A database for the Iraqi Sorani Kurds, specifically focused on the 12 X-short tandem repeat (STR) loci, has been developed to fascilitate forensic and population genetics investigations. The present study involved genotyping 117 unrelated individuals from the Sorani Kurds ethnic group using the Investigator Argus X-12 QS kit. The analysis revealed that the DXS10135 locus exhibited the highest degree of polymorphism, as indicated by a polymorphism information content (PIC) value of 0.94565 and a gene diversity (GD) value of 0.95623. Conversely, the DXS8378 locus displayed the lowest level of polymorphism, with a PIC value of 0.61026 and a GD value of 0.68170. Notably, two individuals were found to possess a rare allele  at the DXS8378 locus, which was not included in the allelic ladder of the kit. Furthermore, a significant linkage disequilibrium (LD) (p < 0.05/117) was observed between the DXS10103 and DXS10101 loci on linkage group 3 (LG3). The ancestral composition of the five primary geographic regions, namely Africa, Middle East, East Asia, Europe, and South America, was determined through the utilization of the  In many mammal species, including humans, the X chromosome is one of the two chromosomes that determines sex. It is only found in one copy in males, sharing this characteristic with both the Y chromosome and the mitochondrial DNA (mtDNA) .The estimated length of the X chromosome is 155 million base pairs (Mb), or about 5% of the estimated size of the human genome . The X chromosome of the male gender is predominantly transmitted to females as a single unit. In contrast, the two copies in females recombine, like autosomes, thus reorganising genetic variation in each generation and increasing haplotype diversity. The newly rearranged chromosome is then passed on to both male and female offspring .In forensic genetics, Short Tandem Repeat (STR) markers on the X and Y chromosomes have been developed as valuable tools that can complement the analysis of autosomal STR and mtDNA . The majThe X chromosome is crucial in understanding the differences in population-genetic studies between male and female individuals. Several factors influence the genetic distances found between males and females, including breeding and death rates, polygamy, and patrilocality. Notably, an important aspect contributing to the gender differences is the uneven distribution of the X chromosome, with females harbouring it for twice as long as males. The enormous genetic information encoded inside the X chromosome has the potential to provide crucial insights that cannot be obtained by analysing either the Y chromosome or mtDNA. Despite having substantially more genetic information than the Y chromosome or mtDNA, the X chromosome gets little attention in population genetic studies , 3.Iraq is a nation located in the Arabian Peninsula, with a population of approximately 40 million people. It shares borders with several countries, including the Arabian Gulf, Kuwait, Saudi Arabia, Jordan, Syria, Turkey, and Iran. The population of Iraq consists of five distinct ethnic groups, with the Arabs and Kurds being the predominant groups. The Kurds, who inhabit a mountainous region spanning Iraq, Iran, Syria, Turkey, and Armenia, have different dialects such as Kurmanji, Sorani, Kirmashani, Zazaki, and Gorani. However, there is limited published data available on the comprehensive diversity of the population in Iraq and the genetic diversity of the Kurdish population –11. ThisFor the first time, a representative database of X-STRs loci for the central Sorani Kurdish population of northern Iraq was presented to the scientific community. This study also looked at potential demographic differences based on genetic distances and allele frequencies between single populations and continental regions. Further, this study included a global investigation of the X chromosome’s ancestral variability, allelic richness, and various populations’ genetic structures.Buccal swab samples were collected from 117 Sorani Kurd males aged 18 years old and above. All work in this manuscript was reviewed and approved following institutional ethical guidelines. The Ethics Committee of the Kurdistan Institution for Strategic Studies and Scientific Research/Department of Biology granted ethical approval on February 15, 2020, with a unique reference number (KI-ET 20/February 2020). 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 DNA was extracted using a PrimePrep Genomic DNA Extraction Kit from Blood (GeNet Bio-Korea) according to the manufacturer’s protocol . The pur® V.5 software (Thermo Fisher Scientific).A set of X-STRs genetic markers were amplified using Investigator Argus X-12 QS kit (Qiagen) . SimultaST calculations based on X-STRs data and multi-dimensional scaling (MDS) plots. MDS analysis is also used to investigate genetic similarities between populations  and Jews [Majorca] (FST = -0.0059), and the farthest relationship was identified between Turkey and Lithuania (FST = 0.0197).A pairwise matrix plot of F 12 loci . The UAEThe average pairwise differences were examined to estimate the corrected genetic differences among 36 worldwide populations. These differences were specifically analysed in three contexts: between the 36 populations as a whole, within each individual population, and between different populations using Nei’s distance.The investigation revealed notable results regarding the average pairwise values obtained by using Nei’s distance. The population of Turkey and the population of Lithuania had the highest average pairwise value, 0.18653. The lowest average pairwise value, -0.05476, was discovered between the Jewish subgroups of Chuetas and Majorca. Furthermore, the Sorani Kurd population had unique average pairwise values. The highest average pairwise value, 0.09900, was found between the Sorani Kurds and the Philippines. The Sorani Kurds and the United Arab Emirates, on the other side, had the lowest average pairwise value (0.00789).The Iranian population had the highest average pairwise difference value within populations (9.44449), while Jews [Sephardim] had the lowest (9.07782). Mongolia and Jews [Chuetas] had the highest corrected average pairwise value (9.47319), while Hungary and Jews [Sephardim] had the lowest (9.09849). The Sorani Kurds had the highest corrected average pairwise value (9.43045) with Iran and the lowest (9.22487) with Jews [Sephardim]. The results are shown in S7 Table  and Fig ST genetic distances of 12 X-STRs loci. The resulting MDS plot was generated using R software, depicting the relationships among samples from 36 global populations. The result is shown in The MDS was calculated based on FTo measure the diversity between populations a phylogenetic tree was constructed based on allele frequency data using POPTREE2 software . FigTreeST = 0.00257) compared to the differentiation between the Middle East and Africa (FST = 0.00206), Far East Asia (FST = 0.00203) and Europe (FST = 0.00125), as shown in ST and the average pairwise differences between and within populations in different continents, in addition to the corrected average pairwise difference between populations of the five different continents were calculated. Nei’s distance results showed that the highest genetic distance was between the Middle East and South America (0.02383) while the lowest genetic distance was between the Middle East and Europe (0.01162). The highest average number of pairwise differences within the population was in Europe (9.27788) and the lowest was in Far East Asia (9.24154). The highest corrected average pairwise difference was between Africa and Europe (9.30024) and the lowest was between Far East Asia and the Middle East (9.26607), as shown in The continental pairwise matrix showed more differentiation between the Middle East and South America . Six clusters have developed. The five geographical regions studied  did not reveal any distinct subpopulation genetic structures .The FSTruct programme was used to analyse the The results showed that the Pairs of bootstrap distributions of This study examined two allelic richness parameters, specifically distinct and private alleles, across five distinct geographical regions. The findings revealed that the distinct alleles demonstrated similarities between Europe and South America, while displaying the highest levels in Africa and the Middle East, and the lowest levels in Far East Asia ). MoreovThe Argus X-12  is the most often used X-STRs kit in the field of forensic genetics. However, because the 12 X-STRs markers in this kit were largely characterised for European ancestry, several of the genetic variants observed in other population groups were missed out. To address this issue, the Argus X-12 QS emerged in the forensic field as an optimised version containing the same markers [There has been few genetic research on the Iraqi Kurdish population, with the majority of previous work focusing on Y-STRs, mtDNA, and autosomal STRs , 65, 66.The DXS10135 locus in the Kurdish population exhibited the highest polymorphism information content (PIC), which is comparable to Bahrain, UAE, Jews, Iraq, Lithuania, Turk, Slovenia, Albania, Guinea-Bessau, Filipino, Mongolia, Eastern Han, Hebei Han, Croatia, and Argentina , 49, 54.A rare allele was observed in two individuals, which is not presented in the allelic ladder of the kit at locus DXS8378 . This off-ladder allele at the same locus, DXS8378, was reported in the earlier study of the German population . In addiWhen MEC values were considered, the forensic efficacy of X-STRs in complex scenarios including kinship and paternity was obvious. These MEC calculations proved crucial in the examination of complex kinship and paternity scenarios, especially when the reference DNA sample came from the child’s putative grandmother, the father’s mother, rather than the alleged father.The phylogenic tree showed that the Sorani Kurds, Saudi Arabia, Turkey, Iran, Ethiopia and Argentina fell into one cluster with the European populations. This study confirms the findings of a previous mtDNA study of the Sorani Kurds, which found that they are genetically related to the European lineage . FurtherEven after applying Bonferroni’s correction, the linkage disequilibrium tests revealed one significant result between the DXS10103 and DXS10101 locus pairs from LG 3. A similar result with statistically substantial LD between DXS10103 and DXS10101 has previously been observed in the Iranian, Filipino, Mongolian, and Chinese Han populations , 43, 44.In comparison to the Y chromosome, which demonstrated a high level of gene diversity across all PPY23 kits in a global analysis , this stUnlike the Y chromosome, the X chromosome population Q-matrices did not reveal a distinct subpopulation genetic structure . In lighIndividuals within populations that have recently undergone admixture differ substantially in terms of their ancestry, with some individuals deriving the majority of their ancestry from one source population and others from a different one. As a result, it might be hypothesised that admixed populations produce more ancestry variability than nonadmixed populations when assessing inferred cluster memberships. This was clearly shown in this study, as the Middle Eastern and African populations had the lowest ancestral variability as compared to European and South American populations.Based on numerous viewpoints on the history of human migrations, we can evaluate patterns of distinct and private allelic richness in relation to our expectations. The higher number of distinct and private alleles in Africa corresponds to the pattern expected for models of human evolution that began in Africa and spread to other regions through a succession of founder events. The combination of the physically contiguous regions of Africa and the Middle East contains the greatest number of alleles. Since many alleles in the founding population would have only moved along part of their migration outside of Africa. The findings of this study on allelic richness and ancestry variability are consistent with the predictions of African-origin models that include serial founder effects throughout outward migrations .The main limitation encountered in this research endeavour was the limited availability of X-STRs data. As seen, this study only included three African countries and two South American countries, necessitating additional data to enhance the understanding of the X-STRs population genetics.In conclusion, this study represents the pioneering investigation into the X-STRs data pertaining to the population of Iraqi Sorani Kurds. The establishment of a comprehensive population database has enabled the utilization of STR markers included in the Argus X-12 Kit in routine forensic casework. The remarkable statistical parameters exhibited by these loci, in conjunction with their independent inheritance patterns, render them an invaluable marker set for both forensic casework and population genetics research. The use of X-STRs loci may prove to be a significant complement to autosomal and Y-STRs markers, particularly in complicated kinship and paternity instances.S1 FileST between the five continents. S9 Table: average number of pairwise differences (FST) within and between continents. 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). S10 Table: (A) Q statistics for the five poulations Middle East, Africa, Far East Asia and South America. (B) Pairwise comparisons using Wilcoxon rank sum test with continuity correction. S11 Table: distinctive alleles and private alleles on a continental scale: Middle East, Africa, Far East Asia, South America and Europe. (A) Distinctive alleles, (B) Private alleles.LG1 = , LG2 = , LG3 = , LG4 = . S2 Table: Allele frequencies and sample size by locus of the Sorani Kurd. S3 Table: Forensic parameters, Number of alleles , Gene diversity (GD), polymorphism information content (PIC), match probability (PM), power of discrimination (PD) and Mean Exclusion Chance (MEC) of the Sorani Kurd males. S4 Table: Pairwise p values of linkage disequilibrium test for all pairs of loci using 117 male samples from Sorani Kurdish population. S5 Table: Forensic parameters, Gene diversity (GD), polymorphism information content (PIC), match probability (PM) and power of discrimination (PD), of the five geographical regions Middle East, Africa, Far East Asia, Europe and South America. S6 Table: Pairwise genetic distances (Fst) based on haplotypes of 36 global populations. S7 Table: Average number of pairwise differences using data from 36 populations. 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: Pairwise genetic distances based on F(XLSX)Click here for additional data file.S2 FileS1 Fig. Plot distribution of allele frequencies per locus using Investigator Argus X-12 QS kit. S2 Fig. Electropherogram shows variant alleles in three samples of the Sorani Kurd males. (A) An off-ladder allele was observed at locus DXS8378 . (B) Duplicated alleles  were found at locus DXS7423. (C) A null allele was observed at locus DXS10079. S3 Fig. P-values for the pairwise exact test matrix for linkage disequilibrium among the 12 X-STR loci. S4 Fig. Ranking of Investigator Argus X-12 QS kit (Qiagen) markers by gene diversity (GD). Rank within continental residency groups, i.e. Middle East (n = 1224), Africa (n = 448), Far East Asia (n = 815), South America (n = 1167) and Europe (n = 2980).(DOCX)Click here for additional data file.


Whole-genome resequencing technology covers almost all nucleotide variations in the genome, which makes it possible to carry out conservation genomics research on endangered species at the whole-genome level.Artocarpus nanchuanensis individuals, we evaluated the genetic diversity and population structure, inferred the demographic history and genetic load, predicted the potential distributions in the past, present and future, and classified conservation units to propose targeted suggestions for the conservation of this critically endangered species.In this study, based on the whole-genome resequencing data of 101 critically endangered A. nanchuanensis generated approximately 2 Tb of data. Based on abundant mutation sites , we revealed that the average genetic diversity  of different populations of A. nanchuanensis was relatively low compared with other trees that have been studied. And we also revealed that the NHZ and QJT populations harboured unique genetic backgrounds and were significantly separated from the other five populations. In addition, positive genetic selective signals, significantly enriched in biological processes related to terpene synthesis, were identified in the NHZ population. The analysis of demographic history of A. nanchuanensis revealed the existence of three genetic bottleneck events. Moreover, abundant genetic loads (48.56% protein-coding genes) were identified in Artocarpus nanchuanensis, especially in genes related to early development and immune function of plants. The predication analysis of suitable habitat areas indicated that the past suitable habitat areas shifted from the north to the south due to global temperature decline. However, in the future, the actual distribution area of A. nanchuanensis will still maintain high suitability.Whole-genome resequencing for A. nanchuanensis into four conservation units and proposed a number of practical management suggestions for each conservation unit. Overall, our study provides meaningful guidance for the protection of A. nanchuanensis and important insight into conservation genomics research.Based on total analyses, we divided the populations of  To reve22.1A. nanchuanensis from Yongchuan District, Qijiang District, Wansheng District and Nanchuan District in southwestern Chongqing City, China  These two populations are located in the same branch range of Dalou Mountains  method . A Qubit2.2A. nanchuanensis (http://picard.sourceforge.net/) were used to sort and delete duplicate reads, respectively. Then, we employed the mpileup + call  and filter modules (-g 3 -G 10 -e “%QUAL<10”) in BCFtools  was conducted by using Plink , and the2.4Artocarpus, we downloaded the resequencing data of five other species of Artocarpus  to explore the phylogenetic status of A. nanchuensis (A. nanchuensis (ser. Clavati), and the last one belongs to the sisters clade (ser. Peltati) of ser. Clavati  and nonsynonymous sites (πN) of each population using VCFtools with a 100-kb sliding window and a step size of 10 kb. Genetic diversity was defined as the average number of pairwise nucleotide differences  in this study (STF) and π ratio values between each population pair.Based on the latest research of  Clavati . These d IQ-TREE . We firsmezianus . In addimezianus , we calcis study . VCFtoolis study  was also2.5A. nanchuanensis (NHZ and QJT), we selected the windows with high genetic differentiation ) as the candidate divergent regions (CDRs) by using Microsoft Excel. Candidate genes were obtained from CDRs and GO enrichment analysis was carried out by using TBtools (A. nanchuanensis were searched and confirmed by HMMER 3.0 (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi). In addition, the genes of the corresponding gene family in Arabidopsis thaliana were downloaded from the Phytozome database   (To reconstruct the detailed demographic history of  module)  was used module)  was used module)  with the 2.6e-9) .2.7A. nanchuanensis, we first defined the dominant alleles in all individuals  as the ancestral allele genotype (It is predicted that deleterious mutations (genetic load) will destroy gene function, which would significantly reduce the fitness of individuals in the population . Moreovegenotype . Then, tgenotype  and SnpEgenotype , respectgenotype .2.8A. nanchuanensis, MaxEnt (version 3.3) (https://worldclim.org/data/v1.4/cmip5_2.5m.html). To minimize the impact of multicollinearity and overfitting on the stability and quality of the model, the 14 bioclimatic variables were analyzed with the Pearson correlation test. When the correlation coefficient was > |0.80|, one of the two variables was removed. Ultimately, six bioclimatic variables were selected to predict the suitable habitat areas for four time periods, including BIO1 , BIO4 , BIO8 (mean temperature of wettest quarter), BIO12 , BIO14 (precipitation of driest month), and BIO15   was usedion 3.3)  with 2.5uanensis . Finally33.1The whole-genome resequencing of 101 individuals generated approximately 2 Tb of data. The average coverage, average depth and primary mapping rate of all samples were 98.00%, 39.23 and 97.57%, respectively  explained 16.2%, 12.9% and 10.7% of the total genetic variance, respectively. PC1, PC2, and PC3 significantly separated the NHZ and QJT populations from all other populations, but did not separate the remaining five populations 3. The a3.3A. nanchuanensis and four species of ser. Clavati  gathered together and formed a sister branch  genes   of A. nanchuanensis began to decline slowly from 10 Ma and encountered the first genetic bottleneck at 2.82 Ma. From 2.2-0.2 Ma, the Ne gradually expanded from 5.4x104 to 1.12x105. However, the Ne has been declining since 0.19 Ma, except for a small increase at 0.09 Ma. Except for the earlier first genetic bottleneck, this species experienced the second and third genetic bottlenecks at 0.14 Ma and 0.06 Ma, respectively. Finally, the Ne of A. nanchuanensis was stable in the range of approximately 6.5 x104 - 8.3x104.The results of PSMC analysis indicated that 3.6Loss of function in a homozygous state could be used as an indicator of species fitness. The statistical analysis of the annotation results for each individual revealed no significant differences in genes containing homozygous LOF sites/PCGs between populations  will still maintain high suitability (The PCA of bioclimate variables showed that the first two PCs (PC1 and PC2) explained 88.5% of the total climate change variation experienced by uanensis 8. Amonguanensis . This su4A. nanchuanensis, covering most of the wild distribution areas, and revealed its complex population structure, demographic history, and genetic threats it currently faces by using whole-genome resequencing data. In addition, we identified several key factors that led to the formation of the distribution pattern of A. nanchuanensis by combining the analysis of niche models and demographic history. Our findings provide important guidance for meaningful conservation actions for this critically endangered WPESP.In this study, we sampled 101 individuals of A. nanchuanensis was lower  The actual Ne found in the field investigation (<200) was significantly smaller than the Ne  inferred from demographic history, indicating that the population of A. nanchuanensis has recently encountered strong pressure. During our field investigation in 2019, three small populations of A. nanchuanensis were found in Qianqiu village (Nanchuan district). When we visited the site again in 2022, we found that only a few young trees remained in one of the populations. The main reason was that its straight trunks of A. nanchuanensis are often used by local people for shipbuilding and furniture. Based on the inferred large Ne value and the evidence that multiple existing populations in the four districts shared very similar genetic backgrounds, we speculated that A. nanchuanensis could be widely distributed in the mountains around the four districts. However, this may not be the case, possibly because human activities such as deforestation, road construction, and the expansion of farmland have drastically reduced the Ne of A. nanchuanensis. Human interference and habitat fragmentation will further accelerate the impact of genetic drift, thereby reducing genetic diversity  in most populations, and the geographical distances between populations usually exceed 10 kilometers. These factors may together hinder the production of outbred seeds, which may be an important way to increase the genetic diversity of the population.Compared to that of other trees that have been studied, the genetic diversity of as lower , which mas lower . This seiversity . (3) Stuminators . HoweverArtocarpus is a genus mainly distributed in tropical regions, and its diversity center is located in Borneo  during most of the Pleistocene (2.2-0.2 Ma)   . In geneountains . In addi history . Therefoistories .A. nanchuanensis were far larger than its actual distribution area , and implement the following protection actions as soon as possible: (1) PCA, genetic structure and phylogenetic analysis revealed the unique genetic backgrounds of QJT and NHZ, which were different from those of other populations  Considering the rarity of adult trees, isolated populations and small STF values between various populations of A. nanchuanensis, artificial cross-pollination among four populations with similar genetic backgrounds should be carried out to maintain the genetic diversity and increase the survival potential of this species. (4) Based on the current status of the extremely small population of A. nanchuanensis, it is strongly recommended to pursue ex situ protection of the populations of the four conservation units to comprehensively preserve the germplasm resources and genetic diversity of this species. Considering the current and future habitat suitability of A. nanchuanensis, large-scale botanical gardens in central China are the preferred choices, such as the Nanshan Botanical Garden in Chongqing City and the Wuhan Botanical Garden of the Chinese Academy of Sciences in Hubei Province.(2) Our field investigation revealed that the individual with the largest DBH (65 cm vs. 63 cm of the listed ancient tree from NHZ) was located in Honglu Town, Yongchuan District, and there is a sand factory under construction around the ancient individual. It is recommended to contact the local forestry administration to confirm the age of the ancient tree as soon as possible and apply 5A. nanchuanensis individuals, we revealed that repeated bottleneck events, human disturbance and habitat fragmentation, as well as excessive self-pollination within populations, together contributed to low genetic diversity. Quaternary climate change, especially temperature change, plays an important role in forming the current distribution pattern and endangered status of the species. The Dalou Mountains provided an important refuge for A. nanchuanensis during Quaternary climate fluctuations. Genetic analysis revealed that the NHZ population not only had a special genetic background but also had specific differentiation in defense-related genes. The analysis also indicated that this species has an abundant genetic load, especially in the genes related to the early development and immune function of plants. Based on the study results, we divided the populations of A. nanchuanensis into four conservation units and proposed a number of practical management suggestions for each conservation unit of this critically endangered species.Based on the whole-genome resequencing data of 101 critically endangered The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI, PRJNA948357 (SRA).HD designed the research. CX, BW and JZ collected the plant materials. CX, YZ and TX performed the research and analysed the data. CX and HZ wrote the paper. MK, XZ and HD revised the manuscript. All authors contributed to the article and approved the submitted version.


Bactrocera dorsalis) by comparing an inbred (monophyletic) original population and an outbred (mixed) invasive population.Changes in population heterozygosity and genetic diversity play important roles in mediating life history traits of organisms; these changes often lead to phenotypic evolution in offspring, which become superior to their parents. In the present study, we examined phenotypic differentiation, the intestinal microbiome composition, and metabolism shift in the oriental fruit fly  in the diet led to an increase in pupal weight of the outbred population. Additionally, transcriptome analysis revealed that the HSPA1S gene was significantly downregulated in the outbred population. HSPA1S was involved in activation of the JNK-MAPK pathway through negative regulation, caused the upregulation of juvenile hormone (JH), and led to an increase in biomass in the outbred flies.The results showed that the outbred population of B. dorsalis. Thus, multiple introductions could lead to invasiveness enhancement in B. dorsalis through population mixing, providing preliminary evidence that changes in the intestinal microbiome can promote biological invasion.In conclusion, the outbred population had an altered intestinal microbe composition, mediating metabolism and transcriptional regulation, leading to phenotypic differentiation; this may be a potential mechanism driving the global invasion of Video AbstractThe online version contains supplementary material available at 10.1186/s40168-023-01664-1. The successful invasion of an alien organism often leads to adaptive colonization, continuous reproduction, and the expansion of invasive populations, which seriously threaten agriculture, forestry, and animal husbandry worldwide and are extremely harmful to economic development , 2. The Arabidopsis thaliana can speed up the initial emergence and flowering time of seedlings and increase biomass and reproductive capacity [Spartina maritima in the UK was crossed with introduced Spartina alterniflora to form allotetraploid Spartina anglica, which had greatly enhanced invasiveness, resulting in displacement of the parent strains in coastal areas and rapid colonization and spread to many new areas [It is generally believed that high levels of genetic diversity facilitate the establishment and persistence of IAS in new habitats . For exacapacity , 8. Furtcapacity . For exaew areas , 11.Bursaphelenchus xylophilus, in China originated from many different countries [Given the strong link between species introductions and global trade and transport, it is not surprising that many IAS are introduced from several sources. The propagule pressure of IAS has been well explored and is related to the bottleneck effect, Allee effects, and founder effect. Multiple introductions  from different geographical populations could provide opportunities for intraspecific hybridization and the evolution of new genotypes , 12, 13.ountries . Maintaiountries , 22.Macrobrachium rosenbergii has been observed [Hypophthalmichthys nobilis, and silver carp, Hypophthalmichthys molitrix, have high fertilization rates and high embryo viability [Some mechanisms, such as gene recombination and invasion evolution acting on individual traits and trait combinations, have been shown to drive the production of key genetic characteristics after interspecific hybridization , 23. In observed . In receobserved , 28. Foriability .Bactrocera dorsalis) is one of the most important and notorious pests worldwide, with the widest host range; it causes major losses in the global fruit and vegetable industries. Previous research revealed that the invasive population of B. dorsalis originated from several geographical populations (e.g. North China) [B. dorsalis in California, USA, were also due to multiple, independent introductions with a wide variety of origins , in addition to the previously known source in Hawaii [2H stable isotope values from B. dorsalis from Beijing were not consistent with those in Beijing rainwater but rather were consistent with the data from Fuzhou in southeast China [B. dorsalis, which is an unexplored field in biological invasion [B. dorsalis comprises hybrid offspring of individuals from several geographical populations; this could increase the invasiveness of B. dorsalis and cause a major threat to agriculture [B. dorsalis were determined to reveal invasiveness traits by using phenotypic omics analyses, and (ii) multiomics  technology was used to examine holobiont and microbiome interactions and explore the potential invasion mechanism of the global invasive B. dorsalis. We hope that these findings will be helpful in guiding prevention and control of B. dorsalis worldwide.The oriental fruit fly  , 30. Forh China) , 32. Then Hawaii , 34; thest China . Furtherst China . Thus, tinvasion . Therefoiculture , 38. We Bactrocera dorsalis [B. dorsalis is mainly distributed in tropical and subtropical regions of the south part [South Asia and Southeast Asia are the major distribution centres of the oriental fruit fly, dorsalis , 39. In uth part , 40. To uth part . The F1 B. dorsalis were reared in an intelligent climate chamber with a 14 h:10 h light:dark photoperiod, a temperature of 28 ± 1 °C, and humidity at 65 ± 5%. After eclosion, the adult flies of each population were placed in a mesh cage and fed an artificial diet (sucrose:soy peptone = 3:1)  outbred populations before the experiments. Mangos were used to evaluate the infestation performance of original (F and H) and mixed outbred populations in the laboratory. We placed mangos in a fly cage containing 10 pairs of wild-caught flies to test the fecundity differences. We also determined differences in pupal weight, ovary size, and hatching rate between the original and mixed outbred populations.One-hundred eggs were incubated on wet filter paper, and the hatch rates of eggs at 24 h, 36 h, 48 h, and 72 h were observed, respectively. Then, hatched 1st instar larvae were individually fed in a 3.5-mm petri dish filled with feed. The developmental stage of each larva was observed every 24 h, and all larval deaths were recorded. The cephalopharyngeal skeleton of 3rd instar larvae was dissected to examine differences between the inbred and outbred groups. When the larvae were reached the final larval stage, 300 individuals were selected and allowed to pupate, and pupal weight and pupation and eclosion rates were measured on the 6th day.The cohort of fly populations (300 newly emerged adults) were maintained to record the number of surviving individuals every day for 60 days; these data were used to calculate the survival rate, sex ratio, and pupal weight. Furthermore, 10 males/females were selected to examine their fecundity capacity. Eggs were collected from mango slices every 24 h for 20 days, and the cumulative number of eggs laid per female was counted. Additionally, the ovaries of female adults were dissected to determine their size under the microscope on days 5 and 15. Survival and reproduction data were used to calculate the parameters of the life table , 43.Final-stage larvae  of the inbred (H and F) and outbred (F♀ × H♂ and H♀ × F♂) populations were dissected to analyse composition of the intestinal microbiome. All samples were stored on dry ice and sent to Shanghai Applied Protein Technology Co., Ltd. for metabolic analysis of amino acids and derivatives and to Shanghai Majorbio Biopharm Technology Co., Ltd. for microbial diversity sequencing analysis and transcriptome sequencing .Bacterial amplification was performed in intestinal samples from the inbred and outbred populations on the Illumina HiSeq platform using the 16S rDNA ITS1 variable region and 16S rDNA V3–V4 variable region, respectively. First, data quality was controlled and screened. Next, the diversity index was analysed according to operational taxonomic unit (OTU) cluster analysis and species taxonomic analysis. Biostatistical analysis was performed to understand the relative species abundance of bacteria and fungi in the samples, and linear discriminant analysis effect size (LEfSe) was used to evaluate the significance of species richness differences. Finally, the intestinal microbes with significant differences between the inbred and inbred groups were identified.The effects of different bacterial groups on pupal weight were verified by exchanging the diet provided to the inbred and outbred populations. We fed larvae of outbred populations a spent diet . Similarly, the larvae of inbred populations were fed a spent diet that had been eaten for 3 days by larvae of outbred populations. We exchanged the spent diets of the outbred populations and inbred population to examine the influence of gut microbiome exchange on the life history of flies. Also, the effects of differential amino acids on pupal weight were verified by adding histidine, arginine, glutamine, glutamate, isoleucine, and valine to the artificial diet of an inbred population see . We alsoHeterozygosity between the inbred and outbred populations was represented by the number of single-nucleotide polymorphisms (SNPs) measured by transcriptomic analysis. Raw data were qualitatively controlled, optimized, and evaluated using SeqPrep, and gene/transcript expression levels were quantitatively analysed using RSEM. Then, DESeq2 was used to analyse the different expression levels of genes between samples to identify the differentially expressed genes to study the function of differentially expressed genes. The differentially expressed genes were divided into different gene sets for analysis according to the up- and downregulation of genes, and the obtained gene sets were subjected to functional enrichment analysis to obtain the main metabolic pathways.P-value of the test for homogeneity of variance was greater than or equal to 0.05 and the data were normally distributed, one-way ANOVA or the T-test was used for biostatistical analysis of the data. If one of these criteria was not met, then nonparametric tests were used.SPSS 25.0 was used for data analysis. The Z-score extreme value standardization method was used to process the original data, and extreme values with an absolute Z score higher than 2 were excluded. Then, the data were tested for homogeneity of variance and a normal distribution. If the B. dorsalis in the different populations. The fitting result had a high fitting degree with the logistic curve, so the logistic curve was used to fit the mortality rate and accumulated offspring quantity in B. dorsalis to determine whether outbreeding affected survival and reproduction. Then, survival parameters, such as the death distribution and death expectation, and reproductive parameters, such as oviposition distribution and oviposition expectation, were calculated using the fitted data. Origin 2021 was used to draw all the resulting graphs.Origin 2021 was used to perform nonlinear fitting for the mortality rate and accumulated offspring quantity of P < 0.001) and ovary size (P < 0.001) than the original population in the mangos  and hatching rates (P < 0.001) were higher in the mixed outbred population than in the original F and H population  and a larger number of SNPs  than the inbred population  than the inbred population, with an increase of 7.32%    and 55.02%  longer and wider than those in the inbred population, respectively.The outbred population had higher genetic heterozygosity  , indicating that the reproductive potential was enhanced after population mixing . The innate rate decreased from 0.33 ± 0.00 in the inbred population to 0.32 ± 0.01 in the outbred population, while the doubling time in the outbred population was 2.82% lower than that in the inbred population (Supplementary Information Table P = 0.174) and food intake (P = 0.275) showed no significant differences between the inbred population and outbred population  Fig. E. RegardP = 0.046)  showed the most significant difference between the outbred and inbred groups .  was the most significantly different between the two populations , Pseudomonadales (5.89%), Burkholderiales (4.62%), Acetobacterales (0.68%), and others (74.48%). The intestinal bacteria in the outbred population mainly belonged to Lactobacillales (45.78%), Acetobacterales (16.44%), Burkholderiales (3.64%), Pseudomonadales (2.17%), and others (31.96%). The abundance of Lactobacillales in the outbred population was 219.70% higher than that in the inbred population  and glutamic acid  were 9.23% and 8.10% higher, respectively, than that in those fed a diet with no amino acid supplementation  and isoleucine  were 14.76 ± 0.04 mg and 14.63 ± 0.05 mg, respectively  when they were feed on a spent diet of the inbred population  and ovary size  than the F population, while the two inbred populations showed no differences  and higher fecundity  than the F population  ≤ 0.05 and fold change ≥ 2 were applied to compare unigenes between the inbred and outbred populations. The results showed that 784 unigenes were differentially expressed in the transcriptomes of the inbred and outbred populations, of which 637 unigenes were upregulated and 147 unigenes were downregulated  (27.21%) and catalytic activity (GO:0003824) (22.45%) were the most enriched. Cellular process (GO:0009987) (19.05%) and metabolic process (GO:0005488) (14.97%) were the most involved biological processes. Cell part (GO:0008152) (23.13%) and membrane part (GO:0044425) (14.97%) were the most upregulated cellular components . The immune system pathway, including the antigen processing and presentation pathway, had the most significant differences between the two populations (P = 0.0019).Based on the KEGG database of 122 metabolic pathways, 60 downregulated genes were obtained, among which 10 pathways were significantly enriched in the outbred population (P = 0.0021) and protein processing in the endoplasmic reticulum pathway (P = 0.0022) and the MAPK signalling pathway (P = 0.0033) showed significant differences. Thus, the expression of the HSPA1S gene in the MAPK pathway was downregulated, which led to upregulation of the JNK pathway. Additionally, the CYP18A1 gene, which regulates the insect hormone synthesis pathway, namely, the ecdysone pathway, was significantly downregulated, but the pathway was not significantly enriched  and mixed outbred populations (invasive population) of B. dorsalis. A, pupal weight; B, ovary size at 15 d after emergence; C, number of eggs laid per female per day; D, hatching rate. Supplementary Information Fig. 2. The effects of amino acid and intestinal microbe on body weight of the oriental fruit fly. A, Cephalo-pharyngeal bone length in inbred and outbred populations of B. dorsalis. B, Food intake of B. dorsalis inbred population and outbred population. C, pupal weight of inbred populations supplemented with different amino acids. D, pupa weight after feed exchange between inbred and outbred populations. Supplementary Information Fig. 3. Phenotypic differences between inbred and outbred populations of B. dorsalis. A, pupal weight; B, ovary size at 15 d after adult emergence; C, survival fraction; D, fecundity. Asterisks indicate significant differences , and ns indicates no significant differences. Supplementary Information Fig. 4. Species compositions of the microbiomes of the intestinal flora and oviposition fluids in F populations of B. dorsalis. A, bacterial composition and relative abundance; B, fungal composition and relative abundance. The inner circle represents the oviposition fluids, and the outer circle represents the intestinal flora. Supplementary Information Fig. 5. Species compositions of the microbiomes of the intestinal flora in the inbred F and the outbred F♀×H♂ populations of B. dorsalis. A, bacterial composition and relative abundance; B, fungal composition and relative abundance. The inner circle represents the inbred F population, and the outer circle represents the outbred F♀×H♂ population. LEfSe diagram of intestinal bacteria (C) and fungi (D) between inbred F and outbred F♀×H♂ populations. Supplementary Information Fig. 6. Species compositions of the microbiomes of the intestinal flora in the inbred H and the outbred H♀×F♂ populations of B. dorsalis. A, bacterial composition and relative abundance; B, fungal composition and relative abundance. The inner circle represents the inbred H population, and the outer circle represents the outbred H♀×F♂ population. LEfSe diagram of intestinal bacteria (C) and fungi (D) between inbred H and outbred H♀×F♂ populations. Supplementary Information Table 1. The artificial diet of Bactrocera dorsalis. Supplementary Information Table 2. Life table parameters of the inbred and the outbred populations ofB. dorsalis.Supplementary Information for methods. Microbiome determination and analysis: DNA extraction and sequencing. Analysis of microbiome data. Diet exchange experiments.


This article presents a comprehensive genetic study focused on pre-Hispanic individuals who inhabited the Aburrá Valley in Antioquia, Colombia, between the tenth and seventeenth centuries AD. Employing a genetic approach, the study analyzed maternal lineages using DNA samples obtained from skeletal remains. The results illuminate a remarkable degree of biological diversity within these populations and provide insights into their genetic connections with other ancient and indigenous groups across the American continent. The findings strongly support the widely accepted hypothesis that the migration of the first American settlers occurred through Beringia, a land bridge connecting Siberia to North America during the last Ice Age. Subsequently, these early settlers journeyed southward, crossing the North American ice cap. Of particular note, the study unveils the presence of ancestral lineages from Asian populations, which played a pivotal role in populating the Americas. The implications of these results extend beyond delineating migratory routes and settlement patterns of ancient populations. They also enrich our understanding of the genetic diversity inherent in indigenous populations of the region. By revealing the genetic heritage of pre-Hispanic individuals from the Aburrá Valley, this study offers valuable insights into the history of human migration and settlement in the Americas. Furthermore, it enhances our comprehension of the intricate genetic tapestry that characterizes indigenous communities in the area. The Antioquia region has played a pivotal role in the pre-Hispanic history of South America, serving as a gateway for the initial human migrations to the continent and as a hub for the development of a sophisticated culture deeply rooted in the utilization of natural resources and advanced technologies . ColombiThe earliest radiocarbon-dated evidence within the Antioquia Department is prominently concentrated in the Medellín/Porce River basin and the Medellín/Porce River high basin  5]. Ear. Ear5]. The first pattern corresponds to the Marrón Inciso ceramic style, spanning from the first century BC to the eighth century AD. The second pattern corresponds to the Ferrería ceramic style, ranging from the fourth century BC to the fourth century AD. The Marrón Inciso style extends throughout the Cauca mountain basin, while the Ferrería style is prominent in the central mountain range of Antioquia. Cerro El Volador, situated within the city of Medellín, has yielded Marrón Inciso-style burials, including an urn containing cremated human remains and a necklace bead resembling a praying mantis, linking ceramics to gold craftsmanship. The second funerary pattern, originating in Antioquia in the fourteenth century AD, is associated with the late ceramic style, spanning from the tenth century AD to the time of the Conquest. This pattern is characterized by burials in intricate and spacious funerary structures known as “pit tombs with a lateral chamber”, commonly found atop hills in cemeteries .Cerro El Volador, for example, revealed twelve tombs featuring rectangular access shafts and expansive chambers of conical and elliptical configurations. These tombs, dated between the fifteenth and seventeenth centuries AD, and contained cremated human remains, indigenous pottery fragments, skeletal remains of cattle and horses, along with European artifacts. The architectural design of these burial chambers mirrors indigenous dwellings or huts. At La Colinita’s summit, three “side chamber well tombs” unveiled cremated and non-cremated human remains, spindle ruffles, gold nose-rings, tumbaga, vessels, and ceramic fragments. Another burial site atop Las Flores contained the skeletal remains of an adult individual, devoid of grave goods but adorned with engravings representing a hut. The “pit tombs with a lateral chamber” in the Aburrá Valley are intricate representations of indigenous dwellings, holding profound significance both in construction and cosmology  These arMolecular biology, leveraging DNA analysis, has played a pivotal role in shedding light on hypotheses concerning the origins of human populations. DNA, the repository of hereditary information and essential protein-coding data, was first structurally elucidated in 1953 by Watson, Crick, and Franklin. Subsequently, Sanger’s sequencing method paved the way for reading and decoding genetic information. Recent advancements in data storage, analytical technologies, and informatics have catalyzed breakthroughs across multiple disciplines, including archaeology. Despite the inevitable degradation of DNA post-mortem, it can adhere to hydroxyapatite in bones, maintaining sufficient quality for reconstructing ancient genomes.Mitochondrial DNA (mtDNA) analysis, particularly in tracing maternal lineages, has emerged as a powerful tool for unraveling population history and genetic ancestry. The exclusive maternal transmission of mtDNA enables the tracking of maternal lineages across generations, facilitating the reconstruction of ancient population movements and migrations.The high copy-number of mtDNA within cells empowers the recovery and examination of genetic information from ancient remains, thereby aiding our comprehension of genetic diversity and population dynamics over time. Maternal lineages, a focal point of mtDNA analysis, are identified through the detection of characteristic mutations that serve as markers for distinct population groups. These findings have significantly contributed to the development of theories regarding the settlement of pre-Hispanic communities in Colombia.Ancient DNA studies have sought to unravel the genetic structure of pre-Colombian populations and establish genetic links between historical and contemporary population groups. These investigations are instrumental in supporting models related to population replacements, migrations, and genetic admixtures. As of now, genetic ancestry data in the Aburrá Valley are scarce.To gain a more comprehensive understanding of population dynamics, migration patterns, and genetic relationships within the region, future studies should consider incorporating complete nuclear and mitochondrial genome analysis. This advanced approach will enable a more detailed and precise assessment of the genetic history of the pre-Hispanic inhabitants in the Aburrá Valley; thereby, enriching our comprehension of the complex tapestry of human settlement and evolution in this pivotal region.This study was centered on the analysis of mitochondrial DNA HVS-I extracted from ten individuals originating from the Aburrá Valley in Antioquia. These individuals were associated with pit tombs characterized by lateral chambers. Gender determination was based on the anthropological examination of the skeletal remains.We conducted research at three distinct archaeological sites: Cerro del Volador, which yielded one individual (bone); Alto de las Flores in Envigado, where we analyzed one individual (bone) in Medellín; and La Colinita, where we investigated eight individuals (molars) in Medellín . The samThe genetic analysis of individuals from bone remains was conducted at the Human Genetics Laboratory within the specialized area dedicated to archaic DNA procedures at the University of Sabana. Stringent authenticity criteria, designed to prevent contamination of ancient DNA as proposed by Cooper, Poinar, and Gilbert , were stTo ensure the accuracy and reliability of the findings, the procedures were independently replicated by two researchers on separate occasions. Furthermore, a subset of the samples was sent to the Department of Genetic Epidemiology and Bioinformatics at the University of Southampton in England.To determine the mitochondrial haplotypes of the individuals in this study, we focused on a 388 bp fragment of HVS-I, specifically nucleotide positions 16024–16408. For amplification, we employed four pairs of overlapping PCR primers that were specifically developed for pre-Hispanic American populations : H15995:To conduct individual analyses, we employed specific haplotypes and constructed phylogenetic trees using unique polymorphisms for each haplogroup. We determined common evolutionary relationships through several approaches, including neighbor-joining algorithms, UPGMA, direct networks using the NETWORK 10 program, and the HaploGrep 2 program .STF genetic distances were computed between populations based on their mitochondrial haplotype frequencies.Our study also encompassed intra- and inter-population analyses, performed with the Arlequin 3.5 program. We calculated various indices to discern population trends, and mean pairwise In this analysis, we considered twenty present-day populations from Colombia and seventy-seven pre-Hispanic American populations. These were categorized into eight groups based on previous research. We obtained distance matrices and visualized them using the MDS ALSCAL algorithm within the SPSS program. This method provides a balanced approach, addressing concerns regarding false positives and statistical power, as opposed to relying solely on phylogenetic trees ,13,14,15In our study, we identified a total of ten individuals, collectively representing the four founding haplogroups: A (60%), B (10%), C (20%), and D (10%) . These hTo classify the mitochondrial haplogroups, we followed the Phylotree hierarchy , which bThe phylogenetic tree  illustraMedian-joining (MJ) analysis was employed to explore maternal relationships between individuals in family graves discovered in the Aburrá Valley . The anaOur study’s results revealed the presence of ten individuals from the Aburrá Valley, representing four founding haplogroups . Each individual exhibited a unique haplotype, indicating the absence of direct matrilineal relationships among them. Haplogroup assignments were made based on specific variants and polymorphisms identified in their sequences.The haplotype network offers valuable insights into the genealogical relationships among haplotypes. It allows us to assess the degree of divergence between different lineages and estimate the distance to their most recent common ancestor. It is important to note, though, that the haplotype network does not directly establish maternal relationships between specific individuals. Instead, it primarily serves as a tool for understanding the genealogical connections and ancestral origins of different haplotypes. Its relevance lies in elucidating the historical relationships and evolutionary patterns within a population rather than establishing direct relationships between specific individuals.Furthermore, the results of population diversity in the Aburrá Valley are presented in In line with the research conducted by Raff , a multiThis similarity is particularly striking with pre-Hispanic populations in Mesoamerica, as well as in the Arctic and Sub-Arctic regions. The study reveals the presence of haplogroups A, C, and D in both current and ancient populations throughout the Americas. Notably, haplogroup B is absent in the Caribbean, while haplogroup X is reported to be in low proportions in the northern part of the continent. Haplogroup M, on the other hand, is exclusively identified in an ancient population in the northwestern United States and not in current populations .The dataset analyzed includes archaeological sites categorized into various geographic zones, namely the Arctic/Subarctic, northwestern United States, Great Basin/southwestern United States, northeastern United States, Mesoamerica, Caribbean, and South America. This regional categorization, as presented in STF values can be found in For more comprehensive information, including the haplogroup frequencies used for the multidimensional scaling (MDS) analysis, please consult The distribution of mitochondrial haplogroups among the individuals revealed several patterns. Five individuals with the C16290T polymorphism were assigned to haplogroup A, with four of them specifically identified as A2. Notably, individual Abu10 was assigned to haplogroup B2s but also exhibited a variant typical of B4. Meanwhile, Individuals Abu4 and Abu7 were assigned to haplogroup C and shared specific variants. On the other hand, the individual with the C16223T polymorphism was placed in haplogroup D, with individual Abu6 classified as D4j1a, based on another specific variant . These fThe findings of this study offer valuable genetic insights into the population history of pre-Hispanic inhabitants in the Aburrá Valley region. This research primarily focused on the analysis of mitochondrial DNA (mtDNA), which has provided significant information about migratory patterns, interactions, and genetic relationships among ancient American populations.Studying ancient populations is a multidisciplinary endeavor that combines genetics and archaeology to unravel the complex tapestry of human history. Haplogroups, which are genetic markers passed down through generations, serve as invaluable tools for understanding ancient population dynamics. In this research, we present a comprehensive table featuring archaeological sites across distinct geographic zones, each holding a wealth of genetic information in the form of haplogroups A, B, C, D, X, M, and H.Variations in haplogroup distribution are evident across these geographic zones, indicating that different regions were inhabited by populations with unique genetic profiles. By examining these archaeological periods, we can observe how haplogroup distributions evolved over millennia; shedding light on genetic adaptations and the ebb and flow of ancient populations. Comparative analysis of haplogroups allows us to reconstruct population movements, migrations, and genetic relationships, providing insights into the interactions between ancient human groups. However, it is crucial to consider sample sizes, as they impact the statistical robustness of genetic findings. Larger sample sizes lead to more reliable insights.Regarding genetic diversity, the Aburráes population in Colombia exhibited a genetic diversity level of 0.644. When compared to various populations from different geographic regions and archaeological periods, this falls within the range of genetic diversity observed across these populations. Some populations displayed higher genetic-diversity values, while others had lower values. Thus, reflecting the intricate tapestry of human genetic variation across different time periods and geographical locations. It is important to note that, in general, a larger number of samples provide a more accurate reflection of the true diversity of the population or community being studied. Therefore, to obtain reliable and representative results, it is recommended to use a significant number of samples in the diversity analysis.It is important to emphasize that the haplotype network does not directly establish maternal relationships between specific individuals. Instead, its primary function is to elucidate genealogical connections and ancestral origins among various haplotypes. Its significance lies in unveiling historical relationships and evolutionary patterns within a population rather than confirming direct relationships between specific individuals.Regarding the Colinita tomb, it’s worth noting that the eight adult individuals found within it are not from the same family. This information holds significant value for archaeologists and anthropologists as it provides insights into burial rituals and helps determine whether the tombs are related to a single family or not.The meticulous examination of various haplogroups, including A2, B2, C1, and D4j1a, has provided valuable insights into the intricate demographic processes that shaped the region. Notably, Haplogroup A2, renowned for its prevalence in the high latitudes of the Americas and its historical ties to Siberian and Eskimo tribes, has emerged as a pivotal contributor to the initial waves of migration into the continent ,74. MoreThis research delves deeply into the dynamics of these populations. The discovery of A2af1b1b within contemporary populations in Panama and Costa Rica carries profound implications, pointing to significant demographic events approximately ten thousand years ago. While this specific haplogroup is presently observed in only two individuals—one from Chiriqui, Panama, and the other from Costa Rica—it originated 4700 years before the present, with a time to most recent common ancestor (TMRCA) of 1000 years before the present .Most intriguingly, the study identifies the presence of the A2af1b1b sub-haplogroup in an individual from the Valle de Aburra. This finding suggests that A2af1b1b has deep historical roots in the region, indicating its presence within ancient populations. Furthermore, the fact that this haplogroup is still present in contemporary populations emphasizes its enduring legacy . This unThe enduring presence of Haplogroup A2ah for over five millennia across various regions offers a compelling narrative of historical population movements. This relatively rare lineage among Native Americans, discerned through comprehensive analyses of complete mitochondrial genomes, is characterized by the uncommon combination of tandem transitions, T16097C and A16098G. Which is rarely encountered in mitochondrial DNA databases . Within the larger Haplogroup A2, A2ah emerges as a distinct branch within the Native American mitochondrial family. This research marks a significant milestone in the field of genetics. Notably, for the first time, researchers have unveiled a mitochondrial lineage in the Andean region that was previously believed to be exclusive to the South American lowlands. Specifically, they have identified the A2ah lineage in the Andean area, even within a pre-Hispanic individual. The implications of this discovery are profound, raising intriguing questions about the potential gene flow between the Andean and lowland regions before the Spanish arrival .Archaeological evidence indicates interactions and trade among diverse populations across various eco-regions during pre-Hispanic times. The presence of trade networks, such as those connected to the Quebrada de La Cueva ravine, which housed the Pukara de La Cueva, suggests extensive pre-Hispanic connectivity. Yet, whether these networks also facilitated gene-flow remains uncertain. This underscores the significance of ancient DNA studies in shedding light on this aspect of pre-Hispanic population dynamics .If indeed gene-flow was a regular occurrence between highland and lowland pre-Hispanic populations, it becomes plausible that certain genetic variants from one region could be found in the other. The identification of an infrequent mitochondrial haplotype from the South American lowlands in the Andean area in this study lends support to this hypothesis. To further investigate this proposition, the researchers recommend expanding genetic analyses to encompass other individuals retrieved from Pukara de La Cueva at the population level. Additionally, isotopic analysis could offer insights into the region where the analyzed individual resided during their early years .The research suggests an approximate origin dating back to 5200 years ago, with some within-clade variations. Geographically, this lineage appears to be primarily concentrated in Bolivia and its adjacent regions with a limited distribution ,99,100.The discovery of the A2ah mitochondrial lineage in the Andean region, as previously discussed, presents an intriguing parallel to the genetic findings in the Valle de Aburra. In both instances, researchers have uncovered the existence of uncommon mitochondrial haplogroups in regions that defied earlier expectations.This shared discovery of sub-haplogroup A2ah among both ancient and contemporary Native American populations emphasizes its profound historical importance and its enduring legacy within the tapestry of mitochondrial DNA. The presence of this lineage in diverse temporal contexts underscores its resilience and persistence through time.By delving deeper into the study of this unique lineage, researchers can unlock invaluable insights into the historical narratives, migration patterns, and genetic tapestry of Native American communities. This connection between seemingly distant regions challenges preconceived notions and beckons for further exploration into the intricate web of human genetic heritage across the Americas.The genetic narrative of the A2i haplogroup, explored in Achilli et al.’s 2008 study within the Valle de Aburra , finds rWithin the complex genetic framework of lineage C1, a thorough network analysis has unveiled a compelling historical narrative. This analysis, which considered sequences from diverse regions such as the Amur River, Japan, and the Americas, lends support to the notion that the foundational haplotype for Native American C1 finds its roots in the Amur River region, echoing previous hypotheses proposed by Starikovskaya .The C1c subclade, an exclusive genetic branch within haplogroup C1 exclusive to Native American populations, stands as a testament to the deep ancestral ties between indigenous peoples and the Americas. C1c plays a pivotal role in genetic and phylogenetic investigations, offering a unique perspective into the evolutionary history and migration patterns of specific groups. This subclade provides invaluable insights into the genetic diversity of Native American populations and their ancestral journeys .The research also delved into broader colonization patterns. The presence of haplogroup B2 across diverse Native American populations adds weight to the idea of an early expansion from North to South America. Additionally, the widespread distribution of haplogroup C1 and its subclades suggests a unified migratory wave that played a crucial role in populating the Americas. The identification of variant 16298C in Abu4 within haplogroup C underscores the distinctive genetic legacy of pre-Hispanic Native American settlers. Moreover, the presence of Haplogroup C1c, with its older coalescent age and evidence of intra-haplogroup variation, in both Mexican Americans and ancient Valle de Aburra populations hints at a captivating connection. This shared genetic heritage suggests that the genetic diversity within Haplogroup C1c, as well as its sub-branches, was firmly established among ancestral populations before their migration and expansion into the Americas. The exploration of this haplogroup’s distribution and variation across populations promises profound insights into historical movements and genetic relationships among these groups, ultimately unraveling the intricate history of Native American origins and migrations.Within the realm of Native American haplogroups, B2 emerges as a distinct lineage exclusive to indigenous populations of the Americas. Setting it apart from the other major founding haplogroups , the basal B2 lineage lacks defining variants in the control region (CR). This unique feature presents a challenge in distinguishing B2 from closely related haplogroups, a critical task for distinguishing Native American from Asian haplotypes .Compelling evidence suggests that the initial wave of people entering what is now Mexican territory carried with them the A2 and B2 haplogroups around 15–18-thousand years ago. The distribution of individuals with higher frequencies of B2 or C1 compared to A2 indicates a specific migration pattern. A2, B2, and C1 appear to have been primarily dispersed along a Pacific coast route, spanning approximately 800 km along the Neovolcanic axis region. Subsequently, they made their way southward along the Pacific coast route, eventually reaching the Yucatan Peninsula via the Gulf coast route.It is noteworthy that the Neovolcanic axis region, characterized by the presence of significant rivers like Lerma and Balsas, acts as a natural barrier against adverse Pacific weather conditions. This geographical feature likely played a pivotal role in facilitating the spread of these haplogroups ,91.Sub-haplotype D4j1a stands out from the norm when it comes to substitutions observed in mitochondrial DNA clusters associated with the southern distribution of aboriginal Siberian peoples. These substitutions are typically linked to major mitochondrial DNA haplogroups prevalent in East and South Asia. The intriguing presence of sub-haplotype D4j1a among both aboriginal Siberian populations and the ancient inhabitants of the Valle de Aburra unveils a fascinating genetic connection spanning vast geographical distances. Importantly, this sub-haplotype diverges from the usual mitochondrial DNA clusters found in aboriginal Siberian populations, aligning instead with major mitochondrial DNA haplogroups prevalent in East and South Asia ,91,92,93The genetic connection between Siberia and the Valle de Aburra holds the potential to illuminate ancient migration patterns and the interactions between diverse populations. To gain a deeper understanding of the historical context and the implications of this shared genetic marker, further genetic and archaeological investigations are imperative .Understanding the genetic connection between Siberia and Valle de Aburra offers a glimpse into the intricate journey of ancient human migrations. It suggests that populations from distant regions might have come into contact, possibly through migratory movements, trade routes, or cultural exchanges.To provide a meaningful context for this genetic link, it is crucial to highlight the fundamental role of archaeological investigations, which, in combination with genetics, offer a more accurate representation of ancient life. The genetic relationship between the Valle de Aburra and Siberia, along with its proximity to Mesoamerica, suggests the diffusion of genetic markers across populations. Mesoamerica’s genetic diversity, a result of its complex history, can provide insights into ancient cultural exchanges and trade routes. A multidisciplinary approach, encompassing genetics and archaeological excavations, is essential for a comprehensive understanding of the history and implications of this shared genetic marker.The revelation of a genetic connection weaving through Siberia, Mesoamerica, and Valle de Aburra is nothing short of enthralling. This revelation acts as a beckoning call, compelling us to embark on an expedition of profound historical significance. Expanding our investigation into Mesoamerica opens doors to unraveling ancient ties, migratory routes, and the intricate web of interactions that wove together diverse populations. In the realm of collaborative genetic and archaeological exploration, we stand on the precipice of unearthing the narratives of our forebears, shedding brilliant light upon the enigmatic pathways of our shared past.Comparisons with other populations across the Americas revealed that the Aburráes population showed the highest genetic similarity with pre-Hispanic populations in America. The results presented in our study contribute to the understanding of the genetic diversity and ancestral connections of the Aburráes population. These findings have implications for the population history and migration patterns in the Americas.Furthermore, the study sheds light on population relationships across regions. The genetic affinity between the Aburráes and Mesoamerican and Arctic–sub-Arctic populations showcase intricate gene flow and migratory dynamics. These findings align with the hypothesis of a coastal migration route and potential interactions between the indigenous Chibchas population and the Mayan population.The primary limitation of this study arises from its exclusive focus on the HVS-I of ancient mtDNA. This choice was necessitated by the inherent challenges in working with degraded and ancient DNA samples. While the results obtained are indeed intriguing and informative, it is imperative to recognize and acknowledge this limitation.Likewise, it is advisable to approach the study’s conclusions with a degree of caution. This caution is particularly warranted due to the relatively small sample size, consisting of only 10 individuals, and the restriction of the analysis to a single mitochondrial region (HVS-I).Despite these challenges, it is noteworthy that the study was successful in elucidating important haplogroups. These findings make a significant contribution to our understanding of the genetic history of the ancient population under investigation.In conclusion, the present study has provided valuable insights into the genetic diversity and population history of pre-Hispanic inhabitants in the Aburrá Valley region. The identification of various mitochondrial haplogroups, particularly A2 and its sub-lineages , Haplogroup B2, Haplogroup C1, and Haplogroup D4j1a, has shed light on the complex migratory patterns and interactions of ancient populations in the Americas.One of the most striking findings of this research is the presence of distinct sub-haplogroups within the larger haplogroups, such as A2af1b1b, A2ah, and A2i. These sub-haplogroups not only reveal local genetic diversification but also highlight the enduring legacy of certain genetic lineages in the region.While the genetic data presented in this study contribute valuable insights, it represents only a starting point in the investigation of the region’s population history. The pursuit of more extensive and refined research will undoubtedly bring us closer to a deeper appreciation of the rich and complex human history that unfolded in the Aburrá Valley throughout the ages.Ongoing research efforts are vital to fully unravel the region’s population history. By continuously exploring and integrating diverse data sources, we can achieve a more nuanced understanding of the region’s past, its connections to broader human migrations in the Americas, and the factors shaping its distinctive genetic landscape.


Iris lacustris, a northern Great Lakes endemic, is a rare species known from 165 occurrences across Lakes Michigan and Huron in the United States and Canada. Due to multiple factors, including habitat loss, lack of seed dispersal, patterns of reproduction, and forest succession, the species is threatened. Early population genetic studies using isozymes and allozymes recovered no to limited genetic variation within the species. To better explore genetic variation across the geographic range of I. lacustris and to identify units for conservation, we used tunable Genotyping-by-Sequencing (tGBS) with 171 individuals across 24 populations from Michigan and Wisconsin, and because the species is polyploid, we filtered the single nucleotide polymorphism (SNP) matrices using polyRAD to recognize diploid and tetraploid loci. Based on multiple population genetic approaches, we resolved three to four population clusters that are geographically structured across the range of the species. The species migrated from west to east across its geographic range, and minimal genetic exchange has occurred among populations. Four units for conservation are recognized, but nine adaptive units were identified, providing evidence for local adaptation across the geographic range of the species. Population genetic analyses with all, diploid, and tetraploid loci recovered similar results, which suggests that methods may be robust to variation in ploidy level. Iris L., Iris lacustris Nutt., “on the gravelly shores of calcareous islands of Lake Huron” .,5,6.I. lMichigan .I. lacustris was initially described, the species was listed as federally threatened [In 1988, 170 years after reatened . The smareatened . Despitereatened , a factoreatened .I. lacustris has been examined to a greater extent than the genetic diversity of the species. To date, only three studies have explored this topic: Simonich and Morgan [I. lacustris, Hannan and Orick [I. cristata Aiton [Iris is quite limited. This genetic paucity is intriguing because I. lacustris and its sister are both putative tetraploids [I. lacustris may have implications for the ability of the species to respond to the changing environment across its geographic range and for various conservation efforts.The ecology of d Morgan  examinedd Morgan  investigd Morgan  examinedd Morgan  found ovd Morgan  also notnd Orick  found thta Aiton , which hraploids , and polraploids ,13,14. II. lacustris, (2) explore patterns of migration, and (3) recognize population clusters for management of this rare species. Given the paucity of genetic diversity identified in previous studies, we hypothesized that there would be limited genetic variation across the species.In order to investigate the population and conservation genetics of the species in a comprehensive manner, we examined multiple populations from across Michigan and Wisconsin, and we used tunable Genotyping-by-Sequencing (tGBS ), a methI. lacustris in Michigan and Wisconsin .Across all datasets, observed heterozygosity slightly exceeds expected heterozygosity, and Fnegative . Pairwisdatasets . Based odatasets . Mantel datasets  with p <tructureSelector, the optimal K values were greater for all loci analyzed together than for either the diploid or tetraploid loci analyzed independently  and discriminant analyses of principal components (DAPC) are similar to those that explicitly consider a priori population structure. With PCA, three to four clusters were recovered corresponding to the same ones from the population assignation analyses, and this was more evident with the MCR90 datasets compared to the MCR50 ones. In all analyses, three populations—MI6, MI16, and WI5—were recognized as most distinct from the other populations. Across DAPC analyses, individuals from populations tended to cluster together, and this is similar to results from other methods. In general, DAPC analyses recover MI6, MI16, and WI5 as distinct units or as a cluster together, with the results for MCR50 all loci being the only exception. In analyses with this dataset, WI5 was included in a cluster distinct from the other two populations, but with WI4 and populations from Michigan. In some analyses, such as MCR50 and MCR90 diploid loci, the divided cluster of central populations was identified. The number of loci under selection in each dataset is in Patterns of migration inferred from BA3-SNPs suggest that migration is minimal, regardless of the dataset analyzed, and that most individuals are from their original population . While tBased on the method of Funk et al. , evolutiThe results of the fastSTRUCTURE, PCA, and DAPC are similar, with one exception. Unlike analyses with fastSTRUCTURE and PCA, where individuals of the same population cluster together, with DAPC, some individuals of the same population are members of different clusters. This is likely due to the large number of clusters identified as optimal, which is particularly the case for MCR90 and MCR50 datasets with all loci.I. lacustris across Michigan and Wisconsin. These clusters are structured geographically, with eastern, central, and western groups, and at higher K values, the central group is subdivided into two groups that are also geographically oriented  hybridization could remain for an extended period of time. It is useful to keep in mind that the island and nearby areas on the mainland are some of the more heavily sampled geographic regions in the present study. This greater sampling could hint at a similar pattern in other areas if individuals were sampled to a larger extent. It was not possible to include representatives from Ontario, Canada in the study, and future studies that add these will likely have greater context for the relationship of the central and eastern populations to those even farther east.Cirsium pitcheri Torr. & A.Gray, a similar pattern was recovered. The populations from the Door Peninsula are also quite distinct from others on Lake Michigan [The four populations in the western cluster  are notable. While these populations form a cluster in most analyses , the twoMichigan , and theI. lacustris because they are situated inland, and this is not the case for the other sampled populations. While other populations can be found a short distance from the shoreline, these populations are ca. 30 km from the current boundary of Lake Michigan. These two populations are consistently recognized as genetically distinct from the other sampled populations, and these both likely became established during higher water level periods of Glacial Lake Algonquin ca. 12,500 years ago [I. lacustris to persist, but without the opportunity to interbreed with other, coastal populations, resulting in their distinct genetic signature . This has resulted in populations primarily being restricted to shorelines where habitat was appropriate. If this is the case, the inland populations, such as MI6, would still represent relicts of a prior time, but this would be due to remnant habitat availability based on adequate disturbance regimes and/or seral stages, not prior establishment during higher water levels of the Great Lakes and subsequent serendipitous survival.An alternative hypothesis for the present geographic distribution of the species also exists. Van Kley and Wujek  and BrotI. lacustris  that was not recovered with the diploid or tetraploid datasets, although hints of this cluster can be seen in the MCR90 2N dataset at K = 5. This cluster is identified in all of the datasets with loci under selection as either one or two clusters  differ from those of ESUs and MUs (The evolutionarily significant units (ESUs) were described above with all loci used for population genomic analyses, and the management units (MUs), which were determined using only loci not under selection, are similar, but not identical to the ESUs; however, the differences are minor . Given t and MUs . The AUs and MUs , and whi and MUs .I. lacustris inhabits three of the landscape ecology regions of Michigan and multiple districts and subdistricts within each region [This local adaptation is, on some level, unsurprising, because even though the species is generally restricted to the same type of habitat presently , climatic, soil, and vegetation differences occur across the geographic range of the species. Indeed, h region ,31. Van h region  also recI. lacustris was collected from 24 locations in Michigan and Wisconsin  for DNA isolation and tunable Genotyping-by-Sequencing (tGBS) to recognize single nucleotide polymorphisms (SNPs) across the populations. Using the restriction enzyme Bsp1286I, paired-end tGBS libraries were created [Leaf material was sent to data2bio ( created  and subs created  after se created . Low-qua created  was emplind/HE statistic [ind/HE < 0.5 and tetraploid loci with Hind/HE > 0.75. Datasets were created for each set of loci (Because I. lacustris is a putative polyploid and many population genetic methods assume that species are (at most) diploid, polyRAD  was usedtatistic ,35 was e of loci . The numF-statistics were calculated with hierfstat [tructureSelector [Observed and expected heterozygosity measurements and ierfstat ,37, and ierfstat . All 24 ierfstat  was emplierfstat , on the ierfstat  and DISTierfstat , was empierfstat . To idenSelector ,45 were tructureSelector were also used for STRUCTURE analyses, with the best K also determined via the method of Evanno et al. [For comparison, and given potential variation in ploidy at loci , STRUCTUo et al.  and Ln Po et al. , and theTo explicitly include geographical data along with SNPs to investigate patterns of population genetics, tess3r  and conSIn addition to analyses for explicit population structure, all datasets were analyzed with principal component analyses (PCA), correspondence analyses (CA), and discriminant analyses of principal components (DAPC) in adegenet , principFST values and a false discovery rate of 0.05.Loci under selection were determined with BayeScan  using 10FST distances, Nei’s distances, and admixture estimates were selected, and the analyses were run for 15 million simulations with a batch size of 1000. Using the results of the training, a random forest analysis was conducted with abcranger [Demographic history and patterns of migration were explored using BA3-SNPs ,53, DIYAbcranger  using 10tructureSelector [Conservation and management units were identified following the three-step method of Funk et al. , in whicSelector , the marSelector , and theSelector . Based oI. lacustris [Iris lacustris will not grow where the habitat has been destroyed by residential, resort, or industrial development”. Therefore, the conservation genetic results are of limited value if management steps are not taken to ensure that individuals of I. lacustris have the opportunity to be successful in situ. This includes not only ensuring intermediate light conditions and limited litter [I. lacustris from Ontario can extend the presented results to investigate the ways in which these populations relate to those in the United States. Given the international geographic range of the species, conservation efforts that are binational would be particularly useful.The present study provides evidence of genomic variation and local adaptation across the geographic range of the species, which is novel given the negligible genetic diversity previously recovered for acustris ,9,10. Hoacustris  stated td litter ,6, but ad litter . Therefo