FST<0.026), with significant differentiation observed only between orchards from different regions, but no isolation by distance was detected. These results are consistent with high levels of genetic mixing in holocyclic Dysaphis plantaginae populations . These findings concerning the adaptation of the rosy apple aphid have potential consequences for pest management.We used eight microsatellite loci and a set of 20 aphid samples to investigate the spatial and temporal genetic structure of rosy apple aphid populations from 13 apple orchards situated in four different regions in France. Genetic variability was very similar between orchard populations and between winged populations collected before sexual reproduction in the fall and populations collected from colonies in the spring. A very small proportion of individuals (∼2%) had identical multilocus genotypes. Genetic differentiation between orchards was low ( Dysaphis plantaginea (Hemiptera: Aphididae) is one of the most serious pests of apple trees in Europe The rosy apple aphid D. plantaginea has resulted in an intense selection regime and the development of mechanisms of insecticide resistance in the field In commercial apple tree orchards, the damage caused by even very low densities of aphids may decrease the commercial value of the crop. This economic loss justifies aphid management techniques, based principally on pesticide use. Recommendations generally suggest the use of several pesticide treatments in apple orchards: in early spring, before flowering and after flowering or in late summer D. plantaginea population may therefore provide essential information about these crucial ecological parameters.Whatever the pest management strategy applied, the likelihood of developing resistance to management depends on the ecological characteristics of the target species: its migration capability, sexual reproduction and clonal multiplication determine, at least in part, its genetic variability and, thus, its capacity to adapt to control measures. An analysis of genetic variation in the D. plantaginea almost certainly has profound consequences for its genetic variability. Like many aphid species, D. plantaginea has a cyclic parthenogenetic (or holocyclic) life cycle Plantago, to the primary host, apple trees, where the gynoparae give birth to sexual females D. plantaginea, in the fall and spring.The life cycle of D. plantaginea.In many species, cyclic parthenogenetic populations coexist with obligate parthenogenetic populations Myzus persicaeCyclic parthenogenetic aphids would be expected to display high levels of genotypic variability, due to the recombination occurring during sexual reproduction D. plantaginea species. The only data available are the preliminary results obtained by Salomon et al. et al. Little is known about the genetic diversity of the D. plantaginea populations function and possible decreases in the genetic variation of D. plantaginea on apple due to cyclic parthenogenesis.The aim of this study was to determine whether the complex mode of reproduction, with a single sexual generation and successive clonal generations, and host shift-related migration events affected genetic variation in this species. In other words, we evaluated the geographic scale over which D. plantaginea and at what level  can this differentiation be detected? (iii) Are the genetic diversity and geographic population structure of D. plantaginea stable at different parts of the life cycle and in different years?More specifically, we used a geographic and temporal sampling scheme and highly polymorphic genetic markers (microsatellite) data to address the following questions: (i) What degree of genetic variability does the rosy apple aphid display at the national scale (over the whole of France)? (ii) Is there any genetic differentiation between populations of Samples were collected according to a geographic and temporal scheme in experimental apple orchards belonging to INRA institute. Here an orchard is defined as a field of apple trees with a given management strategy and a specific tree cultivar. The term “sample” refers to as a group of aphids collected during a specific season and at a specific position in a given orchard. No specific permission was required to sample aphids in these orchards. They were collected at one location in north-western France (near Angers), one location in south-western France (near Agen), and two locations in southern France  . Dependi2O. Eight microsatellite loci for D. plantaginea  Sitobion species  Rhopalosiphon padi (R5.29B) Aphis fabae (AF93) DpL4, DpB10, S24 and Sa4Σ, and the second amplified S3.43, AF93, R5.29B and S16b. Both multiplex reactions were carried out with Qiagen multiplex PCR kits , according to the manufacturer's instructions, in a final volume of 10 µl containing 1 µl of DNA template. The forward primer for each microsatellite was labeled with a fluorescent dye, to allow the detection of PCR products on an ABI 3100 DNA sequencer . We used the following PCR program for both reactions: 95°C for 15 minutes, followed by 35 cycles of 30 s at 94°C, 90 s at 56°C, 1 min at 70°C, and 30 s at 60°C.Template material for the amplification of microsatellites by PCR was prepared from individual aphids with the “salting out” rapid extraction protocol FST) was corrected for null alleles as described by Chapuis et al. AR) on the basis of minimum sample size, with the rarefaction method stat 2.9.3 Within-population genetic diversity was estimated by calculating the number of alleles per locus, and observed and expected heterozygosities calculated with GENEPOP ver. 4.0 ISF for the population.If more than one copy of the same multilocus genotype (MLG) was observed, the null hypothesis of the same MLG being obtained repeatedly by chance through sexual reproduction was tested with Genclone ver. 2.0 K) present in the set of samples was estimated with STRUCTURE X|K), the probability of the observed set of genotypes (X), conditional on the number of genetically distinct populations, K, for values of K between 1 and the number of samples. The program was run for 105 iterations, preceded by an initial burn-in period of 2×104 iterations. Three runs were performed for each value of K, to check that estimates of Pr(X|K) were consistent between runs. The posterior probabilities, Pr(K|X), were then calculated as described by Pritchard et al. Finally, the number of distinct populations  to 34 (locus S24) alleles per microsatellite locus. Within-population genetic variability was high, with mean numbers of alleles per locus (Na) of more than seven for samples with more than 15 individuals. Allelic richness (AR), calculated on a sample of at least 15 individuals for inter-population comparisons, was between 3.9 and 4.3 , and revealed no difference in population variability between samples and between spring and fall . Consistent with this, no heterogeneity of Nei's heterozygosity was detected . All samples displayed a heterozygote deficiency, with many genotypic compositions showing departure from HW equilibrium , suggesting the presence of null alleles at these loci. Loci DPL4, DPB10 and AF93 displayed departure from HW equilibrium eight, seven and five times, respectively, in a total of 26 significant per locus and per sample tests. MICROCHECKER suggested the existence of null alleles for DPB10 and DPB4. No heterogeneity in the proportion of significant HW tests was found between samples or between spring and fall samples . Accordingly, no heterogeneity in FIS value was detected between samples or between spring and fall samples . After removal of the DPL4, DPB10 and AF93 loci from the analysis, the general heterozygote deficiency remained and no heterogeneity was apparent between samples or between spring and fall .We genotyped 532 individuals in total and found the level of genetic variation to be high. There were seven (locus ilibrium . The insNMLG/N = 0.98). Six MLGs were found in multiple copies. Each of these repeated MLGs was found in two individuals sampled from the same orchard on the same date: orchards 65 and 157 in fall 2006, orchards Bio Smoothee and Bio Melrose in Valence in spring 2007, and Agen in fall 2007. These repeated MLGs were probably generated by clonal rather than sexual reproduction . Consistent with the extensive multilocus genotypic variability observed, an analysis of the genotypic disequilibrium between each pair of loci in each sample revealed very few cases of significant linkage. No heterogeneity in the number of significant LD was found either between samples, or between spring and fall .A very high level of multilocus genotypic variability was found. PCR amplification was unsuccessful in some cases. In total, 342 individuals were genotyped with no missing data, and 336 different multilocus genotypes (MLG) were detected in these individuals  and non significant differentiation tests . Parts of orchards and orchards at the same location were therefore pooled by period for analyses of regional genetic differentiation . As the level of genetic differentiation was very low (FST = 4.3×10−3) we decided to pool the samples from each location.As null alleles were suspected for several loci, we also performed an analysis taking these null alleles into account FST<1%) genotypic differentiation was detected between Angers, Avignon and Valence in fall 2006 (FST∼2%). A low level of differentiation was found between Agen and Angers or Valence (FST∼1%) and no differentiation was detected between Angers and Valence. The same overall pattern was observed if null alleles were taken into account: significant, but low to moderate levels of differentiation between locations.Significant, but weak . Comparisons between fall 2006 and 2007 for each location revealed significant but weak  and non significant  differentiation.Only low to very low levels of differentiation were found between samples from the same location collected at different time periods. Almost no difference was found between samples collected at Valence in fall 2006, spring 2007 and fall 2007 .Very similar results were obtained when null alleles were taken into account. In this case, significant differentiation was detected in all comparisons other than that between fall 2006 and fall 2007 at Valence. No isolation by distance was detected between the 16 samples with more than 15 individuals  = 1). This was true for the default model , but also for the admixture and independent allele frequency model. Models without admixture gave inconsistent results (P(K = 2|X) = 1 and P(K = 14|X) = 1 for the correlated and independent allele frequency models, respectively). Evanno's ΔK A Bayesian analysis of population structure grouped all individuals together in a single population, regardless of their location and sampling period  the limited size of the clonal populations sharing the same genotype on secondary hosts compared to the number of different clonal genotypes present on these plants and/or 2) an extensive geographic redistribution of the aphids during their return flight to their primary hosts (but see below), leading the dilution of repeated clonal genotypes.The populations sampled in the fall, before the occurrence of recombination, were produced by lineages that had gone through several parthenogenetic generations since the last sexual event. We therefore expected to find genetic signs of clonal reproduction  in the samples collected in the fall. However, no such signs were observed. This suggests that a single yearly sexual reproduction event is sufficient to generate a high level of genetic variability and to cancel out the genetic signs of clonality, even in the fall, before the occurrence of sexual reproduction. The almost entire absence of individuals with identical multilocus genotypes in samples collected in the fall suggests that the number of individuals from an individual clone of D. plantaginea on its primary host is similar to that found in other cyclic parthenogenetic aphids, such as M. persicae in France S. avenaeR. padiBrachionus plicatilis (Müller), The high level of genetic variability found in We frequently observed heterozygote deficits associated with HW disequilibrium. Possible explanations based on previous findings for aphids include a Wahlund effect, null alleles, inbreeding and selection The Wahlund effect is the unintentional pooling of differentiated populations into a single sample, resulting in excess homozygosity Null alleles were suspected for three loci, and specific statistical treatments were carried out to take this possibility into account. A specific statistical analysis was carried out to detect loci with null alleles, but we cannot rule out the possibility that this problem occurred at a larger number of loci.Inbreeding and selection are often proposed as explanations for heterozygote deficits in sexual aphid populations D. plantaginae populations. We detected no population genetic differentiation at the regional scale or at the intra-orchard or inter-orchard level, for samples located less than 20 km apart. Classically, spatial genetic differentiation results from the balance between migration and genetic drift D. plantaginae, the use of microsatellites to assess spatial population genetic structure also provides information about selection . Our results therefore suggest that the effect of local drift or selection is largely compensated by migration. The fall and spring flights of the aphids mediating host shift are thus sufficient to homogenize genetic variability at a local and regional scale. However, we observed significant levels of population genetic differentiation at the scale of the entire country (France), between different apple-growing areas, with differences observed between Avignon, Agen, Valence and Angers. This genetic differentiation was weak  and no isolation by distance was observed, but these results nonetheless suggest that the emigration and return flights of D. plantaginae are limited by geographic distance, at regional scale at least, in France. D. plantaginea has only one winter host-plant, apple, and this species has a patchy distribution in France. This may account for the spatial limitation of migration. We also found evidence for a local dispersion component in the fall and spring. The sharing of the same multilocus genotype by a pair of individuals on the primary host in the fall, before sexual reproduction, was rare, but nonetheless observed in three instances. In each case, the two individuals sharing the same MLG were found in the same orchard. This strongly suggests that the return flight was local. In other words, this migration may connect secondary and primary hosts located close together, rather than reflecting global geographic homogenization.Another key finding of this study was the very weak spatial genetic differentiation between The situation in spring was similar to that in the fall and provides information about dispersal between primary hosts after sexual reproduction: three repeated MLGs, each shared by a single pair of individuals, were observed in three different orchards, among 118 colonies. One of the repeated MLGs corresponded to individuals collected from the same tree on two different dates and, thus, probably reflected sampling from the same aphid colony. However, in the two other cases of aphids sharing other repeated MLGs, individuals were collected from non contiguous trees, probably reflecting the dispersion of aphids between different trees in the spring. It is unknown whether such dissemination between distant trees is passive (through wind or cropping practices) or active. Overall, these findings suggest that, at the time of sampling in May, i) aphid dispersal between primary hosts occurred but was not frequent and/or ii) dispersion may have been frequent but only a small proportion of the total number of colonies was sampled. A rough estimate of the sampling effort in spring would be one colony sampled per five actual colonies, so the probability of sampling the same MLG twice or more was low.D. plantaginea was very weak or null over short distances and weak but significant over large distances, suggesting that local migration occurs in<-- --> D. plantaginea. This situation is similar to that reported for other aphid species. For instance, in R. padi, no genetic differentiation was found between populations located less than 1000 km apart S. avenaeOverall, spatial genetic differentiation in D. plantaginea is frequently subject to strong demographic bottlenecks, due to pest management practices . We tried to sample D. plantaginea on Plantago close to the primary host sampling locations at Valence, without success. This may be because (i) the populations of D. plantaginea on the secondary host are small, (ii) secondary host colonization is restricted to particular Plantago populations or to plants growing under specific favorable conditions or (iii) Plantago is not the only secondary host of D. plantaginae. It may be important to identify the entire set of actual secondary host plants of D. plantaginea and their distribution, to determine which processes may occur during the asexual phase on the secondary host plant (currently seen as a “black-box”).No such selective forces appear to shape the population genetic structure of D. plantaginea. (ii) Determination of the genetic structure of the population with microsatellites does not reveal genetic structure due to selection, because recombination during sexual reproduction breaks the linkage between adaptive alleles and microsatellite markers. (iii) Migration homogenizes genotypic frequencies, so it is not possible to determine the genetic structure of the population linked to selective forces. The balance between migration and selection was in favor of migration, as discussed below.Our results concerning the genetic structure of the rosy apple aphid population have practical implications for the management of this aphid. We found no genetic differences between samples collected from orchards planted with different cultivars . There are three possible explanations for this result: (i) None of the three apple cultivars was thought to be resistant to the rosy apple aphid, so there is probably no adaptation to these cultivars in D. plantaginea, because adaptation at any one site may lead to the failure of control everywhere. Resistance to carbamate and organophosphate insecticides has recently been found in a D. plantaginea clone collected in Avignon (Southern France) We found that migration had a larger effect than drift and selection in shaping the population genetic structure of this species at various geographic scales. The imbalance in favor of migration was found within orchards, between orchards separated by tens of meters at the same site and between sites separated by one to several hundreds of kilometers. This imbalance has two consequences: local adaptation Table S1Description of Dysaphis plantaginea samples and genetic variation within samples.(DOC)Click here for additional data file.


The Mediterranean region is particularly vulnerable to the effect of summer temperature.Within the CIRCE project this time-series study aims to quantify for the first time the effect of summer temperature in Eastern-Southern Mediterranean cities and compared it with European cities around the Mediterranean basin, evaluating city characteristics that explain between-city heterogeneity.The city-specific effect of maximum apparent temperature (Tappmax) was assessed by Generalized Estimation Equations, assuming a linear threshold model. Then, city-specific estimates were included in a random effect meta-regression analysis to investigate the effect modification by several city characteristics.Heterogeneity in the temperature-mortality relationship was observed among cities. Thresholds recorded higher values in the warmest cities of Tunis (35.5°C) and Tel-Aviv (32.8°C) while the effect of Tappmax above threshold was greater in the European cities. In Eastern-Southern Mediterranean cities a higher effect was observed among younger age groups (0–14 in Tunis and 15–64 in Tel-Aviv and Istanbul) in contrast with the European cities where the elderly population was more vulnerable. Climate conditions explained most of the observed heterogeneity and among socio-demographic and economic characteristics only health expenditure and unemployment rate were identified as effect modifiers.The high vulnerability observed in the young populations in Eastern-Southern Mediterranean cities represent a major public health problem. Considering the large political and economic changes occurring in this region as well future temperature increase due to climate change, it is important to strengthen research and public health efforts in these Mediterranean countries. The Mediterranean basin is one of the areas of the world most influenced by current and future climate change, that is already affecting mortality and morbidity related to temperature increase during warm season ,2. HealtOn the contrary no specific evaluation has been carried out in non-European Mediterranean areas, including North Africa and Middle East countries, usually exposed to more extreme temperatures during summer with respect to the other Mediterranean countries. In fact, summer conditions may vary between different regions of the Mediterranean basin, with prolonged very hot and dry summers along the north African coast and middle eastern cities, and slightly milder and shorter summers in southern Europe and some areas of Turkey.Socio-demographic characteristics of Southern Mediterranean countries are largely different from European countries with a greater proportion of young population, higher infant mortality rates, and high illiteracy rates in the working age populations, especially among women due to cultural and religious restrictions . MoreoveConsidering the large differences in terms of climate, socioeconomic and demographic characteristics between countries surrounding the Mediterranean basin, it could be expected that these characteristics may modify the effect of summer temperature leading to a large heterogeneity in the health impact across populations. However, the contribution of demographic and socioeconomic characteristics has not evaluated yet in the Mediterranean region. The only available evidence comes from US studies reporting population density ,10, raceThis study evaluated the effect of summer temperature in the Mediterranean region including for the first time Eastern-Southern Mediterranean cities, from North Africa and the Middle East. The study hypothesis is that summer temperature has an effect on mortality in these cities and that effects may be different from those observed in European cities around the Mediterranean. Since the European and Eastern-Southern Mediterranean countries are characterized by different climate conditions, demographic and socioeconomic structure and health services organization, we hypothesize that these city characteristics may have a role as determinants of between-city heterogeneity. The study is part of the CIRCE project, an EU funded project carrying out an assessment of the climate change impacts in the Mediterranean and was carried out under Research Line 9 “Human Health”.A time-series approach was followed to estimate the effect of summer temperature on mortality in 10 cities around the Mediterranean: Athens, Barcelona, Bari, Istanbul, Lisbon, Palermo, Rome, Tel-Aviv, Tunis and Valencia and to evaluate the between-city heterogeneity. Although located outside the Mediterranean basin, Lisbon was included as it lies within the Mediterranean climatic zone. The study population includes total population resident in the study cities, for an amount of about 20 million people. Time series data included years from 1991 to 2007 with at least three consecutive years for each city. The study period was restricted to the summer season (April-September) being the focus of the analysis on the effect of summer temperature.For each city, daily mortality data were obtained from local mortality registries. Daily deaths among resident population for all natural causes (ICD-9: 1–799) were considered for the following age groups: 0–14, 15–64, 65–74, 75+ years, and all ages combined. Istanbul and Tel-Aviv data were only for an old period and even the age groups 0–14 years and 75+ years were not available in Istanbul.a priori to better measure the physiological discomfort due to the combined effect of temperature and humidity than temperature alone. Apparent temperature was calculated according to the formula proposed by Kalkstein [For each city, meteorological data collected every 6-hours, were obtained from airport weather stations located closest to the city. As exposure variable maximum apparent temperature (Tappmax) was chosen alkstein , and adaalkstein :For Istanbul, due to the lack of data, the mean apparent temperature was calculated. A delayed effect of Tappmax up to 3 days (Tappmax averaged for day 0 and previous 3 days) was considered for all cities, based on an exploratory analysis throughout Distributed Lag Models (data available on request).Several characteristics at city level were considered as potential factors explaining the geographical heterogeneity in the effect of summer temperature among cities. Socio-demographical, health care and geographical indicators were retrieved from: the World Bank , OrganizThe analysis was developed in two steps, first a common model was applied to each city to obtain city-specific estimates, then results were combined in a random effect meta-analysis to explore the role of potential effect modifiers of the Tappmax-mortality relationship.The city-specific association between Tappmax at lag 0–3 and mortality was assessed by Poisson Generalized Estimation Equations (GEEs), assuming a linear threshold model. The effect of summer temperature was modeled as log-linear increase in risk above the threshold. The city-specific effect estimates of relative risks of dying for 1°C increase of Tappmax (lag 0–3) above the threshold were expressed as percent changes. Moreover, percent deaths attributable to temperatures above Tappmax threshold were calculated from relative risks . The anaThe modeling choice was driven by two main considerations: the first regards the shape of the temperature-mortality relationship and the second deals with the choice of a model able to take into account the autocorrelation structure within data. As for the first aspect, a linear threshold model was assumed to be a good approximation of a more complex non-linear model ,12,21,22A sensitivity analysis was run with different starting points to test the robustness of thresholds identification by Muggeo algorithm.The second aspect driving the model choice is linked to the structure of the data where the observations are restricted only to the summer months allowing to take into account the serial correlation within each summer period and assuming independence between summers.Specifically a first order autoregressive structure for the correlation within each summer was defined a priori based on the previous studies that involved most of the cities included here ,4. FurthModel based variance estimator for the standard errors was used as recommended in the presence of few large clusters .2, holidays, day of the week and calendar month and time to control for seasonality and long time trend respectively. Barometric pressure and wind speed were included as linear terms according to previous studies [2 was considered as indicator of traffic-related air pollution that is the dominant source of air pollution in urban areas. NO2 data were not available for Istanbul and Bari. Other pollutants were not investigated as potential confounders of the Tappmax-mortality association  because of the unavailability of data.The following variables were included as potential confounders: barometric pressure, wind speed, 24-hour mean NO studies ,4. In a Holiday, day of week and calendar month were introduced as indicator variables. Time was included as linear and quadratic term. A sensitivity analysis on alternative modeling choices for controlling seasonal and long time trends was carried out.metareg procedure in Stata [City-specific estimates were included in a random-effects meta-analysis to assess between-city heterogeneity using the maximum likelihood method in the in Stata . Then soin Stata ,13. The th percentile and 75th percentile values of the effect modifier distribution [Results were shown as predicted percent change in mortality at the 25ribution . Meta-reth percentile).To evaluate the robustness of meta-regression results and therefore possible bias in the effect estimates with respect to the choice of temperature threshold values, a sensitivity analysis was performed using city-specific estimates calculated on a more extreme temperature threshold . Turkey and Tunisia have a higher infant mortality rate (greater than 20/1000 live births) and a lower life-expectancy at birth (about 70 years) with respect to the other Nations. These countries also have the lowest hospital bed density (<3/1000 residents). The national GDP per capita divides the countries into three groups: Italy Israel and Spain with GDP around 15,000-20,000 US$, Portugal and Greece with GDP around 10,000 US$ and Turkey and Tunisia with GDP around 2,000-3,000 US$. Regarding health expenditure, a similar pattern to GDP can be observed, with Turkey and Tunisia having only 5% of GDP devoted to health care. A different pattern was observed for unemployment indicator, where Tunisia and Spain having the higher rates.Concerning exposure, Tappmax showed great variability among cities, with lowest mean values observed in Barcelona and Lisbon, and the highest values registered in Tel-Aviv, Valencia, Tunis and Athens  and in the Italian cities of Rome  and Bari , whereas the lowest effect was estimated in Valencia  and Tel-Aviv . Considering the different age groups, the effect was increasing with age in Rome, Barcelona, Lisbon, Athens, Valencia and Palermo, but in the latter two cities the effect reached the statistical significance only in the oldest age groups . In Tunis, Bari and Tel-Aviv the effect was greater in the population younger than 65 years; in particular, in Tunis within the 0–14 age group, while in Bari and Tel-Aviv in the 15–64 age group. In Istanbul the effect was similar in 15–64 and 65–74 age groups being almost twice compared with all ages population.Percent of deaths attributable to Tappmax exposure above threshold varied by cities and by age groups as found for estimates of percent increases  was the maximum Tappmax, while the other variables explained from 36.0% (mean Tappmax) to 10.4% (longitude).th and 75th percentile shows how each city characteristic modifies the effect of Tappmax on mortality. Considering temperature variables , the predicted effect is greater among the cities with a milder climate. For example, in a city with a mean Tappmax of 29.8°C (25th percentile) mortality increased +4.8% while in a city with a mean Tappmax of 34.5°C (75th percentile) the increase was +2.6%. The predicted effect was slightly lower in cities with a greater standard deviation of Tappmax. For standard deviation of relative humidity we observed the opposite, with larger effects predicted in cities with greater variability in humidity values. The predicted effect was greater in cities with lower unemployment, higher health expenditure, and those further at West.The comparison of the predicted percent changes in mortality evaluated at 25City-specific results were robust to different modeling choices regarding the correlation structure, confounders, seasonal and long time trend. Also the thresholds estimation was robust to different starting points. Meta-regression analysis rerun with extreme threshold identified the same effect modifiers (results available on request).The present study is the first to investigate the health impact of summer temperatures around the Mediterranean basin, including cities from both Europe for which some evidence is available -5, as weA possible explanation of the different results by age group in our study could be searched in the different age structure between European and Eastern-Southern Mediterranean cities. In fact, the latter cities are characterized by a proportion of elderly below the European cities and this might explain the lower effect observed in this age group. On the contrary, the strong effect in young population underlines the higher vulnerability of this subgroup that represents a meaningful portion of population in these cities, almost double compared to European ones.The higher vulnerability of older individuals during extreme summer temperatures is well known and can be attributable to the reduced thermoregulatory responses in these subjects and, in some cases, to the presence of chronic diseases, limited mobility and not being self-sufficient . ConsideIn the adult population, the effect of summer temperature is relatively uncommon, unless in developed countries. Therefore results in Bari and Tel-Aviv regarding the effect among the 15–64 year olds are difficult to interpret, although there is some evidence that in this age group the presence of specific chronic diseases or specific occupational risks may increase vulnerability to heat . HoweverOur results suggest that in the warmest cities local populations may have developed a certain degree of acclimatization to summer temperatures. In fact, the high threshold values recorded in Tunis, Tel-Aviv and Valencia underline that in these cities only very high temperatures may still have an effect on mortality. On the other hand, it should be noted that in Tel-Aviv and Valencia where the threshold is very close to the mean Tappmax value, also less extreme temperatures contribute to the estimated effects that actually are the lowest compared with the other cities. According to previous studies, threshold values are influenced by local climatic conditions as well by latitude and local resources in place to prevent health effects related to temperature extremes ,12. In tLocal climatic conditions were also the most important explanatory factor for the large heterogeneity in the effect of Tappmax between Eastern-Southern and European Mediterranean cities. In particular, we observed higher effects among cities with milder temperature (Tappmax and mean temperature) and lower variability in Tappmax. Due to physiological acclimatization, in these populations the milder or less variable climate determines a limited tolerability to extreme temperatures outside the normal range. Multicenter studies from the US found similar results, with greater summer effects in populations with milder temperatures ,10,12,36In the present study none of the socio-demographic and economic indicators was found to be a significant effect modifier of Tappmax-mortality relationship, except health expenditure and unemployment rate. A greater effect was observed in cities with a higher health expenditure that are those of more affluent countries. Similar findings were shown in US cities , while aIn the present study we estimated the effect of Tappmax using a V-shaped model with thresholds estimated using likelihood methods . In otheMore complex nonlinear functions have been used in alternative to the linear threshold model but it has been suggested that they are more suitable when comparing a large range of climates ; this isIn the present study we estimated the effects of summer temperature by using maximum apparent temperature as temperature metric. This indicator was validated in previous European multicentre studies as being able to capture both temperature and humidity effects in a single parameter ,4. OtherAs already demonstrated by previous studies ,4, a lagIn the meta-regression analysis, only few characteristics were found to be significant effect modifiers of the summer temperature effect and this could be attributable to the fact that the available indicators were referred to the national level, thus not appropriately reflecting city-level characteristics. Moreover, the limited number of study cities may have reduced the statistical power and therefore limited the ability to identify the effect modifiers as discussed also in another multicentre study . AlthougThe present study has provided for the first time evidence of the effect of summer temperature for the Mediterranean region including cities from North-Africa and the Middle East never studied before. Results indicate that this area urges more research and public health attention because of the high vulnerability of the youngest populations and the low level of economic resources that limit population capacity to adapt to extreme climate.At the same time, it is worth noting that this combined effect of climatic and socio-demographic determinants, will probably further enlarge the fraction of vulnerable population in the Eastern-Southern Mediterranean area, since large political and economic changes are already occurring , as wellFor the same reasons, future studies should mainly focus on the south of the Mediterranean including a larger number of cities, to identify the major determinants of susceptibility to summer temperature at population level, as well also to monitor them over time.EU: European Union; CIRCE: Climate change and impact research: the Mediterranean environment; US: United States; GDP: Gross domestic product; ICD-9: International classification of diseases, 9th revision; Tappmax: Maximum apparent temperature (°C); Temp: Air temperature (°C); Dew: Dew point temperature (°C); WHO: World Health Organization; OECD: Organisation for economic co-operation and development; GEEs: Generalized estimating equations; GAMs: Generalized additive models; QIC: Quasi-likelihood under the independence model criterion; NO2: Nitrogen dioxide; PM10: Particulate matter with an aerodynamic diameter of 10 μm or less.The authors declare that they have no competing interests.ML designed the study and performed the statistical analysis and contributed to the draft of the manuscript. DD conceived, coordinated and designed the study, and drafted the manuscript. MDS helped draft the manuscript. AA contributed to the statistical analysis and provided the data for Athens. BM is an expert in public health from the WHO, the coordinator of Research Line 9 - “Human Health” of the CIRCE project with ABS. KK provided expert advise on the study design and was leader of WP9.2. FKdD contributed to the meteorological database, discussion of results and draft of the manuscript. XB provided the data for Barcelona. ABS provided the data for Tunis and was the coordinator of Research Line 9 - with BM. EC provided the data for Lisbon. ZD provided the data for Istanbul. CI provided the data for Valencia. CP provided the data for Tel-Aviv. TW collaborated in coordinating the Research Line 9 of the CIRCE project. PM conceived and supervised the study and helped to draft the manuscript. All authors read and approved the final manuscript.Table S2: Country-level demographic, socioeconomic and health care indicators data sources. Table S3: Study period, descriptive statistics of meteorological variables and Tappmax thresholds. Table S4: Percent of deaths attributable to temperatures above Tappmax city-threshold, by age group.City-level population data sources. Click here for file


Salvelinus fontinalis) in Labrador to gather baseline data to facilitate monitoring of future impacts of the recently opened Trans-Labrador Highway. Six-locus microsatellite profiles were obtained from 1130 fish representing 32 populations from six local regions. Genetic diversity in brook trout populations in Labrador (average HE= 0.620) is within the spectrum of variability found in other brook trout across their northeastern range, with limited ongoing gene flow occurring between populations (average pairwise FST= 0.139). Evidence for some contribution of historical processes shaping genetic structure was inferred from an isolation-by-distance analysis, while dual routes of post-Wisconsinan recolonization were indicated by STRUCTURE analysis: K= 2 was the most likely number of genetic groups, revealing a separation between northern and west-central Labrador from all remaining populations. Our results represent the first data from the nuclear genome of brook trout in Labrador and emphasize the usefulness of microsatellite data for revealing the extent to which genetic structure is shaped by both historical and contemporary processes.In conservation genetics and management, it is important to understand the contribution of historical and contemporary processes to geographic patterns of genetic structure in order to characterize and preserve diversity. As part of a 10-year monitoring program by the Government of Newfoundland and Labrador, Canada, we measured the population genetic structure of the world's most northern native populations of brook trout ( The extent and structure of genetic variation across a species’ range are informative about both historical processes, such as the pattern of recolonization after previous glacial maxima , and conSalvelinus fontinalis Mitchill, 1814; Brook trout  following the tissue protocol. Nine microsatellite loci were initially tested: Sfo12, Sfo18 . PCRs were carried out in an Applied Biosystems GeneAmp PCR System 9700 Thermal Cycler  and contained 1X PCR Master Mix , 2 µL 10 mM of each primer and 2 µL genomic DNA (2–570 ng). Four fluorescent dyes were used to label forward primers as follows: PET –Sfo12, SfoD100; VIC –Sfo18, MST85, and SfoC129; 6-FAM –SfoD91, SfoB52, and SfoC86; NED –SfoD75. For primer pairs Sfo12, Sfo18, and MST85 the PCR profile was as follows: 95°C for 4 min; 35 cycles of 95°C for 1 min, 55°C for 45 s, and 72°C for 45 s; 72°C for 7 min. For the remaining loci the PCR profile was: 95°C for 4 min; 35 cycles of 94°C for 45 s, 56°C for 45 s and 72°C for 1.5 min; 72°C for 5 min. Prior to electrophoresis, reactions were diluted (1:4), and 1 µL was added to 8.8 µL of formamide and 0.2 µL of LIZ-500 size standard. Loci were multiplexed as follows: (1) Sfo12, Sfo18, SfoD91; (2) SfoB52, SfoC129, SfoD100; (3) SfoC86, SfoD75, MST85. Samples were electrophoresed in an Applied Biosystems 3730 DNA Analyzer using GeneScan Software . The six loci with the greatest amplification success rate  were selected for subsequent analysis.Genomic DNA was extracted from ∼25 mm2, Sfo18 , MST85  and expected (HE) heterozygosity and number of alleles (NA) in FSTAT 2.9.3 (FIS) at each locus was estimated using the NA, HO, HE) and allelic richness (A) were calculated in FSTAT for each population , as well as each region . The rank-based Kruskal–Wallis test was used to determine if differences between overall HE and A of each lake were significant. FIS values for each locus in each lake were calculated in FSTAT.Diversity at each locus was quantified by calculating observed . If RST > FST, RST will fall outside of the ρRST 95% confidence interval, and differentiation has been influenced by mutation. If it falls within this range, RST does not significantly differ from FST, indicating that mainly drift contributes to differentiation. Since we found that RST (0.153) was not significantly different from FST   , pairwisQUIN 3.1 , were usthan FST .DA distance  was performed in ARLEQUIN 3.1 , with thNei et al.'s (1983)SER 4.05 ) was useFST/(1–FST) were regressed against geographical distance  across all sampled lakes. First we wanted to discern between the possible routes of postglacial recolonization, either from a combination of Atlantic and Mississippian refugia, or from the Acadian refugium. Secondly we wanted to evaluate the overlay of contemporary patterns of gene flow. For each value of K, five iterations were run to assess convergence of the likelihood with a burn-in period of 50,000, followed by 200,000 iterations for values of K= 1 through 20. Each simulation was performed using an ancestry model incorporating admixture and correlated allele frequencies, without prior population information (K was selected in two ways: (1) as the highest value of ln Pr(X|K), as recommended by STRUCTURE 2.1  was usedormation . K was sNA) across all six loci was found, with a range of eight alleles at SfoC129 to 31 alleles at SfoD91 (HO) ranged from 0.528 (SfoC129) to 0.807 (SfoD100), with an average of 0.642. A preliminary indication of genetic structure was detected by deviations from Hardy–Weinberg equilibrium. FIS was significantly greater than zero for four loci  and overall after correction for multiple comparisons (P= 0) (P < 0.0001).A total of 1130 brook trout from 32 lakes within 19 watersheds across six regions of eastern Canada were genotyped at six microsatellite loci. An average of 17 alleles (t SfoD91 . Observes (P= 0) . SignifiHE= 0.727, A= 4.910), and lowest in Dead Dog Pond in central Labrador  (HE= 0.702) and in New Brunswick based on allelic richness (A= 8.347) and lowest in northern Labrador  (P > 0.05). However, there was a significant negative correlation between allelic richness and latitude , as well as a significant positive correlation between heterozygosity and longitude in brook trout populations found in west-central and southeastern Labrador , after outliers were removed. No significant deviations from Hardy–Weinberg equilibrium were observed per locus among lakes or overall (P > 0.05).Levels of genetic variation were highest in Farnham Brook in Nova Scotia (= 2.616) . When po= 6.272) . The KruFST measured among pairs of lakes was 0.139 and comparisons were significantly (P < 0.05) positive except those involving Dead Dog Pond . The significant pairwise FST measures ranged from 0.014 between Gilbert Lake and Feeder Pond (both in southeastern Labrador), to 0.374 between Atikonak Lake  and Alder Brook (Nova Scotia). The average pairwise FST among lakes in a watershed measured 0.070. The isolation-by-distance analysis among all populations revealed a significant correlation between FST/(1–FST) and both overland and current waterway distances, but was more highly significant for overland distances (P= 0 vs. P= 0.002).The mean P < 0.05; P= 0.03) among regions.Both AMOVA analyses detected significant geographic structuring within and among populations and regions (DA)  , the mosDA) . Followi pattern . All watK) (–19197.2) corresponds to K= 14  values plateau once the optimal K has been reached, as was the case here ase here . Bar ploWe investigated population genetic structure of brook trout in Labrador and the broader region of northeastern North America, including insular Newfoundland, Nova Scotia, and New Brunswick, by measuring variation at six microsatellite loci. We have collected the first genetic data from the nuclear genome on brook trout in Labrador, providing us with baseline data about current population structure needed to monitor future impacts of the recently opened TLH. We also make inferences about patterns of post-Wisconsinan colonization of Labrador by brook trout, which are significant in the broader context of postglacial recolonization and the biogeography of fishes in Atlantic Canada.HE= 0.620), are within the spectrum of variability observed throughout the northeastern range  only one mtDNA haplotype (401 bp of the cytochrome b gene) was found among 23 individuals. Similarly a wide-scale survey of brook trout RFLP variation showed that 98% of the fish that colonized northeastern North America were of a single haplotype . Since aplotype . ClearlyA is highest in Nova Scotia and lowest in northern Labrador (P= 0.018); this supports a route of postglacial recolonization through northeastern Canada from more southern refugia, resulting in fewer alleles in northern populations due to a bottleneck effect.As measured by allelic richness, brook trout in northeastern Canada demonstrate the characteristic “southern richness, northern purity”  pattern;Labrador . In factP > 0.05), probably because HE is lowest overall in west-central rather than northern Labrador. Part of west-central Labrador is elevated on a plateau, a type of geography with a number of consequences. Areas of higher elevations remain glaciated for longer periods, and later population settlement means less time to accumulate variation. Higher elevations may also have been more challenging to colonize, resulting in a lower number of founders and smaller effective population size. In addition, populations in higher altitudes are more likely to be isolated by waterfalls, with net emigration downstream again reducing population size and increasing isolation. Reduced heterozygosity at higher altitudes has been reported in a number of fish species  tend to have higher heterozygosities. Previous studies have shown that anadromous fish have higher levels of heterozygosity than freshwater resident fish , a trendFST measures in Labrador revealed similar levels of differentiation  to other studies of brook trout populations, including those in Maine, Québec, and Atlantic Canada . Consistent with this, the average pairwise FST among lakes within watersheds was much lower (FST= 0.070) than among lakes overall. This pattern can be explained by the freshwater resident lifestyle of the majority of brook trout in Labrador, and is consistent with tagging data indicating that brook trout are moving among lakes . By way of contrast, a microsatellite and tagging study of brook trout in Indian Bay, insular Newfoundland, revealed very little movement of brook trout among lakes (Pairwise ng lakes . The patng lakes  includinng lakes  where geng lakes .K= 14 clusters distinguishes the majority of watersheds from one another. One exception to this pattern is that Nikki's Pond (Kenamu River) has a unique cluster assignment, suggesting genetic isolation and differentiation of this lake. This could be due to a natural barrier , or to the TLH which crosses the Kenamu River watershed along a river that isolates Nikki's Pond from Mercier Lake and Brennan Lake . A second exception to the pattern is that the Traverspine River and Eagle River watersheds share a cluster assignment, although each contains a single lake with a unique cluster assignment. The genetic similarity of these two watersheds may be a historical relict reflecting temporal instability of watershed structure; as certain lakes within these separate watersheds are only ∼100 km apart they may have been connected at the time of glacial retreat. Ongoing gene flow of anadromous fish among watersheds is an unlikely explanation, as the river mouths where the watersheds drain are far apart (∼300 km).Support for a strong pattern of differentiation among watersheds was provided by the STRUCTURE analysis in which assignment of individuals into FST= 0.066) and hence membership in the same neighbor-joining dendrogram cluster  that tend to have higher levels of heterozygosity and are hypothesized to contain anadromous brook trout. The connectedness of these coastal populations, also supported by low levels of differentiation (pairwise  cluster , providePrevious studies have reported extensive loss of brook trout from their native habitat due to land development and habitat alteration . The popThe pattern of microsatellite diversity in Labrador brook trout is clearly influenced by contemporary factors, in particular watershed structure. However, a role for historical events is also evident, as despite structure at the level of watershed, certain watersheds are genetically similar, suggesting they were colonized by the same founding population, and there is little genetic differentiation among regions. The isolation-by-distance analysis also indicated that historical waterway distances have been more influential than contemporary ones in shaping the present-day genetic structure of brook trout populations in northeastern Canada. Consistent with this, brook trout populations in the northernmost portion of their range are evolutionarily young and likely not yet in equilibrium between migration and genetic drift. Current patterns of microsatellite variability therefore reflect colonization history overlaid with developing divergence due to ongoing limitations to gene flow.Microsatellites have been successfully utilized to infer colonization patterns , and canK= 2 genetic groups from the STRUCTURE analysis in which we applied the Here we detected evidence for We note that microsatellite variation is sufficient to detect the presence of two refugial sources of brook trout in Labrador, whereas previous mtDNA studies revealed little to no variation (S. Carr, pers. comm.;


A new autosomal recessive genetic condition, the SPOAN syndrome , was recently discovered in an isolated region of the State of Rio Grande do Norte in Northeast Brazil, in a population that was identified by the IBGE (Brazilian Institute of Geography and Statistics) as belonging to the Brazilian communities with the highest rates of “deficiencies” , a term used to describe diseases, malformations, and handicaps in general. This prompted us to conduct a study of consanguinity levels in five of its municipal districts by directly interviewing their inhabitants. Information on 7,639 couples (corresponding to about 40% of the whole population of the studied districts) was obtained. The research disclosed the existence of very high frequencies of consanguineous marriages, which varied from about 9% to 32%, suggesting the presence of a direct association between genetic diseases such as the SPOAN syndrome, genetic drift and inbreeding levels. This fact calls for the introduction of educational programs for the local populations, as well as for further studies aiming to identify and characterize other genetic conditions. Epidemiological strategies developed to collect inbreeding data, with the collaboration of health systems available in the region, might be very successful in the prospecting of genetic disorders. In the 1950's and 1960's, Freire-Maia and his collaborators, as well as other authors, conducted a number of studies on inbreeding levels of Brazilian populations. The overall mean value of the frequency of consanguineous marriages in Brazil was estimated to be 4.8%, corresponding to an average inbreeding coefficient F = 0.0023. As expected, the lowest inbreeding levels were detected in the southern states, whereas in some states of Northeast Brazil these rates varied from 6% to 12% ”] and for promoting family health [“Programa de Saúde da Família (PSF)”], in five municipal districts of the state of Rio Grande do Norte , all of them recently identified by IBGE as belonging to the 50 Brazilian communities with the highest rates of “deficiencies” .These five municipal districts are located in a relatively isolated rural region, 350 to 400 km away from the state capital Natal . AccordiThe data collection to determine the consanguinity levels was performed by community agents of the PACS program, under the supervision of a team of nurses of the PSF program and the scientific staff involved directly in the present study. In a two-day course, health agents and nurses were given the pertinent instructions and training as to interviewing the married couples of the communities and filling out a questionnaire with information regarding the type of possible parental consanguinity, the number of children per couple, as well as diseases, malformations, handicaps and other health problems presented by close relatives. Part of the data was collected as a pilot study, with the aim of clarifying and correcting any mistakes which might have been made by the agents trained to participate in the field study.The degree of biological relationship of the couples was obtained by direct interviews performed by the health agents and classified according to the offspring's corresponding F values , information on all couples was obtained by interviewing one or both members of each couple . In Olho d'Água do Borges and São Miguel, only a sample of individuals was interviewed , after a random selection of homes to be visited .Out of a total of 39,054 inhabitants (number estimated by the IBGE 2007 census), information on 7,639 couples  was personally obtained through direct interview of one or both members of each couple. Considering that the average number of children per married couple in the region is low, presently ranging from two to three children, it can be safely concluded that the data of most families were included in the present study.Validation of the data collected by the health agents was performed using the following method: members of the scientific staff visited some of the interviewed families in the municipal district of Pilões for a second time, and 1/8 of the interviews made by the health agents were repeated, the corresponding questionnaires being filled out again, in order to be compared with the first ones. A negligible error (corresponding to less than 3% of the couples) was detected in the description of the true degree of biological relationship.Of the five districts surveyed here, Olho d'Água do Borges, located beside a main highway and therefore characterized by a population with a higher mobility rate, presented, as expected, the lowest average frequency of consanguineous marriages (about 9%) and the smallest average inbreeding coefficient (F = 0.0032). Pilões, a population aggregate founded more recently by families originating from different communities and settlements of the region, presented the second lowest inbreeding level . Riacho de Santana (F = 0.0049) and São Miguel (F = 0.0073) showed similar average frequencies of consanguineous unions, about 19%. In Serrinha dos Pintos, more than 30% of all couples were constituted by related individuals (F = 0.0095), a fact that can be explained by the relative geographic isolation of its population, living in a highland region (on average 400 m above sea level) in the western part of the state of Rio Grande do Norte.While the frequency of consanguineous marriages is at its lowest in the state of São Paulo in Southeast Brazil , rates as high as 6 to 12% have been recorded in rural areas of Northeast Brazil . In mostIn Serrinha dos Pintos, where 32.5% of all marriages took place between biologically related individuals, the continuation of this practice is directly associated with the manifestation of at least one monogenic autosomal recessive disease, the already-mentioned SPOAN syndrome, whose gene increased in frequency in its population probably due to random genetic drift. In the specific case of this syndrome, the habit of consanguineous marriages has remained unchanged for generations. The individuals of this population are unaware of the fact that there is an association between the practice of consanguineous marriages and the rate of manifestation of genetic diseases .We suggest that the methodology developed in the present study should be applied for analyzing other populations of Northeast Brazil, in order to ascertain their consanguinity levels and also to possibly identify other genetic conditions expressed in these populations, due to the combined effects of genetic drift and inbreeding. The data so collected should be used to inform the families through educational programs on the genetic risks due to unions between close relatives.


Salix koreensisis native to Korea, Japan and China, is a deciduous broadleaved tall tree growing throughout Korea. This species is dioecius and planted along the streets and for landscaping. Along the rivers the species grows well reaching up to 20 meters in height. Trees bloom in April and fruits ripe in May. This study was carried out to investigate the genetic diversity of S. koreensis within and among 10 populations throughout South Korea using ISSR marker analysis.From 10 populations identified throughout the country 8 to 11 individual trees per population were sampled, totalling 84 individual trees.Twigs with leaves were collected and used as material for genetic analysis. DNA extraction was performed using GENE ALL plant SV DNA purification Kit  and amplified by PCR using 5 UBC (University of British Columbia) primers  which showed good results in previous experiments. PCR products were electrophoresed and gels photographed. Amplified fragments were visually scored as present (1) or absent (0). Observed and effective numbers of alleles, Shannon's information index, number of polymorphic loci, Nei's genetic distance were estimated using POPGENE 3. and genetic distance was analysed using UPGMA.S. koreensis is frequently propagated by asexual reproduction. The UPGMA dendrogram based on Nei’s genetic distance did not show any clear geographic pattern. The Seomjin river population with low genetic diversity was genetically more distant from the other populations and the Upo population was the next more distant one. Hanam and Yangchon populations were the ones more genetically related.The overall number of polymorphic ISSR amplicons was 80 and mean per primer was 16. The percentage of polymorphic loci was as follows: Namwon population had the highest level (61.25%), Seomjin river population had the lowest level (37.50%) and the overall mean was 48 %. Shannon’s Information Index indicating genetic diversity ranged from 0.17 (Seomjin river population) to 0.28 (Hanam population) with a mean of 0.24. Shannon’s information index of this species was lower than the one found in other species. This result is likely due to the fact that


The ability of invasive species to adapt to novel conditions depends on population size and environmental mismatch, but also on genetic variation. Away from their native range, invasive species confronted with novel selective pressures may display different levels of neutral versus functional genetic variation. However, the majority of invasion studies have only examined genetic variation at neutral markers, which may reveal little about how invaders adapt to novel environments. Salmonids are good model systems to examine adaptation to novel pressures because they have been translocated all over the world and represent major threats to freshwater biodiversity in the Southern Hemisphere, where they have become invasive. We examined patterns of genetic differentiation at seven putatively neutral (microsatellites) loci and one immune-related major histocompatibility complex (MHC class II-β) locus among introduced rainbow trout living in captivity (farmed) or under natural conditions  in Chilean Patagonia. A significant positive association was found between differentiation at neutral and functional markers, highlighting the role of neutral evolutionary forces in shaping genetic variation at immune-related genes in salmonids. However, functional (MHC) genetic diversity (but not microsatellite diversity) decreased with time spent in the wild since introduction, suggesting that there was selection against alleles associated with captive rearing of donor populations that do not provide an advantage in the wild. Thus, although high genetic diversity may initially enhance fitness in translocated populations, it does not necessarily reflect invasion success, as adaptation to novel conditions may result in rapid loss of functional MHC diversity. Human-mediated global change is causing the local extinction of some species but also the spread of others, resulting in novel species interactions and changes in ecosystem functioning. However, we are still far from understanding why some introduced species become invasive or whether successful invaders are also the most capable of transforming ecosystems  is one of the most widely translocated invasive fishes in the world, having been introduced to 90 different countries and being now present in all continents except Antarctica  and at a functional, immune-related locus  among translocated rainbow in Chilean Patagonia. Along with sexual selection, pathogen-driven natural selection is involved in maintaining MHC diversity in vertebrates (Piertney and Oliver n = 85), (b) recent escapees (n = 33), and (c) hybrids between escapees and naturalized trout (n = 92) as described in Consuegra et al.  fish caught by single-pass electrofishing at 10 different rivers  and 94 farmed fish collected at four nearby fish farms in the Los Lagos Region of Chilean Patagonia during 2007–2009 , 1× buffer, and 2 mmol/L MgCl2. Thermal conditions consisted of 5 min initial denaturation cycle (94°C) followed by 35 cycles of 1 min at 94°C, 1 min at 57°C, 1 min at 72°C, and a final extension cycle of 10 min at 72°C. Amplified fragments were directly sequenced using the same PCR primers and resolved in a 3130 automated sequencer . The resulting sequences were aligned using BioEdit 7.0.5.3  fragment of the exon 2 of the MHC class II-β gene, including part of the peptide-binding region (PBR), was amplified by polymerase chain reaction (PCR) using the forward primer B1RA  was calculated for the 14 study populations (10 free living and four captive) using the individual rarefaction method in PAST , as well as departures from Hardy–Weinberg equilibrium, were assessed using Arlequin 3.5. We employed analysis of molecular variance  to explore how MHC variation compared between free-living and captive populations. The relationship between population diversity at MHC and neutral markers was investigated by examining the magnitude and significance of the Pearson correlation coefficient at AR and Ho and the extent of genetic admixture was calculated using Pielou's J' evenness index  at microsatellite and MHC markers grouping the trout according to the time spent from introduction . AR  was estimated in PAST using an adjusted sample size of 30 individuals/group. Differences in allelic diversity between pairs of groups were determined by resampling 1000 times with replacement a population with the same size as the smallest of the pair  to compare patterns of change in genetic diversity (AR and Ho) between both types of markers in relation to time spent as free living in the wild. To determine which MHC class II-β alleles were contributing most to group separation we used the SIMPER permutation approach . AR and observed heterozygosity (Ho) were not statistically different between captive and free-living trout . In addition, MHC AR was positively correlated with the extent of population admixture (J') in all cases , suggesting that populations with higher neutral diversity and multiple genetic origins were also more diverse at the MHC class II locus.A total of 37 MHC class II-β alleles were identified, 12 of which represent novel sequences  based on MHC class II-β was 0.093 among all samples (P < 0.001). Differentiation was higher among trout living in captivity than among free-living populations , with pairwise FST values ranging from 0.004 to 0.215 (Genetic differentiation (to 0.215 . AMOVA aP = 0.030) and between naturalized and farmed trout (P = 0.001) but not between any of the other pairs (P > 0.9). MHC AR was lower in farmed fish than in escapees and then decreased with increasing time as free living . In contrast, no significant differences in AR  or heterozygosity  were detected among the four trout groups for neutral microsatellite loci, suggesting that diversity at these markers could be less affected by the time spent in the wild. ANCOVA analyses comparing the patterns of variation with time spent as free living between both markers revealed that the slopes between both markers were significantly different for AR (F = 168.5 P = 0.006) but not for Ho . All pairwise comparisons at microsatellite loci revealed significant genetic differentiation between the four groups of fish  and both the FEL and REL methods identified several sites to be under selection. FEL identified three positively selected and two negatively selected sites, whereas REL found 18 positively selected (three of them coincident with those identified by FEL) and two negatively selected sites. Of these, codon 47 was also identified as a potential recombination breakpoint.The Human-mediated species translocations are widely recognized as one of the main threats to global biodiversity  FST, can be problematic and must be interpreted with caution in invasive populations given that the assumption of equilibrium between mutation, drift, and gene flow may not be applicable , similar to those reported for natural populations at the species' native range  and population admixture suggests that populations founded from multiple origins not only display higher levels of neutral genetic diversity  among invasive rainbow trout with increasing time spent in the wild. We suggest that despite the potential effects of bottlenecks and genetic drift associated with invasion and the massive number of escapees and continuous introgression of domesticated trout (Consuegra et al.


We found a significant correlation between genetic distances and dietary differentiation (explaining 46% of the variation) in both the marginal test and crucially, when geographic distance was accounted for as a co-variable. These results, interpreted in the context of other possible mechanisms such as allopatry and isolation by distance, reinforce earlier studies suggesting that diet and associated habitat choice are influencing the structuring of populations in highly mobile carnivores.Recent studies on highly mobile carnivores revealed cryptic population genetic structures correlated to transitions in habitat types and prey species composition. This led to the hypothesis that natal-habitat-biased dispersal may be responsible for generating population genetic structure. However, direct evidence for the concordant ecological and genetic differentiation between populations of highly mobile mammals is rare. To address this we analyzed stable isotope profiles ( Lynx canadensis, three genetically distinct units were revealed throughout the species range, which corresponded to three demographically distinct populations, each with synchronized population cycles, inhabiting three climatic regions Lepus americanus, which de-synchronizes population cycles between regions and leads to the genetic structure et al. Alopex lagopus throughout its range corresponds to two ecotypes, one specialized on lemmings and another feeding on coastal food. Habitat and dietary differentiation has also been shown to correlate with genetic structure in the grey wolf Canis lupusOrcinus orca, it has been proposed that social learning about how and where to exploit reliable prey resources promotes philopatry, and leads to the genetic differentiation between sympatric and parapatric populations that specialize on different prey resources Recent studies on highly mobile carnivoran mammals have revealed cryptic population genetic structures that correlate to transitions in habitat types and prey species composition Haematopus ostralegus) showed no sign of genetic differentiation  It could be argued that differentiation with respect to diet may be expected among populations that have been isolated enough to differentiate genetically, and that there may be no causative relationship. However, it is not always the case that resource specialization is correlated to population genetic structure. For example, sympatric habitat specialist oystercatchers  with the analysis of genetic differentiation demonstrated that conservative habitat selection among individual dispersers served as a proximate mechanism for genetic structuring A candidate mechanism leading to genetic differentiation based on resource specialization is biased dispersal towards familiar resource or habitat. Non-random dispersal, possibly directed by suitable habitat or resource acquisition, may lead to genetic structuring and evolutionary differentiation by genetic drift. For example, a study of a local population of great tits et al.In this study, we analyzed the diet (as reflected in stable isotope profiles) of individual European grey wolves with known genotypes in an area where cryptic population genetic structure has been earlier revealed. Pilot Where diet composition could be tested, the strength of the correlation depended on the genetic markers used . However, because the species composition of ungulates (being the staple prey of wolves) strongly depends on habitat type and climate, genetically differentiated wolves may be expected to differ in diet composition, either due to differential prey choice, or another habitat cue correlating with prey composition. We therefore use stable isotope analysis to obtain discrete information on the individual diet of wolves for which microsatellite and mtDNA genotypes have been obtained in the earlier study δ13C and δ15N values) of muscle tissues of 110 grey wolves from Russia, Belarus, Latvia, Ukraine, and Poland. They represented a subsample of 317 individuals from 15 geographical regions that were analyzed for mitochondrial DNA (mtDNA) control region and 14 microsatellite loci by Pilot et al.We analyzed stable isotope profiles , because this allowed us to assess the diet obtained from an individual within a single locality, and to thereby avoid confounding signals from diet during earlier life stages (as would be reflected in bone collagen). Most wolves disperse from their natal packs, and they are able to move over distances up to 1000 km Alces alces , red deer Cervus elaphus (5), roe deer Capreolus capreolus (5), wild boar Sus scrofa (4), beaver Castor fiber (5), and brown hare Lepus europaeus (9). These species accounted for a majority of biomass consumed by wolves in Eastern European ecosystems, as inferred from feces or stomach content composition δ15N and δ13C stable isotope values) to population genetic structure.Additionally, in order to check whether stable isotope profiles accurately reflect wolf diet, we analyzed muscle tissue samples from the most common wolf prey species in Eastern Europe: moose The samples were dried at 60°C for 48 h and homogenized with a mortar and pestle. To extract lipids, dried powdered samples were placed in glass tubes and immersed in 1.5 ml of 2∶ 1 mixture of chloroform : methanol. Samples were left in a shaker for 24 hours, then left undisturbed for 30 min, centrifuged for 10 min at 8 000 rpm and the supernatant was then discarded. This process was repeated at least three times or more until the supernatant was completely clear and colorless following centrifugation. Then samples were dried at 60°C for 24 h.15N and 13C isotopic composition using an automated nitrogen-carbon analyser (ANCA) coupled to a 20/20 isotope ratio mass spectrometer (IRMS) . Two different analytical quality control samples (AQCs) were also analyzed with each batch for quality control purposes. These two AQCs were glutamic acid (δ15NAir: −5.04‰ and δ13CVPDB: −28.50‰) and leucine (δ15NAir: 10.77‰ and δ13CVPDB: −31.18‰). The international reference materials used for scale calibration were IAEA-CH6 , IAEA-600  and USGS40 . Results of the isotopic analysis were expressed as δ-values relative to the international standards VPDB (Vienna-Pee Dee Belemnite) and Air (atmospheric N) for 13C and 15N, respectively. The analysis was replicated for 11 (10%) randomly selected samples. Each replicate was processed independently starting from the stage of sample powdering. The standard deviation for the replicates was <0.2‰ for δ13C and <0.4‰ for δ15N.One mg of each sample was loaded into tin capsules. The samples were analyzed for their δ13C and 4.6±0.7‰ for δ15N, estimated for Isle Royale wolves The differences in isotopic abundance values for carbon and nitrogen among wolves in different geographical regions and subpopulations were evaluated using Kruskal–Wallis test. Relative consumption of different prey species by wolves was estimated using a dietary mixing model implemented in the program IsoSource We also applied a Bayesian stable isotope mixing model implemented in the software MixSIR FST values between geographical regions calculated based on the data on genetic variability of all 317 individuals from the earlier study δ13C and δ15N values of tissue samples, and geographic distance (latitude and longitude) as predictor variables. We then tested two additional predictor variables: vegetation types and presence/absence of the moose. Moose was the only species for which geographical range did not cover the entire study area, so that the presence/absence criterion could be applied.Stable isotope abundance values were used as a quantitative measure of dietary differences between individuals and populations that could be related to genetic differentiation and geographical distances between them. This relationship was analyzed using a distance-based redundancy analysis, which is a form of multivariate multiple regression that can be performed directly on a genetic distance response matrix forwardUsing the program DISTLM v.5 δ13C and δ15N values as two-dimensional Cartesian coordinates.Additionally, we analyzed the correlation between genetic distance and dietary differentiation between individuals and populations, using the Mantel test implemented in GenAlEx, which allowed us to graphically illustrate the dependence between these variables. In this analysis, we used the same measures of genetic distances between individuals and populations as in DISTLM analysis, a matrix of pair-wise genetic distances between individuals calculated in GenAlEx, and a matrix of isotopic distances between individuals, that was calculated by treating δ15N value for muscle tissue of Eastern European wolves was 9.3‰ (SD 1.0‰) and mean δ13C value was −24.1‰ (SD 0.9‰). The ranges of stable isotope values were broad: from 5.97‰ to 12.14‰ for δ15N and from −20.27‰ to −26.31‰ for δ13C , while differences in δ15N values were marginal , and δ13C ranging between −25.79‰ and −23.19‰ .Mean The composition of the wolf diet inferred in IsoSource based on the mean stable isotope values of the potential prey species is reported as 25th–75th percentile ranges for the relative contribution of each prey. The mean values are given for comparative purposes only and should be treated with caution Significant differences in stable isotope values among wolf subpopulations in Eastern Europe were reflected in substantial differences in their inferred diet composition. The northern subpopulations (NUC1 incorporating MIT1 and MIT2) had a higher share of moose, hare and beaver in the diet as compared with the southern subpopulations (NUC2 incorporating MIT4 and most of MIT3), which had higher share of wild boar .The estimates of wolf diet from MixSIR had wider 25th–75th percentile ranges as compared with IsoSource, and in some cases multimodal distributions see , reflectP = 0.0001 in each case). The conditional test showed that dietary differentiation was correlated with genetic distances over and above the influence of geographical distance (P = 0.0002). However, the forward selection procedure fitted geographical distance before dietary differentiation in the multiple regression model. Both variables altogether explained 10% of the genetic variability (P = 0.0001), while no significant correlation was shown for the vegetation types. In the sequential test, both these variables were significant, and they were fitted after the stable isotopic measure of dietary differentiation in the multiple regression model, with the vegetation types being the least important variable  and geographical distances (P = 0.019), and they explained 46% and 39% of the variation, respectively. The conditional test showed that dietary differentiation was correlated with genetic distances over and above the influence of geographical distance (P = 0.033) and explained 29% of variation. The forward selection procedure fitted dietary differentiation before geographical distance in the multiple regression model. Both variables altogether explained 68% of the genetic variability (P = 0.34), suggesting that there is little variation in the stable isotope signal due to geographic distance alone.The population-level DISTLM analysis showed that genetic distances between local populations were correlated with both dietary differentiation (iability . When laiability . AccordiP = 0.0006) and explained 31.5% of the genetic variation. However, in the sequential test, this variable was fitted after the dietary differentiation and geographical distance, and was non-significant , and differentiation between geographic regions calculated separately for δ13C and δ15N was significantly correlated .Moose presence/absence also was highly correlated with the genetic distance (nificant . VegetatP = 0.008) and population level (P = 0.045), and also showed that at the population level dietary differentiation explains a large percent of genetic variation  from the diet of North American wolves, and from lower δ15N values of the same or sister prey species in North America as compared to Europe In our study, the mean δ13C values for C3 and C4 terrestrial plants over broad latitudinal ranges 4 plants. However, the trophic relationships relevant to our study will be dominated by C3 plants at the base, and do not show a trend over the relevant latitudinal range (50°–60° North) in C3 plants Some portion of wolf isotopic variability potentially could be due to geographic variation  rather than dietary niche differences between populations. For example, there is an observed gradient in δ13C and δ15N is significantly correlated, and each of these two variables separately explains a similar proportion of the genetic variability.Lack of evidence for the correlation between stable isotope composition of wolf tissues and geographic distance is consistent with studies suggesting that variation in ungulate stable isotope signal is not an important factor over the relevant geographic range. We cannot exclude the possibility that plant or soil isotopic values may vary in a non-linear manner, independent of geographical distance, but such variation is much more likely to decrease the strength of the correlation between genetic differentiation and the inferred dietary differentiation than to produce a false signal for a significant correlation. Furthermore, for non-linear variation due to factors other than diet, we could expect inconsistent patterns between the two isotopes see . HoweverStable isotope composition of muscle tissue reflects the diet within a period of several weeks If seasonal patterns or variation in the stable isotope signal of prey species among the geographic regions covered by this study were significantly confounding our analyses, the effect should be to disrupt our ability to detect a particular prey species in multiple regions, and the apparent prey composition in the estimated diets should be inconsistent with data from other sources. The average wolf diet in Eastern Europe during the winter season as estimated from our stable isotope data consisted mainly of ungulates, with an admixture of beaver and hare. Stable isotope studies on North American wolves indicated similar prey choice The differences in the diet composition between local populations estimated using IsoSource also are consistent with these earlier studies, as well as with the data on prey species distribution (though we have not tested for and do not mean to imply a direct match with relative abundance): In north-eastern Europe, the moose is an important part of the ungulate community (in terms of frequency and biomass), and it is an important wolf prey In contrast to IsoSource, the MixSIR model did not give precise estimates of wolf diet due to multimodal probability distributions of prey composition. This was the effect of explicitly incorporating the uncertainty associated with dietary discrimination and isotope signatures, and analysing prey with similar isotope profiles Geographic distance is a common factor associated with genetic differentiation. In this study however, correlation data in support of isolation by distance was weak. For example, large geographic distances from east to west sometimes showed low differentiation while small distances north to south showed stronger differentiation. Even when considering latitude and longitude separately, latitude (which unlike longitude showed a significant pattern) still explained less of the genetic variance than the stable isotope data. Our correlations with geographic distance considered straight-line distances, but there are no credible impediments to wolf dispersal over the study range that would suggest a more appropriate pathway The level of neutral genetic differentiation among populations reflects the long-term patterns of effective dispersal, and an association between non-random dispersal based on prey or habitat preferences and genetic differentiation is a likely mechanism. The preference of dispersing individuals toward habitats similar to their natal habitat has been observed in a broad range of animal species The key role of diet in shaping evolutionary processes in carnivores was also indirectly inferred based on genetic and isotopic analyses of late Pleistocene and early Holocene grey wolves. It has been shown that the Pleistocene wolves in North America and Europe mainly preyed on megafaunal species like large bovids and horses 47,48, and the substantial decline of these prey species at the beginning of the Holocene coincided with complete (North America) or partial (Europe) replacement of wolf mtDNA lineages 44,47.In our study we use a metric related to diet that can be quantified from the same individuals that were also genotyped. This allows a direct comparison between the genetic differentiation among individuals and their diet . We find a stronger signal for diet correlated to genetic distance than found in an earlier study on the same populations We also found a significant correlation between genetic distance and presence/absence of the moose, which supports the hypothesis that differences in availability of particular prey types constitute an important factor shaping genetic differentiation in wolf populations. However, the forward selection procedure in DISTLM classified this variable as less important than stable isotope data, suggesting that overall dietary differentiation explains wolf genetic variability better than the presence/absence of any single species. Vegetation types were not correlated with genetic variability, unlike in the earlier study The integration of ecology and evolution is a necessary step towards major advances in our understanding of the processes that shape and maintain biodiversity Figure S1Distributions of posterior estimates of proportional contributions of prey sources in diet of Eastern European wolves inferred from the stable isotope data using MixSIR for: four subpopulations delimited based on mtDNA variability (MIT 1-MIT 4), two subpopulations delimited based on microsatellite variability , and the entire population.(PDF)Click here for additional data file.Figure S2IsoSource dietary mixing polygon for Eastern European grey wolves. The wolf δ13C and δ15N values are plotted with potential prey. Trophic enrichment values of 1.3‰ for δ13C and 4.6‰ for δ15N  were added to the mean δ13C and δ15N values of potential prey. Stable isotope profiles are presented as mean and standard deviation for the wolf and each prey species.(PDF)Click here for additional data file.Table S1δ15N and δ13C isotope profiles (‰) for 110 Eastern European wolves analyzed in this study.Sample information and (PDF)Click here for additional data file.Table S2δ15N and δ13C isotope profiles (‰) for wolf prey species analyzed in this study.Sample information and (PDF)Click here for additional data file.Table S3Diet composition of wolves inferred from the stable isotope data using MixSIR. For: (A) four subpopulations delimited based on mtDNA variability (MIT 1- MIT 4), (B) two subpopulations delimited based on microsatellite variability , and (C) all individuals pulled together. The result for the subpopulation MIT 4 is based on one individual only and therefore is biased. We report median and 25th–75th percentile (25 and 75%ile) ranges. Because in some cases the posterior distributions are multimodal, these percentiles may not adequately describe the posterior surface of the source contributions. The posterior distributions are presented in the (PDF)Click here for additional data file.Table S4Effects of ecological factors and geographical distance on genetic differentiation of Eastern European wolves. The tests were performed in the same way as those presented in Table for, but additional explanatory variables were considered. For details see (PDF)Click here for additional data file.


Palingenia longicauda (Olivier), which experienced approximately 98% range loss during the past century. Analysis of 936 bp of mitochondrial DNA of 245 extant specimens across the current range revealed a surprisingly large number of haplotypes (87), and a high level of haplotype diversity (Freshwater biodiversity has declined dramatically in Europe in recent decades. Because of massive habitat pollution and morphological degradation of water bodies, many once widespread species persist in small fractions of their original range. These range contractions are generally believed to be accompanied by loss of intraspecific genetic diversity, due to the reduction of effective population sizes and the extinction of regional genetic lineages. We aimed to assess the loss of genetic diversity and its significance for future potential reintroduction of the long-tailed mayfly  Freshwater biodiversity is declining much faster than marine or terrestrial biodiversity Palingenia longicauda  Palingenia longicauda has become a symbol of freshwater conservation efforts in Central Europe, especially in Hungary. This is due to its impressive body size (up to 10 cm) and reproductive behaviour, the spectacular synchronized swarming of millions of individuals  with a recently detected population from the Rába river in Hungary.We hypothesized that i) large-scale range loss will lead to high levels of range-wide genetic impoverishment in a once widespread aquatic species, ii) the rediscovery of this species in formerly lost ranges is due to recent range expansion, raising the hope for its re-establishment across Central Europe. We analyzed genetic diversity within the known refugial populations of We obtained a 936-bp long sequence matrix for 245 extant specimens after combining mitochondrial COI (472 bp) and 16S (464 bp) sequences. There were no gaps in the 16S alignment. We successfully amplified a short fragment (196 bp) of the mtCOI gene in 24 museum specimens . This waWe found high genetic diversity in the populations of both the Tisza and the Rába catchments. The 245 specimens comprised 87 haplotypes, most of them private to either the Tisza (76/77), or the Rába (9/10) catchments . The oveplotypes . Many ofMitochondrial COI fragments of the specimens collected historically in the Netherlands and Hungary contained the same haplotypes as the extant specimens with the exception of a single individual, collected at an unknown location in Hungary . The majIntense demographic changes of the Tisza and Rába populations were indicated by highly significant deviations from selective neutrality (Tajima's We found significant genetic differentiation between the Tisza (228 specimens) and Rába (17 specimens)  P. longicauda populations isolated in the Tisza and Rába catchments. In contrast, we found no genetic differences between the persisting Rába, and the extinct Rhine populations, despite the considerable geographic distance , and the distinct river drainages. The recovered haplotype diversity (but not the nucleotide diversity) is comparable with that of other, still widespread species. Haplotype diversity was Ameletus inopinatus Eaton Drusus discolor (Rambur) A. inopinatus and D. discolor have well diverged, geographically confined genetic lineages in European mountains.We found unexpectedly high haplotype diversity in the P. longicauda. All but one of the haplotypes were private to either the Tisza or the Rába river systems. The geographic confinement of both major Tisza haplotypes, and all satellite haplotypes from both the Tisza and the Rába catchments suggest that the extant populations of these rivers had an independent history. This is supported also by the pairwise Strongly reduced genetic diversity is frequently recorded for species which experienced extensive range losses P. longicauda. The lower Danube drainage is considered as one of the important Pleistocene refugia of freshwater species P. longicauda during the LGM in only a single refugium  cannot be completely excluded. Assuming that mitochondrial mutation rates are not considerably higher in P. longicauda than in other insects, it is difficult to explain the presence of strongly diverged private haplotypes in the Rába .The next question that arises from these results is when did the Tisza and Rába populations diverge? Evidence for differentiation between the Tisza and Rába populations   Eudontomyzon danfordi), and the Danube Gudgeon (Romanogobio uranoscopus)  Probably the most surprising result of our work is the observed high genetic diversity in the present ranges. This is likely the result of a fortunate overlap between two of the species' last glacial maximum refugia, and its current ranges. The middle-lower Danube catchment served as an important LGM refugium for many other European stream and wetland species, e.g. fishes, caddisflies, newts, etc. P. longicauda specimens from the Rhine catchment had haplotypes present today in the Rába and the Tisza systems  refugia.P. longicauda in the Rába suggests that small populations may still survive on the formerly vast distribution range. Recent observations in distant areas (e.g. the Danube Delta) P. longicauda may be just as important for their long-term conservation, as active reintroductions The long-term cryptic persistence of Palingenia longicauda is well-known since ancient times due to its size and brief, highly synchronized mass swarming. The first written record that likely refers to this species originates from Aristotle in the 4th century B.C. from Greece P. longicauda is considered an expansive pontic element, which was present in almost every large European river P. longicauda populations of the Tisza drainage and 18 larvae from the rediscovered habitat on the Rába river . We set up PCR reactions on museum specimens in a laboratory dedicated exclusively for the pre-PCR treatment of non-invasively collected or ancient mammalian material. Amplicons were sequenced on an ABI 3730 DNA Analyzer (Applied Biosystems). We manually edited the tracefiles and aligned sequences in BioEdit We extracted DNA using the DNeasy Blood & Tissue Kit for the extant specimens, and the QIAamp DNA Investigator Kit for the museum specimens , according to the manufacturers protocols. Legs of the dried museum individuals were homogenized in a Qiagen TissueLyser II. We extracted DNA from museum specimens in a laboratory dedicated to the pre-PCR processing of non-invasively collected and historic specimens. The laboratory is physically strictly separated from other DNA-laboratories in order to prevent any contamination from samples with high DNA content. Standard routines for the processing of historic samples were considered, such as strict rules for laboratory access, use of filter tips, and regular decontamination of equipment. In addition, negative controls were included in all extraction and amplification steps as contamination check. We amplified an approximately 600 bp long fragment of the mtCOI gene with the primers Jerry We ensured that the mtCOI and 16S datasets do not contain conflicting phylogenetic information by visually inspecting the topology of two bootstrapped  neighbor-joining trees in SeaView v. 4 We reconstructed changes in effective population sizes using a Bayesian skyline plot We estimated the divergence, lineage sorting, ongoing gene flow and the likelihood of migration of the Tisza (population 1) and the Rába (population 2) in IMa Table S1Collection data of extant specimens. Abbreviations: HNP – Hortobágy National Park; KMNP – Körös-Maros National Park; KNP – Kunsági National Park; BUNP – Balaton Uplands National Park; KM – Kristóf Málnás; BAP – BioAqua Pro Ltd.(DOC)Click here for additional data file.Table S2Collection data. Historically collected P. longicauda specimens processed for amplifying a short mtCOI fragment .(DOC)Click here for additional data file.Table S3Pairwise  values (lower left) and significant ETPD (upper right) results of extant populations. The tests are calculated with combined mtCOI and 16S sequences (936 bp). (XLS)Click here for additional data file.Table S4Pairwise  values (lower left) and significant ETPD (upper right) results of extant and museum (Rhine) populations. The tests are calculated with combined the short mtCOI sequences (196 bp). (XLS)Click here for additional data file.