Theory suggests that different taxa having colonized a similar, challenging environment will show parallel or lineage‐specific adaptations to shared selection pressures, but empirical examples of parallel evolution in independent taxa are exceedingly rare. We employed comparative genomics to identify parallel and lineage‐specific responses to selection within and among four species of North American sparrows that represent four independent, post‐Pleistocene colonization events by an ancestral, upland subspecies and a derived salt marsh specialist. We identified multiple cases of parallel adaptation in these independent comparisons following salt marsh colonization, including selection of 12 candidate genes linked to osmoregulation. In addition to detecting shared genetic targets of selection across multiple comparisons, we found many novel, species‐specific signatures of selection, including evidence of selection of loci associated with both physiological and behavioral mechanisms of osmoregulation. Demographic reconstructions of all four species highlighted their recent divergence and small effective population sizes, as expected given their rapid radiation into saline environments. Our results highlight the interplay of both shared and lineage‐specific selection pressures in the colonization of a biotically and abiotically challenging habitat and confirm theoretical expectations that steep environmental clines can drive repeated and rapid evolutionary diversification in birds. Organisms that inhabit salt marshes are faced with extreme challenges and must cope with harsh, open environments as well as daily influxes of salt water into the system. Our whole‐genome comparisons among four species of sparrows demonstrate the important interplay between species‐specific and parallel signatures of selection in driving adaptation across an ecological gradient. Several lineage‐specific adaptations to salt marshes, particularly related to salt tolerance, suggest that selection of independent pathways may be important for allowing increased osmoregulatory function in salt marsh environments. Our results highlight the utility of a comparative genomics approach in characterizing the genomic basis of local adaptation and may aid in informing conservation strategies for threatened salt marsh endemics through the characterization of evolutionary potential.Steep ecological gradients between saline and freshwater environments represent a potential barrier to the radiation of terrestrial species given the extreme physiological challenges present in saline environments , Nelson's sparrows (Ammospiza nelsoni nelsoni and Ammospiza nelsoni subvirgatus), song sparrows (Melospiza melodia gouldii and Melospiza melodia pusillula), and swamp sparrows (Melospiza georgiana georgiana and Melospiza georgiana nigrescens). For each species comparison, upland populations inhabit freshwater marshes or meadows. The four salt marsh populations inhabit marshes that vary along the salinity gradient , however all of these populations are exposed to saline conditions. By studying replicate comparisons across these independent pairs of phylogenetically related taxa exposed to similar adaptive pressures, we substantially increase our statistical power for inferring the genetic basis of convergent adaptive variation and for testing questions about the scope and pattern of parallel evolution  versus lineage‐specific selection as drivers of salt marsh adaptations across all of these evolutionarily independent sparrow populations. Our comparative genomics approach is based on 80 whole genomes from four independent salt marsh specialists, each paired with their closest upland subspecies Fig. : savannaM. m. gouldii (n = 10), a subspecies of song sparrow that breeds in freshwater marshes along the California coast (Grinnell and Miller M. m. pusillula (n = 10), a subspecies endemic to salt marsh habitats in the San Francisco Bay; Ammospiza nelsoni nelsoni (n = 8), a subspecies of the Nelson's sparrow complex that inhabits freshwater marshes and meadows in the continental interior from eastern British Columbia to northern South Dakota, versus A. n. subvirgatus (n = 9), a subspecies that inhabits brackish and salt water marshes along the Atlantic coast from the Canadian Maritimes to southern Maine (Greenlaw and Rising P. s. nevadensis (n = 10), the subspecies of the savannah sparrow complex that breeds in grassy meadows and pastures in the Great Basin (Wheelwright and Rising P. s. beldingi (n = 10), a subspecies restricted to the Pacific coast salt marshes from Santa Barbara, CA to Rosario, Baja California, Mexico (Van Rossem M. g. georgiana (n = 12), the southern swamp sparrow that breeds in freshwater wetlands within formerly glaciated areas of eastern North America, versus M. g. nigrescens (n = 11), which breeds in brackish salt marshes of the Mid‐Atlantic United Sates  reference genome. We annotated the swamp sparrow assembly with the MAKER pipeline version 2.31.8 . Genes were predicted using SNAP using an iterative process to train the program. This produced a total of 13,733 gene models, which represented 71% of the genes annotated in the zebra finch genome.We used an existing reference genome from a male swamp sparrow . Resequencing data from the 23 swamp sparrows have been previously published and were obtained from Deane‐Coe et al.  and quantified with the Qubit dsDNA BR Assay Kit (Life Technologies). For the 57 song, savannah, and Nelson's sparrows, we prepared individually barcoded libraries using 1 µg of DNA following the TruSeq DNA PCR‐free library preparation kit protocol , with an insert size of 350 bp. We pooled 24 libraries using concentrations of adapter‐ligated DNA determined through digital PCR into one group. The pooled libraries were sequenced on three Illumina NextSeq500 lanes at the Cornell Institute for Biotechnology core facility. The quality of individual libraries was assessed using FastQC version 0.11.5 . The swamp sparrow genome was used as an outgroup to polarize the joint‐SFS for savannah, Nelson's, and song sparrows. For models including the two populations of swamp sparrows, we ran demographic models using the folded SFS due to the absence of a genome from an appropriate outgroup for this pair.To estimate the demographic history associated with each upland‐salt marsh split, we fit a range of demographic models to the joint SFS of the upland and salt marsh population for each of the four species in ∂a∂i version 1.7  based on a 1‐year generation time for all sparrow species Mowbray  and the FST, pi, and Tajima's D). For each species, we simulated 1072, 1 mb regions to approximate the length of the whole genome sequences. For all simulations, we assumed a passerine mutation rate of 3.3 × 10−9  estimated from Estrildidae finches  and Tajima's D for 25 kb windows across the simulated data set within the python package scikit‐allel .We simulated a SNP data set based on the best‐fit demographic model and parameter estimates from ∂a∂i within the coalescent simulator msprime . To do this in a way that accounted for differences in sample size between neutral and elevated windows, we used a permutation test to compare the Tajima's D and pi estimates from the outlier windows to the same number of randomly chosen neutral windows. Finally, differentiated peaks were inspected in Geneious version 9.1.5 . To characterize putative candidate genes, we used ontology information from the Zebra Finch Ensemble database. We also compared genes to several previously published candidate gene lists  for each species pair using the Web‐based GOfinch tool (http://bioinformatics.iah.ac.uk/tools/Gofinch). We provided gene‐level annotations from Ensembl and used Fisher and Hypergeometric tests of enrichment for terms in our input list.We performed GO analyses of candidate genes (mean FST: 0.02 (song sparrows), 0.03 (swamp sparrows), 0.07 (Nelson's sparrows), and 0.26 , we found a strong delineation between all pairs of salt marsh and upland populations, highlighting the clear potential for similar processes of ecological divergence across the freshwater‐saline habitat barrier. Estimates of pi, heterozygosity, and Tajima's D were notably similar between salt marsh and upland populations  of nongenic SNPs  to account for the influence of species‐specific, neutral demography on patterns of genomic divergence. Log‐likelihoods for the top demographic models ranged from –1376.21 in song sparrows to –4812.24 in savannah sparrows with residual error normally distributed largely between –30 to 30. In many species, there were more SNPs distributed along the center of the joint‐SFS than inferred by the top model, suggesting the model may be underestimating migration rate Fig. S1). The best‐fit models from demographic analyses indicate that all salt marsh populations (except for swamp sparrow) experienced fluctuations in effective population size following divergence from upland relatives. Population bottlenecks appear to have occurred recently in most species. This may be linked to anthropogenic development along both coasts  greater than 5 standard deviations above the genome‐wide mean to be elevated , whereas some candidate regions should be interpreted more cautiously. However, several of these regions of elevated divergence exhibit corresponding dips in Tajima's D and pi (see below) and thus offer candidate genes that warrant further investigation. We also accounted for uncertainty in parameter estimates by performing an additional 1000 simulations randomly sampling from a uniform distribution bounded by the 95% confidence intervals around each parameter estimate. For all species, the 95th percentile for each of the 1000 simulated FST distributions fell below the empirical FST cutoff . Several, but not all, of these differentiated windows harbored candidate genes with associated annotations: 43 elevated windows with annotated genes (55%) in Nelson's sparrows, 46 elevated windows (41%) in song sparrows, 67 elevated windows (43%) in swamp sparrows, and 50 elevated windows (22%) in savannah sparrows.shared regions of elevated differentiation between multiple upland‐saltmarsh pairs as signatures of parallel selection. In contrast, we consider any independent regions of elevated differentiation within single upland‐saltmarsh pairs as signatures of lineage‐specific selection. Furthermore, we consider gene regions that exhibit both elevated differentiation and corresponding dips in Tajima's D as our most compelling candidates for both parallel and lineage‐specific selection in this system : 14 (32%) of the regions identified in Nelson's sparrows, 15 (32%) of regions in song sparrows, 23 (46%) of the regions in savannah sparrows, and 23 (34%) of the regions identified in swamp sparrows. More broadly, across all species comparisons combined, we saw a significant reduction in Tajima's D in differentiated windows (mean = 0.75) compared to a random subset of neutral windows  between salt marsh and upland comparisons.We classified regions as shared if multiple paired comparisons identified the same gene within 50 kb of an elevated window. We consider these m Tajima . Severala priori knowledge  and in each species comparison, we observed significant enrichment for genes that participate in pathways important for osmotic regulation, but the specific pathways differ among them. Enriched pathways include the positive regulation of sodium ion transport in Nelson's sparrows (P < 0.001), regulation of Rho GTPase activity in song sparrows (P = 0.0029), regulation of actin cytoskeleton organization in savannah sparrows (P = 0.0024), and regulation of JNK cascades in swamp sparrows (P = 0.0052). This role for novel genetic solutions for osmoregulatory function was further supported by candidate genes identified via examination of genomic regions exhibiting elevated divergence. We documented elevated differentiation for 15 gene regions linked to osmoregulation in savannah sparrows  relative to the empirical FST distribution  and Savannah sparrows . These differences in divergence time are reflected in the greater elevation of genomic divergence and identification of more differentiated windows in Savannah (122 windows) versus Nelson's sparrow (78 windows). However, greater overall genomic differentiation in Savannah sparrows was not associated with the identification of a greater number of candidate genes , which acts as an activator and inhibitor of sodium‐coupled chloride cotransporters and appears to be an important component of an essential pathway for regulating cell volume in response to osmotic stress , which is linked to both drinking behavior and kidney function and is expressed in the region of the brain responsible for regulating thirst in mice ; differential expression of MYOF has been documented in response to being transferred between fresh and salt water in fish , which is involved in sodium ion import across the plasma membrane and was found to be differentially expressed in freshwater versus salt water sticklebacks  to the simulated spectra of the best‐fit model and estimated parameter values (top right).Extended Data Figure S2. PCA plot based on approximately 21 million SNPs for all four species (and eight subspecies) combined.Extended Data Figure S3. Histograms of FST estimates for individual SNPs for each of the four species comparisons.Extended Data Figure S4. Descriptive statistics for coastal and interior populations of savannah sparrows.Extended Data Figure S5. Descriptive statistics for coastal and interior populations of Nelson's sparrows.Extended Data Figure S6. Descriptive statistics for coastal and interior populations of song sparrows.Extended Data Figure S7. Descriptive statistics for coastal and interior populations of swamp sparrows.Extended Data Figure S8. Simulated FST distributions for each species based on neutral demographic history inferred with ∂a∂i.Extended Data Figure S9. Distribution of 95th percentiles of the FST distribution from 1000 simulated SNP data sets.Extended Data Figure S10. Distribution of 99th percentiles of the FST distribution from 1000 simulated SNP data sets.Extended Figure S11. Distribution of Tajima's D estimates for each species comparison for elevated (red) and neutral  windows.Extended Figure S12. Distribution of nucleotide diversity estimates for each species comparison for elevated (red) and neutral  windows.Extended Data Figure S13. Boxplots depicting the percentage of reads mapped to the swamp sparrow reference genome (color coded by species).Extended Figure S14. Different demographic models fit to joint site frequency spectrum of upland (light gray) and tidal marsh populations (dark gray).Extended Data Table S1. Information and sampling locations for individual sparrows analyzed in this study.Extended Data Table S2. Average observed heterozygosity, nucleotide diversity, and Tajima's D for coastal and interior populations of savannah, Nelson's, song, and swamp sparrows.Extended Data Table S3. Parameter estimates for the best‐fit demographic model from ∂a∂i.Extended Data Table S4. List of shared candidate genes in two or three species pairs identified through a comparative genomics approach.Extended Data Table S5. Candidate genes identified through whole genome comparisons of freshwater and salt water populations of Savannah sparrows.Extended Data Table S6. Candidate genes identified through whole genome comparisons of freshwater and salt water populations of Nelson's sparrows.Extended Data Table S7. Candidate genes identified through whole genome comparisons of freshwater and salt water populations of Song sparrows.Extended Data Table S8. Candidate genes identified through whole genome comparisons of freshwater and salt water populations of Swamp sparrows.Click here for additional data file.


Campanula americana, we identified a glacial refugium in the southern Appalachian Mountains from spatial patterns of genetic drift among 24 populations. Populations farther from this refugium have smaller effective sizes and fewer rare alleles. They also displayed elevated heterosis in among‐population crosses, reflecting the accumulation of deleterious mutations during range expansion. Although populations with elevated heterosis had reduced segregating mutation load, the magnitude of inbreeding depression lacked geographic pattern. The ability to self‐fertilize was strongly positively correlated with the distance from the refugium and mutation accumulation—a pattern that contrasts sharply with contemporary mate and pollinator limitation. In this and other species, diversity in sexual systems may reflect the legacy of evolution in small, colonizing populations, with little or no relation to the ecology of modern populations.Colonization at expanding range edges often involves few founders, reducing effective population size. This process can promote the evolution of self‐fertilization, but implicating historical processes as drivers of trait evolution is often difficult and requires an explicit model of biogeographic history. In plants, contemporary limits to outcrossing are often invoked as evolutionary drivers of self‐fertilization, but historical expansions may shape mating system diversity, with leading‐edge populations evolving elevated selfing ability. In a widespread plant,  Following the last glacial recession, many species expanded their ranges from source populations in regions of suitable habitat called glacial refugia. These range expansions have left lasting imprints on the evolution and genetic diversity of populations. Mating systems are highly variable among hermaphroditic organisms ranging from obligate outcrossing to nearly complete selfing, and their evolution is predicted to be strongly impacted by range expansion. Although considerable effort has been made to explain this variation in light of selection in recent time, there is substantial variation not accounted by this explanation. Here, we evaluate the extent to which patterns of historical colonization have shaped mating system diversity by correlating variation in autonomous self‐fertilization with genetic signatures of postglacial range expansion. In the widespread herb American Bellflower, we detected high levels of neutral genomic variation in a putative glacial refugial region and a reduction in diversity outside of this region, reflecting population bottlenecks. Populations farthest from the refugium expressed high genetic load, suggesting that colonization events facilitated the accumulation of deleterious mutations. The ability to self‐fertilize was positively correlated with distance from the refugium and with the amount of genetic drift, linking variation in a key mating system trait to a history of range expansion. Although range expansion has been shown to structure the distribution of genetic variation, we show that it also affects the evolution of reproductive traits. Historical range expansion from glacial refugia is pervasive and has the potential to shape mating system diversity across many organisms. Furthermore, mating system evolution will likely accompany future poleward range shifts expected to occur under globally changing climates.Contemporary species distributions are strongly influenced by past range expansions and contractions. In temperate regions across the globe, range expansion following glacial recession has shaped population genetic diversity and structure Hewitt . For exaAlthough any trait may evolve during geographic range expansion, mating systems are expected to be particularly sensitive to a history of colonization Baker . Two priRecurrent founder events during colonization should reduce effective population size  Does the load of fixed or locally common mutations  increase with distance from the refugium? (3) Does the segregating genetic load  decline with distance from the refugium? and (4) Do populations more distant from the refugium display an elevated capacity for autonomous selfing, and is this associated with fixed and segregating load?If historically small populations present the most conducive opportunities for the evolution of autonomy, this trait should be strongly associated with signatures of genetic drift , is a widespread, autotetraploid herb . Fragments were size selected using gel extraction (300–500 bp range) and amplified by PCR using established protocols . Low‐quality samples were removed, using parameters defined in the available ipyrad parameter file. A minimum of 20 reads were required for each plant at nucleotide sites, and alleles below a frequency of 5% were removed from populations. Contigs absent in six or more populations were removed, as were those with a length below 50 bp. After filtering, populations had a mean of 68.72 kb of nucleotides (Table W) and Tajima's estimators (θπ), in addition to Tajima's D , with serial founding events leading to more pronounced declines. Because random sampling associated with genetic drift causes a deficit of mid‐frequency variants, the site frequency spectrum is expected to systematically shift to more extreme values as populations decline in their effective size  were calculated. Because genetic drift intensifies in regions far from the refugium, pairwise differences in these distances (dij = id − jd) should be linearly related to pairwise differences in the frequency of derived alleles  and a positive slope coefficient. We repeated these analyses using two plants from the Atlantic Coast (VA71) or the Gulf Coast (FL16) to polarize alleles as ancestral  self‐fertilization , (2) outcross fertilization by a donor from the same population (hereafter “within”), and (3) outcross fertilization by a donor from a different population . The paternal plant for the within‐population cross was chosen randomly. For the between‐population crosses, each maternal plant of a given population was crossed with a randomly selected donor from a single population at a roughly similar latitude Table . Betweenn = 1414), we sowed five seeds in each of two cells (n = 10/family) across 18 seedling trays in a randomized fashion . F1 seeds were germinated in the same conditions as the parental generation. We scored the number of seedlings 26 days after sowing in each cell. Proportion germination was quantified as the number of seedlings that germinated divided by the number of seeds planted for each family. We then thinned each cell to a single seedling. Seedlings were vernalized in the same manner as their parents.From fruit produced by hand pollination, that is, a seed family (1 plants (mean = 19.8/cross type/population). For each plant, we scored survival to flowering, the occurrence of bolting, flower production, seed set, and above‐ground biomass. All plants that died prior to flowering and those that did not bolt within three months of transplanting were recorded. We monitored plants three times per week to record the date of first flower. We scored the number of open flowers on each plant once per week for four consecutive weeks beginning one week after initiation of flowering. On each plant produced through selfing and within‐population outcrossing, we applied outcross pollen from a single donor from the same population and cross type to an emasculated flower. For example, all plants from population IN77 in the self were crossed by a pollen donor from IN77 also in the self‐treatment. The resultant seed set thus reflects both maternal and paternal quality of a given population and cross type combination. When mature, we collected the hand‐pollinated fruits and counted seeds produced. We then harvested and dried the above‐ground tissue and measured the dry weight of each plant in grams on a Metler Toledo AG245 balance. Seed production of F1s was not scored for the between‐population hybrids because it could reflect F2 hybrid breakdown due to negative epistatic interactions rather than heterosis  to transplant into the greenhouse. If there were fewer than 16 plants in a cross treatment within a population, we planted a second replicate of some families to achieve the goal of 20 plants per cross type. In total, we collected fitness data on 1424 F1s), but failure to bolt was more common (8.7%). Because reproduction in C. americana depends on bolting and subsequent flowering, we grouped survival and bolting into a single metric. To measure the proportion of plants that reached reproductive maturity , we summed the number of plants that died and did not bolt in each population and cross type combination, and divided this by the number of seedlings transplanted into the greenhouse. We estimated weekly flower production by averaging the sum of the four flower counts. In some cases (4.7% of plants), averages were based on fewer than four counts. Seed set was scored as the number of seeds produced. If a fruit did not set (5%), seed set was scored as zero.Death was exceedingly rare .To assess whether populations near the inferred refugium harbored the most genetic variation, we used the inferred origin of the range expansion based on the site frequency spectrum Fig. A and cal1 progeny produced through within and between‐population crosses. We modeled germination rate, survival, flower production, and biomass using linear models with population, cross type, and their interaction as fixed effects. For proportion germination, we used a beta distribution; for survival, a binary distribution, and for flower production and biomass a normal distribution. Residuals for each model were normally distributed. In the analysis of germination, chamber was included as a random effect because seedlings were germinated in one of three growth chambers. A significant population × cross type interaction indicated that genetic drift load varied among populations. To evaluate the degree of inbreeding depression, we compared fitness of F1 progeny produced through within population outcrossing and selfing. We used the same models and distributions described for drift load. A significant population × cross type interaction indicated that populations varied in the magnitude of inbreeding depression.We tested for drift load, the reduction in fitness resulting from genetic drift /WW, where WW is mean value of the within treatment and WB is mean value of the between treatment. This estimates the proportional increase (heterosis) or decrease (outbreeding depression) in performance when crossing between populations relative to within populations. Using between‐population values in the denominator /WW when WW > Ws and (Ws – WW)/Ws when Ws > WW, where Ws is mean of the self‐treatment . We calculated drift load at each life stage for each population using the equation .C. americana populations is consistent with expansion from a refugium in eastern Kentucky, in the foothills of the Appalachian Mountains  between pairwise differences in site frequency spectra (ψij) and pairwise geographic distances between populations (dij), in a model with this most likely point of origin. Most of the models we tested that had high coefficients of determination placed the refugium in the southern Appalachian Mountains, and populations near this refugium have the largest effective population sizes  was strongly negatively associated with distance from the glacial refugium  in the population closest to the refugium, and significantly increased in populations farther from this putative point of origin  were not significantly associated with autonomous selfing ability at the population level .Estimates of population genomic diversity (θ) varied from 0.004 to 0.011 across r = 0.745, P < 0.001) and biomass  was elevated in populations farther from the refugium but at other life stages did not display a geographic pattern  and negatively correlated with θW .Across populations, heterosis was expressed in between‐population crosses for flower production and plant biomass, and its magnitude for each trait varied among populations Table A. Howeveern Fig. . Cumulatern Fig. A. Cumular = −0.54, P < 0.01, Fig. r = −0.22, P = 0.31; Fig. θW .Inbreeding depression was expressed for all traits measured except germination Table B. HoweveP = 0.0016), however only refugial distance and drift load were significant predictors, each having roughly similar magnitude of effect on autonomous fruit set  in C. americana are negatively associated with distance from this refugium. Increasing values of Tajima's D with distance to the refugium further imply that small populations have lost rare alleles in this species, as would be expected following repeated population bottlenecks during range expansion  is a key component of the mating system. Contemporary C. americana populations  and Gulf Coast (B) populations as ancestral to identify derived alleles.Figure S2: Drift load for proportion germination (A), survival (B), flower production (C) and plant biomass (D) in 24 populations of Campanula americana spanning a range of distances from the inferred glacial refugium.Figure S3: Inbreeding depression for proportion germination (A), survival (B), flower production (C), seed production (D), and plant biomass (E) in 24 populations of Campanula americana spanning a range of distance from the putative glacial refugium.Figure S4: Drift load, inbreeding depression, and autonomous fruit set plotted against average growing season temperature .Click here for additional data file.


Heterodera glycines Ichinohe, is a major pathogen of soybean that was accidentally introduced in most soybean-producing countries. In this study, we explored patterns of genetic exchange between North American populations of SCN and the effect of isolation by geographical distance. Genotyping-by-sequencing was used to sequence and compare 64 SCN populations from the United States and Canada. At large scale, only a weak correlation was found between genetic distance  and geographic distance, but local effects were strong in recently infested states. Our results also showed a high level of genetic differentiation within some populations, allowing them to adapt to new environments and become established in new soybean-producing areas. Bayesian genome scan methods identified 15 loci under selection for climatic or geographic co-variables. Among these loci, two non-synonymous mutations were detected in SMAD-4 (mothers against decapentaplegic homolog 4) and DOP-3 (dopamine receptor 3). High-impact variants linked to these loci by genetic hitchhiking were also highlighted as putatively involved in local adaptation of SCN populations to new environments. Overall, it appears that strong selective pressure by resistant cultivars is causing a large scale homogenization with virulent populations.Determining the adaptive potential of alien invasive species in a new environment is a key concern for risk assessment. As climate change is affecting local climatic conditions, widespread modifications in species distribution are expected. Therefore, the genetic mechanisms underlying local adaptations must be understood in order to predict future species distribution. The soybean cyst nematode (SCN),  The introduction of an organism into a new environment can have unpredictable detrimental consequences, including public health problems, losses in biodiversity and ecosystem services or crop yield losses due to exotic weeds, insects and pathogens, altogether resulting in significant economic impact , RAPD (random amplified polymorphic DNA), and 2-DGE   technologies, sequencing a large number of individual genomes at high coverage in order to perform population genetic studies remains very expensive and may require important quantities of DNA from individuals. Elshire et al.  developeThe main objectives of this study were to (1) investigate the genetic relationships among SCN populations from United States and Canada, (2) detect isolation by geographical distance (IBD) between SCN populations from United States and Canada, (3) detect genetic loci under selection associated to environmental and climatic parameters, and (4) identify the putative gene functions contributing to the adaptation of SCN populations to specific environmental conditions.A total of 64 field populations of SCN, representative of the area currently infested in North America, were sampled or provided by collaborators from 11 US states  and one Canadian province (Ontario) Figure  at the IH. glycines. This pipeline is designed to call SNPs de novo, without a reference genome with high stringency. Only sequences containing a single putative SNP (1-bp mismatch) per sequencing read were kept. Before analysis, the SNPs were further filtered with a minimum coverage (minCov) of 20 reads, a maximum coverage (maxCov) of 10,000 reads, and a minimum allele frequency (MAF) of 0.01.The UNEAK pipeline  in R  values ], as defined by Rousset   test was performed at a 0.95 confidence level.Fixation index  Hegge, . To accePopulations from three states  were selected as case studies to evaluate the local  genetic differentiation. These states were chosen because there were sufficient samples for comparison and because (i) North Dakota shows a recent introduction of SCN and a continuous northward dispersal of the nematode , longitude (LONG), annual mean air temperature (BIO1), maximum air temperature of the warmest month (BIO5), annual precipitation (BIO12), and total precipitation of the warmest quarter (BIO18). All climatic variables were retrieved from the WorldClim global climate database, version 1.4 : BayPass, version 2.1 Gautier, , BayeScap-value (eBPis), a metric measuring the difference between observed data and a simulated set of data (posterior distribution) . The PO threshold to affirm that a locus was under selection, in comparison with a neutral model, was set to 0.91, which corresponds to a strong relationship on the Jeffreys scale  by means of BLASTN with the default parameters, except for a smaller word size of 4, with the Blast2GO application  and processed with Trimmomatic (v0.32) , we explored the genomic regions around outlier loci for genetic hitchhiking in whole-genome sequences from four populations of different origin and distinct virulence profile . Sequencing libraries were prepared using the Nextera XT DNA Library Preparation Kit  and sequenced on a MiSeq sequencer (Illumina) using the MiSeq Reagent Kit v3 (600-cycle). Reads were demultiplexed using the Sabre software program . On the basis of the sequence size used by the UNEAK  pipeline (first 64 bp of each read), the expected horizontal coverage was approximately 0.8% of the total SCN genome, and the vertical coverage at each locus, considering the number of reads obtained, was 400 ×. Before filtering, the UNEAK pipeline identified 3,172 variants. After filtering for low coverage and selecting only SNP variants, two datasets were generated. The first dataset contained 245 SNPs without missing data , and the second contained 804 SNPs with missing data  was performed using the dataset without missing data, which contained 245 SNPs. The PCA plot of the 64 North American SCN populations revealed a geographically ordered pattern Figure . OverallTo better understand the population structure of each population, we used the 804-SNP dataset to conduct a phylogenetic analysis based on Provesti's distance. The inferred neighbor-joining phylogenetic tree Figure  showed tST) of 0.15 ± 0.07. However, pairwise genetic distance was highly variable, with a minimum FST of 0.005 and a maximum FST of 0.53 , North Dakota (ND3 and ND6), South Dakota (SD2), and Ontario .Overall, we observed an average value for Wright's fixation index (FST/(1–FST)] and geographic distance (km) for all pairs of populations revealed a significant (p = 0.012) but weak effect of isolation by distance, with a Mantel correlation (r) of only 0.135. However, when analyzing the effect of isolation by distance at the local scale, a different pattern was found. With respect to US states with recent SCN history, a strong and significant positive correlation was observed between the genetic and geographic distances in North Dakota  and Minnesota . In contrast, no correlation was found among the populations from Illinois .The analysis of the relationship between genetic distance [Frs = 0.535) but significant (p = 0.001) relationship was observed between genetic distance and geographic distance  using the 245-SNP dataset. This analysis included 38 independent populations that were separated by 273 to 1,367 km from MO1 Figure . The gene Figure .Using three Bayesian inference genome scan approaches, we identified 71 SNPs out of 804) that were under selection. Among the different computational methods, 48 loci were identified by BayeScan, 23 loci were identified by BayPass, and 16 loci were identified by BayeScEnv , and five SNPs were located in intergenic regions Table . Two SNPThe whole-genome resequencing of four SCN populations for the exploration of the genic environment of outlier loci highlighted 257 genes containing high-impact variants in a ± 50-kb window around loci under selection. Annotation was available for 148 of these genes, corresponding to 132 different gene functions  approach. Sequencing DNA from pooled samples for each population also has the advantage of keeping the number of redundant sequences low  from those of the central states was only moderate, whereas comparing these recent populations together generated very high FST values (up to 0.53). This corroborates results from the PCA analysis and clearly suggests that populations diverged following two distinct routes, one in the northwest toward North Dakota and one in the northeast toward Ontario. This hypothesis is further supported by the isolation-by-distance analysis. Overall, we observed a significant effect of distance on genetic differentiation at the continental scale. The effect of distance was even more noticeable in newly colonized areas like North Dakota and Minnesota or when each population was compared to a common ancestor. On the other hand, the absence of any effect of distance in Illinois and low FST in the central states clearly suggests that dispersal is frequent enough to weaken signs of isolation by distance after a short period of time. This continuous gene flow combined with the selection of alleles due to the general use of the same resistant soybean lines are thought to cause a rapid homogenization of SCN populations  was strongly associated with precipitation; but the geographic distribution of allele frequencies Figure  at that Most of the 15 outliers identified by at least two pipelines correlated with temperature, reinforcing that temperature is a key factor that acts on the genetic diversity and natural selection of nematode populations. Nematodes are poikilothermic organisms and depend greatly on temperature to complete their life cycle. Thus, any adaptation that facilitates the development and reproduction of nematodes at lower temperatures would be advantageous. On the basis of the distribution of allele frequencies, however, this adaptation does not seem crucial for survival, as the northern populations did not all exhibit similar patterns. This trait is probably not yet fixed in these populations. Nevertheless, six SNPs under selection and associated with temperature were specific to populations from Ontario  or only retrieved from Minnesota and North Dakota , a finding that clearly suggests local adaptations.Caenorhabditis elegans, the DOP-3 protein is well known to modulate chemosensory functions, such as mating and foraging, and to be involved in locomotion  that replaces an aspartic acid with an asparagine, was strongly associated with the annual mean temperature and annual precipitation. This modification was retrieved in two distinct gene sequences (g14642.t1 and g14654.t1), and a synonymous mutation (Pro/Pro) was retrieved in two other sequences (g14639.t1 and g14644.t1) but in a different amino acid, indicating a reading frame shift and a putative upstream indel. These four genes were all annotated as DOP-3 and were probably the result of gene duplication. Gene duplication has frequently been hypothesized to play an important role in adaptation to the environment  coded for G-protein-coupled receptors (GPCR). These proteins are members of a large and very diverse multigene family with hundreds of occurrences in the s et al.  found ths et al. . In our Although some SCN genes were found to be under selection and local adaptation was found to be underway in this study, our results also indicate that there is no critical adaptive event required for SCN establishment in northern latitudes. Consequently, all populations should theoretically survive and multiply at high latitudes. The risk is thus real for new soybean areas where cultivars resistant to SCN are not available at this time. Of course, the method we used does not explore the entire genome, and a pan-genomic study of these populations could reveal other loci under selection in an evolutionary process.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.BM, A-FG, and JB conceived and designed the experiments. A-FG, EL, and P-YV performed the experiments and analyzed the data. A-FG, EL, and BM wrote the paper. All the authors revised the manuscript.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.


FST genetic distances across genome-wide SNP panel, we detected diversifying selection signatures among these two subpopulations and indicated (among others) the significance of DGAT1 and FGF2 genes for milk production traits in these cattle. We also found that among genes being presumably under selection in terms of milk production, there are genes responsible, for example, for mammary gland development  and immune system response . The most important finding of this study is that the most pronounced genetic differences between the analyzed populations were associated with β-defensin genes  located within so-called bovine cluster D on BTA27. The β-defensins are expressed mainly in the mammary gland and are antimicrobial peptides against the Gram-negative and Gram-positive bacteria, viruses, and other unicellular parasites. This suggests that antimicrobial resistance of mammary gland is of high importance during selection towards increased milk production and that genes responsible for this process are selected together with increasing levels of productivity.Polish Red cattle is one of the few indigenous breeds of European red cattle which is characterized by several desired features, such as high disease resistance, good health, longevity, good fertility, and high nutritional value of milk. Currently, Polish Red cattle population is a subject of two independent breeding programs: (i) improvement program and (ii) genetic resources conservation program. The aim of the improvement program is the genetic progress in terms of milk production and body conformation traits, while the conservation program mainly focuses on protection of the genetic resources of Polish Red cattle and preservation of the existing, original gene pool. By the analysis of The online version of this article (10.1007/s13353-019-00484-0) contains supplementary material, which is available to authorized users. Polish Red cattle is one of the few indigenous breeds of European red cattle. It is characterized by several features typical for primitive populations, such as high disease resistance, good health, longevity, very good fertility, easy births, ease of calf rearing, and high biological value of milk. The Polish Red cattle is also characterized by a good adaptation to harsh environmental conditions, which is especially visible in the ability to limit the efficiency , as well as the relatively quick regeneration after condition loss. These features make the cattle of this breed well adapted to mountainous and submountainous living and production conditions  improvement program and (ii) genetic resources conservation program  and “improvement”  breeding programs. The animals were randomly selected and verified to be unrelated for at least two generations. The animals were coming from at least three different herds. All animal procedures were approved by the Local Animal Care Ethics Committee No. II in Kraków—permission number 1293/2016 in accordance with EU regulations. The genomic DNA was purified using Sherlock AX kit  and after quality control was genotyped with the use of BovineSNP50v2 BeadChip assay  according to the standard Infinium Ultra protocol. Only samples with call rate > 0.95 were retained for analysis. The initial markers set included 54,609 SNPs and were further filtered to remove markers without known chromosomal position or located on sex chromosomes. SNPs that had minor allele frequency (MAF) < 0.05, genotyping rate < 0.8 (in joint populations), and deviated from Hardy-Weinberg equilibrium with p < 0.0001 (in each population separately) were also removed. This filtering step allowed retaining for further analysis a common set of 43,165 SNPs with average inter-marker distance of 57.9 kb in UMD3.1 genome assembly.The study material comprised 60 samples of ear tissue collected from cows belonging to Polish Red breed and included in “conservation”  and haplotype block structure at the most divergently selected regions among the studied populations were analyzed using HaploView 4.2  based on individual genotypes and mean or weighted The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.FST distances of 0.0226 and 0.0242, respectively. The PCA showed, however, that both populations form clearly separated clusters of observations with visibly higher level of genetic variation observed in RE population  allowed for detection of 46 overlapped genes. The overrepresentation test performed for those genes showed single enriched GO category connected with defense response to bacterium (adjP = 0.02). The genes associated with this process belonged mainly to the β-defensins family . In the analyzed genes set, we also detected other immune system functioning–related genes, like CD53 and TRAT1 . These two selection signals were analyzed in details to identify their possible meaning and evaluate haplotype structure at these loci. The analysis of the divergently selected region on BTA3 showed that the detected selection signal colocalized with the large chromosomal region of strong LD (31.6–33.5 Mb) and encompassed large haplotype block with high-frequency haplotypes in both studied populations (> 0.5). The detailed analysis of haplotypes showed that in separate populations, the most common haplotypes were composed of alternative SNP variants. Additionally, only haplotypes found in RP breed showed some minor signs of recombination  including one non-coding small nuclear RNA.Two selection signals found in this study included, inter alia, the strongest detected signal on BTA3 (32.5–32.9 Mb) and the signal spanning the largest genomic region (1.3 Mb) among the signals with top 0.1% of The genomic region on BTA27 overlapping with the detected large and strong diversifying selection signal was also characterized by strong LD  and encompassed three haplotype blocks showing common variants with haplotype frequency above 0.5. The detailed analysis of haplotypes again showed signs of diversifying selection among the analyzed populations, represented by haplotype variation and divergent selection of separate variants Fig. . The sigIn this study, we attempt to identify diversifying selection signatures among two subpopulations of Polish Red cattle formed by animals subjected to conservation program (selected mainly towards primitive type and breed standard) and animals used for milk production . These two populations visibly diversified over time and the phenotypical differences between them mainly refer to milk yield traits as well as exterior features which were strongly shifted towards milk production type in the improved cattle Fig. .Fig. 5Twloci which were divergently selected since the implementation of the improvement program. The performed PCA showed that the genomes of both these populations bear signs of differentiation with fraction of individuals being still genetically close. RE population was, however, subdivided into two visible clusters due to the different level of historical crossbreeding present in the pedigrees of the analyzed animals. The fact that both subpopulations originate from the same native population created an opportunity to trace allele frequency differences among them with high probability that they result from application of selection directed to improvement of milk traits in commercial population. This allowed for identification of candidate genes affecting presumably both primitive traits found in conserved population (being lost due to the intensive selection)  confirms their common origin from local population  and FGF2 (Fibroblast Growth Factor 2) , which may be of high importance for the improved population and its milking abilities  controls the size and shape of mammary buds in mice  ablation results in defective mammary morphogenesis and regeneration  was shown to play a role in development and reorganization of the mammary gland  gene. This gene was previously shown to have effect on body size regulation in horses , spanning over 1.1 Mb (32.4–33.6 Mb) in both analyzed populations. Within the detected selection signal, we found only three genes, namely LRIF1, CD53, and KCNA3. These gene functions could be connected with both male fertility and immune system response. Exemplary, LRIF1 product represses the ligand-induced transcriptional activity of retinoic acid receptor alpha (RARA) and disruption of retinoid signaling was shown to impair mammalian spermatogenesis and fertility  affecting milk yield, fat yield, and protein yield in Holstein and other cattle populations , the largest genomic region with signs of divergent selection was identified on BTA27 (1.29 Mb). This signal overlapped with several genes coding proteins belonging mainly to the β-defensins family , and non-lactating cows  has been also presented, where higher concentration of LAP in the milk of cattle infected with Staphylococcus aureus, Streptococcus bovis, Streptococcus dysgalactiae, and E. coli was observed than in uninfected cows  polymorphisms were significantly associated with protein yield and fat or protein contents Genomic regions formed by FESM 2ST values. (XLSX 61 kb)Genes found within top 1% of window-averaged F


Crossarchus platycephalus), a species of least conservation concern, on either side of the River Niger in Nigeria. Ecological disturbances affect the conservation status of many other animals in this region. The two populations were differentiated in the snout and fore limbs, with greater morphological diversity in the western population. We used Restriction site Associated DNA sequencing (RAD‐seq) and identified two genotypic clusters in a STRUCTURE analysis. Individuals from the eastern population are almost entirely assigned to one cluster, whereas genotypes from the western population are a mixture of the two clusters. The population from west of the River Niger also had higher heterozygosity. The morphological and population genetic data are therefore in agreement that the population from west of the River Niger is more diverse than the eastern population, and the eastern population contains a subset of the genetic variation found in the western population. Our results demonstrate that combining morphological and genotypic measures of diversity can provide a congruent picture of the distribution of intraspecific variation. The results also suggest that future work should explore the role of the River Niger as a natural barrier to migration in Nigeria.Geographic barriers can partition genetic diversity among populations and drive evolutionary divergence between populations, promoting the speciation process and affecting conservation goals. We integrated morphological and genomic data to assess the distribution of variation in the flat‐headed cusimanse ( C. platycephalus are small carnivores ; and 17 from east of the River Niger , generally described as Niger Delta Swamp Forest, Cross‐Niger Transition Forest, Cross‐Sanaga‐Bioko Coastal Forest, and Northwestern Congolian Lowland Forest  , following the manufacturer's instructions. DNA quality and concentration was assessed with agarose electrophoresis and with a NanoDrop spectrophotometer .Liver, ear, and skin tissue samples were collected from the 33 individuals of 2.4We used DNA from the 33 samples to construct RAD‐seq libraries following the instructions in a published protocol , 25% of individuals in a population were required to have a genotype for each locus (‐r 0.25), kernel‐smoothed calculations of population statistics was enabled (‐k), and SNP and haplotype‐based F statistics were calculated (–fstats). For output into downstream analyses , we reran the populations program with the option to only write the first SNP from any RAD locus (–write_single_snp) because this reduces the inclusion of linked data in the downstream analyses. Finally, STRUCTURE and GENEPOP were used to determine the genetic structure of the populations and calculate population genetics metrics using the variant calls from Stacks , single‐end run on an Illumina NextSeq500 machine at the University of Houston Seq‐N‐Edit Core, following the manufacturer's instructions. The reads were demultiplexed using the Illumina bcl2fastq software and assembled using the STACKS pipeline , tail length (p = 0.23), or hind limb length (p = 0.43) between the populations. Fore limbs were larger in the east (p = 0.01), and snout length is longer in the west (p = 0.01). We additionally used a principal component (PC) analysis to group individuals based on the five length measurements. Individuals from west of the River Niger have more variation in the first PC  than those from the eastern population  by plotting ΔK (change in the log probability of data with respect to the number of clusters) against successive k values . We calculated a null distribution of FST assuming no population subdivision by randomly assigning the 33 individuals to two populations over 1,000 iterations. The observed FST value does not fall in the upper 5% tail of the null distribution  between the populations from west and east of the River Niger  has a longer snout length than C. platycephalus, and C. obscurus also occurs west of the River Niger . Click here for additional data file.


This study was carried out to assess the haplotype diversity and population dynamics in cattle populations of Ethiopia.b gene of 76 animals from five indigenous and one Holstein Friesian×Barka cross bred cattle populations.We sequenced the complete mitochondrial cytochrome FST) was 0.049539 with the highest (FST = 0.1245) and the lowest (FST = 0.011) FST distances observed between Boran and Abigar, and Sheko and Abigar from the indigenous cattle, respectively. The phylogenetic network analysis revealed that all the haplotypes detected clustered together with the Bos taurus cattle and converged to a haplogroup. No haplotype in Ethiopian cattle was observed clustered with the reference Bos indicus group. The mismatch distribution analysis indicates a single population expansion event among the cattle populations.In the sequence analysis, 18 haplotypes were generated from 18 segregating sites and the average haplotype and nucleotide diversities were 0.7540±0.043 and 0.0010±0.000, respectively. The population differentiation analysis shows a weak population structure (4.55%) among the populations studied. Majority of the variation (95.45%) is observed by within populations. The overall average pair-wise distance (Bos taurus.Overall, high haplotype variability was observed among Ethiopian cattle populations and they share a common ancestor with  Bos primigenius in the southwest Asia (Bos taurus) between 8,000–10,000 YA and south Asia (Bos indicus) [Bos taurus group arrived in these regions, in Ethiopia in particular, around 7,000 years BC [Bos taurus, and Arabian and Indian Bos indicus cattle migration corridor and sometime considered the secondary hybridization zone [Bos indicus cattle descended from the putative cattle domestication center in the northern part of the Indian subcontinent, the Indus Valley [Cattle are believed to have originated from indicus) , and sprindicus) . The Horindicus) ,4. The Byears BC . Those rion zone ,7. The Bs Valley . Convershttp://dad.fao.org/: accessed January 04, 2017) which are classified into humpless Shorthorn, zebu , zenga and sanga types [d-loop region and nuclear genomic information concurred that there has been extensive hybridization among the indigenous cattle populations in Ethiopia and led to have high level of admixture [Being the major entry point of cattle to Africa, the genetic landscape of the current cattle populations in Ethiopia could have been shaped by several introductions of zebu cattle and introgression of the taurine from the Near-East . As a rega types . Both thdmixture ,11,13,14b (cyt b) and d-loop regions of mtDNA play significant role in unraveling the population history of livestock species. Using the d-loop region of mtDNA, history of genetic diversity and maternal origin of 10 Ethiopian indigenous cattle populations have been reported [cyt b region to further uncover the maternal origin and population dynamics despite the fact that Ethiopia in particular and the East African region as whole are considered the hybridization zone for both Bos-taurus and Bos indicus cattle groups [cyt b region aiming to further unveil the genetic diversity, phyogenetic relationship, maternal origin and population expansion of the indigenous cattle in Ethiopia. It also complements information to the findings reported on the diversity of the cattle populations using the d-loop and autosomal DNA information [Cytochrome reported . Howevere groups . Therefoormation ,11,13,14F1 cross between Holstein Friesian (HF) and Barka (Begait synonymously). The cattle populations included in the study are representative of each cattle group previously described by Rege [Blood samples were collected from 76 animals of Abigar, Boran, Guraghe, Horro, Sheko and one cross bred cattle populations. The cross bred population was  by Rege  which incyt b gene  was amplified using forward primer: 5′-CCATAAATAGGTGAA GGTTTGG-3′ and reverse primer: 5′-TTGATGGTGAGAC TGCAGTT-3′ [2O. The PCR cycling profile involved an initial denaturation step at 95°C for 2 min, followed by the first stage of amplification of 30 cycles involving a denaturation step at 94°C for 40 s, annealing at 56°C for 40 s, and extension at 72°C for 90 s. The PCR reaction was completed with a final extension step at 72°C for eight min. The PCR products were analyzed by 1% agarose gel electrophoresis and visualized by ultraviolet illumination after staining with gel rednucleic acid gel stain . The PCR products with good quality were sent to Sangon Biotech in China (http://www.sangon.com) for sequencing.Genomic DNA was extracted using Promega Genomic DNA extraction kit . A complCAGTT-3′ . The ampFST) and Reynold’s distances, neutrality tests and populations expansion events were evaluated using ARLEQUIN. We constructed a neighbor joining (NJ) tree based on the Kimura two-parameter model algorithm in MEGA6 [Bos taurus  and Bos indicus  from England, the Netherlands, USA, South Korea, Japan, and China were included in the phylogenetic network analysis for comparison. The reliability of the tree topologies was assessed by 1,000 bootstrap replications.The sequences were aligned with CodonCode aligner software . The nucleotide and haplotype diversities were generated using DnaSP 10.01  and the in MEGA6  to demonin MEGA6  to furthFS and Tajima’s D neutrality tests which were implemented in 1,000 simulations.In addition, we examined the star-like clustering patterns of the population expansion by mismatch distribution analysis in ARLEQUIN. With the same package, the history of the indigenous cattle was inferred using Fu’s cyt b gene, 1,140 bp, was sequenced for 76 animals representing six cattle populations in Ethiopia. Eighteen (18) segregating/polymorphic sites were detected in the entire cyt b region. The transition to transversion ratio of polymorphic sites is 5:1 and the average codon bias index was 0.566. From the polymorphic sites, seven of them are parsimony informative sites and the remaining 11 are two variant singleton variable sites.The full length of the mitochondrial Hd = 0.8333±0.127) and Sheko (Hd = 0.6282±0.143) cattle, respectively.From the 18 segregating sites, we observed 18 haplotypes , and onlFST = 0.12446), Sheko (FST = 0.11360), and F1 (FST = 0.19437). Similar trends were observed using the Reynold’s distance. However, we observed little or no differentiation between Sheko and Abigar, and Boran and Horro cattle populations with both distance measures. On the other hand, both the phylogenetic network and NJ tree analysis show absence of clear phylogeographic structure among the cattle populations   . Similar alleles .cyt b gene in five indigenous and one cross bred cattle populations in Ethiopian together with published sequences of Bos taurus and Bos indicus cattle. The number of segregating sites (S = 18) obtained in this study is much lower than those reported for Chinese cattle in the same mtDNA region (S = 105) [S = 3 from 18 animals) reported for Leiqiong cattle (Bos indicus type) of China [d-loop region of mtDNA in Ethiopian cattle populations [d-loop analysis of Kenana and Butan cattle populations in the neighboring Sudan [d-loop sequences, 81 haplotypes were identified in 10 Ethiopian indigenous cattle populations [d-loop could be because of high mutation rate of the d-loop region compared to cyt b [In this study, we analyzed the complete mitochondrial S = 105) , but higof China . The traof China . No tranulations . Whereasng Sudan . In the ng Sudan , howeverng Sudan . In 117 to cyt b . For norHd = 0.7540) as well as nucleotide diversity (π = 0.00100)  [Hd = 0.0741) and nucleotide (π = 0.0012) diversities were reported for Leiqiong cattle [cyt b region. Based on the analysis of Y-chromosome microsatellites of north Ethiopian cattle populations, the average haplotype diversity was 0.617167±0.02617 [The current study revealed that the average haplotype diversity (0.00100)  are slig0.00923) . This cog cattle . This cod-loop [d-loop), only 2.4% of variation was observed between Kenana and Butan cattle in Sudan [The population differentiation analysis revealed very weak populations structure (4.55%) among the cattle populations studied and the highest variation (95.45%) is explained by within population variation . Howeverd-loop . With sain Sudan . Very loin Sudan . Very loin Sudan  and 1.1%in Sudan ; single in Sudan ). The vein Sudan ,13,25. Oin Sudan  in whichin Sudan , as we cFST) and Reynold’s distances vary from −0.01113 (between Sheko and Abigar) to 0.12446 (between Abigar and Boran) and zero  to 0.13292 (between Boran and Abigar), respectively (Bos taurus and zebu (Bos indicus) groups [Among Ethiopian indigenous cattle populations, the pair-wise (ectively . However) groups ; and henFST = 0.12446), and Boran and Sheko (FST = 0.1136). This could be because of low gene flow, geographical isolation, ecological factors and morphological adaptation to local conditions. The Boran cattle, a humped breed reflecting indicine introgression, carry genes adaptive to harsh environment and heat tolerance developed by a long-term natural selection, display high resistance to heat and ticks and good productivity on poor forage and low amounts of water, whereas, Sheko and Abigar are found in humid and forest areas where burden of tsetse flies is high [On the other side, we observed highest differentiation between Boran and Abigar  of Bos taurus and two lineages (I1 and I2) of Bos indicus [d-loop analysis of Ethiopian cattle, a major haplotype occurred at the H1 with the highest frequency which showed a broad geographic distribution and represents the possible ancestral haplotype of T1 lineage [We constructed median joining network to reveal the phylogenetic relationships among the cattle populations studied . The netulations . In addi indicus ,29, all  indicus . However indicus . All the indicus . In the  lineage . In the Bos taurus reference sequences clustered with Ethiopian indigenous cattle populations. All these haplotypes are from USA, European and Asian Bos-taurus cattle. In contrast, we did not observe any haplotype from Bos indicus reference sequences being shared or clustered with Ethiopian indigenous cattle despite the fact that four of the indigenous cattle populations included in this study are from Bos indicus group. Despite the proportion of variation of influence, influence of European taurine on North Ethiopian cattle was reported but exhibited a weaker influence compared to Asian taurine origin [Bos taurus and African Bos indicus share the same African set of taurine mitochondrial DNA haplotypes suggesting the pattern of zebu influence on the African continent was a process of introgression rather than replacement of African taurine cattle with unmixed Asian zebu as we also observed in this study. In line with this, the genome-wide analysis unveiled presence of substantial taurine introgression in Ethiopia zebu, sanga, and zenga cattle [Bos indicus) the existing populations, for instance Ethiopian cattle, have been created with different proportions of taurine and indicine backgrounds [Bos indicus than introgression due to adaptation of zebu to harsh environments like tolerance for heat, ticks, drought and poor forage.Interestingly, in the current study, nine of haplotypes of the e origin . Accordie origin , Africana cattle . As a rekgrounds ,9. The nkgrounds . A recenkgrounds , which ckgrounds . Howeverkgrounds  still arBos taurus origin of the reference populations asserts the former finding that shows all African cattle possess taurine humpless longhorns (Bos taurus) [Bos indicus type was observed from 10 indigenous cattle populations analyzed using the d-loop region and attributed to the challenge of recurrent drought and rinderpest epidemics [Over all, the cluster of the haplotypes detected in Ethiopian indigenous and the cross bred cattle populations to the  taurus) ,26. In lpidemics . This copidemics . In contpidemics . OverallSSD = 0.01901±0.009; p = 0.375) and raggedness index  tests were not significant. On the other hand, negative values for all the populations studied and negative and significant values of the coalescent-based neutrality tests  obtained also suggest existence of a population expansion event.The bell-shaped curve of the population dynamics obtained from the mismatch distribution analysis reveals presence of population expansion . HoweverF1 populations which could be because of their taurus background. On the other hand, moderate population differentiation was observed between tsetse susceptible Bos indicus representative (Boran) and resistant Bos taurus group (Sheko) and F1 populations (The absence or little deviation of Horro from Boran could mean that Horro’s zebu line could be Boran. Horro is from zenga group: cross of zebu and sanga cattle . We exhiulations . This obBos taurus ancestral origin given the fact that they are highly influenced by the Bos indicus group. The dynamics of population history showed presence of a onetime but rapid and recent population expansion in all the cattle populations studied.In conclusion this study revealed highest mtDNA variations among Ethiopian cattle populations and the haplotypes clustered into a haplogroup which goes in line with previous reports on East African cattle populations ,24. More


Aphis craccivora Koch (Hemiptera: Aphididae). To do so, we sampled A. craccivora across the Midwestern United States. from two host plants, alfalfa (Medicago sativa) and black locust (Robinia pseudoacacia)—putative source populations for winged migrants. Simultaneously, we sampled winged A. craccivora landing in pumpkin fields where they transmit viruses. Structure analyses supported host‐associated differentiation by identifying two major genotypic groups: an alfalfa group containing a single multilocus genotype and a locust group containing all others. Winged locust‐group aphids landed at a much greater magnitude within focal fields during year 2 than year 1, while those in the alfalfa group remained fairly consistent. Spatial autocorrelation analyses indicated locust‐group aphid movement was characterized by small‐scale dispersal during year 2, likely originating from populations within 10 km. We also detected strong temporal differences in colonization from the two host plants. Early in the summer, most winged aphids (79.4%) derived from the locust group, whereas late in the summer more (58.3%) were from the alfalfa group. Because early crop growth stages are more susceptible to damage from aphid‐vectored viruses, these data implicate locust as the more important source and illustrate how host‐associated differentiation can be used to track dispersal and inform management of heterogeneous pest populations.Small, mobile insects are notoriously challenging to track across landscapes and manage in agricultural fields. However, genetic differentiation among insect populations and host plants acquired through host‐associated differentiation could be exploited to infer movement within crop systems and damage potential. Although many insects exhibit host‐associated differentiation, management strategies for insect vectors of plant pathogens assume a homogenous population. Nevertheless, phenotypic changes derived from host‐associated differentiation could manifest in altered behavior or physiology affecting the likelihood of vector–pathogen–plant interactions, or the subsequent efficiency of pathogen transmission. We used SNPs to assess genotypic structure and host‐associated differentiation in the cowpea aphid,  Our prior research into the epidemiology of cucurbit crop viruses identified A. craccivora as the most important vector among a community of 53 aphid species alighting in fields , winged alates can nevertheless introduce viruses through a stylet‐borne nonpersistent route of transmission. The source for these vectors is completely unknown, but A. craccivora is widely considered a specialist on legumes  and alfalfa (Medicago sativa). Interestingly, prior research has shown that A. craccivora possess unique bacterial symbionts that maintain separate host‐associated populations for these two host plants . This difference may be ecologically significant since ants usually suppress aphid wing formation  at local and regional scales surrounding pumpkin fields. We then used single nucleotide polymorphisms (SNPs) to test for evidence of HAD and, simultaneously, compared the genetic structure of A. craccivora populations with migrant winged aphids flying into focal fields. Finally, we examined geographical patterns in genetic relatedness with spatial autocorrelation analyses to assess patterns in dispersal behavior, predicting that a tendency toward more frequent small‐scale movements among populations would lead to positive local, rather than regional, spatial autocorrelation.Here, we describe the results from a 2‐year field analysis aimed at identifying the source of 22.1A. craccivora from all alates trapped weekly within each field and stored them separately by field and collection date at −80°C in undiluted ethanol.We trapped winged aphids landing in four focal pumpkin fields with pans . Collections from potential source populations occurred within the duration of focal field sampling, except for clover which was only sampled in year 1 due to the difficulty in locating clover fields. Any temporal variation between alfalfa and locust collection dates largely results from variable periods of aphid colonization, as collection attempts were made throughout the sampling period until at least 20 individuals could be collected in a single sampling event. Source population locations were selected to represent a wide variety of distances both locally (ca. 3–50 km) and regionally (ca. 100–700 km) relative to the four focal pumpkin fields . Samples with DNA concentration <5 ng/μl were amplified with REPLI‐g Mini kit (Qiagen). In total, we extracted DNA from 503 aphids collected in year 1, and 465 aphids collected in year 2. We additionally extracted DNA following this protocol from 20 winged 2.2A. craccivora trapped at two pumpkin field sites in 2010 and 2011. Library preparation followed protocol described by Etter and Johnson , cstacks to make a catalog of the consensus loci, and sstacks to match all of the samples in the population against the catalog. We allowed a maximum of two mismatches between stacks within individuals and a minimum depth of coverage of three nucleotides for ustacks, and allowed no mismatches between stacks between individuals for cstacks.The RAD tags were then used to identify de novo candidate SNPs with Stacks v. 1.19 . We subsequently identified both aphid samples and loci with >15% missing data using GenAlEx v6.5 program , but was not found in an A. craccivora transcriptome . Regardless, we conducted analyses using neutral loci only as those undergoing selection can disrupt gene frequency calculations such as Wright’s FST , and running a BLASTX search on those highly similar sequences  to identify putative functions. We then compared open reading frames within highly similar transcriptome sequences with each SNP variant with ORF‐Finder (NCBI) to predict whether they would change resultant protein products.Lastly, we explored the possibility of SNP effects on virus epidemiology by checking for alignment between SNP sequences and an 2.3*D), Simpson evenness indices (V), and the slope of the Pareto distribution (c), and calculated genotypic richness (R: R = (G − 1)/(N − 1), where G = number of MLGs and N = population size). Again, following recommendations for analysis of clonal organisms by Arnaud‐Haond et al. (Ho) and expected heterozygosity (He), inbreeding coefficient (Fis), allele frequencies, and deviation from Hardy–Weinberg equilibrium were calculated with GenAlEx. To assess the likelihood of clonality or recombination within populations sampled, indicating sexual or asexual reproduction within populations, we calculated, a r¯d modified version of the index of association (IA) robust to data with multiple loci  with 999 random permutations. We included geographical distance class end points of 0, 10, 20, 50, 100, 200, 300, 400, and >400 km in analyses. The 0 km distance class was included to distinguish between aphids collected from the same population  vs. from nearby populations separated by some distance within 10 km. Because it is thought most aphid dispersal occurs over short distances , one of alfalfa origin , and two were found only in clover aphid populations. Three repeat MLGs  found in locust were only slightly distinct, with genetic distances of 1–6, and 28 of 31 single MLGs had total allele differences of <10 (Table r¯d) were significant (p < 0.01), indicating sexual recombination seems to be absent within A. craccivora across all populations  and a black locust population (“OH”) exhibiting the highest relative diversity , and very weak negative effects across populations at 10, 50, and 400 km as well , but also across populations at 10 km, and 100 km , and exhibited negative spatial autocorrelation at 20, 50, and 300 km , and in year 1 populations within 300 and >400 km . Negative spatial autocorrelation occurred in year 1 populations within 400 km  and in year 2 populations within 20 km and 300 km .Results were similar following analyses of locust‐group datasets excluding repeat MLGs. Aphids were marginally significant within a given population at 0 km in both years . In total, there were 13 alfalfa group A. craccivora alates in year 1 migrant populations and 8 in year 2, with relatively more landing in focal fields late‐season , whereas locust‐derived aphids differed in early:late proportion and magnitude by year . Thus, alfalfa may be a relatively minor contributor of migrants in focal pumpkin fields with low total numbers overall and biased toward late‐season activity, and the contribution of migrants from black locust may fluctuate more by year, but years with greater migrant production could generate more early‐season dispersal.Genetic grouping of T. incarnatum, T. pratense) are susceptible to WMV  in Wisconsin  and watermelon mosaic virus type 2 , Aphis glycines  found no evidence of dominant clones and contrasted this to M. persicae and other pest species of intensively cultivated monocultures  within the United States varied in their host‐plant associations and ability to transmit five viruses that cause barley yellow dwarf disease (Gray et al., S. graminum if it is a recessive trait. The obligately parthenogenetic nature of A. craccivora reproduction could similarly facilitate the retention of MLGs with enhanced vector competence. Follow‐up studies could examine how relative transmission efficiencies vary among A. craccivora alfalfa‐ and locust‐associated clonal types.It is possible that host race formation within locust‐ and alfalfa‐associated populations has also generated phenotypic changes to behavior or physiology impacting virus transmission efficiency, and/or the likelihood of interacting with virus reservoirs and susceptible crop plants. Although it was evident that many of the study SNP sequences are being transcribed in 5A. craccivora grouping by alfalfa‐ or black locust‐origin, suggesting HAD has generated two host races. Aphids placed into the predominantly alfalfa group by STRUCTURE analysis possessed a single MLG, which was by far the dominant MLG found in aphid populations on alfalfa. Aphids placed in the locust group largely possessed one dominant MLG, but were more diverse and contained several additional MLGs. Due to the lack of diversity among alfalfa aphids, we could not detect genetic structure relative to geographical distance; however, locust aphids supported a prediction for small‐scale dispersal in year 2, with some evidence of moderate migratory dispersal capabilities in both years. Due to evidence for an appreciable dispersal capability, the potential for generating large numbers of migrants, and the early‐season timing of dispersal flights, our data suggest that locust‐origin migrants are more important vectors in the pumpkin virus pathosystem.We observed a pattern of distinct genotypic None declared.https://doi.org/10.5061/dryad.3560pr3.All raw RAD library sequences are available at NCBI SRA: BioProject accession #PRJNA503177, BioSample accession #SAMN10354042. The validated SNPs developed in this study as well as the genotypic, geographical, population and host‐plant or pan collection source data have been deposited in Dryad at  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. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Thrips palmi, in China, which has expanded its distribution range through using greenhouses. Early invaded populations showed a relatively higher level of genetic diversity than recently expanded greenhouse populations. Strong population genetic structure corresponded to a pattern of isolation by distance, with no recent gene flow and low historical gene flow among populations, reflecting limited ongoing dispersal. A genetic signature of population expansion was detected in early invaded populations and three northern populations from greenhouses, suggesting that the greenhouse environments facilitated expansion of this species. Redundancy analysis showed that the independent effects of environment and geography could explain 51.68% and 32.06% of the genetic variance, respectively. These findings point to climate‐ and greenhouse‐related spatial expansion, with the potential for adaptation by T. palmi. They emphasize the contribution of human‐regulated environments on the successes of this invasive species, a situation likely to apply to other invasive species that use greenhouse environments.Biological invasions of pests into climatically unsuitable areas can be facilitated by human‐regulated environments, in which case there may be an impact on genetic structure through population processes and/or adaptation. Here, we investigated the population genetic structure of an invasive agricultural pest,  For invasive species in their introduced areas, population genetic structure can be formed by multiple introductions from genetically differentiated source populations, and demographic events such as bottlenecks and founder effects Barrett, . InvasivFrankliniella occidentalis (Thysanoptera: Thripidae), is a temperate species originating from southwestern USA and has invaded other areas around the world since the late 1970s , was first described in tropical regions of Sumatra in Indonesia .We collected specimens of 2.2T. palmi were used for genotyping  and FO‐AR (5′ TAAACTTCTGGGTGCCCAAAAAATCA 3′) modified from previously published primers  and the unbiased expected heterozygosity (HET)  and standardized expected heterozygosity (HES) among samples with different sample sizes using a rarefaction method in GENCLONE. Allelic richness (AR) and allelic richness of private alleles (PAR) were calculated with a rarefaction approach in HP‐RARE version 1.1  Nei, . Further2.4For microsatellite loci, we used two Bayesian cluster methods and discriminant analysis of principal components (DAPC) to investigate genetic structure across the populations.K) was set to 5, 8, 10 or 14, to ensure convergence and consistency of the results. For each K, 15 repeat runs were performed.The first Bayesian method, Bayesian analysis of population genetic structure (BAPS), was conducted using BAPS version 6.0  were run with 200,000 Markov Chain Monte Carlo (MCMC) iterations after a burn‐in of 100,000 iterations. The optimal value of K was determined using the Delta (K) method  was performed using adegenet version 2.0.1 Jombart,  in the Rcox1 haplotypes were constructed using a split networking method implemented in SPLITSTREE version 4.13.1  for each population and mutation‐scale migration rate (M = m/μ) among all populations, where μ is the mutation rate of genetic markers per generation, were simultaneously estimated with the Bayesian search strategy in Migrate version 3.7.2. Parameter values were as follows: long‐chains = 1, long‐inc = 20, long‐sample = 100,000, burn‐in = 100,000, heating = YES:1:, heated‐swap = YES, and replicate = YES:4. In the first run, θ and M were estimated from FST values, while in subsequent runs, Bayesian estimates of θ and M from the previous run were used.Recent and historical gene flow among populations was estimated using Bayesian methods implemented in BayesAss version 3.0.4  for current population (2Nμ) in a single population model (OnePop). Then, we used a single population model with a single continuous past variation in population size (OnePopVarSize) to estimate mutation‐scaled effective population sizes (θ) for the current population (2Nμ) and ancestral population (2Nancμ) with fixed pGSM as estimated in previous steps. For both demographic models, we first conducted two short iterations with 800 points and 200 runs per point to generate preliminary points. Then, 15 iterations with 800 points and 1,000 runs per point were conducted starting from points of last iteration. In the third round, 3 iterations with 800 points and 20,000 runs per point were conducted starting from points of the last iteration. Estimation of past demographic changes is usually challenging ) with geographic distance using the package ade4 in R, with 10,000 permutations. Pairwise FST values between populations based on microsatellites were calculated with GENEPOP version 4.2.1  was tested in samples from China by correlating pairwise genetic differentiation . Only the first half of positive eigenvectors was retained as explanatory variables of population structure. To avoid high collinearity, we excluded variables with a variance inflation factor (VIF) over 10. We conducted a series of RDAs initially with all variables in the first model, removing a variable with the highest VIF each round, and stopping when all VIF values were below 10. Four PCNM vectors, six environmental variables of habitat and five climate variables remained including three temperature‐related  and two precipitation‐related  variables. Both full  and partial  models of RDA were analyzed in the R package vegan. The independent effect of environment was the variance values for the constrained matrix of geography in the appropriate partial model, while the independent effect of geography was the equivalent for the constrained matrix of environment. The collinear proportion was calculated by subtracting the independent effects of environment and geography from the total amount of variance explained in the full RDA model.Second, we used a multivariate approach of redundancy analysis (RDA) to estimate the extent to which the variance in microsatellite genotypes was explained by climate and geography and by their collinear portion . RDA is a constrained linear ordination method that combines multiple linear regression and PCA . For climate data, we obtained 19 climate variables for each collection site from worldclim.org. To account for the influence of greenhouse conditions, we added variable of habitat with two values of greenhouse and field. Variables of climate and habitat were classified as environmental conditions. Geographic distances among 15 populations reflected isolation‐by‐distance effects on population structure. The matrices of pairwise geographic distances were transformed into principal components of neighborhood matrices (PCNM) using the function 33.1p < .01), while three of 325 locus pairs across all populations showed linkage disequilibrium. Thirty‐seven of 390 loci‐population pairs deviated from HWE (p < .01). However, no locus pair was linked and no locus deviated from HWE in all populations. The Japanese population (JANP) showed the lowest level of genetic diversity based on all parameters. For Chinese populations, two southwestern populations showed the highest level of genetic diversity, five population from central areas showed medium levels of diversity, whereas five northern populations collected from greenhouses and one island population from southern China showed the lowest levels. Similar results were found in estimations based on mitochondrial gene sequence  showed linkage disequilibrium  showed relatively high genetic differentiation from the other populations , and six showed no significant difference between current and ancestral effective population sizes . The effects of geography, the environment, and their interaction explained 17.9% of the total genetic variance. For the explained genetic variance, independent effects of environmental conditions  and geography accounted for 51.68% and 32.06%, respectively, while their collinear component accounted for 16.26%. When environmental and geographic effects were considered simultaneously in the RDA analysis, habitat, three climatic variables , and two geographic variables (PCNM2 and PCNM4) were highly correlated with genetic distance  populations for each population. There was no significant difference in 2Nancμ among the five well‐estimated populations, ranging on average from 0.76 to 1.88, indicating that demographic history may not have had much impact on population differentiation.Size of founder populations may influence the future response of invasive species in newly introduced areas  and maximum temperature of warmest month (bio5) are top variables that contributed to the genetic variation across sites. These results suggest that population genetic structure of T. palmi is related to greenhouse conditions and climate. Environmental conditions may impose direct strong selection pressures on T. palmi as evident from biological studies across the natural distribution range of this species (McDosald et al., T. palmi cannot easily deal with low temperatures. The threshold for the development of T. palmi from egg to adult is about 10–11°C, and a sum of effective temperatures is 189–194 degree‐days (Kawai, T. palmi were successfully eradicated in England after they were introduced (MacLeod, Head, & Gaunt, T. palmi can also be influenced by humidity (Su, Chiu, & Lin, T. palmi is restricted to northern regions with warm temperatures in winter, and its southern distribution may be limited by prevailing aridity (Layland et al., Our RDA analysis showed that environmental conditions could explain more than half the explained variation in genetic distances among the populations, or 9.25% of total variation in s Kawai, . Based oT. palmi to persist despite low temperatures outside, and they also provide humid conditions. The ecological opportunity provided by greenhouses may explain why there was a signature of populations expanding in three of five greenhouse populations. Such ecological opportunities could promote adaptive radiation by generating genetic changes in organisms (Stroud & Losos, These limits point to the likelihood of migration and population size of the thrips being limited by environmental conditions in China. Greenhouse conditions in northern China can help 4.4F. occidentalis and T. palmi are serious invasive pest of agriculture. Nevertheless, our studies showed distinct genetic structure in these two thrips across their distribution range of China. The F. occidentalis thrips showed population genetic differentiation unrelated to its geographic distribution in China, pointing to multiple introductions and human‐mediated dispersal in sporadic directions (Cao et al., T. palmi there was a high level of genetic differentiation across its spatial distribution in China‐related partly to geographic distance. This pattern of population genetic structure is similar to that found in species with stepping‐stone dispersal (Cao, Wei, Wei, Hoffmann, Wen, & Chen, T. palmi was first reported in southern China and occurred in northern areas much later (Yi & Liang, Thrips palmi is particularly abundant on the foliage of cucumbers and eggplants, and dispersal of this thrips may have been slow because these vegetables are transported as fruit rather than foliage (Huang, Thrips are tiny insects that are difficult to detect in quarantine. Invasion and dispersal of such small insects are usually mediated by human activities such as plant transport. Both e Huang, .F. occidentalis to invade a wider range of environmental conditions than T. palmi which originated from the tropics. It is likely that F. occidentalis is pre‐adapted to newly introduced areas and a limited number of individuals may then be sufficient to establish a population. On the other hand, more individuals may be needed for adaptation in T. palmi to provide abundant genetic variation on which natural selection can act. This is consistent with the similar levels of genetic diversity among population of T. palmi when compared to the different levels of genetic diversity among populations of F. occidentalis (Table Distinct genetic structures and different invasion patterns likely reflect differences in the biology of the two thrips species. A temperate origin may allow is Table .4.5F. occidentalis, which can be readily dispersed across a wide range through human activities, dispersal of the tropical species into temperate areas will be restricted by environmental conditions. However, environmental restrictions can be overcome by using artificial environments and there may also be evolutionary adaptation to the relatively constant conditions present in glasshouses. Thus, evolutionary adaptation by T. palmi should be further considered both to cold conditions and to the relatively constant conditions of greenhouses given that adaptive shifts can influence predictions around pest distributions and pest abundance.Greenhouses extend the ability to produce crops in different seasons and regions. The protected conditions in greenhouses lead to serious pest problems that are different from outdoor fields, such as outbreaks of whiteflies, mites, and thrips (Gerson & Weintraub, 5T. palmi. We found that environmental factors and geographic isolation correlated with genetic differentiation. The ecological opportunity provided by greenhouses may contribute to recently expanded populations of T. palmi.In this study, we revealed population genetic structure and potential evolutionary forces affecting genetic differentiation of an invasive agricultural pest, None declared. Click here for additional data file.