Philaenus spumarius is a widespread insect species in the Holarctic region. Here, by focusing on the mtDNA gene COI but also using the COII and Cyt b genes and the nuclear gene EF-1α, we tried to explain how and when its current biogeographic pattern evolved by providing time estimates of the main demographic and evolutionary events and investigating its colonization patterns in and out of Eurasia. Evidence of recent divergence and expansion events at less than 0.5 Ma ago indicate that climate fluctuations in the Mid-Late Pleistocene were important in shaping the current phylogeographic pattern of the species. Data support a first split and differentiation of P. spumarius into two main mitochondrial lineages: the “western”, in the Mediterranean region and the “eastern”, in Anatolia/Caucasus. It also supports a following differentiation of the “western” lineage into two sub-lineages: the “western-Mediterranean”, in Iberia and the “eastern-Mediterranean” in the Balkans. The recent pattern seems to result from postglacial range expansion from Iberia and Caucasus/Anatolia, thus not following one of the four common paradigms. Unexpected patterns of recent gene-flow events between Mediterranean peninsulas, a close relationship between Iberia and North Africa, as well as high levels of genetic diversity being maintained in northern Europe were found. The mitochondrial pattern does not exactly match to the nuclear pattern suggesting that the current biogeographic pattern of P. spumarius may be the result of both secondary admixture and incomplete lineage sorting. The hypothesis of recent colonization of North America from both western and northern Europe is corroborated by our data and probably resulted from accidental human translocations. A probable British origin for the populations of the Azores and New Zealand was revealed, however, for the Azores the distribution of populations in high altitude native forests is somewhat puzzling and may imply a natural colonization of the archipelago. Distribution patterns of animals and plants have faced dramatic changes throughout time and are influenced by ecological requirements and historical factors. In the northern hemisphere, Quaternary long-term glacial (cold) and interglacial (warm) climatic cycles that started about 2.6 million years (Ma) ago Genetic analyses have proven to be useful for a more detailed understanding of post-glacial expansions of several animals and plants Philaenus spumarius   is a widely investigated species, very suitable for genetic and ecological studies. It is a highly polyphagous insect which can be found in a variety of terrestrial plant communities and habitats, being the most common species within the genus PhilaenusP. spumarius have given insights on its evolutionary history suggesting two routes of post-glacial colonization of higher latitudes in Europe and indicating a probable western European origin for North American populations Insects have been widely used as models for animal biogeographical studies e.g., . The meaP. spumarius evolved by (i) providing time estimates of the main demographic and evolutionary events with focus on the populations occurring in the main Mediterranean peninsulas; and, (ii) investigating the colonization patterns out of Eurasia, namely of north-western Africa, North America, and the islands of the Azores and New Zealand.In the present study we tried to explain how and when the current biogeographic pattern of Philaenus spumarius, is considered a widespread species across the Palaearctic and the Nearctic regions, being a crop pest in some locations of USA and Canada. It is not an endangered or protected species.The field sampling was carried out on private lands with owners' permissions. The studied species, P. spumarius were collected or sent by collaborators between 2007 and 2011 from 75 sampling locations across Europe, two from Anatolia, five from North Africa, three from North America and one from New Zealand  was amplified by polymerase chain reaction (PCR) using the primers C1-J-2195 (5′–TTGATTTTTTGGTCATCCAGAAGT–3') and TL2-N-3014 (5′–TCCAATGCACTAATCTGCCATATTA–3') 5'–GTATAGATGTTGATACACGTGC–3') and COI-PspR (5'–TCCAGTAAATAAAGGGTATC–3') was designed to amplify an informative smaller fragment with 300 bp of COI that included the variable sites that differentiate the different haplogroups. Fragments with 500 bp of the mitochondrial genes cytochrome c oxidase subunit II (COII) and cytochrome b (cyt b) were also amplified using the primers TL2-J3033 (5'–GATATGGCAGAAATAGTGCA–3') and C2-N3665 (5'–CCACAAATTTCTGAACACTG–3') and CB-N3665 (5'–GTCCTACCATGAGGTCAAATATC–3') and CB-N11526 (5'–TTCAACTGGTCGTGCTCC–3'), respectively c oxidase subunit I gene was sequenced for all individuals and cyt b and COII were only sequenced in a representative subset. The nuclear gene elongation factor-1α (EF-1α) is widely used in insect genetic studies. Therefore, we chose to sequence it in a subset of individuals that covered all the geographical areas of the study. A 700 bp fragment of the nuclear gene EF-1α was amplified using the primers M3 (5′–CACATYAACATTGTCGTSATYGG–3') and rcM44.9 (5′–CTTGATGAAATCYCTGTGTCC–3') 2, 2.5 µL 5x Colorless GoTaq Flexi Buffer, 0.02U GoTaq DNA Polimerase (Promega) and approximately 30 ng of DNA. The PCR conditions were: an initial denaturation step at 94 °C for 3 min, followed by 35 cycles of denaturation at 94 °C for 30 sec, annealing at 50 °C for 45 sec and extension at 72 °C for 1 min, with a final extension period at 72 °C for 7 min. The same PCR conditions were used for COII and cyt b genes except for annealing temperature where a touch up between 52.5 °C and 56 °C for COII and between 47 °C and 54 °C for cyt b was performed. Nuclear EF-1α gene PCR was performed in a 20 µL reaction volume containing: 0.6 µM of each primer, 0.2 mM dNTPs, 1.125 mM MgCl2, 0.8 µL BSA (10 µg/mL), 4.0 µL 5x Colorless GoTaq Flexi Buffer, 0.05U GoTaq DNA Polimerase (Promega) and approximately 30 ng of DNA. PCR conditions used were: an initial denaturation step at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 45 sec, annealing at 59 °C for 35 sec and extension at 72 °C for 1 min, with a final extension period at 72 °C for 10 min. All PCR products were purified with SureClean (Bioline) following the manufacturer's protocol, sequenced using the forward and the reverse primers with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and analysed on a genetic analyser ABI PRISM 310 (Applied Biosystems).Entire larval stage specimens were used for DNA extraction while in the adults the wings and abdomen were removed and only the thorax and head were used. Genomic DNA was extracted using the E.Z.N.A. Tissue DNA Isolation kit (Omega Bio-Tek) and a 800 bp fragment of the 3'-end of the mitochondrial gene cytochrome equencher v. 4.0.5 (Gene Codes Corporation) and BioEdit v. 7.0.9 afft v. 7.029b  and converted in the appropriate format with Concatenator v. 1.1.0 hampuru v. 1.0 b) and for the nuclear gene EF-1α in Paup v. 4.0.d99 rBayes v. 3.1.2 odeltest v. 3.7 P. spumarius and P. italosignus from Philaenus italosignus was used as outgroup in all phylogenetic analysis. Polymorphic sites and mtDNA haplotypes for COI, COII and cyt b genes were calculated using Mega v. 5.0 etwork v. 4.5.0.1 (Fluxus Technology Ltd. 2004). For COI mtDNA gene, haplotype (h) and nucleotide diversities (π) were calculated for each geographical region  and a piece-wise constant Bayesian skyline tree prior was selected with 10 groups. Two additional analyses of our data with 5 and 15 groups to assess the impact of the number of groups on the final result were conducted. These analyses did not reveal a significant impact on the overall result, whether on the shape of the Bayesian Skyline plot or on the estimation of divergence times. Preliminary runs using the uncorrelated lognormal relaxed clock revealed a posterior distribution of the σr (“CoefficientOfVariation”) parameter consistently abutting 0, suggesting that the COI partitions do not significantly deviate from a strict clock assumption. Therefore, we employed a strict molecular clock for each dataset with a normal prior distribution on the substitution rate with a mean of 0.0354, following the conserved rate of 3.54% per million years as suggested by th iteration, were conducted and combined with LogCombiner v. 1.6.1 racer v. 1.4 We used the software package BDF statistics rlequin. These statistics are widely used with molecular data to detect changes in population size and/or estimating deviations from neutrality, assuming a constant population size at mutation-drift equilibrium. Thus, significant negative values of Tajima's D and Fu's Fs are considered to be evidence of expanding populations. Signatures of population expansion can also be detected through the frequency distribution of the number of pairwise differences between haplotypes and thus statistics based on the mismatch distribution and taking into account the Sudden Expansion Model rlequin and graphics of frequency distribution using DnaSP v. 5 SSD) and Harpending's raggedness index (hg) Neutrality tests of Tajima's rlequin, to time (t) in years using the formula τ  =  2ut, where u is the mutation rate per nucleotide per year multiplied by sequence length (i.e. number of nucleotides), and t is the time since population expansion in years Timing of the demographic expansion as well as the 95% confidence interval for each mitochondrial haplogroup was estimated by converting the expansion time parameter τ, generated by Ab (434bp) genes, 47 individuals were sequenced and a total of 14 (GenBank accession numbers: KF280589 – KF280602) and 18 haplotypes (GenBank accession numbers: KF280603 – KF280620) were found, respectively. As commonly observed for insects A total fragment of 539 bp was obtained for the mitochondrial gene COI in 190 samples. The remaining samples from Finland, Turkey, Canada (GenBank accession numbers: KJ699232–KJ699234) and New Zealand were not included in the main analysis due to their reduced size. In the 190 individuals there were a total of 71 haplotypes (GenBank accession numbers: KC111886 – KC111956) of which 44 occurred only once . Of a toP. spumarius. Almost all sequences exhibited double peaks due to the frameshift resulting from indels (length-variable regions) located at several sites of the intron in the EF-1α gene. Ten individuals were heterozygous in respect to indels and/or to base positions and the phased haplotypes  were differentiated by adding the letter a or b at the end of name . The haplotype H14 belonging to the “western” haplogroup was found in the three New Zealand individuals and this same haplotype was shared with seven individuals from the UK, five from the Azores, three from Canada and four from the USA .clade A, clade B and clade C. However, these groups were not totally congruent to the groups found for the concatenated mtDNA genes. The clade A includes samples from our “eastern-Mediterranean” haplogroup and also from Georgia, Bulgaria, Hungary, Greece and Italy , with the exception for the Azores_1, Italy_2 and Keçan_N1 samples which have one allele in the clade C and the other allele in the clades A or B.In the MP  and BI , except for the Azores (h  =  0.4000) and Slovenia (h  =  0.0000) . Interestingly, Finland and North America also have high haplotype diversity (h  =  0.8971 and h  =  0.8939). Analysing nucleotide diversity, Anatolia, United Kingdom and North America have the highest values . The high values of both haplotype and nucleotide diversities detected in Anatolia and North America are likely a result of the presence of different mitochondrial lineages in these regions. The AMOVA performed for the P. spumarius groupings defined revealed that most of the genetic variation (47% and 43.87%) is explained by genetic differences within populations and not by geographic subdivisions  , that maivisions .The mean ages and 95% highest posterior density (HPD) determined for the TMRCA of mtDNA COI haplogroups are presented in P. spumarius populations was analysed separately for the three COI groups. The distribution of pairwise nucleotide differences (mismatch distribution) showed that the “western-Mediterranean” and the “eastern-Mediterranean” groups exhibited a smooth and unimodal shape while the “eastern” group revealed a slightly bimodal curve , while for the “western-Mediterranean” and the “eastern-Mediterranean” haplogroups it was more recent, at 0.055 Ma ago (0.085–0.021 Ma ago) and 0.058 Ma ago (0.079–0.027 Ma ago), respectively. The spatial expansion for the three mtDNA groups was estimated to be slightly more recent than demographic expansion, at 0.080 Ma ago (0.152–0.035 Ma ago) for the “eastern” group, at 0.056 Ma ago (0.075–0.028 Ma ago) for the “eastern-Mediterranean” and at 0.054 Ma ago (0.074–0.022 Ma ago) for the “western-Mediterranean” group.Philaenus spumariusBiogeographical patterns, divergence time and demographic events in P. spumarius is most likely related to climate changes of the Pleistocene epoch  since this mode of dispersal has already been observed in this species Our time estimates indicate that the evolutionary history of  Balkans . This paThe expansion dates estimated here have wide confidence intervals. However, the lowest boundaries of these dates are about 0.015 Ma ago, suggesting that the demographic and spatial expansion of this species may have occurred earlier than Holocene. Our data and reference P. spumarius but came into contact in some geographic regions  indicating that the north of Europe was colonized by populations that may have survived in several extra-Mediterranean glacial refugia in addition to the “classical” Mediterranean ones Also quite interesting is the uncommon P. spumarius populations and the Iberian Peninsula. This close relationship is also corroborated by the nuclear data. There is evidence that the Strait of Gibraltar has not been an effective barrier to the dispersal, having been, in fact, the route of dispersal for many species from Africa to Europe and vice-versa Cicada barbara, a common cicada in southern Portugal and Spain P. spumarius through Morocco, since this latter can be found in a variety of terrestrial habitats and even at altitudes above 1700m . Although the haplotypes found in samples from Morocco were the same as some of the ones found in the Iberian Peninsula, indicating that they belong to P. spumarius, the analysis of four male genitalia from these populations revealed similarities with P. tesselatusThe presence of the “western-Mediterranean” sub-lineage in Morocco suggests a close relationship between these P. spumarius populations from New Hampshire and adjacent areas of North American exhibit morphological variation in male genitalia features and attributes this variation to hybridization between P. spumarius subspecies from different parts of Europe. Verification of whether there is any correlation between Hamilton's morphological subspecies and haplotype variation would require a parallel investigation of morphology beyond the scope of this work. The colonization of New Zealand and North America was probably recent and resulted from non-intentional anthropogenic introductions. This recent colonization could explain the spittlebug pest status in some locations of the USA and Canada, where P. spumarius reaches high densities, perhaps as result of the lack of competitors and predators Our analyses suggest the presence of at least two mitochondrial lineages in the UK, the “western-Mediterranean” and the “eastern”, and support a British origin for the populations of the Azores and New Zealand, and a multiple origin for the North American populations  and 2. PP. spumarius is one of the most widespread insects in Europe. We successfully provided time estimates of the main demographic and evolutionary events for the populations occurring in the main Mediterranean peninsulas, and in addition interpreted the colonization patterns out of Eurasia, namely of north-western Africa, North America, and the islands of Azores and New Zealand. This combination of a well analysed phylogeographic and demographic pattern with the multiple transcontinental colonization events, some putatively natural, others recent non-intentional anthropogenic introductions, with ecosystem level consequences, make this species well placed for understanding the long term effects of invasive species and their post-invasion evolution.Figure S1Median-joining haplotype network of a set of Philaenus spumarius sampled geographic regions for mitochondrial gene COII (495bp). Size of the circles is in proportion to the number of haplotypes. Branches begin in the centre of the circles and their size is in proportion to the number of mutations.(TIF)Click here for additional data file.Figure S2Median-joining haplotype network of a set of P. spumarius sampled geographic regions for mitochondrial gene cyt b (434bp). Size of the circles is in proportion to the number of haplotypes. Branches begin in the centre of the circles and their size is in proportion to the number of mutations.(TIF)Click here for additional data file.Figure S3Median-joining haplotype network of P. spumarius sampled geographic regions for mitochondrial gene COI (289bp). Size of the circles is in proportion to the number of haplotypes. Branches begin in the centre of the circles and their size is in proportion to the number of mutations.(TIF)Click here for additional data file.Figure S4Mismatch distribution of mtDNA COI haplotypes for each of the three P. spumarius haplogroups. (a) Eastern lineage; (b) Western-Mediterranean sub-lineage and (c) Eastern-Mediterranean sub-lineage. The expected frequency is based on a population growth-decline model, determined using DnaSP and is represented by a continuous line. The observed frequency is represented by a dotted line. Parameter values for the mismatch distribution are given in (TIF)Click here for additional data file.Figure S5Bayesian skyline plots showing the historical demographic trends for each main Philaenus spumarius mtDNA group detected using COI gene. Along the y-axis is the expressed population size estimated in units of Neµ . The y-axis is in a log-scale. Solid lines represent median estimates and blue lines represent the 95% high probability density (HPD) intervals.(TIF)Click here for additional data file.Table S1Analysed samples of Philaenus spumarius with description of the sampling locations and indication of the corresponding mtDNA Cytochrome c oxidase I (COI), Cytochrome c oxidase II (COII), Cytochrome b haplotype/code and Elongation Factor-1α code (EF-1α).(PDF)Click here for additional data file.Table S2Haplotype distribution within P. spumarius geographic regions for mitochondrial gene COI. The total number of haplotypes per geographic region and the total number of individuals per haplotype are also shown. Western Europe: Belgium and France; Italy: Italian peninsula and Sicily.(PDF)Click here for additional data file.Table S3Analyses of molecular variance (AMOVA) among regions of P. spumarius based on COI data.(PDF)Click here for additional data file.Table S4Divergence time estimates in million years (Ma) from the most recent common ancestor of each main Philaenus spumarius mtDNA COI haplogroup estimated using a mean mutation rate of 3.54% per million years as suggested by .(PDF)Click here for additional data file.


Rhododendron ponticum in Ireland are genetically distinct from ancestral populations in Spain and produce flowers which have wider floral tubes. Although Irish populations are spreading and Spanish ones are declining, we found low genetic diversity among individual plants within populations in both regions, and limited between-population gene flow. Wider floral tubes may have evolved in response to novel pollinators in Ireland. Few studies examine invasive species in both their introduced and native habitats, but this approach is needed to understand invasive species evolution and ecology.We demonstrate that invasive populations of  Rhododendron ponticum in its introduced range in Ireland, where it is severely invasive, to determine both genetic and flower width diversity and differentiation. We compared six introduced Irish populations with two populations from R. ponticum's native range in Spain, using amplified fragment length polymorphism and simple sequence repeat genetic markers. We measured flower width, a trait that may affect pollinator visitation, from four Irish and four Spanish populations by measuring both the width at the corolla tip and tube base (nectar holder width). With both genetic markers, populations were differentiated between Ireland and Spain and from each other in both countries. However, populations displayed low genetic diversity (mean Nei's genetic diversity = 0.22), with the largest proportion (76–93 %) of genetic variation contained within, rather than between, populations. Although corolla width was highly variable between individuals within populations, tube width was significantly wider (>0.5 mm) in introduced, compared with native, populations. Our results show that the same species can have genetically distinct populations in both invasive and native regions, and that differences in floral width may occur, possibly in response to ecological sorting processes or local adaptation to pollinator communities.Populations of introduced species in their new environments are expected to differ from native populations, due to processes such as genetic drift, founder effects and local adaptation, which can often result in rapid phenotypic change. Such processes can also lead to changes in the genetic structure of these populations. This study investigated the populations of  Fallopia japonica (Japanese Knotweed) and Eichhornia crassipes (water hyacinth), have been shown to have very low genetic diversity  in expanding introduced populations and compare these with declining native populations. This long-lived woody species was once widely distributed throughout Europe  occurred after R. ponticum was introduced in Britain  quantify genetic diversity both within and among introduced and native populations; (ii) determine genetic differentiation among introduced and ancestral populations; and (iii) quantify floral width (corolla width and tube width) in the introduced and native range. Specifically, we tested the hypothesis that there is genetic and floral width differentiation among populations in Ireland, and between Irish and Spanish populations.We examined genetic and floral morphological diversity within and between populations of R. ponticum within the country, including the west coast , the south-west (County Kerry) and the east (County Dublin). Irish populations were relatively large . Polymerase chain reaction amplification followed , with a precision of 0.01 mm: (i) the width of the corolla at the widest point between the upper wing petals and (ii) the width of the corolla tube at the base. These traits were measured as they represent the extent to which R. ponticum flowers are open to insect pollinators in order to access nectar rewards. Due to logistical constraints, and the fact that these data were collected separately from the leaf material for the population genetic study, only relatively few measurements were taken per population and in only one of the populations  sampled for genetic analysis. Measurements were made in four Irish and four Spanish populations (Table In addition to quantifying genetic variation in native and invasive populations of FIS = 0.1) from the Hardy–Weinberg equilibrium. Statistics of gene diversity were calculated according to P) and Nei's gene diversity (Hj). For the SSR data set, GenAlEx 6.2 (HO) and Nei's expected heterozygosity (HE) were calculated with GenAlEx 6.2, and the average heterozygosity was calculated with PopGene 1.32 . Pairwise genetic distances among populations and their level of significance for both the AFLP and SSR markers were also obtained from the AMOVA (9999 permutations). In addition, a non-hierarchical AMOVA was performed to test population differentiation in Ireland and Spain separately.Euclidean pairwise genetic distances were calculated in GenAlEx 6.2, which allows a common pathway for subsequent statistical analysis for both dominant AFLP markers and codominant SSR markers  was performed in PopGene 1.32 using Nei's genetic distance  to analyn = 5). Analyses were conducted using GMAV5 for Windows . Data were tested for heterogeneity of variances using Cochran's test prior to analysis  and were not transformed. Post-hoc Student–Newman–Keuls (SNK) tests were used to determine which means differed from each other (using the standard threshold of significance α = 0.05).Corolla width and tube width were compared between regions (Ireland and Spain), among populations within regions and among plants within populations, using hierarchical (nested) ANOVA  was similar within both Irish and Spanish populations  and more than half (59–69 %) of loci within populations were found to be polymorphic (Table P < 0.001) but low genetic differentiation was recorded among populations relative to the total (φPT = 0.070), among populations within regions (Ireland and Spain)  and among regions   than among Spanish populations . When individuals were grouped into populations, the UPGMA separated the Spanish population, El Palancar from other populations, and grouped the Las Corzas population with the Irish populations . There were no significant departures from the Hardy–Weinberg equilibrium for any of the markers across populations. All populations had similar percentages of heterozygosity and the effective allele number ranged from 2.00 to 2.92 (Table P < 0.001) and relatively high genetic differentiation among all populations (φPT = 0.243), among populations within regions  and between regions   and no differentiation between Spanish populations . In contrast to the AFLP results, the UPGMA grouped the two Spanish populations with the Irish Glencullen population. Within this group, the Glencullen population grouped most closely with the El Palancar population .A total of 29 alleles were detected within the 83 92 Table . AnalysiφPT = 0.23, among P < 0.05). There were significant differences in tube width between regions, among populations within regions and within populations  or between populations within regions  and invasive introduced (Irish) populations of R. ponticum. However, populations and regions were also genetically differentiated, a trend detected with both AFLP and SSR markers. Such genetic differentiation is expected when genetic drift and inbreeding occur in geographically isolated populations , rather than outcrossing. In fact, observations of the behaviour of pollinators suggest that the levels of geitonogamy (pollen transfer among flowers of the same plant) may be high, since the main pollinators of R. ponticum in Ireland (Bombus spp.) tend to move between flowers on the same plant far more frequently than between flowers on different plants  than invasive Irish ones (which had a mean of six private bands). Spanish populations contained fewer than 30 individual plants possibly due to range contraction and lack of sexual regeneration (The native Spanish Hj), as estimated by AFLPs, and low allelic diversity, as estimated by SSRs. In addition, Irish Glencullen population groups with Spanish populations in both the AFLP and SSR analyses , which reveals the similarity of Irish and Spanish populations. However, the grouping of one of the Irish populations (Glencullen) with the Spanish populations could be due to homoplasy (a similar genetic structure due to convergence): this population also has more private alleles , and other Irish populations may have grouped together as they have fewer private alleles.Our data show that native Spanish populations have probably experienced a recent genetic bottleneck as they have both low overall expected heterozygosity , suggests that the gene flow is limited. This supports findings of R. ponticum, varying between individuals within populations, among populations and between regions, similar to the patterns found for genetic diversity. No previous published studies have described tube width in R. ponticum. However, corolla tubes were, on average, >0.5 mm wider in Irish populations than in Spanish ones. Wider corolla tubes may be associated with increased pollinator visitation rates, because a greater range of pollinating insects can access the nectar from more open flowers with wider tubes. Wider corolla tubes in the introduced range may be an advantage, given that introduced species have to rely on native generalist insects for pollination. Indeed, a range of generalist visitor species were recorded visiting R. ponticum in Ireland, including solitary bees, bumblebees and hoverflies , a Trinity Trust Award, a British Ecological Society Small Ecological Project Grant, a grant from the Percy Sladen Memorial Fund (Linnean Society) and a Science Foundation Ireland Research Frontiers Programme grant (10/RFP/EOB2842). K.J.D. is funded by the University of KwaZulu-Natal Postdoctoral fellowship.This work was funded by an The lead author designed the study, obtained the funding, carried out fieldwork, laboratory work, data analysis and writing; all other authors contributed to study design, fieldwork, laboratory work, data analysis and writing.None declared.Supporting Information is available in the online version of this article –The following File S1. 28 unique genotypes from 61 R. ponticum individuals in Irish populations identified by 4 SSR markers.


Through the PC analysis, HO × JE and HO × BS crossbreeding was detected in the Lowi system. The Conv system appeared to be in between Lowi and CAN + USA populations. Admixture analysis differentiated between the genetic composition of the Conv and Lowi systems, and five ancestry groups associated to sire’s country of origin were identified. The minimum distance between markers to estimate a useful LD was found to be 54.5 kb for the Mexican HO populations. At this average distance, the persistence of phase across autosomes of Conv and Lowi systems was 0.94, for Conv and CAN + USA was 0.92 and for the Lowi and CAN + USA was 0.91. Results supported the flow of germplasm among populations being Conv a source for Lowi, and dependent on migration from CAN + USA. Mexican HO cattle in Conv and Lowi populations share common ancestry with CAN + USA but have different genetic signatures.The Mexican Holstein (HO) industry has imported Canadian and US (CAN + USA) HO germplasm for use in two different production systems, the conventional (Conv) and the low income (Lowi) system. The objective of this work was to study the genetic composition and differentiation of the Mexican HO cattle, considering the production system in which they perform and their relationship with the Canadian and US HO populations. The analysis included information from 149, 303, and 173 unrelated or with unknown pedigree HO animals from the Conv, Lowi, and CAN + USA populations, respectively. Canadian and US Jersey (JE) and Brown Swiss (BS) genotypes  were used to determine if Mexican HOs were hybridized with either of these breeds. After quality control filtering, a total of 6,617 out of 6,836 single nucleotide polymorphism markers were used. To describe the genetic diversity across the populations, principal component (PC), admixture composition, and linkage disequilibrium (LD;  Dairy farms in Mexico are extremely heterogeneous . Conventional (Conv) dairy farms have an average herd size of 230 head and are highly mechanized and milk yield is relatively high. Cows on these farms are typically grouped in pens and rations usually include high proportions of concentrates. Low income (Lowi) systems vary with region with sizes ranging from 3 to 30 cows, with animals usually spending part of the day grazing, but may also be housed in pens . AdditioBefore single nucleotide polymorphism (SNP) data became available, genetic diversity and relatedness was studied through the analysis of pedigree information which, unfortunately, is not always complete. This incomplete data renders results inaccurate or limits the performance of studies of individuals or populations . CurrentPrincipal component analysis was first used in human populations to generate maps summarizing the allele frequency of different geographic areas  and moreAdmixture is defined as the mixing of genomes of divergent parental origins , which iLinkage disequilibrium is defined as a non-random association of alleles at different loci  because To establish genomic evaluations in Mexico, it is important to determine whether multiple HO subpopulations exist. This information determines whether it is necessary to stratify the population by production system and if foreign genomic information, especially from Canada and US, would improve accuracy of predicted breeding values. Thus, the objective of this work was to study the genetic composition and differentiation of the Mexican HO population and the relationship between Mexican cattle with those from Canada and the US. The primary source of stratification of the Mexican population considered was the production system in which those cows performed.Table 1 shows the number and frequency of markers per chromosome included in the analysis.A total of 625 HO and HO like unrelated cows and sires, born on or after 2005 and genotyped with the Illumina BovineSNP50 or BovineLD Bead Chips were used in this analysis. A total of 149 and 303 animals assigned to the Conv and Lowi systems, respectively, and 173 were from the Canadian and US HO populations. In addition, 162 Jersey (JE) and 86 Brown Swiss (BS) sires from Canada and US were included to help determine if crossbred cows were present in the populations. The animals from the Conv system originated from 16 herds in 6 states of Mexico  while the animals of the Lowi system were from 21 herds in 4 states . From the 6,836 common SNP markers in both the Illumina BovineSNP50 and the BovineLD Bead Chips a total of 6,617 SNP were included in the analysis after quality control. Markers with a minor allele frequency less than 2% and call rate less than 90% were excluded. Individuals with a call rate less than 90% were also excluded. The PCA proposed by ik for each individual i. Only Mexican HO animals were used for this analysis. Once the K ancestral populations were determined, the most common sire’s country of origin of each ancestral population was identified based on pedigree information.The ADMIXTURE package  was used2 for alleles at two loci was calculated as:The LD, measured as rD is the difference between the observed and the expected frequency of two loci, based on population allele frequencies and assuming random assortment and can be estimated directly from the allele frequencies , persistence phase of LD was calculated using SAS 9.2 , despite many common ancestors across these groups. The Conv system seems to be intermediate between the Lowi and CAN + USA groups. Neither Mexican system demonstrated a clustering of animals by country of origin of the sire. PCs for all North American HO cattle along with the JE and BS cattle are shown in Figure 2, where the individuals were color coded by breed and population of origin. Crossbred individuals derived from HO and JE or BS were represented by points located between those pure breeds. Crossbreeding was rare in the Conv herds but was much more common in the Lowi systems with a higher proportion of crossing to JE influenced animals than BS.In this study, the first three of 625 components of PCA explained 13% of the observed variation. Population differentiation is observed between the Mexican Lowi and Conv subgroups and the Canadian and US HO cattle  correlated to the sire’s country of origin. Two of the ADMIXTURE strata were directly related to sires originating from Canada and were subsequently combined into group A with the purpose of investigating country of origin influence. Group B consisted of sires most commonly from the United States (USA). The third strata, group C, consisted of sires from both Canada and US. No unique lineages or discernable characteristics were associated with group C. Another stratum was associated to sires registered in the Mexican herd book and assigned as group D. The last stratum included crossbred animals as evidenced by their genetic similarity to BS and JE breeds in the PC analysis and were assigned to group E. Average ancestry contributions, both overall and by production system, can be observed in Figure 4. Population structure showed differences in the genetic composition of the Mexican HO production systems where group C had the largest contribution to the Conv and Lowi populations at 37 and 40%, respectively. In the Conv population, group A (24%), D (19%), B (17%) and E (2.8%) followed while for Lowi animals, group C was followed by D (19%), B (14%), A (14%) and the E (13%). Overall, the contributions of the US, Canada/US, and the Mexican sires were relatively similar among the production systems. The primary differences were within the Canadian and crossbred lineages. The Conv population had approximately 1.7 fold greater contribution from Canadian lineage  and the Lowi population had ∼4.3 fold increase in crossbred lineages .To identify the genetic structure of the Mexican HO systems and its dependency on the Canadian and US populations, an admixture analysis was performed first for the entire Mexican population and then for each sub-population. For both analyses, the value that best explained the stratification of the population was K = 6. Pedigree records of individual Mexican HO identified their sire’s country of origin as Canadian, US, or Mexican, hence providing an indirect measure of the genetic contribution of these countries to Mexican HO. Indeed, the ADMIXTURE strata identified at K = 6  between SNP were calculated for the Conv, Lowi, and CAN + USA populations . At all distances, average r2 was highest for CAN + USA animals, intermediate for individuals from the Mexican Conv farms, and smallest for cattle representing the Lowi systems. The differences between the CAN + USA and Conv populations r2 were quite small (∼0.01) while differences between Conv and Lowi were larger and consistent, ranging from 0.03 to 0.04. The persistence of LD phase between Conv, Lowi, and CAN + USA populations was calculated at the same interval distances as was LD . As expected, the persistence of LD phase decreased when the distance between markers was increased. At all intervals, the highest correlations were between Conv and Lowi populations, with the lowest correlation being between Lowi and CAN + USA. At a distance of <100 Kb (with an average of 54.5 kb), the correlations ranged from 0.91 to 0.94 and for a distance >500 kb correlations varied from 0.75 to 0.81 between the Conv-Lowi and Lowi-CAN + USA, respectively.Mean LD, calculated as rThe results obtained in this study, provide important details to be considered for future genetic research on the Mexican HO cattle and provided the opportunity to measure the genetic relationship of the Mexican animals with the HO populations of Canada and the US.The Conv system seems to be intermediate in most aspects between the Lowi and CAN + USA, and this population may act as a conduit for germplasm flow from Canada and the US to Mexico.When the PCA in the HO breed was explored, a difference in the genetic structure was observed between the Mexican production systems and the CAN + USA population, although population overlap suggests that all three groups share common genetic material. In this case the Conv system seems to be a link between the Lowi system and CAN + USA population because breeders of the Conv system provide genetic material (heifers and semen) to the Lowi one and the Conv system has depended genetically from US and Canada for many years . The LowThe inclusion of JE and BS resulted in individual clusters for both breeds as expected, but more importantly, they helped define the differences between the Lowi and the Conv populations. Results suggest that breeders of the Lowi system occasionally used genetic material of other breeds.Admixture analysis showed that five different populations, linked to origin country of the sires, comprise the ancestral background of the Mexican HO populations. The two systems showed variation in the average proportion of genetic similarity to the different ancestral populations. The results show a substantial influence of the North American HOs on the Mexican population, and agree with previous results in the Conv system reported by Figure 5; 2 estimates for the populations in this study were in the range of those reported in other HO populations (2 averages for the CAN + USA are slightly higher than those reported for the same population in a group of animals born after 1990 at similar distances between markers (2 values among the populations were for the Lowi. These lower values may be the result of breeders in the Lowi system introducing other breeds through crossbreeding and incorporating gene migration and genetic drift. This practice may explain the reduction in LD (2 averages for the Lowi decreased rapidly when the distance increased from <100 to 100 and <200 Kb then decreased slowly at distances >200 Kb.In line with previous studies, decay of LD was observed in this study when the distance between markers was increased (ulations , althoug markers , except on in LD . The r2 2 ≥ 0.20; A practical application of LD is determining the number of markers necessary to perform genome wide associations studies  at a useFigure 6 confirm the results of PCA, because the higher persistence phase was reported between the Conv and Lowi, followed by the one between Conv and CAN + USA ending with confirmation of the lowest relationship between Lowi and CAN + USA. As it was also reported in other studies (r among all populations decreased rapidly when the distance between markers increased. The difference between persistence phases among the Conv, Lowi, and CAN + USA ranged from 0.01 to 0.04, lower than that present in other breeds like Angus, Charolais, and JE (Because the persistence phase or correlation of r among populations show the genetic relationship between them , it was  studies  the corr, and JE  or speci, and JE . At dist, and JE  and at a, and JE .Results showed that the US and Canadian and the Mexican HO cattle of the Conv and Lowi have different genetic structures although these populations share much common ancestry. The main difference between the Mexican HO systems is the result of crossbreeding with other breeds, especially in the Lowi system. If joint genomic studies are to be performed between these populations, stratification of populations is recommended. Joint genetic improvement programs of HO animals across North America, i.e., including Mexico, may be established as these populations share genetic material. The useful LD founded in this populations, will determine the minimum number of SNP markers need if joint genomic studies are to be performed.The considerable similarity between the Conv subgroup with US and Canadian populations means that integration of these groups would be straightforward and should be considered.The authors have made the following declarations about their contributions: Conceived and designed the experiments Adriana García-Ruiz, Felipe de J. Ruiz-López, Curtis P. Van Tassell, and Hugo H. Montaldo. Performed experiments and analyze data: Adriana García-Ruiz Data acquisition and interpretation: Adriana García-Ruiz, Felipe de J. Ruiz-López, Curtis P. Van Tassell, Hugo H. Montaldo, and Heather J. Huson. Wrote the paper: Adriana García-Ruiz. All authors approve the manuscript final version.The Review Editor Ikhide G. Imumorin declares that, despite being affiliated with the same institute as the author Heather J. Huson, the review process was handled objectively. 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.


In sexually reproducing organisms, meiotic crossovers ensure the proper segregation of chromosomes and contribute to genetic diversity by shuffling allelic combinations. Such genetic reassortment is exploited in breeding to combine favorable alleles, and in genetic research to identify genetic factors underlying traits of interest via linkage or association-based approaches. Crossover numbers and distributions along chromosomes vary between species, but little is known about their intraspecies variation.Here, we report on the variation of recombination rates between 22 European maize inbred lines that belong to the Dent and Flint gene pools. We genotype 23 doubled-haploid populations derived from crosses between these lines with a 50 k-SNP array and construct high-density genetic maps, showing good correspondence with the maize B73 genome sequence assembly. By aligning each genetic map to the B73 sequence, we obtain the recombination rates along chromosomes specific to each population. We identify significant differences in recombination rates at the genome-wide, chromosome, and intrachromosomal levels between populations, as well as significant variation for genome-wide recombination rates among maize lines. Crossover interference analysis using a two-pathway modeling framework reveals a negative association between recombination rate and interference strength.To our knowledge, the present work provides the most comprehensive study on intraspecific variation of recombination rates and crossover interference strength in eukaryotes. Differences found in recombination rates will allow for selection of high or low recombining lines in crossing programs. Our methodology should pave the way for precise identification of genes controlling recombination rates in maize and other organisms. Saccharomyces cerevisiae[Solanum lycopersicum[Arabidopsis thaliana[Mus musculus[ZMM family (hereafter referred to as P1), and which are subject to interference [Mus81 gene, and in which there is little or no interference [In sexually reproducing organisms, crossovers (COs) stabilize the pairing of homologous chromosomes during meiosis and ensure their correct segregation. By reciprocal exchange of parental genetic material, the COs also lead to new allelic combinations and thus play an important role in creating genetic diversity. Meiotic recombination occurs during prophase I of meiosis, when DNA double-strand breaks (DSBs) catalyzed by the topo-isomerase-related enzyme SPO11 . In orgaerevisiae,13, Solaopersicum, Arabido thaliana, and Mus musculus. In planrference ,17. The rference .cis and trans genetic factors. In particular, the allelic diversity in the zinc finger domain of PRDM9, a DNA-binding protein, has been shown to influence recombination hot-spot activity [The meiotic recombination rate is known to vary among species ,20. In mactivity ,22. In pactivity . Howeveractivity  and genoactivity , since rZea mays L.) has been the subject of genetic studies for more than a hundred years [® MaizeSNP50 genotyping array comprising around 50,000 SNPs was developed and the first high-density genetic maps of maize were published using two intermated recombinant inbred line populations [Maize  GWRR and chromosome-wide recombination rate, (2) recombination landscape along chromosomes, and (3) CO interference, using high-density genetic linkage maps. We found significant differences of GWRR between individual populations as well as between Dent × Dent versus Flint × Flint populations, but not between the Dent and Flint gene pools in general. GWRR was not correlated with the genetic structure of the lines as inferred from admixture analysis. We analyzed the recombination landscapes in all populations and found significant differences between individual populations and between pools. Finally, we characterized quantitatively the interfering and non-interfering CO formation pathways and found a negative correlation over all chromosomes between interference intensity of pathway P1 and GWRR.Here, we report on the analysis of the variation of recombination rate in 23 populations of maize using SNPs from the MaizeSNP50 BeadChip . All popnduction . DH line1 plants for in vivo haploid induction, so our maps reflect only female meioses. Details on the diversity analysis of the parental lines are given in Additional file We constructed two half-sib panels comprising a total of 24 maize full-sib families of DH lines for the analysis of intraspecific variation of recombination rates. The full-sib families within the two panels represent the diversity of important founder lines of the European Dent and Flint germplasm, respectively Table . In the As a first step in the analysis of recombination, individual genetic maps were constructed for 23 out of the 24 populations and each of the 10 chromosomes. Map statistics are given in Table ga) genes are located  was a cThe average GWRR can be expressed as the ratio of total genetic map length in centiMorgans divided by the genome size in megabase pairs. The genome size of maize is approximately 2.5 Gb, of which 2.1 Gb are sequenced and assembled in the B73 AGPv2 reference sequence. We used the 2.1 Gb B73 AGPv2 assembly as the basis of our calculations. Similarly, the average recombination rate of one chromosome is the ratio between the genetic map length in centiMorgans and the physical length in megabase pairs of this chromosome. The physical lengths of the 10 maize chromosomes in the B73 AGPv2 assembly are between 301 Mbp for chromosome 1 and 150 Mbp for chromosome 10. Genetic map lengths were measured for each population based on the same physical positions of extremities, correcting for particular regions  where mapping information was not available or not reliable. The average GWRR over all populations was 0.74 ± 0.09 cM/Mbp  observed between the GWRR in a population and the average 'flintness' of its parents , indicating that in the founder lines studied here, no general difference in GWRR can be observed between Dent and Flint inbred lines. We conclude that the broad range of variation observed in Figure Nevertheless, we observed large differences in the GWRR of the two crosses of both central lines with B73 . The GWRR in CFD02 was 0.642 cM/Mbp, whereas in CFF02 the GWRR was 0.811 cM/Mbp. We thus asked whether the significantly higher GWRR in the pooled Flint × Flint populations might follow from differences in the central parents F353 and UH007. To test this, we took an additive model whereby the genetic length of a genetic map is the average of effects contributed by each of its parental lines. These parent-specific effects were determined using the fact that the line B73 was crossed to both central lines , thus connecting the two half-sib panels. Then we tested for a correlation between founder line effects on GWRR and their 'flintness'. As shown in Figure In addition to GWRR, we analyzed the patterns of recombination rate along the chromosomes, hereafter referred to as recombination landscapes, and compared them for each pairwise combination of linkage maps and between Dent and Flint pools. We wanted to test whether there were intraspecific differences for recombination landscapes given the large intraspecific diversity in maize and the fact that we observe a clear differentiation between lines in genetic diversity analyses . Significant local differences in the shape of the curves between the pooled Dent and the pooled Flint populations were found, for example, in the centromeric bin 5 and in bin 8 on chromosome 2 or in bin 6 on chromosome 6  the proportion p of COs formed through the non-interfering pathway, hereafter referred to as P2. The parameters nu and p were estimated for each chromosome and each population. These parameters greatly varied among chromosomes and among populations, with values of nu ranging from 2 to 50, and p ranging from 0 to 0.4. Since the population sizes are modest, part of this variation is expected to be due to statistical noise. We thus focused on comparing pooled data, either over chromosomes or over populations.Crossover interference leads to a more regular spacing between adjacent COs than in the absence of interference, where COs are positioned randomly. Interference also affects the distribution of CO numbers per gamete by reducing its variance. In our data, the observed distributions for CO numbers and inter-CO distances departed from the ones expected without interference, allowing us to reject the hypothesis that there is no interference. We also characterized interference quantitatively by estimating the two parameters of the Gamma-sprinkling model : (1) thenu and p when pooling all chromosomes together for each population and for the pooled Dent × Dent and Flint × Flint populations. Confidence intervals for estimates of nu and p from individual populations were large as expected given the population sizes (data not shown). Statistical tests showed that differences between individual populations were not significant at the 5% threshold level - hardly any pairs of individual populations had significantly different values for nu  and 20 (for nu = 8). At 10 cM from the first CO, this reduction factor ranges between 5 (for nu = 2) and 46,000 (for nu = 8). For nu > 3 there is almost no chance to find two P1 COs separated by less than 10 cM. Interference intensity in P1 (nu) was significantly negatively correlated with GWRR for chromosome 7. For other chromosomes, this correlation was not significant, but it was when pooling all chromosomes together .Figure in vivo haploid induction, which reflect a single female meiosis. As expected, the average number of crossovers per DH line in our study (15.1) was about half the number observed in maize RILs (28.9) [Recombination and reassortment of parental genomes lead to an increase of genetic variation. Understanding the mechanisms regulating recombination rate during meiosis would allow their manipulation to increase or decrease recombination rates according to specific requirements described in . Althougs (28.9) . This drAs a prerequisite for our approach, we constructed high-density genetic maps for a large number of populations. Genetic map lengths of the 23 populations varied from 1,180 cM to 1,893 cM with a mean of 1,508 cM, which is in the range observed in other high-density genetic maps of maize ,33. Due A. thaliana (3.6 cM/Mb) [r = 0.66, P value 0.003), similar to what arises in human, mouse and rat [The average GWRR in our populations was 0.73 cM/Mbp, which is in the same range as can be calculated from other maize maps . This va6 cM/Mb)  which ha6 cM/Mb) . We foun and rat . Such coTrans-acting QTL affecting GWRR were identified in A. thaliana, maize, mouse, and wheat [REC8 , and RNF212, a putative homolog of the yeast ZIP3 gene, which is involved in meiotic recombination. RNF212 is also known to be associated with genome-wide recombination in humans [Calculating GWRR assumes constant genome size in maize. Genome size differences were reported in maize, with temperate maize lines having up to 10% smaller genomes relative to B73 and these differences were correlated with the number of knob repeats . Genome nd wheat . In cattnd wheat . For twon humans . Our finn humans . Given tbz1 locus by a factor two [A. thaliana[Medicago truncatula[Not only GWRR but also the landscapes of recombination along chromosomes are highly variable in our populations. We observed characteristic shapes of Marey map curves for each of the 10 maize chromosomes. The overall recombination profiles for each chromosome tend to follow gene density  but thisctor two . For thector two . The extctor two , but evector two ,51,52. Sctor two , althougctor two . Apart fctor two ,55. Due ctor two  it is li thaliana. Thus, fruncatula. In addiruncatula. Our stup of non-interfering crossovers. In the present study, we also found in most cases values of p significantly different from zero, with values averaging 0.1. This conclusion is compatible with the previous estimations that reported an average value of 0.15 [p = 0  may mostly reflect a low power due to limited population sizes: here, individual populations have between 50 and 134 DH lines, whereas in [p of non-interfering (P2) crossovers significantly higher than almost all other populations when considering all chromosomes pooled. To our knowledge, differences in interference features between different genotypes of the same species have not been shown so far. Values of p between 0 and 0.2 for different chromosomes were reported in A. thaliana[p values around 0.3 were found in tomato [nu of interference in the interfering pathway P1, our results indicated values ranging between 2.5 and 8 for all chromosomes pooled, which is similar to the range 4 to 10 found previously in maize [Arabidopsis, nu was in the 10 to 21 range [nu was estimated to be in the 6.9 to 7.9 range in tomato [nu and the genome-wide recombination rate. This result is consistent with the hypothesis that interference may be one of the mechanisms at work to regulate the level of meiotic recombination, biasing the repair of DSBs towards non-COs rather than COs. Compared with the highly significant differences found for GWRR in our study, the variation for CO interference is detected here with much less statistical power. In future works with higher population sizes, providing smaller confidence intervals, but using the same half-sib design it should be possible to estimate the effects of the founder parents alone on interference parameters by removing the effects of the central lines, as we did for GWRR. Similarly as for GWRR, this should also enable the identification and localization of genetic factors influencing interference parameters.Interference was previously shown to occur in maize , based o of 0.15 . The popereas in , the dat thaliana, and bet thaliana. Based on tomato . Considein maize . In Arab21 range , whereasn tomato . FinallyCovering the whole genome using dense genetic maps increases the chance to detect marker-trait associations, both in linkage analyses and association mapping. The construction of high-density genetic maps is now feasible for many crop species in a very cost-efficient way, either by SNP genotyping arrays or through genotyping-by-sequencing approaches ,58. For Meiosis and recombination fundamentally influence genome structure and evolution of species. Although key components of the meiotic pathway are known in plants, further research is needed to better understand the interplay of genetic factors controlling recombination, the role and mechanism of CO interference and the influence of chromatin features such as methylation and structural variation on recombination in plants. Recent advances in high-throughput genotyping and next-generation sequencing offer new opportunities to analyze at a large scale the genome-wide distribution of COs and to characterize sequence motifs around CO hotspots. The present study revealed large intraspecific variation for GWRR and recombination landscapes in maize. Our findings pave the way for the selection of proper crossing parents to analyze genetic factors influencing GWRR and local recombination rates. Finally, using appropriate experimental designs, QTL or association studies will enable the identification of QTL for GWRR.1 plants. The resulting 24 DH populations consisted of 35 to 129 lines  procedure. DNA samples were adjusted to 50 to 70 ng/μl and 200 ng per sample were used for genotyping. DH line purity and integrity was first checked using a custom 96plex VeraCode assay  with genome-wide SNP markers to ensure that the lines carried only one of the parental alleles at each SNP, that they did not carry alleles of the inducer line and that they were derived from true F1 plants. For a subset of DH lines, 13 proprietary SNP markers assayed with the KASP™ technology  were used for testing line purity and integrity. True DH lines were then used for genotyping with the Illumina® MaizeSNP50 BeadChip [® iScan platform. Array hybridization and raw data processing were performed according to manufacturer’s instructions (Illumina®). Raw data were analyzed in Illumina®’s Genome Studio software version v2011 (Illumina®) using an improved version of the public cluster file . SNP da0_B.egt, .Genetic diversity between parental lines was assessed with genome-wide SNP markers by principal coordinate analysis, cluster analysis, and by a pairwise genome scan for polymorphism between the parents of each population. For details, see Additional file Genetic maps were constructed for each individual population as described earlier  using Ca®, San Diego, CA, USA), are based on the B73 AGPv1 assembly with many markers lacking a chromosome and/or position information. We therefore performed a new mapping of the SNPs on the B73 AGPv2 assembly [Chromosome and position assignments of SNPs of the MaizeSNP50 BeadChip supplied by the manufacturer  for any physical position of the B73 assembly. Note that this procedure did not distinguish the regions where these recombination rates were estimated reliably from those where they were not (unmasked versus masked regions). For comparison tests, it was thus necessary to resort to imputation, that is, to infer missing data from other maps in a conservative way.To compare the genetic lengths and the recombination landscapes between two different populations or pools of populations, we used 'imputed' Marey maps where the information missing in the masked Marey map of either population was replaced by the information available in the other population. If a region had masked data in both populations, its content was imputed using the averaged data of all other populations. In all cases, the imputation procedure was designed so that missing or unreliable data in either population to be compared never induced artificial differences. The detailed procedure used for imputation is explained in Additional file For any given population, mapping data led to an estimate of the genetic length for a given chromosome. We examined pairwise differences in genetic length between populations as well as differences between pools of populations and tested them for their level of statistical significance. Such comparisons were performed from the imputed Marey maps with a significance threshold of 5%, using the welch.test function in the R software and the conservative Bonferroni correction for multiple testing. The origin of the information  was taken into account when comparing populations or pools of populations. For details see Additional file 2 specifies what fraction of the variance in the GWRR is explained by the group structure of the parental lines. The function lm also provides the P value for testing the absence of correlation.Genetic map lengths tended to be longer in populations involving Flint parents, suggesting that some alleles of factors controlling recombination rate may be differentially fixed in the two pools. To use a solid and objective measure of degree of 'flintness', we estimated the probability of the 22 parental lines to belong to one of the two main groups (Dent or Flint). To do this, we estimated admixture in a combined analysis of the Dent and Flint diversity panels described earlier , which iThe 23 genetic map lengths we estimated showed a clear positive correlation with the average 'flintness' of the parents in the crosses. However, the two central lines could be driving this correlation. To remove effects coming from the two central lines, we considered an additive model whereby the GWRR of a population produced by a cross is given by the average of two effects, one from each parent in the cross. For each founder line except for B73, there is a single cross in which it is involved. We took the GWRR of that cross and subtracted the GWRR of the cross involving the same central line and B73. This difference gives the individual additive effect of the founder line minus that of B73 up to a constant. This constant does not affect a putative correlation between individual-specific 'flintness' and GWRR. We performed the statistical test for significance of this correlation using the same procedures as in the previous section, central lines and B73 omitted.Just as the genetic length of chromosome maps may differ, two recombination landscapes can have different features (different shapes of the Marey maps). To test whether these differences were statistically significant, we normalized the genetic lengths of the two maps or pools of maps to be compared, by rescaling both of them to their mean value. Then, to compare the shape of both normalized Marey maps, our approach was based on binning the landscapes, representing each as a histogram and then applying a chi-squared test with a conservative Bonferroni-corrected significance threshold of 5%  with significance thresholds of 5%, using variances estimated from the Fisher information matrix obtained at the optimal values of the parameters.To analyze interference in a pool of maps instead of an individual map, we used a similar maximum-likelihood approach, but the likelihood to be maximized was the product of likelihoods calculated for the individual maps. To test for differences between values of cM: CentiMorgan; CO: Crossover; DH: Doubled haploid; DSB: Double-strand break; GWRR: Genome-wide recombination rate; IBD: Identity-by-descent; NAM: Nested association mapping; NOR: Nucleolus organizer region; QTL: Quantitative trait loci; SNP: Single nucleotide polymorphism.The authors declare that they have no competing interests.TA, AC, PF, AEM, MM, JMG, MO, PR, and CCS designed the project. TA, CB, CC, LC, AC, MF, PF, JMG, OCM, AEM, MM, MO, NR, PR, and WS developed the populations or contributed reagents or software tools. EB, CB, CC, PF, NM, NR, WS, and HW performed the experiments. EB, MF, OCM, and CCS designed the data analysis. EB, MF, OCM, RR, and HW analyzed the data. EB, MF, OCM, and CCS wrote the paper. All authors read and approved the manuscript.Maize lines used in this study, assignment to gene pools and origin of the lines.Click here for fileSupplementary results.Click here for fileChromosome-wide genetic lengths and recombination rates for all genetic maps.Click here for fileDetails of all genetic maps, including raw segregation data.Click here for file(A) in all Dent populations and (B) in all Flint populations. The 10 chromosomes are represented along the same horizontal axis. The expected frequency of 0.5 is indicated by a dotted line, and surrounded by solid lines representing its 99% confidence intervals. The x-axis indicates physical coordinates in megabase pairs along the B73 genome. In the bottom of the figure, the heat map represents gene density (low for cold colors and high for hot colors). Gray horizontal line below the heat-map: sketch of the chromosome organization showing centromeres (cen), knobs, nucleolar organizer region (NOR), and known gametophytic factors (gax)  (from ). Color Click here for fileP ≥ 5.10-2, 10-3 ≤ P < 10-2, 10-4 ≤ P < 10-3, 10-5 ≤ P < 10-4, P < 10-5 where P is the P value of the pairwise comparison test, corrected for multiple testing (Bonferroni). Arrows pointing to the right (respectively to the bottom) indicate that the cross listed in the vertical axis  has a higher recombination rate than the cross listed in the horizontal axis . Dendrograms indicate hierarchical clustering of -log10 based on Euclidian distances, and were used to order the populations.Statistical comparison of chromsome-wide recombination rates between the 23 populations of the experiment. Chromosome 'All' (first page) corresponds to the genome-wide analysis. 'DentAll', 'FlintAll', and 'All' correspond, respectively, to pooled analyses of all Dent × Dent populations, all Flint × Flint populations, and all 23 populations together. Dark blue, light blue, green, yellow, and red correspond respectively to Click here for fileMarey maps and recombination landscapes along the chromosomes for three examples illustrating the imputation of regions with missing or unreliable data: CFD01 chromosome 1, CFF07 chromosome 1, and CFF01 chromosome 3. The x-axis indicates physical position of the SNPs on the B73 physical map in megabase pairs. The left y-axis indicates genetic map position in centiMorgans. The right y-axis indicates recombination rate in cM/Mbp. Each black empty circle corresponds to a SNP. Red dots indicate the outlier markers removed from the smoothing analysis. Dark blue dotted line: smoothed Marey map. Red dotted line: first derivative of the smoothed Marey map. Hatched rectangles: regions masked when going from bare to masked Marey maps . Light blue solid line: imputed smoothed Marey map obtained after imputation in the excluded regions, using data from all maps pooled. Pink solid curve: recombination rate computed as the first derivative of the imputed smoothed Marey map.Click here for fileDetailed methods.Click here for fileRecombination rates along maize chromosomes 1 to 10. The x-axis indicates physical position (Mbp) along chromosomes. The y-axis indicates genetic position , recombination rate , and pairwise parental similarity  for 6 of the 23 populations, after smoothing and imputation . Blue lines: Flint × Flint crosses. Red lines: Dent × Dent crosses. In both groups, the solid, dashed and dotted lines correspond to the population with the highest, median or lowest genome-wide recombination rate within its group, respectively. Gray parts of the lines correspond to regions where the information was missing or not reliable , and thus imputed from the other maps . Heat-maps below the curves of recombination rates indicate gene density (low for cold colors and high for hot colors). Gray horizontal line below the heat-map: sketch of the chromosome organization (from ) showingClick here for fileP ≥ 5.10-2, 10-3 ≤ P < 10-2, 10-4 ≤ P < 10-3, 10-5 ≤ P < 10-4, P < 10-5 where P is the P value of the pairwise comparison test, corrected for multiple testing (Bonferroni). Dendrograms indicate hierarchical clustering of -log10 based on Euclidian distances, and were used to order the populations.Statistical comparison of genome-wide recombination landscapes between the 23 populations for all chromosomes pooled as well as for each chromosome. For each pairwise comparison test, both genetic map lengths were normalized to their average value, so the test is not affected by differences in the global recombination rate but only by differences in the shape of the recombination landscape. 'DentAll', 'FlintAll', and 'All' correspond, respectively, to pooled analyses of all Dent × Dent populations, all Flint × Flint populations, and all 23 populations together. Dark blue, light blue, green, yellow, and red correspond respectively to Click here for fileIllustration of the statistical test used to compare recombination landscapes between the pooled data of all Dent × Dent (red) and Flint × Flint (blue) populations. Within regions excluded from the analysis for one population, the data were imputed from the other pool for conservativeness of the test  between individual populations, for all chromosomes pooled together. 'DentAll', 'FlintAll', and 'All' correspond, respectively, to pooled analyses of all Dent × Dent populations, all Flint × Flint populations, and all 23 populations together. Dark blue, light blue, green, yellow, and red correspond respectively to P ≥ 5.10-2, 10-3 ≤ P < 10-2, 10-4 ≤ P < 10-3, 10-5 ≤ P < 10-4, P < 10-5 where P is the P value of the pairwise comparison test, corrected for multiple testing (Bonferroni). Arrows pointing to the right (respectively to the bottom) indicate that the cross listed in the vertical axis  has a higher value of nu than the cross listed in the horizontal axis . Dendrograms indicate hierarchical clustering of -log10 based on Euclidian distances, and were used to order the populations.Statistical comparisons of interference intensity in pathway P1 (Click here for filep) of crossovers formed via the non-interfering pathway P2, for all chromosomes pooled together. 'DentAll', 'FlintAll', and 'All' correspond, respectively, to pooled analyses of all Dent × Dent populations, all Flint × Flint populations, and all 23 populations together. Dark blue, light blue, green, yellow, and red correspond respectively to P ≥ 5.10-2, 10-3 ≤ P < 10-2, 10-4 ≤ P < 10-3, 10-5 ≤ P < 10-4, P < 10-5 where P is the P value of the pairwise comparison test, corrected for multiple testing (Bonferroni). Arrows pointing to the right (respectively to the bottom) indicate that the cross listed in the vertical axis  has a higher value of p than the cross listed in the horizontal axis . Dendrograms indicate hierarchical clustering of -log10 based on Euclidian distances, and were used to order the populations.Statistical comparisons between individual populations, of the fraction (Click here for filenu) and genome-wide recombination rate for the 10 chromosomes pooled together over all populations.Correlation between interference intensity in pathway P1  connect the panels via the central lines F353 and UH007. Both panels are also connected by crossing the central parent to B73 . The panels consist of 11 CFD and 13 CFF full-sib families, respectively.Crossing scheme of the two Dent and Flint half-sib panels. In each panel, a central line was crossed to diverse founder lines and DH lines were developed from the resulting FClick here for file


Culicoides imicola Kieffer, 1913 is the main vector of bluetongue virus (BTV) and African horse sickness virus (AHSV) in Sub-Saharan Africa. Understanding the population genetic structure of this midge and the nature of barriers to gene flow will lead to a deeper understanding of bluetongue epidemiology and more effective vector control in this region.de novo and mitochondrial DNA were utilized in a study of C. imicola populations from Africa and an outlier population from the Balearic Islands. The DNA microsatellite markers and mitochondrial DNA were also used to examine a population of closely related C. bolitinos Meiswinkel midges.A panel of 12 DNA microsatellite markers isolated C. imicola populations occurs. Genetic distance correlated with geographic distance by Mantel test. The mitochondrial DNA analysis results imply that the C. imicola populations from Kenya and south-west Indian Ocean Islands (Madagascar and Mauritius) shared haplotypes while C. imicola population from South Africa possessed private haplotypes and the highest nucleotide diversity among the African populations. The Bayesian skyline plot suggested a population growth.The microsatellite data suggest gene flow between Kenya and south-west Indian Ocean Islands exist while a restricted gene flow between Kenya and South Africa Culicoides into the Islands. Genetic similarity between Mauritius and South Africa may be due to translocation as a result of human-induced activities; this could impact negatively on the livestock industry. The microsatellite markers isolated in this study may be utilised to study C. bolitinos, an important vector of BTV and AHSV in Africa and identify sources of future incursions.The gene flow demonstrated by this study indicates a potential risk of introduction of new BTV serotypes by wind-borne infected The online version of this article (doi:10.1186/s13071-015-1277-4) contains supplementary material, which is available to authorized users. Culicoides (Diptera: Ceratopogonidae) are vectors of a number of arboviruses infecting livestock. Of the 75 arboviruses associated with Culicoides, 15 have been isolated from species belonging to C. imicola complex  using primer pair Bc1 Culic Fm (GTA AAA CGA CGG CCA GTT CWA CWA AYC AYA AAR WTA) and JerR2m (CAG GAA ACA GCT ATG ACC CAA ARA ATC ARA AYA RRT GTT G) . The rest of the African populations seemed to have no genetic differentiation except between the Kenyan population and South African (FST = 0.07) and South Africa and Madagascar populations (FST = 0.06) in the tests employed  the FST value of the South Africa and Madagascar populations was higher (FST = 0.06) than the FST value between the South Africa and Mauritius populations (FST = 0.02) (approximately 3090 km)     Table  which co October  while th October . However2) Table . This coC. imicola as shown by microsatellite results (FST = 0.07)  Table  and the sunlight , 49. A st Africa , 51.C. brevitarsis, another member of the Imicola Complex, in the Australasian region showed no evidence of genetic separation or structure between populations sampled from northern and eastern Australia, approximately 3000 km apart, suggesting a panmictic population in the continent [C. brevitarsis is restricted to fresh dung of wild and domesticated bovids [C. brevitarsis within Australia could be an indication of a recent colonization and expansion into this region [C. imicola in South Africa and Kenya.A phylogenetic study of d bovids . Its disd bovids  suggestid bovids . TherefoC. imicola populations sampled and genetically distinct Balearic Islands and C. bolitinos populations.Model-based clustering method used to infer structure in this study identified that three-cluster was most probable. The K = 3 model scenario suggests a single homogenous population of the African C. imicola, indicative of IBD pattern. Isolation by distance, initially described by Wright [Anopheles gambiae. In the present study, the IBD pattern disappears when the geographically outlying population of Balearic Islands is excluded, suggesting that the pattern may be due to vicariance resulting from reduced gene flow between the geographically outlying Balearic Islands population and the southern and eastern African populations of C. imicola that we have sampled [This study has revealed a significant positive correlation between genetic distance and geographic distance for y Wright , describy Wright . Thus, ny Wright , while Ly Wright  found th sampled .The microsatellite markers isolated in this study were variable. The departure from Hardy-Weinberg equilibrium observed in this study could be as a result of presence of null alleles caused by mutations at locus-specific primer annealing sites, resulting in an excess in homozygosity .ST (0.003–0.004) and slight reduction in the power of STRUCTURE to correctly assign individuals to populations (0.2 and 0.1 % units) [The effect of null allele on genetic test of population structure has been shown to result in small upward biases to F% units) . With thC. imicola genome were also detected in our few C. bolitinos individuals. All the loci were amplified and the alleles were quite varied. This species is more closely related phylogenetically to C. brevitarsis and could be a better vector of BTV than C. imicola [Twelve microsatellite markers isolated in this study from the  imicola . The devCOI gene haplotype network corroborated to a great extent the microsatellite results. Kenya and south-west Indian Ocean Islands (Mauritius and Madagascar) shared haplotypes. Except for a single haplotype which was directly linked to the main haplotype, South Africa population mainly possessed private haplotypes. This could be indicative of a contemporary only modest matrilineal gene flow between east and south of Africa. The Kenyan and south-west Indian Ocean populations shared haplotypes with the eastern Mediterranean populations of C. imicola (Israel and Greece). The western Mediterranean population  had private haplotypes. This could be indicative of different routes of C. imicola dispersal in the Mediterranean region [The mitochondrial n region  as propon region  . HoweveThe Bayesian skyline plot suggested a population growth, compatible with the star-like pattern of the Kenya/south-west Indian Ocean Islands mitochondrial marker haplotype network and the Tajima’s D and Fu’s F test values for Kenya, South Africa and Mauritius. This could be indicative of a recent increase in favourable conditions that increased suitable habitats but also expansions of its distributions in territories where bovids and equids, domesticated or wild were absent for a long time or changing farming practices cannot be ruled out. However the Bayesian skyline plot ought to be interpreted with caution as Bayesian skyline plot appears to slightly overestimate the most recent population . More exC. imicola population as well as the highest nucleotide diversity. This may suggest an ancestral history, reinforced by the closely related species described from the region [This study showed a higher frequency of private haplotypes among the South African e region –65Culicoides- borne virus within the East and South regions of Africa, especially between Kenya and neighboring islands , demonstrated by two types of genetic markers and analysis. This risk is probably due to passive wind-borne infected Culicoides, implicating the monsoon winds in the Indian Ocean. Such vector midge movement could impact negatively on local livestock production and equids industry. On the other hand, we found a reduction in Culicoides genetic flow within the continental mainland, between South Africa and Kenya. The microsatellite markers isolated in this study could be applied in genotyping C. bolitinos, another important vector of BTV and AHSV in the region.The findings of this study demonstrate notable risk of movement of


Podophyllum hexandrum, a highly important anti-cancerous herb, remains endangered. Genetic diversity characterization of 24 populations comprising of 209 individuals representing the whole of the Indian Himalayas revealed that regardless of geographic location, all of the populations are intermixed and are composed broadly of two types of genetic populations. Our findings also suggested that these populations have evolved well in response to the environment. This study will help in the formulation of conservation programs for P. hexandrum populations in this region.Despite its wide distribution across the entire Himalayan range, the current status of  Podophyllum hexandrum is an endangered high-elevation plant species that has great medicinal importance. Here we report on the genetic diversity analysis of 24 P. hexandrum populations , representing the whole of the Indian Himalayas. In the present study, seven amplified fragment length polymorphism (AFLP) primer pairs generated 1677 fragments, of which 866 were found to be polymorphic. Neighbour joining clustering, principal coordinate analysis and STRUCTURE analysis clustered 209 individuals from 24 populations of the Indian Himalayan mountains into two major groups with a significant amount of gene flow (Nm = 2.13) and moderate genetic differentiation Fst(0.196), G′st(0.20). This suggests that, regardless of geographical location, all of the populations from the Indian Himalayas are intermixed and are composed broadly of two types of genetic populations. High variance partitioned within populations (80 %) suggests that most of the diversity is restricted to the within-population level. These results suggest two possibilities about the ancient population structure of P. hexandrum: either all of the populations in the geographical region of the Indian Himalayas are remnants of a once-widespread ancient population, or they originated from two types of genetic populations, which coexisted a long time ago, but subsequently separated as a result of long-distance dispersal and natural selection. High variance partitioned within the populations indicates that these populations have evolved in response to their respective environments over time, but low levels of heterozygosity suggest the presence of historical population bottlenecks.Intraspecific genetic variation in natural populations governs their potential to overcome challenging ecological and environmental conditions. In addition, knowledge of this variation is critical for the conservation and management of endangered plant taxa. Found in the Himalayas,  Throughout history, one of the many ways in which humans have benefited from plant diversity is as a source of traditional medicines. According to the World Health Organization (WHO), as many as 80 % of the world's populations depend on traditional medicine for their primary health-care needs . Most trKnowledge of genetic variation within species, coupled with information about their reproductive biology, is very important when establishing any conservation and management programme . With rePodophyllum hexandrum, an endangered plant species of great medicinal importance. Using amplified fragment length polymorphism (AFLP) markers, we have examined 209 individuals of 24 natural populations of P. hexandrum, representing the wider geographical area of the entire Indian Himalayas ranging from the states of Jammu and Kashmir, and including the Zanskar region, Himachal Pradesh, Uttarakhand to Sikkim. The genetic diversity of P. hexandrum has not been studied for the entire of the Indian Himalayas, and such a large geographical area with a greater number of samples has been analysed for the first time. The present study will comprehensively reveal the overall genetic diversity prevailing in these populations and will also aid in understanding the genetic dynamics of the species. Further, this will also throw light on how these populations are persisting despite their having a small chromosome number and self-pollinating reproductive behaviour.In the present study, we quantified the patterns of genetic diversity within Podophyllum hexandrum  is a species of great medicinal importance. It is confined to the alpine regions of Afghanistan, Pakistan, Nepal, Bhutan, South West China and India , the most common species in the American subcontinent     with a vA total of 24 different geographical locations, ranging from Kashmir to the Sikkim Himalayas and representing most of the Indian Himalayas, were visited for sample collection in the present study during 2008 and 2012  and MseI primers, Taq polymerase, PCR buffer, MgCl2, each dNTPs and deionized water in a final volume of 10 μL. The first selective amplification cycle consisted of 94 °C for 30 s, 65 °C for 30 s and 72 °C for 1 min. The annealing temperature was lowered by 0.7 °C per cycle during the next 12 cycles, followed by 23 cycles at 94 °C for 30 s, 56 °C for 30 s and 72 °C for 1 min. All PCRs were performed on the i-cycler PCR system . 0.5 μL of each selective PCR product was mixed with 0.3 μL of Gene Scan-500 ROX size standard (Applied Biosystems) and 9.2 μL of highly deionized formamide. This mixture was denatured at 94 °C for 5 min, followed by immediate chilling on ice and these denatured products were loaded on an ABI 3730xl automated DNA Analyser  to visualize the amplified fragments.The AFLP protocol was carried out following the procedure described by The software program GeneMapper 3.7 (Applied Biosystems) was used to analyse electropherograms generated by automated genotyping using the ABI 3730xl automated DNA analyser. The large amount of data generated by the automated DNA analyser was checked manually a number of times to exclude unreliable detection and to improve the quality of data. The size range of amplified fragments, peak height threshold in terms of relative fluorescence units (rfu) and bandwidth were considered to be the most important scoring parameters; different sets of parameters were tested, and the parameter set that was optimized for the best fit was used for our analyses. Amplified fragments of 50–500 base pairs having present (1) and absent (0) peaks were extracted using GeneMapper 3.7. The resulting binary matrix was exported in the form of comma-separated text for data analysis.K). The numbers of clusters of the populations (K) were identified by performing six iterations and setting the value of K from 1 to 25 with a burn-in period of 100 000 and 100 000 number of the Markov Chain Monte Carlo (MCMC) repeats after burn-in. The maximal value of LnP(D), the posterior probability of data as per K was estimated through analysis of molecular variance (AMOVA)-based clustering using kMeans software  were conducted using GenAlEx 6.501 , 2012. Ssoftware . To infeGenoDive  was usedGenoDive , was useGenoDive  among al 3.5.1.2 . The inp 3.5.1.2 . The den 3.5.1.2 .P. hexandrum  for seven AFLP primer combinations resulted in 1677 unambiguous fragments in the size range of 50–500 bp, of which 866 (51.65 %) were polymorphic (Table h) estimates recorded in the AFLP analysis were found to be very low, with the maximum observed heterozygosity (0.155 ± 0.008) in the Eastern Himalayan population collected from Sikkim and the lowest heterozygosity in the Sural Pangi population (0.043 ± 0.005). Shannon's information index (I) values also complemented these findings  of the populations, whereas the minimum Fst (0.04) was observed between the Gulaba  and Diankund (Dhauladhar range) populations, and the Dharali  and Diankund (Dhauladhar range) populations.Overall 96 Table . G′st waes Table  showed tNm) was recorded between the populations. On the basis of Fst[Nm = (1/Fst− 1)/4], Nm was found to be 1.02, whereas the value of gene flow on the basis of Gst [Nm = Gst(1 − Gst)/Gst] was found to be 2.13, which indicated a considerable intermixing and low genetic differentiation among populations.Analysis of molecular variance analysis revealed that the majority of the variance was restricted to within-population variation (80 %), whereas variance partitioned among population was 20 %. Significant gene flow -based hierarchical clustering and Bayesian model-based clustering. Principal coordinate analysis  in the P. hexandrum populations and revealed two major groups. A Mantel test between the genetic and geographic distances showed no correlation see Supporting Information][. A significant amount of gene flow was recorded across all the populations of the Indian Himalayas. The fruit is a berry which is not edible initially, but becomes edible as it ripens, and Himalayan birds and grazing animals feed on these fruits, thus facilitating seed dispersal . The seeds remain dormant for up to 3 years , the average number of plants per quadrat (1 m2) decreasing from 2 to 0.6 at Prashar (data not shown). If effective conservation measures are not undertaken soon, we are likely to lose the invaluable genetic resources of this important medicinal plant. Inferences derived from the current study will help to guide management and conservation policies. Moreover, high-throughput sequencing efforts are required to study environmental effects on the adaptation mechanism. This type of study will be extremely helpful in understanding the genes involved in divergent selection and local adaptation.Based on the comprehensive molecular analysis of natural populations of Council of Scientific and Industrial Research (CSIR), Government of India.This research work was funded by the R.K.S. and A.N. designed the study. A.N. collected the populations and conducted the AFLP and data analysis. P.S.A. helped in the coordination of the study. A.N. and R.K.S. wrote and approved the final version of the manuscript. All authors have read and approved the final manuscript.None declared.The following Supporting Information is available in the online version of this article –Table S1. Analysis of molecular variance (AMOVA) between the different mountain ranges.Figure S1. Representative AFLP profile of P. hexandrum samples revealed by E-ACA + M-CAG primer combination using from the automated DNA analyser (3730xl). (A) Lane window screen shot 1–96: Different P. hexandrum samples, (B) green fragments represent the detected fragments while red peaks are the marker fragments indicating size.Figure S2. Pictorial representation of P. hexandrum plant in nature and its fruit showing numerous seeds in it.Figure S3. Mantel test showing no correlation between genetic and geographic distance on the basis of AFLP data in 24 populations of P. hexandrum.


The Tyrolean Iceman is an extraordinarily well-preserved natural mummy that lived south of the Alpine ridge ~5,200 years before present (ybp), during the Copper Age. Despite studies that have investigated his genetic profile, the relation of the Iceman´s maternal lineage with present-day mitochondrial variation remains elusive. Studies of the Iceman have shown that his mitochondrial DNA (mtDNA) belongs to a novel lineage of haplogroup K1 (K1f) not found in extant populations. We analyzed the complete mtDNA sequences of 42 haplogroup K bearing individuals from populations of the Eastern Italian Alps – putatively in genetic continuity with the Tyrolean Iceman—and compared his mitogenome with a large dataset of worldwide K1 sequences. Our results allow a re-definition of the K1 phylogeny, and indicate that the K1f haplogroup is absent or rare in present-day populations. We suggest that mtDNA Iceman´s lineage could have disappeared during demographic events starting in Europe from ~5,000 ybp. Based on the comparison of our results with published data, we propose a scenario that could explain the apparent contrast between the phylogeographic features of maternal and paternal lineages of the Tyrolean Iceman within the context of the demographic dynamics happening in Europe from 8,000 ybp. Tisenjoch Pass in the Italian part of the Ötztal Alps, close to the Austrian border (3210 m s.l.m.). The Iceman is one of the oldest natural European mummies (dated to 5350–5100 ybp), and an extraordinarily well-preserved individual due to a spontaneous freeze-drying process. Scientific interest is further increased by the preservation of parts of his clothes and equipment .The Tyrolean Iceman  was discovered in 1991 on the Vinschgau and Schnals valleys in actual South-Tyrol, Eastern Italian Alps)4This important finding provided unprecedented insights into aspects of the daily life during the Copper Age in central Europe12et al.The Tyrolean Iceman has also been extensively characterized genetically. Whole genome sequencing data indicate that the Iceman was blood group 0, lactose intolerant and genetically predisposed to cardiovascular diseases91011To shed new light on these issues, we analyzed the mitogenomes of 42 K–haplogroup samples from Eastern Italian Alps and compared the Iceman´s sequence with a large dataset of worldwide K1 mitogenomes. The results are discussed with currently available ancient mtDNA data from European Neolithic specimens. Finally, we combine mtDNA and Y-chromosomal data to propose a scenario that could explain the complex genetic history of the Tyrolean Iceman.The mitogenomes of 42 haplogroup K individuals from Eastern Italian Alps were analyzed . The mosn = 1077; Next, we analyzed all K1 mitogenomes available in the literature, scanning databases, in combination with our dataset  in groups from the Mediterranean area, i.e. Sardinians and Corsicans6The direct comparison of the evolutionary history of specific female and male lineages can be complicated by differences in their phylogenetic structure and resolution. However, it is likely that the dissimilarity observed in the diachronic patterns of the Iceman’s genetic lineages are due mainly to the distributions of K1f and G2a in Europe prior to 5,000 ybp and later demographic events that shaped the European genetic structure.Recent ancient DNA studies suggest that both mtDNA haplogroup K1 and Y-chromosome G2a reached the European continent around 8,000 ybp by migrations of Early Neolithic people from the Near East through continental and Mediterranean routes1518191519202122While the Iceman’s Y-chromosome lineage was part of the Early Neolithic genetic substrate, as suggested from current distributions of G2a-L91 , the mtDVinschgau valley), while significant demographic growth started ~2,000 years later during the Middle Bronze Age.After 5,000 ybp, population expansion, migrations and admixture occurred throughout the continent, re-shaping the genetic structure of Europeans8156In conclusion, by combining genetic data from modern-day populations and European Neolithic specimens, we gained new insights into the genetic history of the Tyrolean Iceman. This study demonstrates the continued importance of this unique specimen to shed light on the early migrations in Europe, and marks a first step towards a systematic study of the genetic history of ancient Alpine groups.Azienda sanitaria dell’Alto Adige. Informed consent was obtained from all subjects. The experimental protocol was carried out in accordance with the approved guidelines and was approved by the above-mentioned Ethic Committee.Thirty-nine DNA samples belonging to the haplogroup K were selected from more than 800 individuals from the Eastern Italian Alps  analyzed at low resolution in previous studies252627n = 42) were analyzed for the whole mtDNA genome (16569 base pairs) using the methods described by Torroni and collaboratorsDNA extraction was carried out using a commercial kit . Then, samples were typed for informative SNPs of the mtDNA haplogroup K status  by using the SNAPShot method . Only three samples (~7%) carried the diagnostic variants. Afterwards, the total K samples  and supervised manually. The new sequences were deposit in GenBank (accession numbers from KT749793 to KT749816).In order to classify the sequences in the sub-lineages of haplogroup K, we compared our data with the worldwide human mtDNA phylogenetic tree (PhyloTree Build 16)n = 1042; October 2014). The 1000 Genome Project mitogenomes were processed using in-house scriptsn = 409) were from individuals with reported European ancestry, 440 with unknown geographic origin and the remaining from non-European regions  all available complete K1 mitogenomes from present-day populations (http://eltsov.org) and adjusted manually according to Phylotree Built 16Phylotree Build 16We estimated the Time of the Most Recent Common Ancestor (TMRCA) of clades utilizing a time-dependent clock incorporating a correction for purifying selection-8 as reported inhttp://tree.bio.ed.ac.uk/software/tracer/).The BSP showing changes of the effective population size of haplogroup K1 through time was performed with the Bayesian Evolutionary Analysis Sampling Trees (BEAST)How to cite this article: Coia, V. et al. Whole mitochondrial DNA sequencing in Alpine populations and the genetic history of the Neolithic Tyrolean Iceman. Sci. Rep.6, 18932; doi: 10.1038/srep18932 (2016).