Aedes albopictus is an indigenous primary vector of dengue and Zika viruses in China. Understanding the population spatial genetic structure, migration, and gene flow of vector species is critical to effectively preventing and controlling vector-borne diseases. The genetic variation and population structure of Ae. albopictus populations collected from 22 cities along the Yangtze River Basin were investigated with nine microsatellite loci and the mitochondrial CoxI gene. The polymorphic information content (PIC) values ranged from 0.534 to 0.871. The observed number of alleles (Na) values ranged from 5.455 to 11.455, and the effective number of alleles (Ne) values ranged from 3.106 to 4.041. The Shannon Index (I) ranged from 1.209 to 1.639. The observed heterozygosity (Ho) values ranged from 0.487 to 0.545. The ISF value ranged from 0.047 to 0.212. All Ae. albopictus populations were adequately allocated to three clades with significant genetic differences. Haplotype 2 is the most primitive molecular type and forms 26 other haplotypes after one or more site mutations. The rapid expansion of high-speed rail, aircraft routes and highways along the Yangtze River Basin have accelerated the dispersal and communication of mosquitoes, which appears to have contributed to inhibited population differentiation and promoted genetic diversity among Ae. albopictus populations. Ae. albopictus is an important vector of dengue fever, chikungunya fever, and Zika fever globally [http://www.iucngisd.org/gisd/100_worst.php (accessed on 12 December 2000)). Except for Antarctica, the distribution area of Ae. albopictus expands worldwide [Ae. albopictus is a native mosquito species in Asia, including in most parts of China, where the distribution range of the species includes southern Chaoyang, Liaoning Province, eastern Baoji, Shanxi Province and Tianshui, Gansu Province [Ae. aegypti, which is found only in several provinces in the southern part of China, Ae. albopictus is the main vector of dengue fever in mainland areas and the main blood-feeding mosquito in urban and rural areas of China [globally  and is oorldwide ,4. Ae. aProvince . Compareof China .Ae. aegypti, Ae. albopictus, Ae. japonicus and Ae. koreicus beyond its original distribution to local, regional and intercontinental areas occurs via river boats, sea vessels, automobiles and airplanes [Ae. aegypti is supported by a low diversity of haplotypes [Ae. aegypti is relatively differentiated among 15 seaports in the Philippine Archipelago: low genetic structure and considerable gene flow was found in busy ports, with even distant ports being genetically similar; in small ports, even nearby ports, marine human-mediated long-range dispersal plays a pivotal role in determining the population structure of Ae. aegypti [Ae. aegypti is more affected by cargo shipments than by passenger ships. This indicates that Ae. aegypti gene flow among sub-populations is greatest between locations with heavy marine traffic [In China, the shipping capacity of the Yangtze River exceeds 71,000 km per year, which is 56% of the total inland river. Meanwhile, its passenger capacity and the volume of freight traffic are 180 million people and 4.714 billon tons, respectively, which is 65% of the total passenger capacity of water passenger volume and 70% of the total water freight traffic volume, respectively. Due to its massive transportation capacity, a comprehensive transportation system of waterway networks, on-land traffic, railways and aviation is well developed alongside the Yangtze River. Mosquitoes can disperse through man-aided transport, especially species of Aedes, involving diapause or desiccative eggs and larvae in container water, and expansion of irplanes ,20,21. Rplotypes . Populat aegypti . It has  traffic .Ae. albopictus probes showed worldwide dispersal and invasion patterns at regional and country levels based on microsatellites and SNPs in the genome and mitogenome to reveal population diversity [Ae. albopictus populations in the native range, including China, Thailand and Japan, indicates the maintenance of high genetic connectivity of these populations. Using 13 nuclear microsatellite loci and mitochondrial CoxI sequences, it was determined that Ae. albopictus was most likely introduced to New Guinea via mainland Southeast Asia before colonizing the Solomon Islands via either Papua New Guinea or SE Asia, which supports that the recent incursion into northern Australia’s Torres Strait Islands was seeded chiefly from Indonesia. This study was the first to document evidence of a recently colonised population (the Torres Strait Islands) that has undergone rapid temporal changes in its genetic makeup resulting from genetic drift or representing a secondary invasion [Dispersal patterns and population genetic structure have been analysed to reveal the genetic diversity and gene flow of populations of vector mosquitoes and to supply population genetic information for surveillance as well as for assessing vector competence and integrated management of the species. A series of studies on the population genetics of iversity ,26,27,28invasion .Ae. albopictus individuals can fly a limited distance of 500 m for blood feeding and oviposition, and long-distance dispersal of Ae. albopictus may occur through the convenient transportation system and the broad international, national and regional trade of tires, lucky bamboo and other items, where larvae and eggs could survive in container water [Ae. albopictus in cities and counties along the Yangtze River Basin to reveal its dispersal and population genetic structure under conditions of convenient and heavy transport and to aid in the surveillance and control of the species.Female er water . The preAe. albopictus individuals were collected between July and September 2018 at 22 cities along the Yangtze River Basin, China  and 5′-CCCGGTAAAATT AAAATATAAACTTC-3′ (R-CoxI)) in a 50 µL reaction mixture containing 10 µL PCR buffer, 4 µL dNTPs, 1 µL primers, 0.5 µL Prime STAR HS DNA polymerase, and 34.5 µL ddH20. The PCR amplification program was set as follows: pre-denaturation at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 s, annealing at 54 °C for 45 s and elongation at 72 °C for 1 min, with a final extension at 72 °C for 10 min. All PCR products were separated by 2% agarose gel electrophoresis for detection. The target fragments for CoxI were excised from the gel under UV light and purified with GenElute™ PCR Clean-Up Kit. Each purified PCR product was cloned into the PCR™2.1 vector using TA Cloning™ Kit and selected by bacterial liquid PCR using T7 promoter primers. Finally, at least 20 positive clones for each PCR product were sequenced on both strands using an ABI 3730 × L automatic sequencer .A total of 624 mosquitoes from each of the 22 sample sites were genotyped, and at most 3 to 5 adult mosquitoes were picked out from the same wild breeding place. DNA of individual mosquitoes was extracted following the steps provided with a TAKARA DNA kit and stored at −30 °C until analysis. Individuals were genotyped based on variation at nine microsatellite loci . ApproxiHe), observed (Ho) heterozygosity), the mean number of alleles (Na), and F-statistic parameters  were estimated from allele frequencies with FSTAT 2.9.3.2 [HWE) using GENEPOP’007 [p values were calculated using the Markov chain algorithm with 10,000 dememorizations, 500 batches, and 5000 iterations per batch. Arlequin software 3.5.2.2 was used to assess pairwise differences between populations [ST of each population was calculated from the mean value of pairwise differences between populations. An excess of heterozygosity in a significant number of loci  is an indicator of a genetic bottleneck, whereas the opposite  [D test was tested via Arlequin 3.5.2.2 to determine the population diffusion of each population, while dispersal routes among 7 locations were calculated via the R packages “divMigrate” with the number of bootstrap replicates set to 3, the α value set to 0.05, the Nm method chosen for the migration statistic and the filter threshold value set to 0.15.Polymorphic information content (PIC) for each locus was calculated using POPGENE Version 1.3 and PIC-Calc 0.6. Genetic diversity   suggestsHe, Heq) , and genHe, Heq) ,40. MoreAe. albopictus populations were collected along the Yangtze River Basin . In addition, pairwise FST values between populations range from 0.002 (Anhui and Jiangsu) to 0.108 (Shanghai and Chongqing). Overall, genetic differentiation between Ae. albopictus populations in Shanghai and the other six regions was found to be moderate, whereas the genetic differentiation levels among the other six regions is negligible , Shanghai (SH), Jiangsu (JS), Jiangxi (JX) and Anhui (AH) to Hubei (HB), where gene flow (Nm) among populations was observed to be greater than 0.70, except in Shanghai ; While significant positive correlation (p = 0.009) between the genetic distance and the geographical distance of all individuals, when the Shanghai population was eliminated  are common indices to evaluate the genetic diversity of populations ,43,44, apulation . The avepulation ,46,47. SAe. albopictus populations were adequately allocated to three clades with significant genetic differences. Shanghai was completely separated from the other six regions, and the Fst values > 0.05 in the population of Shanghai and the other six regions indicated a moderate level [Ae. albopictus populations [Ae. albopictus populations in the Yangtze River Basin and the significant bottleneck effect of each Ae. albopictus population, we hypothesised that the rapid expansion of high-speed rail, aircraft routes, and highways along the Yangtze River Basin has not only facilitated trade among different cities but has also accelerated the spread of mosquitoes. Meanwhile, compared with other cities, Shanghai’s disinfection work may be more thorough, resulting in a single diversity of mosquitoes.According to the results of population differentiation analysis, all te level . Previoute level . Genome-ulations . Landscaulations . With thAe. albopictus populations contain 13 polymorphic sites, which is much higher than in other areas. The Shannon index was also richest in Hubei due to the high gene flow (Nm > 0.70) between Hubei and the other collection sites, except Shanghai (Nm = 0.17). This may be due to natural and geographical factors in Hubei, which is known as the “province of thousands of lakes”. Hubei has a rich ecosystem and various geomorphological types, including mountains, hills, and plains. Hubei has a strong high-speed rail hub network, air transport network, highway network and water transport system, directly connecting it with 10 other provinces and cities in China. Ae. albopictus populations can disperse and communicate via convenient transportation [As a transportation hub, Hubei Province connects the upper and lower Yangtze River. According to the results of haplotype diversity, Hubei ortation , which iAe. albopictus displays no relationship with geographic distance in a study in which the mosquitos were sampled from 19 sites across 2512 km2 of volcanic Réunion Island [Ae. albopictus was detected within individuals, at 97.97%. Long-distance dispersal of mosquitoes including Ae. albopictus and Ae. aegypti is driven by human activity, with particular emphasis on human trafficking [In general, the genetic distance of populations and geographic separation are positively correlated; however, no significant correlation between these two variables was observed among all populations in the present study. Moreover, the genetic distance of n Island , with din Island . This mofficking ,50,53,54IS is an index of inbreeding within populations. In present study, all FIS values tested are positive, which means different degrees of inbreeding exist in the populations. Meanwhile, Ho values were lower than He values, which also indicated high gene consistency for the Ae. albopictus populations and that the population might be inbreeding. A combination of anthropogenic and natural factors, such as eradication of breeding habitat, high temperature and decreased rainfall, may lead to breeding habitat fragmentation and reduced population size and therefore increase the likelihood of inbreeding, leading to a heterozygosity deficit [D test results showed that all populations were negative, suggesting that Ae. albopictus populations in the Yangtze River Basin are expanding. This may be due to the influence of hosts and various vehicles in the area.F deficit ,56. NeveAe. albopictus populations in the Yangtze River Basin. All Ae. albopictus populations were adequately allocated to three clades with significant genetic differences. Continuous dispersal and communication, as aided by human activities and transportation, appears to contribute to inhibited population differentiation and to promote genetic diversity among Ae. albopictus populations in the Yangtze River Basin.The present study systematically evaluated the genetic variation, population structure and haplotype-based phylogenetic relationships of


Here, we have evaluated the general genomic structure and diversity and studied the divergence resulting from selection and historical admixture events for a collection of worldwide chicken breeds. In total, 636 genomes (43 populations) were sequenced from chickens of American, Chinese, Indonesian, and European origin. Evaluated populations included wild junglefowl, rural indigenous chickens, breeds that have been widely used to improve modern western poultry populations and current commercial stocks bred for efficient meat and egg production. In‐depth characterizations of the genome structure and genomic relationships among these populations were performed, and population admixture events were investigated. In addition, the genomic architectures of several domestication traits and central documented events in the recent breeding history were explored. Our results provide detailed insights into the contributions from population admixture events described in the historical literature to the genomic variation in the domestic chicken. In particular, we find that the genomes of modern chicken stocks used for meat production both in eastern (Asia) and western (Europe/US) agriculture are dominated by contributions from heavy Asian breeds. Further, by exploring the link between genomic selective divergence and pigmentation, connections to functional genes feather coloring were confirmed. Its domEarlier genomics‐based population genetics studies in livestock and nonmodel organisms have explored population differences, inferred demographic histories, and quantified admixture to provide deeper insights to the origin, evolution, and domestication of a range of species  were generated from libraries prepared using the NeoPrep Library Prep System (Illumina) and sequenced to, on average, 8× genome coverage by paired end (2×150 bp) sequencing on the IIlumina Hiseq X Ten system at CLOUD HEALTH. Table The current study uses both publicly available   package . Local realignment, base quality score recalibration, and joint variant calling on all the samples were done using the IndelRealigner, BaseRecalibrator, and HaplotypeCaller from GATK (version 3.7). Variant filtering was implemented for each autosomal chromosome individually. Small indels and variants with more than 2 alleles were removed, and then, low mapping quality variants that did not meet the criteria  were filtered out using VCFtools  which is based on a hidden Markov model  was used to evaluate the overall population structure across the samples Figure . The firhttps://livestockconservancy.org/index.php/heritage/internal/rired).The individuals of the populations sampled from Tibet and Sichuan (China) were often scattered both between, and sometimes also within, the different sampled stocks. This is likely due to the common admixture events in these regions. Close relationships were sometimes also observed between breeds sampled from different geographic regions. An interesting one is between the Chinese breed Liyang  and the Rhode Island Red (RIR). These as historical records have reported Chinese breeds from this region being important during the creation of RIR , an old American multi‐purpose breed that in this analysis, was found to be related to many of the heavy Asian and Western chicken breeds, in particular the Java (JA), Cochins , and Light Brahma (LB). The Recessive White (RW), Liyang (LY), and Lingnan Yellow (LNY) are closely related, with only minor differences in contributions from different ancestry sources. The Cobb (CB) is a modern western broiler breed and it shares a majority of ancestry sources with the other heavy breeds. As expected, the Langshan populations from the United States (LSUS) and China (LS) are closely related. Also, the different varieties of the Cochin breed are closely related to each other and with Java.3.1.3PopLDdecay 3.31. Overall, the results were consistent with expectations given what is known about them regarding effective population sizes, when they were founded and intensity of artificial selection applied. Populations with slow LD decay were generally found to have been founded more recently, and often also more intensely selected in smaller populations. Most extreme are the Virginia BW lines .Consistent with available historical records Roberts, , the genD = 0.13; DZvalue = 17.0; Figure The Rhode Island Red (RIR) breed was originally developed as a dual‐purpose breed and was later more intensely selected for use as a specialized layer breed. Historical records describe that the RIR originates from crosses of Cochin, Java, Malay, Shanghai, and brown Leghorns Ekarius, . Here, a3.2.3Fst between recessive white and black populations and haplotype selection identified a selection signal at the TYR  and extension (E) alleles. Selective sweep analyses to evaluate the basis for the dominant white was performed by comparing dominant white/recessive white and dominant white/black breeds, and both identified the strongest signal in the region of the ERBB3 gene  family of receptor tyrosine kinases. It is known to be located in the region associated with the dominant white phenotype in several linkage mapping studies  admixture in the later stages of intensive breeding and commercialization of the chicken for food production was explored in detail. Overall, the results show a major contribution by Asian populations to the worldwide chicken population consistent with several waves of introgression followed by population admixture and continued selection. Several detailed findings, where genomics either confirms proposed introgressions or suggest new possible origins of important current breeds, were revealed. Genomic evidence suggests a large contribution by the Shanghai chicken, here represented by the modern descendent the Liyang breed, to major breeds used during the establishment of modern commercial broiler stocks: the Rhode Island Red and Recessive White. The findings here add to earlier work (Dana et al., Except the improvement by the large Asian breeds, the Mediterranean fertile stock Black Minorca also play an important role in modern layer breeding. Evidence from both the historical records Roberts,  and our 4.2The ROH distribution was used to investigate the population demographic history. An increased burden of ROH was common in the analyzed US samples, likely a reflection of a limited effective population size in the past and recent inbreeding. A different pattern was observed for the Virginia BW lines and the White Leghorn with enrichments in both long ROH and the total sum ROH Figure . This su4.3Subgroups were identified in the Red Junglefowl population Figure . The str5Here, we describe the population structure of worldwide chicken breeds and report the most comprehensive genome‐wide comparison between local Asian and modern Western chicken breeds to date. The genome‐wide relationships between the populations were explored to confirm suggested and suggest novel relationships between Asian, Western, and Mediterranean breeds, as well as molecular genetic mechanisms of breed‐defining phenotypes. A large contribution by Asian chicken breeds to Western breeds were found via the introgression of locally developed breeds, illustrating the value of utilizing worldwide genetic variation for the progress of modern poultry breeding.The authors declare that there is no conflict of interest.Supplementary MaterialClick here for additional data file.


Fagus crenata, is widely distributed across the Japanese Archipelago and islands in Japan Sea. Similar to the northern limit of the geographical distribution of F. crenata on the mainland of Hokkaido, the northern limit of the distribution of F. crenata on islands in the Japan Sea is observed on Okushiri Island (ca 42°N). To understand the genetic relationships of F. crenata on Okushiri Island, we examined chloroplast (cp) DNA haplotypes and 11 nuclear microsatellite (SSR) loci among 1,838 individuals from 44 populations from Okushiri Island, mainland Hokkaido, and the northern part of the Tohoku region on Honshu Island. We identified 2 cpDNA haplotypes, which represent not only populations on the Japan Sea coast but also those on the Pacific coast and this suggested the Okushiri Island populations might not be formed by single colonization. Genetic diversity of the Okushiri Island populations of nuclear SSR was not lower than the mainland and the STRUCTURE analysis revealed the Okushiri Island individuals were admixed between Hokkaido and Tohoku clusters. Approximate Bayesian computation inferred that divergence between Tohoku and Hokkaido, and admixture between two populations which generated Okushiri populations occurred before the last glacial maximum (LGM), that is, 7,890 (95% hyper probability density (HPD): 3,420 – 9,910) and 3,870  generations ago, respectively. These inferences were well supported by a geological history which suggested an isolation of Okushiri Island from Hokkaido started prior to the Middle Pleistocene. We discuss the possible persistence of F. crenata during the last glacial maximum on northern islands in the Japan Sea such as Okushiri Island.Siebold’s beech,  Plant populations on islands differ from mainland populations in terms of ecological and demographic processes such as colonization events, population persistence, and expansion because of their geographical isolation. From a genetic perspective, island populations are assumed to have less diversity than their mainland counterparts due to evolutionary processes such as founder effects, limited gene dispersal, and small population size . For exaOn the contrary, several studies of terrestrial plant species revealed that island populations have substantial genetic diversity in spite of their isolated geographic locations which might restrict frequent gene flow . This isAnother interesting issue regarding island populations is the colonization history during the species range expansion . CompareFagus crenata Blume (2n = 24) is monoecious and allogamous, with barochorous and synzoochorous by rodents and birds  representing the northernmost distribution of F. crenata among such islands  started about 6,000 years BP on the mainland  genome for angiosperms and bi-parental inherited nuclear genome provides relevant information such as the colonization history because comparisons of uniparentally (cp) vs. biparentally (nuclear) inherited loci can reveal differences in dispersal between seeds and/or pollen  to infer the species’ population demography on the island, and 3) to discuss the colonization and northern expansion dynamics of F. crenata, considering a possibility of northern persistence of the species during the LGM.The aims of this study are: 1) to evaluate the genetic structure of F. crenata  > 20 cm (11 to 65 trees per population) were chosen at random, and leaves were collected from a total of 1,838 individuals from 44 populations  and reverse (5’-CCGTGCTTGCATCTTTCATTG-3’) primer using Multiplex PCR Kit  containing 10 to 30 ng of genomic DNA in a final volume of 6 µl. Amplification condition was 95°C for 15 min., 35 cycles of , 72°C for 10 min, then hold at 4°C. Amplicons were treated with 0.8 U of ExoI  and 2 U of Shrimp Alkaline Phosphatase  with ExoI buffer  to remove unincorporated primers and dNTPs for 1 hour at 35°C followed by 15 min. at 75°C for inactivation of enzyme, then hold at 4°C. The single-base primer extension was performed using SNaPshot Multiplex Kit  and trnK1754 primer (5’- T14CTAGCATTTGACTCCGCACCACTGAAG -3’). The total volume of the reaction mix was 7 µl which contained 1 µl of SNaPshot Master Mix, 0.7 µl of 2 µM primer, 1 µl of BigDye Terminator 5 x Sequencing buffer , and 2 µl of the first PCR product. The extension was performed under condition of 25 cycles of , then hold at 4°C. SNaPshot extension reaction was treated with 1 U of SAP  to remove unincorporated primer and ddNTPs for 1 hour at 35°C followed by 15 min. at 75°C for inactivation of enzyme, then hold at 4°C. The fragments were detected with ABI PRISM 3130xl Genetic Analyzer  using POP-7 polymer and 36 cm capillary. An aliquot of SNaPshot reaction (1 µl) was mixed with 9.8 µl of Hi-Di Formamide  and 0.2 µl of GeneScan 120 LIZ dye Size Standard . SNPs were determined by GENESCAN for Windows . We first analyzed four individuals from each population to determine cpDNA haplotypes. When four individuals from the same population showed the same haplotypes, the cpDNA haplotype of the population was determined to be monomorphic. When different haplotypes were detected from four individuals from the same population, we further analyzed 12 to 22 individuals from that population to determine the cpDNA haplotype ratio of the population. The cpDNA haplotype are fixed in most of the populations of this species, and we analyzed the limited number of individuals to detect at least 6% of polymorphism.It has been revealed that there are two chloroplast (cp)DNA haplotypes in these regions, that is, A (GenBank: AB046492.1) and B (GenBank: AB046493.1) . We detexl Genetic Analyzer using POP-7 polymer, 36 cm capillary, and GENESCAN for Windows .Eleven nuclear microsatellite (SSR) primer pairs were employed to identify genotypes of all individuals for the following loci: mfc2, mfc12 , FS1-03,The number of individuals used for analyses are shown in HE) (Nei 1987), allelic richness (RS) (FIS) (Wright 1965) using the FSTAT 2.9.3 computer program   obtained through 999 permutations of bootstrapping using GENODIVE  , and the, FSTAT) . SignifiGENODIVE . The degy CERVUS .K, ranging from 1 to 10; as we confirmed that the genetic structure among population could be detected at K < 10 in the pilot run. CLUMPAK server  . All of K server  was emplIn addition, principal coordinate analysis (PCoA) of 44 populations based on the allele frequencies of 11 SSR loci, as well as Mantel test for isolation by distance between the pairwise genetic distance and geographic distances were conducted by GENODIVE . PhylogeK = 3. Then the 50 individuals which showed the highest ancestry within each cluster were selected, and named to Pop1 (the mainland of Hokkaido), Pop2 (Okushiri Island) and Pop3 (Tohoku region). These three representative populations, that is a total of 150 individuals, were examined six simple population demography scenarios  approach and used software DIYABC v2.1 . To makeSince DIYABC requires a population which can be traced back to an ancestral population, we set Pop3 to be the ancestor because the Tohoku region showed higher genetic diversity than the mainland of Hokkaido  , Pop3 to Pop2 (rb), from Pop1 to Pop3 (rc), and from Pop3 to Pop1 (rd) were set at t1, t2, t4, and t5, respectively. As we did not set any orders of these time scale for the admixtures, the time scale order relied on the inference  and it was the same in the following Scenarios (2-6).In Scenarios 3, 4, and 5, the unidirectional admixture was adopted whilst populations on Okushiri Island were assumed to be too small to influence mainland populations by emigrant genes.Scenario 2: Hierarchical split model II: Pop2 was merged to Pop1 at t3 and Pop1 was merged to Pop3 at t6, assuming populations on Okushiri Island were split from the mainland of Hokkaido. Similar to Scenario 1, the directional admixtures between populations after the splitting were set.Scenario 3: Simple split model with admixture: all 3 populations diverged at the same time at t3, allowing admixture from Pop1 to Pop2 (ra) and Pop3 to Pop2 (rb) at t1 and t2, respectively.Scenario 4: Hierarchical split model III: The hierarchical split pattern was similar to Scenario 2. However, we assumed only one directional admixture from Pop1 to Pop2 (ra) at t1.Scenario 5: Isolation with admixture model I: Pop1 was split from Pop3 at t4 and Pop2 was created by admixtures from Pop3 to Pop2 (rc), and from Pop1 to Pop2 (1-rc) at t3. The secondary directional admixture from Pop1 to Pop2 (ra) and from Pop3 to Pop2 were set at t1 and t2, respectively.Scenario 6: Isolation with admixture model II: Although this scenario was similar to Scenario 5, we assumed all possible combinations of secondary directional admixture from Pop2 to Pop3 (ra), from Pop3 to Pop2 (rb), from Pop1 to Pop2 (rc), from Pop2 to Pop1 (rd), at t1, 2, 3 and 4, respectively.HE), the number of alleles (A), and the variance of allele size were used as summary statistics for each of the three populations. HE, A, variance of allele size, classification index  and FST were the summary statistics for each of the population pairs. One million simulations were run for each scenario. After all the simulations had been run, the most likely scenario was determined by comparing the posterior probabilities using the logistic regression method. The goodness of fit of the scenario was assessed by the option ‘model checking’ with PCA in DIYABC, which measures the discrepancy between data sets simulated with the prior distributions of parameters and the observed data as well as data sets from the posterior predictive distribution in the scenario.The prior distributions of these parameters were shown in Two haplotypes were distinguished; namely, A (GenBank: AB046492.1) and B (GenBank: AB046493.1) , among tHT) for all 44 populations was 0.783, and the genetic differentiation for all populations (G’ST) was 0.026. The HE values of Hokkaido, Okushiri Island, and Tohoku were 0.776 , 0.778 (from 0.726 to 0.792), and 0.797 (from 0.731 to 0.800), respectively  values among runs were larger after K = 4. As regards ΔK, even though a hierarchical population scenario is not likely, the highest value can be detected at the uppermost hierarchical level of the population structure  being smaller than K > 4.Although the results from STRUCTURE analysis revealed that Δtructure . Similarin K > 2 . In thisK = 2  were 0.0156 and 0.0275 for Cluster I and II, respectively. The Cluster I was dominant in Tohoku and Cluster II in both Hokkaido and Okushiri Island. Cluster II was more dominant in Okushiri Island than Hokkaido.We compiled the individual coancestry to each population and drew pie diagrams on geographical map  based on the correlation matrix are shown in 2 = 0.198, p = 0.001)  and 8.69 ×10-7 , respectively   were lower than that for the whole geographical range of F. crenata   . This isulations . Our preribution . Althougiversity . On the F. crenata populations on Okushiri Island. In general, the loss of genetic diversity in island populations can be explained by founder effect during population foundation, limited gene flow and genetic drift due to geographical isolation and small population size . HoweverF. crenata , with its dominance expanding northward. This is in concordance of the species increase along the Japan Sea coast of Tohoku reflecting the increase of snowfall in this area. However, during the late-glacial (from 14 ka to 12 ka), Fagus pollen occurred in southern Hokkaido  (Garrulus glandarius brandtii)   and jaysrandtii) . Nishi aas 22 km . Thus, aF. crenata populations examined in this study . An ornamental F. crenata tree at Kuromatsunai Depression flowered at 18 years after the juvenile plantation . Provenance tests of F. crenata at two nurseries of Tokyo University  revealed that the northern originated F. crenata flowered 15 and 17 years after juvenile plantation at the earliest . On the other hand, the age of reproductive trees at the northernmost population was estimated to be 80 years , while newly founded northern marginal populations showed the effects of genetic drift  and Core-to-Core Program (Asia-Africa Science Platforms: JPJSCCB20220007) from the Japan Society for the Promotion of Science and the 27th Pro Natura Fund Grant Program from the Pro Natura Foundation Japan.We thank M. Ooue, M. Takahashi, H. Saitou, T. Nagamitsu, A. Nakanishi, and A. Takazawa for their assistance in the field and laboratory work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Myricaria germanica along the river Isel, Austria, which is part of the Natura 2000 network, and its tributaries. The use of 22 microsatellite markers allowed us to assess the role of tributaries and single populations as well as gene flow up- and downstream. The analysis of 1307 individuals from 45 sites shows the influence of tributaries to the genetic diversity at Isel and no overall isolation by distance pattern. Ongoing bidirectional gene flow is revealed by the detection of first-generation migrants in populations of all tributaries as well as the river Isel, supporting upstream dispersal by wind (seeds) or animals (seeds and pollen). However, some populations display significant population declines and high inbreeding, and recent migration rates are non-significant or low. The genetic pattern at the mouth of river Schwarzach into Isel and shortly thereafter river Kalserbach supports the finding that geographically close populations remain connected and that tributaries can form important refugia for M. germanica in the dynamic riverine network. Conservation and mitigation measures should therefore focus on providing sufficient habitat along tributaries of various size allowing pioneer plants to cope with extreme events in the main channel, especially as they are expected to be more frequent under changing climate.River alterations for natural hazard mitigation and land reclamation result in habitat decline and fragmentation for riparian plant species. Extreme events such as floods are responsible for additional local species loss or population decline. Tributaries might provide refugia and subsequent source populations for the colonization of downstream sites in connected riverine networks with metapopulations of plant species. In this study, we analyzed the metapopulation structure of the endangered riparian shrub species  Centuries of river alterations for land reclamation have resulted in habitat reduction and fragmentation especially for sessile riparian plant species4. Extreme events such as large floods are often responsible for local extinction of plant populations5 and are likely to increase under changing climate8. Tributaries might provide refugia and subsequent source populations for the colonization of downstream sites in connected riverine networks11.Riparian habitats along rivers are of major importance for biodiversity worldwide as they offer high species diversity12, despite plants being highly adapted to changing environmental conditions13. To counteract genetic diversity loss by reduced local density, functional metapopulation networks connecting populations up- and downstream of rivers are necessary14.Connectivity between tributaries and downstream rivers is especially important for plant species inhabiting the ever-changing dynamic riverine zone, as local loss or population decline is frequent already at yearly returning floods15. Many plant species in habitats close to the waterline display seed morphologies suitable for wind and water dispersal16, as well as animal-mediated dispersal mechanisms18. Despite many means of propagation, riparian metapopulations are generally genetically highly structured and e.g. show isolation by distance patterns21. Water mediated dispersal (hydrochory) might enhance connectivity to distant populations downstream22, but gene flow along catchments can be highly impacted by barriers such as canyons or reservoirs23.Studies on genetic diversity have shown the importance of upstream as well as downstream dispersal for riparian species25, if there is local population decline26, or ongoing migration27 despite fragmented habitat. This information is crucial to assess the importance of tributaries and the contribution of single populations to genetic diversity further up- or downstream29.Neutral genetic markers allow to investigate if gene flow and therefore functional connectivity is still present along river networksMyricaria germanica30. This catchment offers a high number of populations of the pioneer plant of the dynamic riverine zon31 despite natural canyons, many hydro-morphological river alterations in the past (land use and flood protection) as well as barriers linked to hydropower. The analysis of 45 sites with both small and large  populations within the catchment allows to assess the influence of tributaries on the genetic diversity as well as the connectivity between sites. If gene flow between populations persists, the tributaries and the main reach should reveal similar genotype compositions, and only low population differentiation.In this study, we investigate connectivity along the river Isel and its tributaries by analysing genetic diversity patterns for the characteristic and endangered riparian shrub species, the German tamarisk, 24, the highly variable microsatellite markers are applied to reveal the primary mechanism of dispersal and if unidirectional or bidirectional gene flow along the river network is more frequent. Moreover, we determine the presence of migrants and migration rates, as ongoing gene flow would support intact functional connectivity along Isel and its tributaries.As isolation by distance patterns and high genetic structure in riparian metapopulations indicate short distance wind-mediated upstream and downstream dispersal to have a higher impact than long-distance water-mediated dispersal33). Populations showed high numbers of polymorphic loci out of the 22 loci analyzed  and between populations within each river . Highest variance was within individuals  and within populations .ST) and distances along rivers, no isolation by distance pattern was detected .The analysis in Structure Harvester revealed that the most likely number of groups of genotypes could be assigned to K = 4 . Of the 45 sites studied, 30 showed individuals which are most likely first-generation migrants from another population (Table p < 0.01). Most migrants per site were detected in two population at Isel (I-08 and I-09), and potential sources were traced to upstream tributaries but also to other sites at Isel downstream. Similar to the findings for Isel, sources of migrants were assigned to both up- as well as downstream sites for all tributaries . Our comprehensive study on both large and small populations shows that genetic diversity is especially high at the large populations at the Isel river where also patterns of population demography indicate ongoing rejuvenation.The German tamarisk displays many populations along the Isel catchment despite changes in river morphology and dynamics by humans , many populations of various size within the catchment show high inbreeding. As the German tamarisk is capable of selfing40, providing valuable insights for conservation measures when linked to demographic and topographic data41. In the headwaters (Kleine Isel) and the tributary Tauernbach, detected reduced genetic diversity is reflecting limited connectivity due to topographical characteristics43. Similarly, the signs of bottlenecks at two sites close to the confluence with Isel of Schwarzach (S-09) and of Kalserbach (K-08) are most probably due to the location downstream of canyons, which are responsible for habitat fragmentation and non-functional connection to upstream populations by wind-mediated dispersal (see in23). However, the genetic diversity pattern at the catchment revealed that downstream transport via canyons  has been possible through the Kalserbachklamm, the Defregger-Klamm and the Prosseggklamm, similar to findings for other riparian species44.Genetic diversity patterns are reflecting habitat fragmentation due to river morphology and connectivity45, studies on woody riparian guildes46, and studies on floodplain meadow species47), the tributaries of Isel do not display unique gene pools or clear isolation by distance patterns, as e.g. found for fish48. Still, some tributaries showed higher percentage of certain groups of genotypes , which has been found for other riparian plant species in the dynamic riverine zone50.Contrary to findings of high differentiation along rivers for other riparian plant species  during extreme flood events52. The genetic structure of populations at junctions can further reveal functional connectivity up- and downstream at a small scale25. At the junction of Schwarzach and Kalserbach with Isel, the populations of the tributaries are more related to the populations at Isel downstream than to the other sites at the tributaries upstream, partly also due to the canyons , but for the German tamarisk it has even been previously detected even between catchments49. Additionally, gene flow over long distances is possible also by pollen mediated dispersal18, and insect pollinators can cross barriers to both water- and wind-mediated gene flow. The current genetic structure indicates that M. germanica can partially overcome both natural canyons and man-made check dams along Isel and its tributaries, although limitations in gene flow might only be detectable after a certain time26.Long-distance wind dispersal seems to be rare , where a large population was established at the small gravel bar within the highly impacted river stretch K-03, see Fig. 36, indirect evidence for ongoing functional connectivity as provided by this study is necessary for management planning for the river stretches within the Natura 2000 area. Although habitat fragmentation is present , the metapopulation network is functionally connected. Still, management strategies are necessary, as M. germanica populations are frequently found in dynamic riverine zones, which are subject to major loss during extreme events (such as HQ30 or HQ100 flood events). As populations with mainly young individuals show less genetic diversity than sites with older plants, the main focus of conservation strategies should be on protecting large populations with diverse age classes. As all tributaries contribute to the gene pool, sheltered and currently more isolated sites (e.g. Daberbach) might provide important refugia and sources for subsequent re-colonization after extreme events as exemplified by58.Due to the lack of a long-term monitoring of the metapopulation at Isel59. Wind-mediated dispersal with a continuous supply of seeds during summer54 together with pollen mediated gene flow mainly allow for shorter dispersal distances60. Smaller populations or less persistent sites in highly dynamic riverine zones might provide important short-term nodes in the metapopulation network52, and currently unsuitable habitat should be restored to provide a better network. The overall habitat loss, e.g. due to hydro-morphological changes like channelization, is the major threat for M. germanica, and therefore, re-introduction projects are often initiated in revitalization projects such as river widenings e.g.63. If human mediated re-introductions of individual plants or seeds are considered, they should only use material originating from the closest populations along the tributary, given the genetic differentiation detected in this study. A future monitoring of sites and population structures as well as changes in genetic diversity would allow to assess the status and development of the German tamarisk along Isel based on the presented study.For management planning, knowledge on species’ dispersal mechanisms is especially important for species inhabiting rivers and riverscapesM. germanica along the Isel and its tributaries provides a first comprehensive overview of the metapopulation, and highlights the importance of tributaries within the catchment for maintaining gene flow. Both large and small populations might play important roles in the metapopulation network, but are equally subject to population declines. Conservation management of the Isel and its tributaries should therefore focus on enabling habitat formation or restoring habitat for the German tamarisk, with a special focus on the migration hotspots at the junctions. This ensures the survival of M. germanica under expected more frequent and more intense extreme events such as floods due to changing climate despite fragmented habitat.Our study on the genetic diversity of Myricaria germanica (L.) Desv., is the main indicator for the protected habitat “Alpine rivers and their ligneous vegetation with Myricaria germanica”, Natura 2000 code 3230 for a study on the habitat, e.g.64. In Tyrol, M. germanica is protected since 2005  and the manipulation of the plant as well as habitat changes resulting in loss of individuals is prohibited . Over the last decade, the species showed a severe decline along European rivers36. As a typical pioneer species on gravel banks and bars, the German tamarisk is threatened, if sediment and hydrological dynamics are restricted.The German tamarisk, 65. Juvenile plants reach age of first flowering after 1–2 years see also64, and the sentenced flowers are pollinated by insects18. Additionally, the plant is reported to be capable of selfing23. As it provides nutrition for insects such as honey bees, recommendations for the cultivation of the shrub along rivers in order to improve the honey yield were promoted in Tyrol in the mid-twentieth century66. Seeds of the German tamarisk have a pappus, facilitating both wind- and water-mediated dispersal67. For wind-mediated dispersal, the majority of seeds were found close to the mother plant, but dispersal kernels show long tails54, with rare long-distance dispersal of several kilometers43. While water-mediated dispersal is known as long distance dispersal for many riparian plants68, dams and canyons are reported to inhibit water-mediated dispersal of M. germanica23.The shrubs can reproduce vegetative by re-rooting of branches, but also produce seeds that germinate within 48 h after seed landinghttps://natura2000.eea.europa.eu/Natura2000, site code AT3314000).This study focuses on the river Isel (ca. 50 km) in East Tyrol, Austria, and its tributaries: North to South: Tauernbach 17 km), Schwarzach (43 km) and Kalserbach (17 km) as well as the small stream Daberbach  and the Schwarzach (Defregger Klamm) display German tamarisks upstream and downstream of the canyons . Following the protocol of70 using Multiplex PCR Master Mix», 2x , 22 microsatellite loci were analysed using PCR. All PCR products were diluted (1:2) with ultrapure water, and 1 µL of the mix was added to 9.5 µL HiDi-LIZ solution  and size standard mixture  for the analysis on a 3730xl DNA Analyzer .For each sample, 15 mg ± 3 mg leave material was lyophilized  prior to total DNA extraction  were calculated to assess genetic diversity. Additionally, the program Arlequin 3.572 was used to calculate FST-values for population differentiation, and pairwise comparisons to detect significantly differentiated populations.Using the software GeneMapper , fragment analysis of the 22 microsatellite loci was performed using scoring bin sets of previous studies for details see73 for populations with at least 10 individuals. We performed all three tests available in Bottleneck, the sign test, standardized difference test73 and the one-tailed Wilcoxon sign-rank test74 to evaluate if the populations showed a heterozygosity excess or deficit. Expected heterozygosity was based on simulations for the genetic distribution for each populations under the assumption of two models, as the microsatellite motifs did not allow to make a prior choice on a single model70: the stepwise mutation model (SMM) and a two-phase model (TPM) allowing for a combination of 90% of SMM and 10% of infinite allele model.To identify if single populations underwent a drastic decrease in effective population size, we used the program Bottleneck72. The genetic structure of the study site was assessed in the program structure 2.3.4, testing for K = 1–45 groups75, with 108 iterations and a burn-in of 104. The output of this Bayesian approach to identify the number of groups the multilocus genotypes could be assigned to was tested for statistical support in the program Structure Harvester76.Molecular variance (AMOVA) within and between populations using Isel and each of its tributaries as a predefined geographic structure (resulting in 6 groups) was performed in Arlequin 3.5ST) of populations with more than 10 individuals and geographic distances along rivers Manteltest in GenAIEx 6.50377. Distances along rivers between the chosen populations were determined using the package “riverdist” in R71. To assess if migration between sites is ongoing, we applied a Bayesian approach27 to identify migrants using the software GeneClass78. In this program, we estimated the likelihood of first-generation migrants by assessing the likelihood of an individual multilocus genotype to originate from the population it was sampled from compared to the likelihood that it is sampled from another population in the catchment see also in78. Probability computations (based on Monte-Carlo simulations) were done using the algorithm of Paetkau79 with 100′000 simulated individuals and a 0.01 type I error rate.Limitations to gene flow in the study sites as seen in an isolation by distance pattern was tested by a Mantel test based on the comparison of genetic differentiation  excluded 084.To analyze the extent of migration between populations, we used the program BayesAss 3.0.4All sampling permits were issued by the Office of the Tyrolean Regional Government (Amt der Tiroler Landesregierung). Sampling in 2014 and 2016 was carried out on behalf of the Office of the Tyrolean Regional Government. Sampling in 2018 was carried out based on the permit (for sampling parts of protected plants) NSCH/N-269/6–2017 from 06.09.2017 (district authority Lienz) and the decision of the State Administrative Court of Tyrol LVwG-2017/41/2267–19 from 23.05.2018.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.


Eleusine indica) is one of the worst agricultural weeds in China. Molecular markers were developed for genetic diversity and population structure analyses. In this study, we identified 8391 expressed sequence tag-simple sequence repeat (EST-SSR) markers from the de novo assembled unigenes of E. indica. Mononucleotides were the most abundant type of repeats , followed by trinucleotides . The most dominant mononucleotide and trinucleotide repeat motifs were A/T  and AAT/ATT , respectively. Fourteen pairs of EST-SSR primers were verified and used to analyze the genetic diversity and population structure of 59 goosegrass populations. A total of 49 alleles were amplified, with the number of alleles (aN) ranging from two to eleven per locus, and the effective number of alleles (eN) ranged from 1.07 to 4.53. The average polymorphic information content (PIC) was 0.36. Genetic structure analysis (K = 2) and principal coordinate analysis divided 59 E. indica populations into two groups in a manner similar to the unweighted pair-group method (Dice genetic similarity coefficient = 0.700). This study developed a set of EST-SSR markers in E. indica and successfully analyzed the diversity and population genetic structures of 59 E. indica populations in China.Goosegrass ( Eleusine indica), an annual weedy grass, is widely distributed in tropical and subtropical regions and has been reported to affect 46 crops which compete for nutrients, water, and light in 60 countries. This troublesome C4, self-fertilization weed has a 5–20 cm tall tufted stem, 2–3 columns of compound spike, and an ability to produce up to 140,000 seeds per plant ), with no other seeds present. The plants were cultured in greenhouses under (20 ± 3) °C/(15 ± 3) °C day/night conditions until they reached the three-leaf stage. The seedlings were then collected, snap-frozen in liquid nitrogen, and stored at −80 °C ) ranged from 0.07 to 0.78, and the lowest and highest values of polymorphism information content (PIC) were 0.05 and 0.56, respectively, with an average value of 0.36 , and Group II contained 18 sites from five provinces  . PopulatE. indica populations could be divided into two major groups (I–II) when the Dice genetic similarity coefficient was 0.70. Group I clustered 41 populations from five provinces , and Group II clustered 18 populations from five provinces  . Genetic relationships were elucidated using a dendrogram based on SSR data. Based on the cluster analysis, the 59 Jiangxi) . AMOVA values showed that a large portion of the variation is attributed to “among populations”. It was shown that 40% of the total variation came from groups clustered using the UPGMA method. A total of 42% of the total variation was due to differences between populations; nevertheless, 18% was attributed to differences between the individuals within the populations .Posidonia oceanica (17.5%) [A limited number of EST sequences are available for use with goosegrass, although screening genic SSR markers utilized in genetic diversity studies via RNA-seq technology has become one of the most efficient methods ,27. In t (17.5%) . The SSR (17.5%) . In our  (17.5%) ,31,32. A (17.5%) ,34. The  (17.5%) ,31.He and PIC values of 0.78 and 0.48, respectively. The average PIC value for all loci was 0.36, and ten had values greater than 0.35. The high repeat number in the dinucleotide locus of SSR216, which was higher than that for the trinucleotide repeats, may be one of the reasons for the greatest diversity, according to a previous assumption [PIC value became greater than 0.56. Yang et al. found that the PIC values for developed markers in C. communis did not exceed 0.50, which is similar to our findings. The PIC value was often higher than 0.50 in some industrial crops, such as Pinus koraiensis, Crataegus pinnatifida, and Curcuma alismatifolia [In this study, the degrees of the polymorphism of different loci for 59 goosegrass populations were measured based on PIC values, which can be used to assess our ability to detect molecular marker polymorphisms. The results showed that locus SSR216 had the highest genetic diversity, with atifolia ,13,14. Gatifolia .C. communis, from which 46 populations were divided into three major groups using 12 SSR markers [The population genetic structure was analyzed according to the structure that was divided by the 59 populations into two groups. The PCA results were consistent with the results of the genetic structural analysis performed using the same EST-SSR markers and individuals. Gene flow is an important factor affecting the genetic structure of plant population structure. Based on the structure result, most of the populations in group I were from north China to the Yangtze River, while most populations in group II were from south China to the Yangtze River. A similar result was also found in  markers . Our datE. indica transcriptome. A total of 14 EST-SSR primer markers were verified, and the population genetic structure and diversity of 59 E. indica populations, which were divided into two groups, were successfully analyzed. These results indicate that next-generation sequencing techniques are powerful tools for SSR development and genetic analysis. In this study, we developed and analyzed a set of EST-SSR markers derived from the


Acropora cervicornis and one population of A. palmata in the Caribbean coast of Colombia. San Andrés was the most genetically differentiated location for both species. An average pairwise FST value of 0.131 and 0.050 between San Andrés and neighboring collection sites was estimated, for A. cervicornis and A. palmata, respectively. Based on population patterns of both acroporid species, we inferred that Magdalena River is not a barrier of genetic connectivity among Colombian populations. Genetic comparisons between the Colombian coast of Caribbean with other Caribbean locations agree with previous studies for both species, where four populations were identified in A. cervicornis and three in A. palmata. Our results support published bio-physical model predictions and highlight the Panama-Colombia gyre as a possible isolating mechanism within the western Caribbean. However, the genetic diversity in both species was about half  than previous estimates in acroporid populations in the Caribbean. The lower genetic diversity as well their relative isolation and high levels of reef degradation may be of particular conservation concern that may require species-specific management coupled with science-based restoration efforts.Using a standardized SNP array, we identified two populations of  Acropora are found throughout the entire Colombian Caribbean; however, they present different states of conservation. While there are still important patches of Acropora palmata, A. cervicornis patches are rare and fragmented, and in some locations they are practically absent  (The population genetic structure of both n region  and the n region . Regiona>500 km) . At leas>500 km) .Regional circulation patterns are important to understand the genetic connectivity of populations, in this regard along the Central and South American coast of the Caribbean, Orbicella have also been studied throughout the Caribbean, and in general the same genetic separation of populations has been observed between the East and West, with a genetic break around the Mona Passage in the North, and in the southern extent they observed a significant level of gene flow between Curaçao and Mexico , a Western cluster  and the central group that identifies the Colombian population with Honduras, Nicaragua, Jamaica, Dominican Republic, Puerto Rico and British Virgin Islands.Species of the reef-building coral genus d Mexico . Foster A. palmata and A. cervicornis in the Colombian Caribbean is necessary to know to what extent the populations of these species in the Southern Caribbean are connected and their importance as a source to contribute to the management and conservation programs. For these reasons we used a Single Nucleotide Polymorphism (SNP) array and standardized analysis work flow for the genus Acropora recently developed by A. palmata and A. cervicornis populations and compare them to those of previously studied Caribbean populations, (2) identify patterns of genetic connectivity of the acroporid subpopulations in the Colombian Caribbean that can provide information for their management and conservation and (3) to analyze different factors explaining the genetic differences among A. palmata and A. cervicornis subpopulations in this region.Estimating the genetic diversity and connectivity of the populations of A. palmata, 86 A. cervicornis and seven A. prolifera colonies were biopsied. At each site, colonies were sampled at least 5 m apart and at depths between 1 and 10 m , Isla Arena, Rosario Islands, San Bernardo Islands, Isla Fuerte, Urabá Gulf and San Andrés Archipelago, Old Providence and Santa Catalina) along the Colombian Caribbean . A totaland 10 m   and quality was quantified using a Nanodrop 2000 and an Agilent 2100 Bioanalyzer. Genomic DNA was submitted to Thermo Fisher for genotyping analysis on the Axiom™ Coral-Algae Genotyping Array . The raw genotype data was processed using the Standard Tools for A. cervicornis genets and 7,078 SNPs from the 159 A. palmata genets.The two Caribbean species were separated for the remaining analyses. For each species, a representative sample for each MLG was extracted and combined with representative samples spanning the geographic range of the Caribbean acroporids ). An addST estimates were calculated using the e, Ho, FIS and nucleotide diversity π) were performed with either hierfstat R package (t-test. Differences in nucleotide diversity (π) between species was performed using a two-tailed Student’s t-test and among regions within a species using a 1-way ANOVA with a Tukey post hoc test. Analysis of molecular variance (AMOVA) was conducted by region and sub-region with poppr.amova function of the poppr R package ) with geographic distances. Geographic distances were computed from the latitude and longitude of the collection sites using the distm function of the geosphere R package  and ADMIXTURE v1.3.0 . DAPC wa package .n = 19,694). PCAdapt correlates each loci with the principal components (axes) retained from a Principal Component Analysis. The number of components retained is equal to the number of PCs that explain the largest proportion of genetic variation. Loci were additionally filtered for linkage disequilibrium ) and minor allele frequency threshold of 5% for the PCAdapt analysis with K = 2. P-values of loci with significant correlations were transformed into q-values using the qvalue R package  for all A. cervicornis subpopulations and some of the A. palmata subpopulations  of 57  hybrids . The gen palmata . ConsistFST between Colombia and Caribbean collection sites were significant but varied regionally, ranging from 0.069 with Florida to 0.243 with Puerto Rico .All pairwise comparisons of rto Rico . Signifiand TNNP . We founA. cervicornis populations across the Caribbean in agreement with previous studies (n = 1), San Andrés (n = 20), Urabá Gulf (n = 2), Curaçao (n = 2), Puerto Rico (n = 4) and USVI (n = 3). The second is composed solely of collection sites within Colombia, including Urabá Gulf (n = 5), San Bernardo Islands (n = 12), Rosario Islands (n = 13), Isla Arena (n = 1) and TNNP (n = 4). There are two populations in Colombia with Urabá Gulf harboring A. cervicornis colonies from both San Andrés and the other Eastern Colombian locations; there is also evidence of gene flow between the two populations  showed Urabá Gulf as a transitional zone between San Andrés and the other localities and variable population separation, with San Andrés separating from the cluster of Rosario Island, San Bernardo Islands, and TNNP . Overlapn = 33 intronic, n = 32 exonic) and seven loci were predicted as missense mutations (n = 4).Between the two populations within Colombia, we identified 584 and 260 candidate loci under divergent selection with PCadapt and Bayescan, respectively . Both meutations . These mFST estimates between Colombia and Caribbean collection sites were significant  and regions within populations .Pairwise nificant . Allelic Islands . Unlike A. palmata populations across the Caribbean regions. Both DAPC and ADMIXTURE analyses supported inferred population cluster size of three   .Acropora species in the Caribbean has been broadly defined between the two Eastern and Western provinces  having colonies from both populations, and evidence of gene flow between Eastern Caribbean colonies and Colombia colonies. Moreover, Curaçao samples also show evidence of admixture between the populations in the Northeast (Puerto Rico and USVI) and samples from Colombia.The population structure of rovinces . Based orovinces . InsteadA. cervicornis, but not for A. palmata is important in terms of the resilience and conservation of the species, because the status of A. palmata is currently better in the Colombian Caribbean, with formations in very good condition, while for A. cervicornis the condition is critical , which could indicate that a higher priority management is required for this species. In A. cervicornis, isolation-by-distance was detected and between 260 and 584 candidates were identified under divergent selection, sharing 165 loci among the methods used (PCAdapt and Bayescan). The presence of two distinct subpopulations in A. cervicornis poses long term conservation risks for the species. Reduced gene flow, in addition to the confirmed poor state of A. cervcornis thickets in Colombia, may jeopardize the resilience of the species under different stress scenarios. However, this scenario could indicate a greater risk for A. palmata in terms of this species being considered in the Colombian Caribbean as an isolated region, genotypically impoverished, which could generate potentially a lower response of the species to local and global environmental stressors. The contrasting result between the geographical distance and the genetic distance within the reefs of Colombia, significant for critical . We founE per site = 0.321 ± 0.012 in A. palmata and 0.369 ± 0.012 in A. cervicornis) than previous studies. Specifically for A. palmata,E between 0.58–0.85, E = 0.389) is less in the rest of Caribbean (HE = 0.701 ± 0.043); a result supporting the assertion by In both species, the genetic diversity estimated was globally much lower , a Western cluster  and a central cluster . Further, they mentioned that the southern extent of the East–West barrier to gene flow across the Caribbean appears to lie between the Venezuelan corridor to the east and the Colombia-Panama gyre to the west and the barrier may be the plume of low salinity runoff from the Magdalena River that lies just north of Cartagena and discharges up to 228 km3 of sediment-laden water into the Caribbean Sea annually, according with FST = 0.002, P > 0.130 in A. cervicornis, and FST = 0.001, P > 0.070 in A. palmata), precisely these localities are separated by the mouth of the Magdalena river ; therefore there has been connectivity between these populations.The joint study of regional circulation patterns related to the genetic connectivity of populations has shown similar patterns of clusters throughout the Caribbean. Acropora cervicornis are regionally connected, and readily share genetic information between Curaçao, San Andrés and in general in the Western Caribbean. According to Orbicella faveolata between Curaçao and Mexico by A. cervicornis by Stegastes partitus revealed evidence of gene flow among populations from the South Caribbean, including San Andrés, Urabá Gulf (Capurganá), Rosario Islands and Santa Marta  an indication of the diversity within populations, the He values were very low in both species, which it also indicates a low level of differentiation throughout the Colombian Caribbean. Therefore, these data suggest that populations of A. palmata and A. cervicornis throughout the Colombian Caribbean share little genetic information.An AMOVA detected significant differentiation between regions explaining 16.66% in ulations . Althouge.g., Acropora palmata stands whereas A. cervicornis stands are uncommon and even absent. We are reporting that the Colombian acroporids harbor significantly lower genetic diversity than in other areas and that the sampled populations are relatively isolated leading us to raise concerns about their conservation status. Significant efforts are being made to understand the early stages of development of Acropora species and the induction of coral settlement in order to improve post-settlement survival (Over 35 years have passed since the disease-induced mass mortalities in acroporids and population recovery is slow, moderate and local e.g., . In Colosurvival , in addisurvival , coupledsurvival  may offe10.7717/peerj.13854/supp-1Supplemental Information 1Lowercase letters that are not shared indicated significant differences between groups using a 1-way ANOVA with post hoc Tukey tes.Click here for additional data file.10.7717/peerj.13854/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.13854/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.13854/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.13854/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.13854/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.13854/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.13854/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.13854/supp-9Supplemental Information 9Click here for additional data file.10.7717/peerj.13854/supp-10Supplemental Information 10Click here for additional data file.10.7717/peerj.13854/supp-11Supplemental Information 11Click here for additional data file.10.7717/peerj.13854/supp-12Supplemental Information 12Click here for additional data file.


Pyrus calleryana Decne.  is a deciduous tree native to China, Japan, Korea, and Taiwan. It is a popular ornamental tree in the United States (US) with early spring blooms and vibrant fall color. There are at least 26 cultivars of P. calleryana available in the US of which “Bradford” is the most well-known. Open-pollinated P. calleryana escapees are becoming one of the most common invasive tree species in the eastern United States. Developing better management practices for invasive P. calleryana requires detailed knowledge about reproductive biology and genetic diversity of the species, however, little is currently known about genetic variability within those open-pollinated populations. We investigated genetic diversity and population structure of non-cultivated, escaped P. calleryana populations within a ∼177 km radius in the southeastern United States. Because P. calleryana exhibits a range of morphological variation with great evolutionary potential, we hypothesized that a high genetic diversity would be manifested among escaped P. calleryana. Using 15 previously developed microsatellite loci, we genotyped 180 open-pollinated P. calleryana individuals that were collected across six naturally occurring sites in Tennessee, Georgia, and South Carolina, United States. Our results demonstrated the presence of a population structure with high genetic diversity, high gene flow, and high genetic differentiation between individuals across collection sites. Our results revealed that P. calleryana populations had differentiated shortly after the introduction to the US, most likely from specimens imported from Asia, consistent with historical records and our prior findings. The high invasive potential of the species is perhaps best underscored by transformation of P. calleryana specimens introduced from Asia into escape populations at continental scale across the United States. Our data also provided novel insight into potential issues that could be problematic for the future as P. calleryana may pose a potential threat to the economy, ecology, and native biodiversity in invaded areas. Invasive species cause great economic, social, and ecological threats to both natural and managed ecosystems  and are A successful plant invasion includes an introduction, establishment of the species in an unintended area, and a lag phase followed by colonization of additional areas . A plantPyrus calleryana Decne.  is native to China, Taiwan, Korea, and Japan  investigate fine-scale patterns in spatial distribution and gene flow within P. calleryana trees in these locations; and 3) infer the evolutionary history of P. calleryana in relation to samples from the species native area and the US-developed cultivars as a source of origin, using Approximate Bayesian Computation.Most research studies in  markers . A few Pve range , howeverve range . Therefove range , we hypoite loci  were usePyrus calleryana leaf samples were collected from individual trees in Tennessee, Georgia, and South Carolina, United States (n = 90) and “South Group” (n = 90). Both “North Group” and “South Group” contained three subpopulations of 30 individuals each, including North Group A; North Group B; North Group C, and South Group A; South Group B; South Group C, respectively (n = 75); the remaining analyses used data generated in this study subdivided into two major groups or six subpopulations.d States . Six to ectively . For oneEach sample of approximately 100 mg of air-dried leaves  was homogenized using a Bead mill 24  and subjected to genomic DNA (gDNA) extraction using EZNA DNA DS Mini Kit , according to the manufacturer’s protocol. Nanodrop  was used to measure the concentration and purity of the extracted gDNA samples.P. calleryana were developed  for eveloped  using ge® DNA Polymerase , 1 μM final concentration of each primer, and 0.5 μl of dimethyl sulfoxide (DMSO). For the data validation, gDNA from P. calleryana var. dimorphophylla  was used as a positive control, and sterile water was used as a non-template control for each primer pair. A touch-down protocol was used for PCR amplification to increase the specificity of the amplified products (The gSSR loci were amplified using polymerase chain reaction (PCR) in a 10 μl reaction mixture consisting of the following: 1 μl of 4 ng gDNA, 5 μl of 2 × GoTaqproducts . The folP. calleryana genotyped dataset of 176 tree samples within the six subpopulations.Amplified PCR products were visualized using the QIAxcel Advanced Capillary Electrophoresis System  and analyzed with a 15/600 bp alignment marker and 25 to 500 bp DNA size marker (Qiagen). Samples that failed to amplify after two subsequent PCR attempts were designated as missing data. Samples that failed to amplify in more than 5 loci were excluded from the study. Consequently, four samples failed to amplify and subsequent analyses included poppr version 2.8.5 (The MS Excel macro FlexiBin  was usedon 2.8.5  in RStudon 2.8.5 . No clonpoppr and hierfstat version 0.04-22 (e as observed in our study), rarefied allelic richness (Ar), observed heterozygosity (Ho), Nei’s unbiased expected heterozygosity -1]) and the presence of private alleles (Pa) in the subpopulations. SPAGeDi (IS), allele fixation index (FST), and their respective repeat number analogues estimated using the allele size and the motif length (RIS and RST)  . Analyseand RST) .poppr with 1,000 permutations. Linkage Disequilibrium (LD) of the 15 gSSRs was assessed in poppr using 1,000 permutations. The pairwise index of association (d) was used for assessing the linkage among loci to identify a possible bias of the patterns of LD due to a single or few pair(s) of loci.Analysis of Molecular Variance (AMOVA) was performed for North/South Groups and six subpopulation dataset subdivisions, to estimate the molecular variance distribution among and within subpopulations using the package ade4 version 1.7-13  to 1 (population structure/complete divergence). Changes in the R2 statistic when the predefined/inferred populations are sequentially removed help infer the contributions of the predefined/inferred populations to the structure of the P. calleryana dataset.The population structure of the on 2.3.4 . Thirty on 2.3.4  with theon 2.3.4  was thension 1.0  was usedP. calleryana dataset using the package adegenet version 2.1.1 , was also used to investigate the genetic structure of the on 2.1.1  in R. A on 2.1.1  using apsion 5.5  in R.P. calleryana populations for North and South Groups. Bottleneck outputs a graph as either an L-shaped graph indicating a stable population or a mode-shift graph indicating a population that experienced a bottleneck. We used a stepwise-mutation model (S.M.M.) and two-phase mutational model (T.P.M.) to test for a recent bottleneck or expansion of the P. calleryana populations. The variance of geometric distribution for T.P.M. was set to 12 along with 95% of S.M.M. Significance of the test under either of these models was evaluated using sign test, standardized differences test, and Wilcoxon sign-rank test with 10,000 simulations. The heterozygosity excess and heterozygosity deficiency of each group was assessed using Wilcoxon sign-rank test, whereas the results of all three basic tests i.e., sign, standardized differences, and Wilcoxon sign rank test, were used to assess the mode-shift in population size.Bottleneck version 1.2.02  was usedP. calleryana was investigated with the Approximate Bayesian Computation approach using the program DIYABC version 2.1 . Based on the geographical location and support by both Structure and DAPC analysis, the collected samples were divided into 3 major populations, i.e., “North Group,” “South Group,” and “Origin Group”.The population history of sion 2.1 . For thi–4  mutations per generation per locus. To explore this parameter space, one million pseudo-observed datasets (PODs) were simulated, and based on the 95% confidence intervals of the results (95% CI) the parameter values for the main runs were established.To assess the parameter values for the main DIYABC run, an initial run that used the entire dataset as one population was computed with the following parameters under uniform distribution: population size ; time , and a generalized S.M.M. with a mean mutation rate of 5 × 10P. calleryana introduction to the United States (–4 (ranging from 5.44 × 10–4 to 8.00 × 10–2 mutations per generation per locus), as per the 95% CI from the initial run. The following summary statistics for each scenario were calculated by the DIYABC program: the mean number of alleles, mean genetic diversity, mean population size variance, classification index, pairwise FST, and distance between pairs of populations (dμ)2. The effective population sizes for each population created within the DIYABC analysis, expressed as median of number of individuals, are mathematically estimated constructs and should not be treated as exact values; rather, the relative comparisons of population sizes provide information of the species demographic changes in time. For each scenario, summary statistics of the PODs were compared with that of the observed dataset of genotyped P. calleryana trees.In the main run, the genotyped dataset was tested using 6 possible evolutionary scenarios devised from the history of d States . Those sd States . ScenariThe logistic regression analysis on 1% of the PODs closest to the observed dataset was used to infer the relative posterior probabilities of the scenarios undergoing comparisons . Two sceP. calleryana individuals in North/South Groups and six subpopulations represented unique multi-locus genotypes (MLGs) and were used for further downstream analyses. Overall, 2.8% of missing data were detected across the entire dataset  was lower than the overall expected heterozygosity (He = 0.74) indicating the presence of high genetic diversity and of population structure (r) detected in both dataset subdivisions suggested the long-term adaptability potential of P. calleryana individuals in the sampled region. In contrast, a positive inbreeding coefficient  suggested that a substantial level of homozygosity observed within the dataset likely resulted from inbreeding (d) was 0.03 (p < 0.001) (d) ranged from 0.01 to 0.06 (p = 0.002), which indicated the absence of linked loci and the genome-wide distribution of the gSSRs  . The valhe gSSRs .est = 0.21), indicating the presence of population structure in the tested P. calleryana dataset. Our data also indicated the presence of high gene flow (Nm = 3.94).In the locus-wise manner, an average of about 12 alleles per locus (ranging from 8 to 19) were detected across the dataset . Our datP. calleryana dataset for both dataset subdivisions, as well as the inclusion of variance within the individuals. In the 3-tier AMOVA, for the six subpopulation data sets, a low proportion of molecular variance was present among populations (6.80%) and the major portions of molecular variance were attributed to within individuals (43.38%) and within populations (49.82%) (p < 0.001) demonstrated the existence of population structure within the P. calleryana dataset regardless of its subdivision.AMOVA was used to assess the proportion of molecular variance partitioned within the (49.82%) . Compara(49.82%) . This vaP. calleryana individuals (p = 0.001), indicating that about r2 = 4.71% of the genetic variance observed can be explained by the geographic distance assessed. The maximum linear distance between samples was approximately 260 km. Across space, there was a non-linear relationship between genetic and geographic distances of P. calleryana individuals, indicating that increased geographic distance between P. calleryana individuals does not necessarily mean increased genetic dissimilarity. The amplitude of the Mantel’s r scores in the correlogram ranged between about −0.10 and 0.15, indicating a relatively a low impact of spatial distancing on the population structure of the P. calleryana dataset.Isolation by distance using the Mantel test was used to determine the correlation between genetic and geographic distances among ividuals . We founP. calleryana dataset (North/South Groups) assessed using SPAGeDi indicated statistically similar results for FST and the mean permuted RST across all loci . This suggested the absence of a phylogeographic signal within populations. To further evaluate the presence of a phylogeographic signal among populations, the slope test  of pairwise RST were also evaluated. No evidence of phylogeographic signal was demonstrated among populations .Additionally, the phylogeographic signals within the Structure indicated an optimum of ΔK = 2 suggesting the presence of two genetically distinct clusters among the studied subpopulations of P. calleryana  in both North and South Groups. The mode shift in the population size was analyzed using the information from all three basic tests . In the cumulative mode shift test, a normal L-shaped distribution was detected in the North Group and South Group signifying no evidence of recent bottleneck events .P. calleryana introduction to US and further evaluated. Of the six scenarios, we found the highest relative support for evolutionary scenario 6  and 1.35 (95% coverage: 0.79), respectively  Benth. and Pueraria lobata Willd.  for our dataset, yet a positive inbreeding coefficient (FIS) was found in contrast to P. calleryana biology. Callery pear is an outcrossing species (IS could signify a founder effect and the alleles deriving from a limited pool of ancestors. Such positive FIS for our dataset could be also the result of human interference, for example with regard to selection, propagation, multiple local introductions, and intentional transportation of P. calleryana leading to the escape of the species. Likewise, insect pollination limiting the long-distance pollen flow and the historically-documented founder effect are expected to influence positive FIS. The positive FIS we observed is consistent with results reported from other studies conducted within P. calleryana’s native range (A high level of genetic differentiation was reported among populations as observed in other studies conducted on lleryana , as wellC. kousa  that hav species  and woulve range .P. calleryana could overcome the limitations imposed by the self-incompatibility and the founder effect. We observed a very high mutation rate for P. calleryana, compared with other species occupying similar ecological niches (P. calleryana invasion.The DIYABC inferences add information about how l niches . This fel niches , exceediP. calleryana. Our data could be enhanced by a broad-scale P. calleryana genomics study that evaluates trees from across a wider geographic range. This genomic approach could help us understand other aspects of P. calleryana not covered in microsatellite-based studies, such as investigating the molecular mechanisms underlying reproductive incompatibility, and genomic features enabling the evolutionary success of P. calleryana despite observed high inbreeding and self-incompatibility. Results from that work are expected to help us compare the genomic characteristics of P. calleryana to our present findings and will help us better understand the invasive character of the species at its molecular background, potentially informing strategies for effective management.Information from this study sheds light on the population dynamics of this invasive species with an outlook to its continued adaptation and spread. Furthermore, our study provides a great prospect for future research on the invasive


Both analytical approaches found significant genetic differentiation within localities, but not among localities nor among provinces. This observation might result from inappropriate agronomical practices involving extensive exchange of plant materials followed by rapid viral diversification within localities. In the Chinese population, both methods found that genetic diversification in AMV was strongly associated with different bioclimatic zones. Rates of molecular evolution were similar in the three countries. The estimated epidemic exponential population size and growth rate suggest that the epidemics grew faster and with higher incidence in Iran, followed by Spain and China. Estimates of the time to the most recent common ancestors suggest that AMV was first seen in Spain by the beginning of the twentieth century and later on in eastern and central Eurasia. After ruling out the existence of recombination breakpoints within the cp gene, a codon-based selection analysis per population was performed and identified many codons under significant negative selection and a few under significant positive selection; the latter varied among countries, suggesting regional differences in selective pressures.Alfalfa mosaic virus (AMV) is the most pervasive epidemic virus affecting alfalfa production. However, detailed investigations on the molecular population genetics and evolutionary dynamics of AMV are scarce. This study aimed to report on a large-scale long-term survey of genetic variability in AMV populations from China and perform a comparative analysis of AMV population genetics in the three most thoroughly studied countries to date: China, Iran, and Spain. The study was based on the analysis of the coat protein gene ( The dicistronic RNA 3 encodes the movement (MP) and coat (CP) proteins, the latter translated from a subgenomic RNA 4 . RNA 3 ccp cistron from 76 new isolates (cp sequences from Spain (56) and Iran (91). The comparison among these three epidemics has allowed the determination of some general principles driving the epidemiology and evolution of AMV.This study aimed to address the population genetics of AMV in China, using the sequence of the isolates S2, and 22.1cp gene fragment by RT-PCR are listed in A total of 516 alfalfa samples was collected from 24 locations of 13 major alfalfa-producing provinces and four climatic regions in China from 2016–2019. The isolate names, their hosts, dates and sites of collection, and climatic regions are shown in 2.22.2.1® PCR Super Mix (+ dye) 25 µL, cDNA 2 µL, pimer cp-F 1 µL, primer cp-R 1 µL, and 21 µL of ddH2O to total 50 µL . PCR cycling conditions comprised an initial denaturation step at 94°C for 3 min, 35 cycles of denaturation at 94°C for 1 min, annealing at 57°C for 1 min, and extension at 72°C for 3 min, followed by a final extension at 72°C for 5 min. The expected RT-PCR products of the cp gene were 666 bp, using primer pairs cp F/cp R  or Beckman (GeXP with Genome Lab DTCS sequencing kit) system. Sequence data were assembled using DNASIS version 3.5 , Laser gene , or BIOEDIT version 5.0.9 , 1 μL gDNA Remover, 1 μL primer AMV-R  were calculated by ARLEQUIN (http://cmpg.unibe.ch/software/ arlequin3)  . The bes2.3.3Sd) and nonsynonymous (Nd) substitution rates for the cp gene calculated by MEGA X  (AI) (MC) (Data were divided into different groups among bioclimatic regions  was usedPS) , the assPS) (AI) , and theAI) (MC) , and to 2.3.5p value < 0.05 were regarded as valid.The data sets from the three countries were analyzed respectively for the recombination using the methods of RDP, BOOTSCAN, CHIMERA, GENECONV, LARD, MAXCHI, SISCAN and 3SEQ implemented in RDP4 in the default configuration  as well 33.1cp cistron. Across the sampling period, the incidence of AMV was estimated to be as high as 57.6% . RT-PCR and sequencing yielded 76 complete sequences of the AMV cp gene from the 23 locations.A total of 516 alfalfa samples was collected from 23 locations in 13 provinces distributed along the main alfalfa-producing Chinese provinces between 2016 and 2019. These provinces encompassed four climatic regions   and one less so (Clades I and II)  , Dali , Qiqihaer , Shihezi , and Hangzhuo    as a discrete trait, no significant associations were found , while viruses isolated from other families were well mixed in the MCC tree    geographic origin, (ii) host species, and (iii) bioclimatic zone. Firstly, using geographic origin as a trait, a highly significant association was found (<0.0001) Table 2.3.2.23.2.3AI = 5.99, P <0.0001; PS = 52.3072, P <0.0001)  (P = 0.0289 (P <0.005)  Table 3,3.33.3.1AMV isolates from Iran and Spain were selected for population genetics analyses since they were the most thoroughly sampled isolates. For the Iranian population, the network suggests a single clade with very few divergent genotypes  , which was mostly driven by isolates from Zaragoza province  forming their own clusters (BaTS found evidence of population structure at the geographic level in both Spain and Iran (Iran: = 0.014) Table 9.3.3.2(P = 0.0026) , while the Chinese population only shows a significant difference at the host family level , Iran (91), and Spain (56) using the whole Both Mushegian and Morris and their respective colleagues stated that all viral contributions to plant ecosystem function must derive from the complex interactions between viruses, plants, and transmission vectors , thus, tA vast territory naturally increases the diversity of the geographical environment, plants, and transmission vectors, and directly leads to the highest evolution rate of viruses to adaptation. In the AMV population of China, there were significant differences in AMV isolates among the provinces and bioclimatic zones. Large differences in topography, landforms, longitude, and latitude among various provinces results in some differences in the microenvironment for the plants , and AMVA significant difference was observed among different locations within the same province for the Chinese and Spanish populations. The geographic distances among locations might explain this phenomenon; moreover, agricultural practices may also be one of the influencing factors. In the past 50 years, the development of agricultural production, frequent introductions of new cultivars, and the changing cropping system may have posed strong selective pressures upon AMV, resulting in rapid evolution. Although there was no difference in isolates among provinces within the same climatic zone, significant differences between isolates at different sampling points in the same province were found. This is likely due to differences in farming systems, which should be the collateral impacts of human activities on these ecological roles and bring a particularly powerful and quantitative effect on plant virus evolution .Within the Chinese population, whether based on the host diversity of isolates or the variability of geographical location and climatic zone, a very rich diversity was observed. The Iranian population, representing the earliest introduction of AMV in Asia and showing the fastest evolution among the three countries analyzed, showed no significant difference among the host plant species. The Spanish population, with the origin of AMV found in Zaragoza, exhibited the slowest spread and the slowest evolution rate among the three countries. The phylogenetic tree was not divided into different populations according to the host species and most of the evolutionary selection was negative selection, with a few sites showing positive selection, which was different from the Chinese and Iranian populations. The significant differences among different isolates in the same location and the significant differences among different sampling sites in the same province were similar to those of the Chinese population.For the Chinese population, there are questions as to whether AMV evolution is a Russian doll model and whether AMV epidemiology is “fractal”. Continents are homogenous while countries are heterogeneous, so do provinces within countries, while locations within province. If the unit of research is set at the country level, the provinces within the country are homogeneous, locations within provinces are homogeneous (depending on the country—they are homogenous for Iran but not for China and Spain), and plants from the same location are heterogeneous (but not for Iran). In addition, there is a further question as to why the AMV populations from China and Spain have the most genetic diversity the lower you go on the geographic scale? With the development of the economy of the world, the economic activities, such as the commercial activities in agriculture, especially the import and export transactions of various countries for alfalfa quality, are important factors affecting the distribution and diversity of AMV isolates. For example, with the development of China’s economy and the improvement in living standards, a large number of imports of alfalfa are needed in China to meet domestic demand; consequently, foreign AMV isolates have entered China from Canada, USA, and Spain, etc. . The difIn summary, AMV populations in China, Iran, and Spain show rich diversity through evolution, and owing to the participation of human activities, the population evolution of AMV will gradually present more new characteristics. This warrants the need for continued research on AMV to facilitate the detection and early warning of plant virus diseases.The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/XW, CL and ZT performed experiments. JZ and RW isolated some isolates. YW and XJ conceptualized experiments. BW designed the study, analyzed data, and wrote the manuscript. All authors contributed to the article and approved the submitted version.