IS). For example, correcting for family structure yielded estimates of FST among patients that were 27 times lower than estimates from the uncorrected samples. These biased estimates would cause one to draw false conclusions regarding these parameters in the adult population. We also found from our analyses that estimates of the number of full sibling families and other genetic parameters of samples of miracidia were highly intercorrelated but are not correlated with estimates of worm burden obtained via egg counting (Kato-Katz). Whether genetic methods or the traditional Kato-Katz estimator provide a better estimate of actual number of adult worms remains to be seen. This study illustrates that family structure must be explicitly accounted for when using offspring samples to estimate the genetic parameters of adult parasite populations.For ethical and logistical reasons, population-genetic studies of parasites often rely on the non-invasive sampling of offspring shed from their definitive hosts. However, if the sampled offspring are naturally derived from a small number of parents, then the strong family structure can result in biased population-level estimates of genetic parameters, particularly if reproductive output is skewed. Here, we document and correct for the strong family structure present within schistosome offspring (miracidia) that were collected non-invasively from humans in western Kenya. By genotyping 2,424 miracidia from 12 patients at 12 microsatellite loci and using a sibship clustering program, we found that the samples contained large numbers of siblings. Furthermore, reproductive success of the breeding schistosomes was skewed, creating differential representation of each family in the offspring pool. After removing the family structure with an iterative jacknifing procedure, we demonstrated that the presence of relatives led to inflated estimates of genetic differentiation and linkage disequilibrium, and downwardly-biased estimates of inbreeding coefficients . One potential problem with this approach is that analyses using the offspring data will be biased because many of the offspring are related and family sizes are likely to be unequal. We show that this sampling issue is relevant in a natural transmission zone in western Kenya and that it yields biases in three important parameters: genetic differentiation, inbreeding coefficients, and estimates of the amount of non-random association between loci (linkage disequilibrium). We also develop a method to remove these biases by removing the sibling structure present in the dataset. Finally, we suggest that our measure of family number, as well as other genetic measures, may be useful measures of the worm burdens in patients. Infectious disease research is rapidly adopting the tools of evolutionary biology and molecular ecology bN (either because few adults were breeding and/or because those that did breed had highly skewed reproductive output). First, the sampling variance in population allele frequencies that arises from sampling the offspring of a small effective number of adult breeders will yield inflated estimates of genetic differentiation among hosts. Second, the strong sibling structure that will exist in a large sample of offspring having a small bN will cause negative deviations from Hardy-Weinberg equilibrium (i.e. downwardly biased estimates of ISF) One challenge associated with using samples of offspring to assay genetic structure is that a sample of offspring may be misrepresentative of the adult population, and can thus give biased estimates of parameters of the adult population b) to census number of breeding adults (Nc) of 0.24. VMR has not previously been measured in natural populations of schistosomes. Therefore, in order to accurately measure important epidemiological parameters, it is essential to determine how large of an effect the above sampling issue will have on population genetic studies of this parasite.The distribution of reproductive output among individuals in natural populations of organisms is usually highly skewed, causing the ratio of the variance of reproductive output to the mean reproductive output, Schistosomes are a substantial public health issue in tropical and developing countries. They are estimated to infect over 200 million people (approximately 1 out of 35) worldwide IS) within hosts, and genetic differentiation (FST and GST) among hosts, and we developed a correction procedure to remove the bias introduced by family structure. We also investigated whether a single fecal sample taken from a human host would give an adequate representation of the genetic composition of worms in that host, or whether multiple fecal samples should be obtained from a host over several days.The primary aim of this study was to investigate whether sampling artifacts are likely to influence population genetic studies of schistosomes from humans when offspring are sampled in lieu of adults. We collected data from 2,424 miracidia noninvasively sampled from 12 humans in western Kenya. To determine the amount of reproductive skew and family structure naturally present in samples collected from humans, we used microsatellite genotype data to cluster offspring into putative sibships. We investigated how family structure influenced LD, inbreeding coefficients (Fadult worms in a host (patient), and “component population” to refer to all the adult worms in all the hosts of a host population Throughout the paper we use “infrapopulation” to refer to the We obtained miracidia from human fecal samples from twelve participants enrolled in a longitudinal study To obtain miracidia, eggs were hatched using standard protocols We used sibship analyses to determine whether family structure was present in schistosomes collected from naturally infected human hosts. COLONY v.2.0 n, we also calculated FSF/n in order to compare among samples. This number gives a relative estimate of infrapopulation size. We employed a commonly used metric, the variance to mean ratio (VMR) for reproductive success, to quantify the amount of family structure in each sample of offspring. Ratios greater than one indicate a skewed distribution in reproductive output or a large variance in family sizes. It is important to point out that this metric will be downwardly biased when the sample size is much smaller than the true number of breeders, thus it is a minimum estimate. We also estimated the effective number of breeders The results from COLONY were first used to calculate the number of families occurring in each offspring sample (FSF = “full sib families”). This number included families with a sibship size of 1. Because the total number of families identified in a sample should increase with the number of sampled miracidia, To correct both empirical and simulated samples for family structure, we randomly sampled one individual per family to create a reduced data set that no longer contained any full-sib individuals. We have named this the “one-per-family” approach. Because there is some inherent stochasticity associated with randomly selecting a single individual from each family, we used custom scripts to automate the process, which allowed us to create a large number of “one-per-family” samples for calculations of genetic parameters. By creating a large number of samples, we can effectively sample all single individuals from a given family and capture the associated mean and variance , and inflate FST among hosts ST (theta), which is unbiased with respect to sample size ST  ST values between patients. This process was repeated 1000 times, after which we calculated the mean and 95% CIs for the one-per-family FST values and compared them to the uncorrected data set. We repeated the above procedure to calculate a global (as opposed to pair-wise) value of Weir and Cockerham's FST using the R package Hierfstat ST (G″ST) ST is often used as an analog of FST because FST is dependent on within sample diversity. For assessment of our correction method, G″ST was also calculated in 10 randomly generated corrected datasets for comparison and tested for significance in each dataset using 10,000 permutations of the data. To calculate within-patient FIS for uncorrected (raw) and 1000 one-per-family samples, we used R scripts to combine observed and expected heterozygosities using the standard equation FIS = (He-Ho)/He We predicted that small Nrandomly without respect to the predetermined family structure. This process allowed for equitable comparisons of LD between the one-per-family data sets and the uncorrected  data sets with the same sample size.We next exported 50 one-per-family data sets from R and imported them into GENEPOP to test for genotypic disequilibrium (a proxy for LD). For each patient we used 1000 batches and 10000 iterations per batch to calculate the percentage of loci pairs that were significantly associated with one another . Because statistical tests can be affected by sample size, we repeated the above procedure on “downsampled” empirical data sets, where we reduced the sample size of miracidia from each patient to the equivalent sample size in the one-per-family data sets from that patient. Unlike in the one-per-family data sets, in the downsampled datasets we removed individuals ST, FIS, and LD. Notice that with these simulated data sets we knew the family structure with 100% certainty, whereas with the empirical data we assumed that COLONY accurately captured all of the family structure. Thus, these simulated data sets allowed us to accurately verify the utility of our approach to correcting for family structure.To further validate the one-per-family approach to correcting for family structure, we also created simulated data sets where we could precisely control which individuals belonged to given families. We used a distribution of family sizes that matched the empirical distribution from the COLONY output to recreate the observed family structure. To create simulated adult schistosomes, we used the empirical allele frequencies to generate multilocus male and female genotypes in accordance with HWE. We next paired adults monogamously and randomly selected one allele from each parent to create offspring in accordance with Mendelian expectation. We generated 1000 offspring per adult pair and then randomly sampled a precise number of offspring per full-sib family in accordance with the COLONY output from the empirical data for family size. For example, if a patient had two families of sizes 5 and 30, then 5 full-sib offspring would be sampled from one pair, 30 full-sib offspring would be sampled from the next pair, and the remaining offspring would be discarded. Each offspring was assigned a unique individual and family ID to validate the downstream analyses. All simulated data sets were constructed with a script written in R 2.15.1 ST, HWE, and parentage analysis  the samples of offspring could be directly compared to the samples of adults for measurements of linkage disequilibrium, FST between temporal samples from the same patient using the raw data and tested their significance via 10,000 permutations of genotypes among samples using FSTAT 2.9.3 ST (F′ST) was calculated and standardized GST (G″ST) was calculated and tested for significance with 1000 permutations using GenAlEx v. 6.5 If reproductive rates are constant, then samples from a single patient should be genetically homogenous over multiple days. However, if reproduction occurs in “bursts”, then sampling a single fecal sample could miss diversity within a patient. For three patients 2–3 fecal samples were collected 1–2 days apart. To determine if these samples differed in genetic composition, we first calculated pairwise FST was calculated among sample days within each of the three patients. Because the reduced sample size should influence significance testing, we performed the same tests on downsampled samples to match the sample size of the corrected samples.Next, for the patients for which the raw data indicated significant temporal differences, we corrected them for family structure using our one-per-family approach. Families were resampled 1000 times and mean pairwise Fb was estimated using the sibling assignment method implemented in COLONY b were performed using Graphpad Prism v. 5.01 . To further determine similarity among the estimators, a multivariate principal components analysis (PCA) using egg output , FSF/n, AR, and both measures of Nb as variables was performed using Systat 11 .Another application of the calculation of the number of full sibling families in a sample of miracidia is to estimate the minimum worm burden within a patient (i.e. minimum number of breeding pairs). However, it is possible that with adequate sampling, this measurement, as well as other genetic parameters, may serve as relative or even absolute measurements of worm burden. The number of families detected, as well as other genetic parameters such as allelic diversity, should be strongly correlated with the number of worms that were reproductively active when the sample was taken. With this aim, we investigated the relationship of several parameters obtained from our genetic data and compared them to the World Health Organization gold-standard estimator of worm burden, the number of eggs per gram of feces as determined by the Kato Katz method b to the sample size was high in samples with VMR<1 . Furthermore, analysis of our simulated samples revealed that the majority of half-sib assignments were incorrect . G″ST indicated significant population subdivision of the uncorrected dataset , and was greatly reduced in the 10 corrected datasets and permutation tests indicated no significant population subdivision . Thus, correction of the dataset reduced standardized G″ST from 0.031 to a mean of −0.0013; and statistical significance was lost with this correction. Furthermore, pairwise FST between patients was greatly reduced in the one-per-family data sets , suggesting a very low level of residual real or artifactual differentiation  that was very similar to the value for the uncorrected data (FST = 0.009). Thus, the change in FST value was due to the removal of family structure.The global Fata sets . In facttion see . The oneIS as indicated by the negative correlation between FIS and VMR    . This re= 0.003) .All of the uncorrected samples of miracidia from humans showed significant genotypic disequilibrium, ranging from 38 to 91% of pairwise comparisons of loci . This peST between the adult schistosome infrapopulations in the two mice was low and not statistically significantly different from zero . In contrast, FST was substantially higher . Although, neither adult nor offspring samples deviated significantly from HWE, point estimates of FIS were more negative in the samples of miracidia, consistent with predictions. These samples of miracidia also showed many pairs of loci in significant linkage disequilibrium, while the adult samples showed no loci in significant disequilibrium.The same sampling artifacts were detected in the samples from mice . We sampled patient 12 on three days. Pairwise tests indicated that the fecal sample collected from day 3 was significantly different from those on day 1 and day 2, but samples from day 1 and 2 were not significantly different .When the temporal fecal samples from the same patient were analyzed in their raw form (no correction for sibships), allele frequencies of miracidia differed significantly among fecal samples collected on different days for two of the three patients  . After correction, there were no significant temporal differences between fecal samples. However, the differences between the uncorrected samples also became non-significant when they were downsampled to match the sample sizes of the corrected samples.We corrected the samples using the one-per-family method only for those two patients for which the raw data showed significant differentiation from the pairwise Fb . However, PCA analysis indicated that all the genetic factors were strongly related and loaded heavily on one factor , with egg counts loading heavily on a second factor (0.99). Factor one (genetic measures) explained 69.1% of the variation and factor two (egg counts) explained 22.2% of the variation. Further examination of univariate correlations indicated that all the genetic measures were strongly correlated (Pearson's r = 0.79–0.83 and P≤0.001)  was not correlated with either the number of full sibling families FSF/n , with AR , or either measure of NP≤0.001) . The strS. mansoni miracidia collected from human hosts can contain large numbers of full-sib families and that not explicitly accounting for this family structure could cause one to draw false conclusions concerning population structure. Importantly, this bias could occur in any system where offspring are sampled in lieu of the adults, and has previously been documented in a variety of systems IS and inflated measures of linkage disequilibrium and FST values. Correcting for family structure in our samples of miracidia had a large effect on estimates of population genetic parameters. For example, the number of pairs of loci in linkage disequilibrium in the empirical samples was dramatically reduced after correction (from a maximum of 91% to a maximum of 0.065%). Similarly, correcting for family structure yielded global FST values that were 27 times lower than that from the uncorrected samples. These results were further corroborated by data simulations. FIS values were also downwardly biased by the presence of family structure, although this bias was not large enough to cause the offspring samples to significantly deviate from HWE (data not shown). Even though this bias appears to be small, it could mask important biological information, such as inbreeding or Wahlund effects, or even important laboratory artifacts , that could otherwise be detected. A heterozygote excess over HWE expectations is predicted for samples that contain many siblings and the extent of the excess is increased in hosts having lower Nb and higher family structure We demonstrated that samples of ST in this study may, at first glance, appear to be modest. However, the documented bias is non-trivial and could result in the misinterpretation of important biological processes. For example, even after Bonferroni corrections, pairwise FST tests among uncorrected patient infrapopulations (i.e. including siblings) show highly significant population differentiation among all patients. Consequently, both the global and pair-wise estimates of genetic differentiation may cause researchers to erroneously conclude that the samples were genetically differentiated. However, because all patients were exposed to the same pool of cercariae both spatially and temporally, and more importantly, because both the simulated and empirical data sets that were corrected for family structure yielded mean FST estimates close to zero , we conclude that there is no real genetic differentiation between patients. This conclusion is further reflected in the data from the mouse system. Even though the adult populations were not differentiated, the uncorrected offspring datasets appeared to be. Thus, biases presented by including siblings in population genetic analyses could potentially affect the interpretation of all of the datasets presented here. More importantly, these biases could affect a wide variety of larger-scale studies that make genetic inferences based on sampling offspring.The upward bias measured in global FST of the pairwise comparisons of empirical data was marginally positive in the corrected samples, the correction yields much improved estimates that are likely to be more biologically meaningful. We hypothesize that the residual positive FST and loci in LD are due to family structure not recognized by the sibship analysis and thus not removed from the dataset after correction. This family structure could be due to failure of the sibship analyses to recognize small sibling families or the presence of small half sibling families in the samples . Half-sibships could theoretically be derived from clonal cercariae  that are transmitted together to the same definitive host and pair with genetically different partners. This mating pattern might occur if the cercariae are unable to move far from their infected snail . However, our results suggest that most cercariae are well-mixed before infecting their hosts. Another way half-sibships could be produced is through mate switching. However, mate-switching is not likely to occur in our samples because each fecal sample represents a small window of reproduction and schistosomes are primarily monogamous Here, we also present a correction method that yields population genetic parameter estimates with this bias removed or greatly reduced. Although some loci remained in LD and the average FST, linkage disequilibrium, and heterozygosity (negative bias in FIS). Previous schistosome epidemiological studies have used a hierarchical AMOVA approach in order to remove the bias caused by family structure in offspring samples We have shown that significant family structure is likely to be present in samples of miracidia collected from human patients. Therefore, using uncorrected genetic data collected from schistosome offspring (miracidia) to infer important epidemiological parameters of the adults is likely to generate false conclusions. Failure to correct for family structure can cause one to overestimate FST>0 between the raw data from two sets of temporal samples that were each collected from a patient over multiple days. Tests using only the raw data would cause one to conclude that genetically different subsets of worms are producing eggs on different days. Family structure and differential representation of families in sequential fecal samples appear to be driving the statistical differences in our data. It should be noted that these biases can occur in samples with both large and small family structure  and our genetic proxies for worm burden is interesting, particularly considering the strong correlations detected with genetic measures even with a low sample size of 12 patients. The Kato Katz methodology for egg enumeration is known to be highly variable between fecal samples collected from the same patient and has been deemed unreliable by some particularly when worm burdens are low e from schistosome offspring populations and allelic richness e may not have been very accurate because there was no correlation between Ne values from the same samples calculated by two techniques and they were estimated with large confidence intervals. This lack of accuracy could be due to the small number of markers used, relatively small sample sizes (10–30 miracidia per patient), and pooling of samples from several infrapopulations rather than using a subdivided breeders model e. The lack of correlation may also be due to saturation of allelic richness since there is a limit to the number of alleles that will be found in a population and a correlation beyond this saturation point is not expected.A previous study did not find a relationship between their calculated NST, and underestimates of FIS. The amount of bias in each of these parameters is positively correlated with the skew in reproductive output of individuals. It should also be noted that sibling structure and skewed reproductive output among individuals  could skew additional population genetic parameters and analyses not evaluated here such as observed heterozygosity (Ho), gene diversity (He) Genetic epidemiology is a powerful tool for infectious disease research. However, in cases where offspring must be collected in lieu of adults, data analysis and interpretation should be carefully considered. We have shown that samples of parasite larvae collected from humans can contain significant family structure, which can lead to inflated estimates of linkage disequilibrium and FMicrosatellite genotype data and annotated R scripts to perform the “leave-one-out” procedure will be made available at Dryad.Supporting Information S1Schistosome infections from a model system: Proof of principle.(DOCX)Click here for additional data file.Supporting Information S2Performance of colony using monogamous and polygamous mating system designation.(DOCX)Click here for additional data file.Supporting Information S3Performance of alternative methods of sibship reconstruction.(DOCX)Click here for additional data file.Supporting Information S4Assessing the variance in the “corrected” one-per-family datasets.(DOCX)Click here for additional data file.Supporting Information S5Temporal sampling: Examining the genetic distribution of miracidia obtained from temporally collected fecal samples.(DOCX)Click here for additional data file.


The distribution of genetic diversity among plant populations growing along elevational gradients can be affected by neutral as well as selective processes. Molecular markers used to study these patterns usually target neutral processes only, but may also be affected by selection. In this study, the effects of elevation and successional stage on genetic diversity of a dominant tree species were investigated controlling for neutrality of the microsatellite loci used.Castanopsis eyrei from different elevations (251–920 m) and successional stages were analysed by eight microsatellite loci. We found that one of the loci (Ccu97H18) strongly deviated from a neutral model of differentiation among populations due to either divergent selection or hitchhiking with an unknown selected locus. The analysis showed that C. eyrei populations had a high level of genetic diversity within populations . Genetic variation increased with elevation for both the putatively selected locus Ccu97H18 and the neutral loci. At locus Ccu97H18 one allele was dominant at low elevations, which was replaced at higher elevations by an increasing number of other alleles. The level of genetic differentiation at neutral loci was similar to that of other Fagaceae species (STF = 0.032, Diversity and differentiation among 24 populations of C. eyrei. The study underlines the importance to check the selective neutrality of marker loci in analyses of population structure.The results indicate higher gene flow among similar elevational levels than across different elevational levels and suggest a selective influence of elevation on the distribution of genetic diversity in  Genetic composition within and among populations is shaped by the interplay of genetic drift, gene flow, mutation and natural selection. Molecular markers have helped to identify the effect of life history traits, phylogeographic history and environmental factors on the genetic structure of plant populations Genetic variation within populations often varies along elevational gradients and among species different patterns have been identified Elevational clines encompass a suite of environmental factors that are either physically linked with elevation like temperature Castanopsis eyrei (Fagaceae) is often the dominant tree species in late successional forests. The long lived evergreen species is native to southeastern China and Taiwan and occurs along a large elevational gradient from <300 m to 1700 m a.s.l. (http://www.efloras.org/florataxon.aspx?flora_id=620&taxon_id=200006236). It is monoecious and wind-pollinated and the acorn seeds are predominantly dispersed by gravity and small rodents C. eyrei populations are expected to have high genetic diversity and efficient gene flow mediated by pollen dispersal should result in low levels of genetic differentiation.In mixed and evergreen broad-leaved subtropical forests of Southeast Asia, C. eyrei populations within a nature reserve of continuous mixed broad-leaved forest across a mountain range. Specifically, we ask (1) whether individual loci are more strongly differentiated than expected from a neutral model, and (2) whether spatial structure, elevation or successional stage affect the patterns of neutral and of putatively adaptive genetic variation, respectively.In this study we examined the distribution of genetic variation in STF value from neutral expectations for locus Ccu97H18  varied from 5.4 to 7.7 (mean  = 6.7). The expected heterozygosity (EH) ranged from 0.68 to 0.86 among populations (mean  = 0.78). At the species level, C. eyrei had a EH value of 0.82. The bottleneck analyses indicated recent reduction in population size in five sites . Over all neutral loci, the multiple regression of allelic richness (RA) against elevation and successional stage showed that RA increased significantly with elevation but succession had no significant contribution . For the putatively selected locus Ccu97H18, similarly only elevation had a significantly positive strong effect on RA in the mutliple regression analysis .Successional stage was significantly interrelated with elevation . However, the standardized STF = 0.316 and STF and STF , but the pattern did not exist for pairwise r = 0.069, P = 0.194). When we checked for a pattern of isolation by elevational distance, both pairwise STF and pairwise STF: r = 0.251, P = 0.002; r = 0.210, P = 0.003). Since elevational distance was correlated with geographic distance , we performed partial Mantel tests to test whether elevational distance was related to genetic differentiation after accounting for geographic distance. For the neutral loci, elevational distance remained significant for pairwise r = 0.129, P = 0.010) but not for pairwise STF . For the putatively selected locus elevational distance remained significantly related to differentiation after accounting for geographic distance in both STF and STF: r = 0.188, P = 0.013; r = 0.201, P = 0.001).Populations were significantly structured as revealed by overall d values . For theral loci  and the C. cuspidata var. sieboldiiC. sieboldii, Ueno et al. Fucus serratusAstronium urundeuvaMicrosatellites are assumed to represent ideal neutral markers, so that only gene flow and genetic drift rather than selection should affect their genetic structure. However, an increasing number of studies indicated the presence of non-neutral loci C. cuspidata with these same loci in a limited number of individuals. In our study, C. eyrei showed many more alleles than C. cuspidata in the original work, possibly due to the larger sample size .At the seven neutral microsatellite loci employed in this study a total of 129 alleles were detected with 10 to 25 alleles found per locus. Ueno et al. C. eyrei was high (EH = 0.82) and similar to that of other congeneric species like C. cuspidata (EH = 0.83 C. acuminatissima (EH = 0.72 Genetic variation at the species level in STF = 0.032) indicating only little differentiation Focusing on neutral genetic variation and thus, excluding the putatively selected locus, overall population differentiation was low . However, additionally significant isolation by elevation was detected in both the potentially adaptive locus and the non-adaptive loci after accounting for the effect geographic distance. This pattern of isolation by elevation suggests higher rates of gene flow among sites at similar elevations than along elevational clines Significant isolation by distance was found for the neutral loci and locus Ccu97H18  were collected, totalling 583 individuals  located in the western part of Zhejiang Province, China . ividuals .C. cuspidata var. sieboldiiGAAATTGAGTTGGGTTAGTTCCAC), Ccu28H18 (FAM), Ccu62F15 (NED), Ccu33H25 (FAM), Ccu90T17 (PET), Ccu102F36 (VIC) and Ccu87F23 (FAM) were in one PCR amplification. Another single PCR amplification was performed for Ccu97H18 (VIC). Thermocycler protocol was one cycle of 95°C for 15 min, followed by 35 cycles of 30 s at 94°C, 90 s at 58°C and 1 min at 72°C, with a final extension of 20 min at 72°C. PCR products from the two amplifications were mixed and separated on an ABI 3130 genetic analyzer (Applied Biosystems) with internal size standard GeneScan™ 600 LIZ. Individuals were genotyped using GeneMapper version 3.7 (Applied Biosystems). C. eyrei is diploid and none of the samples displayed more than two alleles.Total genomic DNA was isolated from ca. 10 mg dried leaf material using the DNeasy 96 plant extraction kit (QIAGEN) following manufacturers instructions. Samples were genotyped at eight microsatellite loci previously developed for C. eyrei microsatellite loci should conform to HWE. Because trans-species amplification of microsatellites often results in null alleles we checked the data for the presence of null alleles under the assumption of HWE using MICRO-CHECKER STF values. This approach corrects for null alleles but restricts the analysis to the visible alleles. Third, we left data unchanged. However, all subsequent analyses showed similar results irrespective of the type of null allele treatment. Therefore, we only present the results of the MICRO-CHECKER approach as it allows the calculation of standard diversity descriptors.Because the study species is a wind pollinated perennial tree of the Fagaceae, many of which exhibit populations in Hardy-Weinberg equilibrium  and only for locus Ccu97H18.We tested the eight microsatellite loci for outliers, i.e. markers potentially under selection, using the program FDIST A), allelic richness  and expected heterozygosity (EH) using FSTAT version 2.9.3.2 STF values based on Weir and Cockerham's θ were calculated using FSTAT. As STF is likely to underestimate genetic differentiation between populations for markers which show high levels of allelic variability, we calculated STF) and pairwise geographic distances (log transformed) was tested using the Mantel test implemented in R 2.8.1 STF and pairwise elevational differences (log transformed) to test for isolation by elevational distance. Since pairwise elevational difference was correlated to pairwise geographic distance, we performed partial Mantel tests to test for effects of elevation after accounting for geographic distance. Because allelic diversity differed between populations and was correlated with elevation, this may have biased the estimates of pairwise differentiation using STF. We therefore calculated standardized pairwise STF values  and the two predictors elevation and successional stage in a multiple regression. We used RA, which corrects for sample size, rather than EH, because sample size varied among populations; however, the results were qualitatively the same for EH. Collinearity of elevation and successional stage was checked by Spearman correlation. All analyses were performed with R 2.8.1 To test the effects of environmental factors on genetic variation, we analysed the relationship between allelic richness (


Bother is versatile in English for the U.S., in that it can describe negative mental states and physical sensations, as well as social disturbances. Bother has many different meanings across cultures, due to this versatility. Alternatives for bother were explored for future PRO instrument development.The objective of this study was to determine the applicability of the term bother resulting from psoriasis was analyzed. This disease can negatively impact patients physically, emotionally and socially. Translations of bother were analyzed to determine its meaning when translated into other languages. Cognitive debriefing was conducted on psoriasis patients with the instrument containing bother. Following cognitive debriefing, a questionnaire was distributed to linguists and cognitive debriefing subjects to collect definitions of bother in each target language, and detail any difficulty with translation.A PRO instrument used to evaluate the degree of bother and demonstrate the breakdown of concepts within bother, translations of the Dermatology Quality of Life Index (DLQI) were analyzed. This instrument was selected for its focus on psoriasis and use of terminology that lacks the ambiguity of bother.To establish alternatives to bother yielded a back-translation that was conceptually different from the source 20% of the time (5/26). Analysis of alternative terminology found in the DLQI revealed much greater conceptual equivalence when translated into other languages.An analysis of back-translations revealed that bother to have a negative connotation, a large variety of definitions and synonyms provided across languages showed a lack of conceptual equivalence. Ambiguity of the term across cultures may result in variation in translation, impacting subsequent international data pooling. Analysis of alternatives revealed that measurement of unambiguous terminology produces the greatest conceptual equivalency across languages and cultures.When developing the wording of PRO instruments, the terminology chosen should be applicable across languages to allow for international pooling and comparison of data. While all linguists and subjects of cognitive debriefing understood  Any report of the status of a patient's health condition that comes directly from the patient, without interpretation of the patient's response by a clinician or anyone else, has been termed a patient-reported outcome (PRO) . DependeThere are many challenges when PROs are included in multinational clinical trials . One chaBother is commonly used in PRO instruments to measure a combination of patient satisfaction and discomfort with the disease and/or treatment. Although generally understood to have a negative connotation, bother can be used to describe physical, emotional and social states in U.S. English. This ambiguity results in a wide range of definitions and interpretations across many languages and cultures. Achievement of conceptual equivalence, therefore, becomes much more difficult, possibly threatening the data validity and pooling in multinational clinical trials [bother when translated into different languages, and to determine the degree of difficulty in achieving conceptual equivalence. Following the analysis of bother when translated, acceptable alternatives were explored for use in PRO instruments.l trials . The purbother were analyzed. Its purpose was to find contexts in which bother is used in US English and to form a foundational definition. 2000 statements that included the word bother were randomly selected from a website called LiveJournal.com. This site contains many users’ public diaries in the form of an online blog entry on a topic of the writer’s choice. The results of this observational study revealed that bother was used to refer to negative emotional feelings, pain in a physical sense, and preoccupation in one’s own thoughts [The findings of a previous observational study of thoughts .bother associated with their erectile ability and ejaculatory dysfunction. The bother assessment for both erectile and ejaculatory dysfunction did not seem to yield additional useful data for assessment of erectile dysfunction. It was also found that throughout the 24-week study, the bother score remained stagnant, while the erection and ejaculatory assessment changed throughout the course of the study.In a study to measure erectile dysfunction due to benign prostatic hyperplasia, subjects were asked to assess their ability to obtain an erection and their satisfaction with their ejaculation . Additiobother to assess severity of symptoms [bother proved to be difficult to measure, due to its many “widespread interpretations.” In order to create clarity for the respondent and to improve data validity and pooling, the PRO instrument was developed with bother appearing in association with specific lower urinary tract symptoms, such as patient’s work productivity impact, mental health and sexual health. Although it was not clear whether data validity improved as a result, this alleviated the issue of conceptual ambiguity.In a study of lower urinary tract symptoms, a PRO instrument was developed which utilizes symptoms . During bother, have been observed in previous studies. A translatability assessment [distress, which was hypothesized to have different meanings in other languages and cultures [Distress was used in the Hospital Anxiety and Depression Scale (HADS), a questionnaire used to evaluate a patient’s views of cancer treatment taking place in Israel, therefore requiring that the instrument be translated into Hebrew and Arabic. Because distress is perceived differently between the two languages, the developers surveyed psychologists, social workers and stress researchers who were fluent in Hebrew and Arabic in an effort to create a definition of distress compatible with both languages. Respondents were asked to define distress in their own language. There was no consensus on a definition for distress among those surveyed. However, the solution was to create two distinct definitions of distress: stress from anxiety, and stress from depression. This resulted in two questions broken down into more specific measures: one concerning a patient’s anxiety with regard to their cancer and treatment, and another concerning a patient’s depression resulting from their cancer and treatment.Solutions to ambiguous terminology, such as sessment  was applcultures . Distresbothered them. The name of the PRO questionnaire, its developer and owner must remain strictly confidential, per contractual agreement. Questionnaire data and key terms have been presented and analyzed as appropriate to honor this agreement. It should be noted that psoriasis was the focus for this study, as the disease may cause physical, emotional and social adverse affects. The PRO instrument went through linguistic validation – the process of forward and back-translation, followed by in-country cognitive debriefing. After completing the linguistic validation, cognitive debriefing data concerning the translation of bother were analyzed.In the psoriasis PRO instrument analyzed as part of this study, patients were asked to what degree their symptoms bother within a Numeric Rating Scale (NRS) was carried out in 26 languages: Arabic (Israel), Bulgarian (Bulgaria), Czech (Czech Republic), Danish (Denmark), Dutch (Belgium), Dutch (Netherlands), English (Canada), English (Israel), English (UK), French (Belgium), French (Canada), German (Austria), German (Germany), Hebrew (Israel), Hungarian (Hungary), Italian , Japanese (Japan), Norwegian (Norway), Polish (Poland), Romanian (Romania), Russian (Israel), Russian (Russia), Spanish (Colombia), Spanish (Mexico), Spanish (Spain) and Spanish (US). Translatability and interpretation of bother were explored through analysis of linguistic validation data. Additionally, linguists and cognitive debriefing subjects were surveyed to determine difficulties in translating the instrument and in defining bother.Translation and linguistic validation  of the pbother can be utilized across multiple parts of speech in English, as a noun, verb, or adjective, only the adjectival form is utilized in the observed PRO instrument (“How bothered are you….”). As a result, all observed translations, back-translations, and cognitive debriefing results refer exclusively to one consistent part of speech. In order to gather feedback on foreign-language use of bother across multiple parts of speech, the follow-up survey provided to linguists and cognitive debriefing subjects asked respondents to define and provide comment on the adjectival form found in the instrument and the term bother itself, which may function as either a noun or a verb. Upon review and comparison of the provided definitions and responses, it was determined that the reported meanings of bother showed no meaningful difference across observed parts of speech in non-English languages.Although bother. The dictionary definition of bother was compared to the dictionary definition of the English back-translation, and discrepancies were recorded. Skin was analyzed as a control term for comparison. After linguistic validation was completed, a voluntary questionnaire was sent out to all linguists and cognitive debriefing subjects. Linguists were asked to provide the term they selected for bother in their language, how they defined bother in their translation, and to elaborate on any translation difficulties. Subjects were asked how they defined bother and in what contexts it may be used in their language.Following linguistic validation, all back-translations and cognitive debriefing data were analyzed, with a focus on the translation of bother.It is theorized that including more specific terminology which identifies the adverse effects of a disease individually will yield greater conceptual equivalency when translated. Examples pertaining to psoriasis were found within the Dermatology Quality of Life Index (DLQI) . The folitchy, sore, painful or stinging has your skin been?• Over the last week, how embarrassed or self conscious have you been because of your skin?• Over the last week, how interfered with you going shopping or looking after your home or yard?• Over the last week, how much has your skin influenced the clothes you wear?• Over the last week, how much has your skin affected any social or leisure activities?• Over the last week, how much has your skin prevented you from working or studying?• Over the last week, has your skin created problems with your partner or any of your close friends or relatives?• Over the last week, how much has your skin any sexual difficulties?• Over the last week, how much has your skin caused © Dermatology Life Quality Index. A Y Finlay, G K Khan, April 1992.bother. The example used to test this hypothesis will be the DLQI.To test the hypothesis that more specific terminology will yield greater conceptual equivalency in translation, the linguistic validation results of the selected terminology were compared to those of The subjects of cognitive debriefing were volunteers who self-reported their psoriasis and gave consent to participate in the interview. Confidentiality was protected as subject names were not collected nor linked to any of the data they provided. No medical data was collected. Subject contributions were solely for linguistic research and to ensure comprehension of the translated text. Additionally, the manner in which participant data was collected was determined to be ethically acceptable by the Institutional Review Board Services of Aurora, Ontario in Canada.bother showed that the term was back-translated as something conceptually different from the source 19% (5/26) of the time. Examples of non-equivalent back-translations were discomfort, embarrassment and anxiety. Words that appeared most frequently as back-translations of bother and their respective definitions are as follows:An analysis of all harmonized translations of the psoriasis instrument containing Trouble: To disturb mental calm and contentment• Annoy: To disturb another in a way that displeases, troubles or slightly irritates• Disturb: To interrupt quiet, rest, peace or order• Discomfort: Absence of comfort or ease, uneasiness, hardship or mild pain [• ild pain discomfort, all of the most frequent back-translations were conceptually similar to bother and therefore contain the same level of ambiguity. The most significant back-translation outlier for bother was anxiety. Anxiety was observed to be the least conceptually similar to bother, as it refers to nervousness or agitation. Anxiety was the selected Russian back-translated term for bother in both Russia and Israel.With the exception of bother by the subjects are summarized in Table Following forward and back translation, the psoriasis instrument underwent cognitive debriefing interviews with five subjects per country. The exception was Japanese for Japan, where only four subjects were debriefed, as recruiters were unable to find a fifth volunteer with psoriasis. Subjects had been diagnosed with psoriasis and were diverse as to age, gender and level of education. The Table bother without replacement, and noted the German word for bother (“stören”) to be a “weak term”. Similarly, a subject in the United Kingdom was uncertain as to whether bother was to be used in a mental or physical context. This is significant in a psoriasis PRO, as the disease may cause physical pain resulting from scaling or abrasions on the skin, or mental anguish due to the skin’s unsightly appearance. In this case, due to the ambiguity of bother, it is not clear whether the severities of mental or physical symptoms of psoriasis are being measured.Several anomalous findings resulted from cognitive debriefing analysis. During cognitive debriefing, one German subject suggested removing bother was understood and paraphrased correctly by all subjects, the dominant issues raised were widespread discrepancies of interpretation between subjects. This variation in meaning is explored below.Although bother in their language, and explain how the term is interpreted in their language and country. Additionally, linguists were asked to provide synonyms in English for their selected translation of bother and note whether they had difficulty achieving conceptual equivalence in their language. Not all linguists involved in the linguistic validation project are represented in the questionnaire results, as participation was voluntary. Table All linguists were asked to provide the term they selected for bother, when translated, varies among different languages and cultures, accounting for the variance in understanding among subjects. For example, the Dutch term for bother describes something that is constant or chronic, while the Norwegian term refers to something that is temporary. In another example, the terms selected in French for Canada and French for Belgium differed conceptually. The definitions provided by linguists were “to disrupt or make a change that is not pleasant” in French for Canada, and “to be troubled, annoyed or embarrassed” in French for Belgium. Overall, linguists reported issues with translating bother. Four out of a sample of 19 linguists attributed translation difficulties to the ambiguity of the term. Three out of this same sample of 19 linguists attributed translation difficulties to the fact that the target language has no direct translation for bother.It was observed that the definition of bother. For example, all Bulgarian for Bulgaria subjects defined bother as troubled and uneasy, while all French for Belgium subjects defined it as embarrassment. In some cases, the same language validated in multiple countries yielded similar interpretations. For example, all Dutch-speaking subjects in both the Netherlands and Belgium interpreted bother as a chronic hindrance and something that is always there. Additionally, a majority of German-speaking subjects from Germany and Austria interpreted bother as discomfort. On the contrary, there were several instances in which a common language yielded different results for each country. While all Spanish for Mexico subjects interpreted bother as a provocation of anger, a majority of Spanish for Colombia subjects interpreted bother as discomfort. Furthermore, all English for Canada subjects defined bother as difficulty or trouble, while all English for the United Kingdom subjects defined it as to irritate or to interrupt.Subjects’ questionnaire responses revealed that the majority of subjects within a single language-country pair had common interpretations of bother as used in the target language for cognitive debriefing, as several of the subjects spoke a different native language, or were bilingual. Table bother. This could be attributed to the fact that English was not their native language, and regional differences in terminology. Definitions provided by a sample of four English-speaking Israeli subjects are as follows:A review of the four languages in this study for Israel  revealed interesting results. As was the case with all subjects in the study, all Israeli subjects were asked to define • To make anxious• To cause inconvenience and disturbance• To interfere and annoy• To make problemsbother. On the contrary, linguists for these languages did not share a common interpretation of bother, as demonstrated below:During cognitive debriefing interviews, subjects for Hebrew, Russian and Arabic for Israel reported a common interpretation of • Hebrew – Negative connotation (that can be used in a number of contexts)• Russian – Disturbance• Arabic – Discomfortbother is still apparent in the results, indicating that a level of bother is a problematic measure when attempting to pool data across residents of Israel.Despite having a native tongue other than the questionnaire’s target language, or bilingualism, a discrepancy in the interpretation of bother revealed that more specific terminology will yield greater conceptual equivalency. The back-translated English words of the specific terms were compared to the source English words of the DLQI, mirroring the analysis used for bother. It was often observed that the specific terminology found in the DLQI back-translation was identical to the source. There were a few instances where the back-translated word was different from the English source, but still conceptually equivalent. Examples of such are interfered back-translated as prevented or disrupted, and self-conscious back-translated as ashamed. Table bother. There were 22 available DLQI translations for analysis and 26 translations of the psoriasis instrument containing bother were available for analysis.Terminology found in the DLQI analyzed as an alternative to self-conscious back-translated as shy and prevented back-translated as caused a problem. Unlike bother, however, those issues were corrected easily after consultation with the linguists.As shown in Table bother, as provided by linguists and subjects with psoriasis in response to the questionnaires, demonstrate that the term does not meet these criteria, and does not maintain conceptual equivalence across languages and countries when translated. Furthermore, as demonstrated by the findings within the four languages of Israel, bother may also present a lack of conceptual equivalence between languages within a single country. As further demonstrated by this research, the use of more specific concepts as an alternative of bother will yield greater conceptual equivalence across languages. To conclude, the use of bother in PRO instruments will threaten the validity of data and hinder data pooling from multinational studies. It is recommended that bother be avoided when developing PRO instruments intended for multinational clinical trials.In development of PRO instruments, the terminology chosen should be applicable across languages to allow for multinational data pooling and comparison. The varied definitions of The authors declare that they have no competing interests.MT conducted analyses of the linguistic validation results, conducted analyses of the questionnaire results, and drafted the manuscript. BB designed the study, created the questionnaires and made significant revisions to the first draft of the manuscript. All authors read, made revisions to and approved the final manuscript.


Fagus sylvatica L.) plots experiencing different disturbance levels. Five plot pairs made up by disturbed and undisturbed plots having the same biogeographic history were sampled throughout Europe. Overall, 1298 individuals were analyzed using four highly polymorphic nuclear microsatellite markers (SSRs). Bayesian clustering within plots identified 3 to 11 genetic clusters (within-plot θST ranged from 0.025 to 0.124). The proportion of within-population genetic variation due to genetic substructuring (FCluPlot = 0.067) was higher than the differentiation among the 10 plots (FPlotTot = 0.045). Focusing on the comparison between managed and unmanaged plots, disturbance mostly explains differences in the complexity of within-population genetic structure, determining a reduction of the number of genetic clusters present in a standardized area. Our results show that: i) genetic substructuring needs to be investigated when studying the within-population genetic structure in forest tree populations, and ii) indices describing subtle characteristics of the within-population genetic structure are good candidates for providing early signals of the consequences of forest management, and of disturbance events in general.The fine-scale assessment of both spatially and non-spatially distributed genetic variation is crucial to preserve forest genetic resources through appropriate forest management. Cryptic within-population genetic structure may be more common than previously thought in forest tree populations, which has strong implications for the potential of forests to adapt to environmental change. The present study was aimed at comparing within-population genetic structure in European beech ( Within-population genetic structure is shaped by the complex interplay of genetic and demographic factors. In forest tree populations subject to anthropogenic influence, management can alter both processes influencing the amount and distribution of genetic variation Populus trichocarpa stand characterized by an extensive gene flow via pollen. The stand was composed of individuals that were unambiguously assigned to two distinct subpopulations that probably originated from different seedling cohorts. Jansson & Ingvarsson The partitioning of within-population genetic diversity into well-distinguished genetic clusters indicates the existence of subpopulations . In each pair, plots were chosen as geographically close as possible to avoid the effect of potential confounding factors. An exhaustive spatial sampling allowed us to perform an in-depth analysis of within-population genetic structure using different approaches  that have proved effective for revealing cryptic genetic structuring. Therefore, the specific aims of the study were: i) to investigate the existence and, in case, the characteristics of within-population genetic structure in the ten plots analyzed, and ii) to check whether disturbance had an impact on within-population genetic structure in each plot pair.The lack of experiments specifically designed to compare the within-population genetic structure between disturbed and undisturbed forest tree stands has retarded our understanding of the fine-scale consequences of forest management Study sites and sampling strategy have been described in details in previous works All 1298 sampled individuals were genotyped at four highly polymorphic unlinked SSRs  taking into account the possible presence of null alleles by using the program INEst, running the individual inbreeding model with a Gibbs sampler of 105 iterations General estimates of genetic diversity as well as genetic differentiation and phylogeographic analyses are reported in Buiteveld et al. Fine-scale within-population genetic structure was investigated following two approaches: classical spatial autocorrelation analysis and Bayesian clustering.r) which minimizes the stochasticity found in single locus or single allele estimates of relatedness. Tests for statistical significance were conducted by i) random shuffling (1000 times) of individual geographic locations to define the upper and lower bounds of the 95% confidence interval for each distance class, and ii) estimating 95% confidence intervals around mean r values by bootstrapping pair-wise comparisons within each distance class (1000 repeats). Analyses were performed using both the even distance classes option (using 20 m wide distance classes) and the even sample size option . The nonparametric heterogeneity test proposed by Smouse et al. Spatial autocorrelation analysis was performed using the multivariate method by Smouse & Peakall Sp statistic Sp is computed as Sp = bF/(F1–1), where bF is the regression slope of the kinship estimator Fij computed among all pairs of individuals against their geographical distances, and F1 is the average kinship coefficient between individuals of the first distance class (0–20 m). Sp has the desirable characteristic of being comparable among stands in a single study and among studies. The statistical significance of F1 and bF was tested based on 1000 permutations of individual locations among individuals. All analyses were performed using SPAGeDi 1.3 The intensity of SGS was also measured by the g) and density, spanning from an unrealistically high SGS for beech (F1>0.09) to very low SGS (F1<0.01). Intermediate scenarios were chosen to mimic SGS in the 2 French plots, according to pollen flow parameters and effective number of pollen donors estimated in the same plots To assess the power of the marker set to detect SGS in the studied stands we performed spatially explicit simulations of a population life cycle  for 64 generations following the simulation approach described in Heuertz et al. i) Comparing the no-structure confidence intervals observed in Fh and Fl with the distribution of simulated F1 values from intermediate scenarios. The rationale is that the four microsatellites have sufficient power to detect SGS if SGS estimates from realistic simulations fall consistently outside the no-SGS 95% confidence interval. ii) Comparing F1 values observed in Fh and Fl with those obtained from the simulated extreme scenarios. The overlap of F1 values from the observed vs. simulated scenarios will inform on the power of the four loci to discriminate between realistic and unrealistic SGS scenarios. iii) Comparing simulations with four and 20 loci. This comparison will help us assess the gain in accuracy and precision of SGS estimates when increasing the number of SSR loci.The simulation results were used to assess possible errors introduced by the low number of loci by: 6 iterations, with a thinning value of 1000. The possible presence of null alleles was explicitly taken into account by using the filter.null.alleles option. Each analysis was repeated 10 times. The highest median number of clusters of the 10 repetitions was chosen as the most representative one for each plot  = (1−FIndClu)×(1−FCluPlot)×. Statistical significance of F-statistics was tested using 1000 randomization of the units defined by the level just below that of interest in the hierarchy.Once reliable Bayesian clustering results were obtained, a hierarchical analysis of the genetic structure of all plots was performed to assess the importance of population substructure relative to structure at higher hierarchical scales. The hierarchical estimates of Nc) in a standardized area of 0.21 ha  in each plot. Since there is very large number of ways to subset an area, we performed the standardization procedure as follows: in each plot, the Nc was recorded within a window of 0.21 ha drawn around each individual and the number of clusters was then averaged for all windows around all individuals. To avoid border effects, Nc was calculated only for individuals more than 23 m from the border . Plots with larger areas are expected to contain a higher number of clusters just by chance. To compare the number of clusters produced by GENELAND among sites with different areas, we calculated i) the F1 values computed from the spatially explicit simulations for scenarios with intermediate strength of SGS fell consistently outside the no-SGS 95% confidence intervals obtained by random shuffling of individual geographic locations, ii) the confidence intervals of observed F1 values in Fh and Fl showed a limited overlap with F1 values from simulated extreme scenarios, with an error rate not higher than 19%, and iii) increasing the number of loci from 4 to 20 moderately increased the precision but had no effect on accuracy: mean values did not significantly differ between 4 and 20 loci scenarios as assessed by t-tests with Welch modification for unequal variances between groups . Therefore, we inferred that the 4 microsatellite loci have high power to detect SGS in a dispersal context that is realistic for the beech stands studied in this work.Spatial autocorrelation analyses based on different methods to estimate pair-wise genetic relatedness were concordant in detecting a significant spatial genetic structure in the first distance class (0–20 m) in all plots with the exception of Al and Ih, where no evidence of SGS was observed . CorreloF1 values were recorded for Fh, Gl and Fl, the last two being the only plots with a statistically significant excess of homozygotes  whereas the lowest Sp values were recorded in plots characterized by an absence of SGS (Al and Ih). In pair-wise comparisons, correlograms of the Fh-Fl and Ih-Il pairs were statistically different. This was mainly due to a large difference in the first distance class, with r1,Fh>r1,Fl and r1,Il>r1,Ih  to 0.124 in the recently colonized plot (Fh), with several plots showing high within-plot differentiation , indicating a negligible influence of the possible presence of null alleles on differentiation estimates. Differentiation among clusters within plots  was actually larger than the differentiation among plots, which is determined by both post-glacial recolonization history at the biogeographical scale and disturbance history at the local scale . FIndClu was 0.186, including the effect of both inbreeding (FIS>0) within clusters and null alleles.The highest median number of clusters into which individuals are grouped by Bayesian clustering (averaged over 10 independent GENELAND runs) ranged from 3 (Ih and NLh) to 11 (Fl) . Within-ntiation . The θSStandardizing the number of clusters over an area of 0.21 ha , we found that undisturbed plots are generally characterized by a more complex structure . The meaF. sylvatica were achieved: i) despite the variety of conditions explored, we found that a large proportion of within-population genetic variation is due to genetic substructuring in all plots, and ii) in plot pairs sharing the same bio-geographic history, disturbance explains most of the difference in the complexity of within-population genetic structure.Our experimental setup allowed us to obtain a reliable set of parameters describing the within-population genetic structure of European beech in a wide variety of ecological and management conditions, spanning from undisturbed to highly managed stands. Two relevant results for the comprehension of within-population dynamics in ed) and dispersal distances d<200 trees ha−1). The lack of a spatial signal in the genetic structure of Ih could have been a result of high local density, which should result in a considerable overlap of individual gene shadows. Conversely, the SGS up to 40 m in Fh, an area characterized by high pollen immigration , ranging from 0.0054 in a low intensity managed stand Sp values between 0.005 and 0.015, confirming the general conclusion by Chybicki et al. Sp in F. sylvatica is around 0.01, which is typical for outcrossing species whose pollen is wind-dispersed. Still, according to Vekemans & Hardy Sp between ∼0.01 and ∼0.035 characterize species whose seeds are dispersed by wind and gravity, respectively. The distribution of Sp values suggests that seed dispersal might not be consistently limited in beech. In fact, although within-population seed dispersal distances are usually limited to a few dozens of meters, long distance dispersal can be strongly enhanced by secondary dispersal by birds, reaching up to several kilometres in beech Quercus robur) population, for instance, seedlings growing under their mothers were responsible for high kinship at short distance whereas dispersed seeds accounted for most of the SGS pattern at distances beyond tens of meters F1 is likely to be mainly determined by seed dispersal, low Sp values can also, in part, be determined by the capability of long distance pollen dispersal in beech.Our results together with previous studies FCluPlot = 0.067 and 0.025<θST<0.124, FPlotTot = 0.045). Notably, a relevant local substructure is present when the spatial signal is weak or even absent, making spatial autocorrelation analysis insufficient to characterize all aspects of population stratification at the local scale. Contrarily to what was previously thought, the presence of a complex within-population structure is emerging as a common characteristic in forest trees, even though until now few studies have addressed this topic and contrasting results have been found P. trichocarpa population whereas, in the same study, a small population surprisingly showed a clear substructure A relevant result emerging from our survey is that local substructure explained a larger proportion of genetic variation  are needed to draw conclusions about the effects of natural and anthropogenic disturbances on the characteristics of within-population genetic structure.The shelterwood system produced weak to negligible genetic effects in the stand pairs analyzed , with no difference in genetic diversity Indices of genetic diversity as well as spatial structure and gene flow parameters are well suited for monitoring in long-term modelling studies, but detectable changes have rarely been found when comparing managed and unmanaged sites in beech Figure S1Bayesian clustering results. Distribution of results from the 10 repetitions of GENELAND analyses for each plot. The highest median number of clusters of the 10 repetitions was chosen as the most representative one for each plot.(TIF)Click here for additional data file.Table S1Stand characteristics of the 10 study plots. For each social class the mean value of tree diameter at breast height and height, the stem number, the basal area and total volume are reported.(PDF)Click here for additional data file.Table S2Heterogeneity tests of SGS between disturbed and undisturbed plots using GenAlEx . The reported values represent the degree of differentiation of SGS between two plots from the same site in each distance class (2t) and total ω.(PDF)Click here for additional data file.Text S1Assessment of the potential impact of null alleles on spatial correlation analyses in the studied Fagus sylvatica stands.(PDF)Click here for additional data file.


Ecklonia radiata and Phyllospora comosa and neither species showed any clear patterns of genetic structuring with geographic distance within or among marine parks. In contrast, connectivity was low for the intertidal, Hormosira banksii, and there was a strong pattern of isolation by distance. Coastal topography and latitude influenced small scale patterns of genetic structure. These results suggest that some species are well served by the current system of marine reserves in place along this temperate coast but it may be warranted to revisit protection of intertidal habitats to ensure the long-term persistence of important habitat-forming macroalgae. Adaptively managing marine reserve design to maintain connectivity may ensure the long-term persistence and resilience of marine habitats and the biodiversity they support.Networks of marine reserves are increasingly being promoted as a means of conserving marine biodiversity. One consideration in designing systems of marine reserves is the maintenance of connectivity to ensure the long-term persistence and resilience of populations. Knowledge of connectivity, however, is frequently lacking during marine reserve design and establishment. We characterise patterns of genetic connectivity of 3 key species of habitat-forming macroalgae across an established network of temperate marine reserves on the east coast of Australia and the implications for adaptive management and marine reserve design. Connectivity varied greatly among species. Connectivity was high for the subtidal macroalgae  Marine reserves are increasingly being promoted as a means of managing coastal resources and protecting marine biodiversity A key reason for designing networks of marine reserves is connectivity Temperate marine reserves worldwide are often dominated by macroalgae that constitutes a major biogenic habitat. Macroalgal habitats play a key role in maintaining marine biodiversity because they support a complex array of associated organisms Ecklonia radiata (C.Agardh) J.Agardh, Phyllospora comosa (Labillardière) C. Agardh and Hormosira banksii (Turner) Decaisne, within and among this network of temperate reserves. Given the strength of ocean currents along this coastline , we predict that connectivity will be reasonably high for all species, but variations will be correlated with differences in morphology and life history. Specifically, we predict that macroalgae possessing the ability for long-distance dispersal (e.g. gas-filled vesicles that facilitate rafting) will have greater connectivity than those that lack such structures. We discuss the implications of connectivity of these ecologically-important macroalgal habitats for marine reserve design on both local (within reserve) and regional scales (networks of reserves).A network of marine reserves has been established along ∼800 km of the coast of New South Wales, Australia. Macroalgae constitute the dominant and most conspicuous biogenic habitats on rocky reefs within most of these reserves 2 and were different distances apart (1 to 21 km). Relevant permits for collection of algae at the 4 marine parks were obtained prior to collection.Marine reserves in New South Wales provide protection on a hierarchy of spatial scales with replicate marine “parks” along the coast each containing a number of fully protected “reserves” or sanctuary zones . We chosEcklonia radiata , the endemic Phyllospora comosa  and Hormosira banksii . Each species represents the dominant form of biogenic habitat on intertidal or subtidal rocky reefs in temperate Australia and supports extremely diverse faunal assemblages Phyllospora and the intertidal Hormosira are both dioecious (obligate-outcrossers) with motile sperm and either sessile (Phyllospora) or negatively buoyant (Hormosira) eggs. In contrast, the kelp, Ecklonia has an alternations of generations life history. As with other laminarian macroalgae, sperm are likely capable of dispersal on small spatial scales (e.g. cm) Phyllospora and Hormosira possess gas-filled vesicles that may facilitate rafting long-distances Ecklonia which does not possess such structures. All 3 species are considered cold temperate macroalgae and are nearing the northern limits of their distribution in northern NSW.We chose the 3 most abundant, perennial species of habitat-forming macroalgae to characterise patterns of connectivity; Phyllospora was only sampled from the southerly parks where it was most abundant (BMP and JBMP). Material was air dried, DNA was extracted and individuals were genotyped using 5 microsatellite loci for Hormosira and 7 for Ecklonia and PhyllosporaHo) and expected heterozygosities (He) and FIS (an estimate of inbreeding within populations) were estimated using GENETIX ver. 4.04 Portions of unfouled thalli of at least 32 mature individuals of each species were randomly collected from each sanctuary zone in 2009 and taken to the laboratory on ice. FST estimates FST) can reflect processes in addition to dispersal and connectivity  meta-analyses have shown that such additional processes rarely overwhelm estimates of dispersal and that FST is still an informative statistic for characterising connectivity FST estimates were also estimated between parks and among sanctuary zones within parks. A sequential Bonferroni correction P<0.05 was used. Tests of isolation by distance were done via a Mantel test on pairwise FST and geographic distance matrices. This was done for all data and for each park separately using the program IBD We infer connectivity from estimates of genetic differentiation using Weir and Cockerham's Phyllospora and all loci were in Hardy-Weinberg equilibrium. There were significantly more alleles over all loci in BMP relative to JBMP . Both JBMP and BMP showed random mating with 13 out of 15 FIS estimates being non-significant  was characterised by inbreeding/selfing (P>0.05). Private alleles were only found in 2 sanctuary zones within BMP.For the kelp, /selfing . There wHormosira showed no evidence of null alleles or linkage disequilibrium at any locus. Tests for Hardy-Weinberg equilibrium over all populations of Hormosira revealed that 3 loci deviated from random mating but patterns were variable among sanctuary zones . Private alleles were found in 3 sanctuary zones within BMP and 1 within SIMP.ry zones . Locus Sbreeding . The meaPhyllospora varied by orders of magnitude between parks with strong structure within JBMP and weak structure within the open coast BMP  being similar to inside/outside comparisons (FST = 0.229).Genetic differentiation of oast BMP . Within oast BMP . PhyllosEcklonia, there was insignificant population differentiation within most marine parks  differed from all others. The only park to exhibit a positive correlation between genetic differentiation and geographic distance between sanctuary zones was PSGLMP , but not within any other park  and weaker structure for Phyllospora (FST = 0.14) and Ecklonia (FST = 0.049). For all species, all pairs of parks were genetically different indicating that dispersal is at least somewhat restricted on these scales, however, the magnitude of these differences varied between parks and species . Patterns were more complex for Ecklonia with varying levels of differentiation among different pairs of parks  but not for Ecklonia . Of these migrants, approximately half were likely sourced from a sanctuary zone within the same park (56–59%). Migrants with a likely source in another park were predominatly from adjacent parks.There was a significant relationship between genetic differentiation and geographic distance over all parks for Ecklonia . For HorEcklonia and PhyllosporaHormosira. These patterns were generally reflected within each park and indicate that Ecklonia and Phyllospora are well served by the current system of marine reserves in place along the NSW coast.Connectivity of habitat-forming macroalgae within and among a network of marine reserves along ∼800 km of Australia's temperate coastline varied greatly among key habitat-forming algal species. Connectivity across the network of parks was high for the large, subtidal macroalgae Hormosira, the loss of localised populations may be particularly problematic for this species. Unlike the subtidal species, Hormosira has a more limited habitat range  and is more vulnerable to direct human interactions. For example, it has been demonstrated that cover can be substantially reduced from trampling, which has flow on impacts to associated communities Hormosira to localised impacts and its highly structured populations, it may be warranted to revisit protection of intertidal habitats at Marine Park zoning plan reviews  to ensure long-term persistence of this important habitat-forming species. It should be noted, however, that the Sydney region has a few intertidal protected areas and aquatic reserves in which macroalgae are protected that augment protection afforded by Marine Parks.Given the highly structured nature of populations of the intertidal Ecklonia and Phyllospora. These subtidal species broadcast spawn propagules directly into the ocean and dispersal may potentially occur over long distances. Further, fertile drift material that is torn from the substratum in storms has been found 100 s km away from its nearest source Phyllospora and EckloniaThe East Australian Current (EAC) is the strongest of Australia's continental boundary currents reaching speeds of up to 3.6 m/s and generates a characteristic cyclonic and anticyclonic eddy field EckloniaPhyllosporaThe EAC is also characterised by seasonal variation in its strength and positioning allowing inshore, north-flowing counter currents that can facilitate bi-directional dispersal Ecklonia and Phyllospora, the current system of marine reserves in place along this coastline is adequate to ensure that connectivity of these ecologically important species is maintained. Indeed, dispersal and mixing of genetic material along the NSW coastline appears to be substantial and conducive to resilience of these species to recolonise following perturbations. With predicted strengthening of the EAC with climate change over the next century Given the high connectivity and lack of isolation by distance for Ecklonia with populations within 2 sanctuary zones differing from all others. This subtropical park is near the northern limit of distribution of Ecklonia and north of the main separation point of the EAC. In this park, kelp forests are interspersed with invertebrate and coral dominated habitat and genetic differentiation may thus arise due to the more fragmented nature of Ecklonia populations relative to their southerly counterparts. Further, relative to populations at higher latitudes, populations of kelp at lower latitudes often undergo intense grazing by herbivorous tropical fish that remove the entire canopy (pers. obs) and are known to exhibit an unusual annual life history Latitude may play a role in structuring populations nearing their northern limit of distribution because of their often fragmented nature and variable population dynamics and ecology. The northern most marine park (SIMP) was the only place to exhibit significant genetic structure of Hormosira with greater allelic diversity (and expected heterozygosity) in parks at higher latitudes relative to parks at lower latitudes. Again, this pattern is perhaps not surprising given that northern NSW is the limit of distribution of this temperate alga and populations are likely to be smaller and more fragmented and experience greater population fluctuations as conditions near their physiological tolerances. Lower genetic diversity may confer a decreased ability to adapt to extreme conditions and populations at the limits of their distribution may be at greater risk of extinction under predicted scenarios of climate change. Multiple species of temperate macroalgae have already been observed to have shifted poleward on the coast of NSW Latitude also correlated with genetic diversity of Phyllospora between the 2 marine parks in which it was sampled. Populations within the large embayment of JBMP displayed reasonably high population structure and are likely isolated from the main flow of the EAC by the protruding headlands and narrow entrance of this bay. Low connectivity as seen within JBMP appears to be atypical of the NSW coastline for this Coastal topography is known to influence patterns of connectivity of marine organisms and is likely to account for the contrasting patterns of connectivity of Phyllospora and Hormosira) did not appear to greatly facilitate rafting and long-distance dispersal as evidenced by high genetic structure in Hormosira and, in some places, Phyllospora (JBMP). Hormosira has previously been shown to have the potential for long distance transport of fertile drift material that is torn from the substratum Hormosira. Nevertheless, approximately half of the Hormosira individuals sampled were classified as migrants that were predominatly from adjacent sanctuary zones in the same marine park suggesting that detached fertile individuals of this species must occasionally disperse on these spatial scales because fucoid fertilisation is generally rapid and zygotes presumably attach quickly to the substratum Hormosira may be, however, related to the intertidal habitat of this species. Intertidal fucoid algae generally have limited dispersal potential Contrary to predictions, the extent to which populations of macroalgae were connected was not related to morphology. The presence of gas-filled vesicles (e.g. Ecklonia and Phyllospora but strong correlations for Hormosira over the entire network of parks. These patterns were consistent within parks except for Hormosira where positive correlations were restricted to 1 park (PSGLMP). The spatial arrangement and distances among reserves are therefore important considerations for ensuring connectivity of Hormosira along this coastline. Clearly, dispersal and subsequent connectivity in this species is often reliant on distances between populations or availability of rocky reef. Therefore, consideration must be given to the design of networks of marine reserves to ensure adequate protection of this species, particularly in light of increasing foreshore modification and development.In designing networks of marine reserves, the relationships between spatial distances among reserves and patterns of genetic differentiation are important considerations. Such patterns may help inform planning of the arrangement and spacing of reserves on both local and regional scales. There was no pattern of isolation by distance for the highly connected Ecklonia which occurs throughout the temperate coastlines of Australia. Connectivity in this species is vastly different among Australia's eastern, southern and western coasts and differences correlate with the peak strength of continental boundary currents With networks of marine reserves being established in temperate regions worldwide there is a critical need for developing predictive models of dispersal and connectivity, particularly where little extant data exists. Generalising connectivity is problematic as spatial patterns of genetic structure can be vastly different among species and among coastlines and thus each may require specific conservation efforts. A classic example is Maintaining connectivity alone will not ensure the long-term persistence of macroalgal or other important marine habitats. With the synergistic effects of forecast climatic changes and increasing anthropogenic stressors there are likely to be large and dramatic effects on important macroalgal habitats


Eugenia uniflora L. (Myrtaceae), a shrubby tree with edible cherry-like fruits which is locally known as pitanga or Brazilian cherry. This species is one of the key species in the Atlantic rain forest geomorphological domain, which includes the Atlantic forest and the adjacent Restinga ecosystem [E. unifloraoccurs in areas of medium and large levels of rainfall and can also be found in different vegetation types and ecosystems. This species present economic and folk medicinal applications and is an important pioneer species in the Restinga ecosystem and has been used to recover and manage disturbed and fragmented areas. Our aim in this study was to investigate the phylogeography and genetic diversity of Eugenia uniflora to help elucidate the evolutionary history of this species as a model for gain insights into past vegetation patterns in the Brazilian Atlantic Forest.The Atlantic Forest (AF) is considered the second largest tropical forest in South America with high species richness and endemisms, harboring a large diversity of animals, plants and habitats types . This bicosystem . E. unifE. uniflora were sampled across the Brazilian Atlantic forest. The samples were collected as leaf material (silica gel dried) from natural populations. Total genomic DNA was isolated using the CTAB method [psbA/trnH and trnC/ycF6) were using for population analysis based on sequence quality and degree of variation. These regions were amplified using universal primers. The individual consensus sequences were aligned through CLUSTALW [http://www.Fluxus-engineering.com). Molecular diversity estimates were calculated using Arlequin 3.1[Forty-six populations of B method . Two cpDCLUSTALW  implemenCLUSTALW , then caequin 3.1 and DNAsequin 3.1. Genetich) ranged from 0 to 0.733 and the nucleotide diversity (π) from 0 to 0.00140. Total haplotype and nucleotide diversities were 0.433 and 0.00088, respectively. The highest haplotype diversity was found in populations ITAP, PALM, CAPI and GRAV from the Atlantic forest South. Populations from Northeast were monomorphic. The most common haplotype was H1, present in 39 of 46 populations. The AMOVA analysis showed a very strong differentiation among all E. uniflora populations . These results can help a deeper understanding of the dynamics responsible for both ancient and more recent events that have shaped the current distribution of genetic variability in Atlantic Forest and also have implications for conservation efforts. For the long-term conservation of the genetic diversity of E. uniflora, it would be important to design strategies that aim to preserve most of its lineages. For such, the South region is a key piece, as it houses many divergent and endemic haplotypes and lineages.The total combined cpDNA matrix presented 1224 sites, which eight were variable. Eight haplotypes were found and the haplotype diversity (Financial support: FAPERGS and CNPq