Lonsdalea quercina from western North America  and compared these populations to other Lonsdalea species found worldwide. Phylogenetic analysis separated Colorado and California populations into two Lonsdalea clades, with genetic divergence near species boundaries, suggesting long isolation and populations that differ in genetic structure and distribution and possibly their polyphyletic origin. Genotypes collected from different host species and habitats were randomly distributed within the California cluster. Most Colorado isolates from introduced planted trees, however, were distinct from three isolates collected from a natural stand of Colorado native Quercus gambelii, indicating cryptic population structure. The California identical core genotypes distribution varied, while Colorado identical core genotypes were always collected from neighboring trees. Despite its recent emergence, the Colorado population had higher nucleotide diversity, possibly due to its long presence in Colorado or due to migrants moving with nursery stock. Overall, results suggest independent pathogen emergence in two states likely driven by changes in host-microbe interactions due to ecosystems changes. Further studies are warranted to understand evolutionary relationships among L. quercina from different areas, including the red oak native habitat in northeastern USA.Understanding processes leading to disease emergence is important for effective disease management and prevention of future epidemics. Utilizing whole genome sequencing, we studied the phylogenetic relationship and diversity of two populations of the bacterial oak pathogen  The complex nature of many forest diseases, often integrating interactions of several organisms in a single environment, makes them difficult to manage and study. However, knowing underlying causes of disease emergence is crucial to facilitate effective disease management and preventing epidemic outbreaks that can have catastrophic consequences for ecosystems health.Global forest ecosystems have been increasingly affected by a variety of disturbances, including altered climatic conditions and increased attack of key tree species by pests and pathogens. While the introduction of alien pathogen species to new geographic areas has been considered one of the main drivers of emerging forest diseases during the last century, other factors such as changes in environmental conditions, new host-vector associations (between introduced insect vectors and native tree pathogens or vice versa), cryptic disease agents , hypervirulent strains of known pathogen species, and/or newly emerging species of unknown origin are also recognized as key factors leading to disease emergence in forest ecosystems around the globeQuercus is one of the most important groups of trees in many regions of the Northern Hemisphere5, with nearly 500 species that have been characterized globally. In addition, oak forests are characterized by high species diversity and great soil fertility6. In North America oak trees compose a significant part of many forest ecosystems. For example, in the eastern USA, oak forest types represent roughly half of all forest land7. However, the biodiversity of oak species varies within the continent, with roughly 90 species described in eastern North America, 20 species in California5, and only one native species in Colorado. In North America, oak species are also often planted as shade trees in urban environments because of the ability to withstand harsh urban environments8. This has led to the movement of oak species to new geographic areas. For example, northern red oak (Quercus rubra L.) that is native to eastern North America, has become a popular shade tree in Colorado, USA. These planted trees may experience new pathogens and/or pathogens may also act as bellwethers of the effects of climate change because they experience environmental conditions outside of their native range.The genus 9. In the United Kingdom, acute oak decline has been associated with galleries of the two spotted oak borer, Agrilus biguttatus Fabricius 1777, and several bacterial species 11. Brenneria goodwinii and G. quercinecans, and Brenneria spp. and R. victoriana are also associated with oak decline in Spain and Iran, respectively12.Oak decline diseases and complex syndromes, caused by combined effects of biotic and abiotic factors, have been reshaping landscapes of deciduous forests in temperate zones around the world13. Recently emerged drippy blight on planted oaks causes abundant ooze on symptomatic tissue and has led to significant increases in tree mortality and removals in public properties in some areas of Colorado13. The main symptoms on infected oaks are leaf scorch and premature leaf drop that is followed by dieback of small twigs and the development of branch cankers, from which copious gummosis/bacterial ooze is produced. Based on phenotypic and genetic analyses coupled with pathogenicity tests it was confirmed that the disease was caused by the bacterial pathogen Lonsdalea quercina Hildebrand and Schroth 1967 in association with scale kermes insect, Allokermes galliformis Riley 1881. In drippy blight studies, symptoms were observed at scale feeding sites13 and investigated insects were contaminated with the bacteria14. Due to the development of abundant ooze on symptomatic tissue, it was called drippy blight disease. The first record of L. quercina was published in 196715 reporting drippy nut disease on native oak trees in California, USA, that infects acorns and acorn caps of native oaks  probably using wounds made by wasps, weevils, and other common oak pests to enter the host tissue.In the early 2000s a significant dieback of northern red oak, pin oak, and Shumard oak of unknown origin appeared in Colorado and was described as drippy blight diseaseL. quercina, three other species of Lonsdalea, all tree pathogens, have been described around the world: L. iberica Brady et al. 2012 causing bark canker and drippy nut disease on native oaks in Spain17, L. britannica Brady et al. 2012 associated with oak decline in Britain18, and L. populi Toth et al. 2013 causing canker disease of Populus in Spain and China20.Besides Lonsdalea spp. oak pathogens) recent emergence remain unknown. To our knowledge, little is known about L. quercina population biology and relationship between the bacterial populations from different areas. Since the only two places in North America with published records of L. quercina causing disease on oak trees are California and Colorado, we studied the relationship between populations of L. quercina from symptomatic oaks in California and Colorado. The objectives of the study were to determine the phylogenetic relationship between L. quercina populations from California and Colorado and other Lonsdalea species found worldwide and investigate population structure of L. quercina in these two locations. Since L. quercina was first documented in California, we also assessed whether the Colorado population originated from California, as well as discuss potential processes leading to the disease emergence in Colorado.While the causal agents of drippy blight have been identified, the causes leading to its . Genome assemblies consisted of on average of 103 contigs (range: 47–252). Total average length of assembled genomes was 3,827,997 bp , with average GC content 55.21% (range: 54.7–55.6%) and average N50 being 345,548 . Our genome assembly and annotation utilized the Prokka and Roary analyses. The pangenome of combined California  and Colorado  populations consisted of 8624 genes, 2370 of which comprised the core genome   Fig. .Figure 1Enterobacteriacea Rahn 1937, Erwiniaceae Adeolu et al. 2016, Pectobacteriaceae Adeolu et al. 2016, and Yersiniaceae Adeolu et al. 2016 families of the order Enterobacterales ord. nov.21, separated the CA and CO populations into two distinct clades grouping them with two other previously published genomes of L. quercina: members of the CA population were grouped with type strain ATTC 29281 previously collected in CA, and CO population genomes grouped with NCCB 100490 previously collected in CO , indicating a clear species boundary between L. quercina from other Lonsdalea species (Table Average nucleotide identity (ANI) above 95–96% is used to define bacterial species boundarieses Table . HoweverComparative analysis of the predicted protein sequences of core genes unique to each of the populations identified 189 orthologous clusters, 186 of which were shared between CA and CO populations Fig. a, suggesThe 245 and 372 core genes unique to CA and CO populations, respectively, were also compared in functional annotation databases dbCAN and PHI-base. Of 245 and 372 core genes unique to CA and CO populations, only four (CA) and three (CO) genes and 26 (CA) and 24 (CO) genes were functionally annotated in dbCAN and PHI-base databases, respectively Fig. –S4. EnzyQ. gambelii Nutt in a natural area; all grouped together with 1 sample from a planted tree of a different species . Due to the small sample size from the natural area, it remains unclear whether bacterial populations are structured by habitat or host type in CO.Based on the gene presence-absence matrix of core and accessory genes CA and CO populations formed two separate clusters Fig. . Both poQ. gambelii  and 1 isolate from a planted Q. palustris (B52) formed a well-supported cluster, distinct from planted Q. rubra isolates. Phylogenetic analyses also supported the gene presence-absence matrix grouping results for CO isolate B28 collected from a planted Q. rubra in a built area as genetically distinct from the rest of CO population.To further study population structure of CA and CO populations, we used an unrooted maximum likelihood phylogenetic tree Fig. a. ConsisQ. gambelii and 1 isolate from planted Q. palustris clustered together on the PCA plot, while the rest of the CO isolates (except B28) collected from planted Q. rubra grouped on the opposite side, further suggesting the presence of population structure within CO. Within CA population, no population structure was observed based on host species or habitat, but some strains clustered separately from the rest also indicating presence of some population structure in that region  based on 2355 thinned core SNPs supported the results of phylogenetic analysis placing CA and CO populations into two distinct groups. CA isolates collected from various species of native oak and different habitats grouped closely together, while CO samples were more spread on the PCA plot Fig.  and 3a, In CA, isolates with identical core genotypes (no SNP variation across the core genomes) varied in their geographical distribution. Some representatives of the same genotype were collected from neighboring trees Fig. a. For ex23 suggested this was due to recombination among 3 isolates from native Q. gambelii, 1 isolate from Q. palustris, and 1 isolate (B28) from planted Q. rubra in the CO population, and recombination between isolates B48 and B165, B44 and B51, as well recombination among several individuals within CA populations . Split decomposition analysisons Fig. a–c. Howeons Fig. .Lonsdalea quercina, a recently emerged pathogen of oak species in association with kermes scale Allokermis galliformis, in Colorado, USA. Using population genomic data, we have revealed that this recently emerged L. quercina population in Colorado is genetically distinct from an established L. quercina population in California, suggesting that an accidental introduction of the bacteria from CA to CO is unlikely. Despite clear population differentiation, the results of our analyses were inconclusive whether the CO L. quercina population comprises a new taxonomic species. Surprisingly, the CO population had a nucleotide diversity that was twice as high compared to the CA population despite a smaller sample size, suggesting bacterial strains isolated from red oak may be moving to CO from elsewhere or that this population has been present in the area for a long time and moved to introduced and planted Quercus rubra (red oak) from native species Q. gambelii (Shumard oak).An increasing number of tree diseases cause significant, long term, threats to wild and human mediated ecosystems. Clarifying processes leading to the emergence of new diseases is a critical step towards their effective management. In this paper, we aim towards characterizing possible causes of emergence of drippy blight disease caused by 24. Based on previous neighbor-joining phylogenetic analysis of Lonsdalea spp. and estimates of average nucleotide identity (ANI) between two previously published genomes of L. quercina . In this study using population data from both locations, maximum likelihood genealogies based on either 20 or 51 core genes placed both populations within the genus Lonsdalea. In the phylogeny, the CA and CO populations formed two distinct and well supported clades that are closely related to L. iberica. Surprisingly, phylogenetic analysis indicated that the L. quercina CO population is closer to L. iberica than the CA population, suggesting that L. quercina may be a polyphyletic group. This pattern was present in both phylogenetic trees, built using either 20 or 51 core genes, however, only the tree based on 51 core genes had a well supported bootstrap highlighting the relationship between L. iberica and L. quercina CO population. ANI, calculated at the population level , indicated higher ANI between CA and CO than the one reported by Li et al. (24). Our analyses demonstrated that these populations show genetic separation just above the proposed ANI species boundaries  and cannot be separated into two distinct species based on this measurement. At the same time ANI between L. iberica and either of studied L. quercina populations is below proposed species boundaries. Overall, the results of phylogenetic analyses and ANI estimates suggest that L. quercina from CA and CO show clear signs of differentiation, likely due to a long period of isolation from one another, and that L. quercina may have a complex evolutionary history. The lack of recombination between the CA and CO populations, based on the results of split decomposition analysis with SplitsTree, is consistent with the hypothesis of a possible polyphyletic origin of L. quercina populations. Our pangenome analysis indicated that while CA and CO share most of the core genome (2370 genes), each population has a unique set of core genes (245 core genes in CA and 372 core genes in CO), yet most of the gene clusters identified in the dataset are orthologous, sharing functionality, between the populations according to the orthology analyses (by Orthovenn3). It is worth mentioning that this result could also be affected by the threshold similarity in Roary analysis that is arbitrary set to 95%. Hence, it is possible that these gene clusters are not unique to each population, but rather are common genes diverged beyond 95% threshold. Some epidemiological differences also exist between the CA and CO populations—L. quercina in CA infects acorns of native oaks , while in CO L. quercina infects branches in introduced and planted Q. rubra and Q. palustris, but acorns in natural areas of native Q. gambelii. Overall, these results remain inconclusive regarding the taxonomy of CA and CO populations, and further phenotypic and biochemical assessments are required to clarify their taxonomic placement, therefore in this study we refer to them as populations.Despite increased access to molecular and genomic data on various microorganisms, including bacteria, taxonomic placement of species in many cases remains a challenging task28, including important bacterial crop pathogens (for example30). Phylogenetic and recombination analyses indicated that both populations of L. quercina in this study are comprised of mostly clonal modes of reproduction. For bacteria that rely on external factors for dissemination, the distribution of identical core genome genotypes may provide insights into organismal population dynamics. In CA where drippy nut disease caused by L. quercina has been established for several decades . There was no association among genotypes with either habitat or host species in CA, suggesting movement of the pathogen between different habitats. These results are not surprising since all oak species in CA are native to the region, and Q. agrifolia is a major component of native forests and is also used as a shade tree in residential areas. To date there are no phytosanitary measures established for L. quercina in CA, making it possible for the inoculum being moved across large distances by insects, birds, small mammals, and human-mediated activities.Many plant pathogens spread via clonal reproductionL. quercina of the same core genotype were always sampled from planted trees growing in a close proximity to each other (often neighboring trees). Albeit a small population of three, L. quercina collected from native Q. gambelii were genetically different from isolates collected from planted Q. rubra. Whether these differences are due to geographic distance or host species remains unknown. It is known that L. quercina from Q. rubra is pathogenic on two other introduced to CO species of oaks, Q. shumardii Buckley and Q. palustris13. In this study, one isolate from Q. palustris clustered together with isolates from Q. gambelii, suggesting possible movement of bacteria among different host species. Interestingly, the location of sampled Q. palustris tree was 45 km away from sampled plants of native Q. gambelii. Based on population genetic clustering, none of the sampled isolates from Q. rubra were genetically similar to the isolates from Q. gambelii, and it remains to be determined whether L. quercina from native Q. gambelii serves as an inoculum reservoir for planted Q. rubra hosts or vice versa. Further studies including isolates of L. quercina from different host species and geographical areas need to be done to understand the pathogen movement clearer.In CO, 33. It was suggested that in CA, L. quercina may enter host tissue via wounds made by acorn weevils, filbertworms, and some cynipid wasps34. Large numbers of free-living nematodes Panagrellus redivivus were often found feeding on ooze produced by L. populi on poplar trees in Hungary19. Lonsdalea quercina is also disseminated by several insect species in CO that may be helping the bacteria spread among host trees14. Among the associated insects in CO, the kermes scale has a well described close relationship with the pathogen to cause drippy blight disease13. It has been suggested that the scale insect may form wounds that allow L. quercina to enter the host plant more readily or that there is a more indirect interaction whereby the scale induces host stress, facilitating pathogen growth and disease development. However, this insect does not travel long distances, and this may be one of the explanations why the distribution pattern of recently emerged L. quercina genotypes is limited to nearby trees.Bacterial pathogens can be associated with insects, whereby entering host tissues via feeding sites, wounds, and/or for dissemination among hostsQ. gambelii are needed to determine the relationship between L. quercina populations infecting native Q. gambelii and introduced Q. rubra hosts in Colorado.Despite more recent emergence, the CO population had ~ 2 times higher nucleotide diversity compared to CA population. This is further unexpected because the sampling area in CA was much larger compared to CO . At the same time, the relative rate of recombination to mutation was the same in both populations. Higher nucleotide diversity observed in CO may suggest the presence of migrants from different source populations or that the CO population is native and is older than the CA population. As mentioned above, analyses including additional samples from Q. rubra) from Boulder and Denver, CO which is widely planted as a shade tree in the state. The disease was first documented in Boulder in 2012 when the number of dead oaks doubled and city foresters assessed tree health13. The disease has since spread to Denver, CO, but is not present on red oak in other cities across the state. In fact, the disease has not been recognized anywhere else on red oak. Further, red oak seedling are sold in local CO nurseries, but often these seedlings are re-sold from suppliers located in northeastern states of the country where this species is native rather than grown in CO. Several bacterial tree pathogens are known to exist in host tissue without causing symptoms and are therefore spread through grafting or nursery stock . Other members within the order Enterobacterales ord. nov. (formerly members of the Enterobacteriacea family21), B. goodwini and G. quercinecans associated with acute oak decline, were found in low abundance in healthy trees35. These two species as well as L. britannica and R. victoriana were detected in healthy tree tissues using metagenomics36, suggesting their possible endophytic behavior. It could be that L. quercina spread via nursery stock or moved from CO native Q. gambelii to introduced Q. rubra and then in older trees, 15–20 years later, began showing symptoms. This hypothesis needs to be tested in future studies, that will include identifying L. quercina populations from northeastern USA, a native habitat of Q. rubra, and populations from CO native Q. gambelii.Most isolates in CO were collected from introduced red oak  growth decline is associated with climate change. Sun et al.39 reported that warming temperatures and increasing drought significantly affect Quercus species richness and distribution in China. Using bioclimatic modeling, Mclaughlin & Zavaleta40 found that saplings of California valley oak (Q. lobata Nee) are especially susceptible to warming and drying conditions. Trees stressed by environment conditions can become more susceptible to other stressors, including pathogens41. It is possible that rapid changes in climate might have altered relationship between Lonsdalea spp. and their hosts. This is particularly likely when considering the drippy blight outbreak in Colorado, as these trees are planted outside of the climatic range where they are native.Bacterial-plant relationships can be altered directly, e.g., via introductions or by changes in environment, or indirectly, e.g., by changes in population dynamics of other organisms with which bacteria interact. According to Filippo et al.A. galliformis), now associated with drippy blight in CO, has been present in the state for a long time but was not recognized as a pest because of a lack of significant damage to oak trees. A spike in insect population size was observed with the first drippy blight outbreak13. Sitz et al.13 hypothesized that interactions between L. quercina and A. galliformis may be direct or indirect, whereby the scale facilitates pathogen movement into the trees via wounds or that the scale is possibly acting as a stressor of the host and facilitating growth and spread of bacteria within the host tissue. A similar observation was reported about the wood-boring beetle Agrilus biguttatus Fabricius, 1777 and B. goodwini relationship, in which presence of the beetle larvae triggers the upregulation of tree damaging genes in the bacterium leading to acute oak decline symptoms in Britain42. Factors leading to the increase of abundance of A. galliformis in CO are unknown. Because of the sessile nature of hard-shelled scale, it seems unlikely that populations are attracted to L. quercina infected trees to create new infestations, but it is possible. Trees infected with L. quercina may be beneficial to the scale, either through reduced levels of host response or changes in phloem sap composition caused by the bacterial infection. It remains to be determined whether the increased scale population and associated increase in the number of entry points through feeding sites has led to the increase of bacterial populations, or vice versa. But it is possible that increase in population size of one organism  could have caused increase in population size of another organism.The kermes scale  states of the USA where red oak seedlings are being produced. Further population genetic analyses, including more populations from Q. gambelii in CO and Q. rubra in NE USA, are needed to test these two hypotheses of the pathogen origin and clarify possible causes and sources of the recent outbreak of drippy blight in CO, USA.This study revealed that populations of Quercus gambelii oozing acorns and acorn caps) were collected from total of 83 (52 CA and 31 CO) trees of various oak species native to North America from trees in undeveloped habitats such as forests, parks, nature preserves, etc.  or developed habitats such as residential neighborhoods of cities, parking lots, yards of private houses in rural areas, etc. (referred here as “built areas”). The tree species were identified by co-author Dr. Ian Pearse  plate. Plates were incubated at 25C for 3–5 days. All cream-colored bacteria were isolated into pure culture on new plates with nutrient agar.In 2018 symptomatic samples with oozing acorns and/or acorn caps  and oozing branches  was used in PCR species verification. PCR conditions followed Sitz et al.14 and were: 94C for 3 min, 30 cycles at 94C for 30 s, 60C for 30 s, and 72C for 1 min, then finally 72C for 7 min using a MJ PTC-200 thermocycler . PCR products were visualized via Sub-Cell GT Wide Mini electrophoresis system  that yielded a single band (286 kb in length) on 1.5% agarose gel.Lonsdalea quercina isolates were incubated at 25 °C for 3–5 days for DNA extraction. DNA was extracted from pure bacterial colonies using Quick-DNA™ Fungal/Bacterial Miniprep Kit  following manufacturer protocol. DNA quality and concentration were estimated with NanoDrop One C spectrophotometer and Qubit 4MT with a dsDNA HS Assay Kit . Total of 83 Lonsdalea quercina strains (one strain per tree) were subjected to whole genome sequencing. Bacterial cultures were put in 15% glycerol for long term storage at −80 °C.Plates with pure cultures of verified Lonsdalea quercina was performed at the Novogene Bioinformatics Institute . DNA libraries were constructed using NEBNext Ultra DNA Library Prep Kit according to the manufacturer’s protocol. Libraries were sequenced on Illumina NovaSeq 6000 platform with 150 bp paired end reads. Raw data cleaning  and quality control were performed at Novogene.Illumina short read genome sequencing of 83 isolates of https://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Genome assembly, assembly quality control, and genome annotation were done using Reads2Resistome pipeline v0.0.244. Within the pipeline, genome assembly was performed using Unicycler v0.4.945, genome quality control was assessed using QUAST V5.2.046 and genome annotation was done using Prokka v1.14.547. The pangenome analyses were carried out with Roary pipeline v.3.13.048 with gff3 input files from Prokka annotation. Three datasets of core genes were used for further analyses that are described in detail below . Reads quality was assessed with FASTQC v.0.11.9 , DIAMOND: CAZy , and HMMER:dbCAN-sub . Only sequences annotated with all three tools were recorded. The annotation of genes involved in host–pathogen interactions within PHI-base was conducted with default parameters except e-value was set to 1e−15 and the percent identity threshold was set to ≥ 50%.Population specific core genes (245 in CA and 372 in CO population) were compared in functional annotation databases Swiss-Prot and GO through Orthovenn352. First, we determined the phylogenetic position of CA and CO populations within the genus Lonsdalea and the order Enterobacterales ord. nov.21. The genomes of members of four families from Enterobacterales ord. nov.  were retrieved from the National Center of Biotechnology Information  samples (CA + CO + E dataset) and with multi-fasta alignments of core genes of each population (mCA + mCO + E dataset), respectively  model54. Tree branch support was assessed via 1000 ultrafast bootstrap replicates55 with hill-climbing nearest neighbor interchange search bootstrap optimization to reduce the risk of overestimating branch supports (-bnni) due to severe violations of the model. The concatenated trees were visualized with iTOL online tool v.5.7 (itol.embl.de56). Average nucleotide identity (ANI) among all strains was estimated with JSpeciesWS using a BLAST-based approach (ANIb)57, using datasets CA + CO + E  for comparisons among L. quercina populations from this study and other species of genus Lonsdalea, and CA + CO , and to estimate ANI among L. quercina strains analyzed in this study.The maximum likelihood phylogenetic trees based on core genes were constructed with IQTREE v.2.0.358 followed by filtering with VCFtools v.0.1.1659: 1 SNP in every 1000 SNPs (–thin 1000) with minimal frequency 0.05 (–maf 0.05). Analysis was run within R package Adegenet v.2.1.360. An unrooted phylogenetic tree of CA + CO dataset was built to assess population structure within samples obtained in this study. The phylogenetic analysis was conducted as described above, with the best-fit model GTR + F + R3 , further analyses were performed on the dataset containing 1 representative of each core genome genotype. Population structure was assessed with principal component analysis (PCA) and phylogenetic analysis. PCA was conducted using 2355 thinned SNPs retrieved from core genes of CA + CO dataset. SNPs were extracted with SNP-sites software23. ClonalFrameML62 was used to estimate relative rates of recombination to mutation (R/θ) in each CA and CO populations and to construct phylogeny with branch lengths corrected to account for recombination. Maximum likelihood phylogenetic tree with CA + CO dataset was used as input for ClonalFrameML.Evidence of recombination was obtained with Split Decomposition analysis and the Phi test implemented in SplitsTreeQuercus agrifolia, Q. parvula, Q. wislizeni. In Colorado, symptomatic plant material was collected from commonly planted as shade trees Quercus rubra and three plants of wild Q. gambelii, an oak shrub commonly growing on the side of roads in the area. Moreover, all methods listed in this study were carried out in accordance with relevant guidelines and regulations.We declare that collection of plant material for this study complies with the relevant institutional, national, and international guidelines and legislations. There were no rare plants used in this study. All symptomatic plant material in California was collected from native to the region, commonly growing oak species Supplementary Information.


In evolutionary studies of human populations based on the Y chromosome, the majority of Native Americans belong to the QM3 lineage. Therefore, to study the history of groups inhabiting northern South America, it is necessary to have a higher resolution of the tree. The objective of this work was to identify new SNPs of the QM3 lineage that would allow the evaluation of the phylogenetic relationships between Andean and Amazonian populations of Colombia. Sequences previously obtained from two Y chromosomes of Amazonian populations were used, from which 13 potential SNPs were selected and typed in 171 Amazonian samples from the Vaupés region and in 60 samples from the Pasto, Nasa, Embera, Arhuaco and Kogüi ethnic groups of the Andean region. In addition, the main SNPs/markers  defining autochthonous Q lineages were typed, along with others defined by different SNPs/markers as reported in the literature . It was found that all the new SNPs are present in the Amazonian samples and only 2 of them are shared with the Embera, Nasa and Pasto, but none with the Kogüi and Arhuaco from the northern Andes, in the Colombian Caribbean. Combining the 13 variants of the present study with 14 previously reported and using TMRCA, a new QM3 tree proposal is generated. This method makes it possible to increase the number of sublineages of QM3 with a higher resolution and to detect differences between the different populations of Vaupés in the Amazon, as in the case of the Kubeos and Pisamiras, the latter of which is in grave danger of extinction. These new sublineages are useful for microevolutionary studies of the Amerindian populations of South America. The American continent was one of the last areas to be colonized by humans. Its settlement has been the subject of extensive debate at the level of evolutionary studies for more than 100 years . A largeInterest in knowing the ancestry of the American population has led to defining highly polymorphic Y chromosome-specific markers , 27–30. The phylogenetic tree of the Y chromosome provides a development to address the issue of population history and movements within geographic territories, especially the colonization of South America, although it is accepted that the ancestors of the Native Americans arrived from Asia, the number of migratory waves, the places of origin and the moment, still represents one of the topics with the greatest debate in evolutionary studies –20, 32.There are two main founding lineages that identify Amerindian populations: Hg C and Hg Q, both of Asian origin . Hg C isUntil a few years ago, most studies for South America only allowed to determine the main haplogroups of the Y chromosome. However, the genomic era and advances in DNA sequencing technologies have made it possible to carry out large-scale analyses, obtaining almost complete sequences of the Y chromosome, which has allowed us to identify some Y SNPs defining Q-M3 sublineages. One of great importance has been described, present at high frequencies and widely distributed throughout the American continent, Q-M848 . DespiteThis demonstrates the need to find new SNP-type variants that allow a greater differentiation of haplogroup Q1b1a1a defined by marker M3 or, failing that, haplogroup Q1b1a1a1a1 defined by marker M848. Therefore, the aim of the present study was to provide new clues about the genetic history of the Amerindian populations of Colombia. Here we present a new set of SNPs obtained from two Amerindian Y chromosomes from Amazonia, which allow us to evaluate the phylogenetic relationships between Andean and Amazonian populations from Colombia.For this study, a bank of indigenous samples from the Human Molecular Genetics Laboratory of the Universidad del Valle was used, consisting of 207 samples from men belonging to different Amerindian communities in Colombia and assigned to haplogroup Q1b1a1a defined by marker M3. From the southeastern region there were 171 samples from the Tucano-Oriental, Kakua and Hupda communities, and from the Andean region there were 36 samples belonging to the Embera Katio, Nasa  and Pasto (Nariño) communities see . All theSpecifically for this research, the approval of the Human Ethics Committee of the Universidad del Valle was obtained in 2010 (document number 197–010) and is still in force (document number 047–021). No samples or data have ever been collected or used without the prior approval of the Ethics Committee.The male sample bank, composed of 207 men belonging to different Amerindian communities in Colombia and assigned to haplogroup QM3, was collected during the period 2012–2015. The 60 new samples belonging to men from Colombian Caribbean communities (Arhuaco and Kogüis) were sampled in 2016.During the period (2012–2019), the data were accessed for research purposes. All sampling was done with the prior informed consent of each study participant and with the approval of the community leaders. This consent was given in writing.We considered male individuals (our target was the Y chromosome) of legal age, in good health and of sound mind, who voluntarily chose to participate in the study, without consanguinity to the third degree and with at least three generations of Amerindian ancestry. Participants were asked to respond to a brief family history survey about the ethnicity of their parents and grandparents. This was to ensure that individuals were not biologically related.For participant identification and privacy, each volunteer was assigned a code that identified him or her in all study documents. This code allows for masking of the participant from whom the sample was taken, protection of information, and confidentiality of results.Personal information (such as name or ID number) that would allow full identification was handled carefully. The researchers have a hard copy and a digital copy of the personal and family information. The hard copy is kept under lock and key at the Human Molecular Genetics Laboratory of the Universidad del Valle. The digital copy was kept in a personal computer used by the researchers for this purpose, with their respective security passwords to prevent access by third parties. The security and responsibility of the data will be the responsibility of the researchers of the project, and for no reason will they be known to third parties, except under legal obligation.This research does not present any legal risks, since it follows the guidelines of Resolution 8340 of 1993 of the Ministry of Health, which regulates these procedures in the communities. Here we present the survey conducted, the informed consent of each participant, the consent of the leader, and some of the ethical guarantees that were used throughout the research. This will protect the people and the researchers.All newly collected samples were typed for eight Y-chromosome SNPs  by mini-sequencing, using the ABI PRISM® SNaPshot battery. ™ Multiplex System ; and for 17 Y-STR , using the Y-Filer kit. The allelic assignment was done with the GeneMapper software to know who belonged to the QM3 haplogroup and how divergent their Y chromosomes were.In a previous study by Rivera et al. , the comAllele-specific primers were designed in the WASP web application  for eachThe 24 selected variants were typed by allele-specific PCR in all samples from the communities studied . In addition to the samples from the Colombian Caribbean (Arhuaco and Kogüi), the 14 SNPs reported by Rivera et al.  were typSNPs CO15, CO16 and CO19 were simultaneously amplified in multiplex 001, SNPs CO24, CO27 and CTS11357 in multiplex 002, SNPs CO18 and CO22 in multiplex 003, SNPs CO20 and CO21 in multiplex 004, SNPs CO23 and Z19483 in multiplex 005, SNPs SA05 and Z19319 in multiplex 006, SNPs L54 and M848 in multiplex 007, SNPs M346 and L56 in multiplex 008, and SNPs CTS11780 and Z780 in multiplex 009, using the following amplification conditions 1X buffer, 4.00mM MgCl2, 0.025mM dNTPs, 0.32μM primers, 0.033U/μL Taq polymerase and 10 ng DNA in a 15μL reaction. Thermocycles are shown in The new variants reported here were validated in the Amerindian populations of the present study by the absence or presence of the derived allele in each sample. In addition, they were validated in a dataset of modern sequences of the Amerindian Y chromosome belonging to haplogroup Q found in the databases , 47, andThe AMOVA statistical test was performed with the program Arlequin Ver. 3.5.2.2. . The anaTo associate the SNPs with the samples and study populations and to observe how they are related, a multiple correspondence analysis (ACM) was performed in the R program version 3.5.3 . The anaIn addition, the geographic distribution of the reported subhaplogroups of 483 Amerindian Y chromosomes belonging to the Q-M3 haplogroup was evaluated. Of this subclassification, 231 were performed on their own samples, while the remaining 252 were performed on samples from other studies , 47, 48.To observe the behavior of the haplotypes of each of the studied SNPs and to observe their frequency and variation, Y-chromosome haplotype networks were constructed using the Network Ver. 5.0.0.1 software . NetworkTwo Y chromosomes belonging to haplogroup Q-M3 were used for phylogenetic analyses. The new SNPs of this work were included, those reported by Rivera et al.  and thosThe tree version proposed by , 41 was To estimate the time to the most recent common ancestor (TMRCA), the calculation of the Rho network statistic was used considering four different mutational rates: 1) EMR (evolutionary mutation rate) considering a mean effective mutation rate of 6.9*10-4/locus/25 years as proposed by ; 2) OMRSOut of a total of 60 samples collected for the Colombian Caribbean, only 24 were classified in QM3. With the addition of these new samples to the existing sample bank at the Human Molecular Genetics Laboratory of the Universidad del Valle, the present study included 231 Amerindian samples.24 SNPs were typed (13 new and 11 reported in the literature) for the populations studied and, in addition to the samples from the Colombian Caribbean, 14 SNPs reported by Rivera et al. , the S4 SNP CO23 and SNP CO20 presented the lowest frequency in the study (2.1%), both are found in the most distal branches of the proposed phylogenetic tree, SNP CO23 was restricted to the Desano and Tukano populations of the linguistic subfamily Tucano-Oriental, while SNP CO20 is exclusively restricted to the Kubeos population. Our language classification is based on .The SNPs CO24 and CO27 presented the highest frequency, they were found in 153 (66.2%) of 231 individuals, they also have a wide distribution, with the exception of the Caribbean populations and the Nasa, they were found in the rest of the populations. SNP CO15, SNP CO19, SNP CO22, SNP CO17 and SNP CO26 have relatively low frequencies and are restricted to the Amazon region, mostly associated with the Tucano and Pisamira populations. On the other hand, SNP CO16, SNP CO18, SNP CO21 and SNP CO25 have higher frequencies, although they are also restricted to Amazonian samples, especially to the Kubeos population, where they have the highest frequency.The SNPs that define autochthonous haplogroups for Amerindians, Q-L56, Q-M346, Q-L54, Q-M848, were present in all study samples and, as expected, the frequency of SNPs Z780 and CTS11780 was null in the study.With the exception of the SNPs that define major Amerindian haplogroups , the other variants studied in this research had a null frequency for the Colombian Caribbean samples. Regarding the five variants reported by Jota et al. , their fFrom the information reported by Arias et al. , data weRegarding the autochthonous Amerindian haplogroups , as expected, 100% of the analyzed samples, that is 252, belonged to these lineages. The sublinaje Q-M848 showed a high frequency, it was found in 147 individuals (58.3%), the SNPs CO10, CO12 and CO24 showed relatively high frequencies, they were found in 59 individuals (23.5%), they also show a high distribution, they were found in samples from Brazil, Ecuador and Colombia. The SNPs CO03 and CO05 were found in 39 individuals (15.5%), the SNP SA05 in 12 individuals (4.8%) from the Colombian Orinoquía. The SNPs CO01, CO02, CO08, CO13, CO14 and CO24 were also found at lower frequencies. It should be mentioned that several of the sequences did not have data in the position where each SNP was found. When we have the complete sequences of the Y chromosome region, it is very likely that the frequency of reported SNPs will increase. See the details of this validation in the Analysis of Molecular Variance (AMOVA) shows significant differences between the study populations; when comparing the Andean populations with the Amazonian ones, it was possible to observe that the highest percentage of variation contributed between regions (53.89%), followed by variation within populations (46.31%), and finally between populations within groups was close to zero. With the exception of the source of variation between populations within groups, in all cases there was statistical significance of each component of variance . When thThe analysis was also carried out in terms of linguistic groupings. However, the separation of the two Amazonian groups was not clearly observed . For this reason, we keep the analysis based on geography.The classification of the subhaplogroups evaluated in the present work in 483 Amerindian Y chromosomes belonging to the Q-M3 haplogroup is presented in Reconstruction of the Q-M3 haplotype network see , based oThe results of SNP genotyping allowed the detection of 13 new variants. The phylogenetic tree of Q, including the new variants discovered in the present study and those described by other authors, is shown in With this study, our laboratory continued the work by Rivera et al.  related The newly reported variants show a distribution almost limited to populations from the Amazon. This makes it possible to identify differences between these populations, as in the case of the Kubeos and Pisamiras, the latter in serious danger of disappearing (currently there are only about 20 active speakers and passive speakers are below 60) . In the According to the results by Jota et al.  and the Considering that Q-SA05 appears to have a relatively ancient origin  and that, in addition, previous studies showed a strong association with Amazonian indigenous populations of several linguistic families , it was However, the same variant was found in 12 samples belonging to the departments of Meta, Guaviare and Amazonas in Colombia, when it was evaluated in the sequences of samples of indigenous people from the northeastern Amazon in the work by Arias et al. . It coulThe CO12 sublineage was one of the oldest SNP type variants described by Rivera et al.  and CO27This is a relatively old lineage, according to the literature, around 11.3 kya it presented a population focus in Mexico, which eventually spread to the southwestern United States, Central America, reaching Colombia and part of the Brazilian Amazon . In the These populations coexist with communities of different languages, such as the Tucanos, where there is a continuous exchange of practices, values and knowledge. This favors the emergence of transethnic identities in which language can be preserved or modified, in other words, processes of "Arawakization" or "Tucanization" occur . Based oThe Piapoco and Achagua indigenous peoples come from an expansion from the mouth of the Negro River to the Isana and Guainia river basins , that isThe 13 new SNPs evaluated show a distribution almost restricted to Amazonian populations, which was expected given the origin of the variants. However, some of them were found in samples from the Andean region . It is important to note that these Andean populations are linguistically close, belonging to the Chibcha-Paezan branch, while the Tukano are linguistically distant. This result supports the conclusion of the study by Barbieri et al. , in whicThis result could be due to possible gene flow or a common ancestor. With regard to the possibility of gene flow, there could be migration of individuals from the Colombian Amazon to the Andean region. Jota et al. , based oIf, however, we consider the main entry points to South America to be the Panamanian isthmus and the Darien area in northern Colombia, then it is possible that the populations were split or separated. Some migrated and settled in the Andes, and others migrated toward the Amazon. By then, both populations had some of the older mutations proposed here; this would indicate a common origin of all ethnic groups, with subsequent expansion and linguistic and biological divergence.Recently, Delgado M et al,  conducteThis could be attributed to demographic processes such as assimilation/mixing between local and extra-regional populations rather than gene flow, and to sex-biased migrations closely linked to changes in social organization . This maThe frequencies of the 13 new variants typed in the present study and of the 14 reported by Rivera et al.  were zerThe TMRCA for each SNP showed differences in the values obtained, due to the mutation rate used. This indicates that the choice of mutation rate is very important, since it depends on the estimation being as close to reality as possible. There are two mutation rates reported as the most used, the genealogical or observed rate (OMR) and the evolutionary mutation rate (EMR), although they have a general pattern, the values estimated with EMR are considerably higher compared to those obtained with OMR.To study major haplogroups, it is suggested to work with the OMR mutation rate, especially when lineages coalesced to the Neolithic or more recent times . In viewOn the other hand, it could be said that the demographic events that were promoted by the diffusion of agriculture could explain the expansion of the other subhaplogroups reported here, whose estimated age ranges between 109 and 2560 years; that is, the most recent SNPs within the phylogeny of haplogroup Q .Our results give us an idea of the relationship between the different sublineages of haplogroup Q, indicating that the oldest SNPs (SNP CO24 and SNPCO27), found in the basal branch of the rest of the lineages, are found in most populations of the study , strengthening the hypothesis of a possible common ancestor in these populations and favoring more than one expansion during the population of the Americas . HoweverThe colonization of South America is not clear. According to Tarazona et al. , there iAlthough this model holds true for some populations, this pattern cannot be replicated for all South American natives. In subsequent studies, the genetic variation of the Y chromosome lacks a clear structure that can be related to geography and linguistics at the continental level. This means that a correlation between genetic and geographic/cultural/linguistic structure can only be expected under very specific conditions .This may be due to variations in the markers used or to non-homogeneity in the populations sampled. And where it would be key to keep in mind the elements that have a direct effect on the genetic structure of a population, we have cultural, environmental, demographic, etc. factors. For example, sex-biased migration , which is very common and can be associated with multiple causes ranging from cultural practices to environmental components. This makes it difficult to decipher local demographic histories and thus to replicate models of population structuring .Another factor that needs to be taken into account is the change in post-marital residence patterns, in which we find patrilocality and matrilocality, which have a direct impact on genetic diversity and structure –66.Taking the Colombian indigenous population as an example, not all of them have the same behavior at the structural level, so it is good to emphasize two main groups: Amazonian indigenous communities and Andean indigenous communities. Based on these two divisions, for the Amazonian populations it is expected that the linguistic component will have a greater weight on their population structure and diversity, since in this area marriages are performed between speakers of different indigenous languages (linguistic exogamy). This increases genetic diversity through a particular genetic flow (asymmetric) in which women are the ones who migrate and men remain settled in their territories , which iRegarding the Andean populations, they behave completely different from the Amazonian indigenous communities, where the geographical position is very important in structuring the populations. Among their geographical positions are the central, eastern and western mountain ranges, which act as a physical barrier to gene flow in the different populations . It is aAccording to the above, there are many factors that must be taken into account when following a model to try to interpret the distribution of genetic variability in the native populations of South America, among them we have the geographical proximity, linguistic, economic, social differences, sexually biased migrations, residence patterns and, in general, everything that encompasses their social dynamics.The AMOVA results are different from those commonly found in Native American populations, where most of the variation is found within populations. In the present research, this variation is being accounted for between regions/groups. In order to understand these results, several aspects need to be considered: 1. The Amazonian origin of the samples that were sequenced and from which the new SNPs that were typed in the rest of the populations were selected, 2. The way in which the populations were grouped within the study and 3. There is no homogeneity between the number of samples for each study region, with by far the largest number of samples from the Amazon region.When comparing the diversity of the Vaupés groups with other Native American groups, it is possible to see that the difference depends a lot on the region. Mesoamerican and Andean groups tend to have moderate to high diversity values in Y chromosome and mitochondrial DNA. While in Amazonian populations whose social structure is based on small family groups, diversity tends to be moderate to low but with greater differentiation between groups , 75.The local social dynamics of these groups have shaped the genetic diversity and structure we see today. For example, exogamy is practiced in this region, which is undoubtedly one of the main differentiating forces between groups, both from a linguistic and Y-chromosomal point of view. They also have a patrilineal social organization, in which married men continue to live in their father’s territory and the identity of the individual is determined by the ethnolinguistic identity of the father , 61.In addition to social organization, wars, forced displacements, the physical landscape, geographic barriers, subsistence strategies, disease, shamanism, and extinction are factors that alter the dynamics of these Amazonian populations and, therefore, their structure.The methodology used in the present study provided 13 new SNPs, which facilitated the proposal of a new version of the phylogeny of Q-M3, which provides an informative male ancestry with new knowledge for research on the history of the peopling of South America.The absence of these variants in the indigenous peoples of the north coast of Colombia allows us to propose the existence of at least two migratory routes to South America. However, the genotyping of a larger number of samples from this region will allow further clarification of this matter.The Andean samples show a clear structuring of the paternal lineage, but it is not fully explained by the SNPs reported in this study, therefore it is necessary to search for new variants typical of the Andean region that will allow the evaluation of phylogenetic relationships between these groups. To do this, a larger population sample is needed in Colombia and neighboring regions to reveal its true distribution among native South Americans.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S1 Questionnaire(DOCX)Click here for additional data file.


ST range 0.03–0.21), with 7% of genetic variance distributed among sites. Allelic richness within some bulbs was nearly as high as that in the total population, suggesting that colonisation of bulbs was not associated with strong population bottlenecks. The significant genetic structure we observed over small spatial scales seems to reflect limited dispersal of mites in soil.Bulb mites are an economically significant pest of subterranean parts of plants and a versatile laboratory animal. However, the genetic structure of their populations remains unknown. To fill this gap in our knowledge of their biology, we set up a field experiment in which we allowed mites to colonize onion bulbs, and then determined the genetic structure of colonisers based on a panel of microsatellite loci. We found moderate but significant population structure among sites separated by ca. 20 m (FThe online version contains supplementary material available at 10.1007/s10493-023-00807-1. Rhizoglyphus robini, have been a subject of numerous eco-evolutionary studies , with a thickened and sharply terminated third pair of legs used for fights, and non-aggressive homeomorphs (scramblers), with unmodified legs , Rr91  and Rr92 . Allelic richness for all loci and populations are shown in Table Mites colonized five out of 14 onions set up  tests (Table S4). Most of these LDs were repeatable between populations , indicating a possibility of two physical linkage groups (Rr51–Rr79 and Rr40–Rr47–Rr91). We therefore retained for further analyses only one locus per linkage group, based on criteria of higher polymorphism, fewer missing genotypes, and lower proportion of null alleles. We thus rejected from further analyses loci Rr40, Rr47 and Rr79. However, results of analyses of such a selected subset of seven loci that we report below were very similar to those based on the full set of 10 loci (not shown).ST values among populations are given in Table ST values except for the lowest of these (between populations 2 and 6a) did not overlap zero. FST among the neighbouring populations 6a and 6b (isolated by ca. 20 cm) was only 0.014  there was moderate, but significant differentiation among populations inhabiting patches of vines located in the same plot   and unrelated individuals. This could select for kin recognition abilities, that can be used to avoid killing and cannibalising relatives  had lower allelic richness, and exhibited the highest FST estimate is the lowest among the ones we obtained and not significant, which could be a result of migration from one bulb to the next, or due to colonisation of both onions from the same source population inhabiting local soil. The former possibility predicts that allelic composition of the recipient onion should be a subset of that present in the source onion, but although we observed some allele sharing between 6a and 6b, private alleles were present on both . This pattern is more consistent with the latter possibility, i.e., independent colonisation of the neighbouring onions from the same source population residing in local soil. This additionally highlights the role of soil as a reservoir of mites colonising bulbs and tubers Below is the link to the electronic supplementary material.


The Y-chromosome has been widely used in forensic genetic applications and human population genetic studies due to its uniparental origins. A large database on the Qatari population was created for comparison with other databases from the Arabian Peninsula, the Middle East, and Africa. We provide a study of 23 Y-STR loci included in PowerPlex Y23  that were genotyped to produce haplotypes in 379 unrelated males from Qatar, a country at the crossroads of migration patterns. Overall, the most polymorphic locus provided by the Promega kit was DYS458, with a genetic diversity value of 0.85 and a haplotype diversity of 0.998924. Athey’s Haplogroup Predictor tool was used to predict haplogroups from Y-STR haplotypes in the Qatari population. In a median-joining network, the haplogroup J1 predominance (49%) in Qatar generated a star-like expansion cluster. The graph of population Q-matrix was developed using Y-STR data from 38 Middle Eastern and 97 African populations , and it demonstrated a stronger sub-grouping of countries within each ethnic group and showed the effect of Arabs on the indigenous Berbers of North Africa. The estimated migration rate between the Qatari and other Arabian populations was inferred using Bayesian coalescence theory in the Migrate-n program. According to the Gene Flow study, the main migration route was from Yemen to Kuwait through Qatar. Our research, using the PowerPlex Y23 database, shows the importance of gene diversity, as well as regional and social structuring, in determining the utility of demographic and forensic databases. Qatar occupies the Qatar Peninsula, which extends northward from the Arabian Peninsula’s eastern edge into the Arabian Gulf and covers 11,427 km2 . Doha, tThe Arabian Peninsula’s inhabitants can be divided into at least four genetic subpopulations that can show the region’s historical migration patterns: Bedouin, Iranians, South Asian, and African. This demographic pattern mirrored on the Qatari population, which included Iranians, Africans, and Bedouins. However, because of its extensive ancestry in the Arabian Peninsula, the Bedouin genetic subgroup was the most prominent –8.The key position of Qatar on the Arabian Gulf was the main reason for the seasonal migration of Arab tribes from the Arabian Peninsula. It was a tricontinental nexus for human migration especially during the bidirectional dispersals between Eurasia and Africa .Short tandem repeats on the human Y chromosome (Y-STRs) markers have useful characteristics for genetic applications such forensic evidence analysis, genealogy research, and historical investigations . AdditioY-STR markers on the Qatari population were studied previously by a limited number of markers. Qataris are organized into patrilineal descent groups, yet little has been done to study the population structure. A larger database of the Qatari population therefore needed to be established, for comparison with other Arabian Peninsula and Middle Eastern databases. The aim of this study is to investigate the genetic variability of the Arab population in Qatar utilizing 23 Y-STR loci from PowerPlex Y23  and to trace human migration in the Arabian Peninsula. Furthermore, we used STRUCTURE to examine the broad-scale population structure of the Middle East and Africa .A total of 379 saliva samples were collected anonymously from unrelated indigenous Qatari males Arabs from Doha. All samples used in this study were obtained for the purpose of the research and ethical approval was granted by the Ministry of Interior of Qatar and the University of Central Lancashire “Ref STEM454”. Informed consent forms were completed by all participants and authors had no access to information that could identify individual participants during or after data collection. The samples were collected taking into consideration relatedness, ensuring that all sample males were separated by at least three generations. The data were collected in June 2016 and made accessible for research purposes in August 2016.Additional information regarding the ethical, cultural, and scientific considerations specific to inclusivity in global research is included in the ® extraction DNA Mini protocol . The extraction procedure was followed in accordance with the manufacturer’s recommendations. The quantification of the collected samples was carried out using the Quantifiler® Trio DNA Quantification kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. All of the samples were quantified according to the manufacturer’s instructions on the 7500 Real-Time PCR System and analysed using the HID Real-Time PCR Analysis Software v1.2.The samples were extracted using the QIAamp® 96-Well Thermal Cycler as recommended by the manufacturer (Thermo Fisher Scientific). PCRs were conducted using half of the recommended volume. Fragments were electrophoresed in eight capillaries (50-cm length) arrays on the ABI 3500 Genetic Analyser using the manufacturer’s recommended protocols (Thermo Fisher Scientific) filled with POP-4™ polymer. GeneMapper IDX software V1.4 was used for allele calling and interpretation.The PowerPlex Y23 kit was used to generate 23 loci: DYS576, DYS389I, DYS448, DYS389II, DYS19, DYS391, DYS481, DYS549, DYS533, DYS438, DYS437, DYS570, DYS635, DYS390, DYS439, DYS392, DYS643, DYS393, DYS458, DYS385a/b, DYS456 and Y-GATA-H4. DNA amplification Reaction setup and thermal cycling were performed according to the procedures described in the PowerPlex Y23 kit User’s Manual. Thermal cycling was performed in VeritiThe haplotype diversity for the Qatari population samples was evaluated by Nei’s formula , HD = n*The population genetic structure in our data was evaluated by the analysis of molecular variance (AMOVA). Molecular data were obtained for the Qatari population using Y-STRs based on the PowerPlex Y23 System and compared with the available data on other Middle Eastern populations –30. CompIn this research study, the methodology employed involved the use of full Y23 haplotypes to allocate haplotypes to their most likely haplogroup, a process which was facilitated through the utilization of Athey’s Haplogroup Predictor. It is pertinent to note that within the scope of this study, DYS543 and DYS533 were excluded from the data due to the unavailability of allele frequency data relating to these markers –36.The batch program utilized 111 markers from the FTDNA set to conduct its prediction, employing the following set of criteria: a fitness score exceeding 15, a probability exceeding 85, and priors attuned to the Near East population. In several instances, the program failed to generate a prediction for certain samples as no haplogroup met the established criteria. Consequently, the haplotypes were subjected to manual examination, yielding results in some of the cases.The present study implemented Median-joining networks using specialized software, namely Network v5.0 and v2.1.2.5 . To prodThe structure of 38 Middle Eastern –30 and 9The STRUCTURE HARVESTER program was employed to process the output and assess the probability values over a broad range of K values, alongside identifying the optimal number of genetic clusters that align with the data ,59. To cThe Migrate software program employs a Bayesian inference framework to estimate several key population parameters, including effective population sizes, historical migration rates between varied populations under an asymmetrical migration model, and population divergences or admixture . A data The Bayesian posterior probabilistic model is commonly utilized in the domain of population genetics to infer the parameters of a given model. Migrate, a widely-used software program, applies this approach to estimate population genetic parameters from the available genetic data. Migrate calculates the posterior probability of various population genetics parameters, such as migration rates and population sizes, by integrating possible relationships within the sample data. This is accomplished through the use of an expansion of the coalescent theory , which eThe gene flow was examined using four models in the Migrate software. Qatar was tested against five countries: Yemen, Saudi Arabia, Iraq, the United Arab Emirates and Kuwait. The present study used four distinct gene flow models to investigate migration patterns. The first model entailed unidirectional gene flow from one population to another. The second model accounted for divergence from a common ancestral population, while the third model incorporated both divergence from the ancestral population and ongoing immigration. The fourth model assumed that both populations belonged to the same panmictic population. To compare the relative strengths of the model fits, the log marginal likelihoods were utilized to calculate the Bayes factors. Magnitudes of the Bayes factors provided evidence for the degree of dissimilarity between the models.To ensure thorough exploration of genealogic space, it is recommended that a single long chain is used in conjunction with a lengthy sampling increment of at least 1,000. This allows for efficient data exploration and facilitates the fitting of different genealogies; ultimately leading to more accurate confidence interval estimations.In accordance with the prescribed methodology, a burn-in value of 5,000 discarded trees per chain was established. Subsequently, based on this value and the aforementioned parameter, each iteration within the sample set underwent an exhaustive evaluation process, totaling 5,000,000 assessments. The experimental approach employed the STATIC heating scheme, which entailed interchanging several chains, in an effort to achieve more comprehensive searches while maintaining an acceptable level of acceptance standards. To optimize the efficacy of the swapping methodology, four chains were utilized, corresponding to respective temperatures of 1.0, 1.5, 3.0, and 10,000, which were arranged in ascending order of thermal intensity. It should be noted that the initial temperature was maintained at a minimum of 1, in accordance with established guidelines .The 23 Y-STRs profiled with PowerPlex Y23 kit were amplified for 379 Qatari males. S1 Table in For the purposes of calculating forensic parameters, 27 haplotypes with duplicated alleles were removed from the Qatari population, bringing the total number of haplotypes to 352. Allele frequency distributions of the 23-STR loci and the most frequent allele for each locus are presented in S3 Table in Genetic diversity and match probability values for each locus are presented in S4 Table in A total of 327 were the unique haplotype and the DC for the kit was 86.2% for the Qatari population. The haplotype diversity for the Qatari population was 0.998924.We compared the performance of the PowerPlex Y23 kit to the previous Y-filer kit (AmpFLSTR™ Yfiler™ PCR Amplification Kit (Applied Biosystems™)) by eliminating the six newly added markers, and the number of distinct haplotypes was reduced to 289 and the DC was reduced to 76.2%. Furthermore, haplotype diversity dropped to 0.995498.Y-STR haplotypes for 379 male samples were obtained for Y-STRs. STR haplogroups were determined using the haplogroup prediction tool by Athey. The majority (48.5%) of 379 Y-haplogroups was assigned to haplogroup J1, a branch of which was the predominant Y-chromosome haplogroup of populations in the Arabian Peninsula; this was followed by J2 (12.9%), R1a (7.7%), E1b1b (5.3%), E1b1a (5%), T (4%), L (4%), G2a (2.1%), R1b (1.1%), and less than 1% for the rest of the haplogroups , then Saudi Arabia [south] (RST = 0.05873), and Saudi Arabia [north] (RST = 0.06009). The farthest relationship is found between Qatar and Iraq [Kurd], Turkey [Dogukoy] and Palestine [Christian Arab] with RST values  respectively.A pairwise matrix plot of R 17 loci . The cloST pairwise value was between Bahrain [Muharraq] and Iraq [Arab North] (RST = 0.19963), whereas the lowest pairwise RST value was (RST = -0.00295) between Jordan  and Jordan .The highest RThe average pairwise differences among Middle Eastern populations were calculated using 17 loci. This average pairwise difference was calculated to show the genetic differences between the 38 Middle Eastern populations and within these populations, in addition to among populations using Nei’s distance .The highest corrected average pairwise value using Nei’s distance between populations was (50.20693) between Bahrain [Muharraq] and Iraq [Sorani Kurd]. Jordan  and Jordan  had the lowest value (-0.9117).Kuwait had the highest average pairwise difference value within populations (241.8143), while Turkey [Dogukoy] had the lowest value (143.64482).The highest average pairwise value between populations was (265.31249) between UAE and Iraq [Sorani Kurd], while the lowest value was (147.99659) between Turkey [Eskikoy] and Turkey [Dogukoy].In the MDS and genetic distances between populations for Y-filer (17 markers) were calculated between all populations using the R statistical program version 4.0.3, to display the relationships among the 38 Middle Eastern populations . Two MidThe second dimension of the plot shows four clusters located in different quarters of the MDS were created. Iraq (Iraqi), Lebanon Israel , Jordan , fell into one cluster in the lower left quadrant, while Iraq (Arab) and Qatar were further away to the lower part of this quadrant. The rest of the Iraqi ethnic groups  were clustered together with Palestine (Christian and Muslims) in the upper left quadrant. Saudi Arabia  formed a cluster with Bahrain , Iran , Yemen (Arab) and Turkey (Eskikoy) extending from the lower right to the upper right quadrant. The fourth cluster occupied the upper right quadrant and formed by Turkey , Iran , Yemen (Jews) and Saudi Arabia (east).Phylogenetic tree was generated using R statistical program version 4.0.3. The 38 Middle Eastern populations were clustered using the R statistical programme version 4.0.3. . Six clusters (K = 6) were identified. As shown in The third cluster consists of four gulf countries: the UAE, Saudi Arabia, Kuwait, and Bahrain, while the rest of the Middle Eastern countries comprise the sixth cluster.The Y-STR graph of population Q-matrix  revealedThe populations of the Middle East form their own cluster. One notable difference was the Jewish Ashkenazi population. The north Africa cluster demonstrates that Egyptians are distinct from other north African populations. Except for the Libyan and Moroccan Jews, who showed similar clusters to the Middle Eastern populations, Libya, Tunisia, and Morocco shared the same pattern. Sudan’s population structure is similar to that of central Africa.With the exception of Cameroon, Central African populations form a consistent cluster. The pattern then gradually shifted towards the west African populations, with the exception of Tanzania and two Kenyan populations (Luhya and other Bantu), which showed the similar clusters as the west African populations. The South African population has its own cluster, with the exception of people of different racial and ethnic backgrounds, including European, Asian, and Ashkenazi Jews. The four African population samples collected outside of Africa, in the United States and the United Kingdom, were not assigned to any of the African regions and are included at the end of the population Q-matrix graph. Their cluster pattern, however, appeared to be very similar to that of west and south African populations.The populations of Jews were seen in three distinct parts of the admixture plot. We have Ashkenazi, Samaritan, and Yemenite Jews in the Middle East; north African Jews in Libya and Morocco; and Ashkenazi Jews in south Africa. With the exception of Ashkenazi Jews in the Middle East, all Jewish populations share the same structure as the Middle Eastern populations.Despite sharing a pink colouring with the North African Arabs of Libya, Tunisia, and Morocco, the Berbers of Morocco  may be distinguished from them by their different genetic profile. The Egyptian Berbers of Swia, on the other hand, shared certain characteristics with the Middle Eastern cluster.Four migration model routes were used to investigate Qatari population movements and their impact on the Arabian Peninsula. Between Qatar and the five countries of the UAE, Iraq, Kuwait, Saudi Arabia, and Yemen, five routes were examined. For each route, four models were used, and the results revealed that model 2 was the most significant, as shown in S8 Table in Qatari inhabitants exhibit two bidirectional migration trends, the first from Qatar to Yemen (Log (ml) = -2244.24), Saudi Arabia (Log(ml) = -2465.67), Kuwait (Log (ml) = -2384.68), and Iraq (Log (ml) = -2496.75), all of which show a minor predominance. The second migratory route was from the UAE to Qatar (Log (ml) = -2444.35), which was slightly more prevalent. The most likely migration routes among these five were between Qatar and both Yemen and Kuwait. However, the route Yemen, Qatar, and Kuwait is considered part of the Arabian Peninsula’s coastal migration path .In the current investigation, an analysis was conducted on Y-chromosomal STR haplotypes utilizing the PowerPlex®Y23 kit (Promega Corporation). As anticipated, this kit demonstrated a robust discriminative power suitable for forensic applications. The robustness of the locus outcomes generated can be attributed to the greater number of markers employed in the kit and the intentional selection of markers with a heightened capacity for differentiation. In particular, it is noteworthy that three out of the top five Y-STRs exhibiting greatest diversity in this examination  were unique to the PowerPlex®Y23, and in agreement with global research findings conducted across various meta-populations ,70.With a proportion of 51.1%, the Qatari population demonstrates the third highest identified frequency of microvariant alleles at the DYS458 marker, trailing behind Saudi Arabia (70%) , and YemThe present investigation determined that haplotypes containing microvariants located at DYS458 among the Qatari population were notably correlated with haplogroup J1. This observation agrees with recent research conducted on Y-chromosome studies within the Arabian Peninsula ,20,25,26The dominancy of the haplogroup J1 in the Arabian Peninsula can be elucidated through previous investigations which have established a correlation between haplogroup J1 and aridity in the Arabian Peninsula, as well as its association with the speakers of Semitic tongues, notably Arabic. In addition to its prevalence among the Middle Eastern populations, this haplogroup has also been traced among Central Asian and South Asian communities. Furthermore, its detection has been documented in contemporary West Asia, North Africa, the Horn of Africa, and Southern Europe –73. NumeThe founder effect presents an additional factor that contributes to the prevalence of the J1 haplogroup, which is accompanied by the process of genetic drift. Moreover, the region in question exhibits significant levels of non-random mating, with cultural practices that foster patterns of patrilocality and polygamy, thereby promoting the preservation of male lineages within the area. Additionally, consanguineous marriages—including those between first cousins—are a notable feature of Middle Eastern societies due to long-standing Muslim traditions, leading to inbreeding phenomena that serve to propagate a particular patrilineage .ST measures. It is noteworthy, however, that these prior works have only limitedly explored a reduced range of 9–11 Middle Eastern populations for comparative analysis, as evidenced by studies such as those outlined in references [In scholarly discourse, researchers have recently endeavoured to compare Middle Eastern populations by utilizing MDS plots and Rferences ,24,30. TThe findings of the Multidimensional Scaling (MDS) analysis indicated that the nations located within the Arabian Peninsula exhibited a distinct clustering pattern. However, the present investigation has revealed that the Qatari demographic shares greater proximity with the Iraqi Arab population and has formed a separate cluster when distinct from the remaining populations residing within the Arabian Peninsula. Notably, the uncovering of valuable archaeological evidence, such as diagnostic pottery sherds belonging to the Ubaid period (6000 B.C. to 4000 B.C.), has further amplified the existence of an interconnected relationship between the regions of the Gulf and Mesopotamia ,76.In recent years, much attention has been paid to populations structure analyses of the Y chromosome. As such, with regards to the Middle East, it is worth noting that there have been two studies conducted on the said topic. The first of these studies, conducted by Lazim et al., undertook a global analysis of Y-STR in 134 populations worldwide, encompassing 21,323 individuals. While this work was able to identify nine specific clusters using 19 markers, its limitations included a lack of representation, with only seven Middle Eastern and ten African populations examined .In contrast, the second study exclusively examined 23 Middle Eastern populations totalling 3833 individuals and provided insight into four distinct clusters, using 17 markers . HoweverIt is in this context that our study presents a significant contribution to the forensic field. In seeking to build upon the insights provided by previous researchers, our study adopted the population Q-matrices of the Y-STRs and harnessed its full potential in a carefully crafted examination of 97 African and 38 Middle Eastern populations. Our study drew upon 17 STR markers in our identification of eight clusters, culminating in an extensive analysis of 11,305 individuals. Ultimately, our work represents a more thorough and precise exploration of the genetics of Africa and the Middle East as compared to earlier studies.Despite the inclusion of two distinct geographic regions, Africa and the Middle East, and the implementation of 17 markers, the population Q-matrix yielded a remarkable graph total of 8 clusters (K = 8). This outcome suggests an unexpectedly high number of distinct geographic and population relationships. It is worth noting that in the preceding worldwide investigation, 9 clusters were discovered through the use of 19 markers. . This coThe African continent boasts of striking genetic, linguistic, cultural, and phenotypic diversity. With more than 2000 distinct ethno-linguistic groups, African languages make up almost one-third of the world’s spoken languages. The African populations exhibits a wide range of subsistence patterns, including various agriculture practices, pastoralism, and hunting and gathering. Notably, African languages have been classified into four primary language families, namely Niger-Kordofanian, Afro-Asiatic, Nilo-Saharan, and Khoisan, with each family predominantly used by certain populations across the African region. The Niger-Kordofanian family, spoken mainly by agriculturalists, covers a vast geographical distribution in Africa. Conversely, Afro-Asiatic is predominantly spoken by pastoralists and agro-pastoralists in northern and eastern Africa. Nilo-Saharan is the primary language spoken by Eastern and central African pastoralists, while Khoisan is primarily used by southern and eastern African hunter-gatherers ,78.The present study’s structure Analysis within African groups have unveiled further substructure, highlighting distinct genetic structures in north, east, west, and south Africa. These findings support the perception of substructure existing between cultures associated with hunter-gatherer and agriculturalist lifestyles ,78.The current study identified another noteworthy observation using STRUCTURE program, wherein a resemblant cluster pattern was observed between the four African population samples located outside of Africa and the populations from the west and south of the continent.A logical explanation for this common genetic pattern could be attributed to the instances of the slave trade from Africa, which hold significant historical importance as one of the most prominent forced migration events. The transatlantic slave trade lasted for approximately five centuries, spanning from 1400 to 1900 and was categorized into four major waves. The final wave of the transatlantic slave trade, which primarily arose along the West African coasts, holds the distinction of being the largest in terms of scale and duration, with over 12 million Africans being coerced to undertake a perilous journey across the Atlantic Ocean .North Africa’s demographic history has been characterized by distinctive features, such as population replacements, extensive gene flow, and diverse admixture from neighbouring regions, particularly the Middle East, which sets it apart from the rest of the continent. The Arab expansion, which began in the seventh century C.E., had a profound and lasting impact on North Africa that reached the westernmost part of the continent .One of the debates revolves around the impact of Arabs on Berber populations, where two contrasting scientific theories exist. The first suggests that there is no genetic differentiation between Arab and Berber populations, as they share similar genetic characteristics ,82. The This study yielded significant insight into the genetic structure of Jewish communities. It was determined that, with the Middle Eastern Ashkenazi Jewish community being the sole exception, the Jewish populations in the study exhibited a genetic structure that is resemble of the Middle Eastern non-Jewish populations. This finding supports the notion of a shared regional ancestry among Jewish communities. Moreover, the results of the present study were consistent with those of other genome-wide analyses of Jewish populations .The STRUCTURE analysis reveals that the Middle Eastern populations form a distinct cluster, corroborating prior investigations on a global scale . The cluThe Arabian Peninsula gene flow confirmed that all migration routes favoured divergence from ancestral populations without an ongoing migration model, which was the dominant model in this study. Furthermore, this study revealed that migration in the Arabian Peninsula occurred along coastal routes and the main migration route was from the United Arab Emirates to Kuwait and Iraq through Qatar. These findings agreed with previous research that looked at gene flow in the Arabian Peninsula as part of out-of-Africa migration . Our genS1 Checklist(DOCX)Click here for additional data file.S1 DataUP: unpredicted haplogroups. DYS389II.I represents the difference between the total repeat number at DYS389II and the repeat number at DYS389I. Fitness score (goodness of fit): a statistical measure that assesses how well a set of Y-STR  values align with the expected values for a specific haplogroup. Probability: a prediction or probability that a haplotype is in a particular haplogroup S2 Table: The quantification information for Qatari DNA samples. S3 Table: Allele frequency of Y-STR loci included in the PowerPlex Y23 kit in Qatari population. S4 Table: Forensic parameters of Y-STR loci included in the PowerPlex Y23 kit. The PowerPlex Y23 specific loci are highlighted in yellow. Gene diversity (GD), polymorphism information content (PIC), match probability (PM) and power of discrimination (PD). *The six loci not examined by the Yfiler kit are highlighted in yellow. S5 Table: Haplogroup Predictor results for the 379 Qatari male samples. *A1, *D, *H1, *O1 these haplogroups only appeared once. S6 Table: The matrix of pairwise genetic distance Rst of Y-STR between the Qatari population and the other Middle Eastern populations based on 17 Y-STR markers. S7 Table: Matrix plot showing population average pairwise differences based on 17 loci. Above diagonal: Average number of pairwise differences between populations (PiXY). Diagonal elements (yellow highlighted): Average number of pairwise differences within population (PiX). Below diagonal: Corrected average pairwise difference (PiXY-(PiX+PiY)/2). S8 Table: Estimation of migration rate in the Qatari population. (1) The migration routes between Qatar and Saudi Arabia; four models were tested. The most probable route was Qatar →Saudi, model 2. (2) The migration routes between Qatar and Yemen; four models were tested. The most probable route was the route Qatar →Yemen, model 2. (3) The migration routes between Qatar and Iraq; four models were tested. The most probable route was the route Qatar →Iraq, model 2. (4) The migration routes between Qatar and Kuwait; four models were tested and the most probable route was the route Qatar →Kuwait, model 2. 5. The migration routes between Qatar and the Emirates; four models were tested and the most probable route was the route Qatar → Emirates, model 2. (5) The migration routes between Qatar and the Emirates; four models were tested and the most probable route was the route Qatar → Emirates, model 2. (6) The most probable migration routes of each of the five countries were tested and the most probable migration route was Qatar →Yemen.(XLSX)Click here for additional data file.