Zingiber kawagoii populations inhabiting varying environments distributed at low to middle elevations (143–1488 m) in a narrow latitudinal range (between 21.90 and 25.30° N). Here, we show evidence of selection driving the divergence of Z. kawagoii. Twenty-six FST outliers were detected, which were significantly correlated with various environmental variables. The allele frequencies of nine FST outliers were either positively or negatively correlated with the population mean FST. Using several independent approaches, we found environmental variables act in a combinatorial fashion, best explaining outlier genetic variation. Nonetheless, we found that adaptive divergence was affected mostly by annual temperature range, and it is significantly positively correlated with latitude and significantly negatively correlated with the population mean FST. This study addresses a latitudinal pattern of changes in annual temperature range (which ranged from 13.8 °C in the Lanyu population to 18.5 °C in the Wulai population) and emphasizes the pattern of latitudinal population divergence closely linked to the allele frequencies of adaptive loci, acting in a narrow latitudinal range. Our results also indicate environmentally dependent local adaptation for both leading- and trailing-edge populations.Ecological and evolutionary processes linking adaptation to environment are related to species’ range shifts. In this study, we employed amplified-fragment-length-polymorphism-based genome scan methods to identify candidate loci among  The altitude-for-latitude model projected the scales of range retractions in altitude and latitude due to warming . LocallyZingiber kawagoii is a species of herbaceous perennial plant in the Zingiberaceae family found in Taiwan and a small offshore island southeast of Taiwan. This species is endemic to Taiwan and is widely distributed along the west sides of the Hsuehshan Mountain Range and the Central Mountain Range in Taiwan, but it is sparsely distributed east of these mountain ranges, at low to middle elevations (140–1500 m), and from the southern to northern tip of Taiwan , 90 km off the southeast coast of Taiwan. Populations of Z. kawagoii are found in different habitats including forest understories, forest edges, slopes, and valleys. Extremely low intraspecific variation and high population differentiation were found based on chloroplast DNA (cpDNA) variation [Z. kawagoii after the last glacial maximum (LGM) has been inferred using ecological niche modeling [Z. kawagoii may evolve locally adapted alleles in association with a leading-front environment. Nonetheless, trailing-edge populations are also important to the future survival of species [f Taiwan . Zingibeariation . A mean ariation . The latmodeling . This clmodeling , limitinvations 10–1500 m,modeling . Althougmodeling . The leavations 10–1500 m, species ,13.Z.kawagoii population divergence related to environmental gradients will advance our understanding of species’ response to global change, particularly in a geographic area only spanning a narrow latitudinal range. Spatial heterogeneity in populations of Z. kawagoii may have played roles in driving local adaptation associated with environmental factors. Although genetic diversity in the latitudinal leading- and trailing-margin populations may be reduced, adaptive loci driven by natural selection may accumulate their frequencies at both range margins of distribution correlated strongly with environment [Z.kawagoii. In this study, we surveyed the genetic variation in 212 individuals from 17 populations of Z. kawagoii using amplified fragment length polymorphisms (AFLPs) [Z. kawagoii related to adaptive evolution along a narrow latitudinal range. Specifically, we asked (1) how do population divergence and environmental variables vary along a narrow latitudinal range? (2) Which environmental variable(s) may have played the main role, and do environmental variables act in a combinatorial fashion in driving adaptive genetic divergence? (3) How do the allele frequencies of adaptive loci vary corresponding to the levels of population divergence?Lower-latitude populations within species may show greater genetic divergence and evolutionary independence, contributing to reproductive isolation and speciation . Moreoveironment ,15,16. NZingiber kawagoii individuals were collected from 17 populations (n = 212) which spanned a latitudinal range of 21.90–5.30° N and an altitudinal range of 143–1488 m  at 37 °C for 1.5 h for restriction digestion. The reaction was deactivated at 65 °C for 15 min. The digested DNA products were ligated to AFLP adaptors (5 µM EcoRI and 50 µM MseI) with 5 U T4 DNA ligase  and 5X ligation buffer (Thermo Scientific) at 22 °C for 1 h in a 10 µL ligation reaction mixture.3–1488 m . Within 2O) was mixed with 12 µM EcoRI (E00: 5′-GACTGCGTACCAATTC-3′) primer, 12 µM MseI (M00: 5′-GATGAGTCCTGAGTAA-3′) primer, 2.5 mM dNTPs, 1 U Taq DNA polymerase , and 10X PCR buffer (Zymeset) in a 20 µL total volume. The PCR for pre-selective amplification was an initial holding at 72 °C for 2 min and pre-denaturation at 94 °C for 3 min, followed by 25 cycles of 30 s at 94 °C, 30 s at 56 °C, and 1 min at 72 °C, with a final 5 min holding at 72 °C. Either three or five additional bases were added at the ends of E00 and M00, and 90 primer combinations were screened initially for proper amplification, the number of fragments were amplified, and the genotyping error rate did not exceed 5%. Finally, 11 EcoRI-MseI (E00 and M00) primer combinations were used in selective amplification (EcoRI selective primer (6-carboxyfluorescein or hexachloro-fluorescein dye labeled) was used in selective amplification. Selective amplification was performed in a 20 µL total volume containing 10 µM EcoRI and 10 µM MseI primers, 2.5 mM dNTPs, 2 U Taq DNA polymerase (Zymeset), 10X PCR buffer (Zymeset), and diluted pre-selective amplified products (1:19 dilution with ddH2O). Selective PCR was an initial holding at 94 °C for 3 min, followed by 13 cycles of 30 s at 94 °C, 30 s at 65–56 °C (decreasing the temperature by 0.7 °C each cycle), 1 min at 72 °C, then 23 cycles of 30 s at 94 °C, 30 s at 56 °C, and 1 min at 72 °C, with a final 5 min holding at 72 °C. Selective amplification products were electrophoresed on an ABI 3730XL DNA analyzer. We used Peak Scanner v.1.0  to score amplification fragments. A fluorescent threshold set at 150 units was used to avoid background noise when scoring AFLP fragments in the range of 100–500 bp. We removed low peaks and fragments within one nucleotide in a ±0.8 base pair window which were recognized as the same fragment. Additionally, amplified fragments that scored higher than 99% or less than 1% of individuals were also removed. Three randomly chosen samples in each population were used to calculate the genotyping error rate per locus. Loci with an error rate per locus greater than 5% were removed [AFLP pre-selective amplification was performed using polymerase chain reaction (PCR). Adaptor ligated product . Apart from the bioclimatic variables downloaded from WorldClim and three topographic variables, twelve other environmental variables not directly defined as bioclimatic and topographic variables were grouped as ecological variables. The twelve ecological variables were the normalized difference vegetation index (NDVI), the enhanced vegetation index (EVI), the leaf area index (LAI), the fraction of absorbed photosynthetically active radiation (fPAR), the relative humidity (RH), cloud cover (CLO), sunshine hours (SunH), the number of rainfall days per year (RainD), the mean wind speed (WSmean), the soil pH, the annual total potential evapotranspiration (PET), and the annual moisture index (MI).Three categories of environmental variables were used in the study. Nineteen bioclimatic variables for sample sites at 30 s spatial resolution (~1 km) were downloaded from the WorldClim 1.4 for information related to temperature and precipitation . Topograhttp://lpdaac.usgs.gov, accessed on 12 June 2021) were obtained for NDVI and EVI , LAI and fPAR , and PET . The monthly mean values of these variables were computed using a maximum-value composite procedure. The monthly mean values of RH, CLO, SunH, RainD, and WSmean at 1 km resolution were calculated using a universal spherical model of the Kriging method in ArcGIS with data obtained from the Data Bank for Atmospheric & Hydrologic Research . Sampling site soil pH values were obtained from the soil data of an island-wide, 1,150 site investigation conducted in 1969–1986 by the Agriculture and Food Agency of Taiwan. Annual potential evapotranspiration values derived from the annual mean temperature and annual precipitation were used to calculate the annual MI.Data from a moderate resolution imaging spectroradiometer (MODIS) recorded during 2001–2020 in the Land Process Distributed Active Archive Center  was explained by the individual variable  with a correlation threshold of |0.7| using the age usdm  in the Rage usdm . The biod WSmean  if more uHE) [P, 95% criterion) based on allele frequencies using the settings of the Hardy–Weinberg equilibrium and non-uniform prior distribution. The per locus uHE was estimated using ARLEQUIN v.6.0 [IA [rD) [ia function of the R package poppr [uHE per locus among and between populations. In LMMs, population and locus were used as a fixed factor and a random factor, respectively, and they were analyzed using the lmer function of the R package lme4 [Anova function of the R package car was based on type II Wald χ2 statistics [uHE per locus with Tukey’s post hoc test were assessed using the lsmeans function of the R package emmeans [AFLP-SURV v.1.0  softwareuHE)  and the IN v.6.0 . The ind.6.0 [IA  and the [IA [rD)  are measge poppr . A lineaage lme4  based onatistics . Pairwis emmeans .ΦST) using the poppr.amova function of the R package poppr. Significance was tested using the randtest function of the R package ade4 [FST was computed using ARLEQUIN, and the significance was tested with 10,000 permutations. Additionally, the level of divergence for each population from the remaining populations was calculated as the mean value of the pairwise FST for each population against the rest of the populations (denoted as the population mean FST). The population mean FST can be used as a proxy of the level of one population diverging genetically from the remaining populations.The analysis of molecular variance (AMOVA) was used to estimate the level of genetic differentiation between populations  in LEA and the Bayesian information criterion (BIC) in DAPC were estimated to determine the optimal number of clusters.Genetic homogeneous groups of individuals were assessed using the sNMF algorithm of landscape and ecological association (LEA)  and disckage LEA . The paradegenet  were usenei.dist function of the R package poppr was used to calculate pairwise Nei’s genetic distances [nj function of the R package ape [aboot function of the R package poppr.A neighbor-joining (NJ) tree was used to assess the genetic relationships among individuals. The istances . Nei’s gkage ape , and booFST outliers were identified using DFDIST and BAYSESCAN. DFDIST is a modification for dominant markers of the software developed by Beaumont and Nichols [FST versus uHE, and loci under selection were identified by comparing them to a simulated neutral distribution. DFDIST parameters include critical frequency = 0.99; Zhivotovsky parameter = 0.25; trimmed mean FST = 0.3 ; smoothing proportion = 0.04; 500,000 resamplings; and critical p = 0.05. AFLP Loci with observed FST against uHE falling above the 95% confidence level of simulated distribution were recognized as FST outliers under directional selection. BAYESCAN v.2.1 [6 iterations were performed in BAYESCAN. Selection is detected when locus-specific component (α) is significantly different from zero. A positive α suggests divergent selection, while negative values suggest balancing or purifying selection. We used a criterion of a logarithmic scale of log10 (PO) > 2 as decisive evidence, corresponding to posterior probabilities between 0.99 and 1 [To detect the signature of selection on AFLP loci,  Nichols . DFDIST AN v.2.1  uses a r99 and 1 , for selp values adjusted using the genomic inflation factor (λ) [p value of 1% was further applied using the qvalue function of the R package qvalue [p value adjustment for both Wald and G scores was used to assess the fit of the model with environmental variables against the null model without environmental variables.To assess the associations of all genetic loci with environmental variables, the latent factor mixed model (LFMM)  and Samβctor (λ) . A falsee qvalue . Samβadastan_glm function of the R package rstanarm [FST outliers, identified using both BAYESCAN and DFDIST, with environmental variables. In stan_glm, the weakly informative priors following Student’s t distribution with a mean of zero and seven degrees of freedom were used, and the scale of the prior distribution was 10 for the intercept and 2.5 for the predictors. All stan_glm models were run with four chains for 2000 warm-up and 2000 sampling steps. The posterior_interval function of the R package rstanarm was employed to estimate 95% credible intervals for the determination of significant correlations of potential FST outliers with environmental variables. In stan_glm analysis, the effective sample size values representing overall sampling efficiencies for each predictor estimated were between 1632.8 and 7199.4, and the convergence diagnostic statistics were all close to 1. These values indicate good priors applied and stable estimates obtained.A Bayesian logistic regression analysis implemented in the rstanarm  was emplforward.sel function of the R package adespatial [p < 0.05) or the global adjusted R2 was exceeded to prevent the overestimation of the explained variance, and significance was determined based on 999 permutations. Environmental variables in the three environmental categories were analyzed separately, and variables explaining more than 5% of outlier genetic variation (adjusted R2 > 0.05) were retained as the final set of environmental variables in the study  . The AICc was used to rank the models and to calculate the Akaike sum of weights (SW) for each model [importance function, was used to assess the relative importance of environmental variables contributing to explaining variation in the outlier loci. However, the SW index is arguably not an appropriate measure, representing the importance of model selection [FST outliers were used to test for correlation with the population mean FST.To test for the most important environmental variables explaining outlier genetic variation, we used the espatial . Forwardhe study . Variablage pscl . GLMs thch model . The SW election . Therefomantel function of the R package vegan [mantel.partial function. Environmental variables and outlier variation were used in a redundancy analysis (RDA). An RDA estimates the relative contribution of environmental variables explaining the outlier variation using the varpart function of the R package vegan. The total outlier variation was partitioned into four fractions attributable to (a) a pure environmental effect, (b) a geographically structured environmental effect, (c) a pure geographic effect, and (d) a residual effect [anova.cca function of the R package vegan with 999 permutations. Sample site geographic coordinates were used as geographic effects in variation partitioning.The mantel test was used to analyze the correlations of the outlier AFLP Euclidean distance matrix with the Euclidean distance matrix of environments using the ge vegan  and the l effect . We testuHE was 0.123, ranging from 0.102 in population TRK to 0.151 in population BTWS. A strong departure from random association between AFLP loci based on the measures of multilocus LD, IA, and rD was found for all populations examined  and in many between-population comparisons    loci was obtained using 11 selective amplification primer combinations . The perexamined . The linparisons . Using t< 0.001)  and pair 0.0001) .K = 18 and K = 8, respectively. However, we observed changes in both the mean minimal CE and BIC elbowed at K = 3, which is consistent with three genetically homogeneous groups observed in LEA and DAPC .Pearson’s correlation test found a significant negative relationship between the population mean latitude a and a slatitude b. Therefas found c. SignifFST outliers  as potential outliers . All 26 rstanarm . We founFST outliers  and the 95% CIs for coefficients of environmental covariates in the best predicting models revealed that environmental variables acted in a combinatorial fashion, influencing the genetic variations in the 26 outliers . Althougoutliers , it was outliers b.FST outliers were significantly explained by a combination of environmental variables. Moreover, environmental variables with high SW values may show no significant effects (95% CIs bracket zeros) on outlier variation. Nine outlier loci were found to have either significant positive  or negative  relationships of allele frequency with the population mean FST  , whereasmean FST . Howevermean FST .The total explainable outlier genetic variation by the seven retained environmental variables was 47.4% based on the 26 outlier loci, of which 24.0% and 18.9% were, respectively, attributed to pure environmental and geographically structured environmental effects. However, only 4.5% was attributed purely to geographic effects .uHE) and latitude  indicates higher levels of population genetic divergence in the low-latitude populations of Z. kawagoii . Thus, we included the LY population as one of the populations in the Z. kawagoii latitudinal distribution.The LY population, located on a small island situated off the southeast coast of Taiwan, had the lowest genetic distance to the population JSY in southern Taiwan . We identified environmental factors , suggesting that multiple environmental factors impose as selective drivers for local adaptation  or with the latitude . The annual temperature range with a higher adjusted R2 than other environmental variables  than that of the Brazilian Z. officinale (average uHE = 0.312) [Zingiber species  [uHE = 0.230) [Z. kawagoii was also reflected in the low average percentage of polymorphism  , three I: 0.367) , and the= 0.230) . The relmorphism  and low kawagoii  is probauHE and latitude: r = 0.021, p = 0.936). It is likely that geographic variation in thermal tolerance limits, consisting of both spatial temperature gradient and warming, can influence the rate of range shifts [Z. kawagoii.Although low-latitude rear edge populations are expected to be small in size and are hence characterized by low genetic diversity , our date shifts . While ce shifts . Moreovee shifts . This stFST along a latitudinal gradient that extended from the south of the Z.kawagoii distribution to its northern distributional margin. The annual temperature range related to the thermal tolerance of local populations could be the major environmental factor influencing outlier genetic variation. Additionally, the combination of various environmental variables may also be important to the local adaptation of Z.kawagoii. This study identified the presence of natural selection acting on adaptive loci at a small latitudinal scale, contributing to understanding how herbaceous species respond to environmental changes. The results broaden the generality of the latitudinal population divergence which is closely linked to environmental gradients in both the latitudinal leading- and trailing-edge populations of Z. kawagoii. Ecological speciation may occur in the low-latitude populations of Z. kawagoii, particularly because of high genetic divergence against other populations with narrower thermal tolerance limits.We studied the population mean


Philaenus spumarius is a cosmopolitan species that has become a major threat to European agriculture being recognized as the main vector of the introduced plant pathogen Xylella fastidiosa, the agent of the “olive quick decline syndrome”, a disease which is devastating olive orchards in southern Italy. Wolbachia are bacterial symbionts of many insects, frequently as reproductive parasites, sometime by establishing mutualistic relationships, able to spread within host populations. Philaenus spumarius harbors Wolbachia, but the role played by this symbiont is unknown and data on the infection prevalence within host populations are limited. Here, the Wolbachia infection rate was analyzed in relation to the geographic distribution and the genetic diversity of the Italian populations of P. spumarius. Analysis of the COI gene sequences revealed a geographically structured distribution of the three main mitochondrial lineages of P. spumarius. Wolbachia was detected in half of the populations sampled in northern Italy where most individuals belonged to the western-Mediterranean lineage. All populations sampled in southern and central Italy, where the individuals of the eastern-Mediterranean lineage were largely prevalent, were uninfected. Individuals of the north-eastern lineage were found only in populations from the Alps in the northernmost part of Italy, at high altitudes. In this area, Wolbachia infection reached the highest prevalence, with no difference between north-eastern and western-Mediterranean lineage. Analysis of molecular diversity of COI sequences suggested no significant effect of Wolbachia on population genetics of P. spumarius. Using the MLST approach, six new Wolbachia sequence types were identified. Using FISH, Wolbachia were observed within the host’s reproductive tissues and salivary glands. Results obtained led us to discuss the role of Wolbachia in P. spumarius, the factors influencing the geographic distribution of the infection, and the exploitation of Wolbachia for the control of the vector insect to reduce the spread of X. fastidiosa. Wolbachia Hertig (α-Proteobacteria) are maternally-inherited intracellular bacteria that establish symbiotic relationships with a wide range of arthropods and nematodes, including 40–60% of the insect species [Wolbachia strains are known to be reproductive manipulators of their hosts and to increase their prevalence within host populations by improving the reproductive fitness of the infected females [Wolbachia to spread within host populations. CI is a form of sterility that occurs when infected males mate with uninfected females  or when mates harbor different Wolbachia strains , resulting in the death of the progeny during early embryogenesis [Wolbachia can increase within the host population and under certain conditions can rapidly reach a stable equilibrium, even close to 100% [Wolbachia can also spread within host populations by inducing female-biased sex ratios of host progeny through parthenogenesis, feminization or male-killing [Wolbachia are beneficial for their host, establishing obligate [ species , 2. Most females . Cytoplaogenesis . Convers to 100% . Wolbach-killing , 6, 7. Oobligate , 9 or faobligate , conferrobligate –15 and mobligate .Wolbachia to interfere with host reproduction and to be pervasive within insect populations makes this endosymbiont promising for the development of innovative methods for biological control of insect pests and to reduce the spread of vector-borne pathogens [Wolbachia-based control methods rely on CI and artificial transfection of the target insects with a Wolbachia strain, they do not naturally carry, expressing a strong CI phenotype. By the Incompatible Insect Technique (IIT) [Wolbachia-infected males are repeatedly distributed in large numbers in the field to determine the suppression of a pest. Because wild females will produce non-viable embryos when mated by infected males, the inundative release of incompatible males, that outcompete wild males in mating wild females, will reduce the size of the target population. Alternatively to IIT, the population replacement approach uses CI to favor the spread of a Wolbachia strain with a pathogen-blocking phenotype  [Wolbachia and on the reproductive advantage that CI provides to infected females. Infected offspring, if able to settle permanently, will lead over time to a progressive reduction in the frequency of the uninfected insect population with the consequent reduction of the vector competence and decline of the transmitted pathogen in the treated area [Nilaparvata lugens (Stål), was transfected with a Wolbachia strains which, in addition to expressing strong CI, inhibits the transmission of a virus that severely damages rice crops, thus attenuating the intensity of the plant disease [Wolbachia-based approach to the control of agricultural pests and the pathogens they transmit [Wolbachia to introduce transgenes into the target population [The ability of athogens –20. To due (IIT) , 22, CI mission) . This stted area . Up to n disease . This sttransmit . Other ppulation , 28 but pulation , 30.Philaenus spumarius L. (Hemiptera: Aphrophoridae) is a xylem sap-sucking insect whose nymphs develop in meadows, feeding on a wide range of host plants, mainly dicots. Adults can remain on the herbaceous vegetation but often disperse onto woody hosts during summer. This very polyphagous species can also feed on crops but has reached the pest status very rarely [Xylella fastidiosa Wells et al. (γ-Proteobacteria) in southern Europe, P. spumarius has become one of the major threat to European agriculture [X. fastidiosa coupled to a wide distribution throughout the Continent [Xylella fastidiosa is now present in Italy, Spain, France and Portugal and has been eradicated in Germany and Switzerland [P. spumarius is the main vector of X. fastidiosa subspecies pauca, the causal agent of the “olive quick decline syndrome” (OQDS). OQDS disease is quickly devastating the olive growing in Puglia with significant economic, environmental and social impacts [P. spumarius is a vector of X. fastidiosa subspecies fastidiosa to grapevine in Mallorca [multiplex and fastidiosa subspecies [The spittlebug y rarely . Recentliculture , 33, dueontinent , 34–37. tzerland . In the  impacts , 40. In Mallorca  and of tbspecies , 42, 43.Philaenus spumarius is a cosmopolitan species originated in the Palearctic region and distributed throughout the Holarctic region [P. spumarius, with contact zones occurring in some region [Philaenus spumarius feeds on xylem sap, a fluid with low amino acid and sugar content. To overcome this nutritional deficiency, it relies on the complementary biosynthetic pathways of two obligate bacterial symbionts [Philaenus spumarius can also harbors facultative bacterial symbionts, namely Arsenophonus, Cardinium, Rickettsia and Wolbachia, whose role in spittlebug populations is not yet known [Wolbachia were detected in P. spumarius populations of different geographic areas [P. spumarius and between the regions of the geographic range [Wolbachia was considered as a possible factor of reproductive isolation, via cytoplasmic incompatibility, between populations of NE and SW lineage in the Carpathians contact zone. However, data on the prevalence of Wolbachia infection within the host populations are limited since very few individuals per population were analyzed by [c region , 44. Accc region –47. Thisc region . The NE e region , 49. Phiymbionts , 51, eacymbionts . Philaenet known . Wolbachic areas –55 and tic range . In the lyzed by . A recenlyzed by .P. spumarius in Italy as vector of X. fastidiosa, there is only little information on the genetic diversity of the Italian populations and the prevalence of Wolbachia infections. The few data available originate from two studies on the global diversity of P. spumarius and suggest that the Italian populations are largely composed of individuals of the SW lineage. However, a clear geographic structure cannot be inferred from these papers as only a few dozen individuals from Italy were analyzed [Wolbachia infection [P. spumarius. Here, we aimed to broaden knowledge about Wolbachia ecology in the populations of P. spumarius of Italy. Understanding the factors influencing the ecology of Wolbachia is of interest to evaluate the applicability of Wolbachia-based strategies to control vector populations and to contrast the spread of X. fastidiosa. Therefore, in order to highlight the factors that may affect the distribution of Wolbachia among the populations of P. spumarius across Italy, the main aims of this work were to: i) study the prevalence of Wolbachia infection in relation to the geographical distribution and mitochondrial genetic diversity of P. spumarius; ii) characterize Wolbachia strains and their association with the host mitochondrial genotype; iii) understand the possible impact of Wolbachia infection on the population genetics of P. spumarius; v) enhance the knowledge on the symbiotic interaction between Wolbachia and P. spumarius through the study of the symbiont tissue tropism.Despite the great importance of analyzed , 46. Simnfection  do not aPhilaenus spumarius populations were sampled from 2015 to 2019 in 75 locations from southern to northern Italy ranging from sea level to 1878 m altitude. Overall, 52 populations were sampled in four regions of southern Italy, including the Puglia region, the focal area of X. fastidiosa epidemics. Four populations were sampled in a region of central Italy and 19 populations in four regions of northern Italy  gene of single individuals was amplified using the DNA extracted from the insect’s abdomen and the primer combination C1-J-2195 and TL2-N-3014 [P. spumarius [P. spumarius sampled throughout Italy were sequenced including 117 from southern Italy, 10 from central Italy and 138 from northern Italy  (Philaenus spumarius sequences were deposited in GenBank under accession numbers MN812359-MN812427 and MW737706-MW737787)  . HaplotyW737787) .P. spumarius populations was studied by a median-joining haplotype network following [COI sequences were aligned using the Muscle alignment tool in Aliview 1.26 [p-distance, that is the number of base differences per site) between and within mitochondrial phylogeographic lineages was calculated using MEGA X [The phylogeographic structure of ollowing . A matriollowing , these liew 1.26  and wereiew 1.26  was usediew 1.26  were geniew 1.26  and visuiew 1.26 . Two netiew 1.26 )  and the 16S rRNA gene of the primary endosymbiont Sulcia mullerii using the primers 10_CFB_FF and 1515_R [Wolbachia screening was also run on DNA extracted from the heads of infected individuals of five populations  and 27 uninfected populations of southern Italy  (P. spumarius populations within and between geographic regions.The sex ratio was assessed in five Sicilia) . The G-tP. spumarius screened for Wolbachia infection and with at least four individuals sequenced for the mitochondrial COI gene were considered for the analysis of molecular diversity. Overall, 27 populations and 226 individuals were analyzed (Hd), number of segregating sites (S), nucleotide diversity (π) and mean number of pairwise differences (k) were calculated for P. spumarius populations grouped on the basis of the geographic region of origin   . An addinfected)  Tables. Wolbachia found in P. spumarius were characterized using the MLST approach suggested by [gatB, coxA, hcpA, fbpA and ftsZ, and subsequent assignation of an allelic profile, the so called sequence type (ST) (http://pubmlst.org/Wolbachia/). As an additional molecular marker, the wsp gene was amplified and sequenced and each Wolbachia strain was typed based on the amino acid sequence of the hypervariable regions of the WSP protein. Primers and PCR cycling conditions were as suggested by [COI gene. Overall, 20 infected individuals with known mitochondrial haplotype were processed for molecular typing of Wolbachia  and were retrieved from GenBank to be included in our analysis. Similarly, alignment of the wsp gene sequences was also examined, although other wsp gene sequences of P. spumarius were not available in both GenBank and MLST database.The strains of ested by  based onested by , while Polbachia . WolbachMN812358 . Alignmeite 3.61  and veriius from ; these sgatB+fbpA, coxA, hcpA and ftsZ. Wolbachia ST-402 (supergroup M) hosted by the aphid Pentalonia nigronervosa Coquerel was used as outgroup. For the wsp gene, the BI tree was obtained by implementing the substitution model GRT+I. The allele wsp-34 of Wolbachia ST-37 (supergroup D) hosted by the nematode Brugia malayi Brug was used as outgroup. Two runs of four Monte Carlo Markov chains (3 “heated” and 1 “cold”) were run in parallel in MrBayes for 15,000,000 generations for concatenated MLST genes and 5,000,000 generations for wsp gene. Trees were sampled every 1,000 generations. Convergence of the separate runs was checked using the average deviation of split frequencies diagnostic and the PSRF parameter . The burn-in value was set at 25% of sampled topologies. Trees were imported into the tree editor TreeGraph 2.14.0–771 beta [Wolbachia phylogeny based on concatenated MLST gene sequences was constructed using maximum likelihood (ML) analysis performed in MEGA X [Wolbachia STs was calculated using MEGA X [Wolbachia can be difficult to interpret using standard phylogenetic analyses [The phylogenetic analysis was performed by Bayesian inference (BI) and the concatenated matrix was exported from Mesquite to Mr. Bayes 3.2.6 . Substit771 beta  for annon MEGA X . ML treeg MEGA X . Becauseg MEGA X –87, evolanalyses , 89. To analyses  using thanalyses  based onanalyses  was condWolbachia localization within host tissues was studied using the probe W2 (5’-CTTCTGTGAGTACCGTCATTATC-3’) [Wolbachia 16S rRNA, labelled on the 5’ end with Cy3. The probe W2 was used in combination with the universal bacterial probe EUB338 (5’-GCTGCCTCCCGTAGGAGT-3’) or the probe CFB319 (5’-TGGTCCGTGTCTCAGTAC-3’) targeting the primary symbiont Sulcia muelleri 16S rRNA [P. spumarius adult females were numb at -20°C and dissected at room temperature in ethanol 70% at a stereomicroscope (40X). The head was separated from the rest of the body and then dissected to extract the salivary glands. Ovaries and bacteriomes were extracted from the abdomen. Ovaries, bacteriomes and salivary glands were separately subjected to the whole-mount FISH method by [2O2 in ethanol for 24–48 h, washed in absolute ethanol, and hybridized for 24 h at 25°C. Hybridization buffer  contained 10 pmol/ml of each fluorescent probe. Then, stained samples were rinsed in washing buffer  and mounted on slides in Vectashield antifade mounting medium with DAPI (1.5 μg/ml) . Slides were observed and photographed at a Leica DM6000 B fluorescence microscope  and images were processed with the Leica Application Suite X (LAS X). The specificity of the observed signals was verified by comparing the probe-treated individuals infected by Wolbachia (sampled from the infected populations of the Alto Adige region) with negative controls made by probe-treated uninfected individuals (sampled from the Wolbachia-free populations of the Campania region) and no-probe controls. The occurrence or absence of Wolbachia infection in each individual used for FISH experiments was confirmed by running PCR with Wolbachia specific primers as described above on DNA extracted from the rest of body parts not used for FISH slide preparation.TATC-3’) , targeti16S rRNA . The lasethod by  with modCOI gene sequencing of 265 individuals of P. spumarius showed the occurrence of 75 haplotypes across Italy including 13 already reported by [P. spumarius into three main mitochondrial groups, namely the eastern-Mediterranean, western-Mediterranean and NE lineages, each with a specific geographic distribution  to the northernmost (Alto Adige) parts of Italy. The other haplotypes of the eastern-Mediterranean lineage were all represented by 1–2 individuals, except for H53 which reached 5.3% and 11.8% of total and eastern-Mediterranean individuals, respectively. This haplotype was recorded in five regions, from south (including Sicilia) to north Italy.The number of haplotypes identified within the western-Mediterranean lineage was lower  than in the eastern-Mediterranean lineage, and three haplotypes  accounted for 77% of total individuals. Haplotype H32 was found in all sampled regions of northern Italy, representing 43.6% of the western-Mediterranean individuals. The haplotype H29 was recorded in six regions, from north to south Italy (including Sicilia) and accounted for 8.8% and 17.6% of total and western-Mediterranean individuals, respectively. The haplotype H34 was recorded only in two regions of northern Italy and accounted for 8.2% and 16.2% of total and western-Mediterranean individuals, respectively.The haplotypes of the NE lineage were the least represented in Italy. They were not found in south-central Italy and represented only 6% of the haplotypes in northern Italy. In particular, they were found exclusively in the northernmost part of Italy (Alps of Alto Adige region) where they accounted for 29% of total individuals. In this area the western-Mediterranean haplotypes were the dominant ones (64.5%) with the most common H24 and H95 restricted to this specific region.P. spumarius were characterized by the occurrence of more than one haplotype and admixtures of different lineages were found throughout northern Italy  resulted uninfected. In northern Italy, Wolbachia was found in 11 out of 17 populations (64.7%) with an average infection rate of 40.5%   . The infnalyzed) , Fig 2 aen sexes  when datthe head  and moreP. spumarius  but showed halved values of nucleotide diversity and mean number of pairwise differences, probably due to the almost exclusive presence of haplotypes of the eastern-Mediterranean lineage. The lowest molecular diversity was found in Sicilia . Similarly, a weak geographic structure emerged when the AMOVA was run by grouping the populations by macro-area of origin . When the AMOVA was run by grouping the populations according to the presence or absence of Wolbachia infection, only a small percentage of genetic variation was explained by the infection status (P<0.001) and 54% of the variation was explained by genetic differences within populations. Similarly, for each geographic area . We were not able to sequence the HVR1 region of the other wsp allelic variant, so it was not formally included in the Wolbachia MLST database. Based on the wsp gene phylogeny  were found for coxA gene, and one (470) for fbpA gene. The five Wolbachia STs with identical wsp allelic variant  also showed invariant gatB, ftsZ and fbpA genes. Only single infections of Wolbachia were identified (no double peaks were detected in gene sequence chromatograms). BI analysis of concatenated MLST gene sequences grouped these STs in a single clade whose phylogenetic relationship was unresolved , including Wolbachia strains of P. spumarius from Carpathians and France, and the symbiont of a leafhopper species. Wolbachia ST-537 diverged by 2.2–2.5% (uncorrected p-distance) from the other Wolbachia STs found in Italian P. spumarius . Finally, the Italian STs from this study diverged from the Wolbachia strains  [gatB, coxA and hcpA genes grouped in or have a sister relationship with the Wolbachia supergroup A   had a sirgroup A . As for ergroups . The phyergroups  supportepA genes . Both ftn events  and the analysis  was congrgroup B . These rrgroup B , mirrorirgroup B .P. spumarius populations infected by Wolbachia   or northern Italy, the sole geographic area with = 0.004) . Howeveron rates , no signWolbachia infection rate differed significantly among Veneto, Piemonte and Alto Adige  , with higher values as longitude (Veneto and Piemonte vs Alto Adige) and altitude  increased  and in southern Italy (Ca1 and Si3). Overall, 72.7% of H29 individuals sampled in northern Italy (n = 11) were infected by Wolbachia, while those sampled in south-central Italy (n = 10) were not .Focusing only on individuals of the western-Mediterranean lineage, the ncreased . Individncreased . The domncreased  both witncreased . WolbachWolbachia strain and P. spumarius haplotype was found to be non-specific. Three Wolbachia STs  were each associated with more than one haplotype, belonging to different mitochondrial lineages  and was associated with five haplotypes of the western-Mediterranean lineage and two haplotypes of the NE lineage. The haplotype H06 was polymorphic for the infection within the Aa1 population, being singly infected by Wolbachia ST-539 or ST-546. Wolbachia STs 537, 538, 545 and 546 were exclusively associated to haplotypes collected in Alto Adige while ST-549 was found in all sampled regions of northern Italy except Alto Adige.The association between lineages . WolbachWolbachia bacteria were spread throughout the telotrophic ovarioles, with the highest concentration in the germarium   did not display signals   Figs. Thriocytes . Finally females . Negativ signals , confirmWolbachia infections in P. spumarius occur throughout the host range [Wolbachia infection in relation to the geographic distribution and genetic diversity of P. spumarius in Italy. In this Country the insect raised to the status of key pest being the main vector of X. fastidiosa subspecies pauca, which is devastating the olive growing [Wolbachia within the Italian populations of P. spumarius, the knowledge of which is of interest for a possible use of Wolbachia to control the spread of X. fastidiosa.st range , 54 but  growing , 40. TheWolbachia infection in P. spumarius populations followed a geographic pattern closely associated with the geographic distribution of the three mitochondrial lineages. Wolbachia was detected exclusively within the populations of northern Italy, where the western-Mediterranean lineage was dominant . Conversely, all populations sampled in south-central Italy were uninfected. In that area, the individuals of the eastern-Mediterranean lineage were prevalent (87%). The exception was Sicilia, the southernmost region, characterized by the dominance of the western-Mediterranean lineage (86%) and by a lower molecular diversity of P. spumarius compared to other regions of Italy. An exception that is explained by the founder effect and geographic isolation, being Sicilia an island. The prevalence of Wolbachia infection varied between the geographic regions of northern Italy and between P. spumarius populations in each region. The highest rates were recorded on Alps in Alto Adige, the northernmost and coldest region, where the populations differed from others in northern Italy due to the specific occurrence (29%) of individuals of the NE lineage (absent elsewhere). We observed a tendency for the infection to increase with the altitude, with the highest rates found at 1200 m (80%) and 1800 m asl (76%). Our results are in line with those by [Wolbachia infection in the populations (SW lineage) of south-central Italy while in north-eastern Italy, on Alps of Friuli-Venezia-Giulia region, two individuals of the NE lineage were infected and two of the SW lineage were uninfected.Across Italy, those by . These aP. spumarius, estimated on the mtDNA, was high and mostly explained by genetic differences within populations rather than by geographic groupings of populations. All populations were admixtures of multiple haplotypes, often unrelated and belonging to two (three in a single case) mitochondrial lineages. Of the 75 haplotypes found in Italy, 12 were shared among populations of different regions, from north to south. For example, the dominant haplotype H49 (eastern-Mediterranean lineage) was distributed in all regions of south-central Italy and two northern regions, from sea level to 1400 m asl. The distribution and frequency of some haplotypes  support the hypothesis [Overall, the genetic diversity of the Italian populations of pothesis , 46 thatpothesis ) are a cpothesis .Wolbachia in the Italian populations of P. spumarius? The spread of vertically transmitted symbionts and the prevalence of their infections within host populations depend on several factors, including the efficiency of maternal transmission, the phenotypic effect on the host and the relative fitness of infected hosts [Wolbachia within host populations and the evolution of the symbiotic interaction. The type of response can vary between Wolbachia strains, which may be susceptible to heat in some host species and cold in others [Wolbachia may vary in the distribution range of a host species and the differences observed between geographic areas may be related to climatic parameters and/or the genetic differentiation of the host populations. Our results would lead to minimize the influence of P. spumarius genetic background on Wolbachia spread. Indeed, if it is true that on the total of the haplotypes analyzed the infection rate differed among the three mitochondrial lineages, the difference between eastern-Mediterranean and western Mediterranean lineage was no longer significant when the analysis was restricted to northern Italy. This result, however, cannot be conclusive, due to the small number of haplotypes of the eastern-Mediterranean lineage sampled in northern Italy. Finally, even focusing the analysis on Alto Adige, the region with the highest prevalence of Wolbachia, the impact of the mitochondrial lineage on the infection rate was negligible, with no significant difference between western-Mediterranean and NE lineage. In a previous study [P. spumarius, with values comparable to those observed here for Italy. This difference emerged also in the Carpathians contact zone, where the infection rate was 93% for the NE lineage and 46% for the SW lineage. This is in contrast to the situation we found in the contact zone of Alto Adige, which could be explained by the genetic composition of the SW lineage, almost entirely represented by western-Mediterranean haplotypes (75% infection rate) and few uninfected eastern-Mediterranean haplotypes. The study by [Wolbachia infection rate between the western-Mediterranean and the eastern-Mediterranean lineages, but graphically showed that in the Carpathians, all haplotypes of the western-Mediterranean and the NE lineages were infected, whilst those of the eastern-Mediterranean lineage had a low infection rate. Coherently, a recent study [Wolbachia or low infection rates (4–15%) in populations of the eastern-Mediterranean lineage sampled from Greece, which were infected by multiple facultative endosymbionts with a prevalence of Rickettsia. This open the question to whether, in P. spumarius, competitive interactions between symbionts [Wolbachia infection, which could be costly to specific host genotypes. Taken together, our Italy-wide data and those of [Wolbachia infection. However, mitochondrial genes alone may not be fully informative to assess the interaction between Wolbachia and host genetic background. Although three analogous main evolutionary branches of P. spumarius emerged from the phylogeographic analysis of both mitochondrial and nuclear genes, a partial discrepancy was found between the two phylogeographic structures [Wolbachia infections [Wolbachia infection and genetic diversity of P. spumarius.What could be the factors influencing the ecology of ed hosts , 94, whied hosts –101. Amon others . Consequus study , the avestudy by  did not nt study  showed aymbionts –105 coulthose of , 54 mighructures , 47, 49.ructures , a procefections , 107. Thfections , 49. Addfections  will neeWolbachia among the Italian populations of P. spumarius. High temperatures, by pulling down the density of symbionts in the host, can negatively affect the efficiency of vertical transmission and consequently the prevalence of Wolbachia infections [P. spumarius populations were always uninfected in south-central Italy, characterized by a Mediterranean climate with a hot summer season. Second, in northern Italy, the infection rate increased with increasing longitude and altitude, i.e. passing from temperate lowland and hilly areas to cold mountain areas. Finally, the haplotype H29 of the western-Mediterranean lineage, although highly infected in northern Italy, resulted always without Wolbachia in southern Italy, in three different regions. By setting up rearings of Wolbachia-infected P. spumarius from high elevation alpine sites under warm and dry conditions of Mediterranean areas would help in identifying the main driver of this symbiosis. It is worth noting that in cold environments ovarian parapause of P. spumarius females is very short or virtually absent and eggs are formed soon after female emergence. On the contrary, in Mediterranean areas, females undergo a long ovarial summer parapause [Wolbachia, if present, are lost and therefore vertical transmission via the egg is inefficient.Our results suggest that among environmental factors, prolonged periods of heat and drought, such as those characterizing the Mediterranean climate of south-central Italy, could play a key role in shaping the geographic distribution of fections , 99, 102arapause , 109, laWolbachia in Italian populations of P. spumarius was characterized using sequence data and phylogenetic analyses of the wsp gene and the concatenated MLST genes [Wolbachia sequence types from P. spumarius. Five of them  were closely related based on concatenated MLST gene phylogeny and shared the same allelic variant of the wsp gene. Wolbachia ST-537, which was found in a single specimen of the NE lineage, had a different variant of the wsp gene and within the MSLT tree, grouped in a distantly related clade, including the Wolbachia strains of P. spumarius from Carpathians and France, and the symbiont of the leafhopper Macrosteles fascifrons (Stål). All six STs were classified in the Wolbachia supergroup B. Contrary to what reported from other geographic areas [Wolbachia strains of P. spumarius from Carpathians, attributed by [hcpA, gatB and coxA gene phylogenies since few alleles grouped in or have sister relationship to supergroup A. The ML phylogeny of concatenated MLST genes was also incongruent with the result of BI analysis, because four of the five Carpathian strains, closely related to supergroup B in BI phylogeny, now showed a sister relationship to supergroup A. The conflicting signals produced by different phylogenies can reflect the presence of recombination events in MLST genes, which can occur between Wolbachia strains of the same supergroup or of different supergroups [Wolbachia strains of P. spumarius split into three groups, including the A and the B supergroups, and an intermediate group consisting of A-B recombinant strains from Carpathians. The phylogenetic position of these strains will be better clarified through the analysis of other non-recombinant genes or a genomic approach [The diversity of ST genes . The combuted by  to superergroups –88. In fapproach , 110.Wolbachia endosymbionts can influence the evolutionary dynamics of mitochondrial DNA of host populations. Wolbachia and mitochondria are simultaneously inherited through the egg cytoplasm, and if the infected individuals have a reproductive advantage over the uninfected ones, the spread of Wolbachia will sweep from the infected populations the mitochondrial haplotypes not associated with infection. The result will be the reduction in the mtDNA diversity, but not that of nuclear markers, of the infected populations and the increased frequency of the mitochondrial haplotype that benefits the most from the association with Wolbachia [Wolbachia selective sweep, the mtDNA will evolve in a non-neutral way and a higher number of low frequency variants are expected in an infected population compared to uninfected one [COI gene molecular diversity and the increasing of Wolbachia infection rate. Comparing the populations of the three analyzed regions of northern Italy, the highest value of genetic diversity was found for populations of Alto Adige, despite showing the highest infection rates. This finding and the absence of significant deviations from neutrality led us to ruling out that a recent selective sweep has occurred in the populations of northern Italy. In accordance, even when the haplotypes were grouped according to the mitochondrial lineage, molecular diversity parameters were not negatively related with the Wolbachia infection rate. Indeed, the highest molecular diversity  was estimated for the NE lineage, the group with the highest infection rate. Furthermore, either considering all haplotypes sampled in each geographic region or focusing the analysis only on the haplotypes of the western-Mediterranean lineage , infected individuals always showed greater molecular diversity than uninfected ones, and no deviation from neutrality. As for the populations of south-central Italy, mainly represented by eastern-Mediterranean haplotypes, the variation in molecular diversity among the four geographic groups of populations (uninfected) was similar to that found among the three geographic groups (infected by Wolbachia) of northern Italy. Consistently, molecular diversity parameters did not differ much between eastern-Mediterranean and western-Mediterranean lineages, despite they differed greatly in the rate of infection. In south-central Italy, neutrality tests showed significant deviations from the condition of neutral evolution, suggesting that demographic events, such us range expansion or population bottleneck, could have determined the observed pattern of molecular diversity. Similarly, given the lack of evidence of a direct relationship between reduction in molecular diversity and increased prevalence of Wolbachia in P. spumarius populations, demographic events are more likely to have determined the pattern of molecular diversity observed in the populations of northern Italy, rather than a Wolbachia selective sweep. Coherently, the AMOVA showed that the component of genetic variability associated with the infection status was negligible in P. spumarius.olbachia , 111. Evcted one , 78, 119Wolbachia may have spread long ago in P. spumarius populations of this geographic area, and since then mutations of host mitochondrial DNA have accumulated. Consequently, evidence of selection has now been lost and cannot be detectable by neutrality tests [P. spumarius, Wolbachia infection is believed to have originated in the NE lineage after the separation of the ancestral lineage into the NE and SW lineages [Wolbachia may have occurred in the populations of the Alto Adige following the contact between the two main lineages and the transfer of Wolbachia from the NE to the SW lineage. In the event of an ancient infection, together with an increase in mtDNA diversity of the host population, the genetic diversity of Wolbachia is also expected to increase due to mutations gained after the last selective sweep, loss of strains and acquisition of new strains by horizontal transfer [Wolbachia STs infecting P. spumarius in Alto Adige. In particular, five out of six STs were closely related phylogenetically, suggesting the origin of new STs by mutation of the ST initially spread. Alternatively, NE haplotypes may have invaded the Alto Adige contact zone repeatedly over time by introducing new closely related Wolbachia strains and adding diversity in the mtDNA. Indeed, many infected haplotypes of the NE lineage were found in Alto Adige, of which four differed in the Wolbachia ST and two shared the same ST. Further, all STs were associated with individuals of the NE lineage and only two of them (ST-538 and ST-539) were shared between the NE and the western-Mediterranean lineage. The most common Wolbachia STs in the populations of Alto Adige were ST-539 and ST-538, with 53% and 27% of infected individuals, respectively. This suggests that one of them could be the strain that started the invasion. Finally, because mitochondrial mutation rates are substantially higher than those typically found in bacteria [P. spumarius, in Alto Adige we found twice the number of infected haplotypes  than the number of Wolbachia STs (five). The prevalent strain ST-539 was associated with the prevalent haplotype H24 and three H24-derived haplotypes . This suggests that Wolbachia strains, because inherited vertically, have persisted in haplotypes derived from the one originally infected. Nonetheless, horizontal transfer may have also played a role in diversifying the correlation between haplotype and infection type over time in P. spumarius populations (see below). The situation reverses in the southernmost regions of northern Italy where Wolbachia infections may have been much more recent and still in the initial phase of spread. We found that 43% of the populations sampled in these regions were not infected and the infection rate was highly variable. Further, only two Wolbachia STs were found and one of these was ST-539. The prevalent one, ST-549, closely related to ST-539, was distributed within populations of three geographic regions but not in Alto Adige, which is suggestive of its differentiation in this area after the beginning of the invasion, e.g. of ST-539. However, an insufficient size of our sample cannot be excluded as a possible cause of the differences in Wolbachia STs composition between the populations of Alto Adige and those of the other regions. Lack of selective sweep in infected populations may be the consequence of Wolbachia’s inability to invade host populations because of imperfect maternal transmission and low penetrance or absence of reproductive parasitism (e.g. CI). These factors limit the spread of symbionts in host populations, unless the symbiotic relationship evolves to increase the fitness of female hosts [Wolbachia infections are maintained in insect populations even without appreciable manipulation of host reproduction [Wolbachia are not strictly necessary for host survival and reproduction and the benefits they provide are frequently context dependent [Wolbachia in P. spumarius is still unknown. We did not find differences between the sex ratio of infected and uninfected populations, ruling out reproductive manipulations inducing female-biased sex ratios. Some authors [P. spumarius are not yet available. In the Italian populations of P. spumarius, the considerable variability of the prevalence of Wolbachia infection among the populations in northern Italy, the absence of populations with fixed infection and the lack of evidence of a recent selective sweep, lead us to exclude a strong CI. Should CI occur it is likely weak and not sufficient to ensure successful symbiont spread because of an imperfect maternal transmission and none or limited direct positive effects of Wolbachia on host fitness. We did not quantify the transmission rate and the density of Wolbachia in P. spumarius, however we did get some evidence of imperfect maternal transmission. Indeed, infected and uninfected individuals of the same haplotype were present simultaneously within infected populations  and Wolbachia did not reached fixation in any of the infected populations. Further, in populations of Alto Adige consisting of admixtures of individuals of the NE and western-Mediterranean lineages, the two lineages did not differentiate based on the infection status, showing the same high infection rate and sharing identical Wolbachia STs. A situation opposite to that observed in the Carpathians [Wolbachia may be a facultative mutualist of P. spumarius able to benefit the host in specific environmental contexts, like those at high altitudes in northern Italy, characterized by low temperature regimes and possibly specific host plants and natural enemies. In these conditions, selective forces act to maintain the infected haplotypes, both of the NE and western-Mediterranean lineages, within populations. Conversely, in temperate or Mediterranean climate environments of Italy, the benefit for the host fitness may be less than the cost of the infection and prolonged periods of high temperatures (e.g. >30°C during summer) may affect dramatically on Wolbachia density and consequently on transmission efficiency and phenotype expression. Therefore, infected haplotypes may be selected against as they are at a disadvantage compared to uninfected ones. As a result, Wolbachia infection is variable and generally low and a diversity of haplotypes is maintained within P. spumarius populations.One possible reason for the lack of data supporting a selective sweep in Alto Adige, despite the high infection prevalence, is that ty tests , 106. Inlineages . Therefotransfer , 113. Cobacteria , consistle hosts , 115. Inoduction , 117. Thoduction , 119, nuoduction  or a betoduction , 121. Hoependent . For exaependent  or envirependent  are beneependent , and theWolbachia and P. spumarius. A genuine vertical transmission of Wolbachia through the egg cytoplasm was found, according to a mechanism already highlighted in other studied systems. Wolbachia symbionts were localized within the telotrophic ovarioles of P. spumarius, with the highest concentration in the germarium, where the differentiation of the female germline cells takes place. Through the infection of germline cells, Wolbachia symbionts and other reproductive manipulators pass into the oocytes during early oogenesis [Wolbachia concentrated at the posterior pole of the oocyte during late oogenesis of P. spumarius. During embryogenesis, the posterior pole of the egg is the site where the germ cells differentiate, therefore the concentration at the posterior pole is a mechanism that increases the chance that symbionts are integrated into the germ cells and are transmitted to the progeny of the infected female [Wolbachia did not localize within bacteriocytes of P. spumarius and their way of transovarial transmission differed from that of primary symbionts. These one, as previously observed in other insects [Wolbachia does, but enter the posterior pole of the developing oocyte and accumulate in the ooplasm forming a characteristic mass . Our FISH experiments also revealed the localization of Wolbachia in the salivary glands of P. spumarius. This finding was also supported by PCR that revealed a positive signal for Wolbachia when DNA was extracted from the head of infected individuals. The localization of facultative bacterial symbionts, including Wolbachia, in the salivary glands has been observed in other species of sap-feeding insects in Auchenorrhyncha [Scaphoideus titanus Ball [Wolbachia  [P. spumarius in both agricultural and natural environments [Wolbachia strains in P. spumarius from multiple unrelated donor species  was first suggested by [P. spumarius, intra- and inter-specific horizontal transmission via plant may be a rampant process contributing to the maintenance of Wolbachia within the infected populations of northern Italy, but probably not sufficient to compensate for the loss of infections, due to imperfect maternal transmission and environmental curing.Although the nature of the symbiotic relationship  is still unknown, FISH experiments in this study confirmed a close relationship between ogenesis –129. Morogenesis , Wolbachd female –133. Wol insects –137, aftrrhyncha –141. In nus Ball , 140, fackettsia ), followckettsia . Wolbach . The latronments , 145. Alronments , thus poested by , 55. Horested by , 147. Acested by –150. In P. spumarius—X. fastidiosa is no exception [P. spumarius are only partially effective and the use of chemicals is associated to unwanted side effects. Wolbachia-based biological control methods could complement existing current tactics in reducing the populations of P. spumarius and therefore the spread and incidence of X. fastidiosa. The IIT strategy [X. fastidiosa subspecies pauca in southern Italy [P. spumarius, almost exclusively of the eastern-Mediterranean lineages, do not harbor Wolbachia and could be adversely affected by the inundative release of Wolbachia-infected males . Crossing experiments are imperative to verify whether CI actually occurs and whether it is sufficiently strong to think about an application of IIT. Alternatively, the population replacement approach [P. spumarius do not seem to allow the spread of Wolbachia infection. Moreover, for the application of the Wolbachia-based vector control, it is important to evaluate if and how Wolbachia can affect the transmission of X. fastidiosa. A prerequisite for the applicability of IIT method should be a low ability of released males to transmit X. fastidiosa to avoid that the benefit of vector population reduction is outweighed by a greater spread of the pathogen soon after the release of incompatible males. Bacterial endosymbionts are key factors regulating the interactions between vectors and phloem-limited phytopathogenic bacteria, having antagonistic or beneficial effects on plant pathogens circulating in the insect body [X. fastidiosa is expected to be less evident due to the absence of an intimate relationship between vector and pathogen. Xylella fastidiosa does not interact with vector’s endogenous factors as it colonizes the surface of precibarium and cibarium regions of the vector’s foregut and is transmitted soon after the acquisition in a propagative and non-circulative way [X. fastidiosa does not seem to vary within a vector species (while there are differences between the vector species) [Wolbachia symbionts are able to localize within the salivary glands of P. spumarius. Watery saliva injected by insect vectors into plants seems to play a role during the inoculation of X. fastidiosa [Wolbachia symbionts of P. spumarius can somehow interfere with the inoculation of X. fastidiosa through antagonistic compounds produced in the salivary glands and released into the host’s saliva is just an intriguing speculation maybe worthy to be investigated.The suppression of vector populations is pivotal for the control of vector-borne diseases, and the system xception , 151. Mestrategy , 22 coulrn Italy , 40. Theect body . Insteadtive way . Indeed,species) . We showstidiosa , 156. WhS1 FigCircle sizes are proportional to haplotype frequency. Colors correspond to the country of origin of the haplotype. Small black dot vertices represent missing or unsampled haplotypes. Diamonds on branches represent the number of mutations. No diamond on branches means one mutation.(PDF)Click here for additional data file.S2 FigWolbachia infection rate in northern (red), central (grey) and southern (green) Italy. The percentage of infected individuals is the slice of cake colored with checkered texture. Similarly, regions with populations of P. spumarius infected by Wolbachia are colored with checkered texture. The big cakes represent the Wolbachia infection rate calculated on the total number of individuals sampled in each of the three macro-areas. The small cakes represent the Wolbachia infection rate calculated on the individuals of each mitochondrial lineage. Individuals of P. spumarius from the colored regions were analyzed to assess the distribution of mitochondrial lineages. The populations analyzed for Wolbachia infection were sampled in checkered, dark grey and dark green colored regions. White regions have not been sampled. The map of Italy by Sinigagl  is licensed under CC BY-SA 3.0; it is similar but not identical (it differs for the colored regions) to the original map and is therefore for illustrative purposes only.Histograms show the frequency of the three mitochondrial lineages each one represented by a different color . Cakes show the (PDF)Click here for additional data file.S3 FiggatB gene. (B) coxA gene. (C) hpcA gene. (D) ftsZ gene. (E) fbpA gene. Alleles found in Philaenus spumarius and sequenced in this work are shown in red, while alleles sequenced by [Wolbachia STs were imported from the MLST database and are indicated on the tree with the name of the host species, the number of the ST and the supergroup letter.(A) enced by  are in b(PDF)Click here for additional data file.S4 FigPhilaenus spumarius and sequenced in this work are shown in bold red. Reference Wolbachia STs were imported from the MLST database and are indicated on the tree with the name of the host species and the number of the ST. Wolbachia strains of P. spumarius by [Wolbachia supergroups.STs found in arius by  are repo(PDF)Click here for additional data file.S5 FiggatB gene. (B) coxA gene. (C) hpcA gene. (D) ftsZ gene. (E) fbpA gene. Alleles found in Philaenus spumarius and sequenced in this work are shown in red, while alleles sequenced by [Wolbachia STs were imported from the MLST database and are indicated on the tree with the name of the host species, the number of the ST and the supergroup letter.(A) enced by  are in b(PDF)Click here for additional data file.S6 FigWolbachia STs sequenced in this work are shown in red . Wolbachia strains of P. spumarius by [arius by  are show(PDF)Click here for additional data file.S7 FiggatB gene. (B) coxA gene. (C) hpcA gene. (D) ftsZ gene. (E) fbpA gene. Alleles found in Philaenus spumarius and sequenced in this work are shown in red, while alleles sequenced by [Wolbachia STs were imported from the MLST database and are indicated with the name of the host species, the number of the ST and the supergroup letter.(A) enced by  are show(PDF)Click here for additional data file.S8 FigWolbachia-specific probe (bright red) and (B) a universal bacterial probe (bright green), contrasting with the autofluorescence background. (C) Merged image of panels A and B, and nuclei of the host cells (blue) counterstained with DAPI, showing Wolbachia bacteria (yellow) stained with both probes and the formation of the primary symbiont ball (green). (D) Developing oocyte of panel C showing the distribution of Wolbachia within the ooplasm. Bars, 100 μm.Samples stained by (A) a (PDF)Click here for additional data file.S9 FigWolbachia-specific probe (red) and a universal bacterial probe (green). (A) Bacteriomes of a Wolbachia-infected individual showing the green signal of the universal probe but no red signal of the Wolbachia probe. (B-D) Ovarioles and developing oocytes of Wolbachia-uninfected individuals with the universal probe showing the primary symbiont penetration into the oocyte and the symbiont ball. (E-F) Salivary glands of Wolbachia-uninfected individuals showing no signal of bacteria and the autofluorescence background. Bars, 100 μm.Samples simultaneously stained by a (PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 TablePhilaenus spumarius (A) and statistical analysis (G-test of independence) (B).Sex ratio in the populations of (PDF)Click here for additional data file.S4 Tablen, number of haplotypes; S, segregating sites; Hd, haplotype diversity; π, nucleotide diversity; k, mean number of pairwise differences; w, group including Wolbachia-infected populations; NE, without haplotypes of the north-eastern lineage.(PDF)Click here for additional data file.S5 Tablen, number of haplotypes; S, segregating sites; Hd, haplotype diversity; π, nucleotide diversity; k, mean number of pairwise differences.(PDF)Click here for additional data file.S6 Tablen, number of haplotypes; S, segregating sites; Hd, haplotype diversity; π, nucleotide diversity; k, mean number of pairwise differences.(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.S8 Table(PDF)Click here for additional data file.S9 Table(PDF)Click here for additional data file.S10 Table(PDF)Click here for additional data file.S11 Table(PDF)Click here for additional data file.S12 Table(XLSX)Click here for additional data file.S13 TableWolbachia STs of Philaenus spumarius sequenced in this study are indicated in bold red. Wolbachia strains of P. spumarius sequenced by [enced by  are indi(XLSX)Click here for additional data file.