Alternaria tenuissima, a pathogenic fungus causing foliar disease in tomato, using next-generation sequencing (NGS) technology. In total, 24,670 unigenes were annotated using five databases, including NCBI non-redundant protein, Swiss-Prot, euKaryotic Orthologous Groups, Kyoto Encyclopedia of Genes and Genomes, and the Gene Ontology. A total of 1,140 simple sequence repeats were also identified for use as molecular markers. Sixteen of the simple sequence repeat loci were selected to study the population structure of A. tenuissima. A population genetic analysis of 191 A. tenuissima isolates, sampled from four geographic regions in China, indicated that A. tenuissima had a high level of genetic diversity, and that the selected simple sequence repeat markers could reliably capture the genetic variation. The null hypothesis of random mating was rejected for all four geographic regions in China. Isolation by distance was observed for the entire data set, but not within clusters, which is indicative of barriers to gene flow among geographic regions. The analyses of Bayesian and principal coordinates, however, did not separate four geographic regions into four separate genetic clusters. The different levels of historical migration rates suggest that isolation by distance did not represent a major biological obstacle to the spread of A. tenuissima. The potential epidemic spread of A. tenuissima in China may occur through the transport of plant products or other factors. The presented results provide a basis for a comprehensive understanding of the population genetics of A. tenuissima in China.A total of 32,284 unigenes were obtained from the transcriptome of  Alternaria tenuissima is an important global pathogen on a large variety of economically important crops, including broad bean, tomato, sunflower, potato, watermelon, and muskmelon  and used to identify large numbers of SSRs. This was done to determine the level of genetic diversity in A. tenuissima populations in China and infer the main evolutionary factors influencing the epidemic outbreaks of A. tenuissima. The distribution of SSR motifs in the transcriptome of A. tenuissima was characterized and the assembled unigenes were functionally annotated.A high level of genetic variation has been reported in  species . High len Russia . Little A. tenuissima isolates were collected during 2015 and 2017 from 34 sampling locations in China  procedure and used for molecular identification and additional SSR assays (1 to identify the Alternaria species.Isolates were identified using the standard procedures reported in our previous study . The proR assays . For theR assays . The PCRA. tenuissima isolates. The mycelia from a culture of “BJ319-1” growing on potato dextrose agar (PDA) plates for 7 days, were harvested for the isolation of total RNA and subsequent transcriptome analysis. Total RNA was extracted using TRIzol reagent . Any traces of DNA were then removed from the RNA extracts using DNase I . The purity of the RNA extract was determined using a Nano-Drop 2000 . Qubit 2.0  and an Agilent 2100 Bioanalyzer  were used to estimate the concentration and integrity of the total RNA. The cDNA library of pooled RNA was constructed with a method described by A. tenuissima cDNA library was sequenced using an Illumina HiSeq 4000 sequencing platform at Beijing Biomarker Technologies Co., Ltd. .One isolate “BJ319-1” was randomly selected from among the 191 collected 2, under the accession number SRP136412. Subsequently, clean reads were assembled using Trinity , Swiss-Prot, euKaryotic Orthologous Groups (KOG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) (To annotate the s (KEGG) . Blast2Gs (KEGG)  was usedA. tenuissima transcriptome sequence data using MISA (MIcroSAtellite identification tool) and SAMtools , 9.5 μL ddH2O, 12.5 μL 2 × T5 Super PCR Mix (TSINGKE Biotechnology Co. Ltd.), and 1 μL each of the two primers (10 μM). The amplification was conducted in an Eppendorf Mastercycler® using the following protocol: an initial denaturation step at 95°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 s; annealing at 57°C for 30 s, and extension at 72°C for 30 s; with a final extension for 5 min at 72°C. The obtained amplicons were then sequenced using an ABI 3730 DNA sequencer (Applied Biosystems).To further characterize the population genetics of the Alleles were aligned using GeneMarker v.2.2.0 software . Sequenced fragments with an identical size originating from the same primer pair were considered as an allele. Multilocus genotypes, defined as having the same alleles at each of the single SSR loci, were detected using GenClone v.2.0 . IsolateFST), summary heterozygosity (H) from each locus  and multilocus linkage disequilibrium values (rd) by 1,000 randomizations according to IA and rd are not significantly different from the expected value of 0, random mating exists in the population  was used in the analysis. The proportion of compatible pairs of SSR loci (PrCompat) was also performed using MULTILOCUS v. 1.3 software  at K = 4 were used as input for CLUMPP_Windows v. 1.1.2  for both individuals and populations were then visualized using DISTRUCT v. 1.1  with ARLEQUIN v. 3.5.2.2 . Statist v. 3.11 .Isolation by distance (IBD) was evaluated by assessing the correlation between pairwise geographical distance and Nei’s unbiased genetic distance  for all emN) and Θ (2eμN), where Ne is the effective population size, m is the constant migration rate between population pairs, and μ is the mutation rate per generation at the locus considered. Likelihood surfaces for each parameter were estimated by simulating genealogies using MCMC approach. The computations were carried out under a Brownian motion approximation of the stepwise mutation model (SMM). The runs consisted of two replicates of 10 short chains  and three long chains , with the first 10,000 genealogies discarded. A likelihood ratio test was used to compare the likelihoods of all models  were obtained from the transcriptome sequence of Collectively, 24,670 unigenes were annotated utilizing five databases, NCBI non-redundant protein (Nr protein), Swiss-Prot, euKaryotic Orthologous Groups (KOG), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Ontology (GO). Among the unigenes, 24,570 (99.6%) exhibited significant similarity to proteins in the Nr protein database, among which 10,562 (42.8%) were also found in the Swiss-Prot database.A. tenuissima. This analysis resulted in the clustering of 7,412 (30.0%) unigenes into 111 pathways. Highly represented pathways included: carbon metabolism , biosynthesis of amino acids , and protein processing in the endoplasmic reticulum . The top 20 biological pathways of the enriched KEGG annotations are presented in Figure A total of 15,503 62.8%) of the unigenes were classified into 25 functional categories according to the KOG functional classification , di- (258 or 22.6%), tetra- (15 or 1.32%), penta- (7 or 0.6%), and hexa-nucleotide (5 or 0.4%) repeat motifs  ranged from 0.309 to 0.582 for the sixteen SSR loci, with an overall average of 0.461. The gene diversity per locus ranged from 0.102 to 0.854  and proportion of compatible pairs of loci (rd), were found to be significant in the four geographic regions for the total sample , indicating that the null hypothesis of complete panmixia was rejected  and q3 . The fifteen populations from Northern China exhibited a high level of admixture, and the isolates were assigned to cluster q4  and q3 , followed by q2 . Most of the isolates from Eastern China were assigned to cluster q1 , and only one isolate was assigned to cluster q2. Most of the isolates from Northwestern China were assigned to q2  and to a lesser extent q3 . Only two Northwestern China isolates were assigned to cluster q4.The Bayesian cluster analysis using STRUCTURE v. 2.3.4 indicated that the number of genetically distinct ancestral populations was best represented by K Figure . The isoBased on the PCoA, the eight populations from Northeastern China clustered within the two right quadrants of the first PCoA axis Figure . The fifP < 0.001) (Table P < 0.001)  performed on the 34 populations indicated that 13.25 and 86.75% of the genetic variation was attributed to variations among and within populations, respectively (FST) between populations were not significant within the geographic regions Northern China, Eastern China, and Northwestern China, except for Baoding city , Northern China , Eastern China , and Northwestern China   and geographical distances (km) for the entire data set . These results suggest that isolation by distance exists among the geographic regions.The strength of the correlation was weak and non-significant within the genetic clusters, Northeastern China (em2N) ranging from 0.54 (Eastern China from Northwestern China) to 3.70 (Northwestern China from Northern China) (Table Considerable levels of gene flow were observed among the geographic regions with an estimated number of migrants per generation M (a) Table . The obsAlternaria have been obtained for A. consortialis (GenBank accession no. BCGG00000000), A. alternata (LMXP00000000), A. arborescens (AIIC00000000), and A. brassicicola (PHFN00000000)  using the Illumina HiSeq 2500 platform in a previous study (Whole genome sequences in the genus 0000000) . A wholeus study . CollectA. tenuissima. Approximately 11.5% of the transcriptomic sequences contained SSR loci. The distribution density of SSRs in A. tenuissima is similar to many other higher plant species, such as rice, wheat, and soybean, which generally express a larger number of genes in a transcriptome than fungi due to the overall size of their genomes. Our results clearly identified a large number of SSR loci in the genes expressed in the A. tenuissima transcriptome.Next-generation sequencing is a highly efficient and low-cost technology that can be used to develop large numbers of new SSR markers . DetectiA. tenuissima populations from four geographic regions. Most of the unigenes with SSR loci had different annotations in GO, KOG, and KEGG databases. The average size of a genome within different Alternaria species is more than 30 Mb  displayed high genetic variation and a lack of random mating. A. brassicicola and suggested that recombination occurred only occasionally. A. solani could be accounted for by mutations rather than by sexual reproduction. These results suggest that random mating is not the main biotic factor that governs the high variation present in the four geographic regions.Sexual recombination is expected to produce high levels of genetic diversity and random association among different loci . In rece. solani , A. brassicicola , and A. lianthin  have beeite loci . The comA. tenuissima causing foliar diseases in China on wheat  Table . There aon wheat , potato on wheat , and waton wheat . These cin China . The genin China .A. tenuissima, however, may account for the observed gene flow between the geographic regions of Northeastern China, Northern China, and Northwestern China  have yet to be determined. Additional population genetic studies of Alternaria are needed for other geographic regions in China and further analyses are needed to determine the population genetic structure of Alternaria isolates over wider geographic regions of China.In recent years, ut China ,b. A comData for this study will be available at the Dryad Digital Repository after manuscript is accepted for publication.NY and XW conceived and designed the study. NY, GM, and KC performed the experiments. NY and XW wrote the paper. XW reviewed and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


During the XX Century, the widespread use of modern wheat cultivars drastically reduced the cultivation of ancient landraces, which nowadays are confined to niche cultivation areas. Several durum wheat landraces adapted to the extreme environments of the Mediterranean region, are still being cultivated in Sicily, Italy. Detailed knowledge of the genetic diversity of this germplasm could lay the basis for their efficient management in breeding programs, for a wide-range range of traits. The aim of the present study was to characterize a collection of durum wheat landraces from Sicily, using single nucleotide polymorphisms (SNP) markers, together with agro-morphological, phenological and quality-related traits. Two modern cv. Simeto, Claudio, and the hexaploid landrace, Cuccitta, were used as outgroups. Cluster analysis and Principal Coordinates Analysis (PCoA) allowed us to identify four main clusters across the analyzed germplasm, among which a cluster included only historical and modern varieties. Likewise, structure analysis was able to distinguish the ancient varieties from the others, grouping the entries in seven cryptic genetic clusters. Furthermore, a Principal Component Analysis (PCA) was able to separate the modern testers from the ancient germplasm. This approach was useful to classify and evaluate Sicilian ancient wheat germplasm, supporting their safeguard and providing a genetic fingerprint that is necessary for avoiding commercial frauds to sustaining the economic profits of farmers resorting to landraces cultivation. Triticum spp.) is the largest primary commodity and one of the oldest and most important cereal crops, being grown on more land area than any other crop, worldwide. Durum wheat (Triticum turgidum L. subsp. durum Desf.) is the second most important Triticum species, next to common wheat (Triticum aestivum L.), and is mainly used to produce semolina and dried pasta. The European Union (EU) holds the largest share in world durum wheat harvested production (8956.58 thousand tons), Italy being the main EU producer with 4303.10 thousand tons . Durum wheat is a crop of primary importance for the Mediterranean region and is mainly cultivated under rainfed conditions, resulting in major yield variations, due to irregular rainfalls. Outside of the Mediterranean region, durum wheat is cultivated in Northern America  and Central America (Mexico), as well as in smaller growing areas of Russia, Australia, Argentina, and Ethiopia.Wheat  and an unknown diploid species  closely related to Aegilops speltoides [Triticum species are native to the Fertile Crescent of the Near East [Granary of Rome.Durum wheat is a member of the eltoides . Thus, dear East . In partear East . Wheat cAt the beginning of the last century, durum wheat breeding was pioneered in Italy when durum and common wheat started to differ more clearly . Many teThe spread of modern wheat cultivars drastically reduced the cultivation of the “old landraces”, confined to the niche areas of cultivation, with their maintenance being entrusted to public research institutions and custodian farmers. Nowadays, these landraces represent a collection of important genetic resources with traits of biological and economic significance, as they have been subjected to several cycles of artificial and natural selections, as reported by De Cillis . The preKnowledge on genetic diversity of durum wheat landraces should be the starting point for their utilization and conservation management. Wheat genetic diversity has been mostly evaluated using morphological and phenological descriptors, established by the International Plant Genetic Resources Institute (IPGRI)  and the Triticum durum, T. aestivum, and T. turanicum, previously collected and described thanks to the effort of De Cillis [Among Mediterranean regions, Sicily is one of the richest in wheat landraces diversity. In Sicily, several durum wheat landraces were recently re-discovered and re-employed because of their adaptability to extreme Mediterranean environments, due to their genetic structure, typical of a self-pollinated population (mixture of different pure lines). The Experimental Sicilian Station for Durum Wheat (ESS) at Caltagirone , is the wheat research station responsible for the conservation of local varieties, breeding and research for wheat growers, and processors in Sicily. The ESS bred and preserved over 200 accessions along the years, including 48 local varieties of e Cillis . The gerThe aim of this study was to evaluate the genetic diversity in a wheat collection from the ESS, using the SNP markers, and agro-morphological, phenological, and quality-related traits. A detailed evaluation and classification of the Sicilian durum landraces will allow their proper preservation, promote their sustainable use, and eventually provide a reproducible genetic fingerprint that can be used for avoiding commercial frauds and sustaining the economic profits of the farmers.Durum wheat collection effectively preserved and documented at the ESS were characterized by their different morphological, phenological and grain quality traits. A heatmap was generated for a simplified representation of the morphological trait distribution .Overall, these traits allowed us to group the germplasm in five main clusters, with 50% of landraces/cultivars belonging to cluster 1 and 2, while 5, 4, 3, and 3 genotypes belonged clusters 3, 4, 5, and 6, respectively. Interestingly, ten traits showing high variability among the samples, as determined by their clusters—six traits , and the plant height ranged from 95 and 135 cm. Nineteen percent of the landraces showed a plant height below 120 cm . Based o920–1950 . These r920–1950 , confirmMains comparison and post-hoc test of grain and wholemeal flour quality-related  traits of wheat genotypes are reported in In durum wheat that have not undergone weathering, test weight (TW) is an excellent predictor of the semolina milling potential. Indeed, this parameter indicated the specific seed weight per unit volume, depending on the filling degree of the seed, was used to indicate the likely milling performance of wheat, and was considered a more reliable predictor of the flour/semolina yield ,30. HereNon-vitreous kernels (starchy), characterized by an opaque area, have a significant impact on the characteristics of kernels during milling, producing a detrimental effect to the end-use quality of durum wheat, and decreasing the semolina extract . The incWholemeal flour quality traits, protein and gluten contents are the most important features useful for characterizing durum wheat landraces/cultivars. Indeed, it is well-documented that the protein content and the endosperm storage protein composition have a decisive impact on the wheat processing quality ,35. HighFrequently, modern durum wheat cultivars show lower grain protein content, compared to the older ones ,39,40, a2 = 0.97) with a high protein content and a wide variability, as already reported [The values of wet and dry gluten content, important for the pasta industry, showed a comparable trend  from the landraces belonging to T. turgidum L. ssp. durum, and the last components were able to discriminate between historical and modern cultivars (T. aestivum L. was characterized by a higher number of starchy kernels (cos2 = 0.75), strongly related to the first component (p < 0.05) among water binding in wet gluten, and dry and wet gluten, with ae higher positive correlation coefficients (0.96) for the last two components and a negative correlation (p < 0.05) between starchy kernels and protein content (−0.64) (p < 0.05).The Principal Component Analysis (PCA) performed on all the quality-related grain traits showed wide differences among landraces . The firultivars . Proteinomponent . These e (−0.64) . LikewisGenetic diversity can be evaluated by biochemical and morphological markers or by the use of pedigree. Unfortunately, these approaches can be influenced by environment or can be erroneous and incomplete, causing frequent misclassifications among the genotypes. By contrast, molecular markers allow the assessment of relatedness at the DNA level, making them necessary for identification of genetic variation among and within landraces/populations, due to the influence of the environment on many traits under polygenic control. Different molecular markers can be used for comparative genomic, phylogenetic relationships, and diversity studies ,54. MoreTo investigate the genetic relationships among cultivars, based on the SNP-data, phylogenetic analysis and PCoA were carried out. Cluster analysis based on Nei (1978) genetic coefficient and the UPGMA algorithm, generated a dendrogram underlining four main clusters across the durum wheat germplasm . Two modPCoA was also carried out to properly describe the clusters reported above, as expected, the first two PCs showed four main clusters, grouping the different genotypes in agreement with cluster analysis . GeneticK) within the collection was determined as K = 7 (K), except bivc, sco4, and tre2. Among landraces, only brc-b1 and bia1 showed a high admixture profile between the gold and green, the purple and blue pools, respectively  values ranged from 0.31 to more than 0.57, the most distinct individual being cuc1 . SelectiGenetic variation in wheat landraces was often analyzed by using morphological and biochemical markers , but theHere, we investigated a collection of durum wheat landraces by using morphological and quality-related traits, together with a large SNP panel . The gerSNPs appeared powerful for evaluating genetic diversity and classifying different durum wheat landraces, due to their reproducibility and the development of relatively high-throughput platforms. Cluster analysis based on the Nei (1978) genetic coefficient and UPGMA algorithm generated a dendrogram underlining the genetic relationships among landraces/cultivars, distinguishing four main clusters across durum wheat germplasm. PCoA confirmed the clusters with the first two PCs highlighting four main groups of genotypes. SNP were also able to discriminate among landraces and within populations by isolating a panel of SNP able to distinct haplotypes. This ability might be useful to define gene-banks conservation strategies by avoiding genetic erosion through selection. The SNP panel will allow us to organize an efficient system for genetic traceability of wheat end products, eluding commercial frauds and sustaining economic profits for the farmers.In light of our results, the interesting panel of investigated genotypes could represent a platform for parental selection to develop high-yield durum wheat lines in breeding programs and for association mapping studies.Twenty-seven durum and one bread wheat Sicilian landrace from the collection of the “Experimental Sicilian Station for Durum Wheat” (ESS) of Caltagirone (Catania), two historical cultivars (Cappelli and Trinakria) parents of many Italian breeding programs, and two modern cultivars (Claudio and Simeto), widespread in Sicilian cereal growing areas were included in this study .2 plots, according to a randomized blocks experimental design with three replicates, adopting the typical wheat agronomic management.Field trials were carried out in 2013 and 2014 in Sicily  in a medium-sandy soil, to assess the main bio-productive traits and quality of the grain and whole grain flours. Genotypes were laid out in the field in 10 mIn accordance with the Community Plant Variety Office descriptors , twenty-Samples of durum wheat grains per each plot were analyzed for the main quality traits. Protein content (% dry matter) was determined by means of Infratec 1241 Grain Analyzer  by near infrared transmittance (NIT), using a calibration based on the Kjeldahl nitrogen method (UNI EN ISO 20483). Calibration was validated in accordance with UNI EN ISO 12099 , using dFurther, representative samples of grains per plot were used to measure the thousand kernels weight (TKW), test weight (TW), and the starchy kernels. TKW was obtained by weighting 8 samples of 100 seeds each and the average weight was compared to 1,000 seeds.TW was determined with a Test Weight Module (TWM) installed under the Infratec 1241 Grain Analyzer . The visual estimation of the starchy kernel percentage was carried out on a representative seed sample (30 g). Kernels were sorted visually into wholly vitreous grains and not (at least two spot) and this last was expressed as a percentage of the total seeds. Worldwide recognized procedures (ISO and ICC standard method No. 129) defined fully vitreous kernels as “those that do not disclose the least trace of farinaceous endosperm” .65 and a* and b* corresponded directly to the red and yellow indices, respectively, while the brown index was obtained as 100-L*. Protein, wet and dry gluten content, WBC, and starchy kernels were analyzed using the arc sine transformation [Grain obtained from each treatment was milled to obtain wholemeal flour by an experimental mill, Cyclotec type 120 , with a sieve of 0.5 mm. Physical and chemical gluten features were analyzed by using a Glutomatic 2200, a Centrifuge 2015, and a Glutork 2020 . Wet and dry gluten content and quality (gluten index) were calculated according to the ICC Standard No. 158 . Water bormation , prior tSamples of wheat grains were analyzed for eleven main quality and commercial traits—protein content, thousand kernels weight, test weight, starchy kernels, wet and dry gluten content, water binding in wet gluten, gluten index, brown index, yellow index, and red index. A Hierarchical analysis of variance (ANOVA) was calculated and a post hoc Tukey’ test was adopted to compare the means of 32 durum and bread wheat landraces/cultivars using XLSTAT version 2018.3. Percentage data were normalized expressing them as arc sin root square. Percentage data of starchy kernels, protein content, wet and dry gluten contents were normalized, and expressed as arc sin root square.p < 0.05) was also calculated among the quality-related traits using the Hmisc R/package (https://cran.r-project.org/web/packages/Hmisc/index.html) and a scatter plot with the correlation coefficients and their significance was developed with the Performance Analytic R/package .Principal Component Analysis (PCA) was performed using the R package FactoMiner . Finally® ND-1000 , respectively. DNAs were delivered to TraitGenetics GmbH  for genotyping. Two hundred nanograms of genomic DNA were used as a template for each reaction, following the manufacturer’s instructions . SNP data matrix was generated from the custom Illumina by wheat 90k SNP array , which assays 81,587 SNPs [Genomic DNA was extracted from the fresh leaves/young seedlings of each landrace and cultivar. The GenElute Plant Genomic DNA Miniprep Kit  was used and the DNA quantity and quality were checked by electrophoresis (1% agarose gel) and NanoDrop587 SNPs .K) available in Sicilian wheat germplasm, FastStructure in the admixture model was used [http://rajanil.github.io/fastStructure/). To assess the genetic distinctness and redundancy among samples, R/AveDissR [Raw data were visualized and analyzed by using GenomeStudio V2011.1 software (Illumina), with the polyploid clustering version 1.0.0. The dataset was filtered and standardized as previously reported . SNPs wiwas used . The anawas used , using tAveDissR  was used


Numerous studies have shown that the phylogeography of many species, including European brown hare, has been affected by the climatic oscillations of the Pleistocene. During this period the Balkans acted as a major refugium offering habitable conditions for many species. However, few studies have focused on the specific role of the Greek peninsula in the phylogeographic history of species in this southernmost margin of Balkans. We, therefore analyzed a 528 bp fragment of the D-loop region of mitochondrial DNA in 154 wild brown hare individuals from unsampled areas from both mainland and island Greece and compared it to 310 available brown hare sequences (including 110 Greek samples). Newly identified haplotypes show characteristic distribution in specific Greek areas reinforcing the theory that Greece can be considered as a subrefuge within Balkans for a number of species, with several “refugia within refugia” spots, holding significant genetic diversity. No haplotypes from wild Greek individuals clustered with the Central and Western Europe group revealing a minimal contribution of this area to the colonization of central Europe. One hundred and ten reared brown hares were also analyzed to elucidate the impact of introductions on local populations. Most of these samples presented close genetic affinity with haplotypes from Central and Western Europe indicating that farms in Greece use breeders from those areas. Therefore, despite human translocation of individuals, the genetic structure of brown hare has mostly been influenced by paleoclimatic conditions and minimally by human actions. Pleistocene glaciation cycles have considerably influenced the current geographic distribution of many European temperate species. During the Last Glacial Maximum . This mammal presents considerable plasticity evidenced by its wide distribution in a range of environments, while it has also been effectively introduced, even in tropical habitats [Pleistocene climate changes are believed to have, also, profoundly affected the phylogeographic structure and the evolutionary history of European brown hare (habitats . Its mobhabitats ) and bethabitats ). Accordhabitats –24. Bothhabitats  and Stamhabitats  confirmeHowever, data on the evolutionary history of brown hare in Greece is missing, especially on populations from Central Greece and the Greek islands, which, some of them, have been shown to be connected to mainland Greece due to sea oscillations during glaciations cycles . We analNo animals were sacrificed only for the purposes of this study. The wild brown hare samples in this study represent material collected opportunistically (after hunting procedure) from animals’ hunter-harvested by members of the Greek Hunting Federation of Sterea Hellas during the hunting seasons, according to the prerequisites of the Greek Legislation . The reared samples included in this study were collected by private veterinarians. No domesticated individual was euthanized during the study and efforts were taken to mitigate animal suffering. There is not an ethics committee in the Aristotle University of Thessaloniki. But, all experimental procedures follow the code of conduct of the Research Committee of the Aristotle University of Thessaloniki. The study was approved through the acceptance of the program by the Research Committee of the Aristotle University of Thessaloniki (Program number: 84430).A total of 154 wild brown hares were collected from various regions of both mainland and island Greece with the assistance of the 4th Hunting Federation of Sterea Hellas. Muscle tissues were obtained during the hunting seasons of 2006 to 2009. Particularly, we conducted a more comprehensive sampling in Central Greece and Aegean islands (including areas which had not been previously sampled or had been poorly sampled) . Additio2 and 2.5 μl of 10 X Reaction Buffer. Thermal cycling amplification conditions were as follows: initial denaturation at 94°C for 3 min, followed by 33 cycles of strand denaturation at 94°C for 1 min, annealing at 50°C for 45 s and primer extension at 72°C for 40 s and a final 3min elongation time at 72°C. The PCR products were purified using the Nucleospin Extra kit  and sequenced by Sanger method from Macrogen Inc.  using an ABI 3730XL DNA Analyzer.A fragment of 528 bp of D-loop  of mitochondrial (mt) DNA (including the entire CR-1) was amplified using primers Le.H-Dloop and Le.L-Dloop . The 5’ L. europaeus retrieved from GenBank: 72 sequences described in Kasapidis et al. [A total of 264 sequences (dataset 1) (Genbank Accession nos: MH842011-MH842084) were aligned using ClustalW  with fins et al. , 69 sequs et al. , 109 seqs et al.  and 18 ss et al. . All abo8 Markov Chain Monte Carlo (MCMC) generations, sampled every 104 generations. Convergence of chains was visualized using Tracer 1.6 [Lepus timidus (Accession number: EF515861), was used as outgroup.The best-fit nucleotide substitution model for this dataset was determined using jModelTest 2.1.10  , 33. Theacer 1.6  discardiacer 1.6 . A D-looe to zero in order to restrict the choice of feasible links in the final network.Additionally, a median-joining network  was consDnaSP 5 [Genetic variability indices of brown hare populations, i.e. the number of distinct haplotypes (h), haplotype (Hd) and nucleotide diversity (π) values, were estimated using DnaSP 5 . To estiDnaSP 5  and plotA total of 74 different haplotypes were identified among the 264 newly analyzed samples, 82.4% of these have never been detected before. Fifty seven haplotypes were found in wild brown hares and 20 in domestic individuals; only three haplotypes were shared between wild and domestic samples. In this dataset 1, the population of Central-South Greece possesses the highest haplotypic diversity (0.938), whereas North Greece possesses the highest nucleotide diversity values (0.038) . Most haThe combination of our haplotypes and the retrieved sequences  resulted in a dataset of 326 unique haplotypes (dataset 2). In this dataset, Greece possessed higher haplotypic (0.982) and remarkably higher nucleotide (0.030) diversity values compared to Central/West Europe. It is remarkable that Greece possesses slightly higher number of haplotypes than Central/West Europe (149 vs 130), despite the fact that its sample size is almost one third of Central/West Europe (287 vs 841 samples). In addition to this, according to the rarefaction curve the plateau has not been reached for the discovery rates of new haplotypes despite the additional extensive sampling in Greece, confirming the great diversity of Greek populations . FurtherThe Bayesian phylogenetic analysis  supporteThe second clade, the “European” one involved subgroups CWE and E1-E5 . This clIn addition to the Bayesian analysis, a median-joining network was constructed to further elucidate the phylogenetic relationships among haplotypes . The groThe combination of our data with previous studies has resulted in a broader and clearer depiction of brown hare genetic diversity distribution and sheds more light on the evolutionary factors that have shaped it.Our data reveal that Greek populations present remarkably high genetic diversity. Although other studies have also illustrated Greece’s role as a biodiversity hotspot , 42, 43 Maculinea arion, [The high diversity values in the Greek marginal populations combined with the low diversity of central European ones do not support the “centre-periphery hypothesis” which includes the idea that core populations present higher genetic diversity than the edge ones , 48, 49.a arion, ), which a arion, , maintaia arion, . This sca arion, , horned a arion, , alpine a arion,  and red a arion,  that havThe fact that high genetic diversity values of Greek populations were maintained for long time periods is also very well presented by the complex structure of the Bayesian phylogenetic analysis and the median-joining network, contradicting undeniably the “centre-periphery hypothesis”. The star-like phylogeny in CWE subgroup combined with the absence of geographic structure support the idea of rapid expansion and colonization of brown hare in Central and West Europe. On the contrary, the Greek haplotypes (mainly included in subgroups E1-E5) do not present very close phylogenetic relationships and there is a clear geographic patterning.Although the concepts of geographic centrality, ecological marginality and centre of origin have been repeatedly presented in the literature, very few studies have focused on all these factors at the same time . AccordiThe identification of haplotypes in newly sampled areas also gives us a more comprehensive view of the phylogeography of the species within Greece. As shown both by the Bayesian and network analyses different lineages  exist, with characteristic geographic structure. Similar results were obtained by Kasapidis et al. , who preApart from historical interplay effects on the genetic structure of the species some other present day factors have to be considered in order to be more accurate in explaining current genetic data. Specifically, the present day isolation of different Greek populations cannot be only attributed to the geographical boundaries as there are no natural barriers between all sampling areas. According to , it can Despite the high genetic variability of Greek populations, Aegean islands presented low haplotypic diversity values , probablConsidering the clustering of Chios with the Anatolian clade, it could be attributed to: i) human induced translocations of individuals from Turkey on this island and/or ii) vicariance when Chios separated from Turkey . HoweveThe fact that many unique haplotypes have been found only in Greece, absent from Central Europe, supports the hypothesis that Greek populations were not involved in the northward expansion of the species. These data reinforce the concept that colonization of Europe took place starting from Central and Northern Balkan refugia , while tOur data indicate that the north part of Greece was the region with the highest mtDNA diversity values . This coDuring the last decades numerous restocking programs have taken place introducing foreign individuals of brown hare into several European countries , 28, 62.The results of the present study indicate that the majority of reared individuals cluster with haplotypes from Central and Western Europe. Since release of such individuals in the environment is a common practice, this constitutes a potential risk to the domestic gene pool of the species. This is aggravated by the fact that farmed individuals can survive in the wild at least for a reproductive period, so it is possible that they transfer their genome . HoweverAlthough during LGM, the Balkan peninsula acted as a major refugium for the colonization of the northern parts of Europe by brown hare , 5, the S1 Table(DOCX)Click here for additional data file.S1 Fig(DOCX)Click here for additional data file.


Arapaima gigas  is the largest freshwater scaled fish in the world, and an emerging species for tropical aquaculture development. Conservation of the species, and the expansion of aquaculture requires the development of genetic tools to study polymorphism, differentiation, and stock structure. This study aimed to investigate genomic polymorphism through ddRAD sequencing, in order to identify a panel of single nucleotide polymorphisms (SNPs) and to simultaneously assess genetic diversity and structure in wild  and captive populations.A. gigas was low with only 2.3% of identified RAD-tags (135 bases long) containing SNPs. A panel of 393 informative SNPs was identified and screened across the five populations. Higher genetic diversity indices  were found in populations from the Amazon and Solimões, intermediate levels in Tocantins and Captive, and very low levels in the Araguaia population. These results likely reflect larger population sizes from less urbanized environments in the Amazon basin compared to Araguaia. Populations were significantly differentiated with pairwise FST values ranging from 0.086 (Amazon × Solimões) to 0.556 (Amazon × Araguaia). Mean pairwise relatedness among individuals was significant in all populations (P < 0.01), reflecting a degree of inbreeding possibly due to severe depletion of natural stocks, the species sedentary behaviour and possible sampling biases. Although Mantel test was not significant , Bayesian analysis in STRUCTURE and discriminant analysis of principal components (DAPC) showed populations of Amazon and Solimões to be genetically differentiated from Araguaia, with Tocantins comprising individuals from both identified stocks.Compared to many other teleosts, the degree of polymorphism in This relatively rapid genotyping by sequencing approach proved to be successful in delineating arapaima stocks. The approach and / or SNP panels identified should prove valuable for more detailed genetic studies of arapaima populations, including the elucidation of the genetic status of described discrete morphotypes and aid in delivery of conservation programs to maintain genetic diversity in reservoirs across the Amazon region.The online version of this article (10.1186/s12863-018-0711-y) contains supplementary material, which is available to authorized users. Over the past 10–15 years the detection and analysis of genome-wide single nucleotide polymorphism (SNP) markers have revolutionized genetic investigations of all types of organisms. As elsewhere, within the field of fisheries science, SNPs are becoming the marker of choice for population genetic studies , their aGenotype by sequencing approaches, including restriction-site-associated DNA (RAD)  and doubArapaima gigas  is the largest freshwater scaled fish in the world with adults reaching up to 250 kg and measuring over 2.5 m in total length [A. gigas includes the basins of the Amazon, Tocantins-Araguaia and Essequibo rivers, which cover Brazil, Ecuador, Guyana and Peru. Also, the species has already been introduced into several non-indigenous water systems [Arapaima gigas is a dioecious and iteroparous species with sexual maturity reached after 3 to 5 years of age [A. gigas and rescuing operations are organised for conservation reasons [The Amazonian pirarucu l length . This eml length . The nats of age . Reprodus of age , 25. Exts of age . After ss of age  with a ms of age , 28. Durs of age . Overalls of age . In some reasons .A. gigas is an easy target for fishermen and natural populations have been historically depleted or even eradicated close to main cities [A. gigas has been included in the CITES red list [A. gigas in captivity is not a routine practice due to complex reproductive traits and dysfunctions in the species , which require further research especially for gender identification and control of spawning [Given its obligate air-breathing behaviour, n cities . It is en cities  and sincred list . While ospawning , 34. Thespawning  and potespawning –37.A. gigas populations. These have involved the use of mitochondrial markers (mtDNA), microsatellite or inter-simple sequence repeats (ISSR) markers to study eight populations from the Amazon, Solimões and Tocantins river systems [Arapaima, which today is supported only by morphological analyses of very few specimens [A. gigas has focused on acquiring tools for gender identification since this species is not sexually dimorphic, a factor that has impeded captive reproduction and aquaculture development. To do so, the species karyotype was characterised (2n = 56) but no apparent sex chromosome dimorphism was observed [A. gigas.To date, limited numbers of studies have been conducted to characterise the genetic diversity and structure of natural  systems , 38, 39, systems  and five systems  populati systems . Overallpecimens –37. Furtobserved , 42, whiobserved . Next geA. gigas was explored. Genetic diversity and structure among five population samples  was assessed and compared with published studies using microsatellite and mtDNA markers. The relevance of the results for future conservation and exploitation of this iconic species is discussed.In the current study the potential of ddRAD to generate an informative SNP panel for population genetic analyses of the endangered A. gigas is summarised in Fig. A. gigas ddRAD generated RAD loci (135 bases long) were identified to contain SNPs. This was 3–8 times lower than observed for other teleosts studied at the University of Stirling using the same methodology and analysis (Table A. gigas samples from different locations varied significantly (P < 0.05); higher polymorphic levels were found in the Amazon (3.5%) and Solimões (2.7%) rivers compared to the Araguaia river (1.0%), whilst fish from the Tocantins river and the Captive stock showed intermediate levels (2.0%) . Analyses of LD indicated significant association between locus 373_82 and 3408_78 in the global dataset after Bonferroni correction (α = 0.05/61463). Therefore, locus 373_82 (lower FST) was removed from further analyses resulting in a final dataset with 392 SNPs. For this dataset , the global FST calculated across all loci was 0.389, and individual locus FST values ranged from − 0.04 to 0.97 with their frequency distribution depicted in Fig. The 393 loci analysed were all found to be in HWE both within individual population groupings and across the global population sample .All pairwise O) (Table P < 0.05), with lower r from the rivers Amazon (0.127) and Solimões (0.125), increased r from Tocantins (0.182) and the r highest from Araguaia (0.323) and the Captive population (0.238) , indicating inbreeding as a potential cause for the loss of genetic diversity in the studied populations.Overall, the population samples from the Amazon basin (Amazon and Solimões) were genetically more diverse than those from the Araguaia and Tocantins rivers in terms of percentage of polymorphic loci, number of private alleles, Shannon’s Information Index (I) and observed heterozygosity (HO) Table . The meaaguaia 0.23 and thThe number of clusters (K) across the five studied population samples was resolved by the Evanno method, to be two Fig. b. AnalysThe DAPC analysis identified three groups K = 3) and agreed with the findings resolved by STRUCTURE. Populations from the Amazon basin (Amazon and Solimões) were clustered together, the genetically “hybrid” area formed by Tocantins was considered as a distinct cluster in DAPC, which also included four Captive individuals. The third cluster grouped together Araguaia and the remaining eight Captive individuals  [Salmo trutta [Acanthopagrus butcheri [Carcharhinus plumbeus [A. gigas in the Amazon floodplains, is likely to have minimised sampling bias [A. gigas populations studied has been influenced by past bottleneck effects, both historic and those more recently documented [The most striking finding of the survey was the extremely low level of genetic variability resolved, both within and between individuals from each of the wild population samples, overall being 3–8 times lower than comparable values for other fish species studied using the same methodology. A low level of detectable variability has also been reported for Northern pike ( lucius)  where veo trutta , the estbutcheri  and the plumbeus . Samplesing bias , 31. Howcumented , 32, whiA. gigas sampled from the Amazon and Solimões compared to the river Tocantins [A. gigas in this drainage.Population samples from the Amazon and Solimões rivers were more genetically variable  compared to fish from the Araguaia. Interestingly, samples from the river Tocantins and the captive stock showed intermediate diversity levels. These observations confirm and complement previous studies using mtDNA and microsatellite markers which indicated higher diversity in ocantins , 38, andocantins , 40. Theocantins , may alsO) and Shannon’s Index, which is indicative of a degree of inbreeding, similar to that reported for mudminnows (Umbra krameri) [A. gigas have been overexploited for more than 200 years, leading to reduced numbers , and even extinction in many localities [FST pairwise comparisons showed clear differentiation among populations. This mirrors findings from other genetic studies of A. gigas populations from Amazon, Tocantins and Araguaia rivers [A. gigas has been regarded as a major factor contributing to low genetic flow, resulting in local population differentiation [A. gigas [A. gigas concluded the existence of high levels of genetic similarity among lakes separated by 25 km, moderate genetic differentiation in sites separated by 100 km and highest genetic differentiation among regions separated by > 1500 km [O), corroborate the hypothesis that genetic drift led to a loss of genetic variability and increased differentiation between populations of A. gigas [Importantly, all five population samples surveyed showed significant levels of relatedness (r). Mean population relatedness was negatively correlated with diversity indexes such as observed heterozygosity (Hkrameri) . An elevkrameri) . Populatcalities , 32. Rescalities , 52. Howa rivers –31, 40, a rivers , 31. Thentiation , 40. EcoA. gigas . Indeed, 1500 km , supportA. gigas , and theA. gigas within the Amazon basin, using two discontinuous mitochondrial DNA regions of 1204 base-pairs (bp) (NADH1 segment) and 1143 bp (ATPase segment) revealed minimal evidence of substructuring, which loosely fitted an isolation-by-distance model [A. gigas predominantly from the mid- region of the Amazon river [Arapaima belongs to a multispecies group based on morphological analyses [FST values, > 96% of the RAD loci identified as polymorphic contained only one or two SNPs. This is a much higher proportion than that observed for a range of other fish species using a comparable methodology. A Stacks-based analysis of crypto species would be expected to reveal higher levels of polymorphisms both among and within RADtags. Therefore, the present genetic data do not provide supportive evidence for genetically distinct species within the samples and populations analysed.An initial population genetic study of ce model . A lateron river . A more on river . In suppanalyses , 36. TheA. gigas. Retaining as much diversity as possible should be a priority, and this can only be realistically achieved through coordinated conservation efforts over a broad geographic scale, and should be informed by regular genetic monitoring. Similarly, the findings of this study have specific implications for the development of commercial A. gigas aquaculture. A concern in all aquaculture enterprises, where high fecundity is the norm, is the potential for deleterious inbreeding. This will require particular awareness and careful genetic management for A. gigas. Also, given the low genetic diversity baseline, the potential for commercial traits gain through selective breeding may be limited. While now being widely employed for improvement of many aquaculture species such as the Nile tilapia (Oreochromis niloticus), the Atlantic salmon , the rainbow trout (Oncorhynchus mykiss) and several others, reviewed by Yue [A. gigas, both in terms of marker development and number of pedigrees required to collect relevant data. It may be prudent to consider establishing farm strains from a wide range of wild populations, which would maximise genetic diversity and also act as gene banks for the species.Clearly, evidence regarding the overall low levels of variability within populations and significant genetic structuring among populations should guide future management and conservation plans for wild d by Yue , the appA. gigas (orange-fleshed and white-fleshed) and the contended species recently described for Arapaima [Though restricted by the unusually low level of polymorphism detected within the arapaima genome, the screening of hundreds of SNPs using ddRAD technology can be considered as a reliable and robust method to determine genetic variability within and between arapaima populations. Furthermore, these genetic markers have a role to play in identifying the origin of animals used in aquaculture, and in informing the maintenance of the genetic diversity of captive reared broodstocks. There is also the opportunity to develop a subset of the most informative SNPs for screening using alternative platforms , which require less labour intensive and less expensive protocols. Such a panel would probably be better suited than RAD for the much more extensive survey of arapaima populations that is required to better understand and manage the species. As a priority, surveys should focus on the different morphotypes of Arapaima , 36 whicThis study analysed samples from the rivers Amazon , Solimões , Tocantins , Araguaia , and also a captive broodstock . Twelve individuals per population / stock were randomly selected from a wider number of sampled specimens, giving a total of 60 individuals Fig. . Samples− 1). The temperature was then increased to 70 °C for 15 min to inactivate the proteinase K, before 5 μL RNAse A (2 mg.mL− 1) was added to each sample and further incubated at 37 °C for 1 h. To precipitate proteins, 160 μL 5 M NaCl (0.7 volumes) was then added, mixed and the tubes left on ice for 10 min. This mixture was centrifuged at 22,000 rcf for 10 min to pellet the proteins. A proportion of the supernatant (c. 300 μL) was then transferred to a new microfuge tube containing an equal volume of isopropanol, mixed to precipitate DNA and centrifuged at 22,000 rcf for 1 min to pellet the DNA. The supernatant was carefully removed and the DNA pellet was then washed in 1 mL 70% ethanol for 3 h, after which the 70% ethanol was replaced and the pellet washed for a further 16–20 h. Finally, the samples were centrifuged at 22,000 rcf for 5 min, the ethanol was discarded and the DNA pellet air dried and reconstituted over a 24-h period in 5 mM Tris (pH 8.0). The DNA quality and concentration were evaluated by spectrometry . Agarose gel electrophoresis (0.8%) was used to check the integrity of the genomic DNA. Finally, DNA concentration prior to library preparation was more accurately quantified by fluorescence assay .For genomic DNA extraction, a fin clip or muscle sample from each of the 60 individuals was initially incubated for 16 h at 55 °C in a lysis solution containing 200 μL of SSTNE buffer , 20 μL 1SbfI  and 0.3 U SphI  high fidelity restriction enzymes  in a 5 μL reaction volume that included 1× CutSmart™ buffer (NEB). After cooling the reactions to room temperature, 2.5 μL of a premade barcode-adapter mix was added to the digested DNA, and incubated at room temperature for 10 min. This adapter mix comprised individual-specific barcoded combinations of P1 (SbfI-compatible) and P2 (SphI-compatible) adapters at 6 and 72 nM concentrations respectively, in 1× reaction buffer 2 (NEB). Adapters were compatible with Illumina sequencing chemistry. The barcoded adapters were designed such that adapter–genomic DNA ligations did not reconstitute RE sites, while residual RE activity limited concatemerization of genomic fragments during ligation. The adapters included an inline five- or seven-base barcode for sample identification. Ligation was performed over 75 min at 22 °C by addition of a further 2.5 μL of a ligation mix comprising 4 mM rATP , and 2000 cohesive-end units of T4 ligase (NEB) in 1× CutSmart buffer. The reactions were terminated by addition of 20 μL PB buffer . Each DNA sample was processed in duplicate (i.e. 120 separate digestion – ligation reactions). All 120 reactions were pooled and column-purified (MinElute PCR Purification Kit) and eluted in 52 μL of EB buffer . The purified pooled sample was separated by agarose gel (1.1%) electrophoresis and fragments ranging from approximately 450 to 650 bp were excised and purified using a MinElute Gel Extraction Kit . The eluted size-selected template DNA (c. 54 μL in EB buffer) was amplified by PCR  using Q5 Hot Start High-Fidelity DNA Polymerase (NEB) and Illumina compatible primers. The PCR reactions were combined  and column-purified with the MinElute PCR Purification Kit , then eluted in 50 μL of EB buffer. The library was then re-purified, to ensure no carry over of PCR primer dimer or other low size range amplicons (< 200 bp) using an equal volume of AMPure magnetic beads  and eluted in 15 μL EB buffer. Finally, the library was sequenced at the Institute of Aquaculture  on a single run on the Illumina MiSeq platform .A ddRAD library was prepared according to a published method  while imA. gigas, the RAD loci were assembled de novo using the following parameter settings: 4 as the minimum read depth to create a stack (m), 2 as the maximum number of mismatches in an individual (M) and 1 as the maximum mismatch between loci for building the catalogue (n). A robust set of SNPs suitable for population analyses was exported from the catalogue in Genepop format [After sequencing, reads were de-multiplexed, low quality reads with missing restriction sites and containing ambiguous barcodes were excluded and sequences trimmed to 135 bases long using Stacks version 1.42 . Given tp format , using tA. gigas samples were compared to ddRAD data of other teleosts generated from other projects undertaken at the University of Stirling using the same methodology. These species and numbers of samples were: Cyprinus carpio L. (n = 85), Dicentrarchus labrax (L.) (n = 26), Oreochromis niloticus (L.) (n = 6), Clarias anguillaris (L.) (n = 5), Sprattus sprattus (L.) (n = 8), Ctenolabrus rupestris (L.) (n = 20) and Melanogrammus aeglefinus (L.) (n = 16). For each species, the mean (± SD) of the following parameters were calculated: number of unique stacks, number of polymorphic loci and number of SNPs obtained. Non-parametric Kruskal–Wallis one-way ANOVA followed by Dunn’s pairwise post hoc tests were used to compare the ratio between polymorphic loci and unique stacks obtained between species. Statistical analyses were conducted using Minitab version 17.3.1  with significance set at P < 0.05.The levels of polymorphism within the FST values for each locus in the global population, to estimate pairwise genetic differentiation (FST) between populations  and to identify outlier loci . Sequential Bonferroni [Initially, tests for Hardy-Weinberg Equilibrium (HWE) and linkage disequilibrium (LD) were conducted for the SNP loci both within populations and across the global dataset  using Genepop version 4.6 . To do snferroni  correctihttps://www.google.com/earth). The software GenAlEx version 6.5 [FST) distances were correlated. GenAlEx was also used to estimate percentage of polymorphic loci, number of private alleles, Shannon’s Information Index (I), expected (HE) and (HO) observed heterozygosity, and coefficient of inbreeding (FIS) from Weir and Cockerham [O using Minitab (P < 0.01).To investigate the hypothesis of isolation by distance, the shortest waterway path among sampling sites (Captive excluded) was measured using Google Earth version 7.1.8  was made using an admixture model and frequencies were assumed correlated. Optimal K-value was determined by Evanno’s method [Population structure was first investigated using a model-based approach, which assumes HWE and linkage equilibrium, implemented in STRUCTURE version 2.3.4 . Initials method  using ths method . Using tfind.clusters was used to transform original data into principal components (PC), retaining 60 PCs in the analysis. The dapc function performed a discriminant analysis using 20 PCs (> 80% of variance explained) and 5 eigenvalues were retained and examined. The assign.per.pop function was used to evaluate the proportions of successful reassignment of individuals to their original clusters.Finally, population structure was explored using a discriminant analysis of principal components (DAPC), conducted in R version 3.3.2. using the package Adegenet v. 2.0.1. DAPC analysis is not a model-based approach, and it optimizes the variance between groups while minimizing the differences within clusters. It requires prior identification of a cluster number, which was made using the Bayesian Information Criterion (BIC). The function Additional file 1:Arapaima gigas: a) the combinatorial inline barcodes used for each individual - DNA samples were processed in duplicate, with a different barcode combination used for each RE digestion - ligation reaction, thus each individual is represented by two sets of identifiers; b) basic RAD locus statistics generated by STACKS for each of the 60 samples; c) mean observed heterozygosity within the 392 loci surveyed for each individual. (XLSX 14 kb)General information and summary data from the ddRAD study of Additional file 2:n = 60). (XLSX 60 kb)Sequence information and associated basic genetic statistics for each of the 393 SNPs used in this study. The position of assayed SNP is indicated by red font within each 135 base RADTag sequence, together with basic genetic statistics (computed with GenAlEx) for the combined sample set . B. Selection of number of clusters was based on Bayesian Inference Criterion (BIC), which indicates 3 clusters for data summarization (elbow drop). C. Membership probabilities  for individuals into clusters, blue crosses indicate the prior cluster provided into DAPC. (TIF 2254 kb)A. Discriminant analysis of principal components (DAPC) using 392 SNP markers in v. 2.0.1  for the


The absence of mucocutaneous candidiasis and undetectable IgE levels until the age of 7 years prolonged the time to molecular confirmation of the cause for the patient's immune deficiency. STAT3 p.L645Q was found to have decreased transcriptional capacity. The patient also had low levels of Th17 cells and STAT3 phosphorylation was impaired in patient-derived cells. Nearly 100 unique mutations in STAT3 have been reported in association with AD-HIES.Autosomal dominant hyper-IgE syndrome caused by mutations in the transcription factor STAT3 (AD-HIES) is characterized by a collection of immunologic and non-immune features including eczema, recurrent infections, elevated IgE levels, and connective tissue anomalies. We report the case of a Qatari child with a history of recurrent staphylococcal skin infections since infancy, who was found to have a novel,  Autosomal dominant hyper-IgE syndrome (AD-HIES) is a rare primary immunodeficiency conventionally characterized by the triad of eczema, recurrent skin and sinopulmonary infections, and elevated serum IgE. Non-immunological features of AD-HIES can include bone fractures secondary to minor trauma, delayed shedding of deciduous teeth, joint hyperflexibility, and vascular anomalies .signal transducer and activator of transcription 3 (STAT3). STAT3 is a transcription factor activated downstream of numerous cytokine signals including interleukin (IL)-6, interferon-α and IL-10, among others  antibiotics and incision and drainage (I&D). He was diagnosed with infantile eczema and a superimposed bacterial infection; wound cultures were positive for Staphylococcus aureus. At age 5 he was admitted to the hospital with pneumonia complicated by a parapneumonic effusion and multiple cavitary lung lesions  was detected . Given his history of recurrent staphylococcal infections and elevated IgE, AD-HIES was suspected. A detected . This noSTAT3 mutations in the SH2 domain , a common finding in AD-HIES. His peak serum IgE has been 10,665 kU/L. At 14 years of age, the patient sustained a non-displaced fracture in his ankle following a ground level fall. Bone densitometry showed osteopenia. He did not manifest other non-immunologic features of AD-HIES such as coarse facies, high-arched palate, scoliosis or joint hyper-extensibility, although he was noted to have retained primary upper incisors. He did have two separate and resolving episodes of facial nerve palsy at age 5 and 15 years not clearly associated with a viral infection.After the diagnosis of AD-HIES, the patient was started on prophylactic cephalexin but continued to struggle with recurrent abscesses. After his response to pneumococcal vaccination was judged insufficient, his prophylactic antibiotic was changed to levofloxacin and he was started on immunoglobulin replacement therapy. He has subsequently experienced a significant decrease in the frequency of his infections and improved quality of life.The study was approved by the Baylor College of Medicine Institutional Review Board. All subjects gave written informed consent in accordance with the Declaration of Helsinki. Written informed consent was obtained from the parents of the patient for the publication of this case report and any potentially identifying information/images. Genomic DNA was extracted from whole blood and sent for targeted sequencing of STAT3.An expression plasmid with the patient's mutation was generated using a plasmid containing the wild-type (WT) STAT3 complementary DNA (cDNA)  altered with QuikChange site-directed mutagenesis (Agilent Technologies) using the primers: Forward: 5′-GCA AAT GAC ATG TTG TTC TGC TGC TGC TTT GTG TAT G-3′ and Reverse: 5′-CAT ACA CAA AGC AGC AGC AGA ACA ACA TGT CAT TTG C-3′. For STAT3 luciferase studies, STAT3-deficient A4 cells were co-transfected  with 250 ng of mutant or WT STAT3 plasmid and a firefly luciferase plasmid under STAT3 transcriptional control with a constitutively expressed renilla luciferase . Firefly and renilla levels were measured after 48 h .Peripheral blood mononuclear cells (PBMCs) from the patient and his parents were isolated using density centrifugation. PBMCs were resuspended in RPMI-1640 media supplemented with 10% FCS, penicillin, streptomycin, and L-glutamine and stimulated overnight using 50 ng/mL PMA and 500 ng/mL calcimycin in the presence of brefeldin A . Flow cytometric analysis of intracellular IL-17 using a BD FACSCanto II was performed after fixation and permeabilization using Cytofix/Cytoperm (BD Biosciences) per the manufacturer's instructions. Antibodies used (BD Biosciences) were anti-CD3-APC-Cy7 (SK7), anti-CD4-PerCP (RPA-T4), anti-interferon-γ-PE (B27) and anti-IL-17-FITC (N49-653). Data were analyzed using FlowJo software.5) in the log phase of growth were left unstimulated or stimulated for 15 min with IL-6 (10 ng/mL) or interferon-α (50 ng/mL). Cells were then fixed in Cytofix and permeabilized using Perm Buffer III (both BD Biosciences) per the manufacturer's instructions. Cells were incubated with phospho-STAT3 (Y705) antibodies (BD Biosciences) for 1 h at room temperature followed by flow cytometric analysis (BD FACSCanto II). Data were analyzed using FlowJo software.Immortalized lymphoblastoid cell lines were generated from PBMCs using standard methods. Patient-derived cells  mutations in STAT3, found in malignancy (somatic) and STAT3 GOF syndrome (germline) are found in all domains (STAT3 mutations are reported outside of the DNA binding and SH2 domains, both these domains are equally represented.Since  AD-HIES . AD-HIES domains . While fThe clinical ability to perform diagnostic genetic testing has dramatically increased. Therefore, it is important for patient evaluation and subsequent treatment to continue to functionally confirm and report novel mutations in known disease genes.All subjects gave written informed consent in accordance with the Declaration of Helsinki. The study was approved by the Baylor College of Medicine Institutional Review Board.NC, JB, and TV: conception or design of the work. JB, AR, AV-H, JO, LF, ME, SP, MA-N, and TV: data collection. JB, AR, and TV: data analysis and interpretation. NC and TV: drafting the article. NC, JB, AR, AV-H, JO, LF, ME, SP, MA-N, and TV: critical revision of the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Cichorium intybus L., well known in Italy with the common name “Radicchio”, is an important leafy vegetable that is prevalently reproduced by allogamy due to very efficient barriers of self-incompatibility. Marker-assisted breeding is widely used by seed firms to develop new hybrid varieties that manifest genetic distinctiveness, uniformity and stability. A total of 29 mapped microsatellite markers were used for genotyping 504 samples of the Red of Chioggia biotype: First, two synthetics, four F1 hybrids and two derived F2 populations were compared to assess the distinctiveness of their gene pool and structure; then, the uniformity and stability of 3 years of production of a commercial F1 variety were also investigated. Genetic similarity and diversity statistics as well as the genetic structure of populations were analysed, including allele and genotype frequencies. The mean estimates and ranges of genetic similarity enabled the molecular discrimination of OP synthetics from F1 varieties and their F2 progenies and the determination of individual plant memberships. Moreover, the genetic structure of F1 hybrids produced in 3 years unexpectedly revealed two main clusters that discriminate the first 2 years from the 3rd, mainly because of the presence of uncommon specific alleles and different allele frequencies. Overall, this molecular information will enable breeders to determine the genetic distinctness, uniformity and stability of commercial and experimental varieties, as well as their genetic relationships and relatedness. Hence, this work provides a useful tool for achieving the molecular characterisation and genetic identification of different radicchio populations. Cichorium intybus subsp. intybus var. foliosum L., 2n = 2x = 18) is the Italian name of an important locally-cultivated leaf chicory belonging to the Asteraceae, one of the largest families among flowering plants. From a reproductive perspective, radicchio is prevalently allogamous due to an efficient sporophytic self-incompatibility system and presents entomophilous pollination [Radicchio  is of particular interest. This genetic map is based on 431 SSR and 41 STS markers and includes nine linkage groups obtained after the integration and organisation of molecular marker data derived from one witloof chicory and two industrial chicory progenies [In this regard, several linkage maps saturated with DNA markers and spanning the entire genome size (approximately 2.6 Gb) are available for leaf chicory ,6,7,8,9.rogenies . Among crogenies . In thisrogenies , who ideA total of 216 samples belonging to two synthetics lines (OP-1 and OP-2), four F1 hybrids  and two F2 progenies  were investigated with 29 SSR markers. Samples were also chosen among short and medium–long term development cycle materials to equally represent the Chioggia biotypes currently available on the market .All SSR markers were polymorphic, and the mean polymorphic information content (PIC) value was 0.61, with a maximum equal to 0.84 for the M2.6 SSR locus. Of note, 24 of 29 SSR markers were highly informative , with PIAdditionally, the mean Ne was higher in the OP populations  than in the F1 hybrids and F2 progenies  of F1 hybrids, considered as a whole, was 50.0% on average  than synthetics (84.3%). In the case of F2 progenies, the genetic similarity within each population was 89.1% and 90.7% for F2-C and F2-D, respectively b. The meAn unweighted pair group method with arithmetic mean (UPGMA) dendrogram was also constructed on the basis of the genetic similarity matrix, whose coefficients were computed in all possible pair-wise combinations of the 216 samples. The dendrogram clustered the entire collection in six main subgroups. Each cluster, with few exceptions, included the whole pool of samples used to represent each of the six commercial lines. Additionally, as shown by the genetic similarity matrix, OP-1 resulted in the most dissimilar group compared to the rest of the samples, and the corresponding branch of the dendrogram was clearly separated from the rest of the tree. Notably, the UPGMA did not enable the full discrimination of the F2 progenies that grouped together with their respective F1 parents .According to the principal coordinate analysis (PCoA), similar to what was highlighted by the UPGMA tree, the two synthetic populations clustered independently from the rest of the groups, although a partial overlap was observed between OP-2 and F1-A. In contrast, F1-C and F1-D formed unique clusters with their respective F2 progenies. The first principal coordinate accounted for 8% of the total variation and clearly separated a group including F1-A, F1-B and OP-2 from the group of F1-C, F2-C, F1-D, and F2-D; the second principal coordinate accounted for 5% of the total variation and separated F1-C and F2-C from F1-D and F2-D. Finally, the hybrid varieties F1-A and F1-B were highly similar genetically, forming a cluster divided into two subgroups .K = 5 such that the 216 samples were grouped into five genetically distinct clusters. Each genotype was plotted as a vertical histogram divided into K = 5 coloured segments representing the estimated membership in each hypothesised ancestral genotype. A total of 212 of 216 individuals showed strong ancestry association (>90.0%), and two samples scored a slightly lower level of association ; only two additional samples had a very low degree of ancestry association , and they were hence considered admixed. Moreover, the two F2 progenies were not distinguished from their original hybrids, and F1-A and F1-B hybrids were grouped as if they had only one common ancestor , the level of genetic differentiation between samples was investigated by calculating the genetic similarity in all possible pair-wise comparisons among all 288 individual plants (96 samples per year).The Rohlf’s coefficient of genetic similarity ranged from 65.0% to 100%, with an estimated average equal to 91%. Additionally, a comparison within and between the 3-year populations was performed to assess the stability of the F1 hybrid variety. Regarding the year 2014, the median genetic similarity observed was 91.8%, ranging from 66.4% to 100%; 2015 went from 77.0% to 100% with a median genetic similarity of 95.4%, while 2016 showed a median genetic similarity equal to 89.7%, ranging within the population from 80.0% to 100%. Moreover, comparisons among the different populations were made. The median values were 92.0%, 89.0% and 90.8% for the 2014 vs. 2015, 2014 vs. 2016 and 2015 vs. 2016 comparisons, respectively. For the minimum genetic similarity data, the lowest value was observed in 2014 vs. 2016 (65.4%), followed by 2014 vs. 2015 (67.0%), and the highest value was in 2015 vs. 2016 (75.2%).With the data obtained from the genetic similarity analysis, a UPGMA dendrogram was also computed, highlighting the clustering in two main subgroups. The first group included individuals belonging to both years 2014 and 2015, while the second group, strongly divided from the previous years, prevalently clustered 2016 plants a.K = 2 coloured segments representing the estimated membership in each hypothesised ancestral , 13.8%, 17.2% and 20.7% of loci  matched for the years 2014, 2015 and 2016, respectively. The year that best represented the third case (A1A1×A2A2/A1A2×A1A2) was 2015  followed by 2014  and 2016 . In contrast, 2016 matched the fourth hypothesis (A1A1×A2A3/A1A1×A1A3) with a percentage equal to 17.2%, which is representatively higher than for 2014 and 2015, and fits 6.9% (2 out of 29 loci) and 3.4% (1 out of 29 loci), respectively. Regarding the last cluster of the bar chart (A1A2×A3A4), where both parental genotypes are heterozygous for different alleles, 3.4% of the loci showed correspondence for 2014 and 2016, while there were none for 2015  matched the hypothetical profile by which both parental genotypes used for crossing are homozygous for the same allele, while for 2016, 48.7% of loci (14 out of 29 loci) matched. Regarding the second putative profile . In contrast, two polymorphisms at the M8.23  and M9.27  loci were detected in 2014 and 2016 but not in 2015  showed new alleles in 2016, with rates extending from 8.6% (M1.3) to 15.5% (M1.2) . Moreove in 2015 . AnotherTraditional methods have recently been integrated with biotechnological methods to accelerate breeding programmes. Marker-assisted breeding (MAB), in fact, is widely utilised for the development of improved lines by firms and research institutes, allowing breeding activity based not only on the evaluation of phenotypes but also on plant genotypes. Moreover, molecular assays have become useful tools for verifying the distinctness, uniformity and stability of varieties (DUS test), three major requirements for the registration of new plant materials. Microsatellite markers can identify essentially-derived varieties (EDV) in the context of variety registration; therefore, these markers represent useful tools for cultivar protection against plagiarism. . In partMarker tools differ in their information content, depending on their polymorphism degree. The PIC calculated for the 29 SSR marker loci showed an average value of 0.61; therefore, the microsatellite markers used in this study were determined to be generally highly informative. According to Botstein et al. , 24 of tA high number of private alleles were detected within the synthetic populations, especially in OP-1, where private allelic variants were found at seven SSR loci; notably, in two cases, the frequency was > 30%. Interestingly, private alleles with high frequencies were also identified in F1-B and F1-C, making them attractive tools for protecting the rights of plant breeders . AdditioThe significant variability, in terms of heterosis, shown by the four F1 hybrid populations, relies on the genetic distance between the relative parents. In fact, information derived from genotyping data are exploited more and more for planning crosses and predicting plant vigour traits  of experimental F1 hybrids on the basis of the overall genetic distance and allelic divergence between parental inbred lines, as an estimate of their specific combining ability ,16. As oOther information regarding the genetic similarity and the genetic structure of the analysed populations were provided by both the UPGMA dendrogram and the PCoA-based centroid, revealing well-separated sub-populations corresponding to the cultivated varieties object of this study. It is worth noting that the high similarity shown by the two hybrids F1-A and F1-B  is also graphically evident in both analyses and is clearly the direct consequence of a common genetic root. The shared origin of these two hybrids is also visible from the STRUCTURE analysis, which confirms a common ancestor. The findings reported for F1-A and F1-B are also transposable to the two F2s (F2-C and F2-D) and their related F1 parents (F1-C and F1-D). The graphical overlapping in the PCoA and the high genetic similarity among the two generations are the expressions of a direct lineage, and their common progenitor further confirmed this finding. The only two samples considered admixed from the structure analysis can be assumed as off-types of these populations; thus, they will be removed from the core collection. Considering that a new variety, before the release on the market, needs to be distinct from all other commercially available cultivars, the results of this study indicated that SSR markers represent an efficient tool to evaluate the distinctness of commercial varieties or to investigate their common origin, as in the case of EDV.From the genetic similarity analysis of the 3 years of hybrid seed production, samples belonging to 2015 showed the highest intra-similarity scores, with a median value of 95.4%, reflecting a generally higher uniformity of this population. In contrast, 2016 exhibited the lowest median similarity within the population, even being the year with the most contained variability among its individuals. At the same time, when considering the pairwise comparison between the different years, 2014 vs. 2015 appeared to be most similar, with a median value of 92.0%, followed by 2015 vs. 2016 (90.8%) and 2014 vs. 2016 (89.0%). Moreover, the 2015 vs. 2016 comparison showed the lowest variability, as the genetic similarity values ranged from 75.2% to 100% .1A1×A1A1 gives p(A1) = 100%; A1A1×A1A2 gives p(A1) = 75% and q(A2) = 25%) among the 29 analysed loci over the 3 seed production years. After this step, all profiles across all loci were matched with the hypothetical profiles and then counted for each year. As described in 1A1×A1A2) showed an average increase of 3.5% per year from 2014 to 2016, rising from 13.8% to 20.7%. This finding was considered to be an increase in the number of heterozygous loci in the parental lines used for the crossing. Additionally, the number of loci fitting the third and the fourth hypothetical profiles scored a substantial decrease in 2016 and increase in 2016, respectively  were present in 2014 and not 2015 and reappeared in 2016 with the same frequencies as in the first year, meaning that a correlation between the parental plants used for the constitution of this hybrid population is certain but with strong differences shown by the presence of specific alleles with elevated frequencies in the 2016 population. Interestingly, these results indicated the presence of a unique allele for marker M9.26 in 2014, substituted by an already existing polymorphism in 2015 and then replaced in 2016 by an entirely new polymorphism. These data enabled us to speculate that in 2014, parental genotypes were A2A4×A1A2; in 2015, they were A2A4×A2A2; and in 2016, they were A2A4×A3A3  among both clusters. Information was also confirmed by the UPGMA analysis, in which the highly different samples outstood the primary roots of the tree. To corroborate the clustering analyses, a comparison between observed allele frequencies at single loci per year and those obtainable in progenies after crossing different hypothetical parental genotypes was performed. Thus, five main patterns were supposed to categorise the different frequency profiles , especially in 2016. Moreover, this second case study demonstrated the efficiency of microsatellite markers in assessing uniformity and stability through the generation of a commercial variety, two crucial requisites for its release and survival on the market.The plant material used in this study, including OP and F1 varieties, represents part of a high-breeding value collection of commercial lots belonging to the “Red of Chioggia” biotype of radicchio. In addition, F2 populations obtained ad hoc for the purpose of this investigation were also used.In this study, 30 samples were used to represent each of the first two hybrids (F1-A and F1-B) and each of the two synthetic populations (OP-1 and OP-2); 36 samples were collected for F1-C (18) and F1-D (18) hybrids, and eventually, the two F2 progenies (F2-C and F2-D) were composed of 30 plants each and obtained by selfing single F1 individuals  from an F1 hybrid variety obtained in an open-field system without physical barriers.® 96 Plant Kit (Qiagen) following the manufacturer’s protocol. The quality, purity and quantity of gDNA were assessed by gel electrophoresis in 1% agarose/1× TAE gels containing 1× SYBR Safe DNA Gel Stain  and a NanoDrop spectrophotometer . DNA samples were diluted to 20–30 ng/µl to be used as a template in a multilocus PCR.On the whole, 504 samples were collected, and approximately 100 mg of fresh leaves were ground to fine powder using a TissueLyser II mill . Genomic DNA was extracted with a DNeasyThe composition of the PCR multiplex reactions was designed to improve a previous panel of 27 SSR markers . A modif®Multiplex PCR Master Mix (Thermo Scientific), 10% GC Enhancer (Thermo Scientific), 0.25 µM non-tailed primer, 0.75 µM tailed primer, 0.50 µM fluorophore-labelled primer, 20–30 ng of genomic DNA and distilled water up to volume. All amplifications were performed in a GeneAmp® PCR 9700 thermal cycler . The following thermal conditions were adopted for reactions of multiplex 1: 5 min at 95 °C, followed by five cycles at 95 °C for 30 s and at 60 °C for 30 s, which decreased by 1 °C with each cycle, and at 72 °C for 30 s; and then 35 cycles at 95 °C for 30 s, at 56 °C for 30 s, and at 72 °C for 30 s. For multiplex 2 and 3, the annealing temperature was modified. Three cycles were undertaken at 95 °C for 30 s and at 56 °C for 30 s, which decreased by 1 °C with each cycle followed by 35 cycles at 95 °C for 30 s, at 54 °C for 30 s, and at 72 °C for 30 s. All reactions were terminated with a final extension of 30 min at 72 °C.Every multilocus PCR was performed in a total volume of 20 µL containing 2X Platinum Finally, the quality of the PCR amplicons was checked by electrophoresis on 2% agarose/1× TAE gels containing 1× SYBR Safe DNA Gel Stain (Life Technologies). PCR products were dried at 65 °C. Capillary electrophoresis was performed in an ABI 3730 DNA Analyzer (Applied Biosystems). The SSR alleles were scored using PeakScanner 1.0 software (Applied Biosystems).i2). Moreover, the PIC for each of the 29 SSR loci, along with the Na, Ne and number of private alleles, was also computed. Among private alleles, we considered only those with a frequency higher than 15% in specific hybrids or populations and absent in others. Finally, marker allele and genotype frequencies for each locus were also determined.All of the analyses described below were performed for both the case study objects of this work with several exceptions that will be specified. Statistical analyses were performed with the GenAlEx 6.5  and POPG1A1×A1A1, A1A1×A1A2, A1A1×A2A2/A1A2×A1A2, A1A1×A2A3/A1A2×A1A3 and A1A2×A3A4). Moreover, the observed allele frequencies for each locus and year were matched to the most representative hypothetical profiles, and a bar chart showing the locus number of correspondences (in percentage) was drawn. Frequencies under 3% were considered deriving from external contaminations.For the comparison of 3 different years of seed production of an F1 hybrid variety, allele frequencies at each locus were also used to hypothesise the putative parental genotypes at the relative loci . All genetic similarity analyses were conducted using the NTSYS software package v.2.21c [The population structure of the sample collection was assessed using the clustering algorithm of STRUCTURE software . All simThe results of this study indicated that our mapped SSR marker-based method developed for genotyping analyses is reliable and informative when applied to the characterisation of the genetic structure of different cultivated populations of radicchio. The comparison among eight different subgroups of plant materials, including two OPs, four F1s and two F2s, allowed us to discover fixed marker alleles and genotypes, which is potentially useful information for assessing the genetic identity of varietal seed lots and for protecting the legal rights of breeders. The comparative analysis of different representative commercial populations also verified that OP synthetics are characterised by lower similarity and heterozygosity than F1 hybrids (which can widely vary for these two parameters) and that F2 progenies usually show intermediate mean estimates of genetic uniformity and diversity. Moreover, from the analyses of 3 production years of an F1 hybrid variety, the same set of SSR markers highlighted significant differences among the commercial seed productions. This finding was observed due to the multi-allelic nature of the derived SSR genotypes and the high PIC values found for this set of marker loci. Furthermore, our panel of SSR markers was shown to be highly informative when exploited to test the stability of an F1 hybrid variety over different production years. Overall, this study provides a cost-efficient method to genotype the Red of Chioggia biotype at different levels of hybrid varieties development, ranging from the pre-selection of parental plants to the post-production verification of the obtained seeds, as well as for the identification of seed and plant lots to prevent potential frauds. In particular, this work supports the exploitation of highly discriminant SSR markers to test whether a new variety possesses uniformity and distinctiveness traits and so can legally gain access to registration and commercialisation. In fact, to be registered and released as new plant variety, some rigorous and specific requirements concerning the distinctness (D), uniformity (U), and stability (S) of the newly bred cultivar need to be satisfied. Although most of the existing methods exploited for DUS testing are expensive and time consuming, in this work, we demonstrated, through two different case studies, that SSR markers can be adopted as a complementary tool for morphological descriptors in DUS tests.


The location of the high mountains of southern Europe has been crucial in the phylogeography of most European species, but how extrinsic (topography of sky islands) and intrinsic features  have interacted to shape the genetic structure in alpine restricted species is still poorly known. Here we investigated the mechanisms explaining the colonisation of Cantabrian sky islands in an endemic flightless grasshopper. We scrutinised the maternal genetic variability and haplotype structure, and we evaluated the fitting of two migration models to understand the extant genetic structure in these populations: Long-distance dispersal (LDD) and gradual distance dispersal (GDD). We found that GDD fits the real data better than the LDD model, with an onset of the expansion matching postglacial expansions after the retreat of the ice sheets. Our findings suggest a scenario with small carrying capacity, migration rates, and population growth rates, being compatible with a slow dispersal process. The gradual expansion process along the Cantabrian sky islands found here seems to be conditioned by the suitability of habitats and the presence of alpine corridors. Our findings shed light on our understanding about how organisms which have adapted to live in alpine habitats with limited dispersal abilities have faced new and suitable environmental conditions. Understanding the evolution of Pleistocene glacial periods in temperate regions has been instrumental in disentangling much of the biogeographic distributions and genetic structure of their biotas. The effect of Quaternary climatic oscillations on the extant genetic diversity in the European and North American biotas is well-documented ,2,3,4. TMountain chains are considered hot spots of biodiversity due to the high levels of unique taxa they host, which is a direct consequence of harbouring a rich range of habitats in a relatively small area ,13,14. BUnderstanding how flightless organisms cope with environmental changes can inform us about how heterogeneous landscapes shape the genetic structure of montane biotas over time. Ultimately, this information will shed light on functional connectivity of disjunct sky islands, providing robust guidelines for conservation managers. Under a recurrent scenario of glacial and interglacial periods, it is not evident how animals with reduced dispersal capabilities respond, although it must be also dependent on the speed of the environmental changes . It is pChorthippus cazurroi). This grasshopper shows a patchy distribution in the Cantabrian Mountains, occurring along a narrow range between 1400 and 2570 m above sea level. Such a narrow distribution appears ideal to make inferences about how landscape features shaped the demographic history and genetic variability of alpine taxa. To achieve our objectives, firstly, we scrutinised the maternal genetic structure from all Cantabrian sky islands where this taxon occurs. We studied mitochondrial data because when the range of a species is exhaustively sampled, it is possible to get robust inferences of its genetic structure due to the absence of recombination and fast coalescent time [With the present study we aimed at unravelling the mechanisms explaining the dispersal process and evolution of spatial genetic structure in an alpine flightless grasshopper  for females (n = 285) and 8.27 ± 0.74 mm for males (n = 156), meanwhile, C. yersini (a relative species capable of short flight and occurring in sympatry) shows longer wing length: 22.07 ± 1.46 mm and 17.88 ± 1.42 mm for females (n = 189) and males (n = 134), respectively. However, both species show closer total body length (19.01 ± 1.83 mm versus 27.20 ± 1.46 mm) for C. cazurroi and C. yersini females, respectively; and 13.53 ± 1.63 mm versus 20.34 ± 1.56 mm for males, respectively. The species is found on grasslands habitats over 1400 m above sea level (a.s.l.) on limestone massifs. This grasshopper species shows the typical univoltine life cycle with nymphs emerging after snow is melted (June–July). After four moults, the nymphs mature to adult stage, which appears to be especially abundant in August, when most reproduction occurs. In controlled laboratory conditions, with food provided ad libitum, females survive on average 21 ± 1 Standard Error days, during which they can lay up to 11 egg pods, each containing on average 8 ± 3 SE eggs [ountains . The verles n = 1, respectChorthippus cazurroi in the Cantabrian Mountains. In particular, the species was found in 22  using the primers LCO1490 and HCO2198 [TM nucleic acid gel stain . We performed sequencing reactions using the Perkin Elmer BigDye v. 3.1  terminator reaction mix in a volume of 10 µL, following the procedures and conditions described in Pato et al. [® 3130xl Genetic Analyzer . Sequences were edited and aligned by hand using BioEdit v. 7.2.5 [We extracted DNA from the hind femur or head using the Qiagen DNeasy kit  and following the manufacturer’s protocol. To determine genetic variability of  HCO2198 . Polymer HCO2198 ,39. PCR v. 7.2.5 . Unique eµ) [We calculated genetic diversity statistics   per locaeµ) . We infeeµ) .We evaluated how two migration models, LDD and GDD  of 0.1 kmeach see  that covWe grouped the 22 localities into seven regions according their spatial distribution . SummaryAs indicated above, we generated 30,000 simulated datasets for each of the two migration models. In addition, we simulated 100 PODS for each model to evaluate the accuracy of the selection method. Considering that the accuracy of the estimation for a particular real dataset may differ among ABC techniques ,55, we aWe estimated the population genetic parameters with the Rrej, Rreg, and Rnn ABC approaches . We usedChorthippus cazurroi’s whole distribution range included 22 localities along the sky islands of the Cantabrian Mountains  for distinguishing between the investigated models (LDD versus GDD) under all the applied ABC approaches . The ana-06 (5.15 × 10−7–9.43 × 10−6). Here we present the parameter values estimated with the highest probability (mode of the posterior distribution), and the variance in the precision of these estimates  for the interpretation of these results.We found that the ABC approaches provided a reasonable power to estimate the population genetics parameters under the GDD model, after using the 100 PODS, although, as expected, the accuracy of this estimation slightly varied among the studied parameters and the applied ABC approaches . The popOur inferred haplotype network suggests a scenario of recent colonisation of the Cantabrian Mountains sky islands, led by a starting population of more than 500 individuals. Our findings support a colonisation by a gradual dispersal process rather than the presence of LDD events such as those caused by upland winds or biotic factors. Such a contemporary expansion is well-matched with the limited genetic structure found, which explains the pattern of incomplete lineage sorting revealed by the haplotype network analysis, and presenting haplotypes shared between massifs. This genetic structure is compatible with a scenario of incomplete lineage sorting, which is common in species with recent differentiation processes ,60.Chorthippus cazurroi started a process of slow and gradual range expansion along the Cantabrian sky islands using, most likely, narrow alpine corridors to achieve its current distribution  surrounding the sky islands. This finding is relevant in order to predict future movements of this species and assess ecological connectivity among disjunct sky islands distributed throughout heterogeneous and unsuitable landscapes. Undoubtedly, this information will be also critical in future management actions with the goal of preserving genetic diversity and ecosystem function. Importantly, our results confirm that this flightless species do not participate in LDD movements or, if they did, such circumstance did not shape its current genetic structure. Long distance dispersal constitutes a recognised strategy performed by diverse organisms with the aim of colonising areas away from the source, rapidly, and without loss of genetic diversity ,29,70. IOur approach provides a framework to improve our understanding, and ecological meaningfulness, of colonisation and diversification processes of non-vagile organisms throughout the sky islands of Cantabrian Mountains. Nonetheless, we are aware that our biologically informed hypotheses hinge upon required assumptions, which can be challenging. Our models assume an ancestral population from which the species spread along the sky islands. We chose such a source population according to our knowledge on the evolution of the last glacial period in the Cantabrian Mountains, the potential habitats where the ancestral population could have occurred, and the high values of genetic diversity found. However, the clear pattern of migration shown by our analyses (GDD) is unlikely to be affected by the origin of the expansion . AnotherChorthippus cazurroi populations established in the Cantabrian Mountains present a simple pattern of progressive and slow gradual migration. We also found that these populations evolved with a slow increase of their population sizes over time, which could be explained by the patchy distribution of suitable habitats and hard environmental conditions present in the region. In conclusion, our findings have shed light on the processes involved in the formation of biodiversity in alpine flightless animals occurring on southern temperate mountains and, importantly, have provided an ideal framework to integrate genetic divergence at the population level with ecological information related to animal differentiation such as morphological and acoustic traits.Overall, we showed that