Emerita analoga, Excirolana hirsuticauda, and Orchestoidea tuberculata to the 27F mega-earthquake/tsunami that occurred in Chile in February 2010. mtDNA sequence analyses revealed a lower haplotype diversity for E. analoga and E. hirsuticauda in impacted areas one month after the 27F, and the opposite for O. tuberculata. Three years after the 27F we observed a recovery in the genetic diversity of E. analoga and E. hirsuticauda and decrease in the genetic diversity in O. tuberculata in 2/3 of sampled areas. Emerita analoga displayed decrease of genetic differentiation and increase in gene flow explained by long-range population expansion. The other two species revealed slight increase in the number of genetic groups, little change in gene flow and no signal of population expansion associated to adult survival, rapid colonization, and capacity to burrow in the sand. Our results reveal that species response to a same disturbance event could be extremely diverse and depending on life-history traits and the magnitude of the effect.Earthquake/tsunamis can have profound impacts on species and their genetic patterns. It is expected that the magnitude of this impact might depend on the species and the time since the disturbance occurs, nevertheless these assumptions remain mostly unexplored. Here we studied the genetic responses of the crustacean species  For example, mega natural disturbances, such as tsunamis, earthquakes, fires, and hurricanes can drive long-term variations in the abundance and distribution of species, community dynamics and ecosystem processes in terrestrial and marine ecosystems14. While the extension of these disturbances is short, they can have deep effects on species. Nevertheless, extreme disturbances not only affect species on an ecological level, but also a genetic level. Provided that genetic diversity is linked to the evolutionary potential of a species to respond to a changing environment, the genetic composition of a population plays an important role for its persistence, since populations with more genetic diversity are less prone to extinction17. Thus, evaluating the impact of disturbances on species’ genetic diversity and their responses on short and long temporal scales is fundamental to understanding the factors and mechanisms that determine population and ecosystem resilience.Large-scale natural disturbances can have profound impacts on ecosystems, affecting several ecological and fitness traits of key species through the reduction in population sizes and habitat destruction (e.g.18). The existing evidence has shown that impacts on genetic diversity appear to be idiosyncratic, depending not only on the magnitude of the disturbance, but also on life history traits associated with species’ breeding systems and their dispersal capacities18. For example, genetic studies have demonstrated a loss in genetic diversity in the tropical tree Pithecellobium elegans after the destruction of its habitat19. On the contrary, no effects were observed in any of the genetic diversity indices of the tree Swietenia macrophylla as a consequence of a century of logging20. This patterns in S. macrophylla was explained by the increase in pollen flow and range expansion as a result of logging, which reduced the loss of diversity due to this species’ dispersal capacity. In one of the few studies available on marine species, authors reported that the genetic diversity of populations of the kelp Lessonia nigrescens in northern Chile was reduced by up to 50% after the El Niño–Southern Oscillation event of 1982/8321. Since then, this species has shown a slow genetic recovery that could be explained by its low dispersal potential. On the other hand, authors studying the intertidal goby Chaenogobius annularisthe and the mud snail Batillaria attramentaria found no evidence of loss in genetic diversity as a consequence of the 2011 Tohoku earthquake and tsunami23. Provided that the scale, magnitude and duration of each disturbance varies widely depending upon the study, generalisations about species’ responses to mega perturbations are difficult to draw. To overcome this limitation, it has been proposed that, when possible, studies should examine the genetic responses of multiple species with contrasting ecological and life history traits to the same ecological disturbance18.Empirical evidence evaluating populations’ genetic responses to disturbances are scarce and mostly focused on the effects of rapid anthropogenic destruction of forest ecosystems , the southern-central coast of Chile was struck by a mega-earthquake (8.8 Mw) and tsunami. The earthquake’s rupture zone covered mainly a region localized between 35° and 37°S24, generating changes in the elevation of the coastline of up to 3 m. As a consequence of the earthquake, massive mortality of intertidal flora and fauna occurred in localities with extreme coastal uplift4. In addition, the tsunami produced by the earthquake significantly affected nearshore marine life, devastating hundreds of kilometres of coast12. The magnitude of the tsunami was variable along the Chilean coast, according to the distance from the epicentre and orientation of the coastline25. In a study of the ecological consequences of the 27F earthquake authors found that the mortality of intertidal invertebrates on sandy beaches was positively correlated with tsunami height4. This study suggested that even in the absence of changes in coastal height, the ecological effects of the 27F tsunami on Chilean marine ecosystems could be significant; similar observations have been reported for subtidal macrofauna in other coastal areas of the world29.On February 27Emerita analoga, Excirolana hirsuticauda, and Orchestoidea tuberculata. These species have diverse life history traits, which have differential consequences on their dispersal ability  inhabiting the lower intertidal zone with a meroplanktonic life cycle and high dispersal ability whose larvae can be found in Antarctica and Polynesia, although adults are only found along the coasts of Chile and Peru31. The cirolanid isopod E. hirsuticauda is a conspicuous endemic component of the middle shore intertidal level of Chilean sandy beaches between 22°S and 42°S33 with juveniles and adult individuals being active swimmers35. The talitrid amphipod O. tuberculata inhabits the upper level of the intertidal zone40 of Chile (35°S–40°S), with females incubating their offspring to the juvenile stage and low dispersal potential. These three species were strongly affected by the 27F mega-earthquake, but their responses varied intraspecific and interspecifically depending upon the particular impacts experienced by the diverse populations (variation of uplift level and tsunami height depending on the beach) and the species studied41. For instance, ecological studies have shown that E. analoga was highly affected during the 27F, but presented a low colonization following the 27F; a contrasting pattern was observed in E. hirsuticauda and O. tuberculata, where a rapid post-disturbance colonization was recorded42.Here we evaluated the population genetic response to the 27F mega-earthquake and tsunami in three common crustacean species of Chilean sandy beach ecosystems: ity Fig. . The sanE. analoga, E. hirsuticauda, and O. tuberculata along the Chilean coast. Comparisons among sampled localities were made through time and among highly impacted areas  in E. hirsuticauda, and 1078 bp (T1 and T2) in O. tuberculata (Genbank Accession number MK914652 - MK917441). No evidence of stop codons were detected in the COI gene, no substitution saturation was found for any species or sampling time, indicating that data sets were suitable for all of the subsequent genetic analyses.The sequence alignment length and concatenation of both genes was 875 bp for T1 and T2, respectively, in E. analoga (from K = 5 to K = 2) and slightly increased in E. hirsuticauda (from K = 4 to K = 6) and O. tuberculata , time (p < 0.002), and site*time  nor for their interaction site*time , but not for their interaction  in T1 Table . At T2,  T2 Fig. . At T2, ion Fig. . For E. hem Fig. . Nucleoties Fig. . At T1, mes Fig. . Differeion Fig. . Contrasved Fig. .(Panmictic-Full Model) >5.0) and showed temporal changes in effective population size (Ɵ) and migration rate among clusters (M). In E. analoga the cluster composed of populations in the impacted area had the lowest value of effective population size in T1  was 12% greater than the gene flow from Colcura to the largest cluster E. analoga. The genetic diversity of E. analoga was lower among populations from impacted areas at T1, showing a significant increase by T2, reaching values similar to those found in non-impacted areas. After the earthquake, E. analoga showed a significant reduction in the number of populations; nonetheless, throughout time, as a consequence of gene flow and population expansion, E. analoga showed a higher genetic diversity at T2 when compared to T1. At T2 the largest cluster, including individuals from 13 of the 14 sampled localities, presented the lowest effective population size (Ne) compared to the populations comprised of individuals from the single locality of Colcura. This lower Ne in the largest cluster could be explained by the observed population expansion signal and increase in the gene flow, leading to the homogenization of the 13 populations at T2, a pattern that has been well documented in theoretical and empirical studies47. Ecological studies analysing the effects of the 27F earthquake in E. analoga have suggested that this species was one of the most affected by the coastal uplift and tsunami during the 27 earthquake41. Consequently, the reduction of the genetic diversity of E. analoga through time could be the result of the combined effect of the removal of recruits and adults during the 27F tsunami as well as a source-sink dynamic. In addition, it has been noticed that the higher recruitment of E. analoga throughout the sampled area occurs near February41, thus the disturbance events from the 27F might have reduced the available habitat for new recruits. Provided that the massive adult mortality was a consequence of the coastal uplift, which may have wiped out most of the rare genetic variants in adults, a lower genetic diversity and higher population differentiation could be expected after the earthquake. However, our results suggest that three years after the disruptive event of the 27F, populations might have recovered, with new recruits settled, reducing the genetic differentiation, increasing the genetic diversity and revealing signals of population expansion in the mismatch analyses. This last finding could be the result of a recolonization of individuals from distant non-impacted areas, which can be explained by the high dispersal potential of E. analoga given its intermediate larval dispersal stage in areas like Polynesia and Antarctic waters31.The negative impacts of the combined effects of earthquake and tsunami on impacted areas through time is evident in the genetic parameters of E. hirsuticauda are in line with the ones of other intertidal species23. This species showed no significant differences in either genetic diversity index at the two sampling times, in impacted and non-impacted areas. However, the analysis of population differentiation throughout time revealed a slight increase in population differentiation, no signal of population expansion, and an increase in gene flow along with a reduction of Ne in the southern non-impacted area at T2. A low or nil impact of mega disturbances has also been reported in other marine species. Though this is the first study assessing the effects of earthquakes/tsunamis on crustacean’s species, studies in other species can also be a matter of interest for contrasting results. For instance, along the coast of Japan, authors have noticed that the intertidal mud snail Batillaria attramentaria experienced a reduction in its effective population size (Ne) after the 2011 Tohoko tsunami23. Nevertheless, the effect of this mass mortality produced no significant reduction in the allelic richness of this mud snail. These authors suggested that the lack of impact on this genetic diversity descriptor may have been the result of a combination of very large populations before the tsunami and a high recruitment of juveniles after the disturbance.The genetic patterns observed in E. analoga and E. hirsuticauda species decreased their Hd in impacted areas and increased their Hd in non-impacted areas, in O. tuberculata the highest Hd was recorded in the impacted area at T1. The same pattern was observed at T1 for π in O. tuberculata, but the increase in π in northern non-impacted areas was so high that it turned out to be the area with the highest Hd at T2. Similar to that observed in E. hirsuticauda, an increase in the number of populations was detected in O. tuberculata at T2, with the differentiated populations located within the southern non-impacted area. Although there are only a few of studies available linking ecology and genetics in sandy beach organisms exposed to disturbances, our results are on the line of previous results with talitrids50. Ecological studies have found a similar trend, with the species E. hirsuticauda and O. tuberculata showing similar abundances before and after the 27F earthquake4. Other authors have found that both species quickly recovered their abundance after the tsunami51, suggesting that the particular life history traits of these species may help to buffer the impacts of the mega-perturbation on their local populations. For instance, a high recovery of Orchestoidea tuberculata has been suggested to be related with its feeding strategy as scavengers and detritivorous , with no decisive answer. Here we have shown that recent massive disruptive events can actually be detected with DNA sequences, most likely due to the magnitude of the event. Nevertheless, it will be necessary to compare these results with other markers characterized by faster mutation rates, such as microsatellite or SNPs53, in order to confirm our conclusion. In a broader perspective, no hypotheses has been made yet on the ecologically meaningful units at which genetic patterns are revealed for sandy beach resident organisms. Our results suggest that we can detect changes in the genetic patterns of sandy beach organisms at population level after a disruptive event, even between relatively close populations. Although more observations are needed, this study can help future researchers to evaluate what major drivers explain the genetic diversity of species (with different life history traits) inhabiting areas frequently exposed to disruptive events.Our study clearly reveal that species’ response to a major disturbance, in this case an earthquake and tsunami, depends on the ecology of each species; and that these distinct responses can influence the genetic diversity of species. The use of highly variable DNA sequences allowed to observe these changes in genetic diversity over a short period of time, only a few generations within each species. Whether DNA sequences can be used for recent processes has been the debate of multiple studies throughout the last decade , impacted area (IA) and southern non-impacted area (SNI) . DNA templates were amplified for the mitochondrial genes COI and 16S using the PCR protocols described by other authors58. Amplified products were then sequenced in Macrogen Inc. . Sequences of both genes were concatenated with Mesquite 3.4 software59 and translated to determine whether stop codons were present in the COI gene.Given that no samples were available before the mega-earthquake that occurred in February 2010, we used an impact-control approach. For this, we sampled 14 localities  with different levels of impacts, according to the uplift level and tsunami strengthNI) Fig. . Samplin60 and adjustment to a neutral model of mutation-drift equilibrium61 were assessed with the software DAMBE 6.4.10062 and DnaSP 6.063, respectively. DnaSP was also used to calculate both haplotype and nucleotide diversity (Hd and π). For each species, mean Hd and π values were compared using a two-way Analysis of Variance (ANOVA), with two fixed factors: area with three levels , and time with two levels (T1 and T2). When significant effects were detected, Tukey a posteriori tests were performed. All two-way ANOVAs were run in the STATISTICA 7.0 program64.The potential existence of saturation65 for each species and sampling time (T1 and T2). Short analyses were carried out with K = 12 for all species, with 5,000,000 MCMC iterations, thinning each 1,000 iterations and burn-in of 10%, using the Akaike Information Criteria in MCMC context (AICM)66. Final simulations were carried out by fixing the number of most probable clusters (K) obtained in the first simulations for each species and sampling time, using 30,000,000 iterations of MCMC, thinning each 1,000 iterations and burn-in of 10%. In each analysis, we computed the Poisson-Voronoi tessellation maximizing the value for the rate of the Poisson process (λ)67. The number of genetic clusters was assessed through probability density distribution graphs, for each species in T1 and T2.GENELAND 4.08 was used to detect genetic discontinuities under a spatially explicit genotypic context68 using the software Arlequin 3.5.2.2 69. Evolution models of the sequences (estimated for each gene independently and for the concatenated data set), and the underlying parameters, were estimated with the program jModelTetst 2.1.1071. To investigate signals of population expansion in the three species at each sampling time (T1 and T2) we computed the distribution of pairwise differences from the segregating sites of the concatenated data set by calculating the mismatch distribution analyses implemented in DnaSP.From the variations in the observed haplotype frequency, the genetic differentiation for each species and sampling time was compared applying an Analysis of Molecular Variance (AMOVA). The design of these analyses was carried out by grouping localities depending on the level of impact of the tsunami/earthquake . The genetic differentiation was assessed through AMOVAs based on matrices of estimated genetic distances according to the better-fitted sequence evolution model72. Therefore, we used this software to calculate the genetic diversity and migration patterns of populations with the parameters effective population size (Θ) and migration rate among clusters (M) scaled by the mutation rate (μ) in Migrate-n 3.6.1176. The analyses were performed using 10 replicates of short chains with 50,000 iterations and one long chain with 1,000,000 iterations and 10% burn-in. The obtained values were used to parameterize the priors of Bayesian runs using six independent replicates for each species and sampling time. Each Bayesian run was performed with the transition/transversion ratio value of each gene obtained from the previously inferred evolution models. In each replicate we computed 5,000,000 iterations with samples taken every 1,000 steps and 10% burning. We compared the parameters obtained based on: (1) better adjustment considering constant or variable mutation rates for both genes, and (2) better adjustment to an exponential or uniform model of a posteriori distribution. For each type of mutation rate and distribution model, the six replicates were merged, and the associated marginal likelihood was compared under the Bayes Factor criterion for the selection of models77 implemented in Tracer 1.6.0 software78 with 10,000 Bootstrap replicates. The mean and standard deviation of the obtained Θ and M distributions were represented in gene flow diagrams using the Excel add-ins PopTools 3.279. Additionally, the inferred population genetic structure was contrasted with a panmictic model, where it was assumed that all of the clusters form part of the same genetically interconnected unit. The adjustment was determined using Bayes Factor criterion estimated from the marginal likelihoods of both models using Tracer 1.6.0 software with 10,000 Bootstrap replicates.It has been recently noted that Migrate-n can provide accurate results of recent migration rates, despite the fact that it has most frequently been used to explain long-term historical patternsSupplementary Information


Aedes aegypti (yellow fever mosquito) collected from eight close‐set, high‐rise apartment buildings in an area of Malaysia with high dengue incidence. We used the SNPs to reconstruct kinship groups across three orders of kinship. We transformed the geographical distances between all kin pairs within each kinship category into axial standard deviations of these distances, then decomposed these into components representing past dispersal events. From these components, we isolated the axial standard deviation of parent–offspring dispersal and estimated neighbourhood area (129 m), median parent–offspring dispersal distance (75 m) and oviposition dispersal radius within a gonotrophic cycle (36 m). We also analysed genetic structure using distance‐based redundancy analysis and linear regression, finding isolation by distance both within and between buildings and estimating neighbourhood size at 268 individuals. These findings indicate the scale required to suppress local outbreaks of arboviral disease and to target releases of modified mosquitoes for mosquito and disease control. Our methodology is readily implementable for studies of other species, including pests and species of conservation significance.Understanding past dispersal and breeding events can provide insight into ecology and evolution and can help inform strategies for conservation and the control of pest species. However, parent–offspring dispersal can be difficult to investigate in rare species and in small pest species such as mosquitoes. Here, we develop a methodology for estimating parent–offspring dispersal from the spatial distribution of close kin, using pairwise kinship estimates derived from genome‐wide single nucleotide polymorphisms (SNPs). SNPs were scored in 162  Wright'NS and dispersal has been investigated using composite methodologies incorporating both individual genetic distances and mark–release–recapture (MRR)  reared in suboptimal larval conditions may settle for ovipositing in suboptimal sites more readily than those reared in favourable larval conditions  to estimate relatedness across three orders of kinship, and then uses the spatial distribution of these kin to estimate parent–offspring dispersal and related parameters. Our method inherits from CKMR, but exploits the fact that the spatial separation distance between each kin pair can be characterized as a set of distances representing past parental dispersal events Figure , provideAe. aegypti, the primary vector of arboviral diseases such as dengue, chikungunya and Zika , with the addition of an RNase A digestion step. Extracted DNA was used to construct two RAD libraries containing 81 individuals each. We followed the double‐digest restriction‐site‐associated DNA sequencing protocol for Ae. aegypti developed by Rašić, Filipović, Weeks, and Hoffmann  and with minor allele frequencies >0.05, and thinned the remaining SNPs so that none was within 250 kbp of another. Thinning at this threshold in Ae. aegypti retains approximately 8 SNPs per map unit, a sampling density shown to largely eradicate linkage effects in SNPs , as the maximum of two weeks between the sampling of any pair of individuals would be insufficient time for the ontogeny of an additional generation , we first used maximum‐likelihood estimation in the program ML‐Relate . To determine cousins, we estimated a lower bound of s Figure . We thenk, we randomly sampled 100,000 simulated k scores from each kinship category. In the initial pool of 13,041 empirical mosquito pairings, ML‐Relate identified approximately 50 full‐sibling and half‐sibling pairs. Assuming that the data contained twice as many first cousin  pairings as sibling  pairings, and twice as many second cousin pairings as first cousin pairings, final sampling distributions were developed as follows: 100,000 unrelated, 2,000 s cousins, 500 full‐cousins, 500 half‐cousins, 250 half‐siblings and 250 full‐siblings, giving a ratio of 400:8:2:2:1:1. This assumption is reasonable if the local population size is approximately constant; for a diploid population of constant size, an average of two offspring from any one individual will themselves have offspring. If we take n to be the number of larvae parented by one individual, the ratio of first cousin to sibling pairings would be approximately nfirst cousinsnsiblings=n2n(n-1), which ranges from 1.1 to 4 for 2 ≤ n ≤ 10. If we allow a slight population expansion with an average of 2.4 reproducing offspring, ratios from 1 ≤ n ≤ 10 produce values from 2.2 to 2.4.Using the theoretical means and standard deviations of k scores from the above sampling distribution and plotted a histogram of this combined distribution and a histogram of the unrelated distribution against a histogram of 10,000 k scores from the empirical data. As the combined distribution matched the empirical distribution much more closely than the unrelated distribution , we adopted it for kinship inference. Note that this unrelated distribution is not completely reflective of a natural population, as it assumes total random mating and zero population structure. It is a convenient null from which various models including this one can be developed and compared.To analyse how closely this distribution approximates the field data, we randomly sampled 10,000 simulated k to define a kinship category for first cousins, we plotted histograms of k scores for each kinship category, following the ratio described above . We assigned k = 0.06 as the lower threshold to describe first cousin relationships. Individual pairs of k > 0.06 that were neither full‐siblings nor half‐siblings were much more likely to be first cousins  than any other category.To determine a lower threshold of 2.4We treated separation distances between pairs of sampled kin as representative of multiple past dispersal events, following Figure Axial standard deviations correspond in form to the dispersal component of NS Wright, . We calc2.5PO, with the following equations:Parent–offspring dispersal is described by Wright  as the dAs we assume a mixed distribution of full‐cousins and half‐cousins in the kinship pairings, these represent the upper (full‐cousin) and lower  bounds of parent–offspring dispersal estimates using the first cousin distribution. Assuming a 50:50 ratio of full‐cousins and half‐cousins in our data, the mean parent–offspring axial dispersal can be approximated by the following equation:σ), 2σ represents the effective radius of dispersal, within which 86.5% of dispersed individuals are expected to be found  to quantify the effects of geographical distance, sampling week and building residency on patterns of genetic distance observed among env”), and for effects of sampling week and building on genetic distance after conditioning for the effects of geographical distance (“env|dist”). We quantified the marginal explanative power of each variable in each of the dbRDAs using ANOVA with 10,000 permutations. The functions capscale and ANOVA.cca were used for dbRDA and ANOVA, respectively.We used dbRDA to build three models. The first (“dist”) tested for effects of only geographical distance on genetic distance. The other models tested for effects of geographical distance on genetic distance after conditioning for the effects of sampling week and building .As dbRDA requires independent variables to have one‐dimensional inputs, we first transformed the matrix of pairwise geographical distances into eight principal components (PCs) using the function Rousset,  as the dNS is estimated as the inverse of the slope of the regression of pairwise genetic distance against the natural logarithm of geographical distance  × b. We used the R function “lm” to perform three of these regressions. The first, using all nonsibling pairs of individuals, we used to estimate NS following the above. For the other two regressions, we considered only within‐building pairings and between‐building pairings, to investigate isolation by distance within and between buildings, respectively.In a population experiencing isolation by distance, Rousset, . That is33.1k > 0.06 as first cousins. Mean separation distance for full‐siblings was 18.1 m; for half‐siblings, it was 48.6 m; and for cousins, it was 75.1 m  and density histograms (3B).Using the 3,939 SNPs retained after filtering, we identified 13 full‐sibling pairs and 34 half‐sibling pairs using ML‐Relate. We also designated 51 pairs with 3.2For each kinship category, we used geographical distances of separation between kin to construct a bivariate normal distribution, from which we calculated the axial standard deviation of dispersal Table . Using tSD in Table Following the process for parent–offspring dispersal, we generated dispersal radii for each kinship distribution. Referencing the axial Pairwise locations of all full‐siblings, half‐siblings and first cousins are shown in Figure 3.3p < 0.006). These PCs were used as the geographical distance covariate in the dbRDAs env|dist and dist|env (Table p < 0.025), env|dist showed that building residency had a significant effect on patterns of genetic structure , while sampling week did not . After conditioning for the effects of building residency and sampling week, the 3 PCs in dist|env showed no significant association between geographical distance and genetic distance . As building residency and geographical distance were necessarily correlated, these results indicate a significant effect of building residency independent of geographical distance, and an uncertain effect of geographical distance.The dbRDA evaluating the effects of geographical distance on genetic structure (“dist”) indicated that 3 of 8 PCs were within the significance threshold , all nonsibling pairs within the same building , and all nonsibling pairs in different buildings , all showed significant positive associations between geographical and genetic distances . These isolation‐by‐distance patterns indicate that Ae. aegypti populations are spatially structured within buildings as well as between buildings, show slightly stronger structuring between buildings than within buildings, and show a clear effect of geographical distance independent of building residency. Considering the size of buildings at Mentari Court, this implies isolation by distance at spatial scales of <200 m. Using the slope of the regression for all nonsibling pairs, we calculated NS as 268 Ae. aegypti . This constitutes the effective number of breeding individuals inhabiting a circle of radius 258 m, which is two times the axial standard deviation of parent–offspring dispersal , median parent–offspring dispersal distance (75 m) and radius of ovipositional dispersal within a gonotrophic cycle (36 m). Our additional analyses with dbRDA and linear regression revealed isolation by distance both within and between buildings and provided an estimate of NS  and between buildings, while the dbRDAs showed that building residency had an effect on structure even after accounting for geographical distance. These results suggest dispersal within buildings is less restricted than dispersal between buildings, and that dispersal between adjacent buildings is more common than between nonadjacent buildings. This interpretation accords with kin observations, wherein most kin pairs found in nonadjacent buildings were cousins  of 129 m, we can estimate the ideal density d of Ae. aegypti breeding adults as 1,281 km−2, using Wright's equation for NS under isolation by distance, NS = 4πσ2d . NS is a parameter representing breeding structure in an isolation‐by‐distance framework and relates directly to dispersal, while eN is dependent on local species abundance and thus habitat availability (Nunney, Ae. aegypti estimated average eN of 400 – 600 across different sites and time points (Saarman et al., eN and may improve our ability to understand Ae. aegypti population characteristics. Census size estimates are often five‐ to ten‐fold larger than those of Ne (Luikart, Ryman, Tallmon, Schwartz, & Allendorf, NS, we would expect to find between 1,340 and 2,680 Ae. aegypti within the dispersal radius, a figure consistent with typical adult census population sizes for this species. All of this emphasizes how combining kinship‐based dispersal estimates with more widely used, distance‐based investigations of genetic structure can improve our understanding of local population and breeding processes in Ae. aegypti.Using the regression between individual genetic distance and the natural logarithm of geographical distance Rousset, , we esti Wright, . In inte Nunney, . These pAe. aegypti on only two sequencing lanes, which provided sufficient read depth and breadth to identify 98 pairs of close kin.Apart from the introduction of novel oviposition sites through ovitraps, our methodology of inferring dispersal through genetic relatedness leaves dispersal processes essentially undisturbed, an advantage over MRR studies. Accordingly, genomic inferences of dispersal may more accurately reflect the true biological processes occurring within the study site. An additional advantage of kinship‐based methodologies is their capacity to investigate population processes such as dispersal among populations in which migration rates have changed too recently to have produced corresponding changes in population genetic structure (Palsbøll et al., Our methodology is applicable to a range of species, including those of medical, economic or conservation significance. However, differences in breeding and dispersal ecologies between species will alter the way that the method is applied. For instance, for species where females mate more than once, additional steps will be required to separate half‐sibling dispersal from maternal dispersal. Similarly, species with sex‐biased dispersal will require additional steps when decomposing axial standard deviations into components. Also, while our method is well‐suited to studying rare species of conservation significance, in which direct population manipulation through MRR may not be possible, these studies will likely have low sample size and involve inbred individuals (Hedrick & Kalinowski, eN from kinship (Skrbinšek et al., The kinship‐based inference processes described here have close affinities with the work of Bravington et al.  and cons5Ae. aegypti from the spatial distribution of 98 pairs of kin across three orders of kinship. We also observed genetic structure within and between buildings at Mentari Court, with most movement between nonadjacent buildings taking place over multiple generations. These findings are being considered in the design of future Wolbachia releases at Mentari Court and other high‐density urban sites, and they will also inform protocols for the effective deployment of resources in response to disease outbreaks. Our methodological approach for investigating dispersal and breeding dynamics will be readily adaptable to future studies of dispersal in a range of species.We developed and applied a methodology to investigate dispersal and breeding dynamics in Research was designed by N.W.A., A.A.H. and S.P.S.; research was performed by M.J., T.L.S. and N.W.A.; manuscript was written by M.J. and T.L.S.; new analytical tools were provided by M.J.; data were analysed by M.J.; supervision was done by T.L.S. and A.A.H.; and funding was provided by S.P.S. and A.A.H.Aedes aegypti have been archived at the Sequence Read Archive at NCBI Genbank, with accession number https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA542421.Sorted .bam files and geographical locations for 162  Click here for additional data file. Click here for additional data file.


In the sport context, imagery has been described as the condition in which persons imagine themselves while executing skills to deal with the upcoming task or enhance performance. Systematic reviews have shown that mental imagery improves performance in motor tasksM = 24.37, SD = 2.85). The participants completed the Vividness of Visual Imagery Questionnaire, the Vividness of Movement Imagery Questionnaire-2, and the Mental Image Transformation Tasks.The aim of the present study was to explore whether imagery vividness  and controllability  differ by sport types . Participants were athletes from team contact and non-contact sports , and individual contact and non-contact sports  between the ages of 20 and 33 years (A 2 ×2 × 2 (gender × 2 contact-no-contact × 2 sport type) between groups MANOVA showed differences in imagery ability by sport type. Practical indications deriving from the findings of this study can help coaches and athletes to develop mental preparation programs using sport-specific imagery. Mental imagery is a multisensory process that combines as many senses as possible to generate a vivid mental image. It plays a central role in the execution of movements and in human functioning. In the sport context, An individual can display high abilities in one type of imagery task, but less or none in others. Image vividness depends on the sensory modalities of the stimulus being imaged, capacity of cognitive processes, and individual differences . ControlSeveral studies have investigated the frequency of imagery use in participants practicing different sports, using the Sport Imagery Questionnaire SIQ; . This inAcross these areas of functioning, the effectiveness of imagery and the way that athletes employ it are influenced by different variables, such as gender, the type of skill (open vs. closed), competitive level , and sport types . Concerning gender, previous researches  found thOpen and closed skills differ in their relationship with the environment. In closed skills, it is easier to figure out the real movement that will be performed. On the contrary, open skills require the ability to imagine external events and changes to the environmental situations. Many individual sports involve closed skills, whereas many team sports involve open skills. Nevertheless, a clear classification of sports as typified by open or closed skills is difficult; indeed, many sports include a combination of both open and closed skills. In a study investigating imagery use by elite and novice athletes in open and closed sports, With respect to the expertise level, a recent study  investigRegarding the differences by sport types, researchers found that athletes playing team sports reported greater use of Motivational general–mastery imagery than athletes practicing individual sports, as found in soccer players . FurtherDespite the large amount of imagery research in sport, very little attention has been devoted to the precise relationship between imagery ability and sport types. Given the limited research, the aim of the present study was to explore whether imagery vividness  and controllability  differ by sport types .Further, we expected athletes of individual sports to show greater imagery ability than those of team sports. The hypothesis was based on the idea that successful performance often depends on the extent to which performers effectively identify, perceive, and use important sensory information . In indiMage = 24.37 years, SD = 2.85 years). All athletes had a minimum of 8 years of practice experience in the sport. They usually trained five or six times per week . The sports chosen were representative of individual contact (karate), individual non-contact (tennis), team contact (rugby), and team non-contact  sports.The participants in this study were 120 injury-free national-level athletes (64 men and 56 women) from four different sports: tennis (16 men and 14 women), karate (16 men and 14 women), volleyball (14 men and 16 women), and rugby (18 men and 12 women). They ranged in age from 20 to 33 years  who had no experience in any sport. Participants did not receive previously mental skills or imagery education. Prior to the beginning of the study, ethical approval was granted from the first author’s university ethics committee. The study obtained ethical permission from the University Enna Kore Internal Review Board for psychological research (28 June 2018). All participants were informed about the procedures of the study and the anonymity of their answers before providing their written consent to participate, in accordance with the Declaration of Helsinki.The athletes were all members of sport clubs and were active competitors. Control participants were 60 university students  in the No image at all (only “knowing” that you are thinking of the object)”; 2: “Vague and dim”; 3: “Moderately clear and vivid”; 4: “Clear and reasonably vivid”; and 5: “Perfectly clear and vivid as normal”. Higher scores indicate greater vividness  self-report instrument for evaluating how vividly individuals perform visual mental images . Once people have imagined a scene, they rate the images on clarity and vividness criteria on a five-point scale: 1: “ividness . Investiα of .89  and a 2-(r = .62  to r = .(r = .86 . Previou(r = .86  reportedperfectly clear and vivid) to 5 . The VMIQ-2 measures three diverse kinds of imagery abilities: 1. External visual imagery (EVI), which denotes to an image obtained by observing movement performed by the subject from an outside perspective; 2. Internal visual imagery (IVI), that indicates an image obtained observing the subject him/herself while performing a movement; 3. Kinesthetic imagery (KIN), that describes the process of simulating the somatosensory experience of executing the task, as if individuals were really running. Previous study . The KIN factor of the VMIQ-2 was significantly correlated with the kinesthetic factor of the MIQ-R . The negative correlations are due to the two measures being scored in opposite directions.The Vividness of Movement Imagery Questionnaire-2 VMIQ-2;  is a selus study  establisus study . The resα coefficient of r = .78 relative to the whole score of the Mental Imagery Test. For the current study, two of the tasks were administered.The validated Italian version of the Mental Imagery Test (MIT) was used . The MITThe picture of a large cube is shown for 30 s; it is composed of nine small cubes per face (3 × 3), and the external faces are coloured. After the stimulus is removed, the participant is asked to state how many small cubes have three external (coloured) faces, how many have two, how many one or none .A digital display with the number 88 composed of small segments is shown for 10 s. Then, another digital display with selected segments of a two-digit number is shown for 10 s. The participant is asked to imagine what two-digit number will remain after subtracting the parts of the new figure from the figure with all digits seen previously .The participants were examined separately, in a quiet room, in individual meetings lasting about 20 min. Measurements were conducted faraway the competitions to minimize any distractions. First, participants completed the vividness of the mental image questionnaires (the VVIQ and the VMIQ-2). For the image transformation task, we began by asking the participants to read the instructions on a computer screen. The participant was required to answer as quickly and as accurately as possible. During the test trials, no talking was allowed, and no feedback was provided. The control group was tested in a separate location near the university at the end of lectures.t-test with Bonferroni correction were then used to examine the locus of significant differences.Statistical analyses were performed using the SPSS v. 25. A 2 ×2 × 2  multivariate analysis of variance (MANOVA) was conducted on the data of the study variables. Follow up analysis of variance (ANOVA) and λ = .837, F  = 3.482, p = .003, λ = .710, F = 7.294, p = .001, Findings revealed significant multivariate effects for individual vs. team sports, Wilks F = 6.160, p = .015, M = 59.30; team M = 54.40), and on External Visual Imagery, F = 6.237, p = .014, M = 25.02; team M = 29.18) than athletes of team sports.Follow up ANOVAs revealed significant univariate differences between individual and team sports on VVIQ and EVI variable scores. Specifically, athletes of individual sports reported higher scores on Vividness of Visual Imagery ability, t-tests confirmed that athletes of individual non-contact sport showed greater vividness for motor imagery combining kinesthetic and visual properties compared with athletes of the other sports. Specifically, significant differences emerged in the interaction between individual/team × contact/non-contact sports regarding the following abilities  = 25.537, p < .001, p < .001; tennis vs. rugby p < .001; tennis vs. volleyball p < .001).External Visual Imagery,  •F = 30.416, p < .001, p < .001; tennis vs. volleyball p < .001).Internal Visual Imagery,  •F= 20.175, p < .001, p < .001; tennis vs. volleyball p < .001; tennis vs. rugby p = .006).Kinesthetic Imagery, p > .05) across groups concerning gender.Scores on the Mental Image Transformation Tasks did not yield significant differences among groups. Moreover, there was no significant difference  and controllability  differ by sport types . These results demonstrate that individual and team athletes have distinct abilities and characteristics when it comes to sport-oriented imagery. Mainly, the statistical analysis supported the hypothesis that athletes practicing individual sports exhibit better imagery ability than those of team sports. Specifically, in the tennis group, significantly higher scores were observed on the variables External Visual Imagery, Internal Visual Imagery and Kinesthetic Imagery. This result was not found in previous studies.Sport type also affects the use of visual perspective, which can be either internal or external depending on the sport . ExternaIn partial confirmation of such findings, Furthermore, kinesthetic imagery ability was found to be higher for tennis athletes. This may be related to the nature of tennis, where the necessity of kinesthetic ability is higher in the actual performance, and in which the movement depends on environmental signals such as the adversary’s body language and feedforward approximations of ball motion or opponent movement. Moreover, in individual sports, such as tennis and karate, the lack of interaction with teammates involves a feeling of higher responsibility in the own actions, as well as lower environmental variability than team sports . These fIn our study, there was no significant difference across groups concerning gender. The results are in line with previous research showing mixed results in imagery ability by gender . MoreoveIn conclusion, results of the present research showed differences in imagery ability across sports.Findings of this study highlight the need for coaches and athletes to recognize the specific requirements of their sport to effectively incorporate mental imagery for performance enhancement in their preparation routines.While the different nature  of the sports considered is a strength of this study, athletes from only four sports were involved due to the difficulty to recruit athletes of similar competitive level and ability. This incomplete representation of sport types is a limitation and, therefore, future studies on imagery ability should recruit a larger number of athletes from a wider variety of sports.Moreover, this study did not consider the potential causal relationships between the practice of different types of sports and imagery vividness and imagery controllability. Future research should be conducted using longitudinal analysis or experimental protocols to assess possible causal relationships. From an applied perspective, the findings of this study can suggest practical indications for coaches and athletes to effectively develop mental preparation programs using sport-specific imagery.10.7717/peerj.6940/supp-1Supplemental Information 1Raw data set of the Vividness of Visual Imagery Questionnaire (VVIQ), External Visual Imagery (EVI), Internal Visual Imagery (IVI), and Kinesthetic Imagery (KI).Click here for additional data file.


Diplodus sargus, L. 1758) in the eastern Mediterranean basin, using a panel of four microsatellite markers. Recorded low STF values within the study area indicate little evidence of genetic differentiation among populations. Results suggest high gene flow which may imply near-panmixia between populations, indicating the possibility of a probable movement of adult migrants, or strong passive drift at sea in early life stages of the species. To this extent, bibliographically speaking, different species within the Sparidae family favor altered population dynamics patterns with respect to local populations and genetic divergence, in the context of the molecular marker used.Population dynamics in the marine realm can shape species’ spatial structure and genetic variability between given geographical areas. Connectivity is an important factor of species’ population structure. In this study, we examined the genetic diversity and structure of white seabream ( Diplodus sargus, L. 1758) populations favor a typical homogeneous mixture of individuals, thus suggesting low genetic differentiation and high levels of connectivity [Oblada melanura, L. 1758), where statistically significant genetic differentiation among populations in the Aegean Sea illustrates a potential small-scale population structure [Population structure at sea is cryptic due to the absence of physical barriers. However, with respect to different marine taxa, population structure can differ, e.g., ,2 dependectivity ,5,6. Howtructure . Also extructure  and disptructure . Howevertructure . Barcelltructure  suggest Inferring demographic connectivity from molecular markers has gained awareness, especially for conservation and fisheries management purposes . FisheriThese two features, a focus on larval survivors and an ability to examine long-term mean population connectivities, are critical contributions that genetic studies can make to marine population dynamics. However, genetic surveys of marine populations also face a number of severe challenges that have limited the impact of these approaches on marine ecology, coastal management, or fisheries preservation. The challenges inherent in such accurate genetic determinations have slowed the use of genetic data to estimate real-time population connectivities and have frustrated the use of genetics in many conservation and fisheries contexts .Neutral markers, such as microsatellites, have been used exclusively to identify the geographic structure of subpopulations and estimate the genetic connectivity. In this study, four microsatellites were used to assess the population structure of the white seabream. The white seabream belongs to the Sparidae family and it is found from the Atlantic to the Indian Ocean, in the Mediterranean Sea and the Persian Gulf . PelagicSamples from local fish markets were collected from six different geographical areas in the Aegean Sea, as shown in EXPH) and observed heterozygosity (OBSH) were carried out using the software Genepop v. 3.4 [STF and ISF , numberribed by . Populatribed by  to test ribed by .99P criterion), as shown in STF) was quite low (0.008 ± 0.010 s.d.) indicating little evidence of genetic differentiation among populations (Among the four loci screened (a subset of the genetic data can be found in the ulations . The numulations , revealiulations .The present study indicated a significant connectivity pattern for contiguous populations of white seabream throughout the study area. The structural analysis result was that of a two cluster assignment of populations which significantly correspond to high mixing of all individuals. Published studies show similar a genetic homogeneity strategy for the species in question within the Mediterranean Sea, e.g., see ,5. The AOn the other hand, according to previously reported studies of other benthopelagic Sparidae species , north aTaking into consideration the results presented here, we suggest a considerably high connectivity for this Sparidae species in the eastern Mediterranean Sea. This may be an effect of the mixing of adult individuals in apparently similar geographic areas, which may also contribute to the low differentiation and high connectivity between samples within the Aegean Sea. Different strategies within Sparidae family, with respect to different larvae phases, seem to shape differential genetic composition and population dynamics, e.g., see . MoreoveHowever, one should take into account that sample sizes and the panel of microsatellite markers are just on the verge of the scientific protocol adequacy. Species’ geographical dispersal might be closely linked to oceanographic features and specific ecological habitats and can provide an efficient tool for distribution and abundance identification of all Sparidae species in a conservation management plan for the eastern Mediterranean basin. Populations of the same species may vary in experiencing different recruitment success or survival rates under different environmental conditions ,41. The These findings reveal complex population dynamics patterns within the Sparidae family and therefore have important implications on differential policies for the effective conservation and management for each species within the family.


Gladiolus palustris (Iridaceae) is an endangered European perennial tetraploid herb with special conservation interest in the European Union. Microsatellite markers can serve as effective tools for the conservation genetics of this species.SSR) regions in a microsatellite‐enriched library. Of all SSR regions, 46 were screened for specific PCR amplification, and 15 were found to be applicable in the target species. We found 1.62–3.08 alleles per population  that indicated moderate to high genetic diversity values (0.28–0.44) in three pilot populations. Cross‐species amplification was less effective in G. imbricatus and G. tenuis.We utilized a 454 pyrosequencing approach to identify simple sequence repeat (G. palustris. They will help us to better understand the conservation genetics of this highly endangered species.The primers reported here can be used for the population genetic characterization of  Gladiolus L. comprises approximately 260 species confined to the Old World, which are mainly found on the African continent. Only seven species are native to Europe, with the highest diversity in the Mediterranean  and is incorporated in the International Union for Conservation of Nature (IUCN) Red List as data deficient  analysis 10, (AAC)8, (AAG)8, (AGG)8, (ACG)10, (ACAT)6, and (ACTC)6. The purified and enriched library was used in the 454 GS‐FLX Titanium library preparation  following the manufacturer's protocols performed by Genoscreen Ltd. . The software QDD version 3.1.2 . Primer pairs were selected from the QDD output according to the following criteria: (1) only pure microsatellites were allowed in the target region with at least six repeats, (2) repeats of (AT)n were excluded, (3) the primer alignment score to the amplified sequence had to be lower than 6, (4) primers had to be at least 10 bases away from the microsatellite motif, and (5) consensus sequences were discarded. Out of the 110 potential primer pairs, 46 loci were selected for screening based on fragment length and melting temperature (Tm) of their forward and reverse primers. All loci were selected based on the criterion that primer annealing temperature (Ta) had to be 64°C.Leaves were dried in silica gel, and genomic DNA was extracted using a modified cetyltrimethylammonium bromide (CTAB) protocol (Doyle and Doyle, Initial testing of the 46 primers for specific PCR amplification was based on a single individual from the Nyirád population Appendix , and resG. imbricatus and three individuals of G. tenuis from various locations across their distribution (Appendix G. imbricatus group.Although we expected all 15 loci to be variable based on visual inspection, only 11 loci proved to be variable. We report population sizes, number of alleles, effective number of alleles, and levels of expected heterozygosity Table . We do nAppendix . This maG. palustris. These 11 polymorphic markers are crucial in population genetic studies in the species, providing information on genetic diversity, levels of inbreeding, and gene flow. Thus, they present a much‐needed tool in the conservation and management of this critically endangered species.We identified the first polymorphic microsatellite markers in APPENDIX S1. Multiplexing groups and amounts of PCR product added to the multiplex mix for each locus.Click here for additional data file.


The plaque reduction neutralization test (PRNT), which measures a subset of immunoglobulin antibodies , and the enzyme-linked immunosorbent assay (ELISA), which measures total immunoglobulin , characterize different aspects of the anti–mumps virus antibody response after vaccination.Data from a recent phase 3 clinical trial (NCT01681992) of 2 measles-mumps-rubella vaccines were used to compare anti-mumps antibody responses measured using an unenhanced PRNT  with those estimated using an ELISA .Of 3990 initially seronegative samples, 3284 (82.3%) were seropositive after vaccination for anti-mumps antibodies in both assays. The Pearson correlation coefficient for double-positive samples was 0.57, indicative of a moderate correlation. Receiver operating characteristic curve analysis showed that an ELISA threshold of 51.7 ELISA units/mL best corresponded to the PRNT seroresponse threshold. There was no obvious vaccine brand effect on the correlation between assays.The moderate correlation between the anti-mumps antibody measurements obtained with PRNT and ELISA reflects different aspects of the serological response. In the absence of a well-defined protective serological threshold, PRNT provides complementary information on the antibody response, whereas ELISA remains a critically useful measurement of vaccine immunogenicity. Data from a phase 3 clinical trial of measles-mumps-rubella vaccines show a moderate correlation between the anti-mumps antibody measurements derived by enzyme-linked immunosorbent assay and plaque reduction neutralization test, reflecting different aspects of the serological response measured by each assay. There are no well-defined protective serological response threshold against mumps virus infection , and serThe PRNT is cumbersome and labor intensive and therefore not readily amenable to high throughput, making it difficult to use in large-scale surveillance and vaccine trials . In contGSK has developed an in-house PRNT that likely provides more discriminative titers and a more accurate measurement of immunogenicity to mumps vaccination than a previously used guinea pig complement- and anti-human immunoglobulin G (IgG)–enhanced PRNT . The newThresholds accepted by the Center for Biological Evaluation and Research within the US Food and Drug Administration, as defining clinically meaningful changes in anti-mumps antibody level following vaccination, were applied in analyses conducted in a recent phase 3 study. This trial assessed the immunogenicity and safety of measles-mumps-rubella (MMR) vaccines, MMR-RIT  compared with MMR II , when administered to toddlers; the clinical results are described elsewhere . In the www.clinicalstudydatarequest.com.)The serum samples used in this study were derived from a randomized, observer-blind study of the immunogenicity and safety of MMR vaccine formulations administered to 4516 children in their second year of life NCT01681992), as described elsewhere /mL, and all subjects with PRNT titers <4 ED50 would have ELISA concentrations below the threshold of 10 EU/mL. The ELISA threshold that best corresponded to the PRNT seroresponse threshold had minimal distance from the perfect threshold marker in the top left corner of the ROC curve (sensitivity = 1 and 1 − specificity = 0), ie, the ELISA concentration that minimized the square root of [(1 − sensitivity)2 + (1 − specificity)2]. Statistical analyses were performed using SAS version 9.3 and SAS Drug Development 4.3 software.A receiver operating characteristic (ROC) curve analysis was used to identify the ELISA seroresponse threshold that best corresponded to the PRNT seroresponse threshold of 4 times the 50% end-point dilution (ED50), 36 were seronegative in both, and 670 were discordant . The correlation between immune responses measured by each mumps assay was calculated using the Pearson correlation coefficient, with the 3284 double-positive samples. This provided a value of 0.57, suggesting a moderate positive relationship.In the analysis of postvaccination seropositivity for antibodies to mumps antigen, of 3990 initially seronegative samples, 3284 were seropositive in both assays  attached to the virus, an enhanced PRNT may provide a value closer to that expected from an ELISA that only measures neutralizing antibodies. Moreover, the unenhanced PRNT measures functional antibodies against mumps wild-type strain Mu-90, whereas the MMR-RIT vaccine strain is derived from the Jeryl Lynn strain, thus preventing bias in the measure, specifically overestimates , and proIn our study, comparison of the unenhanced PRNT and the ELISA in the measurement of mumps virus-specific antibody levels indicates a moderate correlation, despite the fact that the assays measure different aspects of the serological response: the PRNT measures a subset of immunoglobulin antibodies , and the ELISA measures total immunoglobulin . Similar trends were noted in the results for all serum samples and according to the MMR vaccine received, MMR-RIT or MMR II. Overall, the rate of seropositivity was higher with the ELISA  than with the PRNT . As well as reflecting the different antibody populations measured by the assays, these results may have been influenced by the different mumps strain genotype used in each assay .50 and ELISA threshold of 10 EU/mL correspond to seroresponse rates of approximately 80% and 98%, respectively [50 had the same seroresponse potential as an ELISA threshold of 51.7 EU/mL, which is considerably higher than the threshold of 10 EU/mL used in the clinical trial and suggestive of a large difference in assay sensitivity. Differences in assay sensitivity may also have contributed toward the results. The PRNT threshold of 4 EDectively , these rectively . In our A poor correlation between assays that measure mumps neutralizing antibody titers and other serologic methods, such as ELISA and immunofluorescent assays, has been reported elsewhere , 13–20. A limitation of our analysis was that it was only based on serum samples obtained 42 days after the first MMR dose. In a 2-year follow-up study of children vaccinated at age 12–15 months with MMR-RIT or MMR II , the relIn conclusion, though there is a moderate correlation between the anti-mumps antibody measurements derived by ELISA and PRNT, the assays measure different aspects of the serological response. In the last decade, there have been several attempts to identify protective antibody titers , 7, but,


Tetrix subulata and Tetrix undulata pygmy grasshoppers, and summarize findings from observational field studies, manipulation experiments, common garden breeding experiments and population genetics studies.Much research has been devoted to study evolution of local adaptations by natural selection, and to explore the roles of neutral processes and developmental plasticity for patterns of diversity among individuals, populations and species. Some aspects, such as evolution of adaptive variation in phenotypic traits in stable environments, and the role of plasticity in predictable changing environments, are well understood. Other aspects, such as the role of sex differences for evolution in spatially heterogeneous and temporally changing environments and dynamic fitness landscapes, remain elusive. An increased understanding of evolution requires that sex differences in development, physiology, morphology, life-history and behaviours are more broadly considered. Studies of selection should take into consideration that the relationships linking phenotypes to fitness may vary not only according to environmental conditions but also differ between males and females. Such opposing selection, sex-by-environment interaction effects of selection and sex-specific developmental plasticity can have consequences for population differentiation, local adaptations and for the dynamics of polymorphisms. Integrating sex differences in analytical frameworks and population comparisons can therefore illuminate neglected evolutionary drivers and reconcile unexpected patterns. Here, I illustrate these issues using empirical examples from over 20 years of research on colour polymorphic This article is part of the theme issue ‘Linking local adaptation with the evolution of sex differences’. In enThe role of temporal environmental variation is less clear. This is partly because temporal changes may be slow and gradual, periodical and predictable, or irregular and rare. In principle, three responses are possible. Individuals may move to areas where conditions are more favourable ; the pheComplexity is increased even more when environments vary in both time and space, forming dynamic mosaic landscapes. In such systems, genetic drift and rearrangements owing to abundance fluctuations and founder events driven by dispersing phenotypes may constitute important drivers of evolutionary change and contribute to population divergence, besides divergent selection and local adaptations. Dispersal may also translate into genetic admixture, the consequences of which are context-dependent and difficult to predict –23.Evolution in heterogeneous environments is potentially complicated yet further by sex-specific differences. Males and females have different roles, resolve life-history trade-offs differently and are often subjected to opposing selection, leading to sex-specific genetic variation and architecture of phenotypic traits –31. It rHere, I illustrate these issues building on examples from over 20 years of research on pygmy grasshoppers.2.Grasshoppers in the family Tetrigidae (Orthoptera: Tetrigoidea) comprise an old, cosmopolitan group of about 1500 species of small, ground dwelling, hemimetabolous insects variously referred to as ground hoppers, grouse locusts or pygmy grasshoppers, henceforth PGH –34. TheyTetrix subulata and Tetrix undulata carried out by myself and collaborators, as reported in 40 original contributions in scientific journals  and in seven compilation Doctorate theses. The findings synthesized below represent the outcomes of joint efforts and diverse approaches, including comparative field studies, capture–mark–recapture studies, phenotype and environment manipulation experiments, staged predator–prey experiments, detection experiments with humans searching for grasshopper images on computer-screens, split-brood breeding designs, stable isotope analyses, and quantitative and molecular genetics analyses. Brief outlines of the approaches used in the different investigations, together with summaries of the main findings, are included in the electronic supplementary material, table S1. More detailed accounts of the methods can be found in the original publications, included in the electronic supplementary material, table S1. At the start of this review, I conducted two topic searches, for Tetrix subulata and for Tetrix undulata, on 28 December 2017, using Web of Science. These searches generated some hits in addition to my own work that concern ecology and evolution of PGHs of relevance for issues discussed in the present review, also included in the electronic supplementary material, table S1.The present contribution builds on a review of previous work on 3.Studies of PGHs have explored behaviours , physiology , morphology  and life-history traits . Many of the studies have set out to determine whether phenotypic dimensions and responses differ between males and females or according to colour morph or wing morph, and whether they vary between populations and/or change over time within populations depending on environmental conditions . Previous work also includes investigations into population performance, genetic structure and diversity .(a)Tetrix undulata and T. subulata represent sister taxa [p-distance: 7.8% in the mitochondrial ND1 gene [ter taxa , but theND1 gene  and 7.6%In my study areas in south-central Sweden, adult and late instar nymphs hibernate during winter and emerge in April–May when reproduction ensues. Females are larger than males, survive at most one reproductive season, produce multiple pods of egg (less than 35 eggs/clutch) and nymphs develop through five  or six  instars before eclosing . IntraspT. subulata is widely distributed in Europe, Asia and much of North America south to Mexico [T. undulata has a more restricted distribution in Europe, usually occupies drier microhabitats, and it is more often short-winged albeit with some variation among populations [There are some species-specific idiosyncrasies, for instance, in how wing morph frequencies, body size, and genetic structure and diversity respond to similar environments . Moreoveo Mexico ,37 and uulations ,32,38. T(b)One of the most distinctive features of PGHs is that they display an enormous variability in colours and patterns. Intra-specific variation in these animals has been documented and analysed for almost a century ,39–41. G(i)The exuberant diversity in colours and patterns is shared by most, if not all, Tetrigidae grasshoppers, pointing to the conclusion that the polymorphism is beneficial and of phylogenetic antiquity . There i(ii)et al. [T. subulata are plastic. However, because the experimental protocol used was not designed to eliminate other sources of variation, there are alternative explanations to what the authors [It is common among Acridoidae grasshoppers that colour patterns are plastic and respond to conditions experienced during embryonic development and growth ,46. The et al.  state th authors  interpre authors ). A poss(iii)Tetrix species [There are many ways by which colour pattern can contribute to variation in performance and fitness among individuals, and thereby impact the spatio-temporal dynamics of colour polymorphisms . There i species ,66.Invertebrates such as spiders  and endoColour patterns of PGHs affect temperature regulation. During sun basking, darker morphs warm up faster and attain higher equilibrium body temperatures compared with paler morphs ,72. SuchSelection imposed by predators and thermal conditions likely play key roles as drivers of clines, mosaics and dynamics of colour polymorphism in PGHs ,64,72,76(c)T. undulata individuals have higher fluctuating asymmetry compared to paler morphs [Colour morphs of PGHs represent integrated phenotypes ,76,77,80r morphs , possiblPGH colour morphs vary in body size ,49,63,83The genetic underpinnings of this phenotypic integration remain unclear, but viable possibilities include supergenes, genetic correlations via pleiotropic effects and regulatory switch mechanisms. That morph frequencies vary between males and females  supports this conclusion. Different colour morphs do not seem to be fully genetically compatible. Dark individuals born to dark mothers survive better than dark individuals born to paler mothers, and pale individuals born to dark mother survive worse than pale individuals born to pale mothers—indicative of co-evolved morph-specific genetic combinations . Caesar As we have seen, the colour polymorphism in PGHs and other animals can inform about phenotypic integration and local adaptation. In addition, it can have important population-level consequences ,55,88–90(d)T. subulata and T. undulata are wing dimorphic. The wings generally correspond in length with that of the pronotum, and macropterous individuals with a long pronotum have fully developed functional wings [PGHs are sedentary animals that normally move only a few metres per day ,63,95. Hal wings ,39,95,98al wings , aid preal wings , and proal wings ,37,38,95T. subulata than in T. undulata, as shown, for example, by pairwise comparisons in sympatry [T. subulata from [r = 0.84, n = 20, p < 0.0001) and the long-winged morph is equally common in both sexes . The long-winged morph is equally common in males and females also in T. subulata populations in Germany .The macropterous morph is more common in sympatry . Spatialata from , r = 0.8n et al. , paired et al. [F1,194 = 852.70, p < 0.0001; effect of wing morph: F1,194 = 0.27, p = 0.60), but see Steenman et al. [et al. [T. subulata phenotypes in families reared in captivity under controlled conditions differs depending on maternal wing morph but is independent of maternal colour morph , arguing against a genetic correlation between colour morph and wing morph.With regard to phenotypic integration, Berggren et al.  show thaet al.  confirmsn et al. . There in et al. , arguingn et al. . Re-anal [et al.  shows thFemales with functional wings have similar clutch sizes and inter-clutch intervals on average as short-winged females , indicatAssociations of long-winged phenotypes with estimates of population genetic divergence, and with within-population genetic diversity, confirm that gene flow influences the evolution of PGH populations ,18,38  and pass through one additional nymphal instar . FemalesT. undulata from 20 sampling locations indicate that genetic structure based on outlier loci influenced by direct or indirect selection differs according to population and between sexes . On a more general level, sex-specific local adaptations can manifest as geographical variation in the degree of sexual size dimorphism [Ongoing work  based on analyses of DNA  data for morphism ,109, a pmorphism .The colour polymorphism and ‘fire melanism’ in PGHs provide another striking example of phenotypic integration, rapid evolutionary change and local adaptation in a dynamic, spatially heterogeneous and temporally changing fitness landscape that is complicated further by sex-specific differences. This can be visualized by hypothetical fitness landscapes . In PGHsT. subulata [r = 0.78, n = 31 samples, p < 0.0001). This suggests that at an overall level, shared selective environments induce parallel micro-evolutionary responses in the two sexes. However, the black morph is 95% more common overall in males , perhaps in part because the protection against predation offered by different colour patterns depend on body size. Thus, detection experiments show that the relative crypsis of different PGH colour morphs varies depending on the visual background [Spatial variation and temporal shifts in the incidence of the black colour morph are correlated in males and females  can potentially generate insights into sex-by-environment interactions for selection, and can also be applied to colour morphs to identify genes under selection. Recent developments in genomic tools (e.g. RAD-sequencing ) togetheComparisons of colour morph distributions in males and females from populations in stable, disturbed and changing environments  (Y. Yild


DNA sequences (1.61 kbp) to analyse the population structure and the sex‐specific pattern of natal dispersal in the frog‐eating fringe‐lipped bat, Trachops cirrhosus, in Central Panama. Our study revealed that—unlike most of the few other investigated Neotropical bats—gene flow in this species is mostly male‐mediated. Nevertheless, distinct genetic clusters occur in both sexes. In particular, the presence of genetic differentiation in the dataset only consisting of the dispersing sex  indicates that gene flow is impeded within our study area. Our data are in line with the Panama Canal in connection with the widening of the Río Chagres during the canal construction acting as a recent barrier to gene flow. The sensitivity of T. cirrhosus to human‐induced habitat modifications is further indicated by an extremely low capture success in highly fragmented areas. Taken together, our genetic and capture data provide evidence for this species to be classified as less mobile and thus vulnerable to habitat change, information that is important for conservation management.Gene flow, maintained through natal dispersal and subsequent mating events, is one of the most important processes in both ecology and population genetics. Among mammalian populations, gene flow is strongly affected by a variety of factors, including the species’ ability to disperse, and the composition of the environment which can limit dispersal. Information on dispersal patterns is thus crucial both for conservation management and for understanding the social system of a species. We used 16 polymorphic nuclear microsatellite loci in addition to mitochondrial  Wing tissue samples (4 mm ø wing punch) were collected and stored in >95% ethanol until DNA extraction.Bats of both sexes were captured and juveniles were identified by the presence of epiphyseal gaps in the phalanges , on three peninsulas in the Barro Colorado National Monument , Gigante  and Peña Blanca , on the other side of the Río Chagres near the Culebra Cut  and the areas surrounding the village of Gamboa situated at the shore of the Panama Canal , Central Panama . Each multiplex reaction contained 1× Qiagen Multiplex Master Mix and between 0.02 μmol/L and 1.10 μmol/L of each primer (Table genescan 500 liz (Applied Biosystems) and the data were analyzed using genemapper v 5.0 (Applied Biosystems).Genomic DNA was extracted using an ammonium acetate precipitation method  using the primers mt‐DNA‐R3‐F and mt‐DNA‐F2‐R .For the mtDNA, the entire cytochrome 2.32.3.1fstat v.2.9.3  using the sampling site as substructure. Expectations of this test are that the dispersing sex should show (1) greater variance in assignment (vAIC), (2) weaker source‐population‐assignment (mAIC), (3) higher within‐group diversity (HS), (4) a deficiency in heterozygotes due to samples representing a mixture of genetic populations  and (5) lower diversity measures among groups (FST) . As juvenile bats, identified by the presence of epiphyseal gaps in the phalanges  and the second one was comprised of all adult males (dataM). The degree of spatial genetic structuring can be measured by the slope of the relationship mentioned above ; (2) only adult males (dataM)], as the presence of closely related individuals within populations can bias Bayesian multi‐locus clustering methods  thereafter consisted of a total of 121 samples, and the dataset only consisting of adult males (dataM) amounted to 142 samples .Before conducting population genetic structure analyses using the program  F+JUV);  only adustructure was run on each nuclear DNA dataset assuming admixture and correlated allele frequencies using the LOCPRIOR model that allows for the use of sample group information  in the clustering process  to set the threshold of mean membership coefficient in any subpopulation of the dataset to greater than 0.5 and to rather consider MedMeaK or MedMedK instead of MaxMedK or MaxMeaK , the significance of deviations from Hardy–Weinberg equilibrium  and expected heterozygosity (He) for each locus as well as for each population inferred by structure for dataF+JUV were calculated using genetix 4.05.2  and the allelic richness (AR), were calculated for each locus and each cluster using fstat v.2.9.3  and Tonatia saurophila (HG003315); Botero‐Castro et al., Moreover, to estimate mtDNA divergence times, we used one individual of each of the two mitochondrial haplotypes determined, the three 2.3.4N = 29). This sampling took place between 2013 and 2016 within a total of 5380 mist netting hours (mnh). Capture rates were calculated for BCI, BO, GI, PB, all islands within the Panama Canal combined (I) and the forest fragments within the agriculturally managed land close to El Giral (A). Among others, the one individual sampled in A was not captured following the standardized mist‐netting approach and is therefore not represented here. We calculated the capture rate as a standardized measure of relative abundance by dividing the number of bats (recaptures included) captured at each sampling site by the number of mnh employed (1 mnh represents one 6 m mist net open for 1 hr). Relative abundance is then provided as number of bats per 100 mnh.Additionally, capture rates were calculated for a subset of the individuals included in the genetic datasets mentioned above, as only those bats captured in accordance with the standardized mist‐netting approach  and clearly indicated male‐biased dispersal .When regressing geographic distance and relatedness between individuals belonging to respective datasets, weak but significant regression patterns of isolation‐by‐distance were detected , GA with BO (GABOF+JUV) and CUL represents the third cluster (CULF+JUV). For dataM, K = 2 shows the clearest signal, whereas BCI represented one cluster and all other sites were encompassed in the second one  = 0.0392, p = .0167; FST(BCIGIF+JUV/CULF+JUV) = 0.0440, p = .0167; FST(GABOF+JUV/CULF+JUV) = 0.0417, p = .0167). As for dataMFST was calculated between BCI and the combined other sampling sites where adult males occurred. Here, FST value was also significant .Analysing data Goudet, , we dete3.3MH102398, MH102399). Fifty individuals represented haplotype 1, and three individuals, all sampled in CUL, displayed haplotype 2. Throughout the complete fragment of mtDNA analyzed, the two haplotypes differed from each other at 46 sites. Within the cytochrome b alone, the two discovered haplotypes still differed at 23 sites  and I (0.04) compared to the other sites that were mainly dominated by continuous rainforest .According to the capture rates obtained for individuals sampled during the standardized mist‐netting approach, the relative abundance of 4T. cirrhosus shows a pattern of male‐biased dispersal. In terms of gene flow, male‐biased dispersal has various consequences and can thus be identified by comparing the sex‐specific population genetic patterns of a species , compared to dataF+JUV (0.0392–0.0440). Secondly, spatial autocorrelation, or the decrease in genetic relatedness among individuals with geographical distance , one of the haplotypes was only found in three of six individuals from CUL and all other individuals showed the same mitochondrial haplotype. Therefore, no conclusions can be drawn here in terms of sex‐specific dispersal.Several lines of evidence clearly indicate that b, as well as noncoding parts of the D‐loop region, which is well known for its high mutation rate  was estimated to have occurred in the early Pleistocene . However, due to a lack of a more complete geographic sampling across the species range, we can only speculate about the origin of these distinct mtDNA haplotypes.The presence of only one dominant mitochondrial haplotype within the study area is quite surprising for tropical species . We can therefore assume that this species reaches sexual maturity as early as other phyllostomids , even within the male dataset, suggests that gene flow is to a certain extent impeded within our study area. If the former Río Chagres Figure  had impeT. cirrhosus as we find no evidence for genetic structuring between BCI and the Southern side of the Panama Canal, including GI.In this context, it is not surprising that bats can cross between BCI and GI even though due to the flooding of the historical river water is also separating BCI from the Southern mainland side of the Panama Canal. Between BCI and the GI side of the mainland, there are various islands that can be used as stepping stones. The largest continuous water body a bat necessarily has to cross here only amounts to 125 m in the southwest of BCI thereby reaching an island that is only separated from the mainland by 110 m. These distances seem to be traversable for T. cirrhosus. This would suggest that, even within a very short time interval of approximately 100 years, changes in population genetic patterns can be observed within a bat species  and I (0.04) compared to the other sites . This further emphasizes the species’ sensitivity and vulnerability to fragmentation  and telemetry tracking studies (Jones et al., T. cirrhosus moves very little within the landscape, mostly stays within the forest understory, and tends to avoid open areas. In accordance with these findings, our own observations and genetic results confirm the classification of T. cirrhosus as a less mobile species (Meyer et al., Mobility has been suggested to be a good predictor of a species’ vulnerability to fragmentation and altered population genetic structure. In bats, the ability and proclivity for long distance flight and dispersal is mostly reflected in the wing morphology of a species (Meyer et al., 5T. cirrhosus. Moreover, we were able to demonstrate that T. cirrhosus shows male‐biased dispersal. Further research is needed to investigate the driving force for the strong pattern of male‐biased dispersal observed in T. cirrhosus.In accordance with our hypothesis, our results indicate that habitat fragmentation seems to influence both the population genetic structure and the distribution of T. cirrhosus as well as in other bat species within the same habitat. In this context, the phyllostomid family would be particularly interesting as species of this family are relatively closely related, but show high levels of ecological diversity through various differences in morphological, physiological and behavioral traits (Baker, Jones, & Carter, Finally, the effect of the Panama Canal as a potential barrier to gene flow should be further investigated in T. cirrhosus. Knowledge on sex‐specific patterns of natal dispersal is important for understanding the social system of this otherwise well‐studied species and can be used as a basis for future social and behavioral investigations. Moreover, the results from our study may be used to inform effective conservation measures in regions suffering from a high impact of human‐induced habitat fragmentation and to gain additional knowledge on the effects of environmental disturbance on bats.This is the first study to provide population genetic data for The authors declare that they have no conflict of interest.TH and GK designed the study with input from RP and SJP. RP, SB, MT, VF, and TH provided data. TH performed the laboratory work and the data analyses and drafted the manuscript with input from RP, SJP, and GK. SJP contributed further to analyses. SB conducted the analyses on the capture rates at the different sampling sites. All authors have commented on the manuscript and have read and approved the final version of the manuscript. Click here for additional data file. Click here for additional data file.