Dendrolimus punctatus, has caused serious damage to the Pinus massoniana (Lamb.) pine forests. Here, the whole mitochondrial DNA (mtDNA) was employed to analyze the population evolution of D. punctatus and to understand the process underlying its current phylogenetic pattern. D. punctatus populations within its distribution range in China were categorized into five subgroups: central and eastern China (CEC), southwestern China (SWC), Yibin in Sichuan (SC), Baise in Guangxi (GX), and Luoding in Guangdong (GD), with a high level of haplotype diversity and nucleotide diversity among them. The genetic distances between subgroups are relatively large; however, the genetic distances between populations within the CEC subgroup were relatively small, suggesting that many populations were closely related in this subgroup. The mantel test showed that geographic distance had an important impact on the genetic distance of different geographic populations . The neutrality tests, Bayesian skyline plot, and haplotype network showed that D. punctatus experienced a population expansion around 100,000 years ago. The divergence times of GX/SC, SWC, GD, and CEC were 0.347, 0.236, 0.200, and 0.110 million years ago, respectively. The SWC, CEC, and GD subgroups might have evolved from GX or SC subgroups. The population genetic structure of D. punctatus was closely related to its host tree species, geographic distance among populations, the weak flight capacity, and many eco-environment conditions.In southern China, the masson pine caterpillar,  Dendrolimus punctatus Walker (Lepidoptera: Lasiocampidae), is the most destructive insect pest among the >27 pine caterpillar species in China, causing tremendous economic damage annually to the pine forests, especially the P. massoniana Lamb. forests in southern China. D. punctatus is mostly distributed in the provinces south of the Qinling Mountains-Huai River line, and it has multiple generations per year that vary geographically. For example, in Xinyang district of Henan province, D. punctatus has two generations per year, but in Guangdong province, 3~4 generations occur  data for resolving the divergence patterns and histories of geographic populations , which i studied  and sout markers . HoweverD. punctatus collected from its main distribution regions in southern China. Additionally, we inferred the historical population dynamics of D. punctatus and discussed its geographic origin, population evolution trend, and biological characteristics, which are significant factors underlying the genetic variation of D. punctatus in China.Whole mtDNA sequences have much higher resolutions than partial mitochondrial gene sequences. Therefore, in this study, we used the whole mtDNA to explore the population evolution and genetic diversity as well as the differentiations of 48 individuals from 15 populations of D. punctatus were collected from the main host, Pinus massoniana trees at 15 locations from nine provinces in 2016  following the manufacturer’s protocol. NanoDrop 2000  and picogreen  were used to detect the DNA purity and concentration. To assess the DNA integrity, 1% agarose gel electrophoresis was used.http://soap.genomics.org.cn) (http://dogma.ccbb.utexas.edu) (D. kikuchii (NC_036347.1). The complete mitochondrial genome sequences of D. punctatus were submitted to the GenBank, and accession numbers were listed in the Genomic DNA were fragmented with Covaris M220  to 400–500 bp. A 460-bp paired-end library was constructed from each specimen and sequenced to obtain 3 Gb of data using an Illumina Hiseq X Ten platform at Shanghai Majorbio Bio-pharm Technology Co., Ltd. Raw data were sheared as follows in order to make the subsequent assembly more accurate: the adapter sequence in reads was removed; the 5′-terminal non-AGCT base was detached; the ends of reads with low sequencing quality (< Q20) were removed; reads with an N ratio of 10% and small fragments with a length <25 bp were removed. All the downstream analyses were based on clean data of high quality . The clean data were assembled using SOAPdenovo v2.04 (.org.cn) , kmer wa.org.cn)  iterativxas.edu)  and MITOxas.edu)  were usehttps://github.com/broadinstitute/picard). The results were calibrated using GATK (https://github.com/broadinstitute/gatk) (https://github.com/dkoboldt/varscan) to filter out the low sequencing depth and low alignment quality of site, and SNP data sets with high credibility were obtained. The filtering conditions: minimum coverage depth: 30 reads; the minimum mutation frequency: 20%; minimum mutant base number: 15 reads; minimum mass value of mutation detection site: Q20; both forward and reverse reads needed to support mutant sites, and the number of forward and reverse reads varied by less than 10%. The SNPs obtained by combining the data with gff gene annotation information on reference species were annotated using the Annovar program (http://www.openbioinformatics.org/annovar/annovar_gene.html)  using BWA software , and thete/gatk) . SNP Pdene.html) . Populatne.html) . To detene.html) .D. punctatus was analyzed using a median-joining algorithm in the program Network4.6  and maximum likelihood (ML) methods after the correction. MrMTgui software  was usedD. punctatus, we performed divergence time estimation for different populations using BEAST v1.10.4 (COX1 was set as 0.0177 substitutions/site/Myr (s/s/Myr)  and FigTree v1.3.1 (http://tree.bio.ed.ac.uk/software/figtree) were used to calculate and display the results.Based on the 13 protein-coding genes of  v1.10.4 . MrMTguis/s/Myr) , The subD. punctatus among different populations were 892, 1, 0.0107, and 125.8, respectively , Luoding in Guangdong (GD), southwestern China (SWC), Yibin in Sichuan province (SC), and Baise in Guangxi province (GX). Populations of Sichuan Yibin (SCYB), Guangxi Baise (GXBS), and Guangdong Luoding (GDLD) were separate lineages, and the monophyly of these three lineages was well supported; Sichuan Dazhou (SCDZ), Chongqing Nanchuan (CQNC), and Yubei (CQYB), plus part of Guizhou Jinping (GZJP) and Hunan Dongan (HNDA) were clustered in the SWC lineage; CEC lineage included the populations of Anhui Hefei (AHHF), Hubei Dawu (HBDW), Hubei Huanggang (HBHG), Hunan Chenzhou (HNCZ), Hunan Dongan (HNDA), Jiangxi Xingguo (JXXG), Jiangxi Yudu (JXYD), Guangxi Guilin (GXGL), Guizhou Jinping (GZJP) and plus one of CQYB. The phylogeny complied well with the results from the haplotype network. In the topological structure of phylogenetic tree, individuals in GXBS, SCYB, SCDZ, GDLD, and HNCZ clustered together by their separated populations, while individuals in other populations mixed and clustered together among populations, which may be related to certain gene exchanges between populations.Phylogenetic trees (BI and ML) were constructed using the whole mtDNA data of 48 sequences of D. punctatus was constructed by maximum likelihood method, and the phylogenetic analysis of SNPs indicated that their phylogenetic relationships were similar to those of mitochondrial genome. The individuals in GDLD population were clustered into one lineage, two lineages of GD and CEC clustered together, and also there were a few individuals being different from the topology of phylogenetic trees based on mitcochondrial genomes, and five individuals of CEC subgroups clustered into the SWC group. The monophyly of the two lineages—the GX group and the SC groups—was well supported. The results of population structure construction based on SNPs showed that the most reasonable sample classification method was at K = 5 (D. punctatus.The phylogenetic tree of 44 SNPS (The pairwise genetic distances ranged from 0.004 to 0.020 (r = 0.3633 (P < 0.001), suggesting that the genetic distance and genetic differentiation among D. punctatus populations are related to their geographic isolation.The results of the Mantel test showed that there were significant correlations between the genetic distance and geographic distance. The test results had D. punctatus geographic populations showed that the divergence time of SC and GX lineages was 347,000 years ago , which was the earliest divergence time among all five lineages    with a hD. punctatus populations were geographically structured in five lineages: populations of central and eastern China (CEC), southwestern China (SWC), Yibin in Sichuan (SC), Baise in Guangxi (GX), and Luoding in Guangdong (GD). A few specimens belonging to the same population were classified into different subgroups but their clustering boundaries were not obvious, which might be related to the existence of a small amount of gene flow between the subgroups. The formation of these five subgroups may be related to the existence of certain natural geographic barriers between them. Wushan and Wuling Mountains are located at the border of Chongqing and Hubei, hindering the physical exchanges and interactions between the SWC and the CEC lineages. However, the genetic distance between them was small, indicating that the differentiation time due to isolation was not long ago. The Guangdong population locates in the southeast of China, and Wuyi and Nanling Mountains in the north may have blocked the exchanges between the GD lineage and the other lineages to some extent, and GD lineage occurred 3~4 generations more than other lineages, and this lineage might have formed a stable genetic structure, which was also the reason for being monophyletic lineage. The monophyly of SCYB and GXBS populations is also well supported, with mountains and distances between the two lineages blocking cold air from the north and the Indian Ocean, the gene exchanges between the two populations and other populations were also prevented.Structure and phylogenetic analysis of whole mtDNA indicated that D. punctatus’ populations. This is consistent with the results of a study by D. kikuchii.In our study, the genetic distance results were consistent with the results of the phylogenetic tree clustering. The genetic distance between populations within the CEC subgroup is generally small, and these populations are closely related, which may be associated with the geographical characteristics of the subgroup, even though the CEC subgroup is the most widely distributed of the five subgroups. The majority of populations in the CEC subgroup are distributed in the middle and lower reaches of the Yangtze River in China  of the Quaternary glacial period in China , based on its host plants’ distribution .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ruditapes philippinarum is the world’s second most important bivalve mollusk commercially farmed, whose indigenous populations are mainly distributed in the coastal areas of East Asia. However, with the development of commercialization, mixture of populations and loss of local germplasm have become prominent problems. Here, genetic differentiation of seven Manila clam populations from East Asia was investigated through analyzing the polymorphism of the mitochondrial cytochrome C oxidase subunit I (COI) gene as well as 20 simple sequence repeat (SSR) molecular loci. In total, 40 haplotypes were identified, among which 31 were unique. Moreover, two main haplotypes were detected with several radiating derived haplotypes. Populations in Japan-North Korea shared haplotype Hap_31, and populations in China shared haplotype Hap_7, suggesting that the natural geographical isolation of the Yangtze River and the Yalu River might have divided the East Asian indigenous populations into three groups, which were located in South China, North China, and Japan-North Korea, respectively. The Aquaculture breeding activities from South to North in China might have promoted gene exchange among Manila clam populations. Population in Laizhou had the highest genetic diversity and therefore could be an excellent germplasm source.The Manila clam  Ruditapes philippinarum (synonym Venerupis philippinarum)1 is a eurythermal and euryhaline species. Indigenous populations are distributed along the coasts of the Pacific to the Indian Ocean. In 1936, the Manila clam was introduced from Japan to British Columbia and subsequently from North America to the European coasts of France, Britain, Italy, Spain, and Turkey. Because it is characterized by long larval floating time (2–3 weeks), widespread distribution, and strong environmental adaptability, it is now widely distributed and has become the world’s second most important commercially cultured bivalve mollusk5.The Manila clam 2. China is the world’s largest Manila clam germplasm resource, which provides an important genetic basis for studies of population genetic differentiation and genetic diversity of Manila clams. Third, in recent years the northern coastal ecosystems of China have been impacted by economic development, frequent commercial activities, land reclamation from the sea, and artificial breeding of Manila clams using seedlings from southern China. These changes have resulted in mixing of the Manila clam populations and loss of the indigenous germplasm, which have prompted studies of Manila clam population gene exchange and genetic structure changes.The conditions along the coast of East Asia are favorable for genetic studies of the Manila clam. First, the extensive coastline of East Asia, especially the area near the Yangtze River delta, significantly contributes to the biological distribution pattern of the Manila clam in coastal areas. Natural barriers provide unique geographical conditions that are key to investigating the evolution of the indigenous Manila clam population and its geographical and historical patterns. Second, the annual production of Chinese Manila clams is about 3 million tons, accounting for > 90% of the world’ production. This species exhibits numerous genetic variations in growth, temperature and salinity tolerance, shell color, and shell shape6 combined the mitochondrial cytochrome C oxidase subunit I (COI) gene sequence and/or the simple sequence repeat (SSR) gene marker to assess the genetic basis of the Manila clam population introduced from Japan to the North American west coast in 1936. Chiesa et al.7 analyzed the introduction history of European Manila clams using the COI gene sequence. Sekine8 assessed the genetic differentiation between the Japanese and Chinese Manila clam populations through COI gene analysis and found that the Japanese population can be divided into two subpopulations and the Chinese population can be divided into two subpopulations.The genetic diversity of the Manila clam has been studied extensively. For example, Cordero et al.9 and microsatellite loci to analyze the genetic structure of these seven Manila clam populations in East Asia. Our results provide a hints for the conservation and utilization of Manila clam germplasm resources.The genetic structure of the populations in Beihai, Lianjiang, Laizhou, Yingkou, and Tianjin in China as well as the populations in Sinuiju (North Korea) and Hokkaido (Japan) are important wild seed production areas. In this study, we used the mitochondrial COI gene10, which might be due to inbreeding, natural selection, mutation, null alleles, gene flow, genetic drift11. O'Connell and Wright12 suggested that a minimum sample size of 50 individuals per population should be considered for loci with between 5 and 10 alleles. In this study, 50 individuals were sampled for each sample site to ensure a sufficient sample size for an accurate assay. The sampled clams were then dissected in the laboratory and used for subsequent DNA extraction. For each population, ten individuals were used for COI gene sequencing and fifty individuals were used for microsatellite analyses.To investigate the genetic diversity and genetic structure of Manila clams in the context of degenerated germplasm resources and contaminated native populations, seven populations were selected as the experimental materials, including three northern China populations, two southern China populations and two Japan-and-Korean populations Table ; Fig. 1.9. In DUI, male Manila clam has two mitochondrial genomes  and female (F) parent), and the F-type mitochondrial genome is inherited from the female in somatic muscle tissue and the M-type mitochondrial genome is inherited from the male in the gonad. The nucleotide sequence of M-type mitochondrial DNA is up to 30% different from the F-type mitochondrial DNA. Maternally inherited F-type mitochondrial DNA is the best choice for studying systematic classification and population inheritance. Therefore, in this study, foot and adductor muscle tissues were used for population analysis of the Manila clam F-type mitochondrial DNA COI. Genomic DNA of each specimen was extracted from muscle tissues using the Marine Shellfish DNA Extraction Kit  and stored at − 20 °C. Total DNA was used for both mitochondrial DNA and microsatellite analysis.The phenomenon of DUI  has been reported in Manila clam15 were used to amplify a fragment of the COI gene. For each sample, polymerase chain reaction (PCR) was performed in a final volume of 50 μL containing 16 μL of sterile deionized water, 25 μL of Mix containing Takara Taq enzyme, buffer, and dNTP , 2 μL of each primer (20 m mol L−1), and 5 μL of DNA template (100 ng μL−1). The amplification reaction was carried out in a PTC-100 thermal cycler according to the following temperature profile: 5 min initial denaturation at 94 °C; 34 cycles of 94 °C for 30 s of denaturation, 52 °C for 30 s of annealing, 72 °C for 30 s of extension, a final extension of 7 min at 72 °C, and a final hold at 4 °C. All verified PCR products were purified using a Takara MiniBEST Agarose Gel DNA Extraction Kit and then subjected to DNA sequencing in both directions with corresponding PCR primers using an ABI 3730xl DNA Analyzer sequencing system .The universal forward primer COI-F (5′-GGTCAACAAATCATAAAGATATTGG-3′) and universal reverse primer COI-R (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′)R. philippinarum (data not published), 20 pairs of primers , and about 100 ng of template DNA in a volume of 10 μL. The amplification reaction was carried out in the PTC-100 thermal cycler using the following temperature profile: 5 min initial denaturation at 94 °C; 34 cycles of 94 °C for 30 s of denaturation, 50–62 °C for 30 s of annealing, 72 °C for 30 s of extension, a final extension of 7 min at 72 °C, and a final hold at 4 °C. Amplification products were separated by 8% denaturing polyacrylamide gel and visualized by silver staining. A 10 bp DNA ladder (Takara) was used as the molecular size marker.According to the transcriptome data of rs Table  were des16. Arlequin v3.517 was used to estimate the population average pairwise differences among populations and pairwise genetic distance (FST). Haplotype network analysis was performed through POPART19. The confidence threshold for the Manila clam sequence is 95%, and it was used to test whether haplotypes form a single network separate from that of congeneric species21. Data were converted into a nexfile using DNASP software, and we mapped the median-joining (MJ) network using POPART v1.7 (http://www.http://popart.otago.ac.nz/index.shtml) to track the genetic relationship among the identified haplotypes and other breeds from different regions. A MJ network13 was then constructed using POPART v1.7 for Manila clam haplotypes and outpopulations. Additionally, FST values were calculated using MEGA v722 to assess the inter-population relationships for the genetic and geographic distance matrices.DNA sequences obtained were edited and aligned using DNAMAN (Lynnon BiSoft) to obtain a consensus sequence. The haplotype diversity (Hd), number of haplotypes (h), nucleotide diversity (π), and average number of nucleotide differences (K) were computed using DNASP 5.10.0123 and Tracerv1.424 software were used to estimate the divergence time between branches with constant rate based on the degree of divergence between gene sequences and molecular clocks, and the occurrence times of other nodes on the phylogenetic tree also were calculated. The Bayesian Skyline Plot (BSP) method is based on coalescent theory and was used to quantify the relationship between gene sequence pedigree and population geographic history to infer the origin time of related populations and the divergence time of different populations.Beast v2.6.2 software25 software was used for population genetic structure analysis, with K value set to 2–9. The iteration parameters of Burn in Period and Markov Chain Monte Carlo were 100,000 and 50,000, respectively. Structure Harvester (http://taylor0.biology.ucla.edu/struct_harvest/) was used to determine the score of the ΔK value and the optimal number of gene banks . CLUMPP v1.1.2 software26 was used to calculate the individual membership coefficient  based on the optimal K value, and DISTRUCT v1.1 software27 was used to visualize the population genetic structure.STRUCTURE v2.3.3NA), allelic richness (AR), size in base pairs of alleles, expected heterozygosity (HE), observed heterozygosity (HO), Shannon’s Information index (I) and inbreeding coefficient (Fis) were estimated using POPGENE32 software28.For the SSR analysis, each individual electrophoresis band represents a specified locus, and the molecular weights of the loci were determined according to their location. According to the banding migration distance, we used the letters a–z to record each individual’s genotype. Number of alleles per locus 29. PIC is calculated as follows:i and Pj denote the ith and jth allele frequency, respectively. When the PIC value in a population is > 0.5, the locus is highly polymorphic. When the PIC value is between 0.25 and 0.5, the locus is moderately polymorphic, and when it is < 0.25, polymorphism of the locus is low.The polymorphism information content (PIC) is generally used to measure the degree of microsatellite marker DNA variation and as an indicator of The authors followed all applicable international, national, and/or institutional guidelines for the care and use of animals.All necessary permits for sampling and observational 285 field studies have been obtained by the authors from the competent authorities and are mentioned in the acknowledgements, if applicable.The experimental protocols were approved by the institutional and licensing committee by including a statement in the methods section.A 680 base pair sequence of the mitochondrial COI gene from 70 individuals was successfully obtained (accession no: MW093623-MW093692). A total of 40 haplotypes were identified, 31 of which were unique Table . Nine clAll the individual haplotypes could be categorized into two clades. The haplotypes Hap_31 and Hap_7 were the two centers, which were surrounded by the other haplotypes in a radial manner. The clade centering Hap_31 was found in the Hokkaido (JAP-H) and Sinuiju (DPRK-S) populations only, whereas the clade centering Hap_7 was found in the five populations located in China.Our analyses revealed high levels of Hd and π. Hd ranged from 0.9778 to 0.8444 Fig. A, and π Figure FST to compare the seven populations . However, the values differed significantly in pairwise comparisons between JAP-H or DPRK-S and the other populations.Data sets were further used to calculate population pairwise ns Table . The genDNASP software was used to analyze the distribution of population mismatch, and the distribution curves of the mismatch were bimodal or multi-peak Fig. . PopulatNA, AR, HO, and HE, which were used to assess the level of genetic diversity. The NA at the genomic SSRs ranged from 3 to 12 (mean NA 5.3929). The AR at the genomic SSRs ranged from 3.4458 to 4.219 (mean AR 3.8919). The Shannon’s Information index (I) at the genomic SSRs ranged from 1.321 to 1.5331 (mean (I) 1.4322). The HE per locus for genomic SSRs ranged from 0.7011 to 0.7542 (mean HE 0.7276). The highest HE value was detected for RPg15608 (0.8416), and the lowest value was found for RPg16975 (0.6633).All 20 microsatellite loci were polymorphic in all Manila clam populations studied, and the levels of polymorphism varied among loci. In total, 144 alleles were detected at the 20 microsatellite loci analyzed. RPg15608 with 12 alleles was the most polymorphic microsatellite, whereas RPg15130, RPg16260, RPg16876, and RPg16965 were the least variable with only five alleles. Table HO ranged from 0.2803 to 0.5220, and the average HE ranged from 0.7011 to 0.7542. The highest HE value was detected in population CHI-SL (0.5220)  was found in the CHI-SL population (5.70). There was no significant difference in the average AR among seven populations. RPg15246, RPg15130, and RPg16975 showed slightly negative Fis values  Table . The AR es Table .29. Based on the mean PIC, Laizhou population (0.70755) had the highest genetic diversity among the seven populations.The PIC results showed that there were 20 polymorphic loci in this study, and the PIC value of polymorphic microsatellite loci was between 0.39222 and 0.86806 Table . The PICFST, the coastal populations in East Asia can be grouped into South China, North China, and Japan-North Korea populations. Additionally, we detected endemic haplotypes in South and North China and in Japan-North Korea.Based on the comparison of haplotypes of COI sequences of Manila clam populations from different geographic areas using haplotype networks, BMBC, and the 30.We found two main haplotypes in the seven populations studied, and the other haplotypes were distributed radially around these two haplotypes. The Japan-North Korea populations shared Hap_31, and the five populations in China shared Hap_7. Most of the other haplotypes were detected only in an individual area. This phenomenon, which is related in part to frequent overharvesting of Manila clams in coastal areas, also has been detected in the population genetic structure of other invertebratesBMBC analysis based on COI supported the haplotype network tree results. This analysis with k = 3 showed clear differentiation between Manila clam populations in southern China  and northern Japan-North Korea. Cluster 1 was most common in the Hokkaido and Sinuiju samples but was rare in South China. Similarly, Cluster 2 was most abundant in South China but was almost completely absent from the Japan and North Korea Manila clam populations.FST results also revealed obvious genetic differentiation between the Japan-North Korean populations and the South and North China Manila clam populations. This is likely attributable to the natural barrier formed by the Yangtze River delta, which blocks gene exchange of natural populations of Manila clams in South and East Asia. After tens of thousands of years of isolation and population evolution, Manila clams have formed distinctive southern and northern populations, and fresh water flow from the Yalu River further divided the Northeast Asian Manila clam populations into the North China and the Japan-North Korean Manila clam populations. Similar results were observed in previous studies. For example, Cordero et al.6 divided the Chinese and Japanese Manila clam populations into two independent clusters. Kitada et al.31 compared the COI sequence and shell morphological traits between the Japanese and the southern Chinese Manila clam populations and found that the shell morphology and radial ribs of the two populations were significantly different.The 32.We found evidence of genetic exchange among different populations in the southern and northern Manila clam populations in China because of the influence of coastal currents of the Bohai Sea and the South China Sea33. Therefore, genetic differentiation of the three Manila clam populations from Tianjin, Yingkou, and Laizhou in North China from the populations in Guangxi and Fujian in South China was insignificant.The larvae of Manila clam have a long floating time (2–3 weeks), widespread distribution, and strong environmental adaptability, facilitating gene flow within regions and genetic mixing of populations. Therefore, within a certain space, gene flow can homogenize the Manila clamsThe level of genetic structuring among populations is the result of the balance between genetic drift, which promotes differentiation, and gene flow, which promotes homogenization of genetic differences. Gene flow among populations in South and North China might follow the isolation-by-distance model, as the Manila clam is a species with a demand for limited living space, thus, they show no large range movement from birth to death. Each population is spatially continuous. The range of random mating between individuals is limited, the frequency of genes differs little between adjacent populations, and there is a connection between geographic location and genetic distance between different populations. However, we detected clear genetic differentiation between the Japanese and Korean Manila clam populations in Hokkaido and Sinuiju. Results of COI haplotype genetic diversity and DNA diversity analyses suggested that the Hd of the Hokkaido population was high (0.8440) and that π was low (0.00279), which was likely due to rapid population expansion of small populations after experiencing the bottleneck effect. One possible explanation for this pattern is that the Japanese population has experienced overfishing and is located in an island and therefore does not communicate easily with other populations in the Sea of Japan. This may be one of the reasons for the obvious genetic differentiation between the Hokkaido and Sinuiju populations.The haplotype network results for the Chinese populations showed that Hap_7 had the highest frequency and the widest distribution, and it was the most ancestral haplotype. Hap_7 was shared by all the five of the Manila clam populations in China. These results indicate that there is a certain degree of gene exchange between the southern and northern Manila clam populations.6 reported that the Japanese Manila clam population was introduced to the coast of British Columbia in North America in 1936 and then expanded to southern California in the United States after > 30 years of reproduction. Chiesa et al.7 reported that the Manila clams introduced to the European coasts of France, Britain, Italy, and Spain in 1972 exhibited a genetic introgression. In China, Manila clam farming using southern seedlings has caused the southern population to produce a large number of Manila clam larvae in the northern waters of China. The presence of these robust planktonic larvae together with the influence of the Yellow-Bohai Sea coastal current has promoted gene exchange between the Manila clam populations of South and North China.Manila clam breeding activities in China provide conditions for gene exchange between different populations. The main culture areas in China are in the north, but 90% of the breeding seeds come from Fujian and Guangxi in southern China. For instance, the two areas of Zhuanghe and Dandong in the North Yellow Sea annually produce about 1 million tons of Manila clams whose seeds are mainly from the south. Over a decade of continuous aquaculture, up to nearly 10 million tons of the Southern China-originated Manila clams were produced in the sea area of Northern China. The planktonic larvae of southern Manila clams have a strong ability to spread along the Yellow Sea and Bohai Sea offshore, resulting in a certain degree of genetic exchange with local northern indigenous populations. Similarly, Cordero et al.The levels of genetic diversity reflect the capability of a species to adapt to environmental changes. Species with high genetic variation have greater evolutionary potential and ecological adaptability. Conversely, species with low genetic variation generally have lower population resilience. In this study, we found that Hd of the seven populations was high and that the genetic diversity was higher, which showed that the COI gene of the Manila clam was highly polymorphic. Overall, the Manila clam is thought to have high genetic variation and diversity, which confers strong adaptability to the environmental parameters fluctuations and allows this species to distribute widely in coastal waters.R of 3.22–6.15 per locus at the species level. This result was roughly in agreement with findings from previous reports35. This characteristic is expected considering the large population sizes of marine bivalves and the high mutation rates of microsatellites10.Our microsatellite data support this view. All 20 SSR loci were polymorphic, and the number of polymorphic alleles ranged from 4 to 10, with an average A36. Marine bivalves are commonly characterized by a high level of genetic diversity10. For the 20 pairs of primers, a positive value of Fis indicates a loss of heterozygotes, whereas a negative value indicates a loss of homozygotes. Therefore, these results indicate that most of the heterozygotes at the loci are missing, and this phenomenon may be caused by the Wahlund effect.PIC of the seven populations were was consistent with previous reports of the Manila clam populations from North America, Europe, and KoreaIn East Asia, the natural geographical isolation of the Yangtze River and the Yalu River divides the indigenous East Asian Manila clam populations into three geographic populations, the South China population, the North China population and the Japan-North Korea population. Large genetic differences among wild populations may be related to geographic isolation of these populations. The use of Manila clam seedlings from southern China for Aquaculture breeding in northern China, the coastal flow of the Yellow Sea, and the spread of planktonic clam larvae have likely promoted genetic exchange between South and North China populations. We found the genetic homogeneity between populations of Hokkaido and Sinuiju, due to Japan developed aquatic products on the west coast of Korea in 1910s.Therefore, we propose to protect the germplasm resources of the indigenous populations in the north by creating a high-quality stock farm. Natural wild seedlings from the original seed farm could be systematically harvested and transferred to a different area for cultivation following protocols for sustainable utilization of Manila clam germplasm. The Laizhou population had the highest level of genetic diversity, which suggests that it has the greatest potential to adapt to future environmental changes. Therefore, this population is the most suitable for habitat protection and for in situ and off-site conservation and management strategies.The results showed that the distribution curves of the mismatch were bimodal or multi-peak. Population mismatch indicated that population expansion of Manila clams did not occur recently. But Bayesian skyline method showed the population of Manila clams has undergone population expansion at the 12,500 years ago during the long evolutionary process, then the population turn to be stable after 11,000 years. In the future, a more precise study on the geographic population expansion of the Manila clams could be made through increasing the number of individuals from each geographic population or adding the data of simplified genome and genome re-sequencing.Supplementary Information.


Chlorops oryzae is an important pest of rice crops. There have been frequent outbreaks of this pest in recent years and it has become the main rice pest in some regions. To elucidate the molecular mechanism of frequent C. oryzae outbreaks, we estimated the genetic diversity and genetic differentiation of 20 geographical populations based on a dataset of ISSR markers and COI sequences.I), Nei’s gene diversity (H), and the percentage of polymorphic bands (PPB). The mean coefficient of gene differentiation (Gst) was 0.0997, which indicates that only 9.97% genetic variation is between populations. The estimated gene flow (Nm) value was 4.5165, indicating a high level of gene flow and low, or medium, genetic differentiation among some populations. The results of a Mantel test revealed no significant correlation between genetic and geographic distance among populations, which means there is no evidence of significant genetic isolation by distance. An UPGMA (unweighted pair-group method with arithmetic averages) dendrogram based on genetic identity, did not indicate any major geographic structure for the 20 populations examined. mtDNA COI data indicates low nucleotide (0.0007) and haplotype diversity (0.36) in all populations. Fst values suggest that the 20 populations have low, or medium, levels of genetic differentiation. And the topology of a Neighbor-Joining tree suggests that there are no independent groups among the populations examined.ISSR data revealed a high level of genetic diversity among the 20 populations as measured by Shannon’s information index (C. oryzae populations have high genetic diversity at the species level. There is evidence of frequent gene flow and low, or medium, levels of genetic differentiation among some populations. There is no significant correlation between genetic and geographic distance among C. oryzae populations, and therefore no significant isolation by distance. All results are consistent with frequent gene exchange between populations, which could increase the genetic diversity, and hence, adaptability of C. oryzae, thereby promoting frequent outbreaks of this pest. Such knowledge may provide a scientific basis for predicting future outbreaks.Our results suggest that  Chlorops oryzae  is an important pest of paddy rice plants, which inflicts significant economic damage to rice crops throughout Asia. However, the dispersal ability, potential for long-distance dispersal, pattern of migration, ecological amplitude, and population size of C. oryzae are still uncertain. Most research on this species has focused on the physiology and ecology [ ecology –3, and i ecology .C. oryzae outbreaks in recent years have caused the species to become a major pest in some regions. The propensity for outbreaks may itself play an important role in homogenizing genetic variation and intensifying gene flow between pest populations [C. oryzae are associated with the species’ genetic diversity, population demography and high rate of gene flow between populations.Frequent ulations , 6. We hC. oryzae and the level of gene flow between them. We quantified the genetic diversity and degrees of genetic differentiation of 20 different geographical populations, which may provide a scientific basis for predicting future outbreaks.To test this hypothesis, we evaluated the genetic structure of different geographic populations of Bactrocera dorsalis (Hendel) on six offshore islands in South China, which results indicated that these populations have high genetic diversity, frequent gene flow and low, or medium, levels of genetic differentiation. Thus, the geographic isolation of the six islands is no barrier to the dispersal of B. dorsalis [Leucinodes orbonalis collected from a variety of agro-climatic conditions found almost no genetic diversity and no significant genetic variation among the mitochondrial gene Cytochrome Oxidase I (COI) gene sequences of the populations examined. However, a few genetically distinct populations were associated with some specific habitat requirements [COI gene among Iranian populations of Hishimonus phycitis may have been induced by geographical and ecological isolation and may have an impact on the vectoring capability of this insect [We used two effective and promising DNA markers, mitochondrial DNA (mtDNA) and inter-simple sequence repeat (ISSR), to examine between-population differences. Studies of genetic variation between pest populations can not only provide information on their population structure in different geographical regions, but also deduce the demographic history of this species –9. Yi etdorsalis . Researcirements . Similars insect .C. oryzae, but also provide a potential scientific basis for monitoring and controlling this pest.Our results not only provide information on the genetic structure and phylogeography research of COI gene sequence, which defined 47 haplotypes. All 47 haplotypes had 43 variable sites, including 26 singleton variable sites and 17 parsimony informative sites. The mean total nucleotide frequencies of A, T, C and G in the nucleotide sequences from the 20 different populations were 29.98%, 36.56%, 16.95% and 16.51%, respectively, which shows an obvious AT bias (66.54%). The transition/transversion rate ratio was observed to be higher with purines (36.158) than pyrimidines (19.381). The overall transition/transversion bias was 12.022.In total, 432 individuals collected from different locations were used to amplify 684 bp of the Hd) for each population ranged from 0 to 0.71739 and the average number of differences (k) ranged from 0 to 1.35145. Nucleotide diversity (Π) for each population ranged from 0 to 0.00198. Tajima’s D and Fu’s Fs test of neutrality of 19 populations showed a negative value. When all samples were calculated as one population, Tajima’s D and Fu’s Fs values were negative and 1‰ significant, which is strong evidence of population expansion.Genetic diversity parameters of the 20 populations and the results of neutrality tests are shown in Table C. oryzae populations were 0.00012–0.00184 and 0–0.00198  and 1.6460 ± 0.2373 (1.4992 ± 0.3270 to 1.6908 ± 0.2784 among populations), respectively. The H and I were 0.3785 ± 0.0984 (0.2992 ± 0.1588 to 0.3893 ± 0.1196) and 0.5609 ± 0.1151 (0.4547 ± 0.2091 to 0.5692 ± 0.1457), respectively, within all samples  plays a crucial role in genetic differentiation, affects the overall adaption of entire species and adaptative divergence between populations [Rhagoletis cerasi populations (which occupy different habitates in fragmented landscapes) to local habitates, thus preventing them from becoming extinct due to genetic processes [l change . Moreovel change . In thisulations . It has ulations , 30, andulations , 32. Forrocesses , 34.Fst values indicates that most of the genetic variation was resulted from the difference within populations. Furthermore, a Mantel test indicates no significant correlation between genetic and geographic distance. This result is consistent with the findings of Yang et al. [Odontotermes formosanus in different regions. These authors found no significant correlation between geographic distance and genetic distance and no significant isolation by distance. Overall, we found a high level of genetic diversity and a low degree of population differentiation among populations of C. oryzae, and the gene flow was unaffected by geographic distance. Similarly, geographic distance did not appear to affect gene flow between 10 geographically separated populations Oedaleus infernalis [Fst and Gst values for these populations are low, and the gene flow is high, indicating a low level of genetic differentiation and high gene flow among populations [An AMOVA based on g et al.  who compfernalis . Fst andulations . The corulations .C. oryzae, and suggest that high gene flow between populations contributes to the now frequent outbreaks of this pest. However, further research on both additional geographical populations and different genetic markers are necessary before definitive conclusions can be reached. Furthermore, future work can focus on doing a more comprehensive ecological and behavioural research to understand the natural history of C. oryzae in greater detail.Our results provide important, new information on the genetic diversity and genetic differentiation of C. oryzae may due to high gene flow between populations. We have found that these populations have high genetic diversity at the species level, whereas exhibited low genetic differentiation. High genetic diversity and frequent gene flow between populations may enhance the tolerance of populations to environmental variability and increase the adaptability to novel environmental pressures, leading to frequent outbreaks what had happened and what will happen in a large scale.This study showed that the now frequent outbreaks of C. oryzae were collected from different parts of Hunan province, China, and an additional 32 specimens from Zhejiang and Guizhou provinces  and COIR (5′-GGCTAAAACAACTCCTGTTAATCC-3′) primers from isolated DNA. PCR was performed in 20 μL volumes comprised of 10 μL PrimeSTAR Max DNA Polymerase , 1 μL of each primer (10 mmol/L), 1 μL of template DNA solution (70 ng/μL), and 7 μL double distilled water. Amplifications were conducted as follows: 34 cycles of denaturation at 94 °C for 30 s, annealing at 59 °C for 30 s, and extension at 72 °C for 1 min. All the PCR products were checked by electrophoresis on a 1.2% agarose gel and bidirectional sequencing was completed by TSINGKE .The A total of sixteen primers from the University of British Columbia Biotechnology Laboratory Primer kit No.9 were tested for PCR and nine , effective number of alleles (Ne), Nei’s gene diversity (H), Shannon’s information index (I), the percentage of polymorphic bands (PPB), total gene diversity (Ht), genetic diversity within populations (Hs), coefficient of gene differentiation (Gst), and Gene flow (Nm) were calculated using POPOGENE v.1.31 [C. oryzae populations, the geographic distances between them were calculated using the google-maps-distance-calculator   or absent (0) according to the molecular weight (bp) and the resulting matrix of binary values was used for further analyses. The observed number of alleles (E v.1.31 . ClusterE v.1.31 . To esti or absenAdditional file 1. Additional tables.


Strongyllodes variegatus (Fairmaire) is a major insect pest of oilseed rape in China. Despite its economic importance, the contribution of its population genetics in the development of any suitable protection control strategy for the management of oilseed rape crops is poorly studied. It is a much urgent need to prevent its spread to the rest of the world.c oxidase subunit I (COI) and cytochrome b (Cytb) as genetic markers, we analyzed the population genetic diversity and structure of 437 individuals collected from 15 S. variegatus populations located in different oilseed rape production areas in China. In addition, we estimated the demographic history using neutrality test and mismatch distribution analysis. The high level of genetic diversity was detected among the COI and Cytb sequences of S. variegatus. The population structure analyses strongly suggested three distinct genetic and geographical regions in China with limited gene flow. The Mantel test showed that the genetic distance was greatly influenced by the geographical distance. The demographic analyses showed that S. variegatus had experienced population fluctuation during the Pleistocene Epoch, which was likely to be related to the climatic changes.Using the sequences of mitochondrial DNA cytochrome S. variegatus populations in China, which is attributed by the isolation through the geographical distance among populations, their weak flight capacity and subsequent adaptation to the regional ecological conditions.Overall, these results demonstrate that the strong genetic structure of  Strongyllodes variegatus (Fairmaire) (Coleoptera: Nitidulidae), feeds on brassicaceous plant species [Meligethes aeneus [S. variegatus adults chew up flowers, buds and leaves, and create crescent-shaped bites where the mature females lay eggs. After hatching, the larvae feed on mesophyll resulting in irregular bubble-shaped wounds before pupation in soil. The wounded leaves become necrotic and abscise prematurely [S. variegatus population broke out in Hanshan, Anhui province, destructing 97% of oilseed rape leaves [The brown beetle,  species , 2, whics aeneus . The S. maturely , 5. Recee leaves .S. variegatus displays specific ecological characteristics to temperature and photoperiod of geographical regions. In spring oilseed rape areas, it reproduces once or twice a year [S. variegatus has a high reproductive ability [e a year . Howevere a year . In addi ability  and can  ability . In Anhu ability , 6.S. variegatus is generally distributed in the middle and lower reaches of the Yangtze River valley. It was first found on the spring oilseed rape plants in Ningxia, Gansu province, China in 1993 [S. variegatus have not been studied. Consequently, it is in urgent demand to conduct the genetics studies and understand the genetic diversity and structure of S. variegatus populations in order to manage and control this pest. in 1993 , and the in 1993 . For thec oxidase subunit I (COI) and cytochrome b (Cytb) have been widely used among insect molecular markers to study population genetic variation and differentiation of insects, for example, Dendrolimus kikuchii, Chilo suppressalis and Agriosphodrus dohrni [COI and Cytb genes were also used to track the colonization routes of Halyomorpha halys and to identify the places where the insect has originated [Population genetic studies on crop pests can provide information on the spatial scales at which population structure is established and gene flow occurs. Such information can be used in defining relevant strategies for pest control . In addis dohrni –15. The iginated –18.COI and Cytb genes to elucidate for the first time the genetic diversity and structure of 15 S. variegatus populations occurring on the oilseed rape production areas in China. We hypothesize that the populations would have a high level of genetic diversity and a clear genetic structure. At the same time, the efficient molecular data collected are used to assess if historical geographic events and associated ecological adaptations had played an important part in shaping the observed genetic and geographic patterns of this pest in China.In this study, we use COI gene and 67 haplotypes of the Cytb gene were identified from the 15 populations. The S. variegatus COI fragment (652 bp) and Cytb fragment (421 bp) have 45 (6.9%) and 40 (9.5%) variable sites with 28 and 23 parsimony informative sites, respectively (Table Hd) ranges from 0.424 to 0.913 (mean = 0.865) and the nucleotide diversity (π) ranges from 0.00072 to 0.00462 (mean = 0.00427) for the COI gene (Table Hd ranges from 0.464 to 0.833 (mean = 0.834) and π ranges from 0.00119 to 0.00539 (mean = 0.00479) for the Cytb gene (Table Seventy haplotypes of the COI haplotypes (H1-H70) included 34 (48.6%) unique haplotypes  had 35 (52.2%) unique haplotypes, among which 32 were observed in more than one individual  haplogroup , the central China (CC) haplogroup  and the central and eastern China (CE) haplogroup .The haplotype distribution and haplotype network analyses (see below) of both COI gene, there was only one common haplotype (H1) in three haplogroups. The haplotype 2 (H2) was only detected and abundant in the CC haplogroup. The haplotype 3 (H3) was only discovered in the CE haplogroup. There were six common haplotypes (H4-H9) between the NW haplogroup and the CC haplogroup. A total of five missing haplotypes was observed in all populations  in three haplogroups. The haplotype 2 (H2) was most abundant and only detected in the CC haplogroup. The haplotype 3 (H3) was only discovered in the CE haplogroup. The haplotypes 5–6, 7, 8–9  were common in the NW and the CC haplogroups, the NW and the CE haplogroup, the CC and the CE haplogroup, respectively. A total of four missing haplotypes was observed in the CC haplogroup  was highly significant  were significantly higher than those of the comparisons between the regions  ) and the geographical distances among all populations , a rough estimate of the population expansion, were approximately 3.842 (COI data), 2.016 (Cytb data), and 1.595 (COI + Cytb data) mutation units for the CC region. For the NW and CE regions, τ was 1.344 and 0.766 in the data of the COI gene, 3.693 and 0.875 in the data of the Cytb gene, and 2.628 and 1.875 in the combined data of the COI and Cytb genes  region, the central China (CC) region, and the central and eastern China (CE) region. A high proportion of total genetic variance is attributed to the variations within the populations (49.18%) and among the regions (47.01%). This indicates that the largest source of variation might not be due to the geographical barriers among the regions but to the variations among individuals within the populations. It was reported previously that the variations among individuals within the populations had a significant effect on the genetic structure of Chilo suppressalis [Myotis myotis and Plecotus austriacus [Cydia pomonella populations in Switzerland and found that host specificity, geographic isolation, intrinsic flight capacity and anthropogenic measures could all shape the population structure [Based on the analyses of the mtDNA sequences, haplotype distribution, haplotype networks and AMOVA, three genetically diverse and geographically distinct regions of ressalis . This costriacus , 21, whitructure .Nm < 1) was revealed among the regions by the current study. It is known that once populations have become genetically differentiated, their genetic divergence status can be maintained if they have differentially adapted to regional ecological conditions, since geographic variation in selection can act as a strong barrier to gene flow [S. variegatus. It was reported that S. variegatus can fly 30 ~ 40 m in 2 min [S. variegatus is less than tens of kilometres and would not be enough to weaken the isolation-by-distance relationships and to increase the potential for allopatric or parapatric speciation [A limited gene flow  at specific points during the year and low-altitude flights at multiple periods [Plutella xylostella [Gene flow in insects has been reported to increase with mobility, which is more pronounced on herbaceous plants, and this feature is strong especially in agricultural pests . The larape pest , 27–29. ape pest . M. aene periods , resultilostella .COI and Cytb haplotype networks are roughly composed of three “star-like” clusters. Based on 2.3% per site per million years [COI gene and Cytb gene can be estimated to be 104 and 128 ka years ago, respectively, within the interglacial time of the Pleistocene. Vast glaciers developed at that time in Tibetan Plateau, Qinling Mountain and even in the Yangtze River valley [Both the neutrality test and the mismatch distribution analysis indicated a population expansion in the CC region. Furthermore, the phylogeographic patterns of the on years , the expr valley , 34, whir valley –37.S. variegatus have primarily focused on using chemicals. The investigation of the genetic diversity of S. variegatus populations can provide a useful guide for controlling this pest. Furthermore, localized populations with similar genetic structure should be considered as a same management unit for most effective control [S. variegatus in China, and in the rest of the world if the pest would occur in future.In China, the management practices against  control . For isoS. variegatus, a serious pest of oilseed rape crops. The high variability observed using the COI and Cytb molecular markers indicates that the markers are useful for measuring the genetic patterns in S. variegatus populations. The distinct distribution of S. variegatus populations in China could be divided into three genetic haplogroups and geographical regions with the limited gene flow among them. The distribution of this species in oilseed rape production areas in China is mainly structured by the isolation through geographical distance among the populations and their weak flight capacity. The population expansion signature in the CC region might be related to the climatic changes during the Pleistocene. The phylogenetic information obtained from this study could be used to guide the development of suitable protection control strategies against the insect pests of oilseed rape crops.The current study provides the first population genetic analysis of S. variegatus individuals was collected from 15 populations in China . The primers used were LCO-1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and HCO-2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) for the regions of the COI gene and CB1 (5′-TATGTACTACCATGAGGACAAATATC-3′) and CB2 (5′-ATTACACCTCCTAATTTATTAGGAAT-3′) the regions of the Cytb gene in the polymerase chain reactions (PCR) amplification [Total genomic DNA was extracted from each fication .2O, and l μL of template DNA. The procedure for the PCR amplification was 4 min at 94℃, 35 cycles of 30 s at 94℃, 30 s at 48℃, and 1 min at 72℃, and a final extension for 10 min at 72℃. The reaction mixture without DNA template was included as negative control for each set of PCRs.The PCR amplification was performed using Applied Biosystems ABI 3730  in a 25 μL reaction mixture containing 12.5 μL of 2 × Taq PCR Master Mix (BBI), 1 μL of 10 μM forward primer, 1 μL of 10 μM reverse primer, 9.5 μL of ddHThe PCR products were subjected to electrophoresis on a 1.5% agarose gel  containing 10,000 × stock GelRed (Biotium) diluted at 1:10,000, visualized on a BioDoc-it imaging system (UVP), purified from the gel using ExoSAP-IT , and bidirectionally sequenced (using the above primers) on an ABI 3730XL Automated Sequencer using the BigDye Terminator Cycle Sequencing 3.1 Ready Reaction Kit .S), parsimony informative sites, number of haplotypes (Hn), percentage (%) of haplotypes unique to a given geographical area, haplotype diversity (Hd), nucleotide diversity (π), and average number of nucleotide differences (k). To estimate the haplotype completeness a Coleman rarefaction curve was calculated with haploAccum of the spider package implemented in R software [The forward and reverse sequences were assembled, aligned using ClustalW algorithm . The obtsoftware . The Temsoftware , 45.FCT), between the populations within the regions (FSC), and between all populations (FST). The pairwise FST analyses among the populations and the regions were carried out with significance tests based on 1,000 permutations using Arlequin3.5 [FST/(1 − FST) and the geographic distance (ln) between all 15 populations were compared using the Mantel test with 10,000 permutations [The population genetic structure was assessed with AMOVA in Arlequin3.5 according to the degree of differentiation between the regions  and the degree of fit between the observed and simulated data  [p-values for the SSD. The time of expansion was evaluated with the formula τ = 2μkt [The historical demographic expansion was examined with Tajima's ribution –52, as iribution . Tajima'on, SSD) , 54. TheAdditional file 1: Table S1 Geographical distribution of (A) COI and (B) Cytb haplotypes of Strongyllodes variegatus . Table S2 Sample information of Strongyllodes variegatus (Fairmaire) specimens collected for the present studyAdditional file 2: Figure S1 Individual-based rarefaction curves of haplotype diversity of S variegatus of in China.Additional file 3: Figure S2 Pairwise mismatch distributions of (a) COI and (b) Cytb genes for three derived regions. The x coordinate represents the number of pairwise differences among sequences, and the y coordinate represents the frequencies of pairwise differences in each region. The significance values (p) of the parameters were evaluated with 1,000 simulations; PSSD: P value for SSD (sum of squared deviations) PR: P value for Rag (Harpending’s raggedness index); τ: the index of population expansion.


Ovis aries) is not well understood, and little is known about this species’ genetic diversity. The aim of the present research was to explore phylogeography and phylogenetic evolution of Tibetan sheep populations, on the basis of mitochondrial DNA (mtDNA) gene MT-CYB (1140 base pairs). The results of this study will add information to the Tibetan sheep populations and help enlighten upcoming programs related to conservation of Tibetan sheep living in the Qinghai–Tibetan Plateau.The molecular and population genetic evidence of the phylogenetic status of the Tibetan sheep (Ovis aries) have been limited, and little is known about the phylogenetic evolution and phylogeography of Tibetan sheep populations. The aim of the present research was to explore phylogeography and phylogenetic evolution of Tibetan sheep populations, on the basis of mitochondrial DNA (mtDNA) gene MT-CYB (1140 base pairs). Our dataset consisted of 641 MT-CYB sequences from the same amount of animals belonging to 15 populations of Tibetan sheep living in the Qinghai–Tibetan Plateau, China. Haplotype and nucleotide diversities were 0.748 ± 0.010 and 0.003 ± 0.001, respectively. The analysis of phylogeography revealed the presence of two formerly described haplogroups in 15 populations of Tibetan sheep, however only one haplogroup was present in Awang sheep. Moreover, 641 Tibetan sheep were distributed into a minimum of two clusters by clustering analysis. The 15 Tibetan sheep populations and 19 reference populations of 878 individuals were separated into six main groups based on their substitutions per site, from which we constructed a phylogenetic tree. Minor differences in branching order of various taxa between trees acquired from either gene were observed. This study provides insights on the origins and phylogenetic evolution of populations residing in the Qinghai–Tibetan Plateau, which will aid information of future conservation programs aimed at conserving this valuable genetic resource.To date, molecular genetics and population studies in Tibetan sheep ( In the Qinghai–Tibetan Plateau, Tibetan sheep have economic, agricultural, cultural, and also religious significance, providing wool, fur and meat to the local people . The QinMT-CYB gene is one of the genes that is coded by mtDNA, and its gene product plays a key role in the transfer of electrons through the respiration chain [MT-CYB gene has a moderate mutation rate (16.1%) of bases and a clear evolutionary pattern making it well suited for phylogenetic investigations at intra-and inter-specific levels [MT-CYB gene in modern groups of sheep with vast geographical origins, has described diverse patterns of genetic diversity in domestic sheep based on five haplogroups [The on chain . The MT-c levels . Currentc levels . Researclogroups ,12. Genelogroups , howeverlogroups ,15. Haplion rate .1% of baMT-CYB gene variability in Tibetan sheep indigenous to the Qinghai–Tibetan Plateau, China. A total of 15 Tibetan sheep populations were included in population genetics and phylogenetic analyses of the MT-CYB gene. By providing insights into the genetic diversity, maternal pedigree and phylogenetic evolution of Tibetan sheep, our results are relevant to future efforts for conservation and improved management of their genetic diversity.The present study investigated All experimental procedures and protocols were approved by the Institutional Animal Care and Use Committee of the Lanzhou Institute of Husbandry and Pharmaceutical Science of the Chinese Academy of Agricultural Sciences . Blood samples from 641 Tibetan sheep were collected. These animals belonged to 15 Tibetan sheep populations that are distributed through Qinghai Province, Gansu Province, and the Tibet Autonomous Region. The genetic characteristics of these Tibetan sheep populations were analyzed in order to determine the phylogeny and phylogeography of the populations. The examined populations were comprised of the following numbers and analogous population types: 8 Duoma Tibetan sheep (DM), 5 Awang Tibetan sheep (AW), 33 Huoba Tibetan sheep (HB), 10 Langkazi Tibetan sheep (LKZ), 77 Gangba Tibetan sheep (GB), 60 Zashijia Tibetan sheep (ZSJ), 46 Jiangzi Tibetan sheep (JZ), 43 Qinghai Oula sheep (QH), 46 Qilian White Tibetan sheep (QL), 64 Tianjun White Tibetan sheep (TJ), 38 Guide Black Fur sheep (GD), 43 Gannan Oula sheep (GN), 62 Qiaoke Tibetan sheep (QK), 58 Minxian Black Fur sheep (MX), and 48 Ganjia Tibetan sheep (GJ) raised in China. Ten milliliter blood samples via the jugular vein were collected from each animal, and 2 mL from each sample were snap frozen in liquid nitrogen and stored at −80 °C for later genomic DNA extraction . Total DMT-CYB for the 234 individuals in GenBank. These 19 populations were from six global geographic regions i.e., Turkey, India, Italy, Pakistan, Germany, and China , 3 Henan Big Tail sheep (HN), 4 Akkaraman sheep (AKA), 5 Cypriot mouflon (CM), 12 Madgyal sheep (MD), 12 Morkaraman sheep (MOR), 19 Bannur sheep (BN), 21 Garole sheep (GA), 24 Kolhapuri sheep (KO), 29 Deccani sheep (DE), 49 Bulkhi sheep (BU), 27 Sangamneri sheep (SN), 5 Snow sheep (SS), 8 Tuj sheep (TUJ), 11 Argali, and 2 Musimon nd China . MT-CYB gene via Primer Premier 5.0 software [2O (ddH2O). The PCR conditions were as follows: initial denaturation at 94 °C for 5 min, 36 cycles of denaturation for 30 s at 94 °C, annealing for 30 s at 56 °C, and extension for 1.5 min at 72 °C. A final 10-min extension was performed at 72 °C. The amplified products were subsequently stored at 12 °C until use.Primer pairs used for polymerase chain reaction (PCR) and primers for sequencing were designed on the basis of 5′and 3′ conserved flanking sequences of the complete software , and synMT-CYB gene were purified using a PCR gel extraction kit from Sangon Biotech Co., Ltd.  and directly sequenced using BigDye® Terminator v3.1 cycle sequencing ready reaction mix  in an automatic sequencer  and sequenced fragments of the MT-CYB gene were assembled by MITObin software (V 1.7) [2O. The sequencing conditions were as follows: initial denaturation at 95 °C for 2 min, 25 cycles of denaturation for 10 s at 95 °C, and annealing for 10 s at 51 °C. The final extension step was followed by a 190 s extension at 60 °C. The PCR sequencing products were subsequently stored at 12 °C until use.The amplified fragments of the  (V 1.7) . PCR forSTG), and Wright’s subpopulation within total population F-statistic (STF), were assessed using Arlequin version 3.5.1.2 [The sequences were organized using Clustal Omega tool for multiple comparison and were aligned using BLAST and ClustalW 1.2.0 ,22. Thes 3.5.1.2 . The molecular variance (AMOVA) test was estimated using Arlequin version 3.5.1.2 . Four grAll the sequences were aligned with 1140 comparative site and 132 haplotypes were attained from 641 sequenced animals. Sequences from 641 animals (from 15 Tibetan populations) showed considerable length heteroplasmy (the most abundant of which was 1140 base pairs). In total, 157 variable sites were attained from sequences that included 71 singleton variable sites and 86 parsimony informative variable sites. Out of 157 variable sites, 127 were transitions and 30 were trans-versions, among which 15 sites were both transitions and trans-versions. The observed substitutions caused the transition mutations.Whole haplotypes’ nucleotide compositions were: 31.472% A, 27.174% T, 28.441% C, and 12.913% G, 58.646% A + T and 41.354% G + C, while A + T were substantially more common than the G + C haplotype, displaying an AT bias. The ME phylogenetic tree shows that the 132 haplotypes of the 15 Tibetan sheep populations fall into two primarily distinct clusters: haplogroup A and haplogroup B . The maiData are shown in STF to study the genetic differentiation among 15 populations of Tibetan sheep. Estimates for pair-wise STF values , calculated to study the genetic differentiation among the target Tibetan sheep populations, were from 0 to 0.118, p-value from 0 to 0.072, respectively . Moreover, all STF values were less than 0.15 (range: 0 to 0.118), showing that significant genetic differentiation has little differentiation among populations. STF values  was recorded  (coefficient of determination R2 of 0.0703 showed in p < 0.05 shown by *) and decreases for the five populations i.e., GB, JZ, LKZ, HB and DM. We assessed if genetic differentiation between populations can be clarified by altitude. The genetic differentiation between the main population MX and each of the remaining populations were plotted in p = 0.0011.This relationship between genetic differentiation and absolute difference between altitudes is more positive for low altitude populations and more negative for high altitude populations. This relationship between altitude and Pearson correlation coefficients attained between genetic differentiations and absolute differences in altitudes  has r = MT-CYB gene sequences of 875 animals from 15 populations of Tibetan sheep and 19 reference populations  based on complete ulations . The neip < 0.001) .The ME phylogenetic tree shows that the 132 haplotypes of Tibetan sheep populations fall into two primarily distinct clusters: haplogroup A and haplogroup B . Haplogr< 0.001) . The FSTMT-CYB) is shown in The mismatch distribution analysis of the complete dataset  show higher genetic diversity for 15 populations of Tibetan sheep in the Qinghai–Tibetan Plateau . The haplotype diversity was calculated separately for each Tibetan sheep population and was estimated to be 0.992 ± 0.010, which was based on mtDNA D-Loop sequences with 15 Tibetan sheep populations of 636 individuals [Our findings show comparatively greater genetic diversity in 15 populations of Tibetan sheep in comparison to other sheep populations ,31. Mostividuals . We therMT-CYB diversity [MT-CYB diversity are an increased mutation rate (for this gene), mingling of populations from various geographical locations, overlapping generations, natural selection choosing heterozygosis, maternal effects of numerous wild ancestors and subdivision complemented by genetic drift [The Tibetan sheep populations (which have been characterized as the rarest) encountered a genetic bottleneck during the 20th century . Consistiversity . Among fic drift .MT-CYB (1140 base pairs) revealed that 15 Tibetan sheep populations cluster into our sub-clusters. The rich mtDNA diversity in Tibetan sheep populations suggests a non-widespread origin for maternal lineages [Sequencing data of lineages ,35. MoreThe origins of the domestic sheep have been subject to debate. On the basis of studies on the D-loop region of mtDNA in domestic sheep, Hiendleder et al. identified that two maternal lineages are apparent . NJ phylMT-CYB has been used to study phylogenetic relationships at intra-and inter-specific levels, and has been used in gene flow studies [STF characterizes genetic differentiation levels inside a certain population. Differentiation is considered “low” with a value of 0.05, “moderate” with values from 0.05 to 0.25, and “substantial” with values > 0.25 [mN) , refers to the transfer of alleles from one population to another. mN values for haplotype > 1 and for sequence < 1 indicate poor gene exchange, resulting in a situation whereby genetic drift will cause substantial differentiation in local populations [STF values indicate that genetic variations mainly resulted from independent evolution of the isolated populations, with substantial differentiation in local populations caused by genetic drift [ studies ,40. It is > 0.25 . The preulations ,42. In tic drift . Toward ic drift . Thus, aThe present study exhibited that 15 populations of Tibetan sheep belonging to the Qinghai–Tibetan Plateau, China are grouped into two haplogroups: 560 animals belonged to haplogroup A, and 81 to haplogroup B. These genetic groupings are highly consistent with conventional schemes of classification and the findings of other investigations ,13. GeneMT-CYB from the whole mitogenomes created similar phylogenies with completely resolved phylogenetic relationships of wild sheep, however, they were unable to describe phylogenetic relationships among main lineages of domestic sheep. Our findings propose that partial fragments of whole mitogenomes would be troublesome in constructing phylogenetic inferences related to domestic sheep. This issue appears because of the location of diagnostic substitutions elsewhere in the mitogenome [A greater resolution among wild sheep and the main lineages of domestic sheep was displayed by phylogenetic analyses of whole mitogenomes . MT-CYB togenome . So, diatogenome  in this MT-CYB in 15 populations of Tibetan sheep, provides proof of two maternal lineages with greater genetic diversity. Analysis of phylogeny exhibited that two formerly described maternal lineages could be recognized in 641 tested animals of 15 populations of Tibetan sheep. The origin of the maternal line may be Musimon and Argali.Tibetan sheep populations are abundant in China, and there is substantial mtDNA genetic diversity in sheep residing in the Qinghai–Tibetan Plateau. The present study, based on large-scale analysis of the mtDNA


In order to assess the genomic landscape of the United Arab Emirates (UAE) mitogenome, we sequenced and analyzed the complete genomes of 232 Emirate females mitochondrial DNA (mtDNA) within and compared those to Africa. We investigated the prevalence of haplogroups, genetic variation, heteroplasmy, and demography among the UAE native population with diverse ethnicity and relatively high degree of consanguinity. We identified 968 mtDNA variants and high-resolution 15 haplogroups. Our results show that the UAE population received enough gene flow from Africa represented by the haplogroups L, U6, and M1, and that 16.8% of the population has an eastern provenance, depicted by the U haplogroup and the M Indian haplogroup (12%), whereas western Eurasian and Asian haplogroups  represent 11 to 15%. Interestingly, we found an ancient migration present through the descendant of L (N1 and X) and other sub-haplogroups (L2a1d and L4) and (L3x1b), which is one of the oldest evolutionary histories outside of Africa. Our demographic analysis shows no population structure among populations, with low diversity and no population differentiation. In addition, we show that the transmission of mtDNA in the UAE population is under purifying selection with hints of diversifying selection on ATP8 gene. Last, our results show a population bottleneck, which coincides with the Western European contact (1400 ybp). Our study of the UAE mitogenomes suggest that several maternal lineage migratory episodes liking African–Asian corridors occurred since the first modern human emerges out of Africa. There are two potential routes to the Arabian Peninsula, the northern and southern route, which is the first step out of Africa. This is the primary link between Africa and Eurasia. The maternally inherited mtDNA has been used as a marker to relate lineages across geographic origins culminating in African haplogroup L and the Eurasian M and N, which shared a common route with the African L3, and this radiation likely started the Eurasian colonization ,2,3. FurDemographic history, such as effective population size changes, short and long distance migrations, as well as admixture, shape the genetic variation of modern African populations. This is in addition to selection on specific loci, combined with recombination and mutation. For instance, one of the migrations that impacts the genetic variation in modern African populations is the migration of agricultural Bantu speaking from West Africa throughout sub-Saharan Africa 4000 years ago followed by admixture with indigenous populations ,16,17,18Almost in all cases, nuclear DNA is used to describe the signature of selection, while ignoring its effect on the tissue, cells, and subcellular compartments. This is very crucial for mitogenome, which in contrast to the nucleus, serves as a powerhouse for the cell and are present in multiple copies in the cytoplasm that may vary in sequence (heteroplasmy) and quantity among tissues . Each miThe human mitogenome is highly polymorphic, and most of its variants are benign . HoweverIn this study, we analyzed the mitogenome landscape of the UAE population. The UAE is located in Western Asia on the Gulf, south of the Strait of Hormuz at the southeast end of the Arabian Peninsula. It borders Oman (east) and Saudi Arabia (south) and shares sea borders with Qatar (west) and Iran (north). The country is a federation of seven emirates: Abu Dhabi , Dubai, Sharjah, Ajman, Umm Al-Quwain (UAQ), Ras Al-Khaimah (RAK), and Fujairah. Approximately 13% of the population  is Emirati citizens. Al Ain, the site of the current study, is the largest inland city in UAE and is part of the emirate of Abu Dhabi. It is located east of the capital and south of Dubai and has the highest proportion of Emirati nationals (~20%). Citizens of the UAE have diverse ethnicity that includes links to the Arabian Peninsula, Persia, Baluchistan, and East Africa. The society is mainly tribal, and intratribal marriages are fairly practiced. Consequently, founder variants and prevalence of inborn errors of metabolism and genetic disorders are exceptionally high ,38,39.We carried out complete whole mitogenome sequencing, assembly, and annotation of 232 female UAE citizens and highlighted the genetic population grouping (haplogrouping). We characterized variants and their effect and test for selective force acting on the UAE mitogenome. We estimated diversity, population structure, and differentiation among cities and regions. In addition, to characterize variants, we estimated heteroplasmy in the population. Finally, we used mtDNA to construct a portrait of the Holocene and late Pleistocene population size of the UAE population.Al Ain Medical Human Research Ethics Committee approved this study according to the national regulations (#10/09). This study recruited national UAE female students (age range: 18 to 24 years) matriculated at the UAE University. The UAE University is a federal institution and students’ diversity represents all of the seven Emirates. Venous blood samples (5 mL in EDTA-Vacutainers) were collected randomly from 248 consented female students. Total genomic DNA was extracted from blood lymphocytes using the DNeasyBlood and Tissue Kit . DNA quality and concentration were confirmed with NanoDrop and agarose gel, and the samples were stored at −20 °C. We used already published 70 genomes from East Africa in our analysis (accession numbers JN655773–JN655842) for comparative analysis.Mitogenome enrichment is a critical step to reduce nuclear DNA contamination and was accomplished using a specific long-range PCR amplification step directed by two sets of overlapping primers, where each pair of primers flanked 8500 base pairs (bp) . Two PCRp 20 –sensitive -D 15 -R 2 -N 0 -L 22 -i S,1,1.15) [The quality of the raw Ion Torrent PGM fastq files was checked using FastQC. The Geneious software platform  was used,1,1.15) . Homopol,1,1.15) . Geneiou,1,1.15)  using pl,1,1.15) . The ann,1,1.15) .A maximum parsimony tree was generated using coding region sequences from the 232 female mitogenomes  separately, as well as combined with 70 African mitogenomes using MEGA6 . Branch n = 86), Abu Dhabi (n = 18), Dubai (n = 15), Sharjah (n = 15), RAK (n = 39), UAQ (n = 3), and Fujairah (n = 43). Furthermore, Fst was also measured based on the UAE’s geographical regions: Southwest , most-Northeast , most-Northwest , and mid-Northwest .We calculated pairwise population differentiation Fst with vcftools using haploid option. We also estimated Hs (Heterozygosity with structure) and Ht (Heterozygosity without structure) as well as Gst, G’st, and D statistic using R package adegenet . The samN/dS metric implemented in HyPhy using the method BUSTED.Coding sequences for the thirteen mitochondrial genes were codon-aligned in frame using pal2nal (version 14) for the UAE individuals. The number of polymorphic sites per population (S), nucleotide diversity (π), Watterson theta (θ), and Tajima’s D  were calTo reconstruct the demographic history in our samples, we used BEAST version 1.8.0 . The proHeteroplasmy identification was carried out using mtDNA-Server . The mtDn = 232) were deposited in NCBI-Genbank database (accession numbers: MF437054–MF437285), and generated NGS reads were deposited in NCBI-SRA database (SRA ID: PRJNA566159).Preprocessed NGS reads were aligned against the reference mitogenome using the Bowtie2 program. We identified 968 SNPs and 30 indels including 11 MNPs A. We fouFifteen haplogroups were identified in the 232 samples . A netwoHaplogroup R was the second most common haplogroup, found in 15.08% of samples. Sub-haplogroups of R  were identified in 35 samples. Haplogroup M accounted for 12.06% of the total; the generalized African subclade M1a  constituted 14% of the total haplogroup. Haplogroup K represented 11.20% of the total population. Haplogroup J was found in 25 samples (10.77%); sub-haplogroups J1b , J2a , and J2b (J2b1 and J2b1f) were detected in this study .Haplogroup L (Sub-Saharan Africa) accounted for 8.62% of the total mitogenome samples . We also identified one individual with L3x1b sub-haplogroups in the population. Sub-haplogroups L3x1b in L3 is one of the oldest evolutionary steps in the history of out of Africa, and it was previously reported in Kenya, Jordan, Yemen, Ethiopia, and Egypt C.North Africa haplogroup HV was detected in 4.31% of the total, while Western Asia (the Near East) haplogroup H was detected in 8.18% of the total. Haplogroup T constituted 6.03% of the total, a clade that emanated from the Near East and was common among Iranians. The remaining identified haplogroups  constituted approximately 7% of the total samples. The frequency of the different haplogroups per city is highlighted on the UAE map D. The frThe maximum likelihood phylogenetic tree of the different haplogroups colored by cities A shows tst varying from 0.009 to 0.07 as it is shown in  confirm that there is no structure and population differentiation among the different cities. The pairwise population differentiation (Fst) among cities shows no great differentiation with Fshown in  and the  boxplot A. We obsatistics   is higher in Africa compared to different cities in the UAE population. It is also higher in Abu Dhabi and Dubai compared to Alain, which is a province of Abu Dhabi. In addition, we observe the L3 haplogroup in Africa, and the UAE population (π = 0.004) has higher diversity compared to M and N haplogroup. It is also higher even when combined the L haplogroup together C. KinshiN/dS ratio > 1.Looking at the coding sequences of the 13 different mitochondrial genes, we observe higher Watterson theta (θ) compared to Pi (π), as well as a significant negative Tajima’s D . Only ATBEAST output was used to perform an extended Bayesian skyline plot (EBSP) analysis. We reconstructed the distinct demographic epochs, which highlights a significant and transient contraction in population size some 1400 years before the present . Giving His gene at position 12172 A->G, with no Mitomap annotation. This mutation is not at higher frequency (1.5%) in the population compared to the others. The RNA fold server was used to predict the change in the secondary structure of the wild type (A) versus the mutant (G). The results highlight a change in the structure in the mutant versus the wild type for only this mutation  of lineage affiliated with L haplogroups suggests an ancient ancestry in the region, which most likely dispersed through the Gulf corridor towards the Levant and Europe 24–55 Ka . AnotherThe presence of L1, L2, and L3, in our study, points to sub-Saharan mtDNA lineages. Sub-haplogroup L2a, for example, is associated with the Bantu expansion , which iHis reported previously [The mitochondrial genomic landscape of the UAE mitogenomes has 968 variants and the majority is in the D-loop and ND5 gene D. This ieviously  is assoceviously ,78.As it is expected, the mitogenome harbors more synonymous mutations than nonsynonymous mutations A, which p < 0.01) and N  analyzed separately, show low diversity  and significant deviation from neutrality  than UAE (0.0021 ± 0.0012) to (0.0033 + 0.00310). The lower diversity is consistent with the paucity of population differentiation and structure as shown in the Fst for the UAE population, which is also consistent with the genomic result from the UAE population that was recently published . The haputrality , which iThe reconstructed demographic history of the UAE mitogenome sheds light on a bottleneck event around 1400 years ago that coincides with western European contact . The easThis study describes the genomic landscape of the UAE mitochondrial genome and the distribution of haplogroups in different geographic regions in the UAE. The analyzed mitogenomes from 232 female students of UAE University, aged 18–24 years, highlights the high resolution of 15 different haplogroups that share ancestry with Africa, East Asia, and the Near East. Furthermore, it elucidates migration routes to the UAE. The low diversity and population differentiation highlight that the low movement between cities. The Demographic history highlights a bottleneck event that coincides with European contact 1400 ybp. In conclusion, this study also provides a matrilineal history of the UAE and will serve as an asset for genetic counseling, forensic science, and anthropology among other fields.


Cydia pomonella (L.) (Lepidoptera: Tortricidae), is a major pest introduced to almost all main pome fruit production regions worldwide. This species was detected in Chile during the last decade of the 19th century, and now has a widespread distribution in all major apple-growing regions. We performed an analysis of the genetic variability and structure of codling moth populations in Chile using five microsatellite markers. We sampled the codling moth along the main distribution area in Chile on all its main host-plant species. Low genetic differentiation among the population samples (FST = 0.03) was found, with only slight isolation by distance. According to a Bayesian assignment test (TESS), a group of localities in the coastal mountain range from the Bío-Bío Region formed a distinct genetic cluster. Our results also suggest that the codling moth that invaded the southernmost locality (Aysén Region) had two origins from central Chile and another unknown source. We did not find significant genetic differentiation between codling moth samples from different host-plant species. Our results indicate high genetic exchange among codling moth populations between the different Chilean regions and host plants.The codling moth,  Cydiapomonella (L.) (Lepidoptera: Tortricidae), is an invasive species with a widespread distribution in temperate areas with pome fruit cultivation worldwide [The codling moth, orldwide ,2. Origiorldwide .Study of the codling moth population genetic structure has received increasing attention in the last few decades , becauseThe spatial patterns of genetic diversity among codling moth populations may reflect its relatively recent global distribution , which wMalus domestica Borkdhausen), pears (Pyrus communis L.), quinces (Cydonia oblonga Mill.), and less frequently, apricots (Prunus armeniaca L.) and plums (Prunus domestica L.) [Juglans regia L.) as a host plant in the Juglandaceae [The codling moth is an oligophagous species that develops within the fruit of a few species of cultivated Rosaceae, such as apples (tica L.) . In addiandaceae . Singulaandaceae ,19 and oandaceae  Genetic andaceae ,21.FST values [Dominant molecular markers, such as AFLP and random amplified polymorphic DNA (RAPD), have found significant differentiation between populations of codling moth at the regional and local scales in South Africa , centralT values  and littT values , and in T values ,11. TopoT values . For insT values  or develT values , both afT values ,15,16,29The populations of the codling moth from the main apple producing regions in central Chile exhibit a low genetic structure ,31, but No information is available on the genetic structure of the codling moth populations among the main apple production regions, or among populations using different host plants in Chile. We expect that isolated codling moth populations from southern Chile, where no pome fruit industry exists, would show higher genetic differentiation than the populations from central Chile, where apple production for exportation is concentrated. Therefore, the main objective of this research was to characterize the population-level genetic structures of codling moth populations in Chile, all along their distribution area, which covers a latitudinal range of nearly 1400 km. Samples from all host-plant species of the codling moth in Chile were included , with the aim of evaluating whether the development of population differentiation between them was present.Diapausing larvae were collected using cardboard traps wrapped around tree trunks in 2008 and 2009 autumns; there were one to five individual trees per population sample. A total of 34 population samples were collected from 26 locations along a 1400 km transect in Chile . In addiCp1.60, Cp1.62, Cp2.129, Cp5.24, and Cp6.46 [2, one unit of Taq DNA polymerase, and 0.1 mg/mL BSA with 2 µL of DNA template. The forward primer for each pair was labeled with 5′-carboxyfluorescein (FAM) or 6-carboxy-1,4-dichloro-2′,4′,5′,7′-tetra-chlorofluorescein (HEX). PCR products were analyzed on an ABI3730  automatic DNA sequencer using the software GENEMAPPER®, version 4.1.DNA templates were extracted from diapausing larvae according to the salting out procedure . An abdod Cp6.46 , were exd Cp6.46 ,30. FurtAN), allelic richness (a), fixation index or inbreeding coefficient (FIS), and unbiased heterozygosity estimates —were computed with FSTAT version 2.9.3.2 [Na) was estimated using the FREENA program [Data were analyzed for possible stuttering, drop out, and writing mistakes with the MICRO-CHECKER software version 2.2.3 . Basic s 2.9.3.2 . Average program . Deviati program .FST values were tested based on 1000 permutations of the multilocus genotypes.Hierarchical partitions of the genetic variance (AMOVA) were performed using ARLEQUIN version 3.5.1.2  from theFST/1 − FST) and the logarithm of the geographic distances between the sample locations (Log km), and using Mantel’s test based on 1000 permutations of pairs of samples implemented in the XLSTAT software, version 7.5.2 [Isolation by distance (IBD) was tested by regressing the Rousset’s genetic distances between the population samples (on 7.5.2 .max) from 2 to 20 assuming a convolution Gaussian prior for spatial admixture (BYM). For each model, 100 runs were computed with 10,000 sweeps, after a burn–in period of 5000 sweeps [max. Finally, the spatial distribution of clusters was plot using Voronoi Tessellation.Spatial genetic structure was inferred using Bayesian clustering method implemented in the TESS software, version 2.3 . This me0 sweeps . To estiCp5.24) to 16 (Cp.1.62) for Chilean population samples and from three (Cp5.24) to 19 (Cp6.46) for the French population samples. The mean number of alleles per locus ranged from 3.0 to 5.2 over the 34 Chilean population samples, and from 6.2 to 9.0 for the three French population samples . Significant linkage disequilibria were observed between Cp1.60 and Cp.2.129 (p < 0.001); Cp1.62 and Cp.2.129 (p < 0.001); Cp1.62 and Cp6.46 (p < 0.001); and Cp5.24 and Cp6.46 (p < 0.001).The mean expected heterozygosity om Chile . For thend 0.117 . Only thFCT = 0.005, p ≤ 0.05), and between locations in each zone  that together accounted for by a 2.9% of the total genetic variance ) .AMOVA detected a low but significant genetic differentiation between the central and southern Chilean zones (variance . The maivariance . The hie (N.S.))  and whenFST values ranged from 0.171 between CchA2 and SjuW2, to 0.015 between CurP and TalQ . When more than one host-plant species was present in the same locality, a significant differentiation only in three out of 14 possible pairwise comparisons between host plants within the same locality was found (Pairwise comparison between the 34 Chilean population samples showed significant differentiation in 274 out of 561 combinations (48.8%) . Signifiand TalQ . The lowand GulA . Eight pas found . These sas found .FST/1 − FST) = −0.0098 + 0.0172 Log km, r2 = 0.06, p < 0.001), in agreement with an isolation by distance of codling moth populations in Chile (p < 0.001). Our southernmost locality CchA2 on the Aysén Region was significantly different from all remaining locations  . The locFST = 0.03) was one to two orders of magnitude higher than in previous studies of codling moth in Chile (FST = 0.002 − 0.0001), covering a more restricted latitudinal range of approximately 180 km and using only apples as the host plant [Cp1.62 has found significant linkage disequilibrium possibly related with selection of the kdr mutation, which confers resistance against pyrethroid insecticides [Cp1.60 and Cp1.62 are in chromosome 17 at positions 2,792,256 and 17,990,411, respectively. These microsatellite loci were not significantly linked, and therefore they are in rather distant positions on the same chromosome. The remaining three loci were found in different chromosomes , and therefore, they are not physically linked. The significant linkage disequilibrium found in our study could be related with other genetic processes, such as drift or hitchhiking selection.The present study included a wide latitudinal range (approximately 1400 km) that covered the main distribution of the codling moth in the west coast of South America , and thest plant . It is icticides . Furthercticides , from whFST analysis and hierarchical assignment, we detected a group of five population samples from three localities in the Bío-Bío Region, which were significantly different to all other codling moth populations from Chile. These population samples were composed by different host-plant species , suggesting that such a result is not associated with a host-plant-based genetic differentiation. Four of these population samples had less than 10 km between them in the Coastal Mountain Range of the Bío-Bío Region (latitude 37° S), in an isolated area from the main pome fruit production zone in the central valley of Chile.Based on pairwise Pinus radiata D. Don) for the production of timber and paper [Pome fruits in Chile were introduced by Spanish conquerors as early as the 16th century ,50. Howend paper , which mnd paper ,23 or Irnd paper  and desend paper ,11,13.A more recent codling moth invasion process occurred in the southernmost localities included in this study in the Aysén Region, where only during the last decade of the 20th century the introduction of this species was reported . This soFST values only between apples and walnuts (Graneros), pears and quinces (Panguilemo), and walnuts and quinces (Santa Juana). However, another 11 possible comparisons of different host-plant species in the same locality did not present significant pairwise FST values . Previous studies using allozymes in France , microsaT values . FurtherT values . This diThus, overall genetic differentiation between codling moth from different host-plant species was not consistent between host-plant samples within localities. The potential introduction of codling moth populations from different host plants species should have resulted in a more consistent genetic differentiation related to the host plants between the different localities. Furthermore, body mass, wing size, and shape variables studies performed with geometric morphology techniques did not show significant differences between the body size or wing morphology of the codling moths obtained from different host-plant species , further supporting the lack of host-plant strains of this species in the Maule Region of Chile .Finally, the codling moth is regarded as a sedentary pest with rather limited dispersal capacity in the adult stage, with the exception of a few genotypes with inherited long range flight behavior ,58,59,60Low genetic differentiation among the codling moth population samples was found, with only slight isolation by distance. According to a Bayesian assignment test (TESS), a group of localities in the Coastal Mountain Range from the Bío-Bío Region was found to conform to a distinct genetic cluster. We did not find significant genetic differentiation between codling moth samples from different host-plant species. Our results indicate high genetic exchange among codling moth populations between the different Chilean regions and host plants.