Potato virus Y (PVY) is an important plant pathogen causing considerable economic loss to potato production. Knowledge of the population genetic structure and evolutionary biology of the pathogen, particularly at a transnational scale, is limited but vital in developing sustainable management schemes. In this study, the population genetic structure and molecular evolution of PVY were studied using 127 first protein (P1) and 137 coat protein (CP) sequences generated from isolates collected from potato in China and Japan. High genetic differentiation was found between the populations from the two countries, with higher nucleotide diversity in Japan than China in both genes and a KST value of .216 in the concatenated sequences of the two genes. Sequences from the two countries clustered together according to their geographic origin. Further analyses showed that spatial genetic structure in the PVY populations was likely caused by demographic dynamics of the pathogen and natural selection generated by habitat heterogeneity. Purifying selection was detected at the majority of polymorphic sites although some clade‐specific codons were under positive selection. In past decades, PVY has undergone a population expansion in China, whereas in Japan, the population size of the pathogen has remained relatively constant. Its genome has a single‐stranded positive‐sense RNA of ~9.7 kb, encoding a polyprotein that is cleaved into 10 mature functional proteins , the third largest food crop in the world , whereas six subpopulations existed worldwide. Furthermore, unlike results from other regions where the subpopulations all have the same age, it is believed that the Japanese PVYO subpopulation is older than the PVYN and PVYNTN subpopulations.Knowledge of the population genetics and evolutionary biology of PVY, particularly at a transnational scale, is relatively limited compared to other important plant pathogens such as The objectives of this study were to (i) compare the population genetic structure of PVY in China and Japan and (ii) determine the main evolutionary and demographic mechanisms responsible for the observed population genetic structure.22.1Solanum tuberosum) across a range of geographical locations in China including Fujian, Hunan, and Hebei provinces in 2011 and 2012  and amplified with two pairs of degenerate primers as described previously , and 2.0 μl of template cDNA. The PCR program was initially denatured at 94°C for 5 min; followed by 30 cycles of 94°C for 30 s, 53°C (P1 gene), or 55°C (CP gene) for 30 s and 72°C for 1 min; ended with an extension at 72°C for 10 min. PCR products were separated on 1% agarose gels by electrophoresis, visualized using a UV transilluminator and cleaned using a TIANgel Maxi Purification Kit (TianGen).Eighty‐five PVY isolates, confirmed by enzyme‐linked immunosorbent assay (ELISA) with a broad‐spectrum PVY antibody , were randomly collected from potato (Escherichia coli strain Trans1‐T1 (TransGen). Recombinant plasmids were extracted and sequenced in both directions by Nanjing GenScript Biological Technology Co., Ltd. . Due to high mutation rate in RNA viruses, at least four cDNA clones derived from two separate PCR reactions were sequenced for each PVY isolate to ensure consensus. Only the sequence identical in at least three clones was used for further analyses to eliminate potential heterogeneity introduced by Taq polymerase. All sequences generated in this study were deposited in GenBank databases. In addition, 24 sequences of P1 gene and 26 sequences of CP gene from other parts of China  and 34 sequences each of CP and P1 genes from Japan were retrieved from GenBank (Table Purified PCR fragments were ligated into T‐tailed pEASY‐T5 Zero vector (TransGen) and transformed into competent 2.2Hd) and nucleotide diversity (π) were estimated using DnaSP 5.0  and parental sequences were identified using seven methods  implemented in the RDP4 suites  implemented in MrBayes 3.2.5  to synonymous (dS) substitution (ω = dN/dS) was calculated for each gene using CODEML algorithm implemented in PAML 4.7 , the Bayes empirical Bayes (BEB) method  of the parameters were checked using Tracer 1.6.Two different approaches were used to explore the demographic history of PVY populations in the two countries. First, Tajima's  Tajima, . Fu's Falue Fu,  in expanalue Fu, . Samplinalue Fu,  to deteralue Fu, . A Bayes33.1KF722821) and a nucleotide (A) insertion at position 814 (accession number: KF771018), respectively. Moreover, two P1 sequences (accession numbers: KX451346 and KX451344) contained premature termination codons (PTCs) at position 208–210 and 619–621. A single CP sequence (accession number: KC296822) had an 11‐nucleotide deletion at the beginning of the gene. These nonfunctional sequences were removed from further analyses of the population genetic structure. In addition, 34 complete P1 and CP sequences originated from Japan were retrieved from GenBank  was replaced by S (isolates NTNHIR3 and T13) or T (isolate ONGOB6) in the Japanese isolates and the E68 residue was replaced by G (isolates XQ03) or K (isolate Laiwu3) in Chinese isolates  and 96.3% (87.0%−100%), while those from Japan were 89.1% (71.5%−100%) and 95.7% (88.7%−100%), respectively. All typical motifs of potyviruses were detected in the deduced amino acid sequences. However, the conserved Ns Figure .One hundred and seventeen haplotypes were identified in the 127 complete sequences of P1 gene with an overall haplotype diversity of .998 and nucleotide diversity of .119 when the sequences from China and Japan were considered together , AI (p < .001), and PS (p < .001) were detected in both P1 and CP genes when sequences from the two countries were compared  with high posterior probabilities (PPs ≥ .99) and maximum likelihood (ML) bootstrap (BPs ≥ 70%) supports . The temporal scale of samples can impact the estimate of demographic dynamics . However, sequences included in this analysis were relatively limited both in sample sizes and sites. In particular, the temporal scales might be relatively short for analysis of demographic events. Further study with larger, multiple‐location, and longer temporal scale samples may be required to confirm the results and generalize the findings.In summary, our study represents one of a few attempts to understand patterns and causes of spatial population genetic structure across political borders in PVY, a destructive pathogen of potato and many other KF722798–KF722799, KF722801, KF722804–KF722805, KF722807–KF722808, KF722810, KF722812–KF722813, KF722815–KF722818, KF722820–KF722825, KF722827, KF722829–KF722830, KF722832, KF722834–KF722841, KF771008–KF771016, KF771018–KF771020, KX451323–KX451351 (P1 gene), and KC282358–KC282367, KC282369–KC282370, KC296789–KC296790, KC296792, KC296794, KC296796–KC296799, KC296801, KC296803–KC296807, KC296809–KC296815, KC296819–KC296825, KC296827–KC296833, KC296835–KC296859, and KC296861–KC296866 (CP gene).Sequence data obtained in this study are available on GenBank under the accession numbers  Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Berberis trifoliolata), an endemic species to the Chihuahuan Desert. We identify genetic signatures based on plastid DNA and AFLP markers and perform niche modelling and spatial connectivity analyses as well as niche modelling based on records in packrats to elucidate whether orogenic events such as mountain range uplift in the Miocene or the contraction/expansion dynamics of vegetation in response to climate oscillations in the Pliocene/Pleistocene had an effect on evolutionary processes in Agarito. Our results of current niche modelling and palaeomodelling showed that the area currently occupied by Berberis trifoliolata is substantially larger than it was during the Last Interglacial period and the Last Glacial Maximum. Agarito was probably confined to small areas in the Northeastern and gradually expanded its distribution just after the Last Glacial Maximum when the weather in the Chihuahuan Desert and adjacent regions became progressively warmer and drier. The most contracted range was predicted for the Interglacial period. Populations remained in stable areas during the Last Glacial Maximum and expanded at the beginning of the Holocene. Most genetic variation occured in populations from the Sierra Madre Oriental. Two groups of haplotypes were identified: the Mexican Plateau populations and certain Northeastern populations. Haplogroups were spatially connected during the Last Glacial Maximum and separated during interglacial periods. The most important prediction of packrat middens palaeomodelling lies in the Mexican Plateau, a finding congruent with current and past niche modelling predictions for agarito and genetic results. Our results corroborate that these climate changes in the Pliocene/Pleistocene affected the evolutionary history of agarito. The journey of agarito in the Chihuahuan Desert has been dynamic, expanding and contracting its distribution range and currently occupying the largest area in its history.Here we conduct research to understand the evolutionary history of a shrubby species known as Agarito ( The origin and evolution of the biota of the five North American deserts  have been of great interest to scientists since the last century , 2], , , 5], , , and theIn the deserts of North America the uplift of mountain chains occurred in different stages from the middle of the Miocene to as recently as the Pliocene, a series of events known as the Neogene uplift ~15–2 Ma), while climate fluctuations are more recent, through the Pleistocene 5–2 Ma, w, 13], , , 7], , . The fos, . The fIn the Mid-Miocene there was a rapid cooling along with an increased drought associated with volcanic activity, resulting in the expansion of arid and semi-arid land in North America , 2], , , 19], , , 20]. A. A20]. AThe largest and most biologically diverse desert in North America is the Chihuahuan Desert, a warm desert extending from western Texas and southern New Mexico to northern Mexico on the Mexican Plateau , 37], , , 20]. S. S20]. SThe Chihuahuan Desert has been considered an area rich in endemic species and centre of origin of modern desert biota , 6]. Th. Th6]. TBerberis trifoliolata Moric.), a species endemic to the Chihuahuan Desert. We chose this plant because it was an important element of the chaparral of the Madro-Tertiary vegetation in northern Pacific areas from Colorado to Baja California Peninsula  an anD , : t: t2. A cv. 3.3.2  and the v. 3.3.2 .Berberis trifoliolata. We only used packrat middens dating to the LGM  was 1,156 bp. An unreliable hypervariable SSR (position 233–258) was removed from the matrix. Reliable identified gaps were recoded and treated as a single mutation. Forty-five haplotypes were identified in the statistical parsimony network . The relationship within B. trifoliolata haplotypes was not resolved and only one haplotype group with a modest support (PP = 90) that included north-eastern populations  and Mexican Plateau populations (haplotypes H1 and H2) was recovered. No further haplotype groups were supported by the BI analysis.The 50% majority consensus tree (results not shown) grouped all FCT values for groups between K = 2 and K = 5, with the highest FCT values for K = 3  and Real de Catorce  vs. remaining haplotypes occurred in the late Pleistocene at ca. 15 kya . The major intra-specific clades diverged during early Holocene (c. 10 kya to present). Then, the main divergences within North and remaining haplotypes of the Chihuahuan desert including Mexican Plateau haplotypes have occurred at ca. 9–6 kya. Later the haplotypes find in the Eastern of Sierra Madre Oriental diverged from Mexican Plateau haplotypes ca. 4 kya . Divergence estimates for Berberis trifoliolata populations using a coalescent model and assuming expansion population growth are shown in Estimation of time of divergence for π) were calculated for the two geographical groups selected a priori within the Chihuahuan Desert (Mexican Plateau and Northeastern region). Nucleotide diversity values were very low in both groups of populations  (h = 0.86), in contrast to the Mexican Plateau populations with lower diversity (h = 0.79).Parameters of population diversity, including haplotype diversity (h), and nucleotide diversity (ctively) . The higBerberis trifoliolata populations were treated as a single group indicates that 82.64% (P<0.0001) of the variation can be explained by differences among populations . However, when samples were grouped as separated by the geographical regions a significant but smaller proportion of the variation can be explained by differences among populations within groups   . Neithersd>0.05)  providedThe interpolated pairwise genetic distances shown in MseI-CTG/EcoRI-ACA and MseI-CTC/EcoRI-ACA using two randomly selected DNA samples. A high degree of reproducibility (reproducibility score = 98%) was obtained for each of the primer combinations tested (data not shown). All markers shared between populations were non-redundant and had frequencies exceeding 5%.The reproducibility test was carried out with primer combinations Berberis trifoliolata populations shown by the Bayesian assignment based on ΔK values revealed a clear population substructure across hierarchical levels . Some Northeastern populations  formed a second group (dark green cluster), while the SPI, RAZ, ROCA, OJU, PUR, AUST and ART populations in the Northeastern group were assigned to the third group (light green cluster), but slightly mixed with Mexican Plateau genotypes (light blue). The K = 2 groups were formed by RC, RJ, PAB and SSM (yellow cluster), but slightly mixed with the strong green genotype, whereas FCZ was formed mainly by the strong green genotype  indicates a significant but smaller proportion of the variation when the samples were grouped by the Structure results . The  results . HoweverB. trifoliolata during the Last Interglacial period was fragmented, with small areas of suitable habitat in the folds of the Sierra Madre Oriental, and in the north, in Nuevo León and Coahuila  in all three analyses: Current, Last Glacial Maximum and Last Interglacial . Low conRarefaction curves when all populations were considered provided evidence that haplotype richness has reached an asymptote, which could be suggesting haplotype saturation. However the two haplotype rarefaction curves representing each geographic region (Mexican Plateau and Northeastern) provided strong evidence that more haplotypes would be revealed by increasing the number of individuals sequenced see .Berberis trifoliolata, in which incipient speciation has been detected involving the two groups of populations.Our AFLP results showed a clear change of genotypes in close Northeastern populations and between Mexican Plateau and certain Northeastern populations. This is congruent with our previous ecological niche models that identified the same two groups as having different climate preferences . It has Edraianthus graminifolius (Campanulaceae) growing in Mediterranean mountains and it might be related to the inheritance of these markers, uniparental in the chloroplast markers , ..BerberisThe highest level of genetic diversity within agarito populations shown by presence of private haplotypes as well as presence of the most widespread haplotype occurred in the Northeastern group. A set of populations within this group is located at middle elevations, from approximately 1,700 to 2,400 m a.s.l. in the Sierra Madre Oriental, a mountain chain that is complex in both its geology and its climate. Some of these populations have an elevated number of haplotypes others share rare haplotypes, like the most southern and the most northern populations. This diversity and these shared rare haplotypes might be indicative of areas that acted as refuges during the cool climate of the Pleistocene and where populations remained during interglacial periods before expanding again. Furthermore some areas in this Sierra acted as refugia for other plants , 94], , . Therefo, has been stable since the Middle Miocene . O. OBerberS1 Dataset(FAS)Click here for additional data file.S1 Methods(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 FigShading is proportional to genetic distance, with short genetic distances in white and greater distances in black. The white line shows division between the Mexican Plateau and Northeastern populations.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigBerberis trifoliolata fossils found inside packrats middens from the LGM. Range of color from pink to light red to red indicates low, medium, and high probability, respectively.A) LGM projected onto current climate conditions using the CCSM model. B) LGM projected onto current climate conditions using the MIROC model. C) Predicted distribution during the LGM using the CCSM model. D) Predicted distribution during LGM using the MIROC model. Green dots are known occurrence points for (TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.


Blastocystis, differences in infection rates and high genetic polymorphism within and between populations of some subtypes (ST) have impeded the clarification of the generalist or specialist specificity of this parasite. We assessed the genetic variability and host specificity of Blastocystis spp. in wild howler monkeys from two rainforest areas in the southeastern region of Mexico. Fecal samples of 225 Alouatta palliata (59) and A. pigra (166) monkeys, belonging to 16 sylvatic sites, were analyzed for infection with Blastocystis ST using a region of the small subunit rDNA (SSUrDNA) gene as a marker. Phylogenetic and genetic diversity analyses were performed according to the geographic areas where the monkeys were found. Blastocystis ST2 was the most abundant (91.9%), followed by ST1 and ST8 with 4.6% and 3.5%, respectively; no association between Blastocystis ST and Alouatta species was observed. SSUrDNA sequences in GenBank from human and non-human primates (NHP) were used as ST references and included in population analyses. The haplotype network trees exhibited different distributions: ST1 showed a generalist profile since several haplotypes from different animals were homogeneously distributed with few mutational changes. For ST2, a major dispersion center grouped the Mexican samples, and high mutational differences were observed between NHP. Furthermore, nucleotide and haplotype diversity values, as well as migration and genetic differentiation indexes, showed contrasting values for ST1 and ST2. These data suggest that ST1 populations are only minimally differentiated, while ST2 populations in humans are highly differentiated from those of NHP. The host generalist and specialist specificities exhibited by ST1 and ST2 Blastocystis populations indicate distinct adaptation processes. Because ST1 exhibits a generalist profile, this haplotype can be considered a metapopulation; in contrast, ST2 exists as a set of local populations with preferences for either humans or NHP.Although the presence of cryptic host specificity has been documented in  Blastocystis spp. is a common parasite that colonizes the intestines of many animals, including mammals, reptiles and birds. Although this microorganism is the most common eukaryotic parasite identified in human feces, its pathogenic role remains controversial. While some studies have reported that this parasite is associated with the development of cutaneous and intestinal disorders, other studies report no clinical manifestations . P. P27]. P package –29, with package .All sequences were subjected to BLAST search in the GenBank database, and the sequences obtained in this work were accessed with the numbers KT591768-KT591854. Multiple alignments were performed using the CLUSTAL W  and Muscfluxus-engineering.com) with default settings and assumptions. Genetic diversity analyses within and between populations were performed using DnaSPv4 [Nm),genetic differentiation index (FST) and Tajima’s D test were obtained. These indices have been previously applied for population genetics studies in Blastocystis [Nm); and the differentiation between or among populations (FST). To support the data interpretation, populations strongly differentiated have an Nm < 1, whereas those with an Nm > 4 behave as a single panmictic unit. For FST, the following commonly used values for genetic differentiation were considered: 0 to 0.049, small; 0.05 to 0.149, moderate; 0.150 to 0.25, great; and values above 0.26 indicate enormous genetic differentiation. Negative values for Tajima’s D test suggest a recent expansion process or an effect of purifying selection [A Median Joining Network analysis  was perf DnaSPv4 , and indtocystis , 41. Theelection .Descriptive statistics are expressed as the mean and standard deviation (SD). Analysis using Student's t test for unrelated samples and Mantel–Haenszel test were applied, and 95% confidence intervals (95%CI) were also obtained. Data analysis was performed using SPSS software, version 15.0 .All fecal samples were collected using a non-invasive technique after the monkeys defecated. Sampling and procedures were performed in accordance with the provisions of the Regulations of the Environment and Natural Resources Ministry and the Under-Secretary of Management for Environmental Protection NOM-059-SEMARNAT-2010; reference SGPA/DGVS/04725/13. Academic authorities of the Universidad Juarez Autonoma de Tabasco authorized the present study (reference number UJAT-2013-IB-43).A. palliata (59) and A. pigra (166) were collected and analyzed. Blastocystis ST results for the animals from each site; only at site 5  were the monkeys not parasitized. The frequency of Blastocystis spp. was 38.7% (n = 87), 39% for A. palliata and 38% for A. pigra; ST2 was the most abundant (91.9%), followed by ST1 and ST8 with 4.6% and 3.5%, respectively. No association between Blastocystis ST and Alouatta species was observed. In addition, we identified infected howlers with similar frequencies and ST distributions in flooded and non-flooded areas , in contrast with site 8b, which showed FST and Nm values similar to those of the other sampling sites. The populations at sites 2, 8a, 8b and 12 yielded negative Tajima’s D values, but only for site 2, and this result was statistically significant (p<0.01).Blastocystis ST1 populations had lower π values than those of NHP, whereas for ST2, human and NHP parasites had similar variability; despite the disparity between the number of available sequences in the GenBank of humans and NHP for ST1, the genetic diversity values were statistically significant. The Nm and FST values for ST1 and ST2 were inverted, suggesting that Blastocystis ST2 populations from humans and NHP are strongly differentiated with occasional gene flow, while scarcely any differentiation between ST1 parasite populations from humans and NHP was detected.Blastocystis ST in Alouatta monkeys [The few reports on the molecular identification of  monkeys , 15, 43  monkeys , 44–46.Blastocystis ST2 haplotypes have diverged similar to a set of local populations with preferences towards certain host species (humans or NHP). Both population structures have relevance for understanding the prevalence and transmission of Blastocystis ST. According to these findings, the putative metapopulation structure of ST1 implies a link with the processes of population turnover, extinction and establishment of new populations [Blastocystis ST1 may have the capacity to recolonize vacant niches, such as new hosts, even of different species, supporting the zoonotic transmission of this parasite [Blastocystis ST1 in an asymptomatic patient who received anti-parasitic treatment and follow-up three months earlier.In the present study, we observed that ST1 exhibits a generalist profile because there was minimal differentiation between parasite populations from humans and NHP, as well as substantial gene flow between them, suggesting that ST1 landscapes resemble a metapopulation (ensemble of interacting populations with a finite lifetime) –50. In culations , i.e., Bparasite –9, 11–14parasite  documentBlastocystis was the unicellular parasite most frequent (60%) in samples of A. palliata aequatorialis howlers from Ecuador. Subsequently, in another study performed in similar populations of these howler monkeys, 68% of these animals were infected with Blastocystis ST8, while humans living in close proximity were infected with ST1, ST2 and ST3 [A. palliata and A. pigra howlers, respectively, were primarily infected with Blastocystis spp., and of these, 91.3% of A. palliata and 92.1% of A. pigra howler monkeys were harboring Blastocystis ST2. Other relevant findings regarding the host specificity of ST2 were derived from the phylogenetic tree of parasites from Rubondo Island, Tanzania [Blastocystis, those from humans and those from NHP.However, the existence of ST2 as a set of resident populations locally adapted by each parasite population would support the preference for infecting either humans or NHP. Such a process of local adaptation is supported by Helenbrook et al.  who obse and ST3 . Interes and ST3 , 41. In Tanzania , in whicIn addition, as cited above, local adaptations are relevant for the maintenance of genetic diversity and population diversification. The high degree of intra-ST genetic polymorphism has been reliably documented, particularly for ST1 and ST3 , 43–44, Blastocystis and other parasites [It has been observed that the use of more than one genetic marker has facilitated the clarification of the differences between organism populations. However, some studies of genetic variability focused only on the SSUrDNA gene analysis have shown that this marker is sensitive enough to resolve phylogenetic relationships, population differentiation events and cryptic infections in both arasites , 56–58.Since mixed ST infections are common , 59–60, Blastocystis has not been identified, it has been suggested that transmission occurs orally through water, food or direct contact with the parasite; here, Blastocystis transmission apparently occurs through water [A. palliata and A. pigra feeding and traveling on the ground, in small patches in locations with scarce trees, and during the dry and wet seasons in Balancan, Tabasco [Although a definitive mode of transmission for gh water , but wilgh water , suggestgh water –64, wet  Tabasco .A. palliata and A. pigra with locally adapted Blastocystis ST1 and ST2 populations exhibit distinct generalist and specialist types of host specificity. In addition, these data suggest that the Blastocystis ST2 populations in humans are highly differentiated from those of NHP, while the ST1 populations are only minimally differentiated. The host generalist and specialist specificities exhibited by ST1 and ST2 Blastocystis populations are thus distinct processes. Because ST1 exhibits a generalist profile, this haplotype can be considered a metapopulation; in contrast, Blastocystis ST2 exists as a set of local populations with preferences for either humans or NHP.The results of the present study demonstrate that howler monkey populations of Furthermore, it is important to highlight the use of population genetics studies in the epidemiology of this eukaryote because these studies may clarity some aspects of host-parasite relations; for example, how genetic variability within a subtype is reflected in phenotypic and functional variability and its potential role on the symptoms of the parasite carriers .S1 FigBayesian phylogenetic tree using a fragment of SSUrDNA sequences; the values of the nodes indicate posterior probabilities values using 10 million generations. ST and GenBank accession numbers are shown, as well as identification of each sample.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.


Abies taxa, which include both widely distributed species and taxa with minuscule ranges, represent a good model to study the impacts of range size and fragmentation on the levels of genetic diversity and differentiation. To assess the patterns of genetic diversity and phylogenetic relationships among eastern‐Mediterranean Abies taxa, genetic variation was assessed by eight nuclear microsatellite loci in 52 populations of Abies taxa with a focus on those distributed in Turkey and the Caucasus. Both at the population and the taxon level, the subspecies or regional populations of Abies nordmanniana s.l. exhibited generally higher allelic richness, private allelic richness, and expected heterozygosity compared with Abies cilicica s.l. Results of both the structure analysis and distance‐based approaches showed a strong differentiation of the two A. cilicica subspecies from the rest as well as from each other, whereas the subspecies of A. nordmanniana were distinct but less differentiated. ABC simulations were run for a set of scenarios of phylogeny and past demographic changes. For A. ×olcayana, the simulation gave a poor support for the hypothesis of being a taxon resulting from a past hybridization, the same is true for Abies equi‐trojani: both they represent evolutionary branches of Abies bornmuelleriana.The eastern‐Mediterranean  The genus itself is the second‐largest in the family Pinaceae with the number of species varying between 48 and 59 depending on particular taxonomic revision  Spach are unanimously classified into the section Abies. A. cilicica (Antoine & Kotschy) Carr. with two subspecies (subsp. cilicica and subsp. isaurica) was assigned by Liu . Distribution area of A. bornmuelleriana is located south from the Black Sea, mainly in the Köroğlu, Ilgaz, and Küre Mountains. Finally, the nominate subspecies A. nordmanniana subsp. nordmanniana (further A. nordmanniana s.s.) occurs near the southeastern and eastern coast of the Black Sea, from the Pontic Mountains to the western part of the Greater Caucasus. Far south from the A. nordmanniana populations, A. cilicica subsp. cilicica (further A. cilicica s.s.) grows in Central Taurus, Anti‐Taurus (Aladağlar), and the Amanos Mountains in Turkey, the Alawi Mountains in Syria and the Northern Mount Lebanon Range in Lebanon. Area of A. cilicica subsp. isaurica is located in the Western Taurus in Turkey.d Cullen , it growAbies that included A. nordmanniana and A. cilicica , A. cephalonica , A. nordmanniana s.s. , A. bornmuelleriana , A. equi‐trojani , A. ×olcayana , A. cilicica s.s. , and A. isaurica . An overview of geographical positions of these populations is shown in Figure A total number of 1,529 individuals from 52 indigenous populations were sampled; mostly in managed, naturally regenerated stands, less frequently in national parks, and nature reserves  with Q solution according to manufacturer's instructions and approximately 50–100 ng DNA. Concentrations of primers were 0.20 μmol/L NFF3, 0.10 μmol/L NFH15, 0.20 μmol/L NFH3, and 0.10 μmol/L Ab15 in multiplex A; 0.15 μmol/L AfSI16, 0.15 μmol/L SF1, 0.30 μmol/L SFb4, and 0.15 μmol/L NFF7 in multiplex B. The singleplex reactions for primers SF 324, SF333, and ABF18 were done in 5 μl mixtures containing 0.2 U of Hot FirePol DNA polymerase , 1× PCR buffer, 3 mmol/L MgCl2, 0.2 mmol/L dNTP, 0.8 mol/L BSA, and 0.3 μmol/L of the corresponding primer. Amplification programs consisted of initial step at 94°C for 15 min., followed by 35 cycles of 30 s denaturation at 94°C, 90 s annealing at 58°C, and 90 s elongation at 72°C. A final elongation step was performed at 60°C for 20 min. 0.9 μl of multiplex A product with 0.1 μl of size standard GeneScan 500 LIZ  and 9 μl of formamide was loaded as a first batch for separation on capillary analyzer ABI 3130 (Applied Biosystems). Second batch consisted of the singleplex products (0.6 μl of each) together with 0.8 μl of multiplex B product, 0.1 μl of size standard, and 7.3 μl of formamide. genemapper 4.0 (Applied Biosystems) was used to analyze the raw data and produce genotypes.DNA extraction protocol according to Doyle and Doyle  was used2.3micro‐checker 2.2.3  was used  , A. bornmuelleriana, A. cilicica s.l., A. cilicica s.s., and A. isaurica populations. The presence of barriers against gene flow was tested using the Monmonier's maximum‐differences algorithm implemented in the barrier 2.2 software , starting from 1, until new barriers with a bootstrap support of at least 50% were appearing.Isolation by distance was tested according to Rousset , regressSlatkin,  against Slatkin,  was useddiyabc v. 2.05  for specific segments of the speciation sequence displayed in Figure A. nordmanniana s.l. grows in similar communities with similar stand structures  for the conversion.For the recalculation of the number of generations into a time scale, a reasonable estimate of generation time needed to be chosen. In silver fir, isolated trees start flowering at 20 years of age and trees in a canopy at 60 years Wolf, , but thi3A. alba cannot be considered representative of the whole species but for the other species and subspecies, sample sizes are sufficient to provide a realistic view. Both at the population and taxon level, the subspecies or regional populations of A. nordmanniana s.l. exhibited generally higher allelic richness and expected heterozygosity compared with A. cilicica s.l., while the difference in private allelic richness was nonsignificant. Within A. nordmanniana, neither allelic richness nor gene diversity within populations varies substantially and there is not visible difference pattern between the stenoendemics and the more widespread taxa or regional groups. On the taxon level, however, the endemics A. equi‐trojani contains clearly less alleles and private alleles  than the other subspecies . Expected heterozygosity did not show any geographical trend, nor it was associated with the range size: Endemic and geographically proximate A. equi‐trojani and A. ×olcayana exhibited contrasting values. In A. cilicica, the subspecies A. cilicica s.s. showed genetic variation values comparable to A. nordmanniana ; in contrast, A. isaurica was distinctly less diverse, with lower allelic richness (A[140] = 10.50) and private allelic richness (P[140] = 0.12).Table Abies species and subspecies, A. cilicica subsp. isaurica was substantially more differentiated (FST = 0.0562). In contrast, absence of differentiation within A. equi‐trojani (FST = −0.0027) indicates that the three studied samples were in fact drawn from the same population . While the first and the second clusters joined several taxa, the third and the fourth clusters split the A. nordmanniana s.s. populations into two groups: northern (located around the Greater Caucasus) and southern . The fifth and the sixth clusters very clearly distinguished A. cilicica subsp. isaurica and A. cilicica subsp. cilicica  cluster into the respective species. The second cluster covering northwestern and northern Turkey was subdivided into A. equi‐trojani and the rest, while A. ×olcayana was not clearly differentiated from A. bornmuelleriana. In the northern A. nordmanniana s.s. (fourth) cluster, the analysis rendered the Georgian population (Ann16) as different from the rest. This population is located further from the rest of the cluster and near the border with the southern group; otherwise the two clusters in A. nordmanniana s.s. did not exhibit any clear geographical pattern. In A. isaurica (fifth) cluster, the population Aci3 that formed an individual subcluster is located further from the rest of populations. Again, the other two subclusters were not clearly differentiated. Finally, in A. cilicica s.s., the secondary analysis singled out the three eastern populations that showed an admixture from the A. isaurica cluster.Nevertheless, the multimodality of the Δly Table a,b. ThisAnalysis of molecular variance showed that there is highly significant variation both at the species and the subspecies level, even though the respective variance components both represent around 5% of the total variation Table . As specA. cilicica subspecies, both from the rest and from each other  were positioned separately. The northern A. nordmanniana s.s. group was the most distinct, while the A. equi‐trojani was the least distinct group, close to A. bornmuelleriana.Discriminant analysis of principal components showed a clear and strong differentiation of the two her Fig. . To studA. cilicica were by far the most differentiated groups. They were followed by the A. alba and A. cephalonica populations. The A. nordmanniana groups were more mixed. The northern populations of A. nordmanniana s.s. represented the most distinct group in this cluster. However, it was possible to identify all the other groups except for A. ×olcayana.The neighbor‐net Figure  similarlp < .0001, Mantel test), the RMA slope was 0.1685  but RMA explained less than 5% of the total variation only (R2 = 0.0478). Otherwise, a significant IBD was observed across A. nordmanniana s.l. (p = .0036) and in the southern group of A. nordmanniana s.s. (p = .0207) (Table Isolation by distance across the whole population set was significant (7) Table .barrier analysis coincided well with both distance‐based and model‐based approaches. The Monmonier's algorithm yielded 10 barriers with a bootstrap support of >0.5 , no new barriers appeared. The strongest barriers appearing already at the lowest number of groups K were those separating A. cilicica from the remaining species and separating A. cilicica subspecies from each other. Moreover, well‐supported barriers appeared also among the populations of A. isaurica. Other barriers separated European species from the Asian, A. equi‐trojani from the rest of A. nordmanniana s.l., and the Caucasian A. nordmanniana s.s. from the populations of Asia Minor. The easternmost A. bornmuelleriana population (Samsun‐Vezirköprü) and both easternmost A. nordmanniana s.s. populations in Georgia (Ritsa 2 and Ritsa 3) also formed separate groups.The 0.5 Fig. , whereasNe) and divergence times are shown in Table A. ×olcayana, the simulation gave a poor support for the hypothesis of being a taxon resulting from a past hybridization between A. equi‐trojani and A. bornmuelleriana but rather it is a clade belonging to the latter subspecies. The same is true for A. equi‐trojani: It represents an evolutionary branch of A. bornmuelleriana, which diverged from the parental line around 300–400 ky before present, rather than being a hybrid of A. alba and A. bornmuelleriana. The two alternative scenarios for A. nordmanniana s.l. had quite equivalent posterior probabilities (0.608 vs. 0.392) indicating that the intention of drawing a scenario for the whole species based on the available data was too ambitious. Here the simulations yielded earlier separation of A. equi‐trojani from A. bornmuelleriana (621 ky BP) and a very similar divergence time of the North‐Caucasian A. nordmanniana s.s. populations from the South‐Caucasian and Anatolian ones (692 ky BP). In A. cilicica, extreme differentiation and low allelic richness of A. isaurica suggest that this subspecies may have experienced a severe bottleneck in its history. However, the pure‐divergence model with stable effective population sizes in both subspecies  performed better than the model including Ne change and indicated an early split of the subspecies (695 ky BP).ABC simulations were run for a set of scenarios of phylogeny and past demographic changes in specific sets of fir eastern‐Mediterranean taxa, addressing the questions formulated in the Introduction section. In spite of a careful tuning of model priors, the performance of simulations was suboptimal Table , Fig. S644.1In contrast to continental Europe, the Mediterranean basin as a hotspot of species and genetic diversity . The ranges of these two taxa are not completely continuous but they form generally large local forest complexes  is not substantially different but the more rugged topography may contribute to isolation of local populations  and lowest interpopulation differentiation were observed in A. equi‐trojani and the putative hybrid A. ×olcayana are separated by a large gap from the rest of A. nordmanniana s.l. Moreover, the Kazdağı subpopulation suffers from logging, acid rain, and degradation of the habitat  which, seen from the perspective of conservation, is a good news.The populations of the westernmost subspecies A. cilicica (notably in A. isaurica) were generally lower compared with the remaining taxa. The reasons may be associated with biogeography. The range of A. cilicica is more fragmented and the whole Taurus mountain range suffers from long‐term forest degradation, especially at lower elevations  within most taxa. Along with a cline‐like pattern of structure group proportions, this indicates that a certain level of gene exchange between neighboring populations occurs in Abies nordmanniana s.l. The intrataxon differentiation level in Ponthic firs is clearly correlated with the range size: no differentiation in stenoendemic A. equi‐trojani vs. moderate levels in A. bornmuelleriana and regional populations of A. nordmanniana s.s. Unexpected was a strong differentiation between the North‐Turkish/Georgian and Russian populations of A. nordmanniana s.s. In contrast to our study, Hansen, Kjær et al.  allowed getting an insight into phylogeny and demographic processes in eastern‐Mediterranean firs.Even though the set of analyzed nSSR loci offers a quite poor coverage of the genome, the combination of distance‐based  and model‐based Bayesian approaches  levels of admixture in all populations, exhibiting a geographical trend only across A. nordmanniana s.l. and partly across A. cilicica s.l., which may reflect recent gene flow. A relatively high proportion of A. alba/cephalonica gene pool in the southern A. nordmanniana populations is more plausibly due to shared ancestral polymorphisms, as this gene pool is mostly absent in geographically intermediate A. bornmuelleriana. At the same time, neither structure nor ABC simulations supported the hypotheses of hybrid origin of A. × olcayana and A. equi‐trojani, suggested by Ata and Merev  as suggested by Ata and Merev , except A. cilicica s.l., which is Near Threatened as a whole, although local populations (mainly outside Turkey) are considered Critically Endangered. The outcomes of our study generally support this view. Even though genetic variation at neutral loci does not directly imply high adaptive variation, the observed relatively high levels of allelic richness and gene diversity within the studied taxa, including endemic and quite isolated A. equi‐trojani, are promising for their further survival. The only exception is A. isaurica; limited allelic richness and a strong differentiation from all remaining taxa make it a primary potential target of gene conservation efforts.The studied species are generally classified as Least Concern in the IUCN Red List (None declared.LP designed and organized the study, MH, DG, and DK made the analyses and statistical treatment, SK, HS, ITu, and ITv designed sampling and provided materials, MH and DG wrote the first draft, all authors contributed to the final text. Click here for additional data file.


Daktulosphaira vitifoliae, is a gall-forming insect that feeds on the leaves and roots of many Vitis species. The roots of the cultivated V. vinifera cultivars and hybrids are highly susceptible to grape phylloxera feeding damage. The native range of this insect covers most of North America, and it is particularly abundant in the eastern and central United States. Phylloxera was introduced from North America to almost all grape-growing regions across five of the temperate zone continents. It devastated vineyards in each of these regions causing large-scale disruptions to grape growers, wine makers and national economies. In order to understand the population diversity of grape phylloxera in its native range, more than 500 samples from 19 States and 34 samples from the introduced range  were genotyped with 32 simple sequence repeat markers. STRUCTURE, a model based clustering method identified five populations within these samples. The five populations were confirmed by a neighbor-joining tree and principal coordinate analysis (PCoA). These populations were distinguished by their Vitis species hosts and their geographic locations. Samples collected from California, Europe and South America traced back to phylloxera sampled in the northeastern United States on V. riparia, with some influence from phylloxera collected along the Atlantic Coast and Central Plains on V. vulpina. Reproductive statistics conclusively confirmed that sexual reproduction is common in the native range and is combined with cyclical parthenogenesis. Native grape phylloxera populations were identified to be under Hardy-Weinberg equilibrium. The identification of admixed samples between many of these populations indicates that shared environments facilitate sexual reproduction between different host associated populations to create new genotypes of phylloxera. This study also found that assortative mating might occur across the sympatric range of the V. vulpina west and V. cinerea populations.Grape Phylloxera,  Daktulosphaira vitifoliae (Fitch) is an aphid-like root and leaf-feeding insect that is now found in most of the world’s vineyards. Phylloxera gained the reputation as the most important viticultural insect pest after they were accidentally introduced from their native home in North America into France during the mid 1800s [Vitis vinifera. The leaves of V. vinifera grapes are relatively tolerant to phylloxera feeding, but their fine and structural roots are highly susceptible [V. vinifera cultivars onto rootstocks derived from resistant North American Vitis species, which allow feeding on young root tips and leaves, but prevent destructive feeding on the structural roots, was the only effective means of control.Grape phylloxera, id 1800s . There tceptible . The intceptible . From FrThe native range of grape phylloxera extends from southern Canada to northern South America, but they are most common in the eastern and central United States from Texas to Canada and east to the Atlantic Ocean. Grape phylloxera can also be found in geographically isolated locations in the southwestern United States. The classic life cycle of phylloxera is comprised of cyclic parthenogenesis with temporal polyphenism , 4. The V. arizonica and six sites in New York on the host species V. riparia. The samples collected from Arizona were strongly associated with collection site and specific host plant. Later, Downie et al. [Across its native range, phylloxera feed on young leaves and root tips of American grape species , 12, 13.V. vinifera [DNA marker based studies from samples collected in the introduced range also show evidence of the selective pressure of different host plants , 9, 18. vinifera , indicatVitis species hosts across the grape phylloxera’s native range. Samples from the introduced range in the United States  and from other countries in Europe and South America were also included. The objectives of the study were to assess the genetic diversity of grape phylloxera in its native range and to define boundaries of host and geographic associations; to evaluate grape phylloxera’s mode of reproduction in relation to variable host plants, climates and geographic regions; and finally to establish and evaluate the point of origin of grape phylloxera currently present in California, Europe and South America.Grape phylloxera is an appealing insect for the study of the evolution and maintenance of sex because of its ability to have both sexual and asexual life cycles . They arVitis species. However, samples collected from Arizona, New Mexico, South Dakota and Utah had more triploid data points compared to samples from other regions. When more than two alleles were observed, data was considered missing for that genotype at that marker. In next stage, a total of 47 samples were removed from the study that had missing data for more than three markers. From the remaining 502 samples, a total of 466 unique MLGs were identified with data from 26 SSR markers ; four samples that were collected from different sites and separated by large distances accounted for 2 unique MLGs ; lastly two samples collected from South Dakota from the same site but different plants constituted 1 MLG  and not the result of independent sexual events (data not shown).A set 549 samples collected from phylloxera’s native range in the USA, and samples from California, South America and Europe were genotyped with 32 SSR markers. Six markers were eliminated for reasons explained in the Methods section. Three alleles were observed in 134 samples with one or more markers resulting in 176 triploid data points . More th markers . Four hued 1 MLG . We ruleV. vulpina west population, and the other grouped in association with the V. riparia population.The first STRUCTURE output indicated a total of 4 populations within 466 MLGs supported by both delta K and Ln P(D) plateau. A closer examination of the individual replicated STRUCTURE runs indicated that population assignment of unique MLGs in two populations was stable and did not change within different replicated runs. However, the position of 34 samples switched between two other populations during the replicated STRUCTURE runs. A second STRUCTURE analysis with 175 MLGs from the two populations in question divided the samples into three distinct populations. The 34 samples that switched groups in the first STRUCTURE analysis formed a new population. We combined the STRUCTURE results from the two analyses and designated five populations within the 466 unique MLGs. Four hundred twenty-five samples (91.2%) were assigned to one of the 5 populations with a STRUCTURE q value of 0.90 or above. An additional 17 samples (3.6%) were associated with one of the 5 populations, but at a lower STRUCTURE-based probability (q was between 0.70 and 0.89). Only 24 samples (5.1%) were admixture samples with no population assignment  including four samples that did not group consistently across the analyses. Two of the admixed samples grouped with the V. vulpina west population, and other two were associated with the V. riparia population group. Samples from Hungary, Austria, Brazil and Uruguay were all members of the V. riparia population. California SAL samples that were taken from the rootstocks 101-14Mgt (101-R1 and 101-R2) and Freedom (Fre-R1 and Fre-R2B) were also members of the V. riparia population. Samples from Peru, Argentina and two other California SAL lines (Vin-R1 and AxR-R1) clustered together as a small group near the V. riparia population in the PCoA . The California foliar sample (WEO4802) was identified by STRUCTURE as an admixture between the V. riparia and the V. vulpina west populations  and was not associated with other samples in the PCoA. The samples identified as admixtures by STRUCTURE were found outside the 5 distinct populations when the PCoA was carried out on all unique MLGs . The V. cinerea population contained 72 percent of the samples collected on V. cinerea, and 100 percent of the samples collected on what appeared to be V. cinerea x V. vulpina hybrids. Similarly, the V. vulpina east and V. vulpina west populations contained 97 percent of the samples collected on V. vulpina.A strong host species influence on the population structure of the phylloxera in its native range was observed . Ninety-V. vulpina did not cluster with either the V. vulpina east or west populations. One of the outliers collected on V. vulpina was collected on the border of the V. vulpina east and west populations and was identified as an admixture between the two populations by STRUTURE. The remaining three outliers from V. vulpina all came from one plant collected in Texas and were identified by STRUCTURE as associated with the V. riparia population or were an admixture of the V. riparia and V. vulpina west populations. Three samples collected on V. riparia clustered with the V. vulpina west population. One of the samples was from a site in Indiana where members of both the V. riparia and V. vulpina west population were identified; while the other two samples were found at a site in Tennessee halfway between Nashville and Memphis. Three of the samples collected on V. arizonica at one site in Silver City, NM clustered with the V. vulpina east population. These samples were collected in an urbanized area and these V. arizonica vines may have been hybrids with other species. Six of the samples collected on V. cinerea at one site in Florida were all identified by STRUCTURE as being admixtures between the V. cinerea and V. vulpina east populations.Only 16 samples collected across the four main host species appeared to be outliers. Four of the samples collected on Vitis species sampled in this study  were identified at this collection site. While we did not identify V. riparia plants in this region, earlier work placed V. riparia in this part of Texas [V. vulpina east population samples found in New Mexico, and the V. arizonica population samples found in South Dakota, were likely the result of the movement of infested plant material by humans. The V. arizonica population samples collected in South Dakota were from a vineyard associated with South Dakota State University where phylloxera may have been accidentally introduced. Similarly the samples from the V. vulpina east population collected in New Mexico were found in downtown Silver City along a city park/river walk where human importation or movement of grapes from the southeastern United States would be possible.There were four sites where one of the populations appeared to be outside of their geographic region . The firof Texas . The V. V. vulpina west and V. cinerea populations had a large genetic distance (1.3781) even though they have a sympatric range. The divergence between the two populations can also be seen in their FST values  per locus varied between 3.19 and 7.85, while the effective number of alleles (ne) ranged from 1.72 to 2.93  ranged between 0.249 and 0.415, while expected heterozygosity (He) ranged between 0.293 and 0.501. For all five populations He was greater than Ho. The V. arizonica population had the greatest disparity between He and Ho with a difference of more than 0.2. The remaining populations were more similar with differences in He and Ho values of less than 0.09.Values for Nei’s genetic distance among the 5 populations varied from 0.6909 to 1.9360 . InteresT values . The ove to 2.93 . ShannonV. arizonica population–V. arizonica and V. vulpina–V. vulpina and the samples collected in Arkansas and Tennessee) were identified as having a structured population with multiple sub-populations within the group, a situation that can disrupt HW equilibrium [V. arizonica population and samples collected on V. arizonica as the only groups where HW equilibrium could be rejected.The evidence for sexual reproduction was analyzed at multiple levels. ilibrium . RemovinIS values were generally positive and ranged between -0.108 and 0.506. Only six groups  had negative FIS values. The highest FIS values were detected in the three individual collection sites in Arizona  ranged between 0.688 and 1.000. All groups related to the V. arizonica and V. cinerea populations had R-values of 1, indicating that no clonal MLGs were sampled. Additionally, samples from Indiana, Minnesota, northern Massachusetts and southern Florida also had R-values of 1. The lowest R-values  came from groups associated with North Carolina, Tennessee, and Virginia on V. vulpina and to a lesser extent from samples collected on V. riparia in Pennsylvania. Climatic data were also evaluated to determine whether temperature plays any role in increasing or decreasing clonal diversity. No trend was observed as clonal samples were identified in both warm and cold climate sites.F Arizona , samples Arizona , samplesrizonica , and thepulation , indicatMore than two alleles were observed in 134 samples with one or more markers. Occurrence of multiple alleles indicates that either the primer sequences had multiple priming sites due to a lack of sequence specificity, or that genomic regions represented by these primers were duplicated in the grape phylloxera genome, or that the samples were mix of two or more genotypes. Sample contamination could have happened at all stages from phylloxera extractions, DNA isolation and fragment size analysis. Sample contamination had occurred at some point with the original FreR1bulk isolate that was verified by single adult DNA extractions with results suggesting that cross contamination with the AxRR1 line had occurred. When the remaining three SAL lines that showed three alleles  were retested, genotypic data remained consistent, indicating that these lines have real third allele at the PhyIII_19 marker. The identification of more than two alleles with 20 SSR markers in 134 samples is the first indirect glimpse into the genomic complexity of phylloxera. The grape phylloxera genome is approximately 400 Mb and initiatives are underway to fully sequence the genome  to improV. riparia conform to one population both with RAPD [V. riparia were placed in the V. riparia population. This is an important result and shows that phylloxera populations adapted to V. riparia are stable for at least the 12 years between two studies. The other host-based populations identified earlier were less consistent within those studies [V. riparia and V. arizonica- based samples. The concept of host-based selective pressure is common in grape phylloxera both within their natural range [V. vulpina-based samples into east and west populations identified for the first time in this study indicates that the host may not be the only factor distinguishing phylloxera populations.In this study we utilized three different analyses  to identify the approximate number of genetic clusters within the sample set. The program STRUCTURE  has beenith RAPD  and mitoith RAPD . In this studies  and resual range . The hosal range , 29, 30.Vitis species is not easy. Most Vitis species are associated with distinct geographic ranges. For instance, V. riparia is most commonly found in the north central and northeastern United States and V. arizonica is restricted to the southwestern United States and northern Mexico. There was a geographic distinction within the samples collected from V. vulpina. These collection sites were separated by the Appalachian Mountains and were grouped into an eastern and western population. The climatic conditions on either side of the Appalachian range are distinctly different with relatively colder climate on the west side, an additional factor capable of increasing the selective pressure driving divergence and specialization on the same host plant. Geographic correlations were also found in previous studies of grape phylloxera’s native range [Separation of host- and geography-based associations within ve range , 13. In ve range , and on ve range . The effve range , 30, 31.ST and Nei’s genetic distance varied widely among the 5 populations, while other parameters were less variable. When the smaller V. cinerea population was excluded, the remaining four populations had similar values for the number of alleles, effective alleles and information content of alleles. These results indicate that although the four larger populations are genetically distinct from each other but they have comparable levels of diversity within each. The major differences among the populations were lower observed heterozygosity in comparison to their expected values indicating that inbreeding is playing a role. Specifically, V. arizonica and V. cinerea-based populations had significantly lower Ho than He as a result of their more isolated geographic locations and potential for increased inbreeding.Population statistic comparisons of 5 phylloxera populations also showed distinct patterns. Both FV. riparia population , showed association with the pulation . In an eV. riparia and V. vulpina east populations. Previous work by Downie [V. vulpina along the Atlantic coast. The matching of mitochondrial DNA from the Atlantic coast in Downie’s work [V. riparia and the V. vulpina east populations based on SSR markers may indicate the source of these samples. Our results suggest that the root phylloxera samples from Argentina, Peru and the CA SAL lines VinR1 and AxRR1 tested in our study are the result of sexual reproduction between the V. riparia and V. vulpina east populations with the female coming from the V. vulpina east population. The matching of mitochondrial haplotypes was also used to confirm the presence of the same phylloxera clone in California and Peru [In this study, phylloxera from Argentina, Peru and California (AxRR1 and VinR1) appeared to be admixtures based on STRUCTURE and PCoA results . These ay Downie  using mie’s work  and the and Peru . CarefulV. riparia and V. vulpina west populations, and was similar to phylloxera collected in Indiana—where these two populations overlap. Further work is needed to determine how this phylloxera strain got to California and whether it exists in other parts of the introduced range.Foliar phylloxera has been rare in California, until the discovery of a widespread outbreak of leaf-galling phylloxera that occurred in grape rootstock nursery plantings in Yolo and Solano counties. One hundred and twenty-two of the 170 leaf-galling phylloxera samples tested in a previous study  were detIS values, major departures from HW equilibrium and high numbers of clones with low clonal diversity at any one site.The occurrence of sexual reproduction was expected in phylloxera’s native range as active sexual morphs have been identified in the southwestern, central and eastern United States , 33. HowV. vulpina west and V. vulpina east populations, especially from sites collected in North Carolina, Virginia and Tennessee had more clonal MLGs .Samples were also collected from central Arizona in 2010 and from eastern Arizona and western New Mexico in 2011. Sampling was more sporadic in these trips, as the region’s dry climate forms natural barriers to the distribution of grape, and thus phylloxera. In addition, several collaborators from New York , Indiana, Minnesota, South Dakota, southern Texas, Utah and western Florida, sent phylloxera samples to include in this study. Samples from Indiana, Minnesota, South Dakota and the western Florida sites were made at local vineyards growing American To get a representation of phylloxera from other countries, samples were obtained from Europe (Hungary and Austria) and South America . With the exception of Argentina, samples from other South American countries arrived as dried DNA pellets extracted from foliar phylloxera taken from leaf galls. These samples were re-suspended in 60μl of pure water prior to being stored at -20°C. Samples from Europe and Argentina arrived as multiple adults collected from roots and suspended in alcohol.V. vinifera cv. Chardonnay at the University of California, Davis vineyard. AxR-R1 was collected from the roots of AxR#1 rootstock in Mendocino County. 101-R1 and 101-R2 were collected from the roots of the rootstock 101-14Mgt from different vineyards in Sonoma County. Fre-R1 and Fre-R2 were collected from the rootstock Freedom in separate Napa County vineyards. The WEO4802 sample was collected from a foliar gall on St. George rootstock at the National Clonal Germplasm Repository, Winters CA.Six single adult lineage (SAL) lines were developed from root feeding behavior experiments on various rootstock hosts in California. Each line was started with single adult phylloxera growing on an excised root in a petri dish following procedures modified from De Benedictis and Granett . The eggIndividual galls on each leaf were opened with sterile equipment to examine their contents with 10X magnification. Ideally, galls containing one adult with 10 to 100 eggs were collected for DNA extraction. If galls with single adults were unavailable, galls containing multiple adults or galls containing only crawlers were used. The contents from selected galls were placed in a 1.5mL centrifuge tube and stored in a -20°C freezer until DNA extraction. Whenever possible 3, and up to 10, samples were isolated from each sample bag. Bags were kept sealed at all times to avoid cross contamination and left over plant material was autoclaved to prevent dispersal of phylloxera. The SAL phylloxera samples were composed of 50 or more adults from multiple generations. DNA was extracted from all samples using the protocol described in Lin and Walker . After D2 solution. All SSR primers were amplified at a 56°C annealing temperature, keeping all other conditions of the protocol constant: 10 minutes at 95°C; 35 cycles of 45 s at 92°C, 45 s at 56°C, 1 minute at 72°C; with a final extension of 10 minutes at 72°C.A total of 540 samples were genotyped with 32 fluorescently labeled  simple sequence repeat (SSR) primers published in previous studies , 10, 42.PCR products of up to four primers were mixed taking into account label color and fragment size. One μl of mixed products was added to 11μl of HD-formamide and 0.2μl of the internal size standard GeneScan-500 Liz . The mixture was denatured at 92°C for 2 minutes prior to being run on an ABI 3500 Genetic Analyzer  using a 50 cm capillary filled with POP-7 polymer . Allele sizes were determined using GeneMapper 4.1 software .Six markers were discarded due to missing data with more than 20% of samples, and/or inconsistencies in genotyping due to one base pair allelic variation and weak amplifications in different PCR groups. Many samples showed a third allele with one or more markers. In that case, the data were considered missing for that genotype at that marker. In the next step, all samples that had missing data for more than three markers were removed. The FreR1 line was found to be mix of two separate multi-locus genotype (MLG), and was replaced with FreR1A and FreR1B to identify two separate MLGs. The microsatellite tool kit software  was usedsex). Because the program is unable to accommodate missing data, two runs were performed. The initial run consisted of the 215 samples with no missing data at 26 loci. The second run consisted of the remaining 286 samples and 22 loci; four markers  were removed due to missing data. The program was run using both HWE and FIS models at 1,000 simulations each. Analysis of this set identified seven possible clonal MLGs.To investigate whether matching/clonal samples were the result of asexual reproduction (a true clone) or the result of independent sexual events the program MLGsim  was usedThe software STRUCTURE V2.3.1  was usedSamples were considered “in” a cluster when the q value generated from STRUCTURE was 0.90 or above. Samples were considered to be “associated with” a genetic cluster if their q value was between 0.70 and 0.89. To evaluate the STRUCTURE output results, sample sets were also analyzed with the DARwin software . The disA total of 175 samples showed membership in two populations based on the assigned q-values from the first STRUCTURE run. A second STRUCTURE run was carried out to better assess the clusters within these two populations in the absence of outliers. The criteria described above were used except that the K value ranged from 1 to 6. The number of clusters was determined by both delta K and the plateau point of the Ln P(D) and groupings were also confirmed with a neighbor-joining tree and PCoA. The results of the two STRUCTURE runs were used to color code the samples to reflect their population assignments. The neighbor-joining tree and PCoA analyses were used to verify the refined genetic clusters in the entire set of 466 MLGs and to display the results.ST values [A comparison of the geographic range and host-based association was conducted using species assignment given to the host plants when collected. To test for differences among the populations. Nei’s genetic distance  was calcT values  between T values  to evaluIS [IS.Phylloxera’s reproductive mode was investigated and compared within groups delineated by population, collection site, State, host, and hosts within States. Any group with less than 8 MLGs was excluded from the analysis. Clonal diversity (R) of each group was calculated as (G-1)/(N-1) where G was the number of MLGs present inside of a specific group and N was the total number of samples in that group . Clonal IS  was testhttp://www.ncdc.noaa.gov/data-access/land-based-station-data/land-based-datasets/climate-normals/1981-2010-normals-data). Clonal diversity was then compared to the lowest minimum and highest maximum temperatures for each collection site.Weather stations closest to each collection point were identified and records were downloaded from the National Climatic Data Center (NCDC) a unit of the National Oceanic and Atmospheric Administration (NOAA). Weather reports containing the average minimum and maximum temperature per month averaged between 1981 and 2010 for the identified weather station were obtained from an online database (S1 Table(XLSX)Click here for additional data file.S2 TableND reflects no data.(XLSX)Click here for additional data file.S1 FigV. vulpina west population by STRUCTURE, but were grouped with the population in the neighbor-joining tree and PCoA. Samples in squares were considered to be part of the V. riparia population by STRUCTURE, but were not grouped with the population in the neighbor-joining tree and PCoA. All admixed samples that were not placed in any one population are coded by black color.The X-axis accounts for 21% of the variation, while the Y-axis accounts for 8.27%. Each population is circled and labeled with a corresponding color. Samples in triangles were not considered part of the (TIF)Click here for additional data file.


Aframomum corrorima (Braun) P.C.M. Jansen] is a spice crop native to Ethiopia. Understanding the extent and partitioning of diversity within and among crop landraces and their wild relatives is among the first steps in conserving and measuring their genetic potential. The present study is aimed at characterizing the population genetic structure and relationships between cultivated and wild korarima in the southwestern part of Ethiopia.Korarima [We analyzed a total of 195 individuals representing seven wild and fourteen cultivated populations. Eleven polymorphic simple sequence repeat (SSR) markers were used. We observed a total of 53 alleles across the eleven loci and individuals. In total, 32 alleles were detected in the cultivated populations, whereas 49 alleles were detected in the wild populations. We found higher genetic diversity in wild populations than in the cultivated counterpart. This result implies the potential of wild korarima as a possible source for novel alleles contributing to the improvement of cultivated korarima. Analysis of molecular variance (AMOVA) showed significant but low differentiation between cultivated and wild korarima populations. Similarly, neighbour-joining and STRUCTURE analyses did not group cultivated and wild populations into two distinct clusters. The lack of clear differentiation between cultivated and wild populations could be explained by historical and contemporary gene flow between the two gene pools.Aframomum species. From the five administrative zones considered in this study, the Bench-Magi and Sheka zone showed populations with high genetic diversity, and these populations could be used as a potential starting point for in-situ and ex-situ germplasm conservation and korarima improvement through breeding programs after proper agronomic evaluation.The 11 SSR loci developed in this study could be employed to examine genetic diversity and population structure of korarima in other countries as well as other The online version of this article (doi:10.1186/s12863-017-0540-4) contains supplementary material, which is available to authorized users. Aframomum corrorima (Braun) P.C.M. Jansen) belongs to the monocotyledonous family Zingiberaceae [native to Ethiopia [Korarima  to evaluate the genetic variation of wild and cultivated korarima populations in southwestern Ethiopia (2) to determine the presence of indirect evidence for crop-wild hybridization by testing for admixture of SSR alleles in cultivated and wild korarima populations from the same regions (3) and to determine potential factors shaping the population genetic structure of cultivated and wild korarima in Ethiopia.The genus For this study, 195 individuals from 21 korarima populations (14 cultivated and 7 wild) were sampled across five zones/areas  in the southwestern part of Ethiopia . QIAamp® 96 DNA QIAcube® HT Kit was used to extract DNA from samples that we failed to isolate in desired quality and quantity using the CTAB method.Total genomic DNA was isolated from about 1 gram of pulverized leaf sample following a modified CTAB method employing triple extractions to yield optimal amounts of high quality DNA . GenomicAframomum in the NCBI nucleotide database, we used DNA sequences of Alpinia in GenBank to develop genomic SSR markers for Aframomum. Alpinia was used as it is the most closely related genus to Aframomum within the Zingiberaceae family [Alpinia species in NCBI for mining SSRs. DNA sequences that contain SSR with two to six nucleotide repeat motif were identified using WebSat web-based software [. The analysis revealed that only 1.5% of these sequences contain SSRs and they all were from Alpinia oxyphylla. After evaluating the suitability of these sequences for designing primers, 23 SSR primer-pairs were designed using the Primer3 primer designing program [Since there are no microsatellite containing DNA sequences of e family  which hasoftware . The anaAframomum corrorima cultivated and wild populations. The remaining 11 primer-pairs successfully amplified their target loci in A. corrorima populations and hence used for genetic diversity analysis , 0.3 mM dNTPs, 0.3 μM forward and reverse primers, 1.5 mM MgCl2 and 1 U Dream Taq DNA polymerase  was used for PCR reactions. The reactions were performed using a GeneAMP PCR system 9700 thermo cycler (Applied Biosystems Inc. USA) in 96-well plates. PCR amplification reaction consisted of 3 min preliminary denaturation at 95 °C, 30 s touchdown denaturation at 94 °C for nine cycle, 30 s annealing at 58 °C  and elongation for 45 s at 72 °C, afterward 29 cycles of denaturation at 94 °C for 30 s, annealing at 48 °C for 30 s, and elongation at 72 °C for 45 s. Following completion of the 29 cycles, a 20 min final elongation at 72 °C was included to allow completion of the reactions. Quality check on the PCR products were conducted with gel electrophoresis and PCR products were stored at 4 °C until further use.A total volume of 25 μl containing 25 ng genomic DNA, 1 For each locus, amplification and quality was confirmed by running 7 μl of the PCR products mixed with 2 μl of 6× loading dye on ethidium bromide containing 1.5% agarose gels. A DNA ladder with the size of 50 bp (GeneRuler™ Fermentas Life Sciences) was used as fragment size marker. The PCR products were multiplexed into three panels where markers with different fluorescent dyes and sizes placed together. ABI Prism 3730 DNA Analyzer (Applied Biosystems) was used for subsequent analysis of the multiplexed PCR products at Swedish University of Agricultural Sciences, Alnarp, Sweden.A. corrorima (one cultivated and one wild) amplified by the 11 primer-pairs were sequenced. Before the PCR products were sent for sequencing to Eurofins Genomics (Germany), they were purified using the Qiaquick PCR purification kit. Then, each purified PCR product was mixed with corresponding forward primer, which was used as sequencing primer. The final edited sequences were searched in NCBI through Basic Local Alignment Search Tool (BLAST) to find out if it hits with the original SSR containing microsatellite sequence or any other similar sequence in the database. Similar sequences were aligned using Clustal X version 2.1 software [To confirm that the amplified products are the target microsatellite loci, the PCR products of two individuals of software , and thesoftware .Allele peak and its size was identified using GeneMarker® V2.2.0 software  based on the Genescan-500 LIZ internal size standard. After pre-analysis of peaks using default settings in GeneMarker and visual examination, peaks with threshold intensity of 200 were accepted for scoring.ST. Analysis of molecular variance (AMOVA) was conducted using Arlequin 3.0 according to Excoffier et al. [For estimating observed heterozygosity, expected heterozygosity, gene flow and allele frequency based genetic distance analyses, POPGENE software version 1.31  was usedr et al. . A neighThe pattern of population structure and detection of probable introgression was visualized using a Bayesian model based clustering method implemented in STRUCTURE software version 2.2 . The sitAfco_8 and Afco_14, only one allele was detected within cultivated populations  and within wild populations , respectively, whereas the remaining nine loci were polymorphic within both groups of populations. Therefore, only the 10 polymorphic loci within each group were used to compute genetic diversity parameters when cultivated and wild populations were analyzed separately.In the present study, a total of 11 genomic-SSR loci was developed. Detailed information of these SSR- loci such as source sequence accession numbers, forward and reverse primers, repeat motifs, expected fragment size and observed fragment size range was described in Table A. oxyphylla sequences used for designing the SSR primer-pairs except in the case of Afco_2. The sequencing of the PCR product amplified by Afco_2 failed. All sequences that matched the original A. oxyphylla sequences contained the target microsatellites except in the case of Afco_8 which had poor sequence quality in the microsatellite containing region and hence trimmed off  that are unique to a single population. In the case of the cultivated populations, only one population (Mizan-Teferi_C1) produced a private allele, which was at locus Afco_11 , whereas private alleles were detected in three wild populations . The highest number of private alleles (9) was detected in the Mizan-Teferi_W population across six loci but with a low frequency ranging from 6.3% to 33.3%  to 2.39 (Afco_11) for cultivated populations and from 1.42 (Afco_8) to 4.87 (Afco_2) for wild populations. The Shannon diversity index (I) per locus ranged from 0.06 (Afco_19) to1.02 (Afco_2 and Afco_11) for cultivated populations and from 0.63 (Afco_8) to 1.72 (Afco_2) for wild populations. The average Shannon diversity index for all loci was 0.76 and 1.06 for cultivated and wild populations, respectively. Observed heterozygosity (Ho) ranged from zero at locus Afco-19 to 0.88 at locus Afco-11 in cultivated populations and from zero at locus Afco-19 to 0.86 at locus Afco-11 in wild populations. The average observed heterozygosity across all loci was 0.63 and 0.48 for cultivated and wild populations, respectively. Expected heterozygosity (He) ranged from 0.02 at locus Afco-19 to 0.58 at locus Afco-11 in cultivated populations and from 0.30 at locus Afco-8 to 0.80 at locus Afco-2 in wild populations. The average expected heterozygosity for all loci was 0.48 and 0.57 for cultivated and wild populations, respectively  per locus was from two to five in cultivated populations and from four to eight in wild populations. The effective number of alleles (ne) per locus varied from 1.02 (Percentage of polymorphic loci (PPL), observed and expected heterozygosity, Shannon diversity index was determined for each population. The maximum PPL per population was 100%, which was observed in the Mizan-Teferi_W from the Bench-Maji Zone, while the lowest was 60% for Masha_W (Sheka zone), both of which are wild populations. In the cultivated populations, the highest PPL was 90% which was recorded in seven of the 14 populations  and the least was 70% for the Masha_C1 population Table .Table 3PI = 1.03) and the lowest for Masha_W (I = 0.52), whereas in the cultivated populations, the highest was for Masha_C2 (I = 0.82) and the lowest was for Masha_C1 (I = 0.52). The highest and lowest observed heterozygosity was scored for Bonga_C1 (Ho = 0.78) and Tepi_C2 (Ho = 0.50) in the cultivated populations, and Metu_W (Ho = 0.73) and Masha_W (Ho = 0.30) in the wild populations. The expected heterozygosity in Masha_C2 (He = 0.56) was the highest and the lowest was 0.40 (for Jimma_C3) among the cultivated populations. Among the wild populations, the highest and the lowest expected heterozygosity was recorded in Mizan-Teferi_W (He = 0.64) and Masha_W (He = 0.38), respectively . In the case of AMOVA, the total genetic variation was partitioned into three hierarchical levels: among groups (cultivated vs wild), within groups among populations and within populations. Pair-wise FST was estimated for all populations as well as for cultivated and wild populations, separately.Two complementary approaches were used to explore the genetic diversity structure: analysis of molecular variance (AMOVA) and pair-wise fixation index (FP < 0.001) but low level of genetic differentiation (FST = 0.04) between the wild and cultivated populations. Of the total genetic variation, 5.7% was attributable to the variation between cultivated and wild korarima. The analysis revealed absence of differentiation among the cultivated populations as well as wild populations . However, in the case of wild populations significant differentiation was obtained at population , region  and zone  levels  to 1.56 (B_C1 vs M_W). The genetic distance within cultivated populations ranged from 0.01 (MT_C2 vs M_C1) to 0.19 (B_C3 vs M_C1). The genetic distance within wild populations ranged from 0.04 (Ma_W vs B_W and Ma_W vs JG_W) to 1.64 (M_W vs Ma_W) (Table ST) is presented in Table ST showed that M_W was significantly differentiated from all other wild populations as well as from all cultivated populations.The comparison of the extent of genetic differentiation within and between cultivated and wild korarima populations  . Primer equences . All of lla Fig. , which sNearly half of the loci developed for the diversity study amplified alleles exclusive to a single population . The mainly self-pollinating nature of korarima  could beZingiber officinale) accessions genotyped with eight SSR markers [Curcuma longa [Piper nigrum [A. corrorima and Z. officinale could be attributed to their diploid chromosome number, as C. longa and P. nigrum are triploid and tetraploid, respectively. In the present study, Afco-2 and Afco-11 were the most polymorphic loci both containing eight alleles. Comparable number of alleles per locus was reported in Z. officinale , C. lonalleles; ) and P. alleles; ). Based alleles;  reportedO) obtained in this study was 0.54  and in , . Likewim  and in a . The reOur study showed higher levels of diversity in wild populations compared to cultivated populations in terms of average number of alleles/locus, Shannon diversity index and expected heterozygosity. The average number of alleles/locus and Shannon diversity index of wild korarima were higher than that of cultivated korarima, 4.9 vs. 3.2 and 1.06 vs 0.76, respectively. The expected heterozygosity (He) showed the same trend with 0.57 and 0.48 for wild and cultivated korarima, respectively  possessing high percentage of polymorphic loci and high Shannon diversity index compared to other cultivated populations. This may indicate that farmers in this area grow a mixture of landraces of korarima or have their own unique culture of conservation. The lowest diversity found in the other two Masha populations (Masha_C1 and Masha_W) in the Sheka zone, which are cultivated and wild in that order, was rather unexpected. The decreased genetic diversity in cultivated Masha_C1 could be due to strong genetic drift which results in a rapid decline of population size or sampling error during individual collection. However, the reduced diversity observed in Masha_W is most likely due to human interference such as digging the rhizomes for planting and other traditional practices. These will result a decline in population size. There is a real risk that if the trend towards a decline in population size is left unchecked, genetic consequences associated with small isolated populations, including genetic drift and inbreeding, will reduce most of the genetic diversity in the Masha wild population. Thus, there is an urgent need to assess the distribution and size of ST = 0.04; P < 0.001). Pair-wise FST also showed the presence of low level of crop-wild genetic divergence with each wild population showing a significant differentiation from only less than half of the cultivated population. Several factors can be included in attempts to account for the observed genetic structure in cultivated and wild korarima. The present-day cultivated A. corrorima may have been directly introduced to cultivation from wild progenitors inhabiting the same localities and thus may have maintained most of their initial genetic diversity. In Southwest Ethiopia, the cultivation area of A. corrorima overlaps with the distribution of natural habitats of wild populations. Because of the sympatric distribution pattern of the wild and cultivated populations, migration from wild to cultivated populations by natural or artificial means may be an ongoing process. Therefore, subsequent gene flow from wild ancestors into cultivated populations  whereas wild (non-crop) populations showed significant population differentiation . The absence of clear differentiation among cultivated populations can be explained by the movement of genetic material between growers within and among the major production areas, which result in a higher degree of gene flow among cultivated populations  with FST value of 0.06 and 0.09 respectively  Passport data of Additional file 2:Aframomum corrorima  (DOCX 16 kb)Characteristics of 23 microsatellite primers developed for Additional file 3:A biplot detected the maximum peak at K = 2 (the optimum number of clusters) based on Evanno et al. 2005  predictiAdditional file 4:Neighbor-joining tree based on pair-wise simple matching dissimilarity index showing the genetic relationships among 101 individuals randomly re sampled from cultivated and wild korarima populations (19 populations were represented by five samples each and two populations were represented by three samples each). Blue color indicates individuals from cultivated populations whereas red represents individuals from wild populations. (DOCX 22 kb)Additional file 5:UPGMA dendrogram based on pair wise simple matching dissimilarity index showing the genetic relationships among Ethiopian cultivated and wild korarima populations using SSR markers. The analysis resulted in two major clusters shown as “I” and “II”. (DOCX 27 kb)Additional file 6:Allele frequency data for all korarima populations across the 11 SSR loci. (XLSX 18 kb)


Lymantria dispar populations from Far East Asia using microsatellite loci and mitochondrial genes. In the microsatellite analysis, the genetic distances based on pairwise FST values ranged from 0.0087 to 0.1171. A NeighborNet network based on pairwise FST genetic distances showed that the 20 regional populations were divided into five groups. Bayesian clustering analysis (K = 3) demonstrated the same groupings. The populations in the Korean Peninsula and adjacent regions, in particular, showed a mixed genetic pattern. In the mitochondrial genetic analysis based on 98 haplotypes, the median‐joining network exhibited a star shape that was focused on three high‐frequency haplotypes  connected by low‐frequency haplotypes. The mismatch distribution dividing the three groups was unimodal. In the neutral test, Tajima's D and Fu's FS tests were negative. We can thus infer that the Far East Asian populations of L. dispar underwent a sudden population expansion. Based on the age expansion parameter, the expansion time was inferred to be approximately 53,652 years before present (ybp) for Group 1, approximately 65,043 ybp for Group 2, and approximately 76,086 ybp for Group 3. We propose that the mixed genetic pattern of the inland populations of Far East Asia is due to these expansions and that the inland populations of the region should be treated as valid subspecies that are distinguishable from other subspecies by genetic traits.We analyzed the population genetic structure and demographic history of 20  The sequencing was performed with a MiSeq Sequencer  by the DNA sequencing company DisGene . The resulting contigs were assembled in CLC workbench .For NGS, we extracted genomic DNA from an egg mass of 2.2® Blood & Tissue Kit  according to the manufacturer's instructions.For polymerase chain reaction (PCR) analysis of polymorphisms with the developed microsatellite markers and for genetic structure analysis, 552 samples were collected from 20 sites in Mongolia 1), Russia (1), China (1), Korea (12), and Japan (5) using pheromone attraction traps . For the markers showing polymorphism in the electrophoresis, each forward (sense) primer for genotyping was labeled with 6‐carboxyfluorescein at the 5′ end . PCR products were checked using 1% agarose gel electrophoresis. The PCR products were purified and sequenced using the sequencing services of Macrogen  and Bionics . The obtained sequences were submitted to NCBI GenBank  was then estimated with Harvester  to estimate the number of genetic groups  on selected markers were checked with MICRO‐CHECKER ver. 2.2.3 . FST distances among all pairs in the population were used to assess the genetic structure of L. dispar asiatica. Population pairwise FST values were calculated using the Kimura two‐parameter model in Arlequin ver. 3.1  , their presence causes a slight underestimate of the average exclusion probability at a locus; however, this is usually not of sufficient magnitude to warrant great concern. For p > .20, however, the mean “estimated with null” exclusion probability can be much higher than the “true” and “estimated without null” values  of each regional population were estimated with eight markers. We determined the allele type frequencies based on microsatellite loci for each population . Deviations from HWE were not detected in the exact p‐values; however, some markers were identified as relatively low exact p‐values in each population . Testing for genotyping errors at each locus revealed that one marker  showed evidence of null alleles , yet showed a high genetic distance of 0.1030 when compared to a Jeju regional population (Site 31). A Chinese regional population (Site 39) was more similar to a Korean inland population (Site 28) with a relatively large geographical distance (genetic distance: 0.0050 and geographic distance: 776.35 km) than to a Russian population (Site 33) with a relatively short distance (genetic distance: 0.0199 and geographic distance: 289.10 km). A Kyushu regional population (Site 41) with a small sample size had a genetic distance of 0.0307 to 0.0964 from other populations. Lastly, the Korean inland populations (Sites 1–30) ranged from −0.0087 to 0.0358 and were similar in genetic structure. In the analysis of isolation by distance, genetic distance increased with increasing geographic distance .The population genetic structure of 3.3.2FST genetic distances, the 20 regional populations could be divided into five groups: Group 1, Hokkaido ; Group 2, Kyushu (Site 41); Group 3, Jeju Island (Site 31); Group 4, Korean Peninsula and adjacent areas ; and Group 5, Mongolia Selenge (Site 34)  Figure . We ther3.3.3K was estimated by varying it from two to eight, and the ad hoc statistics ∆(K)  and L. dispar japonica, based on mitochondrial DNA and microsatellite analysis . In the Chinese and Russian regional populations, however, the blue genetic content was higher than other genetic content types. Several individuals . Mitochondrial DNA sequence divergences obtained from the 480 samples ranged from null to 0.5%, with 98 haplotypes distinguished by 85 polymorphic sites ; Group 2, Korean southern region ; and Group 3, Hokkaido region  . The results of the analysis of IBD were similar to the microsatellite results . In particular, haplotype H90 appeared only in Hokkaido regional populations and was connected with haplotype H1 by haplotype H95, which is another Hokkaido haplotype  were connected to each other by low‐frequency haplotypes , the equation 4L. dispar in Far East Asia has been debated  was clearly distinct from the Asian two subspecies; however, the Asian subspecies were difficult to distinguish from each other. The Japanese subspecies, L. dispar japonica, was genetically similar to the populations from the southern end of the Korean Peninsula, and the Korean populations had mixed genetic content   was calculated to be three, the same as the number of groups examined in the mitochondrial genealogy. The genetic diversity of the regional populations was higher in the Korean Peninsula than in other regions, with the Korean Peninsula populations showing the same mixed pattern reported previously  may have taken place in the middle of the Würm glacial stage I , a period with a cold and dry climate that might have led them to move to more southern regions. The southern lineage of the Korean Peninsula (Group 2) might have expanded during the late Würm glacial stage I . During this period, the gypsy moth populations might have dispersed into the southern part of the Korean Peninsula because of the cold climate. Lastly, Group 1 might have dispersed into the Vladivostok area, the middle region of Korea, and even Mongolia because the estimated expansion period is approximately 53,652 ybp, which is known as the subinterglacial stage I , a period with a hot and wet climate. We can thus infer that the gypsy moth populations dispersed from Far East Asia into middle Asia. In the Korean Peninsula, however, they may not have dispersed southward because the Noryeong and Charyeong Mountains were formed in the Miocene , although the analyzed samples did not cover the full distributional region of the species in Far East Asia. Taxonomically, 15 nomino‐subspecies have been assigned to L. dispar: L. dispar dispar Linnaeus, L. dispar erebus Thierry Mieg, L. dispar asiatica Vnukovskij, L. dispar praeterea Kardakoff, L. dispar hokkaidoensis Goldschmidt, L. dispar koreibia Bryk, L. dispar kolthoffi Bryk, L. dispar andalusica Reinig, L. dispar mediterraneae Goldschmidt, L. dispar bocharae Goldschmidt, L. dispar chosensis Goldschmidt, L. dispar japonica Motschulsky, [1861] (TL: Japan); L. dispar umbrosa Butler, L. dispar hadina Butler, L. dispar obscura Goldschmidt, L. dispar nesiobia Bryk, L. dispar dispar and L. dispar japonica, by Schintlmeister . We partly agree with Schintlmeister's ; DNA sequences of COI gene are available in GenBank ; DNA sequences of ATP6/ATP8 gene are available in GenBank (KX945522–KX946001).Tae Hwa Kang participated in the correction of the main idea of the study, coordinated the experiment, collected samples, participated in the analysis on the genetic diversity, and drafted the manuscript; Sang Hoon Han participated in the correction of the main idea of the study, coordinated the experiment, and participated in the analysis on the genetic diversity; Heung Sik Lee participated in the design of the main idea the study, collected samples, and managed funding. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.