Paspalum intermedium to study population-level genetic and morphological variation and ecological differentiation in polyploids. Using flow cytometry, amplified fragment length polymorphism (AFLP) genetic markers, environmental variables, and morphological data, we assessed variations in ploidy, reproductive modes, and the genetic composition in 35 natural populations of P. intermedium along a latitudinal gradient in South America. Our analyses show that apomictic auto-tetraploids are of multiple independent origin. While overall genetic variation was higher in diploids, both diploids and tetraploids showed significant variation within and among populations. The spatial distribution of genetic variation provides evidence for a primary origin of the contact zone between diploids and tetraploids and further supports the hypothesis of geographic displacement between cytotypes. In addition, a strong link between the ecological differentiation of cytotypes and spatial distribution of genetic variation was observed. Overall, the results indicate that polyploidization in P. intermedium is a recurrent phenomenon associated to a shift in reproductive mode and that multiple polyploid lineages from genetically divergent diploids contributed to the successful establishment of local polyploid populations and dispersal into new environments.Polyploidy plays a major role in plant evolution. The establishment of new polyploids is often a consequence of a single or few successful polyploidization events occurring within a species’ evolutionary trajectory. New polyploid lineages can play different roles in plant diversification and go through several evolutionary stages influenced by biotic and abiotic constraints and characterized by extensive genetic changes. The study of such changes has been crucial for understanding polyploid evolution. Here, we use the multiploid-species  Polyploidization in plants is a recurring and pivotal evolutionary phenomenon that brings both short- and long-term benefits for plant diversification . Comparavia seeds) is a reproductive strategy almost exclusively coupled with polyploidy. Since apomictic plants skip meiosis and fertilization and form clonal euploid seed embryos, apomixis can be particularly beneficial during the establishment of new polyploids by shielding triploids from the negative effects of unbalanced chromosomal segregation, low density, and parental introgression, thus aiding new polyploids to overcome reproductive exclusion   explainsCytotype coexistence implies the possibility of spontaneous exchanges of genetic information between ploidy levels. Inter-cytotype gene flow will result in patterns of genetic admixture, like observed in different polyploid apomictic complexes . In geneStudies of extant genetic variation in polyploid populations compared to their putative diploid parents not only provide evidence for the dynamic formation and fate of polyploids in natural populations ; they alPaspalum intermedium Munro ex Morong is a grass species of the sub-family Panicoideae with two major cytotypes: self-sterile sexual diploids (2n = 2x = 20) and self-fertile apomictic autotetraploids (2n = 4x = 40)  . Despite4x = 40) , a recen4x = 40) . TherefoP. intermedium populations; 2) to evaluate the differences in population structure between cytotypes with divergent reproductive modes; 3) to examine the genetic composition of sympatric, parapatric, and allopatric populations in the contact zone; and 4) to search for associations between genetic variability parameters and distribution patterns which may support alternative ecological hypothesis and population attributes of different cytotypes that may help to clarify the recent evolutionary history of the species.In the present study, using flow cytometry, amplified fragment length polymorphism (AFLP) markers, and ecological data, we endeavor 1) to assess extant levels of genetic variability in P. intermedium . Leaf materials were collected in silica gel from 35 populations and between 20 and 30 individuals from each population. Flow cytometry ploidy evaluations data are from our previous analyses published in Sampling was done covering the core and peripheral distribution of The genome DNA of all the collected samples were extracted using QIAGEN mini plant DNA extraction kit , from 50 mg of silica-dried leaf materials. The DNA concentrations were quantified and used for AFLP analysis.For AFLP, we followed the methodology described by EcoR I (5 Units) and Mse I (1 Unit) , and T4 DNA ligase  (1 Unit) with EcoR adapter pair (5 pmol) and Mse adapter pair (50 pmol) with the presence of NaCl (0.05 M) and BSA (0.05 mg/ml) in 1X ligase buffer . The direct selective amplification reaction mixture consisted of 1X PCR buffer , 2.5 mM MgCl2 , 0.2 mM dNTPs , 4 pmol of each EcoR and Mse primers, 1 Unit Taq polymerase , and ca. 80 ng of digested DNA in 25 μl final volume. The PCR was done in a Thermal Cycler  with the following program. Denatured at 94°C, 2 min, 9 × , 23 × , 60°C, 30 min. Amplified fragments were analyzed in an ABI 3130xl Genetic Analyzer  with the 500 ROX size standard . A total of 887 individual fingerprints were retained after the initial analysis of all the individuals . Genotyping and binary presence–absence matrices were assembled in GeneMarker 2.6.0 , with a threshold of 75 RFU for scoring bands of size range of 100–510 bp  indicating admixture in almost all individuals. This was also tested with ad hoc statistic ΔK based on the rate of change in the log probability of data between successive K values described by Bayesian model-based clustering implemented in the “find.clusters” function of R package ADEGENET was used to determine the potential number of clusters that can describe the data best. Here, BICs (Bayesian Information Criteria) are calculated using k-means algorithm . A forward selection with 95% collinearity was used to sequentially remove intercorrelated variables, and only four variables were retained . An environmental distance matrix was obtained using these variables, and a regression analysis of the environmental distance and the genetic diversity of populations was performed to detect isolation by environment.We also performed environmental association analysis of genetic diversity among populations using bioclimatic data. Bioclimatic data were downloaded from mate.org , and poiP. intermedium individuals.During plant material collections, at least three plants were collected from all the studied populations for morphological assessment, and the collected vouchers were deposited in several herbaria in Argentina  and Germany (B and GOET). Overall, 42 morphological characters  of both diploids and tetraploids were studied from the voucher specimens to discern any morphological variation among cytotypes and populations. A principal coordinate analysis (PCoA) was performed to identify morphological clusters among diploid and tetraploid populations. Similarity percentage analysis (SIMPER) was performed using the software PAST 3.23  to assesn = 41) than in diploids (n = 34) (p = 0.02).In the combined binary matrix, a total of 189 fragments were scored; 84 were from the EcoR-Mse (ACA- GAAC), 58 from EcoR-Mse (AATG- AAC), and 44 from EcoR-Mse (AGA- ACA). Out of this, 66.2%  of fragments were polymorphic. The number of bands per individual ranged from 84 to 91. Cytotype-specific fragments were overall significantly higher in tetraploids  in both genotype and gene diversity . One such population  and a single effective genotype (Nei’s diversity = 0.0) and hence were classified as pure clonal populations  showed that all diploids have significantly higher values  is found within populations, the rest accounting for among-population variation . When ‘cytotype’ was assigned as the preferred hierarchy, within-cytotype genetic variation was 36% . Among-population variation increased to 64.8% in tetraploids when cytotypes were analyzed separately, while the values for diploids did not change noticeably (36%).r2 = 0.163, p = 0.031). Contrary to diploid populations, all but three tetraploid populations  formed compact groups within each cluster . Furthermore, when the overall distance found among 4x populations and genetic clusters of the studied individuals were plotted against distance intervals . This is expected in apomictic species where random mating—even within populations—is limited as apomixis imposes substantial reproductive isolation among individuals (see Discussion for details). Our IBE analysis showed that the observed genetic diversity among all populations shows a strong correlation to the environmental distance showing substantial isolation by environment  were observed in the unrooted NJ tree , and fiv cluster . Two out cluster  formed a cluster . Mantel The PCoA revealed higher genetic similarity among diploid populations compared to tetraploid ones . Despitek = 3) wherein most pure diploid and tetraploid populations were segregated each in individual clusters, while a few pure populations were clustered together with all mixed-ploidy populations in a third cluster   .K-means clustering with K = 3 (based on the number of clusters observed in the genetic variation) showed that the morphology of individuals makes three clusters with substantial overlap among clusters  and restricted gene flow , such studies take a new dimension for understanding natural variation and the evolutionary fate of cytotypes within polyploid complexes. The present analysis revises local and regional standing genetic variation in modeling , the preP. intermedium are autopolyploids  or through backcrosses (2x × 4x). The finding of only one triploid individual among the four mixed populations  instead of an expectedly higher number of triploids from backcrosses suggests that 1) there is likely an interploidy barrier between cytotypes (either pre- or post-zygotic) and 2) that rare triploids are more likely to arise from diploids through an unreduced gamete, thus providing support to the hypothesis of de novo formation of polyploids via a triploid bridge, as postulated for other Paspalum species , suggesting that regular gene flow occurs between diploid individuals among populations and there is minimal isolation by distance. Despite the noticeable number of cytotype-specific fragments among genotypes (21.6%), a considerable proportion of fragments (33.8%) were shared between diploids and tetraploids. Since tetraploids in lyploids , the obsalum spp  and othealum spp  and are alum spp . The levermedium  point to species  and rece species . The facP. intermedium . Our results—and particularly the neighbor-joining analysis—further indicate that the observed among population variation in genotype diversity is due to independent polyploid founder events, plus the posterior sporadic occurrence of sexuality. Low proportions of sexual reproduction (<16%) had been detected through embryology on ovules and flow cytometry analysis on seeds of individuals from all tetraploid apomictic populations included here  populations are expected to hold high levels of genetic admixture facilitated by regular random gene flow between individuals . The stuP. intermedium populations point to the occurrence of distinct levels of genetic admixture among populations depending upon the ploidy, the occurrence of multiple cytotypes, and the geographic location of the population  among the three main genetic clusters of our dataset  evidence of introgression from tetraploids, diploids can inject genetic variability into a tetraploid population through recurrent formation of new polyploids. This is very likely the case in all four mixed-ploidy populations that show a higher number of genotypes compared to pure polyploid populations and intermediate fixation indices   further supports the evidence of genetic isolation by distance among these populations and significant spatial dissociation (001) see . While s001) see , in polyKnautia arvensis agg.  shows a more complex pattern in our Neighbor-Joining tree whereby the diploids exhibit high genetic similarity to pure diploid populations M31/1W, Hojs423/1R, and Hojs468/2S, suggesting gene flow between diploids rather than a possible secondary contact with conspecific tetraploid migrants. While inter-cytotype crosses are commonly observed in secondary contact zones in sexual plants . While lack of morphologically discrete cytotype groups can be expected in grass species , provides support to the view that the polyploid complex is evolutionarily young. Alternatively, the paucity in genotypic variation despite the observed rates of residual sexuality might be explained by a high formation/extinction rate of polyploid lineages.The analysis of morphological variation found in 42 quantitative and qualitative characters displays the formation of arbitrary groups with substantial overlap among the apomictic populations of es e.g., , or in oes e.g., , cases oes e.g., , includiPaspalum . Such moPaspalum . PolyploPaspalum . In P. id niches , the muld niches  or in thggregate . The facP. intermedium shows that apomictic autotetraploids are of multiple independent origins, indicating recurrent polyploidization and subsequent genotype diversification through facultative apomixis. The substantial among-population genetic variation and genetic similarity analyses further point to polyploidization events from genetically distinct diploid populations. The contact zone existing between diploids and tetraploids is primary in origin where tetraploids are persistently formed from diploids. Such polyploids might become established or outcompeted by coexisting diploid parentals, and yet, because of wider ecological tolerance, some tetraploids manage to disperse southward and establish new polyploid populations, clonal or mostly clonal. Apomictic populations display reduced genotype and genetic variability, strict geographic isolation, and high genetic differentiation which support a recent evolutionary origin for the polyploid complex.The present study on genetic variation in 35 natural populations of


Poecilia reticulata) provide a unique opportunity to study this phenomenon. Guppy populations that were transplanted from high‐predation (HP) to low‐predation (LP) environments have been shown to evolve toward the phenotypes of naturally colonized LP populations in as few as eight generations. These changes persist in common garden experiments, indicating that they have a genetic basis. Here, we report results of whole genome variation in four experimental populations colonizing LP sites along with the corresponding HP source population. We examined genome‐wide patterns of genetic variation to estimate past demography and used a combination of genome scans, forward simulations, and a novel analysis of allele frequency change vectors to uncover the signature of selection. We detected clear signals of population growth and bottlenecks at the genome‐wide level that matched the known history of population numbers. We found a region on chromosome 15 under strong selection in three of the four populations and with our multivariate approach revealing subtle parallel changes in allele frequency in all four populations across this region. Investigating patterns of genome‐wide selection in this uniquely replicated experiment offers remarkable insight into the mechanisms underlying rapid adaptation, providing a basis for comparison with other species and populations experiencing rapidly changing environments.Although rapid phenotypic evolution has been documented often, the genomic basis of rapid adaptation to natural environments is largely unknown in multicellular organisms. Population genomic studies of experimental populations of Trinidadian guppies ( The genomic basis of rapid adaptation to new environments is largely unknown. Here, we take advantage of a unique replicated experiment in the wild, where guppies from a high predation source were placed into four low predation, previously guppy‐free localities. Earlier reports document census size fluctuations and rapid phenotypic evolution in these populations. We used genome‐wide sequencing to understand past demography and selection. We detected clear signals of population growth and bottlenecks at the genome‐wide level that matched the known history of population numbers. We then identified candidate regions of selection across the genome, some of which were shared between populations. In particular, using a novel multivariate method, we identified parallel allele frequency change at a strong candidate locus for adaptation to the low predation environment. These results and methods will be of use to those studying the growing number of examples of rapid evolution.Historically, evolution in natural populations was thought to happen on a long timescale, one that could not be observed in real time  are unlikely to result in the same evolutionary changes in independent populations. Here, following Lee and Coop , we use Poecilia reticulata) to investigate the early stages of adaptation. We first examine patterns of genetic variation with whole genome sequence data to understand the impacts that neutral processes may have had on the populations. Then, we investigate signals of selection across the genome and instances of genomic convergence among the four replicate populations with a combination of selection scans and a multivariate analysis of allele frequency change vectors.Here, we capitalize on a unique set of rapidly evolving replicated experimental populations of guppies  to low‐predation (LP) environments within rivers , Lower Lalaja (ILL), Taylor (IT), and Caigual (IC). The common ancestors of the fish placed in these four locations were fish from a high predation site in the Guanapo River (GHP), which is downstream from the four populations in the same drainage. There were two independent collections of GHP fish, one per introduced pair, and the introductions were designed to introduce the same distribution of genetic variation into each member of a pair. ILL and IUL were introduced with much lower initial population densities due to fewer introduced fish and larger stream size than either IC and IT. IUL and IT had artificially thinned tree canopies Fig. , which iA unique advantage of our experimental system is that both census population size and phenotypic change have been carefully monitored since their introduction, using monthly mark‐recapture data and annual common garden phenotypic evolution studies    was performed on a linkage‐pruned VCF (–indep‐pairwise 50 5 0.2) in Plink version 1.90b6.7 . Therefore, we chose to use two statistics based on haplotype homozygosity that are more suitable for detecting very recent directional selection. Our reasoning was that strong selection will sweep a haplotype to high frequency before recombination or mutation will break up the haplotype. First, we used cross‐population extended haplotype homozygosity (XP‐EHH), which was developed to detect selective sweeps that are nearly fixed in one population but still polymorphic in the entire metapopulation  between GHP and each of the introduced populations. This was done by calculating the change in minor allele frequency in nonoverlapping windows of 75,000 bp, chosen based on LD decay Fig. . The intX) of allele frequency changes (ΔAF) per SNP between the founder and each introduced population:n is the number of SNPs in a window (in this case 200) and m is the number of introduced populations (in this case four). Each row was normalized to unit length (length = 1). For each of these matrices, we then computed the correlation matrix describing correlations of normalized allele frequencies among the vectors of each HP‐LP pair, by multiplying matrix X with its transpose:N = 200) along the genome, and allowed for a random start position for windows. For each null permutation, all individual IDs were shuffled and allele frequency vectors recalculated between random pairs. This null permutation approach therefore captures potential bias associated with features of the genomic landscape and the sampling design, but for randomized vector trajectories. We applied the same transformations described above to each window and ran 10,000 permutations. Windows were identified as outliers if they were above 99.9% of our randomized null. Finally, we investigated where the allele frequency changes are parallel and where they are antiparallel by examining the loading of each population on the first eigenvector. Lineages with the same sign loading can be interpreted as evolving in the same direction, and lineages with opposite signs are evolving antiparallel. The code to perform this method is available as an R package: AF‐vapeR (https://github.com/JimWhiting91/afvaper). The method is fully described and validated with simulated data in Whiting et al.  and sampling (pτ). The aim here was to produce a neutral distribution of standardized allele frequency change under drift for each population to compare with observed standardized allele frequency change, calculated on the GHP minor allele, to determine whether these are significantly nonzero. For observed data, we assume the allele frequency observed in GHP is representative of the founding frequency given the large size of this population and short time between sourcing and sampling. Alleles are polarized to the GHP minor allele, so our standardized allele frequency change measures include information on direction and magnitude of change. We therefore summed these estimates within SNPs across populations, such that the summed value is maximized by allele frequency change that is of large magnitude and in the same direction.For each mutation, for each simulation, we calculated the selection coefficient based on a diploid single locus model, to standardize for starting allele frequency according to:Experimental populations showed limited divergence from their source , with the strongest difference being that between IT and GHP , whereas IUL experienced very slight increase of He  and a significant increase in ILL . Similarly, IT and IC experienced a decrease in Ho . ILL also decreased in Ho  but IUL increased . Median nucleotide diversity (π) decreased in IT and IC  and was slightly, but significantly, increased in ILL and IUL . Tajima's D was shifted to a more positive value in IC and IT compared to GHP . This suggests a loss of rare alleles compared to the source, which is consistent with a sudden population contraction. In ILL and IUL, Tajima's D has decreased slightly compared to GHP, but stayed positive , implying an increase of rare alleles that could be related to rapid population expansion.The introduction of guppies from GHP to ILL, IUL, IC, and IT led to very minor changes in genetic diversity within the introduction populations Fig. . IT and N = 83) and IT, which experienced a population crash in the first year after initiation, had nearly three times more ROH than the other introduced populations .The patterns of ROH are consistent with known history: GHP, the source population, had the lowest number of ROH  were significantly higher in IT compared to the other introduced populations , again confirming recent inbreeding in this population. The remaining three introduction populations did not significantly differ from each other , but did have slightly higher values compared to GHP .Inbreeding coefficients , followed by ILL (N = 39), IUL (N = 17), and finally IC (N = 14) , followed by ILL (N = 39), IC (N = 36), and IUL (N = 19)  Fig. –S10. For19) Fig. –S14.N = 4, for both), followed by ILL‐IUL (N = 2), with no overlap between IUL and IC , followed by IC‐IT (N = 6), IUL‐IC (N = 5), ILL‐IUL (N = 4), and no overlap between IUL and IT. Taken together, shared signals of selection are not driven by introduction year or canopy thinning.Similarly, for iHH12, there was little overlap in outliers, and no outlier windows were common to all four comparisons. However, among the three‐way overlapping outliers, ILL‐IC‐IT overlapped the most  were similar to observed distributions in each of the experimental populations (particularly ILL and IUL), which means that much of the genome‐wide allele frequency change from GHP to each experimental population is broadly in‐line with neutral expectations  , where many unlinked loci showed parallel change in addition to change in a large haplotype present in the founder  could cause correlated differentiation landscapes among the populations, which results in a constrained axis of allele frequency changes Burri . HoweverIt is widely appreciated that demographic changes associated with changes in population size can create patterns that resemble the consequences of selection. Therefore, studies of rapid evolution ought also to explore past demographics. Unfortunately, most demographic inference methods are underpowered at recent timescales  methods. Although IT had a decrease in k Tajima . All fouk Tajima . SubsequIt could be expected that, as a result of the bottleneck, IT might have sustained a chance loss of genetic variation that caused it to have an altered evolutionary trajectory relative to the other three replicates. The natural population of guppies found downstream from the IT population had a higher mortality rate than IC in the first year. This elevated mortality has been attributed to a stronger influence of flood‐induced mortality during the wet season and perhaps disease —reference numbers: PRJEB42705  and PRJEB10680 (GHP). All scripts and associated data are available on Github repository: mapping and SNP calling ; population genomics and haplotype scans (https://github.com/bfraser‐commits/Rapid_genomic_adaptation_guppies); the software for multivariate AF analyses (AF‐vapeR) (https://github.com/JimWhiting91/afvaper); and simulation analyses (https://github.com/JimWhiting91/fibr_simulations).The data that support these findings are openly available at: European Nucleotide Archive (supplementary Tables and Figures informationClick here for additional data file.supplementary Tables and Figures informationClick here for additional data file.


Vitis rotundifolia syn. Muscadinia rotundifolia) is an American grape species native to the southeastern United States that has been cultivated for centuries. Muscadines are one of three grape species in subgenus Muscadinia with a chromosome number of 2n = 40 , making them genetically distinct from the European wine and table grape (Vitis vinifera) and other species in subgenus Euvitis. Crop improvement efforts have been continuous since the late 19th century, yet the germplasm that served as the foundation for early muscadine breeding efforts was sourced from a relatively small portion of their native range, mostly in the coastal plains of North Carolina. This study used the rhAmpSeq Vitis core panel haplotype markers to genotype 194 Muscadinia accessions from five cultivated populations and 15 wild populations collected across their native range. Wild populations from the western half of the native range were generally less genetically differentiated than hypothesized, but were genetically distinct from the material used in both past and present breeding efforts. One population collected from coastal North Carolina grouped closely with V. munsoniana accessions despite being well outside the reported range for that species. Principal coordinate and structure analyses revealed three main groups within the 194 accessions: one for cultivated material, one for wild V. rotundifolia, and one for V. munsoniana and V. popenoei. At K = 5, structure results showed that more recent muscadine cultivars are further differentiated from wild accessions and varieties. These analyses confirmed our hypothesis that muscadine cultivars are genetically differentiated from their wild counterparts. This study also showed that genetic diversity in V. rotundifolia is not equally distributed across its native range and that the limited number of genotypes used in crop improvement efforts has not fully utilized the genetic diversity within the species.The muscadine ( Vitis rotundifolia Michx. syn. Muscadinia rotundifolia Simpson ex Munson) is a member of the grape family (Vitaceae) native to the southeastern United States. For centuries, this species has been used for both wine and fresh market production. The native range of the muscadine is from Maryland west to Texas and south to the Gulf Coast. Perhaps, the most important factor limiting the range of the species is cold hardiness. Although there are documented instances of muscadine vines surviving temperatures of −23°C, regions that regularly attain winter lows of −18°C are considered unsuitable for commercial muscadine production (The muscadine (oduction .Vitis can be split into two subgenera based on differences in chromosome number: Euvitis or bunch grapes (2n = 38) and Muscadinia (2n = 40). Major synteny between the Euvitis and Muscadinia genomes has been observed despite the difference in chromosome number and barriers to hybridization. Collinearity between chromosomes 7 and 20 in muscadines and chromosome 7 in bunch grapes suggests a chromosome fusion in Euvitis grapes (Euvitis such as the European wine and table grape (Vitis vinifera L.). Muscadines are resistant to many pathogens, such as Pierce’s Disease , grapevine downy mildew [Plasmopara viticola (Berk. and M.A. Curtis) Berl. and De Toni], and powdery mildew [Erysiphe necator syn. Uncinula necator (Schw. Burr)] that make V. vinifera cultivation in the southeastern U.S. difficult  have been used in some breeding efforts  collected from Veracruz, Mexico, affording the species a minor presence in previous molecular studies , wild V. rotundifolia varieties , historical cultivars released prior to 1970 , recent cultivars released since 1970 , and unreleased selections from the UA muscadine breeding program . Varieties were defined as cultivated vines selected from the wild, while cultivars were defined as cultivated vines resulting from controlled crosses in formal breeding programs. The Muscadinia (MUS) population was composed of wild V. munsoniana accessions , one accession with V. popenoei, V. munsoniana, and V. rotundifolia parentage (“Fennel’s 3-way hybrid”), and the single V. popenoei (DVIT 2970) accession maintained by the NCGR. The 14 individuals within the Arkansas selections (ARK) population are advanced, unreleased genotypes from the UA breeding program. Each of these selections is preceded by the code “AM” (Arkansas Muscadine).In order to quantify the genetic diversity of cultivated muscadines as well as compare between cultivated and wild populations, 72 additional muscadine accessions were included in this study . These vYoung leaf tissue was collected from wild muscadines for DNA extraction and genotyping and immediately placed in labeled 2 ml centrifuge tubes. Locational data for wild populations were recorded using OnX mapping software . Observational data, such as plant health, flower sex, and growing environment were noted at the time of collection when possible. Fresh tissue from the 72 additional accessions was sent to UA for extraction. DNA extraction followed a modified CTAB protocol . The quaV. vinifera genomes and de novo assemblies of seven Euvitis genomes, including wild American species such as Vitis cinerea and Vitis rupestris, although V. rotundifolia was not included in the development process , a method that specifically targets amplicons within the genome . The acc process . The 2,0adegenet. A minimum read depth threshold of five reads per allele call was used. Monomorphic loci and loci with >20% missing data were removed. Loci putatively under selection were searched for using the R package dartR’s implementation of OutFLANK (structure 2.3 software (K) was run from 2 to 10 without a priori assumptions of population delineations and an admixture model. A burn-in period of 300,000 iterations was followed by 800,000 Markov-Chain Monte Carlo (MCMC) repetitions. The simulation was run three times for each K value. The most likely number of populations present in the data was determined using the delta K statistic  was performed following poppr to investigate genetic variation based on the a priori assumption that each collection site sampled represented a distinct population. A dendrogram was created using an unweighted pair group method with arithmetic mean (UPGMA) tree based on Nei’s genetic distance using the packages poppr and ape  was conducted using the package ade4. The package ade4 was also used to conduct a Mantel test of the de novo wild populations using a genetic distance matrix calculated using hierfstat and a physical distance matrix calculated using geosphere  and 37 loci were not polymorphic in this dataset. After filtering loci for missing data, 30 of the 152 wild accessions failed to amplify at >90% of loci and were removed. The failed reactions disproportionately affected the final sizes of the ARK3 (n = 4), TEX (n = 3), and NC3 (n = 4) populations . Therefore, the final dataset was composed of 194 unique genotypes and all 687 polymorphic haplotype loci.In this study, 152 wild ulations . ApproxiOutFLANK . To ensuV. munsoniana, which was likely the result of an error during laboratory preparation of samples for genotyping. “Spalding” and “Stuckey” were excluded because initial results showed that they grouped with V. munsoniana despite there being no indication of V. munsoniana in their reported pedigrees. The cultivar “Oh My!” was developed from a seedless V. vinifera cultivar crossed with V. rotundifolia that was then backcrossed to V. rotundifolia multiple times, resulting in a cultivar with 23.1% of its genetic makeup from V. vinifera (Observed heterozygosity ranged from 0.26 to 0.39 across the 15 wild and five cultivated populations . The ARKvinifera . Due to K (2–5) are presented in this research. The statistic of K = 3  and Muscadinia (MUS) populations formed the first cluster, while the wild populations from west of the Appalachian Mountains  formed the second. Five populations  collected within 250 km of the Atlantic or Gulf coasts showed moderate levels of hybridization between the two clusters. Each of the vines from the NC1 and FLA populations consistently had approximately 20% assignment to the second cluster, while the admixture levels in the MIS and NC2 populations were more variable from individual to individual.At K = 3, the cultivated populations generally grouped as one cluster with some notable exceptions. The historical cultivars “Spalding” and “Stuckey” had over 90 and 50% assignment, respectively, to the same cluster as MUS. The ARK population showed no levels of admixture with any other cluster while the other cultivated populations showed varying levels of admixture within each accession. The FLA, MUS, and NC1 populations were assigned to the second cluster with low to moderate levels of admixture with the other two clusters. Populations from Alabama, Arkansas, Oklahoma, Texas, and Tennessee , generally from the temperate regions of the southeastern U.S., formed a third cluster with low levels of admixture compared to other populations. The KIL, NC2, and NC3 populations appeared to be mixtures of all three hypothetical populations assumed for structure simulation. Despite being geographically equidistant from the populations collected in North Carolina and the populations collected in Arkansas, the ALA and TEN populations showed no admixture with material from North Carolina and grouped exclusively with material from Arkansas, Oklahoma, and Texas , Florida (FLA), and coastal North Carolina (NC1) populations from the cultivated populations and the wild populations collected in Arkansas, Alabama, Oklahoma, Tennessee, and Texas. Three populations  that showed significant admixture in the structure analysis grouped closest to the wild populations but were distinct from the tightly-grouped cluster of populations from Arkansas, Alabama, Oklahoma, Tennessee, and Texas.Principal component analysis (PCA) produced three clusters corresponding to the at K = 3 . The firFst values ranged from −0.01 to 0.31, indicating that there was significant structure among the populations in this study . The NC1 population had pairwise Fst values greater than 0.20 for most wild populations collected west of the Appalachian Mountains. Both FLA and MUS had pairwise Fst values ranging between 0.11 and 0.24 when compared to the 18 other populations used in this study. The populations collected in Alabama, Arkansas, Oklahoma, Tennessee, and Texas were generally undifferentiated from one another, the majority of comparisons having Fst values of less than 0.15. The cultivated populations in this study also showed relatively little differentiation. Historical cultivars (HIS) and wild varieties (VAR) had the lowest observed Fst of all the comparisons of −0.01. Pairwise Fst values for the ARK compared to other cultivated populations decreased from the wild varieties  to historical cultivars  to recent cultivars . AMOVA results supported the high Fst values between populations, showing that just over 14.5% of genetic variation was among populations, 4.7% of variation was within populations, and 80.8% of variation was within individuals. The Mantel test showed a significant positive association between genetic distance and physical distance .Pairwise is study . The higV. popenoei sample (DVIT 2970) and “Fennell’s 3-way Hybrid,” which is one half V. popenoei, one quarter V. munsoniana, and one quarter V. rotundifolia by pedigree. The FLA population grouped most closely with the V. munsoniana material included in this study. The NC1 population, consistent with both the structure and PCA results, was a separate clade despite being closely related to the MUS and FLA material. Cultivated and wild materials generally formed separate clades. Only one cultivated accession (“San Rubra”) grouped with wild material. Two genotypes from the NC2 population (NC2-1 and NC2-12) grouped within the cultivated material.An UPGMA tree was constructed using genetic distances calculated following V. rotundifolia genetic diversity study conducted to date. One hundred and ninety-four individuals and 687 loci were used to evaluate population structure and quantify genetic diversity of wild and cultivated muscadine populations. Prior genetic diversity work in muscadines focused mainly on cultivated accessions or the relationship between muscadines and Euvitis grapes  after a data imputation process  and historically important cultivated varieties (VAR) that were largely sourced from the North Carolina Coastal Plain. In contrast, V. munsoniana. V. munsoniana was almost as cold hardy as V. rotundifolia and had fruited for multiple years his vineyard in Denison, Texas . Thus, it seems possible that the far southern Outer Banks could be a sort of isolated refugium for V. munsoniana or an underexplored intermediate bridge between the two species or subspecies. This hypothesis should be explored further by sampling coastal populations in South Carolina and Georgia.Results from the PCA, n origin 4. One un origin –5. The NVitis rotundifolia has a large native range, and it was hypothesized that genetic diversity within the species would be evenly distributed across the southeastern U.S. The structure and PCA results showed that even populations such as ALA and ARK3 that were collected more than 800 km apart were genetically similar  showed no admixture with cultivated populations despite its proximity to the muscadine vineyard used by the UA breeding program. Admixture between cultivated and wild populations has been shown to decrease the unique genetic diversity of wild populations in a process known as genetic swamping. This phenomenon has been observed in Vitis previously, such as regular hybridization between V. californica Benth. and V. vinifera leading to genetic diversity being lost in the former  showed more admixtures in the els of K , likely Muscadinia germplasm from Florida has also been important in muscadine breeding. The “H1” source of hermaphroditism in muscadines was selected from a cross between North Carolina varieties and wild Florida pollinizers and used extensively in subsequent breeding efforts  showed the wide gulf between the material collected in Alabama, Arkansas, Oklahoma, Tennessee, and Texas and both recent cultivar releases and the advanced breeding selections within the Arkansas muscadine breeding program . This fi efforts . The impAlthough the wild populations from the western portion of the native range of muscadines had lower genetic diversity than the coastal populations  sampled in this study, the presence of unique alleles within each population indicates the potential for beneficial alleles in these populations. Cold tolerance is of particular importance to the UA muscadine breeding program because of its location near the northern limits of the native range of muscadines. Damage from winter temperatures was rarely observed on wild muscadine vines during tissue collection in this study. The ARK5 population was collected from a site further north and at higher elevation than the UA breeding program vineyard in Clarksville, AR. Vines at the ARK5 collection site were regularly observed to survive past juvenility, indicating that this was an established population adapted to local winter conditions. Crossing with wild muscadine vines from the southern Ozarks could help achieve a major breeding goal of the UA muscadine breeding program by serving as a source of cold hardiness. It would not be the first time that wild material was found to contain beneficial traits. Bronze-fruited muscadines are considered superior for winemaking due to a lack of diglucoside anthocyanins that cause unsightly browning during the aging process. Using HPLC, Results from the PCA show a steady differentiation over time of cultivated material from the two clusters of wild populations . HistoriV. rotundifolia, V. popenoei, and Euvitis material in its pedigree -National Institute of Food and Agriculture (NIFA) Specialty Crop Research Initiative, award No. 2017-51181-26829. Additional funding for this research came from Hatch Project ARK02599.The genotyping was funded by The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Torenia fournieri Lind., an annual from tropical Indochina and southern China, is a popular ornamental plant, and many interspecific (T. fournieri × T. concolor) hybrid lines have been bred for the international market. The cultivated lines show a pattern of genetic similarity that correlates with floral color which informs on future breeding strategies. This study aimed to perform genetic analysis and population structure of cultivated hybrid lines comparing with closely related T. concolor wild populations.The wishbone flower or Torenia. These included 15 cultivated lines of three series: Duchess ; Kauai ; Little Kiss  and two wild T. concolor populations (Q and R). PCR products from each individual were applied to estimate the genetic diversity and differentiation between lines/populations.We applied the retrotransposon based iPBS marker system for genotyping of a total of 136 accessions from 17 lines/populations of Genotyping results showed a pattern of genetic variation differentiating the 17 lines/populations characterized by their specific floral colors. The final PCoA analysis, phylogenetic tree construction, and Bayesian population structural bar plot all showed a clear subdivision of lines/populations analysed. The 15 cultivated hybrid lines and the wild population Q that collected from a small area showed the lowest genetic variability while the other wild population R which sampled from a larger area had the highest genetic variability.T. concolor populations could be due to our varied sampling methods. The two wild populations  and the cultivated hybrid lines  are genetically more closely related, but strong positive correlations presented in cultivated lines A, C, E, M, and N. These results could be used to guide future Torenia breeding.The extremely low genetic variability of 15 cultivated lines indicated that individual line has similar reduction in diversity/heterozygosity from a bottleneck event, and each retained a similar (but different from each other) content of the wild genetic diversity. The genetic variance for the two wild Torenia species. Our study revealed that wild Torenia populations sampled from broad geographic region represent stronger species strength with outstanding genetic diversity, but selective breeding targeting a specific floral color decreased such genetic variability.The genetic variation and population structure found in our study showed that cultivated hybrid lines had similar reduction in diversity/heterozygosity from a bottleneck event and each line retained a similar (but different from each other) content of the wild genetic diversity, especially when strong phenotypic selection of floral color overlaps. Generally, environmental factors could induce transposon activation and generate genetic variability which enabled the acceleration of the evolutionary process of wild  Torenia belongs to the class Magnoliopsida, order Scrophulariales and family Scrophulariaceae. There are more than 50 species native to tropical and subtropical Asia and Africa   released the Crown series with pink, white, and reddish-purple color lines. Selected from an interspecific cross between T. fournieri and T. concolor in 1995, a trailing type cultivar called the Summer Wave series was later released by Suntory Ltd. . For more than 30 years, many hybrids have been released as patents or trademark series with a full range of floral colors from white with yellow throats to blue, cobalt, lavender, and violet, making Torenia a popular ornamental plant on the international market (Torenia could aid future selective breeding of this important genus.The genus d Africa . Accordid Africa . Wishbononcolor) . T. fourl market . As a coT. fournieri as an ideal model plant for micromanipulation of artificial fertilization. Two major characteristics of the Torenia species included the exceptional protruding embryo sac  resulted from gene duplication through insertion of an En/Spm-related transposable element into a floral homeotic gene  and two wild T. concolor populations sampled from two different sizes of area. Our overall aim was: (i) to estimate the genetic variation and population structure for T. fournieri cultivated lines which could be influenced by breeding selection; (ii) to explain the variation of the genetic diversity between the two wild populations could be differed from the sampling method we used; (iii) to provide insight into the effect on genetic variation in a population experiencing a bottleneck in which strong phenotypic selection overlaps; (iv) to provide a practical model for the genetic analysis of Torenia populations so as to better enable selective breeding in the future.Specifically, for a practical transposon marker system, T. hybrida seed lines from Shanghai Yuanyi Seedling Co. Ltd. These 15 colorful seed lines included three commercial series. Two wild T. concolor populations (Q: Lipu and R: Xichou) were collected from two different subtropical habitats in southern China, Lipu in Guangxi Zhuang Autonomous Region and Xichou in Yunnan Province , B (Duchess: Burgundy), C (Duchess: Deep Blue), D (Kauai: Burgundy), E (Kauai: Rose), F (Kauai: Deep Blue), G (Kauai: Blue and White), H (Kauai: Magenta), I (Kauai: Lemon Drop), J (Kauai: White), K (Little Kiss: White), L (Little Kiss: Burgundy), M (Little Kiss: Blue and White), N (Little Kiss: Rose Picotee), P (Little Kiss: Blue), Q (wild: Lipu) and R (wild: Xichou). The origin of the plant materials used in this study are listed in Province . To reprR Plant Genomic DNA Kit  in accordance with the manufacturer’s protocols. The quality and quantity of DNA examined using 2.0% agarose gel electrophoresis by comparison with the standard λ DNA concentrations. A portion of the isolated DNA was diluted with distilled water to 1 ng μL−1 concentration and used as the template for later PCR experimentation.About 100 mg of fresh young leaves from each accession were used for DNA isolation. Genomic DNA for each plant was extracted using the EasyPureAccording to the sequence designed by 2O. The PCR profile included an initial hot start at 95 °C for 3 min, 40 cycles of denaturation at 94 °C for 30 s, annealing at 39–43.5 °C for 30 s and with an extension at 72 °C for 2 min. There was a final extension at 72 °C for 5 min and the program was terminated by holding at 4 °C. Using 0.2 mL tubes or 96-well plates, all the reactions was performed in a LIFE ECO Thermal Cycler . Final PCR products were electrophoresed at 70 V for 3.5 h with 1.5% (w/v) agarose gel in 1 × TAE buffer . The standard Dongsheng LD DS5,000 ladder  was used as the marker to estimate fragment lengths. GoldView Ⅰ Nuclear Acid Dye   was used to post-stain the gels for 20–30 min and photographs were taken using the GenoSens 1860 model with Clinx GenoSens Capture Software .The PCR experiments were performed in 20 μL reaction mixtures containing 1 μL (1 ng) genomic DNA, 10 μL 2 × Bench Top Taq Master Mix , 1 μL of 10 μM primer (single primer), and 8 μL of ddH2 + (1 − f)2], where f is the frequency of the dominant marker in the data set. Accountable bands consistently appeared in all the repeated experiments.For the process of scoring, the DNA bands were carefully sized, visually examined and all faint bands were excluded. Only clear bands were included and same sized bands were treated as a single locus. The data record for each locus used ‘1’ for presence of a band and ‘0’ for absence so as to form a binary matrix. The polymorphic informative content (PIC) value for each primer was calculated by PIC = 1 − , the optimal number of genetic clusters for these 136 accessions was determined to be five  was similar to the 15 cultivated lines, but was significantly different from the other wild population R (selected across a much larger area). This most likely indicates that these differences in genetic diversity were resulted from bottleneck/drift effects when bringing a small number of individuals into cultivation during breeding practices. Thus, this Phi-statistics method could be used further to compare the genotype data of the parent lines against the hybrid lines so as to estimate such bottleneck/drift effects mentioned above. On the other hand, it needs to be noted that when considering wild populations within their natural habitats, sampling methods are one of the major factors affecting the conclusions being drawn about the population genetic structure.We know that population structure analyses using dominant markers such as the iPBS system can be weakened if the heterozygosity remains undetected or underestimated. To prevent such an issue in our analysis, we adopted T. fournieri and T. conclor, and our two wild T. concolor populations had blue flowers, all the cultivated lines were bred for a specific floral color. In our study, clustering information among cultivated lines and wild populations via our PCoA analysis showed interesting patterns, and much of the variance in genotype among lines is due to differences between certain sets of lines which are most distant from one another. For instance, among cultivated lines I, K, M, N, P and the two wild populations Q and R, there were similar reduction in diversity/heterozygosity from a bottleneck event, and each line/population will retain a similar (but different from each other) content of the wild genetic diversity. As Torenia breeding.Since blue is the common wild type of floral color for Being useful for reconstructing the evolutionary history of lines/populations from population genetic analysis, the population genetic distance can be interpreted as a measure of genetic divergence between species or between lines/populations within a species, whether the distance evaluate the time from common ancestor or the degree of differentiation. Generally, species or wild populations of a species with many similar alleles exhibit small genetic distances due to having had a recent common ancestor. Results from our study indicated that the wild population Q is genetically more closely related to lines M, N, P and they are likely therefore to have had an overall genetic similarity. For the purpose of expanding heterozygosity, these closely related lines/populations should not be used as parent candidates for any inter-line/population crosses, especially those targeting introgressive traits from wild populations. On the contrary, it would be better to cross these closely related lines/populations with other lines/populations containing more genetic differentiation such as A–F.Torenia lines/populations studied through the 136 accessions. A large number of specific bands deserve special attention as they were private alleles specific to the line(s)/population(s). The genetic information from these specific alleles indicated significant genetic relationships that separated certain lines/populations, as presented in the above PCoA analysis, phylogenetic dendrogram, and the final population structural bar plot. Furthermore, such population specific polymorphic bands can be excised, then purified from the gel and sequenced to provide more thorough genomic information relevant to the genetic analysis of the population. These specific molecular markers could also be associated with particular phenotypic traits of interest, such as floral color.As previously indicated, this study revealed an extensive genetic diversity and structural pattern within and among the 17 Torenia populations tested, 15 commercial lines and the wild population Q had low values of Na , Ne , I (Shannon’s information index), He (expected heterozygosity), and uHe (unbiased expected heterozygosity). Line J has the lowest value of records whilst wild population R had the highest values. Since wild population Q was sampled from a smaller area, and population R was sampled across a much larger area, the different results of these two wild populations could be due to our sampling practices, as suggested above. Furthermore, the mean value of I (0.0758), He (0.0513) and uHe (0.0547) of all the 17 Torenia populations tested are considerably less than that reported previously in other genera of plants by Torenia series that have been released, and in the wild population Q. This suggests either some kind of bottleneck/drift effect by selection during breeding practices.Regarding the heterozygosity for 17 Torenia species have been used previously for frontier research on fertilization and gene engineering of ornamental characteristics within horticultural ecosystems, our research conclusions could complement and expand its use as a model plant for new interests in plant population genetic analysis. Therefore, the results from this study could have two main flow-on effects. The first could be information applicable to selective breeding for commercial purposes. A second application could be with endangered species because the population genetic analysis would be able to index the strength of the population of the species in its natural habitat.Our results also support the view that the retrotransposon-based iPBS system we used could also be used to estimate genetic diversity and structure of other plant species. This would enable population genetic analysis on other biological phenomena such as adaptation, speciation, and population structure. Whilst T. concolor populations and T. fournieri cultivated lines with different floral color. For cultivated lines and wild populations, there were similar reduction in diversity/heterozygosity from a bottleneck event, and each line/population retained a similar (but different from each other) content of the wild genetic diversity. Variances in genetic diversity/heterozygosity for the two wild populations could be explained by differences in sampling method. Environmental factors could induce transposon activation and generate genetic variability which enabled the acceleration of the evolutionary process of wild Torenia species, but selective breeding targeting a specific floral color decreased genetic diversity. As a consequence, this study also provide insight into the effect on genetic variation in a line/population experiencing a bottleneck in which strong phenotypic selection overlaps, but wild Torenia populations sampled from broad geographic region represent stronger species strength with outstanding genetic diversity. Using of the iPBS marker system not only revealed genetic variation and population structure for wild and cultivated Torenia plants, but this result could guide future selective breeding especially for commercial purposes.The iPBS markers showed interesting patterns of genetic variation among wild 10.7717/peerj.11702/supp-1Supplemental Information 1T. fournieri commercial seed lines for population A to P were purchased from Shanghai Yuanyi Seedling Co. Ltd., and two T. concolor populations  were collected from their natural habitats (Note: 15 habitats .Click here for additional data file.10.7717/peerj.11702/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.11702/supp-3Supplemental Information 3In the first three axes or components, high eigen values are marked as bold and the low values are indicated by a subscript. A, Duchess Pink; B, Duchess Burgundy; C, Duchess Deep Blue; D, Kauai Burgundy; E, Kauai Rose; F, Kauai Deep Blue; G, Kauai Blue and White; H, Kauai Magenta; I, Kauai Lemon Drop; J, Kauai White; K, Little Kiss White; L, Little Kiss Burgundy; M, Little Kiss Blue and White; N, Little Kiss Rose Picotee; P, Little Kiss Blue; Q, Lipu and R, Xichou.Click here for additional data file.10.7717/peerj.11702/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.11702/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.11702/supp-6Supplemental Information 6PCR banding patterns of 136 accessions of Torenia using seven primers.Click here for additional data file.


Spartina pectinata Link) is a native perennial warm-season (C4) grass common in North American prairies. With its high biomass yield and abiotic stress tolerance, there is a high potential of developing prairie cordgrass for conservation practices and as a dedicated bioenergy crop for sustainable cellulosic biofuel production. However, as with many other undomesticated grass species, little information is known about the genetic diversity or population structure of prairie cordgrass natural populations as compared to their ecotypic and geographic adaptation in North America. In this study, we sampled and characterized a total of 96 prairie cordgrass natural populations with 9315 high quality SNPs from a genotyping-by-sequencing (GBS) approach. The natural populations were collected from putative remnant prairie sites throughout the Midwest and Eastern USA, which are the major habitats for prairie cordgrass. Partitioning of genetic variance using SNP marker data revealed significant variance among and within populations. Two potential gene pools were identified as being associated with ploidy levels, geographical separation, and climatic separation. Geographical factors such as longitude and altitude, and environmental factors such as annual temperature, annual precipitation, temperature of the warmest month, precipitation of the wettest month, precipitation of Spring, and precipitation of the wettest month are important in affecting the intraspecific distribution of prairie cordgrass. The divergence of prairie cordgrass natural populations also provides opportunities to increase breeding value of prairie cordgrass as a bioenergy and conservation crop.Prairie cordgrass ( Panicum virgatum L.), big bluestem (Andropogon gerardii Vitman), indiangrass (Sorghastrum nutans L.), and prairie cordgrass have shown potential use for conservation practices and potential bioenergy production [Prairie grass species native to North America, such as switchgrass  grass that once dominated North American tallgrass prairies. The habitats of prairie cordgrass cover wet to moist prairies and low areas alongside rivers and tributaries ,9. MobbeSpartina species [Cytotaxonomic studies of prairie cordgrass revealed different ploidy levels existing among populations, including tetraploid (2n = 4x = 40) populations distributed from Southern Canada and the Eastern USA, and octoploid (2n = 8x = 80) populations distributed across Midwestern USA ,17,18. A species ,24.To fully investigate the genetic variation and phylogeography in this outcrossing species, nuclear molecular markers with single nucleotide polymorphisms (SNPs) should be jointly used with organelle molecular markers ,26. WithUndomesticated species have often gone through extensive inter-specific gene flow, lineage splitting, and genetic drift, resulting in incongruences of genealogical information carried by each gene ,34,35. Ehttps://www.census.gov/geo/reference/gtc/gtc_census_divreg.html, accessed on 28 January 2018) . In addition, more than 100 rhizomes of each of two cultivars (‘Kingston’ germplasm (KST), ‘Southampton’ germplasm (STP)) were obtained from the USDA-NRCS Big Flats Plant Material Center, NY. Seeds of ‘Red River’ prairie cordgrass, a cultivar developed by interpopulation open pollination among vegetative propagules obtained from east central Minnesota (Grant County), northeastern South Dakota (Day County), and east central North Dakota (Cass and Grand Forks Counties) [−1 quinclorac  before the emergence and 0.79 kg ae ha−1 2,4-D ester  in the growing season from 2011 to 2013. All plots were also fertilized with 112 kg N ha−1 in April of 2011, 2012 and 2013.From 2009 to 2011, seeds of 96 prairie cordgrass natural populations were collected from New England , the Middle Atlantic (New Jersey), the East North Central , the West North Central , and the West South Central (Oklahoma and Louisiana) regions  . For theounties) , were alLeaf tissue samples from each genotype were collected and bulked for DNA extraction in 96-well frozen plant format using a standard CTAB protocol . A minimTo estimate ploidy level of each population, flow cytometry was performed on the main tiller of four clonally propagated plants collected from one genotype for each population when one or more secondary tillers were initiated. Nuclear DNA content was determined using a procedure modified from Rayburn et al.  and Kim fastSTRUCTURE, a Bayesian-based algorithm [fastSTRUCTURE was run from K = 1 to K = 10 using default parameters for 96 samples. Geographical distribution of populations was mapped on the US state map using ggplot (‘ggplot2’) [fastSTRUCTURE and the Bayesian information criterion (BIC) score provided by DAPC to infer the best number of demes supported by the data For population and genetic diversity analyses, we selected data from one genotype from each population to represent each population. Samples with higher than 15% missing rate were also avoided. A total of 96 samples were selected. Single nucleotide polymorphisms data were first imputed using the LD-kNNi algorithm in Tassel V5 ,45 and slgorithm , and thelgorithm  to visuagplot2’)  based onhttps://www.ncei.noaa.gov/products/us-climate-normals, accessed on 16 May 2019). Three geographical variables are longitude (LONG) and latitude (LAT) (expressed in hundredths of degrees) and altitude (ALT) (expressed in meter). The environmental variables were then calculated to generate more biologically meaningful variables using a ’biovar’ function in the R package ‘dismo’ [p > 0.05). A total of 17 environmental variable including an EcoregionIII Factor (EF), 8 temperature variables and 8 precipitation variables over 30 years (from 1987 to 2017) were collected. The EF was created according to Omernik [To assess the association of environmental and geographical variation with the genetic variation of natural prairie cordgrass populations, a 30-year normals for temperature and precipitation were collected from National Oceanic and Atmospheric Administration (NOAA) weather stations located closest to the collection site  matrix was calculated using the genet.dist function in ‘hierfstat’ package, in which the Weir & Cockerham approach was used [F-approximations of Wilks’ Lambda using p.asym function in ‘CCP’ package [To evaluate the influence of environmental adaptation on the formation of subgroups within prairie cordgrass, we conducted mantel tests and canonical correlation analyses using the SNP data, environmental and geographical data. For mantel tests, the environmental distance matrix was created based on 17 environmental variables, using the vegdist function in ‘vegan’ package in R ,56. To cwas used . The manwas used ,59,60,61was used ,63. In owas used . The sta package .A total of 240 million single-end sequence reads were produced from the Illumina Hi-Seq2000 platform. The minimum and maximum lengths were 32 and 90 base pairs (bp), respectively. A total of 29.7 million reads were used to build the pseudo-reference genome based on available computational power. The final pseudo-reference genome contains 371,332 sequences and 19.1% of them were non-singletons which were filtered in the downstream analyses. The average length of the reference sequences was 82 bp with a standard deviation of 13.5 bp. The initial assembly and SNP calling without filters yielded 211,294 SNPs. A final subset of 9315 SNPs was retained and genotyped in 213 samples after applying restrictions on read depths and allele frequencies. The average read depth was 36.4 × per SNP. The distribution of sample read depth is also provided There were three DNA ploidy levels found in this study: tetraploid, hexaploid, and octoploid cytotypes . The intfastSTRUCTURE and the BIC score from DAPC suggested two genetic demes , based on 9315 nuclear SNPs (The simulation result from ear SNPs . The fir†) was considerably different. In West deme, populations were less variable/more tightly clustered than populations from East deme on PCOA1 and PCOA2. There were only four tetraploids in West deme. There were several populations from MO, NE, IA, IL, IN, CT, and KS that could equally likely be categorized in East deme or West deme. Furthermore, most of these populations are geographically adjacent to both populations from East deme and from West deme. These populations were also distributed along the mixed- to tallgrass prairie border. This provides support that populations from these states  are in areas where intraspecific breeding occurred. Populations from both demes were tightly distributed on PCOA3, except for eight populations from MA, ME, IL, NJ, and CT (p = 0.77) The first two principal coordinates separated East deme from West deme, indicating two major gene pools . Althoug, and CT . Those pUsing 9315 SNP markers, analysis of molecular variance showed that SNP marker variance was significant among ploidy levels, among populations within ploidy levels, and among samples within populations . VariancHo), average expected heterozygosity (He), and overall genetic diversity (Ht) across all populations were 0.27, 0.22, and 0.24, respectively. Ho, He, and Ht within East deme were 0.21, 0.19, and 0.20, respectively. Ho, He, and Ht within East deme were 0.35, 0.26, and 0.27, respectively. Inbreeding coefficients (Fis) across all populations, within East deme, and within West deme were −0.212, −0.133, and −0.369, respectively. The fixation index (Fst) across all populations, within East deme, within West deme, and between two demes were 0.05, 0.045, 0.053, and 0.079, respectively. Since a population-based imputation method (LD-kNNi) was used, the heterozygosity calculated in our study could be underestimated.Average heterozygosity was calculated across a whole population panel, within each deme, and between two demes . Averager = 0.25) and the geographical distance (r = 0.33) (p < 0.001). The partial Mantel test generated a significant correlation between genetic and environmental distance  after controlling for geographical distance. This indicated that it is necessary to dissect the relationship between specific environmental variable and genetic distance. Following the Mantel and partial Mantel tests, CCA showed significant coefficients from five pairs of canonical variables   ranged from 0.71 to 0.92. The top three canonical axes explained 73.5% variance cumulatively. Therefore, we selected the top three canonical axes and presented their correlations with genetic (PCOAs) and environmental/geographical variables in r = 0.618), (II) (r = 0.578), and (I) (r = 0.947), respectively. This indicated that populations separated by PCOA3 in PCOA were largely contributed by canonical variables on canonical axis (I). Populations separated by PCOA1 and PCOA2 were largely contributed by canonical variable on axis (II).The Mantel tests showed significant correlations of the genetic distance with both the environmental , which resulted in separating East deme, West deme, and the New England populations . The ALT and SDAT were also contributing to the pattern in a relatively low magnitude . In canonical axis (II), ALT, MTWM, MPDM, and EF were significantly correlated with canonical axis (II) , which resulted in separating East deme, West deme, and a widely scattered pattern in East deme populations. The LONG also showed a moderately high correlation with canonical axis (II) (r = −0.419). In canonical axis (III), a complex of environmental and geographical variables was correlated with PCOAs, especially PCOA7 and PCOA8. The LAT, MAT, MTWM, MTCM, MPSP, MPSU, MPAU, and MPWI were the top contributors in canonical axis (III). Notably, several precipitation variables were associated with LONG in canonical axis (I) while temperature variable were associated with LAT on canonical axis (II).In canonical axis (I), LONG, MPDM, MPSP, and MPWI were significantly correlated with canonical axis (I) (He, Ho, Fis, and Fst to evaluate the degree of divergence within and among demes. The populations within West deme exhibited higher heterozygosity than that in East deme, as indicated by both the fixation statistics and the principal component analysis between and within demes. However, this is likely due to a larger number of octoploidy populations in the West Deme compared to that in the East Deme. Although dismoic SNPs calls were used for samples of all three ploidy levels, we still observe a higher heterozygosity level in the octoploidy dominant West Deme than that in the tetraploidy dominant East Deme. Similar to switchgrass, the two identified potential regional gene pools have a dominant ploidy level, either tetraploid or octoploid [Using nuclear molecular markers, significant genetic diversity and population structures were found within perennial grass species ,68,69. Ictoploid . Comparectoploid , we inclctoploid  study, tctoploid ,71. Howectoploid ,72,73. Ictoploid ,74,75.Phenological and morphological differentiation due to geographic isolation and climatic gradients was observed within several tallgrass species native to North America ,77,78,79Our research reported the population genomic variation and potential diversity centers in prairie cordgrass based on the analysis of 9315 SNPs from 96 natural populations. Two distinct genetic groups were identified which were associated with ploidy levels and geographical and ecotypic separation. Analysis of intraspecific variation among and within genetic groups and ploidy levels revealed evidence of adaptation history of prairie cordgrass in the Midwest and Eastern USA. The major gene flow in prairie cordgrass could be a consequence of geographic, climatic events, and human activity. Future studies on local landscape variation in prairie cordgrass could provide further information on the adaptation strategies in perennial grass species. From a standpoint of improving prairie cordgrass, both for biofuel production and conservation purpose, the identification of divergent genetic resource could provide opportunities to combine breeding value from different gene pools.


Anthonomus grandis Boheman (Coleoptera: Curculionidae), is an important pest of commercial cotton across the Americas. In the United States, eradication of this species is complicated by re‐infestations of areas where eradication has been previously successful and by the existence of morphologically similar variants that can confound identification efforts. To date, no study has applied a high‐throughput sequencing approach to better understand the population genetic structure of the boll weevil. Furthermore, only a single study has investigated genetic relationships between populations in North and South America. We used double digest restriction site‐associated DNA sequencing (ddRADseq) to resolve the population genomic structure of the boll weevil in the southern United States, northern Mexico, and Argentina. Additionally, we assembled the first complete mitochondrial genome for this species and generated a preliminary whole genome assembly, both of which were used to improve the identification of informative loci. Downstream analyses revealed two main lineages—one consisting of populations found geographically west of the Sierra Madre Occidental mountain range and the second consisting of populations found to the east—were revealed, and both were sub‐structured. Population geographic structure was consistent with the isolation by distance model, indicating that geogrpahic distance is likely a primary mechanism driving divergence in this species. Boll weevil populations from Argentina were found to be more closely related to the eastern lineage, suggesting a recent colonization of South America by the eastern lineage, but additional sampling across Mexico, Central America and South America is needed to further clarify their origin. Finally, we uncovered an instance of population turnover or replacement, highlighting the temporal instability of population structure.The boll weevil,  In addition to representative specimens from multiple populations across the species’ North American distribution, we have also sampled populations from South America and inferred the genetic relationship of these populations to their North American counterparts. We also sampled collection localities temporally to assess the stability of the population genetic structure over time.Accurately describing the population structure and dynamics of species is fundamental to understanding their geographic distributions and evolutionary history. This information is especially important for integrated pest management, which needs to consider pest evolution , across the Americas. Native to Mesoamerica, it is generally accepted that the most recent common ancestor of the boll weevil originated in southern Mexico and Central America and diverged from the sister species, Anthonomus hunteri Burke and Cate, during the Pliocene . The weevil underwent at least one host shift to one or more endemic Gossypium L. species and later shifted to G. hirsutum after its cultivation began in the Americas. The species has since expanded its geographic distribution over time, likely in association with the expansion of the cultivation of commercial cotton. In the late 1800s, the boll weevil underwent a northward range expansion through Mexico and eventually across the entire cotton‐producing region of the southern United States (US), where it became an infamous agricultural foe ; (2) the Thurberia weevil (A. g. thurberiae), which has traditionally been regarded as a host‐race associated with Arizona wild cotton, Gossypium thurberi Todaro ; and (3) the Mexican boll weevil, an intermediate form that has never been given any formal subspecies designation . A total of 292 weevil specimens were collected and processed across 20 spatiotemporally distinct collections , the lower Rio Grande Valley along the United States–Mexico border (RGV), the Chihuahuan Desert ecoregion in Mexico (CMX), and Sonora, Mexico (SMX). The fifth geographic region was southern Arizona where wild cotton, ns Table . Weevil ns Table , whereasG. thurberi) localities in southeastern Arizona. In 2017, the Sonora locality was resampled, but for the central Mexico (CMX) geographic region, we collected weevils from Coahuila, Mexico, rather than the previously sampled Durango and Chihuahua localities due to variation in weevil presence from year to year. The Coahuila and Durango localities were only 55 km apart and are both part of a contiguous cotton production region known as La Laguna. These two populations were thus expected to be representative of the same geographic population, though this was not assumed a priori in downstream analyses. Specimens from Argentina were collected in 2017 from five localities in the four cotton‐producing provinces of Chaco, Salta, Santiago del Estero, and Formosa, using cone traps baited with synthetic boll weevil pheromone .Weevils were first collected in 2014 in Mexico from the cotton‐producing states of Sonora, Chihuahua, Durango, and Tamaulipas, as well as from the lower Rio Grande Valley (RGV) cotton production area in Texas, USA, just north of Tamaulipas along the United States–Mexico border. In 2016, the Texas and Tamaulipas localities were resampled, and an estimated 14–20 generations were expected to have occurred between sampling events. Weevil specimens were also collected in 2016 from six wild cotton (2.2The Gentra Puregene Cell and Tissue Kit (Qiagen) was used to isolate genomic DNA from whole weevil specimens. Individuals collected in 2014 were processed and delivered for sequencing in 2015, and individuals collected in 2016 and 2017 were processed and delivered for sequencing in 2017. DNA was isolated from all individuals using the same protocol  was maximized. Fragments were size selected using a Pippin Prep (Sage Science) and were then ligated with standard Illumina adapters, multiplexing indexes, and sequencing primers, albeit with a single notable exception; the R1 reads  were ligated with a custom sequencing primer that contained the 5′ restriction site remnant. In 2015, libraries were sequenced on a HiSeq 2500 using 2x125 sequencing cycles; 2017 libraries were sequenced on a NovaSeq using 2x150 sequencing cycles. Potential differences in sequence batches were addressed during the bioinformatic analyses as described below.Genomic DNA isolated from weevils was delivered to the Texas A&M AgriLife Genomics and Bioinformatics Service (TxGen) for purification, library preparation, and sequencing. DNA was purified using the Agencourt AMPure XP purification system (Beckman Coulter) prior to library preparation. Library preparation for the ddRADseq was nearly identical in 2015 and 2017, but 2015 libraries were prepared for a HiSeq 2500 (Illumina), and 2017 libraries were prepared for a NovaSeq (Illumina). To prepare the ddRADseq libraries, purified genomic DNA was digested using the 2.3A. g. thurberiae  were collected on December 27, 2019, from the same collection locality as AWC‐Bi2 (2016). Individuals were flash frozen in liquid nitrogen and stored at −80°C. The genome size of each was estimated as described in Johnston et al. (Drosophila virilis reference standard (1C = 328 Mbp). Nuclei were released by grinding with 15 strokes of the loose “A” pestle at a rate of three strokes every 2 s. The released nuclei were strained through 45 µm nylon mesh, stained with 50 µg/ml propidium iodide for at least 1 h in the cold and dark, then scored for the relative fluorescence of the 2C nuclei from the sample and standard using a CytoFLEX flow cytometer (Beckman/Coulter). DNA content was determined as the ratio of the mean fluorescence of the sample and standard times the 1C amount of DNA in the D. virilis standard. A minimum of 1000 nuclei were counted for each 2C fluorescent peak, with a CV <2 for each peak.A total of 10 0 parents were collected in the lower Rio Grande Valley of Texas and reared for three generations at the boll weevil rearing facility at the Texas A&M AgriLife Research and Extension Center in Weslaco, Texas. Six F3 progeny were flash frozen in liquid nitrogen and shipped overnight to Dovetail Genomics. The Dovetail team utilized a proprietary hybrid Illumina‐HiRise approach to assemble the genome sequence. Using this approach, Illumina reads generated by whole genome shotgun sequencing are mapped to intermediate‐range scaffolds and long‐range physical maps generated by Chicago  as a seed sequence. The mitogenome was annotated using the web‐based program MITOS version 1.0  that may experience different evolutionary pressures than the mitochondrial parent  across populations for each locus and removed loci with a mean FIS < 0. PGDSpider version 2.1.1.3  was used to manually check the phylip for abnormalities prior to uploading to CIPRES. Next, PartitionFinder version 2.1.1 . Geographic distances were measured as straight‐line distances (in kilometers) between pairs of GPS coordinates and then transformed by the natural logarithm. The adjusted values of FST and straight‐line distances were extracted from the Isolde output and plotted in Microsoft Excel to calculate the slope and intercept of the linear regression and calculate the R2 value.R/genepop version 1.0.5  has undergone a rearrangement into the middle of the control region, differing from the ancestral arrangement found in most insects. Similar rearrangements have been found in Sitophilus oryzae and S. zeamais , a Thurberia weevil (A. g. thurberiae), and an intermediate Mexican boll weevil; and (2) a two‐form genetic hypothesis wherein there exists a distinct lineage distributed west of the Sierra Madre Occidental mountain range and a distinct lineage distributed to the east and stretching down into Central and South America. The results of our study were more consistent with the two‐form hypothesis, though it does not fully describe the genetic variation we observed across all sampled populations. There are two primary pieces of evidence that supported this conclusion: (1) the long internal branch separating the two main lineages in the unrooted phylogenetic tree  a three‐form morphological hypothesis wherein there exists a southeastern boll weevil  are typically indicative of species‐level differentiation or greater, and despite some consistency with previous measurements made for boll weevil populations (Alvarado et al., FST values are relatively consistent with our other analyses, and other types of genetic markers have yielded similarly high estimates (Alvarado et al., A. g. thurberiae, to warrant subspecific taxonomic status. Though the authors recognize the unique association of some populations in the western lineage with G. thurberi, those populations, in our study, were not more distinct from populations infesting commercial cotton in Sonora than populations in the lower Rio Grande Valley were from those in the Chihuahuan Desert. If those G. thurberi populations warrant subspecific status, then so should many of the other sampled populations. Despite the host association in those populations, their genetic divergence from other populations could just as easily be explained by IBD (Figure Gossypium, boll weevil has been documented in association with other Malvaceae including Hampea spp., Cienfuegosia spp., and Thespia populnea (Burke et al., Though both the phylogenetic reconstruction and the discriminant analysis of principal components pointed to further substructure in both lineages, there is clear evidence for a major genetic divide between the western and eastern lineages. In the phylogenetic reconstruction Figure , the intD Figure . It is a4.2Consistent with Scataglini et al. , we foun4.3G. thurberi in Arizona were not so different from those infesting commercial cotton in Sonora (Figures Our results, based largely on weevils from commercial cotton, showed that there was a large amount of genetic diversity across geographic sampling regions. However, within geographic sampling regions, diversity was much lower, suggesting that in a given region, there is typically a single, contiguous population Figures  and 3. M Figures  and 2, aMore broadly and perhaps most critically, managers must recognize the reality of the temporal instability of pest population genetic structure (Choi et al., Genetic bottlenecks could be particularly threatening to effective management if the surviving individuals do so due to resistance to management. Were this the case, it could lead to a selective sweep. Fortunately, in the lower Rio Grande Valley, boll weevil populations have remained susceptible to malathion, so a selective sweep due to management is not expected to have caused the population turnover we have documented here. Nonetheless, a turnover event or replacement does appear to have occurred, which highlights the dynamic nature of the population genetic structure even within a single growing region. Managers in any growing region would be best served by a robust understanding of where genetic populations occur and what reservoirs exist that could possibly contribute to re‐infestations. Population genetic approaches have previously been effective for determining the sources of re‐infestations (Barr et al., 4.4Temporal instability of population genetic structure is not a novel discovery. Studies of economically or ecologically important fish species have long monitored changes in allele frequencies over time (Garant et al., The authors have no conflict of interest to declare.TJR carried out the majority of the analyses and wrote the manuscript. CMD provided custom scripts and generally supported the bioinformatics used for the population genetic analyses. LCP conducted the mitochondrial genome assembly and annotation and the relatedness analysis. AET contributed to the bioinformatics and downstream analyses in R. CPCS, RRA, TNB, and MRF coordinated and supported sample acquisition and contributed intellectually. JSS supported the genome size estimation. GAS provided resources and laboratory space for the study, collected samples, helped write the manuscript, and contributed intellectually. All authors were given opportunity to review and contribute to the manuscript before submission into consideration for publication.Supplementary MaterialClick here for additional data file.