Alternaria linariae or Alternaria solani and leaf blight (LB) caused by A. alternata are economically important diseases of tomato and potato. Little is known about the genetic diversity and population structure of these pathogens in the United States. A total of 214 isolates of A. alternata (n = 61), A. linariae (n = 96), and A. solani (n = 57) were collected from tomato and potato in North Carolina and Wisconsin and grouped into populations based on geographic locations and tomato varieties. We exploited 220 single nucleotide polymorphisms derived from DNA sequences of 10 microsatellite loci to analyse the population genetic structure between species and between populations within species and infer the mode of reproduction. High genetic variation and genotypic diversity were observed in all the populations analysed. The null hypothesis of the clonality test based on the index of association Alternaria spp., suggesting that recombination can play an important role in the evolution of these pathogens. Most genetic differences were found between species, and the results showed three distinct genetic clusters corresponding to the three Alternaria spp. We found no evidence for clustering of geographic location populations or tomato variety populations. Analyses of molecular variance revealed high (> 85%) genetic variation within individuals in a population, confirming a lack of population subdivision within species. Alternaria linariae populations harboured more multilocus genotypes (MLGs) than A. alternata and A. solani populations and shared the same MLG between populations within a species, which was suggestive of gene flow and population expansion. Although both A. linariae and A. solani can cause EB on tomatoes and potatoes, these two species are genetically differentiated. Our results provide new insights into the evolution and structure of Alternaria spp. and can lead to new directions in optimizing management strategies to mitigate the impact of these pathogens on tomato and potato production in North Carolina and Wisconsin.Early blight (EB) caused by  Alternaria has been divided into 24 sections based on morphological and molecular data1. Among them, the Alternaria sect. porri produces long beaks and large conidia and includes A. porri, A. solani, and A. tomatophila1. Early blight (EB) is caused by A. solani sensu stricto (Ellis and Martin) Jones and Grout in tomato (Solanum lycopersicum L.) and potato (Solanum tuberosum L.)3. Alternaria tomatophila has also been reported to cause EB in tomatoes4. Based on a concatenated multigene phylogeny, A. linariae (Neerg.) (syn. A. tomatophila) Simmons, A. solani, and A. grandis cluster together in one clade1. Under conducive environments, yield losses attributed to these pathogens on tomato and potato range from 40 to 80%6. Alternaria spp. have been known to cause EB and leaf blight (LB) in tomato and potato, and these disease epidemics have increased recently10. Members of the Alternaria genus are haploid and heterothallic fungi11. The asexual cycle produces conidia that are mainly dispersed by rain splash and dew over short distances11. The relative importance of these pathogens has increased recently, and they pose a serious threat to tomato and potato production in North Carolina (NC)7 and Wisconsin (WI)8.The genus Alternaria spp. Due to its short life cycle and rapid growth rates via asexual reproduction12, the reduced sensitivity of strains of Alternaria spp. to quinone outside inhibitor (QoI) and succinate dehydrogenase inhibitor (SDHI) fungicides has been reported13. The planting of resistant varieties is the most economical, sustainable, and environmentally friendly approach to controlling these plant pathogens. However, the effectiveness of resistant varieties and fungicides inextricably depends on knowledge of the evolutionary potential and population genetic structure of Alternaria spp.15. Furthermore, investigating the population biology of plant pathogens can help us understand the origin, sources of inoculum, migration, and phylogeography of the pathogen15. It also provides useful insights into the patterns of genetic diversity, which can assist in selecting target pathogen isolates for screening germplasm for resistance and developing durable resistant tomato and potato varieties16. Information on the genetic diversity, genetic structure, and reproductive biology of Alternaria spp. populations are necessary to understand the impact of evolutionary forces, such as mutation, selection, genetic drift, recombination, and migration, on the evolutionary trajectory of pathogen populations over time17. Understanding the factors that are responsible for creating pathogen variability and structure is of practical significance in epidemiological studies and in optimizing pathogen management strategies. For example, plant pathogenic fungi with a mixed reproduction system of clonality and recombination have a higher potential for mutation and gene flow than asexually reproducing fungi16. The recombination process creates genetic diversity and enhances the propensity of pathogens to evolve rapidly in response to changes in management strategies16.Several fungicides are currently available to control Alternaria spp. in tomato and potato are unknown in NC and WI. Alternaria alternata, A. solani, and A. linariae are excellent candidates for investigating the model of genetic divergence and speciation because of their intimate association with tomato and potato. Alternaria spp. are known to reproduce primarily by asexual reproduction, and a high degree of clonality is dominated by a few genotypes, low gene diversity, and a significant degree of nonrandom association among unlinked alleles16. Although no sexual stage has been found in Alternaria spp., signatures of recombination in clonal lineages of A. alternata (the citrus brown spot pathogen) and high levels of genetic and genotypic diversity have been reported in a few Alternaria spp.20. Sexual reproduction in most plant pathogenic fungi, including Alternaria spp., are controlled by two mating type (MAT) idiomorphs: MAT1-1 and MAT1-222. Using a polymerase chain reaction (PCR)-based approach, the MAT1-1 and MAT1-2 idiomorphs of approximately 1.9 and 2.2 kb, respectively, were cloned and sequenced in A. alternata21. Subsequently, MAT1-1 and MAT1-2 idiomorphs have been identified in several Alternaria spp.23. This has prompted the analysis of population genetics using molecular markers to indirectly infer the hypothesis of clonality or recombination26.Currently, the genetic variation and reproductive strategy of A. alternata and A. solani from tomato and potato28. However, these markers have limited discriminatory power to differentiate closely related isolates within populations of the pathogen. Microsatellite loci or simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) are preferred markers17 because they are co-dominant, reproducible, and are located in known sequences in the genome compared to RAPDs, AFLPs, and other markers. SSRs are tandem repeat motifs29 that occur mostly in the intergenic and non-coding regions of the genome31. Microsatellite loci have been widely used in population genetic studies to analyse the genetic structure and evolutionary biology of several pathogenic fungi35. The use of SNPs and high-throughput sequencing to investigate the genetic diversity of plant-pathogen populations has gained attention in recent years, partly owing to the reduced cost of DNA sequencing, the proliferation of tools for assembly and annotation, and the possibility of simultaneously genotyping thousands of molecular markers in multiple individuals37. Importantly, several limitations of gel electrophoresis and capillary-based methods38 can be overcome by the sequencing of microsatellite loci (SSR-seq). Subsequently, SSR-seq offers a more precise and statistically powerful tool to resolve the genetic diversity and population structure of pathogen populations39.To assess genetic diversity, molecular markers such as amplified fragment length polymorphisms (AFLPs), isozymes, random amplified polymorphic DNA (RAPD), and random amplified microsatellites (RAMSs) have been used in Alternaria spp. sampled from tomato and potato in NC and WI. We tested the hypothesis of population subdivision to determine whether local populations are genetically structured by geographic location or tomato variety and the evolutionary factors that shape the population structures of different populations. We further tested the hypothesis of recombination within Alternaria spp. to discern evolutionary relationships among isolates in each population and to examine the role of random mating in creating diversity in these species. Stratified by geographic location or by tomato variety, we conducted analyses to answer the following biological questions. Do SNPs from microsatellite loci detect the genetic structure within a species and are the populations of Alternaria spp. from NC and WI genetically differentiated? If so, which of the species is more diverse? What are the main factors (geographic location or tomato variety) structuring populations within a species? Is there genotype sharing between species and between populations within a species, which could be an indicator of gene flow? What is the implication of the observed high genetic and genotypic diversity in disease epidemics? We also explored evidence of random mating by examining MAT idiomorphs distributed between species and between populations within a species. Does clonal or random mating occur or is there a mixed reproductive system? What are the implications of investigating this for pathogen management strategies?In this study, we examined SNPs from 10 microsatellite loci to detect genetic variation in three Alternaria isolates were classified into two groups. The isolates of group I were characterized by the formation of obclavate, dark brown, and short septate conidia with a short beak and branched chains. These morphological features of 61 isolates were similar to those of A. alternata described previously4. Group II isolates produced large conidia with long beak(s) and developed brownish-black pigment on A-PDA. These isolates were identified as either A. linariae or A. solani. Alternaria linariae isolates also tended to produce some conidia with branched beaks. In our observations, approximately 30 to 50% of them were branched.Based on the characteristics of single spores developed on acidified potato dextrose agar (A-PDA)  plates that were amended with two antibiotics, ampicillin (0.06 g/L of medium) and rifampicin (0.024 g/L of medium), to minimize bacterial growth, all A. alternata. The primer pairs OAsF7 and OasR6 amplified a 164 bp PCR product from all DNA samples specific to A. solani, whereas the primer pairs OatF4O and atR2 produced a 483 bp amplicon from all A. linariae isolates. In all, 61, 96, and 57 isolates were confirmed as A. alternata, A. linariae, and A. solani, respectively, and were included in genetic analyses  Pi and θw in A. alternata. Tajima's D values were negative for most of the microsatellite loci across populations , SSR391 (MAD-Al population and SWA-Al population), SSR457, SSR534 (HAY-Al population), and SSR534 (SWA-Al population) also yielded very low Tajima's D values (0.0 to 0.01) within populations of A. linariae.Statistics of nucleotide diversity and neutrality tests varied with microsatellite loci and populations within a species  was high for all Alternaria spp. For both geographic populations and tomato variety populations, E5 ranged from 0.69  to 0.96 (HAY-Al and Picus-Al population) , and the Heirloom-Aa population had the highest (Hexp = 0.43).In general, all genotypic diversity statistics were higher in ni Table . The higni Table . Similarn) Table . In the Alternaria spp. with 46/61, 70/96, and 46/57 MLGs of A. alternata, A. linariae, and A. solani, respectively . Comparatively, the eMLG value for A. solani was slightly higher (n = 46.00) than that for A. linariae (n = 45.00) and A. alternata (n = 43.50). The WAUA population had the highest eMLGs (n = 46.00), and the SWA-Al population had the lowest eMLGs (n = 7.58). eMLG values in tomato variety populations varied from 7.58  to 9.57 (Picus-Al population), with an average value of 8.69  . These populations were not significantly differentiated (P < 0.05)  Table . Stratif5) Table . Similar83 Table . AMOVA aA. alternata, A. solani, and A. linariae were positioned in different quadrants, suggesting that these species are genetically differentiated  and two discriminant functions . Three distinct genetic clusters of A. alternata and A. linariae in the sampled locations was investigated using approximate Bayesian computation. These scenarios supported our DAPC results, which showed the closeness and lack of clustering of three A. alternata populations . Scenario 3 was proposed to be the PSW-Aa population as the most likely progenitor of the STO-Aa and BHT-A populations  between species and most populations within a species  deviated from the hypothesis of recombination.We calculated the standardized index of association es Table , and S13MAT1-1 fragment or a 280-bp MAT1-2-specific product from any DNA sample ; however, skewing towards MAT1-1 was observed in A. linariae  from a 1:1 ratio. The null hypothesis of random mating cannot be rejected for these populations. However, the mating type ratios deviated from 1:1 for the remaining geographic location populations and tomato variety populations within a species. The mating type ratios in seven populations  were skewed towards MAT-1–1, whereas in two populations , it was skewed towards MAT1-2  tests suggested potential recombination in two A. alternata geographic location populations (BHT-Aa and PSW-Aa) and four A. linariae geographic populations . In contrast, the A. solani population (WAU-As) and two A. alternata populations (STO-Aa and Heirloom-Aa) exhibited clonality. A few geographic location populations (STO-Aa and WAU-As) and one tomato variety population (Heirloom-Aa) contained the two mating types, and their distributions were not significantly different from a 1:1 ratio, suggesting random mating. The occurrence of both mating types along with high levels of genetic diversity, the large number of singleton MLGs, and random mating provide evidence for most populations undergoing sexual recombination. Our findings corroborate previous studies that also reported a similar conclusion in asexual and sexual fungi54. We also found an unequal frequency of mating types or a biased distribution in some populations within a species. These results could be due to crop management practices such as the application of fungicide or the use of resistant cultivars that can favour one or the other mating type, as demonstrated in other studies23. Alternatively, nonrandom mating55 and the selective advantage of the isolate virulence in the host might contribute to causing skewed mating type ratios57. The findings of a mixed reproductive mode and high genetic diversity of Alternaria spp. based on SNPs are parallel to those of other pathosystems50, where the impact of mixed reproduction is well documented.Multilocus linkage disequilibrium ; A. linariae = 32.9 Mb, and A. solani = 34 Mb30. Furthermore, these genomes are comparable to recently sequenced and annotated genomes of several other species of Alternaria30. A larger genome size usually generates higher genotypic diversity because more alleles could emerge through mutation and random genetic drift61. One of the limitations of our study is the reliance on small sample sizes of A. alternata populations collected from tomatoes in NC. Similarly, the A. solani population (WAU-As) was collected from a potato from one location in WI. To overcome these limitations, geographically more diverse and larger samples are needed to collect and analyse population genetics parameters. We expect that the distribution of sample sizes may not be representative of the populations being sampled and is likely affected by the accuracy of genotypic diversity and population differentiation analyses. Our results must be interpreted cautiously when comparing genetic diversity and population structure with global populations of A. alternata and A. solani.The assessment of population differentiation using Jost’s A. solani populations causing EB on tomato and potato were not differentiated from each other3. The hypothesis of a lack of host specialization needs to be confirmed by cross-inoculation of A. linariae and A. solani isolates on tomato and potato varieties. We also found a few signals of admixed members attributed to the movement of the common MLGs through the dispersal of airborne conidia and infected planting material62. Although the founder sample history is unknown, our neutrality test showed a signature of population expansion. When populations were stratified by geographic location or tomato variety, > 85% of the genetic variation was distributed in individuals within a population. Our findings of high genetic diversity in three Alternaria spp. were similar to previous studies64, who also reported little or no evidence of population differentiation based on geographic origin or host of origin. The centre of origin of any pathogen is likely to possess a population with higher genetic variability than recently established populations54. In this study, the A. alternata and A. linariae populations from tomatoes in NC had higher genotypic diversity than the A. solani population from potatoes in WI. It appeared that the populations from tomatoes could have been established earlier than the A. solani population from potatoes.Our analyses revealed three genetic clusters corresponding to the individual species. The SWA-Al population from Swain County was slightly different from the other three populations HAY-Al, MAC-Al, and MAD-Al. Likewise, A. solani population (WAU-As) was sampled from EB-resistant potato genotypes such as Atlantic, Russet Burbank, a wild relative S. berthaultii, F1 hybrids, advanced breeding lines, and parental genotypes8. These genetically diverse potato genotypes might have some effects on the pattern of differentiation within the A. solani population from WI. All A. alternata populations analysed were sampled from hybrids and heirlooms with unknown genes for resistance. In contrast, A. linariae populations were collected from commercially grown tomato varieties such as Picus , Testi Lee65, and Plum Regal66, and these varieties have some levels of resistance to A. linariae. These resistant varieties could exert selection pressure on the pathogen populations, thus promoting high genotypic diversity in A. linariae populations in NC. It is worth noting that the SWA-Al population was more variable than other populations and was collected from ‘Plum Regal’. This variety was developed from a cross between the EB-resistant tomato breeding lines NC 25P and NC 30P66.The Alternaria species diversity and population structure with respect to species, geographic location, and affected hosts in NC and WI. Our analysis revealed three distinct genetic clusters that corresponded to each species. The effect of geographic location and tomato variety on populations within a species was low. We used several population genetics tools to test for the occurrence of random mating to further infer the mode of reproduction of the studied populations. The presence of high genotypic diversity, high and diverse MLGs, linkage equilibrium based on the Alternaria spp. can maintain the sexual pathway in the studied populations23. Plant pathogen populations with a mixed reproductive system  are considered the highest risk16. The process of random mating may increase the frequency of the evolution of new MLGs through the random association of alleles, whereas asexual reproduction maintains the propagation of well-adapted MLGs. Thus, gene flow may occur through the movement of these MLGs between geographic locations or through infected tomato transplants. Importantly, careful intervention must be exercised when applying different fungicides and deploying single-gene resistance varieties because pathogen populations with high evolutionary potential are more likely to overcome host genetic resistance16. Tomato and potato breeding strategies that combine quantitative resistance should be a valuable approach for developing durable resistance to these fungal pathogens in NC and WI.In conclusion, we used SNPs from 10 microsatellite loci to gain insights into Alternaria spp., depending on the size of the fields and the number of tomato cultivars planted between 2008 and 2014. To isolate the fungi from tomato, all samples were brought to the Department of Entomology and Plant Pathology, North Carolina State University, Raleigh, NC. Three segments (~ 4 mm2) were cut from each infected leaf and submerged in 70% ethanol for 30 s. The leaf segments were further sterilized by dipping in 3% Clorox  for 1 min and rinsed three times in sterile distilled water before plating. The segments were transferred with sterile tweezers into A-PDA plates. The plates were sealed with parafilm and placed under light at 25 °C for 5 days to induce sporulation. In this study, a ‘sample’  herein is defined as an experimental unit used for measurement, and ‘population’ comprises groups of isolates defined by geographic locations or tomato varieties. Geographic locations are defined by an administrative boundary, such as a county or a region within NC and WI, and represented by prominent features, such as soil type, weather conditions, and cropping patterns in the vicinity. Tomato genotypes grown in NC are diverse, with different combinations of fruit size and growth habits, and are resistant to multiple plant pathogens. Tomato types are mainly represented by Cherry, Roma, Grape, Plum, Heirlooms, and Hybrids. The commercial tomato varieties were Picus, Plum Regal, Red Defender, and Tasti Lee. In this study, ‘fungal populations’ were grouped into three strata. ‘Species’ refers to isolates identified based on morphological features and molecular characterization and belonged to A. alternata (Aa), A. linariae (Al) and A. solani (As). ‘Geographic location populations’ refers to isolates collected from a county or geographic region within NC and WI. ‘Tomato variety populations’ indicates isolates collected either from a certain tomato type or variety and were only within the population.Tomato and potato leaves with symptoms of EB and LB were collected from farmer fields, commercial plots, or research plots in NC and WI Fig. . We usedA. alternata, a haphazard sampling strategy was used in geographic regions and spanned ~ 370 km from south to north and ~ 650 km from east to west in NC. Alternaria alternata isolates were collected and isolated (as described above) and grouped according to counties within a similar geographic region and tomato type  was collected from grape-type tomatoes66 from Buncombe, Henderson, and Transylvania (BHT) counties in the western mountain region, NC. The PSW-Aa population (n = 11) was collected from the commercial hybrid tomato cv. Florida 47 (Fla 47) and Roma from Pender, Sampson, and Wake (PSW) counties in the inner coastal plain and Piedmont region in NC. The Stokes (STO)-Aa population (n = 40) was sampled from Stokes county in the northwestern Piedmont region in NC. The STO-Aa population was collected mainly from heirloom tomatoes such as Aunt Gertie’s Gold, Cherokee Purple, German Pink, Mortgage Filter, Mr. Stripey, Pink Brandy Wine, Snowstorm, and Verna Orange. Alternaria alternata populations were grouped according to tomato type and represented by the Grape-Aa population (n = 10) from BHT in southwestern NC; the Hybrid-Aa population (n = 10) was from PSW in central and eastern NC, and the Heirloom-Aa population (n = 38) was from STO in northwestern NC.For ype Fig. . Among t67 to collect A. linariae isolates from four major tomato-growing counties in NC: Haywood, Macon, Madison, and Swain. These counties spanned 165 km from east to west. Each site was divided into strata or rows approximately 250 m long. In each row, three-leaf samples were collected ~ 40 m apart. At least 10 rows were sampled, and up to 60 samples were collected from each county (Supplementary Table A. linariae isolates were collected and represented by HAY-Al (n = 21), MAC-Al (n = 33), MAD-Al (n = 21), and SWA-Al (n = 21) from Haywood, Macon, Madison, and Swain counties, respectively , Tasti Lee-Al population (n = 54), and Plum Regal-Al population (n = 21)  between 2008 and 2009 at the Hancock Agricultural Research Station, Waushara County, WI  were cut from the margin of EB lesions on the same leaflet and placed onto clarified V-8 (CV8) medium  amended with streptomycin (25 μg/mL) and kanamycin (50 μg/mL). Some of the isolates of A. solani from potatoes represented by the WAU-As population were also characterized previously by RAPD markers8 and were obtained from the U.S. Department of Agriculture, Agricultural Research Service, Vegetable Crops Research Unit, Madison, WI under an import permit .We used random sampling to collect  WI Fig. . Potato 4. To maintain the fungal isolates, an 8 mm diameter mycelial plug was cut from each A-PDA plate after 7 to 10 days of incubation using a cork borer. Mycelial plugs were dried under laminar flow overnight, transferred into 2 mL screw-cap cryogenic vials, and stored at − 80 °C. In total, 214 isolates of three Alternaria spp., A. alternata (n = 61), A. linariae (n = 96), and A. solani (n = 57)  Fig. , were anTo extract genomic DNA, isolates were revived on A-PDA plates and incubated at 25 °C for 7 d. Fungal tissues were scraped with a sterile razor blade and placed into 2 mL screw-capped tubes. The tubes were freeze-dried  for 72 h with the following cycle: − 25 °C for 40 min; − 30 °C for 60 min; − 20 °C for 600 min, and − 10 °C for 600 min with a pressure of 400 mTorr. The lyophilized freeze-dried fungal tissues were ground to a fine powder using a microtube homogenizer  at 400 rpm for 3 min. The glass beads were removed, and genomic DNA was extracted using the DNeasy Plant Mini kit  following the manufacturer’s recommended protocol. The DNA concentration was quantified using a fluorometer . The final concentration of DNA at 10 ng/µL was prepared for each sample for all PCR assays and SSR-seq.A. alternata can be distinguished morphologically from the other two species, species-specific primers have been used to differentiate between A. linariae and A. solani and A. alternata55. Alternaria solani-specific primer pairs OAsF7 (5′-CGACGAGTAAGTTGCCCTCA-3′) and OAsR6 (5′-TGTAGGCGTCAGAGACACCATT-3′) amplified the Alt a1 genomic region in A. solani, whereas another primer pair, OAtF4 (5′-TGCGGCTTGCTGGCTAAGGT-3′) and OAtR2 (5′-CAGTCGATGCGGCCGTCA-3′), amplified a DNA fragment from the calmodulin-encoding gene of A. linariae and some other large-spored Alternaria species excluding A. solani55. To identify the isolates, the genomic DNA of the 214 isolates of Alternaria spp. was amplified using the two sets of species-specific primer pairs synthesized by Invitrogen Life Technologies . Genomic DNA of A. solani isolate # BMP 0185 from potato30 was kindly provided by Barry M. Pryor, University of Arizona, and used as a positive control. PCR assays were conducted in a 25 µL reaction volume containing 15.22 µL of sterile deionized water; 2.5 µL of 10 × PCR buffer ; 1.25 µL of 2 mM dNTP mix, 1.8 µL of 25 mM MgCl2 , 0.1 µL (5 Units/µL) of Taq polymerase , 1 µL of 10 nM each reverse and forward primer, 1 µL (10 ng/µL) of genomic DNA; and 0.125 µL (20 mg/mL) of bovine serum albumin . PCR amplification was performed in a thermal cycler  as described previously55. Amplification with the primer pairs OAsF7 and OAsR6 had an initial step of 94 °C for 2.5 min, followed by 30 cycles of 92 °C for 20 s, 68 °C for 30 s, and a final extension of 72 °C for 3 min. The program for the amplification of primer pairs OAtF4/OAtR2 had the same features except that the final extension was 72 °C for 40 s. The PCR products were resolved on 1.5% agarose gels in 1 × TAE  buffer stained with GelRed . Each well was loaded with 6 µl of PCR product mixed with 3 µL of 5 × loading dye . A 50-bp DNA marker  was loaded at the beginning of each row as a molecular weight marker. The gel was run under 100 V for 90 min, and an image was captured by a molecular gel imager .Although A. solani # BMP018530 were kindly provided by Tobin Peerver and Lydia Tymon, formerly at the Department of Plant Pathology, Washington State University, Pullman, WA, USA . Reference genome sequences of A. solani # BMP0185 were used30 to identify single nucleotide polymorphisms (SNPs) in each microsatellite locus using Geneious ver. 7.1.7 . The minimum call rate per microsatellite locus was estimated to be 0.1 (10% missing data per microsatellite locus × isolate), and such inferior-quality sequences, if present in a few isolates, were resequenced. Final sequences were further checked for missing nucleotides and, if there were any, missing values were coded accordingly in the datasets. In total, 220 SNPs were used to analyse the population genetic parameters for three strata and formatted in GenAlEx 6.568 These included (1) between species collected from tomato and potato in NC and WI, (2) between geographic populations within species, and (3) between tomato variety populations within A. alternata or A. linariae collected in NC , the number of haplotypes (h), haplotypic diversity (Hd), nucleotide diversity (Pi), and Watterson's Theta (θw) per site from the total number of mutations (Eta)69 between species and populations within species in DnaSP ver. 670. We used SNP data sets and calculated Tajima’s D (TD)71 and probability (P) values to test the hypothesis of deviation under a standard neutral model of molecular evolution. The significance of these values was tested using 10,000 random permutations. A negative TD value indicates population expansion71. A genotype accumulation curve was produced with the R package poppr73 to determine whether SNPs were sufficiently informative to discriminate between individual multilocus genotypes (MLGs). The average gene diversity (HT)74 for each locus was calculated using GeneAIEx ver. 6.568.Data for population genetic analyses were based on the haploids and the presence and frequency of SNPs from 10 microsatellite loci. We computed the number of segregating sites  based on rarefaction was calculated from numbers of MLGs expected at a sample size equivalent to the population with a minimum of 10 samples using 999 bootstrapping in the R package poppr ver. 2.8.175. Genotypic evenness (E5) is a measure of the relative abundance of each genotype in the populations76. To assess the distribution of genotypes within each species and between populations within species, E5 was calculated as the ratio of the number of abundant genotypes to the number of rarer genotypes75. E5 values ranged from 0 to 1, with 0 indicating that a population is dominated by a single MLG and 1 indicating that all genotypes occur at the same frequency76. We computed multilocus genotypic diversity in poppr ver. 2.8.175 by calculating Shannon–Wiener’s index (H)77, Simpson’s genotypic diversity index (λ)78, and Stoddart and Taylor’s genotypic diversity index (G)79. To compare the genotypic diversity within a population of different sample sizes, we analysed the indices E5, G, and λ following random sampling of the data using rarefaction in poppr ver. 2.8.175 with 1000 jackknife replicates as described previously52. Unbiased expected heterozygosity was defined as Hexp = (1 − ∑pi2) × (n/n − 1), where p is the SNP frequency at a locus, and n is the number of observed SNPs at each locus75 and was calculated between species and between populations within species using the poppr package ver. 2.8.175 in R80. For further analyses, sequence data were clone-corrected on all population hierarchical levels using the clone correct function in poppr ver. 2.8.175, and isolates that shared the same SNPs were regarded as clones.We assume that populations of D differentiation index81 implemented in R using the mmod 1.3.3 package82. The statistical significance of Jost’s D was calculated by a randomization procedure in which all isolates in the respective populations were reassigned randomly to the populations, and Jost’s D was recalculated from the randomized dataset. Jost’s D was estimated under the null hypothesis of no differentiation between populations within a species. Analysis of molecular variance (AMOVA)83 was calculated using Arlequin ver. 3.583 and hierfstat ver. 0.5-784 implemented in the R package to test the following null and alternative hypotheses. In this context, we computed three F-statistics to address the biological questions: (1) FST partitions the genetic variation among populations within a species. If the FST value = 0, then there was no genetic differentiation, whereas if the FST value > 0, then the populations were subdivided. (2) FIS partitions the genetic variation among individuals within populations of a species, and (3) FIT indicates the genetic variation within individual isolates in a population. FIS and FIT may be positive or negative, with a negative value being indicative of excess heterozygosity. The significance of F-statistics at each level was evaluated based on 1000 random permutations, and significant genetic differentiation was declared when P < 0.05 in the R package ade4 ver.1.7.585. Alternaria solani (n = 57) has only one population, and thus, this species was not included in AMOVA for comparison between and within populations within a species.Genetic differentiation between pairs of populations was assessed in clone-corrected data using Jost’s Alternaria spp. using clone-corrected data to test the hypothesis of population subdivision between species and populations within a species. Population structure was analysed by conducting a discriminant analysis of principal components (DAPC) and using the function xvaldapc in the R package adegenet ver. 2.1.286. Because DAPC assumes no prior knowledge of group membership, our main goal in using this analysis was to display clustering of genetically related populations while minimizing variation within clusters. Another advantage of using this approach is that DAPC does not make any assumptions regarding the data structure or underlying population genetics model. To estimate the optimal number of genetic clusters of MLGs in the data sets, the K-means clustering algorithm procedure was implemented in the function ‘find. clusters87. This model relies on data transformation using principal component analysis (PCA) to delineate clusters and then uses discriminate analysis based on the number of principal components retained by the user to maximize the separation between populations87. We identified the alleles that best discriminate a set of defined clusters of Alternaria spp. A minimum spanning network (MSN) was constructed with clone-corrected data to calculate the genetic relatedness among MLGs in populations within a species using the imsn function in poppr80 and Nei’s74 genetic distance. The network was visualized using the igraph package ver. 1.2.188.We assessed the genetic structure of the combined populations of Alternaria spp. These scenarios were investigated using the approximate Bayesian computational (ABC) approach and conducted in the clone-corrected dataset using DIYABC v 2.1089. The observed MLG data were compared with large numbers of simulated datasets (one million per scenario) based on evolutionary scenarios (models). Possible scenarios were set with conditions as follows: t4 > t3 > t2 > t1. The posterior distribution of each of the three scenarios was estimated by conducting linear regressions on the 1000 simulated datasets after a logit transformation. The posterior probability of each scenario was computed by direct and logistic regression methods to identify the scenario with the highest posterior probability.The DAPC clusters were used to inform and develop historical scenarios describing the evolutionary relationships among populations of řd2)90 on clone-corrected data using the poppr package in R to infer the mode of reproduction  between species and between populations within a species. The significance of řd was tested by comparing the observed řd values against 10,000 randomized data sets to the null hypothesis of no LD; that is, alleles observed at different loci are unlinked and in random association in these populations90. Sexual populations are expected to have linkage equilibrium owing to no linkage among loci, whereas clonal or asexual populations are expected to have significant LD owing to linkage among loci90.We calculated the standardized index of association 91. MAT1-2 was amplified with primer pairs MCHMG1 and MCHMG222. The 25 µL PCR mixture used for amplification was prepared as described above. The PCR conditions and cycles for mating type analysis were conducted as described previously91. The amplified PCR products were separated in a 1.5% agarose gel in 1 × TAE buffer stained with 0.001% (v/v) GelRed . A 100-bp DNA marker  was loaded in each gel to determine the PCR product size. Clone-corrected data were used to estimate the frequency of the mating types between species and between populations within species according to the formula χ2 = ∑[(o − e)2/e], where o is the observed value of the mating type, and e is the expected value. The null hypothesis was that the observed mating types to the expected show equal frequencies (1:1 ratio) under random mating. The chi-square values for goodness-of-fit were calculated in SAS V9.4 .We carried out two PCR assays to amplify the Supplementary Information 1.


Leptonycteris yerbabuenae, the lesser long-nosed bat is an abundant migratory nectar-feeding bat found in most of Mexico, and in some areas of northern Central America and small sections of southwestern USA. We analyzed the distribution of the maternal and paternal lineages of this species with phylogeographic methods based on two mitochondrial markers, Cyt-b and D-loop, and a marker located in the Y chromosome, DBY. We obtained tissue samples from 220 individuals from 23 localities. Levels of genetic diversity  were high . No clear patterns of population genetic structure were found for mitochondrial markers, while male genetic differentiation suggested the presence of two lineages: one from Mexican Pacific coast states and another from central-southern Mexico; in accordance to strong male philopatry and higher female migration. We used genealogical reconstructions based on Bayesian tools to calculate divergence times, and to test coalescent models to explain changes in L. yerbabuenae historical demography. Our results show that recent demographic changes were consistent with global climatic changes  and divergence times dated from molecular genealogies exhibited older divergence times, Cyt-b (4.03 mya), D-loop (10.26 mya) and DBY (12.23 mya). Accordingly, the female lineage underwent demographic expansion associated to Pleistocene climate change, whereas the male lineage remained constant. Climate oscillations have global and local effects on distribution, demographic and genetic diversity patterns . In partLeptonycteris yerbabuenae, (Glossophaginae) is considered the most widespread nectar-feeding bat in Mexico, pollinating at least 64 plant species, including many Agave species, columnar cacti and Bombacoideae trees  have concluded that there are different genetic groups along the species distribution . WilkinsL. yerbabuena encompasses several biogeographic regions  and control region (D-loop), which are maternally inherited and variable in most mammal species. We also sequenced the region of Dead box associated to the Y chromosome gene (DBY) which is located in the Y chromosome and is paternally inherited, this marker has been used before for phylogeographic studies in phyllostomid bats , 0.2 µM of dNTPs (10 µM) and 7.325µl of H2O. The PCR profile for Cyt-b was: 5 min of initial denaturation at 95 °C, followed by 35 cycles of 30 s at 96 °C, 1 min at 53 °C, 2 min at 72 °C, and a final extension of 7 min at 72 °C; and for D-loop: 3 min at 95 °C, followed by 35 cycles of 30 s at 94 °C, 45 s at 54 °C, 2 min of 72 °C, and a final extension of 10 min at 72 °C in an ABI Veriti 96-Well Thermal Cycler .We obtained 1121 bp of CAAGAC 3′. For theTTTATG-3′. PCR forDBY) with primers 5′-CCGTTACTTCCATTTTCAAAA-3′and 5′-GCTAAAACCAACGAGATTGGT-3′(2 (25 µM), 2.5 U of Taq DNA polymerase (Promega) and 7.7µl of H2O. Amplification was carried out as follows: 10 min of an initial denaturation at 94 °C, 36 cycles of 45 s at 94 °C, 30 s at 54 °C, 2:30 min at 72 °C, and a final extension of 5 min at 72 °C in an ABI Veriti 96-Well Thermal Cycler . We sequenced each genetic region with forward and reverse primers at Macrogen USA’s Maryland headquarters (http://www.macrogenusa.com).We amplified a 450 bp fragment from the Dead box associated to the Y chromosome gene .Differences in the final sample number among markers resulted from PCR artifacts,  for DBY . All seqWe assessed the quality of DNA sequences and assembled forward and reverse sequences with Consed 29.0 using the default settings . We aligHd) and nucleotide diversity (π) for each sampled locality using DNAsp 5.10.01 , number of haplotypes (h), haplotype diversity , D-loop (n = 43) and DBY (n = 70). We conducted separate analyses for each gene, because each region shows a different mutation rate; thus, allowing to make inferences at different time scales and for each sex. We determined the substitution model with best fit to our data with jModelTest 2 , and Leptonycteris 12 mya (1.0 SD) were derived from detailed Bayesian analysis previously reported  using GTR+G+I in a Piecewise-linear nucleotide substitution model , Museo de Zoología “Alfonso L. Herrera” , Colección de Mamíferos UAM-I (Universidad Autónoma Metropolitana), Colección Mastozoológica ENCB ) and GBIF (We obtained coordinates for the presence of and GBIF  databaseand GBIF . To avoihttp://www.worldclim.org) to perform a variance inflation factor analysis to eliminate correlated bioclimatic variables, leaving a set of uncorrelated informative variables to be used in environmental niche modeling, and to obtain Mahalanobis distances. The later was used along with occurrence data to identify outlier records that could be a source of error. These points were removed from further analyses. All analyses were performed with R 1.4.1106  scores, where values above 0.85 are considered useful  for xEnt 3.3  for fourxEnt 3.3 . The datxEnt 3.3 . A binarCyt-b ; 828 bp for D-loop  and 450 bp for DBY . Genetic diversity (Hd) values were lower for maternally inherited mitochondrial regions  than for paternally inherited DBY  ; Fig. 1.BY 0.91; , a similBY 0.91; .Cyt-b and D-loop networks showed a main group with most haplotypes from all localities connected, and smaller groups consisting mainly of members from the Peninsula of Baja California-West of Balsas localities  connected by several mutational steps  exhibited a well-defined genetic structure between the Isthmus of Tehuantepec-East of Balsas and West of Balsas (Pacific Coast) with values ranging from 0.6 to 0.98.Pairwise  markers –S2 but hers (DBY . For CytCyt-b (log likelihood = −10512.121) and D-loop (log likelihood = −10990.658) detected two genetic clusters  groups. In the case of the DBY gen, BAPS detected three different genetic groups, congruent with the geographic distribution of sampled localities  for s K = 2; , consistDBY clustering provided a slightly better explanation of genetic variance   .Cyt-b . In contrast, for mitochondrial D-loop .The Bayesian genealogy for Cyt-b  and D-lo  indicate a late Pleistocene demographic expansion, starting at ∼130,000 ya for Cyt-b  and ∼500 .Tajima’s L. yerbabuenae  to build distribution models. The potential distribution models for babuenae ; 8 showebabuenae  suggestsbabuenae  and LGM babuenae  models dbabuenae .L. yerbabuenae had a marked contraction specially in Central America, in contrast with the expansion observed in Central Mexico, West Balsas (and Pacific Coast of Mexico) and south of Baja California Peninsula , south of the peninsula of Baja California, the states of Oaxaca and Chiapas on the Isthmus of Tehuantepec zones   . The con history , and to ilopatry ; there iilopatry .i.e., Tadarida brasiliensis . In paruantepec .L. yerbabuenae females move more than males, and that males show philopatric behavior, conducting only local and altitudinal movements  and one of the maternally (Cyt-b) inherited molecular markers, although there are some similarities. Genealogical relationship among L. yerbabuenae and sister species is not clear, with D-loop and DBY markers of L. curasoae and L. nivalis forming a monophyletic group. While in the case of the genealogy based on Cyt-b, L. nivalis and L. curasoae form independent branches, and L. nivalis is established as the closest species to L. yerbabuenae clade. In addition, we observe some disagreement in haplotype relationships depicted in the maternally inherited Cyt-b and D-Loop genealogies. In this sense, high homoplasy has been reported for the D-loop in several species  and the chromosome Y associated DBY marker  were older than those obtained for Cyt-b . Dates of divergence for DBY and D-loop are consistent with the origin of ecologically related taxa, such as Agave sensu lato  signals of demographic expansion, probably associated with Pleistocene climate changes as supported by demographic analyses.The demographic history of populations also influences the shape of ultrametric trees, where a genetic bottleneck can increase the rate of coalescence of lineages, while a demographic expansion could produce isolated long branches . AccordiD results also support demographic expansion for mtDNA. These results are further supported by past distribution models showing an expansion from the LIG through the Holocene.High haplotype diversity and medium-low nucleotide diversity, in addition to a star-like haplotype network, further suggests population expansion . SkyLineAgave towards the north of Mexico . Furthermore, this result can overlap with current conservation programs, as “Batfriendly” which consists of promoting the conservation of agaves grown in strategic ecological and economic areas to support the migratory movements of bats .Click here for additional data file.10.7717/peerj.12168/supp-5Supplemental Information 5* Values calculated from a sample smaller than three individuals.Click here for additional data file.10.7717/peerj.12168/supp-6Supplemental Information 6Personal ID, Genbank ID and GenBank accession number for sequences used in analysesClick here for additional data file.10.7717/peerj.12168/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.12168/supp-8Supplemental Information 8Each sequence represents an individual tissue sampleClick here for additional data file.10.7717/peerj.12168/supp-9Supplemental Information 9Each sequence represents an individual tissue sampleClick here for additional data file.10.7717/peerj.12168/supp-10Supplemental Information 10Each sequence represents an individual tissue sampleClick here for additional data file.


Microsatellite DNA analysis is a powerful tool for assessing population genetics. The main aim of this study was to assess the genetic potential of the peregrine falcon population covered by the restitution program. We characterized individuals from breeders that set their birds for release into the wild and birds that have been reintroduced in previous years. This was done using a well-known microsatellite panel designed for the peregrine falcon containing 10 markers. We calculated the genetic distance between individuals and populations using the UPGMA (unweighted pair group method with arithmetic mean) method and then performed a Principal Coordinates Analysis (PCoA) and constructed phylogenetic trees, to visualize the results. In addition, we used the Bayesian clustering method, assuming 1–15 hypothetical populations, to find the model that best fit the data. Units were segregated into groups regardless of the country of origin, and the number of alleles and observed heterozygosity were different in different breeding groups. The wild and captive populations were grouped independent of the original population. Falco peregrinus population in Europe occurred mainly in the 1950s and 1960s [F. p. pealei. It is assumed that this was due to the different diet of this cliff-dwelling subspecies [The peregrine falcon can be found on all continents except Antarctica. A decrease in the nd 1960s , leadingbspecies ,4,5.Falco p. peregrinus) presence all over Poland, the population size has never been very large [Falco p. peregrinus) as the urban ecotype. The ecotype of a falcon is determined by the nesting site: falcons of the tree-nesting ecotype nest in trees, while birds of the urban ecotype prefer sites similar to cliffs or mountainside-like tops of skyscrapers or industrial chimneys. Despite the fact that the conditions conducive to tree nesting occur in places other than the above-mentioned fragment of Europe, there is no evidence of tree nesting in other areas. Furthermore, there is no evidence of phylogenetic differences between ecotypes [Despite the peregrine falcon’s  population in Poland.” [The development of methods for breeding falcons in captivity in the 1960s made reintroduction programs possible . In somebspecies ,12. The Poland.” . The aimPoland.” . By 2009Poland.” .Falco cherrug) and the low genetic variance among this species. In Scandinavia, low genetic variance was found in a contemporary population as well as in a historical population from museal collections [Some peregrine falcon populations have been subjected to genetic variance analysis. Using samples from a Scandinavian population, Nesje et al.  describelections . Bryndovlections , analyzelections , but an lections .n = 262) and wild birds (n = 91) of the wild bird tree-nesting ecotype (n = 34) and urban nesting (n = 57) ecotype. Birds were assigned to ecotypes on the basis of observation of the place of nesting. The wild individuals originated from the Polish population and were collected from 19 nesting sites, 9 forest nests and 10 urban nests, as shown in n = 86), Czech Republic (n = 91), Germany (n = 54), Slovakia (n = 21), and Denmark (n = 10)—prior to the birds being released into the wild as part of a restitution program. For genetic variation analyses, individuals were divided into 6 groups, 5 containing captive birds divided by country of breeding and 1 containing wild birds.Peripheral blood was collected from wing veins by needle puncture, and this was inserted into test tubes containing 96% ethanol . Blood samples and feathers were collected from 353 peregrine falcons, both captive  in accordance with the manufacturer’s instructions. For DNA extraction from feathers, the duration of incubation with proteinase was extended to 12 h.F. peregrinus [®), 0.93 µL of H2O, and primers in the following volumes : NVHfp5, 0.056 µL; NVHfp13, 0.056 µL; NVHfp46_1, 0.05 µL; NVHfp54, 0.070 µL; NVHfp79_4, 0.056 µL; NVHfp82_2, 0.042 µL; NVHfp86_2, 0.105 µL; NVHfp89, 0.056 µL; NVHfp92, 0.056 µL; NVHfp107, 0.042 µL. The reaction was performed in a Biometra T3 thermocycler. The cycling conditions were as follows: 14.5 min at 95 °C; followed by 15 cycles at 95 °C for 30 s, 58 °C decreasing every cycle by 0.2 °C for 30 s, 72 °C for 1 min and then 20 cycles at 95 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, and then 60 °C for 30 min. The samples were then kept 4 °C until collection.Microsatellite genotyping was performed for all individuals. Microsatellite markers were amplified with primers for regrinus ,18,19,22regrinus ). Based ® genetic analyzers, which analyzes the quality of obtained fragments and assigns the analyzed fragments to specific loci based on the length of the fragments and the type of dye. Lengths of obtained fragments represent specific alleles.PCR products were mixed with size standard and formamide loading buffer. An ABI3500 DNA analyzer was used to visualize the PCR products. Allele sizes were assigned using GENEMAPPER 4.0 software produced by Applied Biosystems Inc. GENEMAPPER 4.0 is a fragment analysis software for Applied BiosystemsGenotypes were tested for departure from the Hardy–Weinberg equilibrium using GenAlEx v6.5 software  ,24 by peThe basic analysis, featuring genotype comparisons, heterozygosity calculation, and the estimation of allele numbers, was performed using GenAlEx and Excel 2016 v16.0.12901.20462 (Microsoft) software. The probability of the occurrence of null alleles was estimated using Cervus 3.0.7 .The genetic distance among populations was calculated using Nei’s  genetic In STRUCTURE v2.3.4 ,31,32,33Multilocus genotypes were obtained in 10 loci. A total of 326 out of 353 individuals were genotyped in all loci, 23 in 9 loci and 4 in 8 loci. No null alleles were found. Every individual had a unique multilocus genotype. The number of alleles per locus in the analyzed groups ranged from 2 to 9. The mean heterozygosity observed for each country of breeding and the wild population ranged from 28.9% to 41.2%, with an average value of 35.1%. Private alleles were observed in five samples across four populations in five different loci. The marker NVHfp5 was found to be monomorphic in the Danish population.ST varied from 0.008 (for the Poland captive-Czech Republic pair) to 0.081 (for the Poland wild-Denmark pair) with an average value of 0.035. The highest FST values were obtained for pairs containing Denmark . For each of these pairs, the calculated FST was greater than for any other pair . This may have been caused by the low number of individuals from Denmark included (n = 10). In addition, one of these individuals was properly genotyped in nine loci, while the genotype with the NVHfp92_1 locus was not obtained.Genetic variability factors across all samples are presented in ST, highest Nei’s genetic distance values were obtained for pairs containing Denmark . Nei’s genetic distance varied from 0.018 (for the Poland captive-Czech Republic pair) to 0.207 (for the Poland wild-Denmark pair) with an average value of 0.082. Similar to FThe best fit for the data determined by analyzing the STRUCTURE results with the “Evanno” method obtained for K = 2 groups . As FiguThe genetic distance tree  containiWhen only wild birds divided by ecotype (urban or tree-nesting) were analyzed, according to the STRUCTURE Harvester, the variability in the population was best described by division into two groups (delta K = 383.190615). Forty out of 57 urban ecotype individuals were classified into group 1, while 23 out of 34 tree-nesting individuals were classified into group 2 .The division into two groups is even more visible on the genetic distance tree （Compared to the Scandinavian population of peregrine falcons that was studied by Nesje et al. , who anan = 38), captive (n = 20), and contemporary (n = 44) wild populations. Compared to the historical populations originating from Denmark and Norway, for 6 out of 10 markers, the number of alleles was greater in our study, and for two markers, the number of alleles was lower. The observed heterozygosity was greater in the historical Scandinavian population for seven markers. Compared with captive and contemporary populations, the number of alleles in our population was greater for seven markers in both these comparisons. The observed heterozygosity was lower for seven markers; however, only one marker had a greater observed heterozygosity in both captive and contemporary populations.In another study, three groups of Scandinavian birds were analyzed : historiSimilarly, as with the Czech falcon population , the PolThe microsatellite analysis revealed a low level of genetic variation in the Fiji falcon population associated with a lack of gene flow from populations inhabiting the nearest islands and leading to differences among the analyzed populations . MicrosaA low level of heterozygosity was observed across all analyzed samples in loci NVHfp82_2, NVHfp79_4 and NVHfp5  in our sFulica atra) showed that long-established urban populations differ significantly from the forest population, although no significant differences between newly established urban populations and the forest population were identified [The main aim of the Polish reintroduction program has been to restore the tree-nesting population. Our wild population analysis shows genetic differences between ecotypes. The genetic distance tree shows individuals representing both ecotypes. Their diversity is mainly due to isolation—birds from the urban ecotype do not migrate to forests. However, the reintroduction process may change this relationship. Birds introduced in urban areas show greater diversity than tree nesting birds. However, no correlation between the genetic distance and the distance of the studied nests was found, which clearly indicates that in most cases there is no natural gene flow. An analysis performed on the Polish population of the Eurasian coot . Some d


Lycorma delicatula is native to China and invaded South Korea, Japan, and the United States during the last decade, causing severe damages to the fruits and timber industries. However, its global phylogeographic pattern and invasion history are not clearly understood. We applied high‐throughput sequencing to obtain 392 whole mitochondrial genome sequences from four countries to ascertain the origin, dispersal, and invasion history of the spotted lanternfly. Phylogenomic analyses revealed that the spotted lanternfly originated from southwestern China, diverged into six phylogeographic lineages, and experienced northward expansion across the Yangtze River in the late Pleistocene. South Korea populations were derived from multiple invasions from eastern China and Japan with two different genetic sources of northwestern (Loess Plateau) and eastern (East Plain) lineages in China, whereas the each of Japan and the United States had only one. The United States populations originated through single invasive event from South Korea, which served as a bridgehead of invasion. The environmental conditions, especially the distribution of host Ailanthus trees, and adaptability possibly account for the rapid spread of the spotted lanternfly in the native and introduced regions.Biological invasion has been a serious global threat due to increasing international trade and population movements. Tracking the source and route of invasive species and evaluating the genetic differences in their native regions have great significance for the effective monitoring and management, and further resolving the invasive mechanism. The spotted lanternfly  However, in its new habitats, it feeds on grapevines and other fruit trees, leading to a huge decline in the quality and yield of grapes and causing severe economic losses to the fruits industry. The invasion of the SLF into other country was first confirmed in 2004, when it was found in western South Korea. This is followed by its rapid spread across the country , ina Liu, . In ChinSeveral phylogeographic and population genetic studies have partially revealed the invasion process of the SLF between East Asian countries .A total of 392 SLF specimens were collected from 40 populations in China, South Korea, Japan, and the United States, covering the native and invasive regions of this species Figure , Table 12.2http://www.geneious.com/) with parameters of up to 5% mismatches, a maximum gap size of 5 bp, and minimum overlap of 40 bp with at least 95% similarity. Due to the distant relationships of the three species and the strict similarity and continuity requirements of the parameters, only reads of SLF were allowed to be isolated and mapped onto the reference genome from the sequencing readpool. The consensus sequences were generated as novel individual mitogenomes under threshold of 75% uniformity each site. The mitogenomic assembly of L. meliae also followed this workflow with additional mapping iterations to fish reads to known regions until the whole sequence was constructed  to reduce the sequencing expense after several preliminary experiments , the number of haplotypes (H), haplotype diversity (Hd), and nucleotide diversity (π) were calculated based on the PRT dataset using DnaSP software. We used Arlequin software to calculate the genetic differentiation FST values and to obtain p‐values for FST values using 3,000 permutations. MEGA7 software  were plotted with 999 replicates in the ade4 v. 1.7 module of R with the introduced populations excluded.To test genetic diversity divergence across geographic populations and phylogenetic lineages in our study, the number of polymorphic sites  and Harpending's raggedness index (r) were used to study the difference between observed and expected distributions . The introduced populations of South Korea  and Japan  showed much lower diversity , a total of 244 haplotypes were defined from 755 segregating sites. The overall Hd was 0.9892 and FST and genetic distances (p‐distances) between 40 populations were −0.0499 to 1 and 0.0001 to 0.0183 respectively, which were lowest between and within populations of eastern China and South Korea. Populations in different topographical areas, especially those separated by Yangtze River, had larger values (Tables p < .01) between the genetic and geographical distance in SLF populations  using ML and BI methods contained six phylogeographic lineages Figure . The disπ values, followed by Southeast Hills and Guizhou Plateau lineages  had higher diversity than those from northern Yangtze regions Table . Yunnan es Table . For thees Table .FST among the six lineages varied from 0.5275 to 0.9677. The largest value was detected between East Plain and Yunnan Plateau lineages with the smallest variation between East Plain and Sichuan Basin lineages  varied from 0.00085 to 0.01737 between the different lineages, being smallest between East Plain and Sichuan Basin lineages and largest between Southeast Hills and Yunnan Plateau lineages, which were both lower than 0.06021–0.06229 between two species  of SLF diverged into Yunnan Plateau and other lineages with the MRCA age of 191.4 ka (kilo‐annum) and 482.5 ka. The other lineages then diverged into two clades located in southern and northern Yangtze regions Figure . The sou3.3The median‐joining networks supported similar results of the phylogenetic relationships. For the East Plain (EP) lineage, several typical star‐like topologies were identified for 22 populations, indicating the recent expansion in this lineage Figure . The H5 For the Loess Plateau (LP) lineage consisting of 13 populations, two central haplotypes were found with small sample size. H24 was connected with most haplotypes from middle LP  and two East Plain (EP) populations  by less than five mutations Figure . Only onFor the Southeast Hills (SH) lineage including eight populations, no shared haplotype was found among different populations except H11 Figure . Unlike 3.4p < .05) of Tajima's D and Fu's Fs were detected in four lineages, East Plain, Loess Plateau, Sichuan Basin, and Southeast Hills lineages (Table p > .05). In the mismatch distribution analysis, the observed and simulated curves did not significantly differ from each other in all lineages with the parameters of SSD and r (Table p > .05). Hence, the population expansion process was not rejected in any of the lineages. In the BSP analyses, the trend of population expansion was only shown in the East Plain and Southeast Hills lineages  analyses were used to explore the demographic history of the six lineages. Significant negative values  and Loess Plateau lineages, whereas each of the United States and Japan had only one. Although the Korean Peninsula is bordering on northeast China in land, our network results did not support the dispersal of SLF across the land from northeast China, because the mutation step between northeast China and South Korea was large Figure  and SoutOf all individuals in East Plain, five possessed haplotypes of Loess Plateau lineage, which suggest another route of SLF invasion from Loess Plateau to South Korea and Japan via East Plain. In addition, the only one South Korea haplotype in Loess Plateau lineage motivated us to track the invasion scenario between Japan and South Korea, because it had the sister relationship with the monophyletic Japan haplotypes. When SLF was first recorded in South Korea, researchers had predicted that it would be introduced into Japan soon, and the first Japan record was obtained three years thereafter. A single invasive event from a small population of South Korea with the haplotypes of Loess Plateau lineage may be a reasonable explanation for the origin of Japan population. However, considering the higher haplotype diversity in Japan Table  and the The genetic source of population in the United States was inferred to be East Plain lineage. The lowest genetic diversity supported the single introduction hypothesis, and their closest relationships with South Korea haplotypes suggested the origin of the introduced population in the United States to be South Korea. In our study, South Korea was inferred to serve as a bridgehead in the global invasion history of SLF. The similar invasive pattern was also found in the soybean aphid (Kim et al., It is important to note that in the invasion history of SLF, the introduced population expanded quickly outward in South Korea (Han et al., 4.4Biological invasion represents one of the natural rapid evolutionary processes in contemporary time scales. The genetic diversity of introduced populations might increase largely due to different invasion scenarios (Garnas et al., 5Based on the detailed analysis of mitogenomic phylogeography, we explored the invasion history of SLF from China, to South Korea, Japan, and the United States. We revealed that the invasion of SLF into the United States may be a secondary invasion from a bridgehead population in South Korea. Our study supports the great potential of mass mitogenome sequences for resolving not only the phylogeographic pattern but also the invasive source and route of the pest species. Using high‐throughput sequencing and metagenomic approach, multiple invasive species could be analyzed together to reveal shared or different invasive patterns in defined regions. Mitogenome normally under strong purifying selection pressure offers little information for key genes behind invasion. Nevertheless, with whole genome sequence of SLF currently available (Kingan et al., The authors declare no conflict of interest.H.L. and W.C. designed the study. Z.D., Y.W., Z.C., L.C., T.I., S.K., and F.S. contributed to the sample collection. Z.D. performed the molecular experiments and phylogenomic analyses. All authors discussed the results, and Z.D., T.S., L.T., W.C., and H.L. wrote the paper.Supplementary MaterialClick here for additional data file.


Malus resistance genes (R) has not been previously studied for Venturia inaequalis fungus, the causal agent of apple scab. In this study, the sequences of two housekeeping genes encoding elongation factor alpha (EF-1α) and β-tubulin and two previously unstudied effector genes of V. inaequalis from mannosidase and glucosidase families of 100 strains collected from apple cultivars with Rvi6, Rvi1, and Rvi17 and without known scab resistance genes were submitted to the analyses. Based on the phylogenetic and diversity data, as well as recombination analyses of the sequenced regions, we assessed the phylogenetic relationships and genetic structure of the pathogen within the species and the evolutionary forces that are currently acting upon this microorganism. The topology of the obtained phylograms demonstrates the lack of a relationship between the phylogenetic position of the strain and the host cultivar and the geographical origin or race of the strain. The isolates from different hosts were differentiated but did not form diagnosable, distinct phylogenetic groups. These results suggest that the analyzed genes may be too conserved to reflect the adaptation of pathogens to apple genotypes with different R genes; thus, they do not adequately reflect race discrimination. In contrast, based on variation and gene flow estimation, genetic divergence was observed among strains virulent to apple trees containing Rvi6. The results of this study confirmed a lack of free recombination between strains and demonstrated that the analyzed regions are in linkage disequilibrium and contain non-random polymorphisms associated with the strain.The relationship between housekeeping and pathogenicity-related genes and virulence or avirulence towards the primary  Venturia inaequalis (Cooke) G. Winter is responsible for the most severe disease affecting apples, as well as many ornamental Malus species in many countries, including Poland: apple scab. Despite the consistent progress in breeding for resistance to apple scab, effective protection against the disease still primarily depends on repeated fungicide application in spring and leaf litter management in autumn [V. inaequalis races have been reported on Malus spp. [R). In recent decades, molecular population genetics have been widely utilized to demonstrate the patterns of genetic diversity within and between V. inaequalis populations and to trace the histories of divergence and speciation in pathogens by genotyping neutral microsatellite markers [Rvi6 apple gene [Rvi6 cultivars divide V. inaequalis populations into two genetically different subgroups, Rvi6-virulent (named race 6) and Rvi6-avirulent, while this structuring effect was not observed for other R genes. It is assumed that two pathogen lines exist: populations able to or not able to affect Rvi6 hosts, respectively. These presumably diverged and were separated by distance long time ago [Rvi6, Rvi17 or Rvi1), on other genomic regions of V. inaequalis, including the core genome with housekeeping genes or genes encoding pathogenesis-related proteins, has not been studied to date.Apples are among the most economically important fruit tree crops in temperate zones, including Poland. The ascomycete n autumn . To datelus spp. , with ea markers ,4,5. Sevple gene ,9,10,11.V. inaequalis genome that are not under direct selection pressure from fungicides or from host resistance genes [V. inaequalis to examine the changes in nucleotide diversity and to reveal intraspecies variations in this taxon. It was demonstrated that introns in the 18S rDNA gene and alleles of ITS1 provided genetic markers for subdividing populations of V inaequalis [V. inaequalis that are responsible for scab on Malus and Pyracantha [V. inaequalis strains derived from apple, pyracantha and loquat demonstrated that the strains were differentiated but did not form distinct phylogenetic species [Rvi6-virulent and -avirulent V. inaequalis populations to date, as was reported from multilocus microsatellite loci analyses [V. inaequalis fungus growing in vitro and in planta [The new gene combinations produced in the sexual stage are spread via abundantly produced spores of the asexual phase of fungus  which gece genes . Aside faequalis , while sracantha . Analysi species . In contanalyses . In paran planta , the uprMultilocus sequence analysis (MLSA) is a technique that enables the recognition of phylogenetic relationships between taxa based on genic regions. As reported by Hanage et al., 2006, the genes selected for the MLSA approach should be ubiquitous to the studied taxon and present in a single copy within the genome . SeveralV. inaequalis or in terms of their diversity in relation to the virulence abilities of V. inaequalis races.Housekeeping genes, as slowly evolving regions that encode proteins essential for microorganism survival , are suiV. inaequalis, one from mannosidase and one from the glucosidase family, were investigated to infer phylogenetic relationships and genetic structure within the species and to detect whether there is any relationship between genetic variation and host specialization among the strains of V. inaequalis species.In this study, two housekeeping genes, elongation factor alpha (EF-1α) and β-tubulin, and two previously unstudied effector genes of Rvi6 cultivars belong to distinct phylogenetic lineages; (b) the genetic divergence among groups of strains to identify intraspecific populations; (c) the evolutionary forces that are currently acting on this microorganism; and (d) the possible occurrence of recombination events within the species. To investigate these aims, we applied phylogenetic, diversity and recombination analyses to the sequence data of four genetic loci.The aims of the study were to assess (a) the phylogenetic structure of the pathogen within the species to answer the question of whether the populations that are virulent or avirulent to V. inaequalis ; thus, Although slightly different relationships between leaves among dendrograms obtained using Maximum Parsimony, Maximum Likelihood and Neighbor-Joining) criteria were observed, the genealogy of each locus was best resolved  by a single rooted NJ tree. The topologies of the trees representing all the regions and combined datasets were similar in the NJ and Bayesian analyses; therefore, only NJ trees are presented a–f, withRvi6, as was expected from previous studies. Moreover, no relation between strain position and its origin, i.e., plant cultivar or geographic localization, was detected. The visual inspection of well-supported nodes exhibited no particular topological congruence among individual gene trees. The majority of nodes on trees were supported with low bootstrap values (below 50), indicating low variation among strains.The phylogenetic analysis of EF-1α, β-tubulin, mannosidase and glucosidase regions or combined datasets of the housekeeping- or pathogenicity-related genes indicated slightly different topologies between the studied gene fragments. None of the genealogies resolved the isolates from different cultivars as distinct clades according to virulence type towards The lowest average sequence similarity within the analyzed fragment was observed for EF-1α (93.3%), while the highest was observed for mannosidase (98.9%). The G+C content was similar for all analyzed regions and ranged from 0.481 to 0.569, and the lowest G+C content was observed in mannosidase (below 50%). The highest percentage of parsimony informative sites and segregating sites (S), as well as the number of haplotypes per locus, were counted for EF-1α, although this gene fragment was the shortest one analyzed. Similarly, gene diversity (Hd) and Theta-W values were the highest for this region.Rvi6-virulent and -avirulent populations were compared for single loci, the percentage of segregating sites (S) and Theta-W values were higher in Rvi6 populations, with the exception of glucosidase  values for these regions. For EF-1α, β-tubulin and glucosidase, values of Tajima’s D test were higher for Rvi6 populations, although only for EF-1α were they significant.Only for EF-1α was the null hypothesis of neutrality rejected in both the Tajima’s D and the Fu and Li’s D tests. A significant negative value of D, but in only one of the tests, Fu and Li’s D test, was also observed for the β-tubulin gene. Positive or negative values of D statistics, although not significant, were observed for mannosidase and glucosidase, indicating that these regions are evolving close to mutation–drift equilibrium, and no evidence of selection can be observed. In concatenated dataset analysis, the number of parsimony informative sites, the percentage of segregating sites (S) sites and haplotypes as well as two parameters of nucleotide diversity (π and Theta W) and gene diversity (Hd) were higher for combined housekeeping genes than for pathogenicity-related genes b. Only fcosidase c. A valuThe results of the McDonald–Kreitman test, conducted on coding sequences, revealed that in EF-1α and glucosidase gene fragments, there is a twofold increased number of non-neutral polymorphisms compared to the number of neutral polymorphisms , while iRvi6-virulent and -avirulent populations, were highest for EF-1α and lowest for β-tubulin (XY) was the highest for EF-1α, while the average number of nucleotide differentiations between populations was the highest for glucosidase.Gene flow estimates of Nei , represe-tubulin . AdditioRvi6-virulent populations and the other containing Rvi6-avirulent populations, demonstrated that the largest genetic variation was partitioned between individuals within populations , while the lowest values were observed among groups (0.6–3.2%) (Rvi6-virulent or Rvi6-avirulent populations in the group), the greatest genetic variation was again partitioned between individuals within populations (from 77.0 to 96.6%), regardless of the gene and group, but the variation among populations was significantly higher in the Rvi6-virulent group than in the Rvi6-avirulent group for the analyzed genes with the exception of the β-tubulin region. In this instance, variation among populations was higher for the Rvi6-avirulent group than for the Rvi6-virulent group.Analysis of molecular variance (AMOVA) among the two groups, that is, one containing only .6–3.2%) . The excRvi6-virulent and -avirulent populations, significant FST values were higher for the two analyzed housekeeping genes, 0.34 and 0.23 for β-tubulin and EF-1α, respectively, than the values obtained for genes related to pathogenicity: 0.16 and 0.08 for mannosidase and glucosidase, respectively , while Rvi6-virulent populations were more distanced  split decomposition and recombination detection analyses, b) index of association (rBARd) and c) the four gametes test for the detection of the minimum number of recombination events (MR).The extent of linkage disequilibrium and recombination between and within p < 0.05) in glucosidase and mannosidase genes but not in EF-1α and β-tubulin loci. Additionally, recombination signals were detected using two out of seven algorithms under RDP4 for the glucosidase locus only. Consequently, evidence for recombination in the phi test, as well as recombination signals, were detected in the concatenated mannosidase and glucosidase dataset only .(a) Split decomposition analyses revealed a lack of reticulation among strains for all studied regions analyzed as separate or concatenated datasets, suggesting a lack of recombination between strains. However, the phi test did find statistically significant evidence for recombination  for all analyzed regions and combined pathogenicity-related regions (p > 0.05). Complete panmixia, related to rBARd values near 0, is expected for fully recombining populations, while in linkage disequilibrium, a deviation from panmixia is observed, related to an rBARd value significantly greater than 0.(b) The random recombination hypothesis was tested both for all strains without any grouping assumptions, as well as for the strains divided into two assumed populations:  regions , showingMR) needed to explain the alleles was ≥1 [(c) Data were considered to have failed the simple four gamete test if the minimum number of recombination events  based on pathogenicity-related regions. In contrast to microsatellite markers [Although it was reported that sequences of genes encoding pathogenicity factors, such as endopolygalacturonase and exopolygalacturonase, have been used and proved to be helpful to reveal the relationship of genetic divergence with pathogenicity variation , in our  markers , both ho markers , which lRvi6 populations for three of four analyzed loci, while in Rvi6-avirulent populations, they were higher for mannosidase. Tajima’s D may be higher in populations that have experienced a recent bottleneck because of the preferential loss of low-frequency variants [Rvi6 strains were the highest for the EF-1α gene and the lowest for β-tubulin. This finding may suggest that housekeeping genes may be under selective pressure to retain function, although the pressure can act on some regions only. In addition, the McDonald–Kreitman test conducted between the two groups of strains demonstrated diversifying selection acting on EF-1α and glucosidase gene fragments, while the β-tubulin region was under neutral selection, and the mannosidase protein fragment was the most evolutionarily conserved in the study. More detailed studies are necessary to establish which genes are relevant for the adaptation of V. inaequalis strains to the defined virulence type.To demonstrate the genetic diversity, population divergence and neutrality of mutations, we used Tajima’s D and Fu and Li’s D tests. Values of Tajima’s D test were higher in variants . The expvariants . HoweverRvi6-virulent populations than in Rvi6-avirulent populations, which was congruent with gene flow FST estimates, indicating that populations in the Rvi6-virulent group were more distanced, while those in the Rvi6-avirulent group were closely related. Meanwhile, higher gene diversity was observed for two regions in Rvi6-virulent populations, while for two other regions, it was observed in Rvi6-avirulent populations. Usually, a higher number of segregating sites, nucleotide diversity and Theta-W values were found in Rvi6-virulent populations than in Rvi6-avirulent populations for three loci. Selection might not be the main reason for the severe reduction of diversity in Rvi6-avirulent populations, although the effects of selection cannot be excluded entirely. The reduced level of genetic diversity may reflect the consequence of evolutionary processes, such as a recent speciation event, population bottleneck or selective sweep for a preferred genotypic variant in V. inaequalis [Rvi6-virulent populations were characterized by low gene diversity, even demonstrating a clonal structure [Rvi6 populations are the descendants of a few genetically different virulent individuals that migrated relatively recently to Poland from non-agricultural reservoirs, and they are not abundantly represented in our county at present. In contrast, Rvi6-avirulent populations, commonly occurring in our agrosystem for decades and which are evolutionarily older, were characterized by higher allelic and private allelic richness, as well as higher genetic diversity [Rvi6-avirulent individuals are only a small representation of the whole Rvi6-avirulent population in Poland and that the rate and mechanism of evolution are different for genic and microsatellite regions, and their effects cannot be compared. These genes were studied to assess another level of genetic variation between pathogen populations. However, the examined regions are not sufficient to resolve differentiation among Rvi6-virulent and -avirulent populations, while generally, genetic diversity between populations is supposed to be a factor facilitating adaptation of pathogens to specific resistance genes of the host both by increasing risks of avirulence losses and the spread of new virulences.Moreover, in most cases , higher variation was observed among aequalis . This fitructure ,11. It hRvi6-virulent and -avirulent, which indicates that some loci can be associated. Theoretically, linkage disequilibrium is affected by various evolutionary and demographic factors, including selection, recombination, population admixture, inbreeding and bottlenecks [Rvi6 cultivars). However, this reproductive barrier or significant genetic differentiation between groups cannot be explained by geographic isolation, since strains of different virulence were sampled in the same orchard in half of the cases (strains from Lublin and Nowy Dwor). Based on the data obtained in this study, we did not find evidence of gene flow after secondary contact in the analyzed regions, as was previously observed for virulent and avirulent Rvi6 populations based on microsatellite markers [Depending on the algorithm used, only in glucosidase or in the glucosidase and mannosidase concatenated dataset were recombination signals detected. On the other hand, estimation of the index of association based on four single loci demonstrated that random mating was restricted both among all strains and between assumed groups of strains, that is, tlenecks . The res markers . Moreove markers . In our Malus x domestica, in addition to one location where leaves of crab apple trees were obtained, F1 seedlings of Malus x zumi var. “Colocarpa” originating from open pollination  were collected. The sampled apple trees represented both mono-R-genic cultivars  and cultivars without any known sources of apple scab resistance (hereafter designated Rvi0). The scab lesions were sampled and prepared for the analysis as described previously [V. inaequalis, which were divided into 20 groups according to cultivar and/or location, each containing five strains, and the DNA was subjected to further analyses  located in the central, southern and northern parts of Poland. In all the orchard trees of eviously . In totaanalyses .V. inaequalis (“Venin1”), provided by Cecilia Deng at the New Zealand Institute for Plant and Food Research Limited, available at https://genome.jgi.doe.gov/Venin1/Venin1.home.html (accessed on 3 February 2021), was searched for CWDE sequences. Among these sequences, two contigs, one complementary to mannosidase and one to glucosidase of Venin1, were selected, and primers were designed for them using PrimerSelect software of the Lasergene v. 8 package . The specificity of designed primer sets was tested in conventional PCRs using DNA from 100 isolates of V. inaequalis pure culture . After the optimization process was undertaken, two primer sets were selected and used for the amplification of part of mannosidase and glucosidase gene sequences, VinManno1 and VinManno2, and VinGluco1 and VinGluco2 ). Alignment was then visually refined, and the consensus sequence for each gene was employed as a query in Nucleotide Basic Local Alignment Search Tool (BLAST) to search the global GenBank sequence database, available at https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 3 February 2021), and the V. inaequalis reference genome sequence. Alignments per gene with all sequences trimmed to the same length, as well as concatenated alignments of housekeeping or pathogenicity-related genes, were exported to nexus files. All sequences used in the study were submitted to the GenBank database, and accession numbers were assigned for them  and sequenced in both directions with the same primers used for amplification . The obtained sequences for each gene fragment were assembled and initially aligned using MEGA v. 6.0 software  version 4.0b10  to ensurThe percentage of similarity between sequences was calculated for each gene fragment using MegAlign software from the Lasergene v. 8 package.For each gene, the number of haplotypes (h), number of polymorphic (segregating) and parsimony informative sites, G/C content and haplotypic diversity (Hd) were calculated, and 2 parameters of nucleotide diversity, polymorphism , the average number of nucleotide differentiations between groups and the average number of nucleotide substitutions per site  were calculated.When two main groups of populations were assumed, that is, http://bioinf3.uab.cat/mkt/MKT.asp, accessed on 3 February 2021) was applied to coding sequences of the studied gene fragments to provide evidence of selection in the case of non-neutral polymorphism [Rvi6 and non-Rvi6 populations of V. inaequalis. All nucleotide sequences were converted to amino acid sequences according to an adequate reading frame using the EXPASY translate tool  and were subsequently utilized to construct neighbor-joining dendrograms  to visualize the topologies of amino acid sequence-derived trees with DNA sequence-derived trees.Tajima’s D genetic diversity and population divergence test  and Fu amorphism . The tesXY) and the average number of nucleotide differentiations between populations were calculated between assumed populations (Rvi6-virulent vs. Rvi6-avirulent) using the DnaSP program.Gene flow estimates of Nei , the aveRvi6-virulent and -avirulent populations were also assessed based on the fixation index (FST) measuring the population differentiation among strains, applying 100 permutations using ARLEQUIN 3.5.1.2 [ST, can range from 0 (no differentiation between populations due to unrestricted gene flow) to 1 (complete isolation due to the absence of gene flow) [Rvi6-virulent populations and the other containing only Rvi6-avirulent populations. The total amount of differentiation among the groups of populations, among populations within the groups and individuals within populations was evaluated by applying global hierarchical analysis of molecular variance (AMOVA) using 1023 permutations and a significance level of p ≤ 1 for single loci.The gene flow estimates between  3.5.1.2 . Estimatne flow) . Each ofThe best fit evolutionary model of DNA substitution for four datasets was calculated using jModelTest v. 2.1.1 . The staPhylogenetic analyses were performed by a heuristic search for optimal trees under three optimality criteria: parsimony (MP), likelihood (ML) and distance (NJ) using MEGA6. NJ trees were constructed based on model-based corrected distances, as indicated by jModelTest for each dataset.The strength of the internal branches of the resulting trees was tested with bootstrap analyses using 1000 replications in MEGA6.MrBayes v. 3.2.6  was usedThe split decomposition method  was usedRvi6 cultivars and one containing the rest of the strains. The significance of the test was assessed by comparing the original observed dataset with those in which alleles were resampled without replacement to simulate recombination in 100 randomized datasets.Association among alleles, as expected under clonality, was investigated using a generalized measure of multilocus linkage equilibrium (rBARd) ,57, whicMR).Finally, the simple four gamete test was performed within each locus and combined dataset using DnaSP to estimate the minimum number of recombination events in the history of each gene sample (Rvi6 host plants, and these results confirm higher genetic distance and variation among Rvi6-virulent populations than among avirulent ones and are in contrast to some results from previous studies based on microsatellites concerning the level of genetic diversity in Rvi6-virulent and -avirulent populations. Discrimination between V. inaequalis populations of different virulence types is achieved primarily by testing their pathogenicity on apple cultivars with defined R genes [V. inaequalis races.The four loci examined in this study are only a small part of the total genome. In our study, the total information obtained from these data did not demonstrate one consensus phylogenetic resolution for the analyzed strains; probably, involvement of several additional regions would be required to obtain more informative phylogenetic trees. On the other hand, these data provided some indications related to evolutionary forces affecting the strains belonging to different pathological races, either virulent or avirulent to  R genes ; neverth


Culex tritaeniorhynchus Gile is a major vector of Japanese encephalitis in China. The population genetics study is crucial as it helps understanding the epidemiological aspects of mosquito-brone diseases and improving vector control measures. Here, the genetic population structure of C. tritaeniorhynchus in the mainland China were estimated using the cytochrome c oxidase subunit 1 (COI) DNA barcodes region. 485 individuals of C. tritaeniorhynchus were collected from 38 sampling sites in 21 geographic populations in the mainland China. In total, 485 sequences were used to explore the population structure and genetic diversity. The results showed that the populations of C. tritaeniorhynchus had high haplotype diversity , low nucleotide diversity (p = 0.02245) and high gene flow (Nm = 47.11) with two maternal lineages and four groups. An AMOVA indicated that 98.8% of the total variation originated from variation within populations. In addition, the population genetic structure exhibited by C. tritaeniorhynchus filling the vacant of the genetic structure in the mainland China. Human activities may also assist mosquito movement and migration. Gene flow among the populations of C. tritaeniorhynchus can facilitate the spread of insecticide resistance genes over geographical areas, and it will be a challenging for controlling the populations. Culex tritaeniorhynchus Gile is the principal vector of Japanese encephalitis (JE). JE is a mosquito-borne zoonosis caused by infection with Japanese encephalitis virus (JEV). JE usually manifests as mild central nervous symptoms, primarily in children and adolescents. In addition, it can also cause sow abortion and equine encephalitis, bringing serious economic losses  DNA barcodes region is considered as a valuable and reliable diagnostic tool for studying the genetic structure and genetic diversity of mosquitoes  and India (KM350664.1-KM350669.1).Mosquitoes were collected from the following 38 sampling sites in 21 geographic populations: Liaoning (LN), Zhejiang (ZJ), Shanghai (SH), Jiangsu (JS), Fujian (FJ), Hebei (HB), Jiangxi (JX), Beijing (BJ), Hubei (HUB), Henan (HEN), Guangdong (GZ), Hunan (HUN), Hainan (HAN), Guangxi (GX), Shaanxi (SX), Chongqing (CQ), Guizhou (GZ), Gansu (GS), Ningxia (NX), Sichuan (SC), Yunnan (YN) in the mainland of China, July to September from 2014 to 2020 . Adult f2O. The amplification program consisted of one cycle at 94 °C for 5 min, followed by 35 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, and one cycle at 72 °C for 5 min  and a DNA/RNA Extractor-32 system (BioTeke). A 658-bp fragment of COI region was PCR-amplified using the Folmer primers LCO1490 and HCO2198 , C. interrogator (JX259909.1), C. tarsalis (GU908102.1), C. salinarius (GU908096.1), C. nigripalpus (JX259910.1), and C. pipiens pallens (FN395206.1), C. pipiens (GU908084.1), C. quinquefasciatus (HQ398883.1), C. quinquefasciatus (GQ165798.1) were included as out-group taxa. A mtDNA COI haplotype network was constructed using the TCS inference method  were showed in C. tritaeniorhynchus populations. A total of 303 haplotypes were identified which the most common haplotype was H2, H55 and H130. Genetic distance was calculated based on the Kimura 2-parameter model. The results showed that the intraspecific genetic distance was 0.000–0.069 among 21 C. tritaeniorhynchus population. The average genetic distances between intraspecific genetic distance was 0.024.The COI gene amplified 658 bp sequence with 160 variable sites, 94 parsimony-informative sites and 66 were singleton variable sites for C. tritaeniorhynchus was due to genetic variation within populations. According to the topology of NJ tree and ML tree, the phylogenetic tree showed the clustering of these samples into two maternal lineages and four groups, group one includes Singapore, LN, FJ, HB, BJ, HEN, GZ, HUN, HAN, GX, HUB, CQ, SC, group two includes India, YN, GZ, group three includes JX, JS, ZJ, SH, group four includes GS, NX, SX .The AMOVA results are displayed in , NX, SX . The res, NX, SX . The 21 C. tritaeniorhynchus in the mainland China with sampling spanning many of provinces. It was found that C. tritaeniorhynchus with high haplotype diversity and low nucleotide diversity, we assumed that C. tritaeniorhynchus population had experienced a bottleneck effect, accompanied by rapid population expansion and accumulation of variation . AMOVA analysis showed that the genetic differentiation of C. tritaeniorhynchus was mainly between individuals within the population (98.80%), and there was little difference between different populations (1.20%). This may due to the long flight distance of C. tritaeniorhynchus, with an average maximum flight distance of 2.2 km and a maximum flight distance of 7.5 km , Ningxia (NX) and Shannxi (SX) are grouped into one branch, NX was in the north of China which occurred an outbreak of JE in 2018 (Liu et al. C. tritaeniorhynchus may increase the risk of JE in these areas. With the movement of people and trade, it will enhance the gene flow between adjacent areas which leads to the formation of groups three and four. C. tritaeniorhynchus is the main vector of JE with a wide distribution in China. Except the use of bed nets, window screens and repellent, insecticides are also effective for vector control. Gene flow among the populations may assist the spread of insecticide resistance genes over geographical areas (Barnes et al. C. tritaeniorhynchus population in the mainland China which providing guidance for vector control of JE disease.According to the topology of NJ tree and ML tree, COI sequences of C. tritaeniorhynchus has a certain pedigree and geographical structure in the mainland China. The results showed that the populations of C. tritaeniorhynchus had high haplotype diversity, low nucleotide diversity and high gene flow with two maternal lineages four groups. Human activities and the use of pesticides will affect the mosquito populations which may develop insecticide resistance gene and a challenging for controlling the mosquito vector populations.In this study,