small and large drift angles (Φ), indicating that the direction of the heading was not related to the track and seasonal PDM . By contrast, the autumn mass migrations showed a highly skewed distribution of α, with 83% of the values in the total dataset being positive, and this distribution was significantly different from 0° with a mean value of +28° . Further examination of the autumn data showed that values of α were random with respect to the track and PDM when drift angles were less than 10° , but when Φ was greater than 10°, the hoverfly mass migrations showed a highly skewed distribution of positive values (87% of headings lay closer to the PDM than the tracks) with a mean value of +30° . This result indicates that as the degree of wind-induced lateral drift away from the seasonal PDM in autumn (198°) increased, hoverflies showed a greater offset of their headings from the windflow in an attempt to partially correct for the drift.Secondly, we compared the frequency of mass migrations when the mean heading was closer to the seasonal PDM than the mean track , with 50% of occasions with speeds between 4 and 9 m s−1 (a), and they were significantly slower than winds during non-mass migration days . Winds were slightly faster in spring than autumn ; however, the effect of season on wind speed was not significant , although there was a significant interaction between season and migration , indicating that hoverflies selected weather conditions with the slowest winds during autumn mass migrations (a). Displacement speeds were somewhat faster than wind speeds, signifying that much of the self-powered flight vector was directed in the downstream direction. However, the combination of selection of slightly slower winds, plus a greater offset of the heading vector from the downwind owing to correction for lateral drift, resulted in significantly slower displacement speeds during the autumn than the spring .Finally, we investigated the impact that seasonal differences in wind selectivity and orientation performance had on the displacement speed of the migrant hoverflies. The mean wind speeds during mass migrations were relatively low , despite the unfavourableness of the wind. The first stage is that mass migration only occurs on days on which the wind direction is more favourable than the average situation (i.e. with a greater southward component). They also showed a tendency to migrate on days with slower winds than the average, and although this was true in both seasons, the effect was most pronounced during the autumn. The selection of relatively slow winds, blowing closer to the seasonal PDM, provides some benefit to the autumn migrants, but on its own is not enough to allow them to migrate towards the south. In order to achieve this, the autumn migrants also showed a strong pattern of collective orientation towards their seasonal PDM. Our estimate for the autumn PDM (198°) is very similar to previous observations of flight directions of hoverflies migrating close to the ground across western Europe . Insectet al. ,27. The et al. , and onlDrosophila) is able to maintain a fixed course along an arbitrary  direction by visually navigating with respect to both celestial patterns of polarized light [Our results indicate that migrant hoverflies must have: (i) an internal compass that allows them to identify their seasonal PDM, and (ii) a mechanism for identifying the wind direction. The nature of the compass mechanism has not been elucidated in hoverflies as yet. Another dipteran (ed light  and the ed light . Given ted light , it seemed light ,37, and Greater uncertainty surrounds the marked seasonal difference in orientation performance. It has been previously predicted that in species which seasonally invade higher latitudes, natural selection for mechanisms which enable migrants to select favourable winds and orient appropriately will be much stronger for autumn emigrants escaping deteriorating conditions than the original spring immigrants . Indeed,Migrant hoverflies are just one component of the highly diverse and hugely abundant meso-insect community engaging in seasonally directed diurnal migrations above the UK ,5, and i


Pinus koraiensis clones grown in the Naozhi orchard in northeastern China. All clones were significantly different in most traits. Most of the phenotypic characteristics showed great genetic variation among clones, while the genotypic differentiation was weak between the selection sites of clones. The SSR markers showed a relatively high level of genetic diversity  of 0.574), with higher heterozygosity as an indication of a lower probability of inbreeding events in the orchard. Despite weak correlation coefficients between dissimilarity matrices (r(A/B), range equal to 0.022, p-value < 0.001), the genetic and phenotypic classifications congruently subdivided all the clones into three major groups. The patterns of phenotypic trait variations and genetic diversity are valuable to effectively select materials in breeding programs of P. koraiensis.Combining phenotypic and genetic characteristics in a genetic variation study is of paramount importance to effectively orient the selection of producers’ elite trees in a seed orchard. In total, 28 phenotypic characteristics and 16 microsatellite loci were used to analyze the clonal genetic variation, to characterize the genetic diversity, and to refine the genetic classifications of 110  Pinus koraiensis Sieb. et Zucc (Korean pine) is an important economic timber species that dominates the mixed broadleaf-conifer forests in northeastern China [P. koraiensis naturally occurs in a reduced area within northeastern China, north of North Korea, central Japan, and southeastern Russia [2 sequestration, water regulation and soil protection for plantations on slopes [rn China . Korean rn China . P. koran Russia . It is wn Russia . The nutn Russia . Becausen slopes , the Korn slopes . Currentn slopes .P. koraiensis breeding program is considered among the oldest and largest forest tree improvement programs in China. Phenotypically superior Korean pine trees have been selected from natural forestlands and have been established in seed orchards since the early 1960s [P. Koraiensis populations [P. koraiensis spp. were selected based only on their growth traits or wood properties to meet the demand for timber over the past several years [P. koraiensis [The ly 1960s . Many stulations . Improveal years . Howeverraiensis ,12. Consraiensis .P. koraiensis trees and elite materials has traditionally been based on phenotypic characteristics, many genetic markers have been developed to characterize Korean pine genetic diversity [Although the selection of superior iversity ,15 and aiversity ,17. HoweSpecies genetic diversity highlights a pivotal role for trees under breeding programs to develop well-adapted tree varieties and heighten genetic gain for a multitude of useful traits ,19. To aThe genetic diversity parameters measure the degree of resemblance and difference in the genetic characteristics of individuals or the population and could therefore guide assisted hybridization processes by preventing associations of genetically closed individuals . Much reThe pairwise relatedness measurement displays a significant role in the genetic resource conservation. For instance, in a seed orchard, substantial effort would be made to ensure that close relative trees are not crossed to reduce inbreeding and reduce the loss of genetic variation by random genetic drift . Using mP. koraiensis clones in the Naozhi seed orchard. The data of 16 codominant microsatellites were combined to compare genetic variation with phenotypic variability. Hence, the objectives of this study were to (i) analyze the clonal phenotypic and genetic variation, (ii) compare and combine phenotypic and genetic distances to assess the genetic diversity of P. koraiensis, and (iii) establish the genetic relationship among clones/genotypic relatedness based on phenotypic and molecular markers.The present study analyzed the phenotypic variability and genetic diversity of different clones (parent trees) and sought to establish their genetic similarities to lay the foundation for understanding vigor in offspring from possible combinations of different clone genotypes. In total, 28 phenotypic traits  were investigated and analyzed using 38-year-old P. koraiensis forest and broad-leaved mixed forest are typical forest vegetations of this region and occur at an altitudinal range between 700 m and 1100 m. The area experiences a continental monsoon climate type. In this region, the summer temperature is moderate because of the altitudinal gradient and rains, while the winter is long and cold. The average annual temperature and average annual rainfall range between 4 °C and 6 °C and 750 mm to 1000 mm, respectively. The frost-free period is 135 days. The soil type corresponds to the Albi-Boric Argo sols, according to U.S. soil taxonomy [Data were collected from the Naozhi forest seed orchard located on the western hillside of Changbai Mountain in Linjiang city, Jilin Province, northeastern China . The taxonomy , and is taxonomy .P. koraiensis clones (PK1-PK110). In 1979, superior Korean pine trees (based on their size) were selected from the natural P. koraiensis forestland in Linjiang, Jilin Province  (PKJ-1 to PKJ-90) and Liaoning Caohe, Liaoning province  (PKL-91 to PKL-110). These clones were grafted in the year following their collection, and plantations were established with four-year-old seedlings in the spring of 1984. The experimental design consisted of a complete randomised block design using single tree-plots, with 10 blocks containing 110 different clones, and ramets of each clone were planted at inter plant and row spacing of 7.0 m × 7.0 m. Tree measurements and plant materials were collected from surviving ramets of the 110 The following traits were measured on living ramets in July 2018: 11 growth traits, including tree height (Ht), basal diameter (BD), diameter at breast height (DBH), diameter at 3-m height (DIAM-3m), bark thickness (BTH), stem volume (Vol.), stem straightness degree (SSD), branch angle (BA), crown breadth (CB), crown height (CH), and branch number (BNN); three wood traits, including wood density (WD), fiber length (FL), and fiber width (FWd); and 14 cone, seed, and nut traits comprising cone number (CN), cone length (CL), cone width (CWd), cone weight (CW), layer number (LN), seed number per cone (SNC), seed length (SL), seed width (SWd), seed weight (SW), nut length (NL), nut width (NWd), nut weight (NW), seed coat thickness (SCTH), and 1000 seed weight (1000 SW).Ht and CH were measured with a meter ruler; BD, DBH, and DIAM3 were measured using a caliper; SSD was estimated and squared before calculation using the method of . CB was From each clone, wood cores were collected from all ramets, following the altitudinal south-north orientation at breast height level (1.3 m) using an increment borer and were then taken to the laboratory for investigations of their wood properties. The WD and fiber dimensions (FL and FW) were measured according to the method used by Yin et al. .For fruit-related traits, all the cones were collected from each ramet after reaching maturity. Cones were counted (CN) and directly weighed (CW). To obtain a balanced number of cones per clone, cones from previous years (2014 to 2018) were taken into account, and the calculated average was used in the analysis. Meanwhile, the cone size seeds and nuts were measured only in the year 2018. CL and CWd (perpendicularly at the broadest part) were measured with a digital caliper. After taking dimensions and weights from cones, all the seeds were removed from cones, and the number of seeds per cone per ramet was counted; the average value was used as cone SNC. To calculate the 1000 SW, seeds from different ramets were mixed. Then, 400 seeds were randomly chosen and divided into four equal parts to serve as replicates. Each group of 100 seeds was weighed, and the thousand seed weight was obtained by extrapolation from the weight of 100 seeds. Fifty seeds from each clone were randomly chosen to determine seed characteristics and measure SL, SWd, and SW. Finally, nuts were extracted from measured seeds, and NL, NWd and NW were assessed. SCTH was measured 10 times from different sides for each seed using a Vernier caliper, and the average values were used.P. koraiensis needles using a modified cetyltrimethylammonium bromide (CTAB) method, according to Allen et al. [Fresh needles were collected from each clone from an accessible branch. The collected needles were directly chilled under ice, transported to the laboratory and stored in a freezer at −80 °C for subsequent DNA analysis. Genomic DNA was extracted from n et al. . The DNAn et al. ,37) and 2O. The PCR was performed according to the following cycling program: denaturation step at 95 °C for 2 min; followed by 35 cycles of 95 °C for 20 s, annealing at 53~58 °C for 20 s , and 72 °C for 20 s for extension; and a final extension at 72 °C for 10 min. The reactions were then kept at 12 °C. All forward primer sequence markers were labeled with four fluorescent dyes: HEX (green), FAM (blue), TAMRA (yellow), and ROX (red). Amplified SSR fragments were analyzed using a capillary electrophoresis sequencer ABI 3730 XL DNA analyzer . The allele sizes were recorded using Gene Marker software V2.2.0 . DNA amplification (PCR reaction) was carried out in a total volume of 25 μL containing 2.5 μL of ExTaq buffer, 2 μL of dNTP (2.5 mM), 1 μL of MIX primer, 1 μL of plant DNA, 0.2 μL of ExTaq , and 18.3 μL of HAverage clonal performances were evaluated, and the genotypic and environmental/random effects were estimated by the following linear mixed model, n 3.6.1) , with Yiectively . Two-wayectively  to deterg et al.  by dividg et al. . To measg et al.  was usedP. koraiensis genotypes was performed using an agglomerative hierarchical clustering (AHC) method [https://www.xlstst.com). [Clonal phenotypic performances were standardized by subtracting the mean value and dividing by the standard deviation to construct an individual pairwise genetic distance using the Mahalanobis distance of dissimilarity. The dissimilarity matrix was then used to compare the genetic distances between phenotypic and molecular characteristics . Finally) method . The unwst.com). . biology-assets.anu.edu.au/GenAlEx) [ST) was retained from the analysis with GenAlEx software to test whether the two collection sites of clones were genetically different and to link genotypic differences to observed phenotypic characteristics. Genotypic data of clones were recorded by SSR markers  using codominant values. First, clone kinship estimation using the SSR genotypes was done according to the protocol of Wu et al. . The allGenAlEx) . The genThe Bayesian model of  integratwww.itol.embl.de) [A pairwise comparison of individual genetic distances was performed using Nei’s genetic distance method  in Powerembl.de)  was usedembl.de) . The comp-value ≤ 0.001) based on ANOVA  for K  for K a. Howevea) for K b. A few a) for K c. The 11a) for K c—Clustera) for K d based op-value < 0.001), suggesting that the molecular and phenotypic categorizations of P. koraiensis were nearly identical.To determine the correspondence level between the dissimilarity matrices based on phenotypic characteristics and SSR markers, a Mantel test of correlation was performed. The correlation result is shown in In general terms, phenotypic characteristics are considered the basic elements for detecting dissimilarities/similarities between individuals from the same or different tree species . In treeP. koraiensis clones. Similar findings were reported on many tree species, indicating that selection based on phenotypic variation results is feasible [P. koraiensis variation studies [The mean growth performances showed a significant difference among clones for most growth traits, indicating the effects of clone genotype were significant, and this result was supported by a clonal repeatability greater than 70% for these characteristics. This result is promising for the selection of feasible ,61,62,63 studies ,10; this studies . The genetic variation parameters evaluated in this work on phenotypic traits seem to be higher, especially in fruit-related traits, with repeatability values up to 90%. A previous study of  reportedPrunus avium L. [Festuca arundinacea Schreb. [Medicago sativa L. [All 110 clones exhibited a strong significant difference in most of the investigated traits. However, the clustering dendrogram  showed tavium L.  and Fest Schreb. , along wativa L. , suggestP. koraiensis clones in this study showed high genetic diversity based on SSR marker analysis. The average number of alleles and effective alleles per locus  and the mean He (0.62 ± 0.02) and uHe (0.63 ± 0.02) were higher compared to the diversity parameters reported for Pinus thunbergii [P. koraiensis population containing seeds, male and female trees using 14 SSR markers. Our results corroborated previous studies [Since phenotypic markers are affected by the environment, they are less useful for characterizing neutral genetic diversity to effectively capture the genetic diversity required to maintain a wide genetic base in future breeding programs ,71,72,73unbergii . Howeverunbergii ,77, whic studies ,79, whicP. koraiensis is an essential factor for improving the efficient selection of genotypes that might resist different environmental hazards  [P. koraiensis was greatly affected by wind direction during the pollination season. The Fixation Index (F) of −0.11 ± 0.03 that we found in this orchard complex suggested strong inbreeding. Similar results of limited heterozygotes in seed orchard materials have been reported by other workers , and Fixation Index (F) were 0.5736 ± 0.294, 1.15 ± 0.07, and −0.11 ± 0.03, respectively, and were comparable to the findings of  that anas, etc.) . Severals, etc.) ,82,83. Os, etc.)  demonstrd et al. ).P < 0.001)  . This fi< 0.001) , the Bay< 0.001) c, and ne< 0.001) d showed The Mantel test , which cP. koraiensis, which will be used to improve growth and seed production and to maintain a wide genetic diversity in future breeding programs. All the clones were phenotypically different at moderate to high levels of significance. The phenotypic difference was stronger due to the genetic properties of clones than to the environment and physiological effects. A significant relationship was observed between clone classification based on the phenotypic and genetic distances matrices. The results based on the SSR techniques revealed a high level of genetic diversity in the P. koraiensis clones, which is essential for effective selection regarding the reduced and nearest area in the collection sites and the number of individuals in this study. Three main groups were identified by both genetic and phenotypic data.The present study focused on the variation in phenotypic traits and genetic relationships that exist among a large number of genotypes from clones from plus trees of P. koraiensis cloned trees at the Naozhi orchard were determined. The classification dendrogram allows identification of whether selectable clones display similar performances in terms of genotypic/clonal values on the ordination axis that may be done on growth performance-based volume and seed weight. For instance, a 10% slice of high performing materials, which later would serve as selection objects for wood products or seed qualities, showed relatively low to high similarity coefficients ranging between 0.22 and 0.82. The different genotype groups allow one to determine whether these potentially selectable materials are genetically near or distant from each other in order to prevent inbreeding, which could impact the genetic properties of seeds that are produced and the long-term future improvement of the species.Based on this work, growth genotypic values and genetic characteristics of 110