Colletes inaequalis in an urbanized landscape. First, we developed a predictive spatial model to detect suitable nesting habitat for this ground nesting bee and to inform our field search for nests. We genotyped 18 microsatellites in 548 female individuals collected from nest aggregations throughout the study area. Genetic relatedness estimates revealed that genetic similarity among individuals was slightly greater within nest aggregations than among randomly chosen individuals. However, genetic structure among nest aggregations was low (Nei’s GST = 0.011). Reconstruction of parental genotypes revealed greater genetic relatedness among females than among males within nest aggregations, suggesting male-mediated dispersal as a potentially important mechanism of population connectivity and inbreeding avoidance. Size of nesting patch was positively correlated with effective population size, but not with other estimators of genetic diversity. We detected a positive trend between geographic distance and genetic differentiation between nest aggregations. Our landscape genetic models suggest that increased urbanization is likely associated with higher levels of inbreeding. Overall, these findings emphasize the importance of density and distribution of suitable nesting patches for enhancing bee population abundance and connectivity in human dominated habitats and highlights the critical contribution of landscape genetic studies for enhanced conservation and management of native pollinators.Bees are the primary pollinators of flowering plants in almost all ecosystems. Worldwide declines in bee populations have raised awareness about the importance of their ecological role in maintaining ecosystem functioning. The naturally strong philopatric behavior that some bee species show can be detrimental to population viability through increased probability of inbreeding. Furthermore, bee populations found in human-altered landscapes, such as urban areas, can experience lower levels of gene flow and effective population sizes, increasing potential for inbreeding depression in wild bee populations. In this study, we investigated the fine-scale population structure of the solitary bee  Bees are the main pollinators of flowering plants in natural and agricultural areas ,2. AboutFor many bee species, genetic population structure at fine geographic scales is unexpected due to their high mobility and potential for long distance dispersal. Even though species in highly fragmented habitats can show moderate to high levels of genetic differentiation ,15, studFine-scale population structure as a result of philopatric behavior can provide evolutionary benefits under certain conditions. For example, to maintain local adaptation to environmental conditions , decreasMany characteristics of bees make them particularly prone to inbreeding depression under conditions of low genetic diversity or systematic inbreeding . As haplOne possible mechanism of inbreeding avoidance in bee species is pre-mating kin recognition through identification of cuticular hydrocarbons, or pheromones ,41. BeesAnother contributor to greater inbreeding in wild populations is reductions in population size due to habitat loss . Urban aColletes inaequalis. This bee species is a wild pollinator of early blooming trees such as red maple and willow trees, and it is potentially important for blueberry and apple pollination in eastern North America [C. inaequalis is solitary, meaning each female independently builds a nest  individuals within a nest aggregation are inbred and more genetically related to each other than would be expected at random, (2) females within a nest aggregation are more genetically related than males, and (3) distribution and patchiness of suitable nesting habitat influence the distribution of genetic variability among nest aggregations in an urban landscape. Due to the ecological characteristics of this species, that include strong female philopatric behavior, short life spans and highly patchy distribution of nesting resources, we have three predictions about the fine-scale genetic structure of this species: (1) relatedness is higher within rather than among nest aggregations, (2) dispersal is male mediated, and (3) distance among nest aggregations is an important explanatory variable of genetic connectivity. Results from this study are relevant for bee conservation, especially as they provide insights into the mechanisms of inbreeding avoidance in solitary bees and how these play out in a human-modified landscape. Our findings also reveal landscape features necessary to maintain, and potentially enhance, bee population connectivity, minimize the risk of inbreeding depression in urban/suburban populations, and preserve the vital ecosystem services these bees provide. Finally, the management recommendations based on results from this study also have implications for many native species that share similar phenology and nest habitat specialization.In this study, we investigate the fine scale genetic structure (<4 km) of C. inaequalis is not an endangered or protected species. Private land owners: Daniel Peck (N10), James Liebherr and Ann Hajek (N7), Mary Smith (N9). No permission was required for N1, N2, N3, N4, N5, N6, N8, and N11. Nest names (N1-N11) and their GPS coordinates are detailed on Research sites sampled in this study included Cornell University property , private  and public land . Permissions to sample sites located on Cornell University and privately owned lands were respectively granted by the Cornell University grounds department and landowners. No permit was required for sites located on public land. Bees were not sampled from national parks or protected areas. C. inaequalis in urban/suburban areas of Ithaca, Geneva, and Rochester   surrounded by rural areas consisting of agricultural fields, extensive forests, and scattered residences. The urban area and perimeter contain a large number of gardens and forested areas [2, ca. 13,000 people) but, unlike Ithaca, it is surrounded by agricultural land dedicated mainly to apple, grape and pumpkin production. Rochester is a heavily urbanized area  with a dense city park system within the perimeter of the city. The climate in Central New York is characterized by large variations in temperature throughout the year with cold winters reaching temperatures of -18°C, and warm and humid summers that reach 32°C.We studied nest aggregations of rk, USA) . Ithaca ed areas  that proC. inaequalis requires at least three basic components to nest in a particular area: sparsely grassy areas, sandy soils [vs unsuitable) for four landscape components: grassy areas, slope, aspect of the slope and soil type. To identify areas of the landscape with sparse grass, we performed an unsupervised classification of high resolution orthoimagery of the Ithaca area from 2007 , with a spatial resolution of. 3 m and. 6 m in urban and rural areas, respectively. We used ERDAS IMAGINE v.9 to classify the landscape into 20 categories based on intervals of wavelength in the grayscale. We chose four categories, as indicators of suitable habitat, based on the categories where two nest aggregations were located prior to model development. In addition, USGS digital elevation models, with a 10 m resolution, were used to classify areas with slope greater than 5° and aspect between 135° and 215°, as suitable nesting habitat for bees. To identify areas of suitable soil type, we used the Soil Survey Geographic Database (SSURGO) to select areas with >30% proportion of sandy soil. However, the soil type for the city of Ithaca is classified as suburban soil, with no further information about percent sand in the soil. Therefore, this fourth landscape variable was not included in our final model. All binary layers were overlaid in a simple multiplicative fashion in ArcGIS v.10.2, whereby the lack of suitability in any single spatial layer rendered a location unsuitable for nesting. The final map output was a suitability model that indicated suitable areas for nesting for C. inaequalis throughout an area approximately 40 km2 surrounding Ithaca, NY.We informed our sampling scheme in the city of Ithaca by building a spatial suitability model to predict the distribution of nesting sites in the area. We chose biologically meaningful variables for our spatial model based on literature of nesting biology ,52 of thdy soils . Thus, wC. inaequalis. At each nest aggregation, we collected and mixed soil from three 30 cm core samples, after removing the top 5 cm from the sample to avoid confounding factors from topsoil additions. Subsequently, a soil texture analysis was performed to determine composition and percent sand in all the nesting sites.Due to the lack of fine-scale data for soil type inside the city, we analyzed soil samples in 11 nest aggregations in the city of Ithaca to gauge the soil type specificity of C. inaequalis nesting sites. First, we searched for 1) presence of a nest aggregation in areas predicted by the model, and 2) absence of nest aggregations in areas not predicted by the model. Twenty random points of each category were selected within our study area using Hawth’s tools [We used two sampling approaches to estimate sensitivity (true positives) and specificity (true negatives) of the suitability model and to inform our field search for ’s tools ; all 40 ’s tools , we visiC. inaequalis across the landscape and indicate a greater likelihood of finding nests when using our predictive model.To further validate our spatial model, we performed a bootstrap analysis to test whether the use of our spatial model increased the probability of finding suitable nesting sites. First we estimated the total area of suitable nesting habitat, as predicted by our model, in the vicinity of all known nesting areas, using 100 m and 300 m buffers. Then, we randomly chose 10,000 sites of equal area through a resampling procedure performed in Python 2.6, and compared these to the areas around the known nesting sites using a Kolmogorov-Smirnov (K-S) test performed in R. We ran the K-S test 10,000 times and calculated the proportion of statistically significant values indicating whether the amount of predicted suitable area was greater around known nesting sites than at randomly chosen sites. A higher proportion of significant values would support our choice of environmental variables that determine the distribution of C. inaequalis using insect nets. Specimens were placed in vials with 95% ethanol and stored at -20°C until laboratory analysis. Sampled nest aggregations included sites within the suburban/urban areas of Ithaca (n = 9), Geneva (n = 1) and Rochester (n = 1)   . We usedrk, USA)  in AprilC. inaequalis  that were multiplexed according to López-Uribe et al. [DNA was extracted from individual antennae using 150 μl of a 10% Chelex 100 solution, 5 μl of Proteinase K and incubated at 55°C for 1 h and at 99°C for 30 min . Superna1 = 0.5, Δ2 = 0.5), paternal halfsibs , maternal halfsibs  and unrelated  individuals. We ran 106 simulated pairs of genotypes and set the confidence level at 0.05. We regenotyped 18% of our samples to estimate genotyping error, which varied from 0 to 0.02 depending on the locus (We estimated the power of the microsatellite markers to infer family relatedness using the software KinInfor v.1 . The powhe locus . To idenNa), observed and expected heterozygosity (HO and HS), using the software GenoDive v.2.0b25 [FIS) were estimated in FSTAT v.2.9.3.2 [Presence of null alleles and large allele dropouts were detected in MicroChecker v.2.2.3 . Hardy-Wv.2.0b25 . Allele .2.9.3.2 . Sibship.2.9.3.2  by runniGST, Hedrick’s GST, Jost’s Dest) in GenoDive v.2.0b25 [GST was estimated using the fstat function in the R package “adegenet” [QGt) because of its robustness to differences in the number of alleles per locus, the allele frequency distribution, sample size, and the presence of inbreeding [We used three different methods to test the hypothesis of non-random mating between individuals within and between nest aggregations. First, we tested for the overall population genetic structure using three G-statistics  using Mantel tests, as implemented in program zt [FIS) and (3) genetic composition  of C. inaequalis within the Ithaca study area. The sPCA analysis, performed in the R package “adegenet” [We calculated the area of each nest aggregation as the minimal area that encompassed all nest entrances at each site, and statistically tested this variable as a predictor of within nest aggregation genetic diversity . Pairwise Euclidean distance between nests was tested as a predictive variable of genetic differentiation  of the particles in the soil samples .Our predictive spatial model indicated that 24% of the area in the city of Ithaca could be suitable nesting habitat for aequalis . Using t samples . Our modSignificant evidence of null alleles was detected for loci CI35 and CI87 across all nest aggregations ; therefoNa) of 7.73 (±0.443), allele richness (Ar) corrected for sample size of 6.38 (±0.191), and observed and expected heterozygosity of 0.568 (±0.055) and 0.615 (±0.061), respectively , suggesting deviations from random mating across the study area. For pairwise Nei’s GST, 10 out of the 36 comparisons were significantly different than zero .All G-statistic estimators indicated significant (p<0.005) but weak genetic structure (Nei’s han zero . When neQGt = 0.003± 0.0134). Overall, average relatedness was higher among individuals within nest aggregations than among randomly chosen individuals (P< 0.01). However, the difference in relatedness between groups was variable when each nest was assessed independently (Mean levels of relatedness within nest aggregations were low and exhibited large variances (endently . The spaendently .mean = 60). Other methods of estimation resulted in ‘infinite’ population sizes, suggesting that larger sample sizes would be necessary for accurate parameter estimation [(P< 0.0001)  .r = 0.71, P = 0.03), but not with number of alleles or expected heterozygosity. The relationship between FST and geographic distance showed a positive trend but non-significant trend, partially explaining the genetic differentiation between nest aggregations across the landscape  (We found a significant correlation between size of nesting patch and effective population size (= 0.096) . The plo= 0.096) . However= 0.096) . Rather,= 0.096) . Results= 0.096) .C. inaequalis mate with individuals from the same nest aggregation more frequently than expected under panmixia. In fact, we detected significant genetic differentiation among nest aggregations sampled within a ~14 km2 area, demonstrating that solitary bee populations can show genetic structure at small geographic scales despite their ability to fly long-range [Our results from G-statistics, relatedness, and spatial autocorrelation support the hypothesis that ng-range . These rng-range ,78–80. HOne factor that could reduce the long term stability of bee populations across the landscape would be high levels of inbreeding. Indeed, presence of intranidal mating in several solitary bees with gregarious nesting ,81 suggeC. inaequalis nest aggregations were small  and significantly lower than census population sizes. In most sampled nest aggregations, we observed number of individuals (census population sizes) that varied between hundreds to thousands per site (M. M. López-Uribe pers. obs.). Presence of negative assortative mating, the second possible mechanism of inbreeding avoidance, has been demonstrated in the congeneric European species C. cunicularius [C. inaequalis as males of this species are attracted to females of the closely related species C. validus [The first mechanism, maintenance of large effective population sizes, is not strongly supported by our data. On average, effective population sizes of cularius . However validus . TherefoC. inaequalis females are receptive for most of their lives and can probably mate more than once [C. inaequalis may dramatically increase coancestry of individuals at natal sites and males have the capacity for kin discrimination [We hypothesize that the most likely demographic process that explains the observed pattern of low genetic structure and lack of inbreeding is male-based dispersal. Within nesting sites, females were more genetically related than males, indicating a sex bias in migration rates. Sex-biased dispersal is a common behavior in mammals, birds and reptiles ,82 and hhan once . Becausemination , we intemination  and longmination .C. inaequalis, and possibly other ground nesting solitary bees, disperse less than males and may have limited colonization capacity, especially where suitable nesting habitat is limited and patchily distributed. Management strategies for bee conservation generally focus on improving habitat quality for females because they provide the majority of pollination services, while males are short-lived. Our results draw attention to the important role of males for bee population dynamics and long-term persistence, as they are likely the mediators of population connectivity.Three aspects of our results highlight the importance of maintaining high densities of suitable nesting patches distributed across the landscape for the enhancement of solitary bee populations in urban areas. First, we detected a positive trend between genetic structure and geographic distance among nest aggregations, suggesting that nest aggregations too far apart could become genetically isolated. Second, effective population sizes of the nest aggregations were positively correlated with the size of the nesting patches. Thus, it is likely that increasing the amount of available suitable nesting habitat in the landscape may increase bee population abundance and maintain long term population stability. Third, females of Urbanization can negatively impact local and regional levels of gene flow in native bees . Even thS1 TableParticles are expressed in percent sand, clay and silt.(DOCX)Click here for additional data file.S2 TableNa), observed heterozygosity (HO), heterozygosity within subpopulation (HT), inbreeding coefficient (HDEF), genotyping error and significance of null alleles. Stars denote levels of significance for the presence of null alleles .Number of alleles ((DOCX)Click here for additional data file.S3 TableGst values based on 15 microsatellite markers (below) and pairwise distance among nest aggregations (above). Numbers in bold denote significant differentiation at the alpha-level 0.05.Nei’s (DOCX)Click here for additional data file.


Lupinus angustifolius L., a potential forage crop. In the study ten L. angustifolius genotypes, including three Polish cultivars, two Australian cultivars, three mutants originated from cv. ‘Emir’, and one Belarusian and one Australian breeding line were evaluated. The highest seed coat percentage was recorded in cultivars ‘Sonet’ and ‘Emir’. The lowest seed coat thickness percentage (below 20%) was noted for breeding lines 11257-19, LAG24 and cultivar ‘Zeus’ . Despite having low seed weight, the Australian line no. 11257-19 was characterized by a desirable proportion of seed coat to the weight of seeds. In general, estimation of the correlation coefficient indicated a tendency that larger seeds had thinner coats. Scanning Electron Microscopy images showed low variation of seed coat sculpture and the top of seeds covered with a cuticle. Most of the studied genotypes were characterized by a cristatepapillate seed coat surface, formed by elongated polygonal cells. Only breeding line no. 11267-19 had a different shape of the cells building the surface layer of the coat. In order to illustrate genetic diversity among the genotypes tested, 24 ISSR primers were used. They generated a total of 161 polymorphic amplification products in 10 evaluated narrow-leaved lupin genotypes.The paper investigates seed coat characteristics  in  Lupinus luteus, the narrow-leaved lupin (NLL) L. angustifolius is the main lupin species currently cultivated in Poland and world-wide. As compared with the yellow or white lupine Lupinus albus, the narrow-leaved lupin has grown in importance during the recent years due to its improved yield, lowered seed alkaloid level and acquisition through selection of forms characterized by field tolerance to anthracnose Besides the yellow lupin Lupinus angustifolius mutations from cv. ‘Emir’ and subsequent selection, it was possible to obtain forms of 24–30% proportion of pod walls in a pod Lupinus to be divided into two sections, smooth-seeded and rough-seeded, confirmed also by scanning electron microscope (SEM) studies Research on lupins and also that on other leguminous plants, such as the pea, bean or soybean, involves studies which are aimed at acquisition of thin-walled pods and the thinnest possible seed coat The breeding process of lupins requires genetic analysis and application of the new biotechnological methods to be more intensified and accelerated. The knowledge about the lupine genome organization and genetic background of important genes is still limited For the study, seeds of ten narrow-leaved lupin genotypes were chosen, which included three Polish and two Australian cultivars, three mutants of cultivar ‘Emir’, an Australian and a Belarusian breeding line .Experiments with selected genotypes of lupin were conducted in two consecutive years, 2006 and 2007, according to the method of randomized block design in three replications. For each genotype, samples of pods with seeds were randomly collected  at the stage of physiological maturity. The seeds were removed from the pods, and then from each sample ten healthy seeds were selected randomly and weighed. The 1000-seed weight was calculated. Evaluation of the seed coat proportion was conducted according to Clements et al. Seeds were hulled for each replicate. Ten healthy seeds were randomly selected and weighed. They were then placed in Petri dishes in a small amount of water at 4°C for 24 h. From swollen seeds the seed coat was removed so that the cotyledons and embryo were not damaged. Prepared samples of the seed coats, seed and cotyledons with embryo were dried separately for 48 h at 105°C, and then dried to dry matter. The dried seed coats and embryos with cotyledons were placed in a desiccator to cool and then weighed separately.The seed coat proportion was reported as a percentage relative to the dry weight of seed coats of whole seeds.ijkl is the weight of 1000 seeds and seed coat of the levels k of genotype (G), i of year (Y) factors, R(E)j(i) is the effect of the j block within the i years, GYik is the interaction effect of the k genotypes with the i years, eijkl is the random error component. In A variance analysis was carried out in order to verify the hypothesis of lack of difference in the means of the genotypes studied with respect to the 1000-seed weight and the seed coat proportion. The analysis of variance (ANOVA) model for the individual data is as follows:The mean values of these two traits were grouped using the Duncan test. To show the relationship between the traits under analysis, a correlation coefficient and linear regression were calculated.The seed surface and cross-sections were observed by SEM in four replications (seeds).Specimens were coated with gold using a JEOL JFC 1200 ion coater and observed in a JEOL JSM – 5310 LV scanning electron microscope under 20 kV. In 2 4 mM , 0.53 µM primer and 45 ng of DNA using a Biometra thermal cycler for 40 cycles. After initial denaturation for 5 min at 94°C, each cycle was composed of 30-sec denaturation at 94°C, 30-s annealing at 43–53°C depending on the primer  method of Doyle & Doyle e primer , 45-s exFor grouping objects based on genetic distances cluster analysis (CA) was performed using the unweighted pair-group method with arithmetic mean (UPGMA). Each fragment that had been amplified was treated as a unit character and scored in terms of a binary code (1/0 = +/−). Data were entered in the Qiaxcel Screening Gel Software  and in a Microsoft Excel spreadsheet to create a binary matrix. The basic genetic diversity parameters were calculated: the polymorphism of amplification products (P – %), the mean number of observed alleles (na), the mean Nei gene diversity index (Dh) and PIC (Polymorphism Information Content), using the PowerMarker Software The dendrogram was developed with the Treecon ver 1.34b software The performed variance analysis revealed significant diversification of the genotypes studied with regard to the proportion of the seed coat to the seed size. What is more, the climatic conditions were found to significantly affect the analyzed traits . The thiAn analysis of dependence between the size of seeds and the percentage of seed coat based on the correlation coefficient  revealed a negative correlation , similarThe formation, structure and morphology of the seed coat in legumes have been widely analysed  to 0.311 (between cvs ‘Zeus’ and ‘Yorrel’) . The genGMA tree . Two maiGMA tree , Table 5LupinusAlso, available literature provides sample information on the use of molecular markers, including ISSR markers, for evaluating genetic diversity in the genus L. angustifolius, L. luteus and L. albus. The genetic distances calculated with the Jaccard coefficient ranged from 0.37 to 0.96. Range of diversity obtained in this was lower. Yorgancilar et al. Yorgancilar et al. amplified fragment length polymorphism) and ISSR markers to evaluate the genetic diversity among 88 genotypes of L. angustifolius. Molecular markers grouped modern cultivars as sub-clusters within the wider diversity of wild germplasm. The three ISSR primers considered for analysis gave a total of 25 bands (8.3 bands per primer and 5.6 bands per primer and accession), producing a total of 6 monomorphic bands . The similarity between accessions ranged from a minimum of 0.5185 to a maximum of 0.9767, the average amounting to 0.7681. In our studies mean value of bands per primer was higher, similar as percentage of monomorphic products. These differences could resulted from higher number of primers used in our experiment. Talhinhas et al. - consisted domesticated and wild accession. We tested only cultivated genotypes.Talhinhas et al. L. angustifolius gathered in the collection at the Department of Genetics, Plant Breading and Seed Production, Wrocław University of Environmental and Life Sciences. A whole set of UBC ISSR primers were pre-tested. Finally, using a set of 24 primers, ten genotypes were tested. The high repeatability of ISSR markers Lupinus. ISSR markers are thought to be particularly useful for study of closely related individuals that exhibit low levels of polymorphism L. angustifolius. This set of primers could be used in further studies on the genetic diversity of L. angustifolius in the breeding materials. The outcome of such research would facilitate the selection of appropriate genotypes for production of hybrids with a thin seed coat and for development of a mapping population. Subsequent research efforts should focus on the identification of quantitative trait loci (QTL) responsible for this trait in order to increase the breeding intensity with the application of marker-assisted selection (MAS).The undertaken study was the first attempt to investigate the genetic diversity of Breeding lines no. 11257-19 and LAG24, mutant line no. 41H and cultivar ‘Zeus’ can be used in breeding programmes as parental forms, to enhance the values of the seed coat thickness in the segregating progenies or further generations. Through inducing mutations and subsequent selection, desirable genotypes with a low percentage of seed coat in seeds have been obtained.Mutant line no. 41H, derived from the cultivar ‘Emir’, had a significantly thinner seed coat as compared with the initial cultivar.Moreover, the shape of cells that build the surface layer of the seed coat in lupins is species-specific and can be used as a diagnostic character not only in the taxonomy of two sections: smooth-seeded and rough-seeded. The exeption is line 11267-19 which was characterized by different shape of the cells building the surface layer of the coat.L. angustifolius breeding, and for development of a mapping population. Subsequent research efforts should focus on the identification of molecular markers (MAS) correlated with the thickness of the seed coat.Molecular and phenotypic studies facilitate the selection of favourable genotypes for the production of hybrids with the thickness coat in


Ginkgo biloba and the co‐occurring Cercidiphyllum japonicum (published data). Phylogenetic, coalescent, and comparative data suggest that Ginkgo population structure is regional, dichotomous (to west–east refugia), and formed ˜51 kya, resulting from random genetic drift during the last glaciation. This split is far younger than the north–south population structure of Cercidiphyllum (~1.89 Mya). Significant (recent) unidirectional gene flow has not homogenized the two Ginkgo refugia, despite 2Nm > 1. Prior to this split, gene flow was potentially higher, resulting in conflicting support for a priori hypotheses that view isolation as an explanation for the variation in postglacial range limits. Isolation hypotheses (with or without gene flow) are thus not necessarily mutually exclusive due to temporal variation of gene flow and genetic drift. In comparison with Cercidiphyllum, the restricted range of Ginkgo has been facilitated by uncompetitive life history traits associated with seed ecology, highlighting the importance of both demography and lifetime reproductive success when interpreting range shifts.Postglacial expansion to former range limits varies substantially among species of temperate deciduous forests in eastern Asia. Isolation hypotheses (with or without gene flow) have been proposed to explain this variance, but they ignore detailed population dynamics spanning geological time and neglect the role of life history traits. Using population genetics to uncover these dynamics across their Asian range, we infer processes that formed the disjunct distributions of  Ginkgo, Metasequoia, Cercidiphyllum, Davidia) presently extends from southwest China eastward across the Yangtze Valley to the east coast and as far as southern Japan  as habitat loss (and gain) in different areas of temperate and subtropical China is thought to have occurred   may explain the variation in the degree of postglacial expansion to former range limits between temperate deciduous forest plants in eastern Asia. Qian and Ricklefs  hypothesGingko biloba and Cercidiphyllum japonicum, two wind‐pollinated dioecious trees, offer a means of testing the same set of hypotheses of genetic isolation and the possible roles of life history traits with data gathered from codistributed species. Both trees are flagship species of warm‐temperate forests in eastern Asia, co‐occurring in habitats with rocky outcrops in proximity to streams subject to frequent flooding disturbance . We date the Ginkgo split to a narrow window during glacial periods and compare the degree of genetic isolation experienced by the two species during these episodes. The effects of gene flow and several life history traits on the observed genetic structure are discussed, as are the degree of postglacial expansion to former range limits in eastern Asia and the variance in expansion observed among temperate tree taxa.Using phylogenetic, coalescent, and comparative methods, we infer the processes responsible for the formation of disjunct distributions in these two species  , and 50 ng genomic DNA. Cycling conditions were 94°C for 5 min, followed by 35 cycles of 94°C for 1 min, annealing for 1 min , and 72°C for 1.5 min . Amplicons were purified using PCR Clean‐Up Kit . Both strands were sequenced using Taq dye deoxy terminator cycle sequencing kit on an ABI 377XL DNA Sequencer (Applied Biosystems). Sequence alignment was performed using GENEIOUS 4.8  (Table S2 in Appendix S1).Genomic DNA was extracted from ~100 mg of silica gel‐dried leaf material using a modified CTAB protocol from Doyle and Doyle . Three cHamilton , and atpμL containing 1 μL of 10× PCR buffer, 1 mmol/L dNTPs, 0.5 μmol/L each primer, 0.5 U of Taq polymerase, and 30–50 ng genomic DNA. The forward primers were labeled with a fluorescent dye . Amplification conditions were as follows: 94°C for 5 min followed by 35 cycles of 94°C for 1 min, the optimized annealing temperature (Table S3 in Appendix S1) for 30 sec, and 72°C for 45 sec . Fragment analyses were performed on a MegaBACE 1000 autosequencer . To reduce scoring errors, allele sizes were manually scored twice with reference to an internal size standard (ET‐ROX 400) using Genetic Profiler 2.2 .All 761 individuals were genotyped at 14 nuclear microsatellite (nSSR) loci  and nucleotide diversity (π) were estimated for each population  and at the species levels  using the same program.Each indel and inversion was treated as a single mutation, and coded as a nucleotide substitution, after Caicedo and Schaal . PlastidTo quantify the proportion of total genetic variance explained by differences between regional groups of populations, we applied analyses of molecular variance (AMOVAs) based on Tamura and Nei's distance  with 10,Cycas taitungensis as the outgroup . The best fitting substitution model, HKY + G, was determined according to Akaike Information Criterion (AIC) using jModelTest 0.1 . Significance levels were adjusted using the sequential Bonferroni correction for multiple comparisons (Rice NA), allele richness (AR), expected heterozygosity (HE), and observed heterozygosity (HO), was estimated per locus and per population using FSTAT 2.9.3 (Goudet RA) was manually calculated following Hartl and Clark  were tested for each locus–population combination using GENEPOP 4.2  was inferred using Evanno's ΔK method  4.0.5  the genetic clusters revealed in BAPS and (2) the phylogeography of cpDNA haplotypes see . The tweθW and θE) diverge from a common ancestor (of size θA) at time t and continue to exchange migrants at rates m0‐1 and m1‐0. As the complete data set was too large to analyze using an MCMC approach, we generated a smaller subset consisting of 25 individuals from each regional population (by random sampling of both the nuclear and the chloroplast data sets). IMa2 was also used to conduct likelihood ratio tests of nested models to test whether models with unidirectional gene flow are a significantly better fit to the data than models with/without bidirectional gene flow by the method of Nielsen and Wakeley (We used an isolation‐with‐migration (IM) model  = 100, split time t = 1, and migration (m0 and m1) = 10. We adopted an average mutation rate for cpDNA of 2.70 × 10−10 substitutions per site per year (s/s/yr) for cycads  were run for 2,500,000 steps beyond a 500,000‐step burn‐in, and trees were sampled every 100 steps. Four runs with different seed numbers were implemented independently.After exploratory runs, the priors were as follows: population size parameters  and rejecting those that are significantly lower scored than the full model based on a likelihood ratio test. To convert the parameter estimates to demographic units, we assumed a generation time of 160 years for Ginkgo. This is in line with the average age (as a proxy for generation time) of one refugial population (from which our population JF was sampled) based on a direct measurement of increment cores of 46 Ginkgo trees with DBH greater than 5 cm  and Harpending's (HRag) was tested using parametric bootstrapping   compared to western ones with only 3/8 and 2/7 populations being polymorphic, respectively. Nine of the twelve known cultivated populations were monomorphic  and fixed with the widespread haplotype H1.Chloroplast sequences also indicate that more than half of the analyzed populations 14/25) are polymorphic  using prior information on spatial coordinates (BAPS) revealed that the best genetic partition within the data had a clear west–east spatial component Fig. B, supporK peaked at K = 2 . Individuals from western China  were assigned to one cluster (“green”), while those from the eastern population (TM) were assigned to a different cluster (“red”)  and biparentally inherited nSSR  level for both the maternally inherited cpDNA  . Analysis of the nucleotide frequency spectra in cpDNA indicated no significant signal of population expansion; Tajima's D was slightly negative but not significantly so (Table S7 in Appendix S1).From the IMa2 analysis, we also inferred extremely small effective population sizes for the derived (west and east) daughter populations (in the order of 100's), which is an order of magnitude smaller than their census sizes. The magnitude of these effective population sizes may be indicative of demographic changes and thus genetic bottlenecks. The latter, for example, may have resulted in the loss of (rare) genetic variants; detectable in a skew in allele frequency spectra. Nevertheless, Ginkgo biloba with our (in‐house) published results of Cercidiphyllum japonicum  and for each nSSR locus . Gene flow is expected to reduce or eliminate this differentiation. Low levels of migration  are sufficient to prevent population differentiation by drift  we observe from our cpDNA haplotype and nSSR data. This timing (0.4Ne generations) in turn corresponds to approximately 39,000 years ago (using an average estimate for Ne from western and eastern populations and a generation time of 160 years). The simulations suggest that the relatively high FST we observe between the regional populations dates from ~39 k years ago and may mirror the lag before FST reaches equilibrium between the lineage sorting (random genetic drift) of population divergence and the homogenizing effects of asymmetrical gene flow.The IMa2 analysis suggests significant evidence for unidirectional gene flow from west to east. Nevertheless, significant population differentiation between west and east is observed for each chloroplast locus . We may thus infer that the demography of Ginkgo includes recent fragmentation of a formerly larger population with probably long‐term in situ persistence  confirm that genetic population structure (and apparent isolation) can persist in the presence of (recent) gene flow. Harrison et al. (This illustrates that isolation hypotheses (with or without gene flow) fail to account for the temporal dynamics of both isolation and gene flow among populations during glacial and interglacial periods. A model which includes isolation with migration is a good fit to the n et al.  and Qiann et al.  hypothesGinkgo is also apparent in numerous other temperate woody plants from eastern Asia, for example, Taxus wallichiana  or shifted clockwise along a northwest–southeast axis between Sino‐Himalayan and Sino‐Japanese forests . Among all of the taxa, the ages of these regional divisions varies substantially, from the youngest in Ginkgo ~51 kya to the oldest in Cercidiphyllum 1.89 Ma. In between these ranges, the genetic structure has been dated to 1.45 Ma in Q. variabilis and to 0.28–0.74 Ma in K. septemlobus. Interestingly, particularly for inferences regarding in situ persistence versus expansion hypotheses, postglacial expansions to former range limits differ among these species. Temporal and spatial incongruences suggest that these four trees did not respond to climate‐driven vegetation shifts in the same manner. One explanation for the incongruences may be differences in life history traits which we examine in detail below for two of the more ecologically associated taxa, Gingko and Cercidiphyllum.The west–east dichotomous regional genetic structure that we observe in uth Fig. . HoweverGinkgo and Cercidiphyllum, the range expansion observed in Cercidiphyllum following glaciation may imply comparatively poorer seed dispersal in Ginkgo. Indeed, the fleshy (heavy) seeds of Ginkgo are probably dispersed less efficiently than the wind‐dispersed seeds of Cercidiphyllum. Knowledge of the seed dispersers in Ginkgo has been speculative  mediated gene flow. The split time (~51 ka) for the regional population structure far predates that of human‐mediated introductions (<2 ka), and is too recent to have allowed new mutations to generate five newly derived haplotypes specific to eastern China. We thus consider animal mediated gene flow via seeds to be limited.Given the maternal inheritance of cpDNA in both Ginkgo is also at a comparative disadvantage to Cercidiphyllum in disturbed riparian rocky environments because its large seeds with slowly developing embryos require a level of stability less often available in habitats with frequent disturbance and flooding  regional Ginkgo populations in China. Life history traits can impose restrictions on the range dynamics and population genetic structure of temperate plants by their influence on lifetime reproductive success , may explain this variance. However, proving that isolation occurred continuously throughout a glacial period is challenging. Gene flow and random genetic drift can vary both temporally and spatially throughout glacial periods. Both isolation hypotheses are thus not necessarily mutually exclusive, and they also neglect the effect of life history traits on range dynamics and population genetic structure via their influence on lifetime reproductive success. Using population genetics to uncover the dynamism of postglacial history, this study illustrates the inadequacies of “isolation” hypotheses that span geological time where population dynamics are the norm. The comparison between Ginkgo and other taxa, particularly Cercidiphyllum, sampled across the same ecosystem indicates that co‐occurring species may not have congruent range dynamics. This highlights the importance of both demography and life history traits when interpreting range shifts and the evolution of ecological communities.Two competing isolation hypotheses (with or without gene flow) have been proposed to explain the variance of postglacial expansion to former range limits among warm‐temperate deciduous Asian tree species. During glacial periods, isolation with admixture at lower altitudes , observed (HO) and expected heterozygosity (HE) for fourteen microsatellite loci analyzed in 25 populations of G. biloba.Table S4. Chloroplast DNA sequence polymorphisms detected in the three regions of Ginkgo biloba identify 13 haplotypes (H1–H13).Table S5. Plastid and nuclear diversity in 25 G. biloba populations sampled across its complete natural range in China.Table S6. Tests of nested models for the WEST versus EAST group for the three cpDNA loci and 14 nSSR loci data set.Table S7. Summary of neutrality tests and mismatch distribution parameters for groups WEST and EAST of G. biloba. Estimates of mismatch distribution were obtained under models of spatial expansion using ARLEQUIN.Fig. S1 Likelihood values across multiple values of the cluster number in Bayesian assignment analyses, K (1–10) using Evanno's methods .Click here for additional data file.


Phylloscopus trochilus trochilus, P. t. acredula) with distinctive migratory strategies occurs in central Sweden. These subspecies exhibit differences in migratory direction and distance, resulting in geographically distinct wintering areas in Africa. The subspecies may have diverged from a common refuge after the last ice age, and neutral genetic markers are homogeneous across their range. By contrast, several phenotypic traits and genetic markers of two chromosomal regions previously identified show steep clines across the divide. The evolutionary forces that maintain this migratory divide remain unknown. Here we use plumage colour, morphology, genetic markers and feather stable nitrogen-isotopes (δ15N) to assess if assortative mating between migratory phenotypes could be acting as a possible mechanism for keeping the two forms genetically separate and maintaining the migratory divide. We colour-ringed a willow warbler breeding population in the central part of the hybrid zone and observed the breeding population to assess phenotypic and genotypic traits of social pairs.In contact zones, genetic mixing of two taxa can be restricted by prezygotic (e.g. assortative mating) or postzygotic (lower fitness of hybrid offspring) barriers, or a combination of the two. A hybrid zone between two willow warbler subspecies (δ15N between social mates.Our data suggest that wintering area and genetic ancestry had an effect on male arrival time to the breeding grounds which could contribute to assortment. However, evidence for assortative mating could not be detected based on a comparison of plumage colour, morphology and This finding was strengthened by analyses of subspecies-specific genetic markers, which allowed us to identify the presence of a large proportion of potential hybrids and backcrosses at the study site. Our results supported the hypothesis that pre-mating isolation in willow warblers is weak, resulting in extensive hybridisation across the migratory divide. Sylvia atricapilla) ]. Di]. Di3]].apilla)  and Swaulatus) . In addulatus) ]. Fr]. FrSylvulatus) ), e.g. ulatus) ].].Sylvia 1 hybrid offspring demonstrated an intermediate migratory direction compared to the parental phenotypes ]. D]. D13]].notypes ]. I]. I1 hybnotypes ].].1 hybriPhylloscopus trochilus trochilus in the south, and P. t. acredula in the north) with distinctive migratory behaviour meet and form a migratory divide. Each migratory phenotype likely possesses distinct endogenous migratory traits, including timing, migratory direction and distance, but presumably also different physiological adaptations such as fuelling decisions ]. T]. TST ≈ ST ≈ 0) ]. I]. T]. TS300 km) .Previous work demonstrated that the central Scandinavian migratory divide is maintained by selection, as geographic clines for both phenotypic and genotypic traits are narrower than expected from a neutral diffusion model assuming random mating between the subspecies since the time of secondary contact ]. R]. R22]].We quantify the proportion and different classes of hybrid genotypes in the migratory divide. We also assess to what extent assortative mating is occurring and how it is related to the observed degree of hybridization.We addressed this question by studying a breeding site in the centre of the hybrid zone where we expected both subspecies to occur. We assessed the degree of assortative mating based on a comparison of the characteristic phenotypic traits for pairs captured on their breeding territories. In order to estimate the genetic ancestry of individuals, we made use of subspecies-specific genetic variation segregating at two newly identified loci that had been detected through analyses of transcriptome sequencing data . These The key objectives of our study were to identify evolutionary processes and selective forces within our focal breeding population by (i) characterising the distribution of phenotypic and genotypic traits in a breeding population of willow warblers in the hybrid zone, in order to subsequently (ii) assess the role of prezygotic selection in maintaining the migratory divide.We captured individuals of 25 breeding pairs in 2011, and 15 breeding pairs in 2012. In addition, we captured 13 additional males holding territories in 2011, and 22 in 2012. In 2012, late winter weather delayed spring arrival and breeding, which accounted for the lower number of breeding pairs captured.With few exceptions, individuals in both subspecies-specific reference sets were assigned to each of the two population clusters with high probabilities using the newly identified subspecies-specific genetic markers  to arrive at differing schedules. We found no significant correlation of male δ15N feather values with capture date in males    Figure A. Howeveδ15N values (indicator of wintering grounds) there was no relationship between the values of the social mates     there was no relation between the values of the social mates. Body size PCA results and trait values for all social pairs are summarised in the Additional file r = 0.22, n = 38, p = 0.08) could be detected between the genetic ancestry of social mates.We tested for assortative mating for 40 confirmed pairings based on comparisons of phenotypic values using Pearson’s correlations . The lWe further observed no evidence for assortative mating in the hybrid zone based on a comparison of several characteristic phenotypic and genotypic traits. Careful within-pair comparison of morphological traits , genotype, and migratory direction, which all show steep clines across the migratory divide, indicated no pattern of assortative mating between the pairs. A similar pattern has been found in the hybrid zone of yellow-rumped warblers, where extensive hybridisation in the absence of assortative mating occurs . Our reOther ecological (e.g. habitat) or sexual (song) traits can be excluded as likely factors in stabilising the willow warbler hybrid zone. This is unlike, e.g. Swainson’s thrushes, where both co-occurring subspecies occupy distinct ecological niches , or blaBased on the SNP data in the two sequenced genes we were able to assign probabilities for each individual to the two population clusters (i.e. northern and southern subspecies) identified by STRUCTURE . As expThe use of only two diagnostic markers was insufficient to detect more complex hybrids, such as advanced backcrosses, which would result in an underestimation of hybridisation. The low number of markers also prohibited reliable classification of individuals into discrete hybrid classes, such as backcrosses, F1 and F2 hybrids . Even tP. t. trochilus genotypes at the study plot, suggesting that our focal breeding population was situated slightly south of the centre of the migratory divide, if we assumed a smooth cline in genetic ancestry across the divide. The possibility that our study site may indeed be situated south of the divide may have limited the statistical power to detect the possible occurrence of assortative mating. However, none of the characteristic traits investigated showed any correlation between pairs implying that lack of assortative mating was the most parsimonious conclusion for our data.Our sample of 40 pairs of willow warblers were collected over two years from one breeding population in the centre of the migratory divide. The site was carefully chosen because of its location based on previously described trait clines . HoweveThe lack of assortative mating in willow warblers combined with our previous work further supported the suggestion that postzygotic selection against hybrids rather than prezygotic selection (mate choice) is the major force maintaining this willow warbler migratory divide. The degree of intrinsic reproductive isolation generally accumulates with the genetic distance between the hybridizing taxa . In birOur data suggest that without a clear separation in arrival time, habitat choice, song or some other phenotypic traits that would result in assortative mating, hybridisation will remain common throughout the willow warbler migratory divide. Given that mating in willow warblers appears to be random, future research will focus on understanding the mechanisms governing postmating isolation. The only potential postzygotic isolation factor so far identified is the presence of a migratory divide. If postmating isolation is present in our system, a narrow zone of overlap should be associated with random or near random-mating, and our study provides evidence for this. In order to understand putative reproduction and/or survival consequences in hybrids, we should focus on long-term observations of colour-marked breeding populations in the hybrid zone, allowing for direct assessment of fitness and survival consequences. Experimental approaches that will better allow us to better understand the role and mechanisms of postzygotic factors may include an improved and more robust genotyping protocol containing more subspecies-specific markers that allows the detection and reliable quantification of different genotype classes . These data could in turn be used to derive more reliable estimates of fitness parameter, survival and reproduction for different genotypes. Further, genomic analyses characterising divergent chromosomal regions and most importantly the identification of candidate genes that are driving the differentiation pattern are likely to be involved in the reproductive isolation and knowledge of their function may help to suggest the mechanism by which reproductive isolation is maintained. In addition to improved genomics resources, future studies will also benefit from more accurate phenotypic measures, e.g. tracking the individuals’ migratory strategy and timing decisions across their annual cycle if technology further advances, especially with relevance for correlating these measures with individual survival and reproduction.δ15N (proxy for migratory direction), and one AFLP derived genetic marker whose allele frequency coincides with migratory direction. These data revealed four steep coincident trait clines ,,[9,[22,[22sewhere . PlumagNBEA and C11orf41 gene, which are located in regions on chromosome 1 (NBEA) and chromosome 5 (C11orf41); previously identified to be highly differentiated between the subspecies [[We developed a genotyping scheme to allow for an assessment of each individual’s genetic ancestry to subspecies, and to identify potential hybrids and backcrosses within the population. Individuals were genotyped for variation in the species . For easpecies  and ampAmplification was performed using a touchdown PCR starting with an annealing temperature of 63°C, which decreased with 0.5°C per cycle for 12 cycles, followed by 28 cycles with an annealing temperature of 57°C. Each cycle contained an elongation step of 1:15 minutes. PCR reactions were performed in a 25 μl volume consisting of 0.125 mM of each dNTP, 1.5 mM MgCl2, 0.4 μM of primer, 0.5 units of Taq Polymerase  and 10–50 ng of DNA template. PCR products were quality checked on 2% Agarose Gels and precipitated with 11 μl Ammonium Acetate and 37.5 μl 95% Ethanol. Sequencing was performed using BigDye® Terminator v.1.1 cycle sequencing kit  on an ABI 3130 prism robot .Sequencing the full length of the PCR products was complicated by the occurrence of multiple length polymorphisms in many individuals. Sequencing efforts were therefore focused on the initial few hundred base pairs of each gene. An internal set of primers  corresponding to both subspecies. We justify this choice by the existence of two subspecies of the willow warbler in Scandinavia and a hybrid zone between these populations, which has previously been characterised by cline analyses of genetic and phenotypic data in Central Sweden . The opTo obtain ancestry estimates for each individual with two population clusters, the admixture model was first ran without explicitly using the samples from the two reference populations to assist clustering of samples at the study site. As expected, the samples from each of the reference populations showed a high assignment probability to each of the two population clusters, respectively. In the final run, we specifically used the reference samples to aid clustering of samples from our study site and used them to update allele frequencies in each of the population clusters throughout the simulation. The model was run with 500 000 burn-ins followed by 500 000 steps of Markov Chain Monte Carlo (MCMC) iterations. We otherwise used default parameters, including the assumption of correlated allele frequencies between the population clusters.−6. The clustering performed by the linkage model was generally accompanied by slightly more uncertainty, but the individual genetic ancestry estimates were very strongly correlated with those calculated from the admixture model .The admixture model assumes that the genetic markers within populations are in linkage equilibrium within each population cluster. This could be violated if the markers are tightly linked to each other, such as polymorphisms within the same gene used in this study. Although STRUCTURE does not specifically model tightly linked genetic markers , we cheδ15N values or the probability of northern ancestry to arrival (capture) date, we calculated Pearson’s correlation coefficients (r). We limited our sample of males to those captured in 2011 as the 2012 season started two weeks late due to extended winter conditions. Next, we summarised morphometric data using a principal components analysis where PC1 explained the largest percentage of the variation in our data and was used for subsequent analyses  and genotypic (genetic ancestry) traits between social mates using Pearson’s correlation. All statistical analyses were carried out using R [[To determine if there were any correlations between male using R .The authors declare that they have no competing interests.SB, SÅ, KWL and MLu participated in the study design. KWL, MLu, MLi and AG conducted the field work. MLu and MLi carried out the genetics analyses. KH and LIW carried out the stable isotope analyses. KWL statistically analysed the data. MLi wrote the manuscript. All authors read and approved the final manuscript.Table S1. Internal primers used for amplification and sequencing of samples whose genotypes could not be determined by using the standard primers for each locus. Table S2. Samples used for STRUCTURE analysis and the resulting assignment probability (genetic ancestry) for each individual to a northern (acredula) subspecies population cluster. “Population” indicates individuals caught in the distribution range of the southern (Phylloscopus trochilustrochilus) or northern (Phylloscopus trochilusacredula) subspecies, or within the hybrid zone. Table S3. Eigenvalues and proportion of variance explained by each principal component using four morphological measurements, i.e. wing chord, tail, tarsus, and bill-head length, separately for males and females.Study site: Flatruet. Click here for fileTable S5. Sequence data for short reads (< 200 bp) for polymorphic regions on chromosome 1 .Deposited sequence data (of at least 200 bp) in GenBank for polymorphic regions on chromosome 1  and chromosome 5 . Click here for file


In recent years, several new ELISAs for the detection of antibodies against the porcine reproductive and respiratory disease virus (PRRSV) in pig serum have been developed. To interpret the results, specificity and sensitivity data as well as agreement to a reference ELISA must be available. In this study, three commercial ELISAs  were compared to a standard ELISA (IDEXX PRRS X3 Ab Test - ELISA I). The serum of three pigs vaccinated with an attenuated PRRSV live vaccine (genotype 2) was tested prior to and several times after the vaccination. Furthermore, serum samples of 245 pigs of PRRSV positive herds, 309 pigs of monitored PRRSV negative herds, 256 fatteners of assumed PRRSV negative herds with unknown herd history and 92 wild boars were tested with all four ELISAs.ELISAs II and III were able to detect seroconversion of vaccinated pigs with a similar reliability. According to kappa coefficient, the results showed an almost perfect agreement between ELISA I as reference and ELISA II and III (kappa > 0.8), and substantial agreement between ELISA I and ELISA IV (kappa = 0.71). Sensitivity of ELISA II, III and IV was 96.0%, 100% and 91.5%, respectively. The specificity of the ELISAs determined in samples of monitored PRRSV negative herds was 99.0%, 95.1% and 96.4%, respectively. In assumed negative farms that were not continually monitored, more positive samples were found with ELISA II to IV. The reference ELISA I had a specificity of 100% in this study.All tested ELISAs were able to detect a PRRSV positive herd. The specificity and sensitivity of the tested commercial ELISAs, however, differed. ELISA II had the highest specificity and ELISA III had the highest sensitivity in comparison to the reference ELISA. ELISA IV had a lower sensitivity and specificity than the other ELISAs. The porcine reproductive and respiratory syndrome (PRRS), caused by the PRRS virus (PRRSV), is responsible for significant economic losses worldwide . The PRROne cost effective method is the serological detection of antibodies against PRRSV by ELISA. Several ELISAs have recently been developed, most of them detecting antibodies against both PRRSV type 1 and type 2 -17. SomeThe objective of the study was to test three different commercial ELISAs for the detection of antibodies against PRRSV in serum and to evaluate their specificity and sensitivity in comparison to the IDEXX PRRS X3 Ab Test.A total of 923 serum samples of 905 pigs were included in the study. The pigs were divided into 5 groups. Group 1 consisted of 21 samples of three pigs from a PRRSV negative farm  that weAll 21 samples of group 1 were analysed by real-time RT-PCR for the presence of PRRSV viral RNA. Positive samples were sequenced. Within group 2, all serum samples of the boar stud and the Southeast Asian farm, as well as several tissue samples from the Russian farm, were analysed by PCR. Pigs from group 3 were from farms with continuous PRRSV monitoring by PCR and ELISA with no positive results. The 104 fatteners of group 2 and the samples of groups 3–5 were not tested with PCR in this study.RNA extraction was performed using the Freedom EVO® 150  automated platform and the Nucleospin® 96 Virus and the Nucleospin® Virus Core kits  for serum and tissue samples, respectively, following the instructions of the manufacturer. The samples were then analysed by a commercial real-time RT-PCR assay that allows the simultaneous detection and differentiation between PRRSV type 1 (EU) and type 2 (NA) genotypes  on the ABI 7500 Fast Real-Time PCR System (Life Technologies).Representative PRRSV positive samples were further typed using the corresponding ORF5 gene modified methods ,20. AnotAll serum and residual blood samples were analysed with four different commercially available ELISAs: ELISA I was the IDEXX PRRS X3 Ab test. Samples with sample-to-positive (S/P) ratios ≥0.4  were considered positive for antibody against PRRSV. ELISA II was the INgezim PRRS 2.0 . The cut-off value for this ELISA was 0.4 like in ELISA I. ELISA III was the Priocheck® PRRSV Ab porcine . In this ELISA the cut-off value was at the S/P ratio 0.3. ELISAs I, II and III are able to detect antibodies against both type 1 and type 2 of the PRRSV. ELISA IV was the CIVTEST suis PRRS E/S PLUS . The cut-off value of this ELISA was 0.2. ELISA IV is able to detect only antibodies against PRRSV type 1 . All ELISAs were conducted according to the manufacturer’s instructions.Positive and negative samples of group 2 were classified into two-by-two contingency tables. Sensitivity of the ELISAs compared to ELISA I was tested using the samples from group 1 and 2. The specificity of all ELISAs was estimated using group 3. The agreement of ELISA II, III and IV with ELISA I was determined using the samples of group 1 to 4 with the kappa coefficient (κ). Positive and negative predictive value, as well as the accuracy of the ELISAs were determined using group 1 to 4 with ELISA I as reference test.Before vaccination, all three pigs from group 1 were negative by real-time RT-PCR for both genotypes 1 and 2. Between day five and day 12 after vaccination, all three pigs were viral RNA positive for PRRSV type 2 RNA and remained negative for genotype 1. From day 18 on, all pigs were negative by PCR. Because it was known that the pigs were vaccinated with a PRRSV type 2 live vaccine, sequencing was not performed. In group 2, the boar samples were PRRSV type 1 subgroup 1 (EU-1) positive. Several field PRRSV strains belonging to PRRSV type 1 subgroup 2 (EU-2) were found in the samples originating from the Russian pig farm. Only highly pathogenic PRRSV type 2 strains were found in the Asian samples analysed.All three pigs seroconverted between day nine and day 12 after vaccination, as is seen in Figure The S/P values of ELISA I for group 2 to 5 are displayed in Table The results of the samples of PRRSV negative farms of group 3 are shown in Table In the wild boar samples group 5), only a few PRRSV antibody positive samples were found . Two of the ELISAs (ELISA II and III) were recently developed. No published data exists concerning the specificity and sensitivity or data of agreement for these ELISAs. One recent study is available that refers to ELISA IV in comparison to ELISA I in experimentally infected pigs and 205 samples of pigs from PRRSV negative herds . HoweverELISA I, II and III detected a seroconversion in all three pigs of group 1 at day 12 after vaccination with an attenuated PRRSV live vaccine. ELISA I and III, however, were able to find PRRSV antibodies in one pig as early as day 9 after vaccination. Seroconversion can usually be first detected between day 9 and 15 after vaccination or inoculation ,22-25. TAccording to the kappa coefficient, an almost perfect agreement (κ > 0.80) was found between ELISA I on one hand and ELISA II and III on the other hand. No published reference data are available for ELISA II and III, but the newly developed ELISA II seems to be much more precise than the former INgezim PRRS Universal (Ingenasa) . ELISA IIn our study, ELISA I as reference test had a specificity of 100%, which agrees with the manufacturer’s declaration (99.9%) and was also found in another study . Other sIn group 4 on the other hand, some positive results were found with ELISA II and some more with ELISA III and IV, whereas ELISA I remained negative but with S/P values in some samples slightly beneath the cut-off. Since no anamnestic information is available on the herds, a PRRSV infection or previous vaccination cannot be excluded in farms of group 4 by a single testing for PRRSV antibodies. Since the sensitivity of ELISA I is, according to manufacturer, 98.9%, it may well be that the sensitivity of ELISA II and III is higher than this. On the other hand, antibody response detectable by ELISA after a PRRSV infection has a duration of at least 120  to 137 dA slightly lower sensitivity (91.5%) of ELISA IV was found in our study, which is in agreement to the results of other studies ,26, althBlood samples of wild boars are usually of poor quality. Therefore, the sensitivity, but more so the specificity of the ELISAs must be very high to produce reliable results. In our study, only a few wild boar samples in ELISA I, II and III were PRRSV antibody positive. This confirms the usually low seroprevalence between 0% and 3.8% in European wild boars that was shown in other studies -30 and cThis study shows that the newly developed ELISAs tested in this study are able to reliably detect antibodies against PRRSV in vaccinated and naturally infected pigs. The agreement to the reference ELISA I was almost perfect. ELISA II stood out due to a high specificity (99%) and sensitivity (96%). Occasional false positive results, however, need to be considered in all of the tested ELISAs, even in ELISA I that was used as reference method in this study. ELISA III has an especially high sensitivity (100%) that is probably superior to the reference ELISA I, but a lower specificity (95.1%). In ELISA IV that is on the market for several years, a substantial agreement to ELISA I was found. Both sensitivity and specificity of this ELISA were lower than in the other ELISAs.


Eunicella singularis is one of the most common gorgonian species in Northwestern Mediterranean Sea, playing an important role as ecosystem engineer by providing biomass and complexity to the coralligenous habitats. It has a wide bathymetric distribution ranging from about 10 m to 100 m. Two depth-related morphotypes have been identified, differing in colony morphology, sclerite size and shape, and occurrence of symbiotic algae, but not in mitochondrial DNA haplotypes. In the present study the genetic structure of E. singularis populations along a horizontal and bathymetric gradient was assessed using microsatellites and ITS1 sequences. Restricted gene flow was found at 30–40 m depth between the two Eunicella morphotypes. Conversely, no genetic structuring has been found among shallow water populations within a spatial scale of ten kilometers. The break in gene flow between shallow and deep populations contributes to explain the morphological variability observed at different depths. Moreover, the limited vertical connectivity hinted that the refugia hypothesis does not apply to E. singularis. Re-colonization of shallow water populations, occasionally affected by mass mortality events, should then be mainly fueled by larvae from other shallow water populations.Gorgonian species show a high morphological variability in relation to the environment in which they live. In coastal areas, parameters such as temperature, light, currents, and food availability vary significantly with depth, potentially affecting morphology of the colonies and the structure of the populations, as well as their connectivity patterns. In tropical seas, the existence of connectivity between shallow and deep populations supported the hypothesis that the deep coral reefs could potentially act as (reproductive) refugia fostering re-colonization of shallow areas after mortality events. Moreover, this hypothesis is not so clear accepted in temperate seas.  Marine modular organisms exhibit a large morphological variability, with phenotypic plasticity a likely source of this variability  can influence organisms’ morphology and drive genetic differentiation . Each paet al. [Eunicea flexuosa exposed to high water motion in shallow habitats compared to those living in deeper habitats are taller, have bigger calices, thicker branches and developed on a single plane. Shallow and deep colonies of E. flexuosa belong to two genetic lineages [Seriatopora hystrix [Montastraea cavernosa [Occurrence of high connectivity between shallow and deep populations gave rise to the hypothesis that deep coral reefs can potentially act as (reproductive) refugia , fosteriet al.  showed tlineages , while wlineages . Adaptat hystrix  and Montavernosa .Eunicella are among the most representative ecosystem engineers providing biomass and structural complexity in sublittoral communities. Among the 18 recognized Eunicella species, 6 species are present in the Mediterranean Sea: E. singularis, E. cavolinii. E. verrucosa, E. filiformis, E. gazella and E. labiata. The distribution of the last three species is limited to the Strait of Gibraltar and to the Alboran Sea, and they are considered mainly Atlantic. E. cavolinii and E. singularis occur both in the western and eastern Mediterranean Sea, being the later also present in few locations along the Atlantic coast near the Strait of Gibraltar. E. singularis is the only species showing a high morphological variability along a depth gradient from 10 to 70 m in the western Mediterranean Sea [Gorgonian corals of the genus nean Sea ,15 .E. singularis have been identified . Eight further samples were collected in the same area along about 15 km of coast at 15–20 m depth to assess the horizontal pattern of genetic connectivity, and to be able to compare the 2 patterns. Finally, the phylogenetic relationships between E. singularis, the Mediterranean species E. cavolinii and E. verrucosa, and the Atlantic E. racemosa, were analyzed to scale their genetic distances.A shallow and a deep morphotype of ied  at 18–20 m depth, E. verrucosa at Tarragona  at 13–17 m depth, and E. racemosa along the Morocco coast  at 15 m depth. All the collected material was preserved in 80–100% ethanol and stored at 4°C pending analysis.The study was planned at Cap de Creus , where  m depth , Table 1 m depth ; Table 1Total genomic DNA was extracted from three to five polyps per colony fragments following the cetyl trimethyl ammonium bromide (CTAB) procedure . After D2, 0.5 μm of each primer, 0.4 mm dNTPs and 1 U of Taq polymerase (Promega). Amplifications were performed on a GeneAMP PCR System 2700 (Applied Biosystems) as follows: an initial denaturation at 95°C for 3 min, 30 cycles including 95°C for 30 s, 57°C for 30 s and 72°C for 60 s. A final extension at 72°C for 7 min was added. PCR products were sent to Macrogen (South Korea) for purification and sequencing since some sequences contained several heterozygous sites, ITS1 sequence types were estimated using PHASE 2.1 [For ITS1 sequence analysis, only individuals belonging CCR1, CCR2, CCR3 (10–60) and CCR4 were amplified. Polymerase chain reaction (PCR) amplifications of the ITS1 region were carried out using the primers ITS1-new-F and ITS1-new-R . Each 25HASE 2.1 , which iID) [O) and expected (HS) heterozygosity using the GENETIX software package v. 4.05 [IS values were estimated using Weir & Cockerham’s F [http://genepop.curtin.edu.au/), with the level of significance determinate by a Markov-chain randomization. The presence of null alleles was estimated using the expectation maximization (EM) algorithm of [Linkage disequilibrium analysis for pairs of loci was based on the likelihood ratio test with the EM algorithm  through ID)  was comp v. 4.05 . Allelic v. 4.05 . Single-rham’s F , and sigrham’s F  as implerithm of  in FREENrithm of . BOTTLENrithm of  was usedrithm of  were tesrithm of ) was userithm of .FST estimator in ARLEQUIN. Genotypic differentiation among populations was tested with an exact test implemented in GENEPOP . Since the presence of null alleles, genetic divergence among samples at microsatellite loci was estimated in FREENA using the FST estimates of Weir [FST estimates, computed excluding null alleles. To evaluate the number of clusters (K) in the dataset without prior information regarding the geographic distribution of the samples, a Bayesian method implemented in STRUCTURE v. 2.3.4 [http://taylor0.biology.ucla.edu/structureHarvester/) [For both molecular markers, the genetic divergence among populations was estimated using Weir & Cockerham  FST esti of Weir  and foll of Weir  which prv. 2.3.4 ,43 was uvester/) . Moreovevester/)  to obtaivester/)  was perfvester/) . Hierarcet al. [E. singularis among sites, the numbers of first-generation migrant were also calculated through the program GENECLASS v. 2.Significance of F-statistics was achieved using 99,999 permutations. Individual assignment tests were performed using the program GENECLASS v. 2 . This Baet al.  set at 1Eunicella, a dataset with all the individual ITS1 sequences from the different species here examined was created. Distance matrixes of sequence divergence among the species were calculated as p distance (Dp) in MEGA v. 5.05.To perform a preliminary phylogeny of the genus Corallium rubrum (GenBank accession number: FJ87608282 and FJ87608283) were used as outgroup.The best-fit substitution model for the dataset was calculated using JMODELTEST v. 1.1  Mac softp > 0.05), confirming that all loci are independent markers. Six shared multilocus genotypes (MLGs) between colonies were found. Five MLGs were encountered twice in CCR3_40, CCR3_50 and CCR3_60. Within CCR3_50 one MLG was encountered five times. In CCR3_50 the probability that each of these genotypes was produced by chance alone was high (PID = 4.6 e-02).301 individuals were genotyped for the 6 loci. Tests of linkage disequilibrium between loci within populations indicated no significant association of alleles  and observed heterozygosity (HO) were lower in CCR3_50  and higher in CCR4 . Three  out of 13 samples showed significant deviations from HW equilibrium with values of FIS positive and higher than 0.173  based on a minimum sample size of 22 genes, ranged from 2.24 (in CCR3_50) to 4.25 (in CCR7), while the private allelic richness (Ap) ranged from 0 (in CCR3_20 and CCR3_30) to 0.46 (in CCR7). Expected heterozygosity (Han 0.173 .p = 0.005). The pairwise test located a threshold of genetic variability at 40 m depth .Estimated null allele frequencies ranged between loci from 0.022 ± 0.035 in EVER9 to 0.0748 ± 0.087 in C21. The Wilcoxon’s signed rank-test, performed in BOTTLENECK showed no demographic population changes in the recent past for all samples (data not shown). The one-way PERMANOVA showed that the total genetic variability differed by depth  and along the vertical gradient in CCR3 (CCR3_10 to CCR3_60) were analyzed using ITS1 sequence polymorphism. In total, 286 ITS1 sequences corresponding to 143 individuals were analyzed. The length of the amplified ITS1 fragment was 200 bp with 9 variable sites at the positions 14, 21, 114, 119, 158, 159 and 173, corresponding to 9 sequence types . The sequence type network showed a star-shape pattern with a high frequency dominant haplotype with at the center. Derivatives with lower frequencies were connected to the dominant haplotypes by only one mutation step  to 0.482 (CCR3_50 vs. CCR3_60). After FDR correction, all the pairwise comparison with CCR3_40, CCR3_50 and CCR3_60 were significant  to 0.548 (CCR3_40 vs. CCR3_50), and 15 out of the 36 pairwise FST estimates were not statistically significant after False Discovery Rate correction. As for the microsatellites, also the AMOVA performed using the ITS1 dataset supported the clustering structure   .E. singularis were aligned with the sequences of E. cavolinii, E. verrucosa and E. racemosa. The Atlantic species E. racemosa differed from the others for the presence of an insertion of 5 bp at the position 22–26. All the 5 colonies of E. verrucosa and E. racemosa showed identical sequences. Three colonies of E. cavolinii present the same sequence of E. singularis ST1 and the PHASE analysis implemented in DNAsp showed that one colony of E. cavolinii presented 2 different sequence types: ST1 and ST7 (GenBank accession numbers KX002245-KX002255). The p-distance matrix showed that the greatest divergence among sequences was observed between E. racemosa and all the other species with values ranging from 4.52% to 5.02%  were close to values of the mean genetic distance between E. singularis and E. cavolinii . On the other hand, mean p-distance values among shallow  and deep sites  were 0.000 and 0.243 ± 0.0002, respectively. Mean difference between shallow and deep sites was 0.0016 ± 0.0012 . Moreover, E. verrucosa and E. cavolinii clustered together with the E. singularis sequence types (E. cavolinii sequence types could be detected (each PP = 0.95), the first one clustering sequence types ST3 and ST4  and the second one composed by the sequence types ST7 and ST9  (see All the sequences types found in to 5.02% . Moreove± 0.0012 . Howeverce types . Only a _60) see .Eunicella singularis in Cap de Creus [The present study reveals that shallow and deep morphotypes of de Creus ,17 correEunicella are more complex than expected, and current morphological and molecular taxonomy cannot clearly discriminate the three 3 common Mediterranean species. Further studies using new polymorphic and variable markers are needed to shed light on the evolutionary history among Eunicella lineages.Observed phylogenetic patterns within the genus Corallium rubrum populations in Cap de Creus [C. rubrum decreasing genetic variability has ascribed to the present of recent bottleneck, probably as a consequence of the past overexploitation of deeper populations. In E. singularis no recent bottlenecks were observed in any population. In fact, in Cap de Creus, rocky bottoms below 40 m depth are characterized by high density of medium-sized and sexually mature colonies [FST estimates using microsatellite loci, and AMOVA on ITS1 sequences revealed a high degree of genetic differentiation between populations above and below 40 m depth. Structure analysis suggested the presence of a barrier to gene flow across 40 m. Among the deepest populations, CCR3_50 seems the most divergent with a high rate of self-recruitment, suggesting that a nonlinear increase of genetic distance with depth. DAPC and structure analysis with K = 4 also support this hypothesis, showing a higher similarity between colonies at 40 and 60 m depth compared to colonies at 50 m depth. Pey et al. [E. singularis and Pivotto et al. [E. cavolinii did not found any genetic differentiation comparing colonies from 10–15 m to 35–40 m depth, confirming absence of barrier to gene flow in populations above 40 m depth. While C. rubrum colonies collected along a 20–70 m depth gradient revealed a threshold in connectivity at about 40 m depth [E singularis in two morphotypes  differing in color, colony shape and sclerites features described by Théodor [et al. [Seriatopora hystrix [Montastraea cavernosa [Eunicea flexuosa [Significant differences in the genetic variability were observed between shallow and deep populations. This variability was mainly explained by the presence of high values of private alleles observed in the populations below 40 m depth. These results are in contrast with the decreasing genetic variability along depth gradient observed in de Creus . In C. rcolonies ,53, suggy et al.  in E. sio et al.  in E. ca Théodor  and Gori [et al. . Limitedavernosa  and in tflexuosa , stressiE. singularis, a gonochoric brooding species, releases mobile planulae larvae with a short pelagic larval duration [Seriatopora hystrix, populations and associated symbiotic algae are highly structured across habitats on a single reef [E. singularis colonies collected at 40–60 m depth [In the study area, a seasonal thermocline is located at about 40 meters depth ,57. Largduration ,59. The duration ). Nevertduration  genetic duration ,12,13,61gle reef . Endosym m depth . The cor m depth , because m depth .E. singularis along the coast of Cap de Creus [et al. [Eunicella verrucosa [Eunicella species are thought to have a limited dispersal, with larvae settling mainly close to the maternal colonies [E. singularis, together with the observed high horizontal connectivity, suggests that marine coastal currents are likely to be the main vector for larval and or gamete dispersal in this species. In fact, in Cap de Creus the general circulation pattern is dominated by currents usually flowing from North to South, but intense and persistent Catalan eddies have also been registered in summer [No genetic structuring was observed among shallow populations of de Creus . Distanc [et al. , while cerrucosa . Both Eucolonies . Howevern summer , when lan summer .Eunicella singularis [E. cavolinii [Callogorgia delta and C. americana). Within Mediterranean species of the genus Eunicella speciation events were not detectable by the available genetic markers and may be recent or still in progress. This former hypothesis is in agreement with the opening of the Strait of Gibraltar approx. 5.32 My, when marine conditions were restored by the massive flow of Atlantic water and species into the Mediterranean Sea [Both microsatellites and ITS-1 showed a low level of the genetic variability. The observed number of alleles was in the same range as reported for ngularis ,54 and Eavolinii  populatiavolinii . Quattriavolinii  suggest nean Sea .E. racemosa and the three Mediterranean Eunicella species, which clustered together in a single group. Similar results were obtained using mitochondrial markers MutS, COI and Igr1 segments, having the same sequence in E. singularis, E. cavolinii and E. verrucosa, while differing from E. racemosa . It is worth to notice that despite the large morphological differences between E. racemosa and the Mediterranean Eunicella species . Further studies should include additional species from Ibero-Moroccan Gulf and the near Atlantic African coasts in order to get insights into the diversification of the Eunicella genus, and the origin of the Mediterranean endemism.Contrary to ITS2 , ITS1 semosa see . E. racesee also ), mitochShallow benthic communities are commonly exposed to higher environmental stress compared to deeper ones, e.g. thermal stress events and storm-induced disturbance . In tropEunicella in the Mediterranean Sea, low levels of mitochondrial genome evolution [Eunicella species. The large morphological variability of Eunicella genus, as in other Octocorallia genera, leads uncertainty regarding species boundaries and classification. Methods based on next-generation sequencing . Howevevolution , and lowuencing, ) may proS1 DatasetE. singularis populations.The allele sizes in 6 loci studied in 13 (XLS)Click here for additional data file.S1 Materials and MethodsEunicella species and PCR amplification of the three mitochondrial markers and their genetic variability.DNA extraction of the (DOCX)Click here for additional data file.S1 TableEunicella species including the sampled populations of Eunicella singularis.Pairwise genetic p distance (Dp) among (DOCX)Click here for additional data file.


A central question in evolutionary biology is how gene flow and natural selection shape geographic patterns of genotypic and phenotypic variation. My overall research program has pursued this question in tree populations through complementary lines of inquiry. First, through studies of contemporary pollen and seed movement, I have studied how limited gene movement creates fine‐scale genetic structure, while long‐distance gene flow promotes connectivity. My collaborators and I have provided new tools to study these processes at a landscape scale as well as statistical tests to determine whether changes in landscape conditions or dispersal vectors affect gene movement. Second, my research on spatial patterns of genetic variation has investigated the interacting impacts of geography and climate on gene flow and selection. Third, using next‐generation genomic tools, I am now studying genetic variation on the landscape to find initial evidence of climate‐associated local adaptation and epigenetic variation to explore its role in plant response to the climate. By integrating these separate lines of inquiry, this research provides specific insight into real‐world mechanisms shaping evolution in tree populations and potential impacts of landscape transformation and climate change on these populations, with the prospective goal of contributing to their management and conservation. Darwin “In considering the distribution of organic beings over the face of the globe, the first great fact which strikes us is, that neither the similarity nor the dissimilarity of the inhabitants of various regions can be accounted for by their climatal and other physical conditions… The dissimilarity of inhabitants of different regions may be attributed to modification through natural selection… The degree of dissimilarity will depend on the migration of the more dominant forms of life from one region into another… Thus, the high importance of barriers comes into play by checking migration; as does time for the slow process of modification through natural selection”. The Origin of Species, and those who have followed  have continued to grapple with the roles of these countervailing forces. Gene flow and natural selection are two central, and usually opposing, evolutionary forces: gene flow distributes, homogenizes, and maintains genetic variation that can act as the ‘stuff of evolution’, while natural selection reduces genetic variation to the variants that favor survival and reproduction. For tree species, which are often highly outcrossed with extensive gene flow, genetic drift plays a lesser role in their population genetics, except in small or substantially isolated populations  that flag candidate genes underlying traits experiencing selection created by local environments, even for nonmodel species without reference genomes , and administered through the Missouri Department of Conservation , and didn't worry about finding the pollen sources? To do this, we developed a two‐generation procedure, dubbed twogener, which combines the simplicity of a population structure approach with the parent‐/offspring‐deductive aspects of the parentage approach . This number is typically much smaller than the total number of pollen donors because a few local pollen sources dominate, with rare long‐distance pollen flow from multiple different sources. From an evolutionary perspective, this result explains how local adaptation is compatible with long‐distance gene flow. From a conservation standpoint, estimating Nep focuses on the diversity of the pollen pool as a critical element for conservation strategies because it reflects risk of genetic drift.For most tree populations, gene flow occurs primarily through pollen. When I first began this line of research, the primary method of studying pollen movement was to identify the paternity of progeny sampled from a maternal source , and only about two effective pollen donors  within local patches and for the local site  and genetic diversity across patches within the area (γ). We presented an elaborated treatment of diversity statistics in an NEM or α‐diversity, it is valuable to be able to complement this information with direct documentation of seed movement. Taking advantage of the fact that first‐year valley oak seedlings remain attached to the maternally inherited acorn tissue and that we have a well‐mapped study site at the UC Santa Barbara Sedgwick Reserve, with every adult genotype identified, we were able to track seed dispersal into valley oak seedling patches away from an adult canopy. We observed that most of the seedlings came from nearby trees, but many were dispersed from a seed tree 1–4 kilometers away, putatively by scrub jays , based on kinship coefficients  was greater than nondispersed seedlings (Ne = 3.6), largely because the nondispersed seedlings had only one maternal source. Thus, we provided important new evidence that seed dispersal has a substantial impact on the local neighborhood size of newly established seedlings despite the homogenizing contribution of the pollen donors  γ‐diversity does not change, but the α− versus γ‐diversity does; (ii) the α‐ and β‐diversity values are not affected, but the overall γ‐diversity is changed; (iii) α‐, β‐, and γ‐diversity measures are all affected. If statistical tests show that a disturbance affects cases (ii) or (iii), one can reliably conclude that the disruption is jeopardizing genetic diversity.The geographical patterns of genetic variation in a species are the outcome of demographic history of population expansion and contraction, migration history, and the impact of natural selection both spatially and temporally. By putting genes on a map, it is possible to extract inference on these processes from the geographical patterns of allele frequencies. The analysis of spatial patterns is a form of discovery‐based science that provides insight into the environmental context of the organism and its evolution. With my early ecological training, understanding the environmental context of my study systems is inherent in how I think about evolution. My research has always utilized a spatially explicit framework, and the landscape context can play such an important role in the distribution of genetic variation. This work has been done in collaboration with a geographer and plant ecologist, Frank Davis, a forest geneticist, Robert Westfall, postdocs, Cyril Dutech, and especially Delphine Grivet and Paul Gugger. Our studies fall into three types of approaches: spatial autocorrelation, geographic and climate associations of genetic gradients, and phylogeography.The simplest form of spatial analysis of genetic variation is to examine the scale of spatial autocorrelation of alleles, particularly appropriate for examination of the extent of gene dispersal. Our early work in this arena involved a study of seed dispersal by tropical avian frugivores  and that the distribution of CG‐SMP FST values included more values at the upper end of the distribution (Fig. To test whether epigenetic variation differs across populations of valley oak, we sampled individuals from three southern populations with divergent elevations, levels of rainfall, and seasonality of rainfall (Platt et al. ion Fig. . The finion Fig. . It willThe interaction of pollen and seed dispersal vectors with landscape features shapes contemporary gene flow. My research has shown that pollen movement in oaks is responsible for maintaining connectivity among populations, yet local pollination is sufficiently frequent that it is possible to create fine‐scale genetic structure that could facilitate local adaptation. Seed dispersal is more likely to be restricted, so it accounts for much more of the fine‐scale genetic structure than does local pollen movement. Dispersal agents, such as western scrub jays, facilitate long‐distance acorn movement that would allow colonization of new sites, but because they visit few trees, the genetic diversity of patchy seedling populations is reduced during this phase from that introduced through pollen dispersal. The generality that has emerged from this work and that of others for many tree species is that localized gene movement of pollen and seeds will create sufficient genetic structure to allow the evolution of local adaptation, while long‐distance gene flow will maintain high levels of genetic diversity and spread favorable alleles over large spatial scales.The analysis of contemporary gene flow in plants provides valuable information on the extent to which human disturbances are jeopardizing future genetic diversity by isolating populations. However, conservation scientists need to test hypotheses about the impact of human disturbance. My collaborators and I have also introduced statistical tests for landscape scale questions to determine whether landscape change through fragmentation isolates or enhances gene movement or whether dispersal vectors differ in their impact on the genetic structure of populations and thus their loss would jeopardize genetic diversity. Thus, our studies are part of a growing body of literature in landscape genetics exploring whether plant populations are threatened by human activities.The geographic patterns of genetic variation reflect the evolutionary history of a species that is the foundation for current responses to environmental conditions. Valley oak populations are likely to be several hundred thousand years old with high genetic diversity within populations and high gene flow among populations. While nuclear markers indicate extensive connectivity across the species range, chloroplast haplotypes indicate that colonization is local. This restricted dispersal would allow evolution of local adaptation. In addition, the species‐wide spatial pattern of genetic variation reflects an influence of climate through either gene flow or selection against migrants into populations. Regardless, this pattern explains the prevalence of genetic and phenotypic differences among tree populations shown here and which is likely to be true for many forest tree species.The emergence of next‐generation sequencing tools now allows the direct analysis of candidate genes and epigenetic impacts that could shape phenotypic response to the environment. Our work demonstrates extensive genetic differentiation across populations for certain genes and a correlation between genetic and climate gradients that provides initial evidence of local adaptation. However, future work needs to also control for background genetic structure that may be confounded with environmental associations. Our evidence of significant epigenetic differentiation at methylated sites further provokes the question of the extent to which epigenetic processes might shape phenotypic response to local environments.We have also demonstrated that the homogenizing influences of long‐distance gene flow are not sufficient to ‘swamp out’ the impact of selection on tree populations. Instead, genes show a strong impact of selection likely due to both the strength of selection and the presence of fine‐scale genetic structure.Valley oak populations have already experienced a reduction in regional coverage due to habitat transformation and problems with regeneration. Restoration efforts will need to consider future climates as they consider the impact of climate change on the future environments of their restoration sites. It is my goal to develop and ‘ground‐truth’ landscape genomic approaches that can be used to inform future conservation strategies when long‐term common garden and reciprocal transplant experiments are not feasible. This information will not only help to preserve this and other California oaks, but also provide a case study for other tree species of conservation concern.My research efforts have taken place in the larger context of landscape genetics and genomics, plant evolutionary biology, forest genetics, and conservation science. My studies are built around the valley oak study system is poised to complement other exciting forest genetic research to address the emerging questions about the ability of long‐lived species to face and survive anthropogenic challenges. Maintaining healthy tree populations is not only important to their survival, but also important to sustaining the ecological processes of the ecosystems they define (Hughes et al. Gene movement on the landscape. Unlike studies of animal landscape genetics that have focused on the impact of landscape features on migration, landscape genetic studies of plant populations initially focused more on geographical and environmental associations of genetic variation (Manel et al. Evolution of local adaptation. To understand how local adaptation evolves, we need first to identify candidate genes underlying phenotypic traits under selection and the fitness consequences of those traits (Mitchell‐Olds et al. FST outlier tests or environmental association analysis is one way to identify the candidate genes for local selection, when controlling for demographic history (for example, see Eckert et al. Evolutionary and ecological response to climate change. How plants are going to respond to rapid climate change is a critical issue (Parmesan Parmesan . For lonParmesan . Are mosParmesan ? Is locaParmesan ? Will plParmesan ? Does epParmesan ? My longIn short, the integration of 21st century genomic approaches, quantitative and population genetics, and recently developed geographic methods of modeling environmental data provides exciting and unprecedented opportunities to understand the evolutionary and ecological processes of plants and important applications.No new data are reported in this manuscript. Archived data details are available through original publications.