Taurulus bubalis, Euphrasen 1786) belongs to the family Cottidae and is a rocky shore species that inhabits the intertidal zones of the Eastern Atlantic since Iceland, southward to Portugal and also the North Sea and Baltic, northward to the Gulf of Finland, with some occurrences in the northern Mediterranean coasts eastward to the Gulf of Genoa. We analysed the phylogeographic patterns of this species using mitochondrial and nuclear markers in populations throughout most of its distributional range in west Europe. We found that T. bubalis has a relatively shallow genealogy with some differentiation between Atlantic and North Sea. Genetic diversity was homogeneous across all populations studied. The possibility of a glacial refugium near the North Sea is discussed. In many, but not all, marine temperate organisms, patterns of diversity are similar across the species range. If this phenomenon proves to be most common in cold adapted species, it may reflect the availability of glacial refugia not far from their present-day northern limits.The longspined bullhead ( One of the major concerns in studies of phylogeography of northeastern Atlantic organisms has been the location of unglaciated refugia, where marine inshore organisms could survive the successive glacial peaks of the Pleistocene . These refugia must have acted as propagule sources for the recolonization of more northerly areas during the interglacials The inshore fish that now occur in west Europe vary in their thermal tolerance and biogeographic origin. Several distributional patterns can be observed. Warm temperate and tropical eurithermic species rarely breed in the North Sea and must have survived the glaciations in the warmer waters of the Mediterranean, south Iberia and/or western Africa . More cold tolerant species (cold temperate) presently live and breed north of the English Channel in the North Sea, reaching northern Scandinavia. Artic species occur mainly in or near the Arctic and are very rarely found south of the English Channel Symphodus melopsAtherina presbyterPomatoschistus micropsSyngnathus typhleHomarus gammarusNephrops norvegicusSprattus sprattusLipophrys pholisWhen we turn to genetic diversity, the available data reveal, again, many contrasting patterns. Some results conform to a pattern of high level of genetic diversity in the southern  part of the range, with genetic diversity decreasing to the northern limit of the species distribution. In these cases, few haplotypes will be present; near the northern limit they would be widespread in large areas and often derived from alleles present in the south, private haplotypes being rare or absent. Examples in inshore fish include Taking all studies together, the phylogeographic patterns uncovered in the northeastern Atlantic hardly fit a single explanation. We suggest that the differences in thermal tolerances mentioned above may have an important role in explaining a substantial part of the variability in the results. Multiple refugia, varying in latitude and probable SSTs during glacial peaks likely mean that warm water species must have survived in warm areas, while cold temperate species may have survived in low temperature refugia, closer to the ice sheets. In order to assess this hypothesis, however, the number of phylogeographic studies of western European inshore fishes must be increased using fish with contrasting thermal preferences in order to provide data for multi species comparisons.In the first years of marine phylogeography much emphasis was placed in thermophilic species since much of the focus in studies of European marine ichthyofauna involved the assessment of the role of the Mediterranean as a refugium and the level of differentiation between Atlantic and Mediterranean populations Taurulus bubalis, Euphrasen 1786) belongs to the family Cottidae and is a rocky shore species that inhabits the intertidal zones (tide pools and inshore waters) of the Eastern Atlantic from Iceland, southward to Portugal, the North Sea and the Baltic northward to the Gulf of Finland, with some occurrences in the northern Mediterranean coasts eastward to the Gulf of Genoa The longspined bullhead  data set consisted of a total of 425 characters , representing a total of 27 haplotypes . The ampThe CR haplotype network shows two dominant haplotypes, separated by only two mutational steps . The mosFor the CR fragment and S7 intron, the genetic diversity indices for each population are summarized in x2 = 5.930, p = 0.431).For the CR data, haplotype diversity values were not significantly different among populations according to the X2 test developed by CT  for two groups: the North Sea locations versus the three Atlantic locations.SAMOVA (CR dataset) yielded a maximum FFSTs and corrected average pairwise differences shown in The pairwise FSTs and SAMOVA we decided to pool together Egersund, Oslo, Gullmar Fjord and Helgoland vs Roscoff, Spain and Portugal for further analyses. A hierarchical AMOVA for the S7 data using the same two groups  yielded a non significant difference among groups with most variation being within populations: percentage of variation among groups = 2.89 (p = 0.296); percentage of variation among populations within groups = 14.56 (p<0.001) and percentage of variation within populations = 82. 56 (p<0.001) (FST = 0.1744).Given the concordance between the pairwise p<0.05; Fs = −4.532, p<0.05), while for the Galicia population only Tajima test yielded a negative and significant value . In the absence of negative and significant values for the neutrality tests in most populations the application of mismatch analysis was assumed to be inappropriate.Tajima and Fu’ Fs tests, for the CR dataset, yielded negative and significant results only for the Roscoff population , reaching a Nef of 13 million fish in the present day. The Time for the Most Recent Common Ancestor (tMRCA) for T. bubalis is 12.701 kya, with the southern population having a higher tMRCA (6.205 kya) when compared with the northern population (5.425 kya).ef than the northern one. The migration rate was higher northwards , than in the opposite direction . We could not perform estimations of population age because the model including growth did not converge.Lamarc analyses yielded higher theta for the southern population (average 0.012) in comparison with the northern population (average 0.005), suggesting that the southern population may have a larger NT. bubalis. The genetic diversity is similar throughout the entire species range and, although there is statistically significant genetic differentiation between the Atlantic and the North Sea, the northern populations display a mixture of widespread haplotypes with others that are almost exclusive of the North Sea.The results presented above highlight two main features of the phylogeography of in situ accumulation of mutations in the north. This possibility seems however less probable because of the clade which is exclusive to the North Sea and nearby Roscoff represents almost half of the fish sampled in the North Sea (where it is widespread), but gave rise only to 5 haplotypes a single mutational step away.The presence in the North Sea of a mixture of widely distributed haplotypes with an entire clade almost exclusive of this area, from which a number of private haplotypes derive, is consistent with the hypothesis that the North Sea was colonized from at least two different sources. Indeed, these results are compatible with a scenario with two components: 1) survival in the Atlantic with an expansion to the north as the climate ameliorated after the end of the last glaciation, an expansion that is possibly still in progress; 2) persistence in periglacial conditions of at least one high latitude population that could take advantage of its proximity to the retreating ice to colonize quickly the new habitats that were becoming available. We cannot discard the possibility that the high genetic diversity in the North Sea resulted from an expansion from the Atlantic population with T. bubalis is a cold water adapted species that breeds at very high latitudes  and HDL1 (5′- CCTGA AGTAG GAACC AGATG CCAG - 3′)  TGG CCT CTT CCT TGG CCG TC - 3′) and S7RPEX2R (5′- AAC TCG TCT GGC TTT TCG CC - 3′) http://www.stabvida.net/).Total genomic DNA was extracted from fin samples preserved in 96% ethanol with the REDExtract-N-Amp kit (Sigma-Aldrich) following the manufacturers instructions. Voucher specimens are deposited in ISPA (ethanol preserved tissues). PCR amplification of mitochondrial control region (CR) and the first intron of the nuclear S7 ribosomal protein gene (S7), were performed with the following pairs of primers: dloop – LPro1  approach ARLEQUIN software package V.3.5 a priori, allowing instead the groups to emerge from the data. The most likely number of groups was identify by running SAMOVA with two to six groups and choosing the partition scheme with the highest FCT value. Because SAMOVA identified two groups which maximized FCT (see the results section) the data of the populations included in each group were pooled and mismatch analysis FsFor the CR, the spatial analysis of molecular variance (SAMOVA 1.0) T. bubalis was inferred with the CR data using both mismatch distributions and the extended Bayesian skyline plot (BSP) tMRCA) and the median and corresponding credibility intervals of the BSP were depicted using Tracer v.1.4 et al. 2003; 15%–20%/MY for Indo-Pacific sardines, T. bubalis we address the uncertainty by tentatively assuming a within lineage mutation rate of 5%/MY, which is within the range of values found by Past population demography of


Heterandria formosa, a wide-ranging regional endemic, presents an ideal system to test the expansion-contraction model within this biota. By integrating ecological niche modeling and phylogeography, we infer the Pleistocene history of this livebearing fish (Poeciliidae) and test for several predicted distributional and genetic effects of the last glaciation.Climatic and sea-level fluctuations throughout the last Pleistocene glacial cycle (~130-0 ka) profoundly influenced present-day distributions and genetic diversity of Northern Hemisphere biotas by forcing range contractions in many species during the glacial advance and allowing expansion following glacial retreat ('expansion-contraction’ model). Evidence for such range dynamics and refugia in the unglaciated Gulf-Atlantic Coastal Plain stems largely from terrestrial species, and aquatic species Pleistocene responses remain relatively uninvestigated. Hd = 0.934; mean π = 0.007) consistent with rapid, recent population expansion; a lack of mtDNA isolation-by-distance; and clinal variation in allozyme diversity with higher diversity at lower latitudes near the predicted refugium. Statistical tests of mismatch distributions and coalescent simulations of the gene tree lent greater support to a scenario of post-glacial expansion and diversification from a single refugium than to any other model examined (e.g. multiple-refugia scenarios).Paleoclimatic models predicted range contraction to a single southwest Florida peninsula refugium during the Last Glacial Maximum, followed by northward expansion. We inferred spatial-population subdivision into four groups that reflect genetic barriers outside this refuge. Several other features of the genetic data were consistent with predictions derived from an expansion-contraction model: limited intraspecific divergence (e.g. mean mtDNA p-distance = 0.66%); a pattern of mtDNA diversity (mean H. formosa fits the classic Pleistocene expansion-contraction model, even as the genetic data suggest additional ecological influences on population structure. While evidence for Plio-Pleistocene Gulf Coast vicariance is well described for many freshwater species presently codistributed with H. formosa, this species demography and diversification departs notably from this pattern. Species-specific expansion-contraction dynamics may therefore have figured more prominently in shaping Coastal Plain evolutionary history than previously thought. Our findings bolster growing appreciation for the complexity of phylogeographical structuring within North America’s southern refugia, including responses of Coastal Plain freshwater biota to Pleistocene climatic fluctuations.Congruent results from diverse data indicate  Present-day distributions of many of Earth’s biotas reflect the profound influence of climatic and sea-level fluctuations during the glacial-interglacial cycles of the Pleistocene , passively displaced , or went extinct  and geographic distances [ln (km)] among sites at rangewide and FL-region levels . We used straight-line geographic distances, not river distances, because several sites were bodies of water (e.g. lakes) that are isolated from any nearby rivers. Based on our niche models (see Results), we predicted much of the range would lack isolation-by-distance; however, under vicariance-northeast colonization, only ACP populations should lack isolation-by-distance  in the Early-Middle Pleistocene. The tMRCA estimate for subclade 'a’ was 0.377 Ma . Bayesian posterior distributions for these tMRCA estimates strongly rejected LGM divergence . Thus, the deepest population structuring (interpopulation divergence) within H. formosa appears to have initiated before the LGM. We estimated divergence between Poeciliidae sp. and H. formosa at 8.478 Ma  in the Late Miocene-Early Pliocene; confidence intervals for this split were much wider than the tMRCAs above but we still statistically rejected Pleistocene divergence (P < 0.05). Parameter-trace ESS scores >600 indicated thorough Markov chain mixing. Our BEAST runs estimated a geometric mean evolutionary cytb rate of 7.12 × 10-9 substitutions/site/yr, and an RPS7 rate of 1.01 × 10-9 substitutions/site/yr.The model resulting from multilocus coalescent-dating in BEAST ; however, Bayesian posterior Nefm distributions peaked at the lower limit of resolution, with 95% confidence intervals including zero. Setting ancestral Ne equal to the Nef estimates above, we ran separate simulations testing three hypotheses modeled as hypothetical population trees , and subsequent colonization and diversification of local subpopulations  while simultaneously expanding from a source population , thus our gene tree is more consistent with a scenario of LGM population contraction and postglacial re-expansion than either of the alternative vicariance models.By summing regional group Our results failed to reject most expansion-contraction model predictions  compare  or within local habitats. If any secondary refugium existed, the most likely candidate area would seem to have been the Tallahassee area, where we also found one private allozyme allele.Of course, it is difficult to discount the possibility of 'microrefugia’  in smallH. formosa are characterized by shallow genetic divergences and a single population-lineage , suggesting local H. formosa populations have not evolved independently but that gene flow has importantly shaped their evolution [Nem within and across two peninsular Florida rivers ranged >3-400 [Nem suggesting zero on-going mtDNA gene flow (see Supplement S1); and ACP populations as indistinct from other regions , and higher gene-flow rates [Nem estimates and Ne estimates seem preferable in the case of H. formosa  among populations nearing migration-drift equilibrium, and large Ne, has influenced H. formosa phylogeographical structuring. This could partly explain the low observed genetic divergences, but can we reconcile this scenario with the isolation-by-distance findings, taken at face value? Taking such a scenario as compatible with the expansion-contraction model assumes most population structure has arisen since the LGM and that sufficient time has passed for H. formosa allozyme loci to reach equilibrium. This is supported by the data if we assume Nm = 10 . Given our Nef (~6 × 105-3 × 106) and ~1.2 Ma intraspecific tMRCA estimates, the number of generations required to approach equilibrium is recent, ~60,829 generations, and since the LGM based on the equation G = 1/(2 m + 1/(2Ne)) [Such nuclear-mitochondrial discordances are traditionally explained by several factors. The first and most common explanation is balancing selection (e.g. during genetic hitchhiking) mainly on allozymes , some ofow rates ,101,102.ow rates ,9. Regar species ). Still,d Baer’s  as indicmilar to , or highmilar to ) and tha1/(2Ne)) . Thus, t1/(2Ne)) .Heterandria formosa exhibit strongly female-biased sex ratios due to higher male mortality rates, as demonstrated by predation trials [FST) are not estimable from the mtDNA and allozyme-frequency data, and low RPS7-gene sampling restricts possible mtDNA-nDNA comparisons. Last, consistent with the observation that deep coalescences appear to influence the H. formosa mtDNA/gene tree , as in H. formosa fits the expansion-contraction model is also reinforced by mismatch analyses and mtDNA genetic equilibrium tests , which fail to reject demographic expansion models  between the phylogeny and network analyses congruently follow expectations derived from the model  that are inferred to have experienced postglacial population expansions e.g. [H. formosa expansion has been both spatial and demographic  and three sunfishes —this pattern has been linked to vicariant isolation in upper reaches of different drainage basins that were reduced in size by eustatic high-stands 50-80 m ASL of the Pliocene-Early Pleistocene interglacials [H. formosa. Variation at protein loci in H. formosa also departs from this vicariance model, revealing shallowly diverged and geographically overlapping population groups/lineages without deep genetic breaks at predicted barriers. While the common pattern of Gulf drainages containing Atlantic-coast haplotypes (but not vice versa) is present in the allozymes . This indicates that recent isolation within constraints of Pleistocene-Holocene (rather than earlier) drainage geomorphology, e.g. drainage divides, has influenced genetic variation of both H. formosa (i.e. genetic distinctiveness of mtDNA at AMOVAs structured by Florida drainages) and A. calva. However, additional studies will be necessary to test whether these species diversification has actually been synchronous, and to evaluate finer-scale patterns relative to drainage basins. It would also be interesting to examine whether Coastal Plain freshwater fish species that are presently codistributed with H. formosa responded to Late Pleistocene disruptions in climate and sea-level with similar range-shifting responses, possibly involving refugia; however, the only species in which effects of Pleistocene expansion-contraction dynamics have been rigorously tested is H. formosa (this study). Here, the eastern/Atlantic intraspecific lineages (Florida peninsula-Atlantic seaboard) present in many freshwater taxa will likely present good candidates for testing for range expansion-contraction dynamics similar to H. formosa. For example, the eastern/Atlantic lineages recovered by Bermingham and Avise [tMRCAs  resulted from unique histories of dispersal and range-shifting in response to Pleistocene climatic fluctuations have been advanced [Fundulus seminolis, Jordanella floridae, etc.), and crayfishes from the Florida peninsula will therefore permit testing the generality of the scenario of historical range-shift and population dynamics uncovered within H. formosa, as well as general predictions of the expansion contraction model [Our estimated nd Avise  exhibit te mtDNA ), and phadvanced ,31,32, ton model , within Heterandria formosa indicates southward LGM range contraction and large-scale postglacial expansion, as predicted under the Pleistocene expansion-contraction model [Understanding historical responses of species to the climatic and sea-level fluctuations of the Late Pleistocene has emerged as a major goal of ecology, evolution and biogeography ,4,15,29.on model .b, and KF633115-KF633133, RPS7). Sequence alignments and phylogenetic trees are available in the TreeBASE repository: http://purl.org/phylo/treebase/phylows/study/TB2:S14718.Sequences are deposited in GenBank  and Figures (S1-S3) and an additional description of methods and results (Supplement S1).Click here for file


Eucalyptus globulus. Using two common garden trials we examined variation in PSMs at multiple genetic scales; among 12 populations covering the full geographic range of the species and among up to 60 families within populations. Significant genetic variation in the expression of many PSMs resides both among and within populations of E. globulus with moderate  to high  narrow sense heritabilities and high coefficients of additive genetic variation estimated for some compounds. A comparison of Qst and Fst estimates suggest that variability in some of these traits may be due to selection. Importantly, there was no genetic by environment interaction in the expression of any of the quantitative chemical traits despite often significant site effects. These results provide evidence that natural selection has contributed to population divergence in PSMs in E. globulus, and identifies the formylated phloroglucinol compounds  and a dominant oil, 1,8-cineole, as candidates for traits whose genetic architecture has been shaped by divergent selection. Additionally, as the genetic differences in these PSMs that influence community phenotypes is stable across environments, the role of plant genotype in structuring communities is strengthened and these genotypic differences may be relatively stable under global environmental changes.Understanding among and within population genetic variation of ecologically important plant traits provides insight into the potential evolutionary processes affecting those traits. The strength and consistency of selection driving variability in traits would be affected by plasticity in differences among genotypes across environments (G×E). We investigated population divergence, selection and environmental plasticity of foliar plant secondary metabolites (PSMs) in a dominant tree species,  For example, narrow sense heritability is a useful within population genetic parameter to estimate the proportion of phenotypic variance that is due to additive effects (and thus is the variance selection can act upon). This matched with the coefficient of additive genetic variance (the additive genetic variance standardised by the trait mean) to assess the ‘evolvability’ of a trait, provides a clear methodology for understanding the evolutionary potential of traits Phenotypic variation in traits across the geographic range of a species can be the result of a variety of environmental effects and/or genetic processes such as local adaptation and drift Plant/herbivore systems represent a model approach to investigate patterns of evolutionary population divergence across the geographic range of a species and the evolutionary processes affecting relevant traits within populations. Major drivers of plant/herbivore interactions are plant secondary metabolites  Eucalyptus globulus is a dominant tree species of ecological significance in south-eastern Australia, and is rich in foliar PSMs including terpenes and phenolic based compounds such as formylated phloroglucinol compounds (FPCs). These compounds affect rates of herbivory by many marsupial herbivores and structure foliar arthropod communities E. globulus is well understood, however, previous work has focused on among population genetic variation E. globulusWe advance previous research on broad-scale geographic genetic variation in PSMs in No permits or approvals were required for this field study as only small samples of an abundant tree species were taken. Effects to individual and ecosystem health were negligible. The land accessed is not protected. No protected species were sampled.Eucalyptus globulus has been classified into a genetic hierarchy of 13 geographically and genetically distinct races and 20 sub-races across its full geographic range, based on differences in 35 different morphological traits assessed in a range of common environment trials E. globulus sub-races. Foliage was collected from these progeny trials located at Togari  and Salmon River  in north-west Tasmania. Both field trials were established in August 2005 by Forestry Tasmania. Sites differed markedly in their soil profiles. Salmon River was yellow brown mottled clay on Precambarian mudstone whereas Togari was red brown clay on Cambrian inter-layered mudstone, siltstone and sandstone. Each trial comprised plants grown from open-pollinated seed collected from 310  and 244 (Togari) families collected from throughout the native, natural distribution of E. globulus , three leaf pairs back from branch tips. This ensured that fully expanded juvenile leaves (but not yet entering senescence) were sampled and that leaves sampled were the same age across all plants. Trees harvested from Salmon River represented 60 families from 12 sub-races while samples were taken from trees representing 53 families from the same 12 sub-races at Togari. These were collected across 20 and 15 replicates from Salmon River and Togari, respectively. In total 749 samples were collected from the two sites . Sub-racMammal browsing damage was scored at Togari in March 2006. Trees at Salmon River were not browsed. Browsing damage was scored as percentage of total foliage removed from the whole plant on a scale from 0 to 6, where 0 = 0%, 1 = 1−5%, 2 = 6−25%, 3 = 26−50%, 4 = 51−75%, 5 = 76−95%, and 6 = 96−100%. Due to generally low browsing (∼27% of trees received damage), scores were converted to a binary presence/absence for analysis. Diameter at breast height  was assessed in February 2010 after 5 years growth to relate browsing and foliar chemical variation with a surrogate measure of tree fitness.E. globulus) and total phenolics. Near infrared reflectance spectroscopy has been used extensively in ecology and other fields to enable rapid analysis of samples on a scale that would be impractical using conventional laboratory techniques  at a resolution of 4 cm−1. The resulting four spectra taken from the two leaves were averaged.We predicted the concentration of six different chemical constituents in the leaves using near infrared reflectance spectroscopy (NIRS). These constituents were nitrogen, two formylated phloroglucinol compounds , total oils, 1,8-cineole (the dominant oil in 2 0.66–0.90 (SEP: 0.22–5.21) for the six chemical traits; the external validation r2 ranged from 0.69–0.86 (SEP: 1.29–5.05).Near infrared reflectance spectroscopy models were developed to predict the concentration of all chemical constituents. Prior to model development, principal component analysis (PCA) was carried out on the spectra of all samples, to enable selection of a subset  of samples for wet chemical analysis (see below) for model validation purposes. These selected samples were representative of the spectral variation present in the entire sample set. Half of these samples (n = 50) were added to existing NIRS models −1 equivalents of cineole using a 1,8-cineole standard. Total phenolics were assayed with a modified Prussian blue assay for total phenolics using gallic acid standards −1 equivalents of gallic acid. Sideroxylonal A and macrocarpal G were assayed by high performance liquid chromatography (HPLC) following Wallis & Foley −1. Results for macrocarpal G concentration were expressed as mg g DM−1 equivalents of macrocarpal A . Freeze dried ground leaf samples were re-ground to a fine powder using a ball grinder (Retsch MM200) and analysed for nitrogen using an elemental analyser (Thermo Finnigan EA 1112 Series).Wet chemical analysis was carried out on 50 samples per site. Total oils and 1,8-cineole were assayed by gas chromatography-mass spectrometry (GCMS). The method was modified from that reported in O'Reilly-Wapstra et al. Statistical analyses were conducted using ASReml™ To examine the variation in foliage chemistry across the two common environment trials, among sub-races and families within sub-races, as well as genetic-by-environment interactions, using only families from the native range  we fitted the following model with the PROC MIXED procedure of SAS:Y = μ + SITE + SUBRACE + SITE*SUBRACE + REP + FAM (SUBRACE) + SITE*FAM (SUBRACE) + RESIDUALREP is the random replicate within site effect, FAM (SUBRACE) is the random family within sub-race effect, SITE*FAM (SUBRACE) is the random site by family within sub-race interaction effect, RESIDUAL is the residual. Fixed effects of SITE and SUBRACE, including the interaction term, were effectively our ‘treatment’ effects.whereby, Y is the phenotypic observation, μ is the mean, SITE is the fixed site effect, SUBRACE is the fixed sub-race effect, SITE*SUBRACE is the fixed site by sub-race interaction effect, Discriminant analysis separating sub-races was conducted on a data set comprising the native-forest family means  for the six chemical components using the PROC DISCRIM procedure of SAS. Mahalanobis distances among sub-races and their significance levels were calculated and summarised by UPGMA clustering undertaken with PROC CLUSTER.Genetic parameters for each chemical constituent and DBH were estimated for each trial using the following univariate linear model:native + FAMother + RESIDUALnative + RESIDUALotherY = μ + GROUP + SUBRACE + REP + FAM (SUBRACE)REP is the random replicate effect, nativeFAM (SUBRACE) is the random family effect (nested within sub-race) for the native forest progeny, otherFAM is the family effect for the non-native progeny, that is, seedlots from breeding programmes and commercial seed-orchards, native,RESIDUAL is the residuals for the native progeny, and otherRESIDUAL is the residuals for the non-native progeny. The utilisation of all available data  in the analysis of individual sites allowed the effect of REP to be more precisely estimated, but only the results from native forest progeny are presented.where Y is the phenotypic observation, μ is the mean, GROUP is the fixed population  effect, SUBRACE is the fixed native-forest sub-race effect, E. globulus, maternal effects are minimal CVa) Due to mixed mating systems in eucalypts, additive variance (nativeFAM (SUBRACE)]; nativeRESIDUAL); r is the coefficient of relationship, fixed to equal 0.4 to account for an assumed selfing rate of 30% where REP and RESIDUAL effects. Furthermore, for each pairwise combination of traits, a multiple-site multiple-trait (i.e. four variates: two traits by two sites) analysis was conducted to estimate inter-trait genetic correlations. The multivariate models used for this purpose combined the aforementioned bivariate models and accounted for covariance between the REP, FAM and RESIDUAL effects for traits within sites. Two-tailed likelihood ratio tests (LRTs) were conducted to test if inter-trait genetic correlations were heterogeneous across sites and to test whether the inter-trait and inter-site genetic correlations were significantly different from zero REP) and genetic terms  from the model.Bivariate analyses were undertaken for each trait to estimate pair-wise inter-site within sub-race family correlations  QST calculated as follows:To estimate the quantitative inbreeding coefficient (QST estimates were significantly greater than a published estimate of the mean FST (0.09) which was based on eight microsatellite markers and used a similar suite of E. globulus sub-races Fst study . We similarly tested whether the QST estimates were significantly greater than the maximum of the FST estimate for any microsatellite locus published for this species  or divergent (QST>FST) selection having impacted on the genetic architecture of the trait where Significant differences among sub-races were evident for all chemical constituents , with soCluster analysis based on the Mahalanobis distances  amongst 9 =  6.1; 1-tailed P<0.001). Single site Qst estimates for sideroxylonal, macrocarpal, cineole and DBH were also significantly greater than the maximum Fst estimate for individual loci  was significantly different between (both data sets) and within  sub-races. The significant DBH by site interaction term at the sub-race level was due to poor growth of the St Helens sub-race at the Togari site. We also examined correlations between the chemical traits and DBH and browse damage . There wE. globulus, (ii) the expression of this genetic variation is stable across environments, and (iii) that diversifying selection is likely to have acted on some PSMs.In this paper we link chemical variation with evolutionary dynamics and ecological interactions across different levels of organisation to demonstrate that (i) significant genetic variation in the expression of many PSMs resides both among and within populations of E. globulus have been based on broad sense heritability estimates  that inform us of how responsive PSM traits may be to selection E. globulus. While other factors, such as strong genetic correlations amongst traits and correlated responses to selection cannot be dismissed E. globulus, and identifies the FPCs  and cineole as candidates for traits whose genetic architecture has been shaped by divergent selection Indeed, our examination of differences in the Qst and Fst estimates suggest that sub-race divergence in concentration of foliar PSMs have already evolved through selection. Differences in quantitative trait and neutral molecular estimates have been calculated in a number of plant species to indicate patterns of selection for a range of life history traits E. globulus may play a role in numerous ecological functions including defence against mammalian herbivores and fire promotion E. globulus are under selection by mammalian herbivores because mammalian browsing preferences differ amongst E. globulus populations and these differences can be explained, in part, by variation in PSMs among those populations, particularly the FPCs, sideroxylonal and macrocarpal E. globulus survival is size dependent E. globulus are under selection and that that mammalian herbivores may play a role. The correlated nature of the compounds Plant secondary metabolites in Obviously, fitness estimates are difficult to obtain in a long lived tree species and hence surrogates of fitness (such as DBH and reproductive output at a snapshot in time) are often our best option. While we have only assessed DBH in this current study, we have shown that repeated browsing by mammalian herbivores greatly reduces individual plant reproductive fitness (number of buds and capsules) at 10 years of age compared to those plants that were not browsed Broad-scale population divergence in the expression of PSMs in this study appear to follow some clear geographic patterns. The two mainland Australian regions, the south-western Victorian sub-races (Eastern and Western Otway) and the south-eastern Victorian sub-races  show divergent patterns of PSM expression. If one region is high in concentration of PSM then the other region is low . These two distinct groupings are also reflected in the UPGMA clustering of sub-races based on variation in PSMs presented in this current paper and also in Wallis et al Another clear pattern is the steep latitudinal cline in most PSMs  from Flinders Island, Southern Furneaux through to the north-eastern Tasmanian sub-race of St Helens and where Flinders island groups more closely to the south-eastern Victorian sub-races. Clinal patterns in quantitative traits have become a point of interest in recent literature due to probable effects of large scale environmental variation (e.g. climate change effects) on local adaptation of populations Finally, based on a chloroplast DNA study, Freeman et al. E. globulus is stable across sites. We did not detect a genotype by environment interaction (G×E) for any of the chemical traits, despite highly significant genetic and site main effects. Previously in nutrient trials, we detected a G×E on mammalian herbivore responses to E. globulus in a captive feeding trial, but did not detect a G×E with any of the PSMs we assessed in the seedlings. Additionally, Andrew et al. Eucalyptus tricarpa at the population level, but not within populations. In some recent work, we manipulated CO2 levels in the glasshouse with different populations of two eucalypt species, E. globulus and E. pauciflora, and found that CO2 had minimal effect on the expression of PSMs and no G×E was detected Variability in the genetic expression of traits across environments provides an indication of the relative strength of genetics in affecting ecologically important traits E. globulus2. Additionally, as many eucalypt species are important commercial forestry species (E. globulus is the most widely planted hardwood species in temperate regions worldwide) the knowledge that genotypes may be stable in the expression of PSMs across different plantation sites give confidence to tree growers for artificial selection for these traits in order to reduce losses from pest damage What is becoming apparent in eucalypt systems is that while the levels of PSMs may change with environment, the relative differences amongst genotypes remains stable. Low or absent G×E for eucalypt PSMs has important ecological and applied management implications. If these genetic based traits that influence community phenotypes are stable across environments, the role of plant genotype in structuring communities as observed in Table S1Table of pair-wise Mahalanobis distances amongst E. globulus sub-races and their significance. The Mahalanobis distances were calculated using the PROC DISCRIM procedure of SAS and data set comprising the native-forest family means  for the six chemical components analysed.(DOC)Click here for additional data file.


Porcine reproductive and respiratory syndrome (PRRS) is a viral disease that has a major economic impact for the swine industry. Its control is mostly directed towards preventing its spread which requires a better understanding of the mechanisms of transmission of the virus between herds. The objectives of this study were to describe the genetic diversity and to assess the correlation among genetic, Euclidean and temporal distances and ownership to better understand pathways of transmission.. Significant positive correlations were observed between genetic and ownership, genetic and Euclidean and between genetic and temporal binary distances. The relationship between genetic and ownership suggests either common sources of animals or semen, employees, technical services or vehicles, whereas that between genetic and Euclidean binary distances is compatible with area spread of the virus. The latter correlation was observed only up to 5 km.A cross-sectional study was conducted on sites located in a high density area of swine production in Quebec. Geographical coordinates (longitude/latitude), date of submission and ownership were obtained for each site. ORF5 sequencing was attempted on PRRSV positive sites. Proportion of pairwise combinations of strains having ≥98% genetic homology were analysed according to Euclidean distances and ownership. Correlations between genetic, Euclidean and temporal distances and ownership were assessed using Mantel tests on continuous and binary matrices. Sensitivity of the correlations between genetic and Euclidean as well as temporal distances was evaluated for different Euclidean and temporal distance thresholds. An ORF5 sequence was identified for 132 of the 176 (75%) PRRSV positive sites; 122 were wild-type strains. The mean (min-max) genetic, Euclidean and temporal pairwise distances were 11.6% (0–18.7), 15.0 km (0.04-45.7) and 218 days (0–852), respectivelyThis study suggests that transmission of PRRSV is likely to occur between sites belonging to the same owner or through area spread within a 5 km distance. Both should be considered in the perspective of prevention. Porcine reproductive and respiratory syndrome (PRRS) is a viral disease representing one of the most challenging threats to swine industry. This disease has a major economic impact, inducing late-term abortions, stillbirths, mummified and weak piglets in breeding herds and respiratory disease, increased mortality rate and poor growing performances in growing pigs ,2.Arteriviridae within the genus Arterivirus and the order Nidovirales[PRRS is caused by a spherical, enveloped, single-stranded and positive-sense RNA virus belonging to the family of dovirales. An impodovirales,5. This dovirales-8. Both The virus can be transmitted between herds through several mechanisms, including introduction of infected animals or semen -14. ContBased on the premise that PRRSV strains sharing a high level of homology is suggestive of a common source of infection, molecular epidemiology can help improving our understanding of mechanisms of PRRSV dispersion over space and time ,26. The 2[A cross-sectional study was conducted in the Monteregie administrative region in the province of Quebec, Canada, between February 2005 and June 2007. Within this region, a high density area (HD) was purposely targeted for a larger project on transmission and control of PRRSV, and corresponded to 10 adjacent municipalities covering approximately a 20 km-radius area  with 3542. A site 2. All sit2 for meth2. PCR proORF5 sequences having ≥98% pairwise homology with MLV  Ltd.) or ATP  Ltd.) commercial vaccines strains were excluded from further analyses whereas the remaining sequences were considered as wild-type strains.For each pair of sites, the genetic, Euclidean, temporal and ownership distances were calculated. Pairwise genetic distances were defined as the percentage of homologous nucleotides between sequences. It was calculated from nucleotides using Juke and Cantor substitution model in Bionumerics software , following a pairwise alignment of ORF5 sequences. For the Euclidean distances, geographical coordinates (latitude/longitude) of the sites were transformed in metric units using Quebec Lamber Conic Conform projection in ArcInfo . Pairwise Euclidean distances between sites were then calculated in SAS  using the projected coordinates . PairwisDescriptive statistics on herd characteristics were performed in SAS. Means of genetic, Euclidean and temporal pairwise continuous distances were calculated. Number of pairwise combinations of strains having ≥98% homology over total number of combinations was computed according to different Euclidean distance thresholds  and ownership (same vs. different). The Euclidean distance thresholds were defined according to the literature. Most mechanisms of transmission acting on a local scale as aerosols, insects, mammalian species and fomites are more likely to occur at a distance of less than 5 km, whereas transmission occurring at more than 10 km apparently necessitates human interventions, since infectious virus was not identified in aerosols at more than 9.1 km from a population source .Bivariate Mantel tests for genetic, Euclidean, temporal continuous distances and ownership were computed in R  using vegan package and Pearson correlation coefficient . BivariaAn ORF5 sequence was identified for 132 out of the 176 (75%) infected sites. ORF5 sequences having ≥98% homology with MLV and ATP vaccines strains were observed for seven and three sites, respectively. On these latter sites, the use of commercial vaccination was confirmed or highly suspected. Consequently, 10 sites composed of two farrow-to-finish and eight weaners and/or finishers operations were excluded from further analyses, the remaining 122 sequences being considered as wild-type PRRSV strains. Table All wild-type ORF5 sequences belonged to the North American genotype and most sequences 92%) had a length of 603 bp with the exception of 7 strains presenting insertion (606 bp) or 3-base deletions (600 bp). The mean (min-max) genetic, Euclidean and temporal pairwise distances were 11.6% (0–18.7), 15.0 km (0.04-45.7) and 218 days (0–852), respectively. Among the 122 wild-type PRRSV strains, 62 (51%) did not show any ≥98% pairwise genetic homology with other (s) sequence (s), 34 (28%) showed this latter homology with one strain only and 26 (21%) with more than one strain. Using dichotomized distances, proportion of pairwise combinations of strains sharing ≥98% homology over total number of combinations is reported in Table % had a lBivariate correlations between genetic, Euclidean and temporal continuous distances were computed on the 122 PRRSV wild-type strains using 7381 different pairwise combinations. Results are shown in Table m) obtained between binary matrices of genetic and Euclidean distances for different Euclidean thresholds. Significant positive correlations were observed up to 5 km, whereas non significant correlations were noted for any other higher thresholds (up to 40 km). Positive correlations between genetic and temporal binary distances were observed for 11 months, being not significant thereafter , which is almost the double of the 6.5% genetic diversity obtained for 55 sequences submitted by 48 farms from the Midwest of United States . The max%, which A correlation between genetic distance and ownership was observed, which to the best of our knowledge has never been explored before. About 16 times more pairwise combinations of strains sharing ≥98% homology were observed for sites having the same owner compared to those having different owners Table . ResultsSimilarly to other results , we did 2The positive correlation observed between genetic and temporal distances is compatible with genetic evolution of the virus in the area over a two year period of observation and was also reported by others . InteresThis study attempted to include all sites located in a restricted geographical scale to obtain more precision in exploring area spread compared to other studies ,26. HoweThe study brought useful information regarding PRRSV epidemiology in a perspective of prevention. Results support the concept of area spread and also highlighted the role of ownership in viral transmission through different potential mechanisms such as common personnel, vehicles and source of animals.The authors declare that they have no competing interests.MEL participated in the design of the study, collected all data, performed the statistical analyses, interpreted data and wrote the initial draft of the manuscript. JA provided valuable expertise and support on statistical analyses, participated in data interpretation and critically reviewed the manuscript. ZP provided valuable expertise and support on statistical analyses, and participated in data interpretation. SD participated in the design of the study and data interpretation and helped to draft the manuscript. All authors read and approved the final manuscript.


Levels, folding, and function of the infamous cancer and inflammatory disease-related signaling molecule Stat3 are regulated by interaction with the chaperonin TRiC; manipulation of this interaction is a therapeutic avenue for exploration. in vitro and in vivo. TRiC binding to Stat3 was mediated, at least in part, by TRiC subunit CCT3. Stat3 binding to TRiC mapped predominantly to the β-strand rich, DNA-binding domain of Stat3. Notably, enhancing Stat3 binding to TRiC by engineering an additional TRiC-binding domain from the von Hippel-Lindau protein (vTBD), at the N-terminus of Stat3, further increased its affinity for TRiC as well as its function, as determined by Stat3's ability to bind to its phosphotyrosyl-peptide ligand, an interaction critical for Stat3 activation. Thus, Stat3 levels and function are regulated by TRiC and can be modulated by manipulating its interaction with TRiC.Signal transducer and activator of transcription 3 (Stat3) transduces signals of many peptide hormones from the cell surface to the nucleus and functions as an oncoprotein in many types of cancers, yet little is known about how it achieves its native folded state within the cell. Here we show that Stat3 is a novel substrate of the ring-shaped hetero-oligomeric eukaryotic chaperonin, TRiC/CCT, which contributes to its biosynthesis and activity  Stat3 is a multidomain transcription factor that contributes to many cellular functions by transmitting signals for over 40 peptide hormones from the cell surface to the nucleus. Understanding how multidomain proteins achieve their fully folded and functional state is of substantial biological interest. As Stat3 signaling is up-regulated in many pathological conditions, including cancer and inflammatory diseases, insight into what controls its folding may be useful for the identification of vulnerabilities that can be therapeutically exploited. We demonstrate that the major protein-folding machine or chaperonin within eukaryotic cells, TRiC/CCT, is required for Stat3 to fold during its synthesis and for Stat3 to be fully functional within the cell. We also find that TRiC can refold chemically denatured Stat3 and provide evidence that the CCT3 subunit of TRiC binds to the DNA-binding domain of Stat3. We also show that Stat3 activity is decreased by down-modulating levels of TRiC and can be increased by increasing Stat3's interaction with TRiC. TRiC therefore regulates both Stat3 protein levels and its function, making Stat3 modulation by manipulation of its interaction with TRiC a potential approach for the treatment of cancer and inflammatory diseases. Signal transducer and activator of transcription 3 (Stat3) is a member of a family of seven closely related proteins responsible for the transmission of peptide hormone signals from the extracellular surface of cells to the nucleus de novo folding of approximately 10% of newly synthesized proteins in the eukaryotic cell and for refolding proteins that become denatured following stress For the majority of eukaryotic proteins, folding to the native and functional conformation requires the assistance of elaborate cellular machinery composed of proteins known as molecular chaperones in vitro and within cells. TRiC binding to Stat3 was mediated, at least in part, by TRiC subunit CCT3. Stat3 binding to TRiC mapped primarily to its β-strand-rich, DNA-binding domain (DBD). Addition of a second TRiC-binding domain (TBD) to the N-terminus of Stat3  further increased the affinity of Stat3 for TRiC and its function. Thus, the structure and function of Stat3 is regulated by the major eukaryotic chaperonin TRiC/CCT and can be modulated through manipulation of TRiC levels or substrate affinity.Here we demonstrate that TRiC binds the oncogenic transcription factor, Stat3, and contributes to its biosynthesis, refolding, and activity To determine if Stat3 interacts with TRiC, we performed TRiC immunoprecipitation in a cell-free mammalian translation system used previously To assess if the TRiC interaction with nascent Stat3 polypeptides is coupled to their biogenesis, RRLs were immunodepleted of TRiC and tested for the ability to generate Stat3. TRiC depletion of RRLs  markedlyin vitro, Stat3 protein chemically denatured in guanidine hydrochloride was added to RRLs before and after ATP depletion and the levels of refolded Stat3 assessed by native PAGE  Having established that TRiC interacts with newly translated and denatured Stat3 in RRLs, promoting its synthesis and folding To determine the contribution of TRiC to the levels of total and activated Stat3  within cells, we used shRNA targeting CCT2 to reduce the levels of TRiC within two cells lines (HS-578T and HepG2), both previously demonstrated to have constitutively activated Stat3 p = 0.0026; p = 0.0056; p = 0.0072; p = 0.0176; p = 0.0124; Knockdown of TRiC in HS-578T cells decreased constitutively activated Stat3 (pStat3) levels by 74% (p<0.05 for both). These results indicated that TRiC contributes to maximal levels of ligand-activated Stat3 within cells at sub-maximal ligand concentrations. Thus, levels of both constitutively activated and ligand-activated Stat3 are sensitive to decreased TRiC levels within cells.To determine if levels of cytokine-activated Stat3 are affected by TRiC reduction in addition to constitutively activated Stat3, we examined the levels of pStat3 in IL-6 stimulated control and TRiC knockdown HepG2 cells. Use of the HepG2 control and TRiC knockdown cells, as opposed to their HS-578T counterparts, allowed us to examine the levels of cytokine-activated Stat3 independent of levels of total Stat3. HepG2 cells were treated for 30 min with increasing concentrations of IL-6 in the most sensitive portion of the dose–response curve  . Levels Stat3 consists of six distinct functional domains  each tagged with mCherry into MEF cells stably expressing GFP-tagged Stat3α (MEF/GFP-Stat3α) r = 0.512) did not change with IL-6 receptor stimulation (Rr = 0.493); in contrast, Pearson's correlation of co-localization within the nucleus for CCT3 increased markedly from Rr = 0.457 without IL-6 receptor stimulation to Rr = 0.800 with IL-6 receptor stimulation. In addition, ImagePro analysis of multiple fields of cells co-expressing GFP-Stat3α and either CCT3 or CCT7 and stimulated with IL-6/sIL-6Rα revealed a significantly higher Pearson's coefficient of co-localization within the nucleus for cells expressing CCT3 (0.90±0.06) versus CCT7 .To confirm and extend these results The TBD of pVHL previously mapped to a 55 amino-acid residue, β-strand-rich in vitro translated vTBD-Stat3 to wild-type Stat3. We previously demonstrated that Stat3 bound with high affinity to a phosphotyrosyl-dodecapeptide (p1068) through its Src-homology (SH) 2 domain p<0.0028; To assess if increased interaction of Stat3 with TRiC affects the function of Stat3, we compared the activity of in vitro and within cells. TRiC binding to Stat3 was mediated, at least in part, by TRiC subunit CCT3. Stat3 binding to TRiC mapped primarily to its β-strand rich, DBD. Addition of a second TBD vTBD further increased its affinity for TRiC, as well as its function. Thus, Stat3 levels and function are regulated by TRiC and can be modulated through manipulation of substrate affinity and TRiC levels within the cell.Our understanding of how transcription factors achieve their folded and functional conformations within cells is incomplete. Here we demonstrate Stat3, a transcription factor activated in up to 50% of cancers, binds TRiC/CCT chaperonin and that TRiC is required for Stat3 biosynthesis and activity Approaches used successfully to identify TRiC clients have included both directed and global strategies. The earliest recognized TRiC clients—β-actin Taking a more global approach Our report represents the most comprehensive demonstration that an oncoprotein, Stat3, is a TRiC client. As noted above, earlier investigators There have been few studies that simultaneously compare the effects of targeting TRiC on the levels and function of multiple TRiC clients. In this regard, it is noteworthy that despite 90% reduction of TRiC within cells, levels of the TRiC clients, β-actin, GAPDH, and β-tubulin were not reduced . These rEarlier reports have supported a contribution of chaperones to the biology of Stat3. Stat3 previously was identified within the cytosol of unstimulated cells (HepG3), predominantly within high-molecular weight complexes in the size range of 200–400 kDa (Statosome I) and 1–2 MDa (Statosome II) We have shown by immunoprecipitation studies in RRLs that the interaction between TRiC and Stat3 occurs predominantly through the CCT3 of TRiC binding to the DBD of Stat3. Other domains of Stat3, including the N-terminal domain, linker domain, and SH2 domain, also were pulled down with TRiC, although at lower levels than the DBD indicative of lower affinity interactions. Of these domains, only the SH domain contains β-strands.We demonstrate in this report that chaperonin–client interactions can be modulated not only to reduce client function but also to increase it. Knockdown of TRiC using shRNA reduced total Stat3 protein levels, reduced constitutively activated Stat3, and reduced the sensitivity of Stat3 to IL-6–mediated activation. These findings provide proof-of-principle that targeting TRiC, or more specifically targeting the interaction between TRiC and Stat3, may provide a novel approach to reducing levels of activated Stat3 for therapeutic benefit in cancer. Alternatively, addition of a new TRiC binding site to Stat3 through a covalent modification significantly improved its affinity for TRiC and its function. This result serves as proof-of-concept that future development of noncovalent modulators that increase TRiC–Stat3 interactions may be a novel approach to increasing Stat3 activity in the setting of acute injuries such as myocardial infarct 2 in DMEM with 10% fetal bovine serum, 1% penicillin/streptomycin, and Glutamax. MEF/Stat3Δ/Δ cells were provided as a generous gift from Dr. Valeria Poli Δ/Δ cells stably transfected with GFP-Stat3α were generated as described All cell lines were maintained at 37°C in 5% COThe coding sequence for human Flag-tagged pVHL and Stat3 were subcloned into a modified pSG5 vector as XhoI/HindIII and HindIII/NotI fragments, respectively. Stat3 was also engineered to contain the 55 amino-acid TBD of pVHL (vTBD) at the N-terminal end to generate vTBD-Stat3. The sequence encoding the 55 amino acids was generated by PCR using VHL cDNA as template and subcloned into the mammalian expression vector pSG5. For mapping the TBD within Stat3, sequences encoding each of the six domains of Stat3 were generated by PCR and cloned into pSG5 vector. All inserts were sequenced to ensure fidelity.In vitro transcription and translation reactions (50 µl) were carried out using an RRL system  with 1 µg of pSG5 vector containing the insert of interest according to the manufacturer's instructions for 30 min at 30°C, and terminated by the addition of 2 mM puromycin, 5 mM EDTA, and 1 mM azide. Protein translation was monitored by SDS-PAGE followed by autoradiography. Where indicated, RRLs were depleted of ATP by incubation for 10 min at RT with apyrase (50 U/ml).Δ/Δ cells) 2. The lysates were either used immediately or snap frozen in liquid nitrogen and stored at −80°C. Lysate (100 µl) was incubated on ice for 1–2 h with 3 µl each of rabbit antibodies to CCT2 and CCT5 or rabbit IgG anti-human control antibody. Magnetic protein A beads  were resuspended in the antibody/lysate mixtures and incubated for 10 min on ice. Beads were washed two times with 500 µl of buffer A and two times with buffer A containing 1% Triton ×100. Bound proteins were separated by SDS-PAGE and immunoblotted.TRiC immunoprecipitations were performed as described 35S-methionine-labeled TRiC subunits were mixed with RRLs containing either an unlabeled Flag-tagged Stat3 construct or no expression construct; each RRL mixture then was incubated with the reversible protein cross-linker DSP (dithiobis[succinimydyl-propionate]) at a final concentration of 2 mM. Samples were quenched by 10-fold dilution in 20 mM Tris pH 7.5 containing 10% glycerol prior to immunoprecipitation with M2 anti-Flag antibody-bound agarose beads. The immunoprecipitated and cross-linked protein complexes then were incubated with β-mercaptoethanol (5 mM) to reverse the cross-linking and analyzed by SDS-PAGE and autoradiography.RRL reactions containing Biotin-dodecapeptide 1068 or biotin-phosphododecapeptide p1068 (10 µl at 10 mg/ml) were synthesized commercially based on the sequence within the cytoplasmic portion of the EGFR surrounding Y1068 previously shown to bind Stat3 through its SH2 domain To reduce TRiC levels within cells, MISSION shRNA lentiviral transduction particles targeting CCT2 sequence (SHCLNV-NM-006431) and control shRNA lentiviral particles were obtained from Sigma-Aldrich. HepG2 and HS-578T cells were transduced with CCT2 shRNA or control shRNA lentiviral particles at an MOI of 5 and grown for 48 h. Stable TRiC knockdown cells were established by replating the cells in selection media containing puromycin at 3 µg/ml.2. The cells were then treated with IL-6 at 0, 0.1, 0.3, and 1 ng/ml for 30 min at 37°C, then lysed in M-PER lysis buffer (Thermo Fisher Scientific) supplemented with phosphatase and protease inhibitor cocktails.CCT2 knockdown or control HepG2 cells were cultured in six-well plates overnight in DMEM supplemented with 10% fetal bovine serum  containing 2 mM Glutamax (GIBCO-BRL), 10 mg/ml penicillin, and 10 mg/ml streptomycin (GIBCO-BRL) at 37°C in 5% COFor immunoblotting, total protein concentration was measured within M-PER lysates using the Coomassie assay kit  with BSA as a standard. Total cell lysates containing 20 µg of total protein were separated by 7.5% SDS-PAGE and transferred onto PVDF membranes. The membranes were blocked with 5% BSA in 0.1% TBS-T (0.1% Tween-20 in TBS) for 1 h and incubated overnight at 4°C in blocking buffer with one of the primary antibodies against total Stat3, pStat3 (BD-Biosciences), β-actin, GAPDH, CCT1, CCT2, and CCT5 (Abcam); washed; incubated with appropriate secondary antibodies conjugated to horseradish peroxidase; washed; and developed with ECL substrate (Thermo Scientific). For Luminex bead assays, cells were lysed and assayed for pStat3, total Stat3, and GAPDH using Millipore (Milliplex) kits as described by the manufacturer. Levels of each protein analyte were determined and analyzed using the Bio-Plex suspension array system (Bio-Rad).2) at 4°C, rinsed 3× with PEM buffer, quenched in 1 mg/ml NaBH4 (Sigma) in PEM buffer for 5 min, then permeabilized with 0.05% Triton X-100 in PBS for 5 min, counterstained with 4′,6-diamidino-2-phenylindole , mounted onto slides, and imaged using confocal fluorescence microscopy.Transformed MEF cells stably expressing GFP-Stat3α(MEF/GFP-Stat3α) were plated on polylysine-coated glass coverslips. After 12 h the cells were transfected with mCherry-CCT3 or mCherry-CCT7 fusion constructs using GeneJuice reagent (EMD4 Biosciences) according to the manufacturer's instructions. Forty-eight hours later, cells were treated with or without IL-6/sIL-6R (∼250 ng/ml) for 30 min. The coverslips were rinsed with ice-cold PBS twice and fixed for 30 min with 4% paraformaldehyde in PEM buffer  expression vector. The transformed cells were induced with 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) at an optical density of 0.7 at 600 nm. After growing for 5 h at 37°C, cells were harvested; resuspended in buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM DTT, 1% Triton X-100, and 100 µg/ml lysozyme; and lysed by sonication. The insoluble fraction containing Stat3 as inclusion bodies was collected by centrifugation and washed extensively with a buffer containing 2 M urea. The washed pellet was solubilized in 0.1 M NaH2PO4, 10 mM Tris-HCl, pH 8.0, 300 mM NaCl, 5 mM imidazole, 10% glycerol, and 6 M guanidinium hydrochloride (Gn-HCl). After centrifugation, the supernatant was applied onto a Ni-NTA column (Qiagen) equilibrated in the same buffer. The column was washed successively with buffer A  and buffer B (buffer A at pH 5.9). Proteins were eluted from the column with buffer C (buffer A at pH 4.5), concentrated, and buffer exchanged into buffer C containing 1 mM DTT. Proteins were stored at −80°C until use.TRiC was purified from bovine testes essentially according to previously established procedures Figure S1Targeting of CCT2 within cells using shRNA reduces levels of other CCT subunits. HS-578T cells stably expressing CCT2 or control shRNA were immunoblotted for the indicated proteins using specific antibodies to each and the results of a representative experiment shown.(TIF)Click here for additional data file.


Sogatella furcifera (Hemiptera: Delphacidae), is a serious pest of rice in Asia. In the present study, inter-simple sequence repeat (ISSR) markers were employed to investigate the genetic diversity and differentiation of 47 populations sampled from 14 prefectures of the Greater Mekong Subregion. A total of 14 selected primers yielded 121 bright and discernible bands, with an average of 8.6 bands per primer. According to the hierarchical analysis of molecular variance (AMOVA), the genetic variation among geographic regions (79.84%) was higher than that of among populations within region (20.16%), and the FST value was 0.72, indicating a high level of genetic differentiation. Neighbor-Joining cluster analysis of the 47 populations showed two major clusters, one consisting of mostly southwestern Yunnan Province and Myanmar populations; and the other one consisting of southeastern and central of Yunnan Province plus Vietnam and Laos populations. No significant positive correlation was observed between genetic and geographic distances by Mantel test , indicating the role of geographic isolation did not shape the genetic structure of the sampled S. furcifera populations. This paper provides useful data for understanding and speculating the migration of S. furcifera and reveals available information to develop sustainable strategies for manage this long-range migratory pest.The white backed planthopper,  Sogatella furcifera (Horvath) (Hemiptera: Delphacidae), is widely distributed throughout Asia and is considered a major pest of rice in the region. The nymphs and adults suck the plant sap and reduce plant vigor, delay tillering, stunt, yellow leaves, and shrivel grains  markers, which are cost-effective, rapid and efficiently sensitive, are extremely useful for assessing genetic variability in some species . In conthypogaea , two speinymphae , and the aegypti .S. furcifera in China has not been suppressed may be due to the ineffective coordination of implementing the control strategy, as well as the lack of comprehensive knowledge (including population structure and diversity) of the pest. A sound understanding of the genetic diversity, migration/dispersal patterns, and the environmental adaptability of S. furcifera is essential to the development of rational control strategies. The present study was designed to use ISSR analysis for investigating the genetic structure as well as diversity of S. furcifera among the geographic regions in the partial Greater Mekong Subregion. The main objectives of the study were to (i) assess bio-geographic relationshipsand genetic similarities across several populations in Yunnan Province and its adjacent southeastern Asian countries; and (ii) provide useful information in modeling and forecasting outbreaks of S. furcifera and in designing sustainable strategies to manage the pest.The fact that the population of S. furcifera were sampled across 11 geographical regions in Yunnan Province and three Southeast Asian countries, including Vietnam, Laos, and Myanmar. The longitude, latitude, and altitude of each sampling population, sample size, and collection dates were recorded . All samples were stored at -20°C until needed.Total genomic DNA was isolated from S method . The DNAwww.biotech.ubc.ca) and synthesized by Sangon Biological Engineering Technique & Service, Co. Ltd. (www.sangon.com). These primers were initially screened, and the 14 primers that produced bright, clear, and reproducible fragments were utilized for further study (Sixty-seven ISSR primers were selected from the ISSR primer set (UBC primer set #9) developed by the University of British Columbia Biotechnology Laboratory , and 30 ng DNA templates in a total volume of 20 µl, and 2.0% of deionized formamide was added to the PCR mixture to increase band clarity. Amplification was performed in a Mastercycler Gradient  under the following cycle profile: 4 min at 94°C, followed by 35 cycles of 1 min at 94°C, 1 min annealing (temperature depending on primers used) (www.beckmancoulter.com)Each PCR amplification reaction mixture consisted of 2 µl reaction buffer, 2.5 mM/L Mgrs used) , and 2 mwww.biorad.com). Amplified fragments were scored for the presence or absence of bands . Since ISSR markers were dominantly inherited, each band was assumed to represent the phenotype at a single biallelic locus . In order to estimate the congruency among dendrograms, cophenetic values (rcp) based on the results of the NJ cluster and UPGMA cluster analysis were calculated to measure the quality of the clustering  program 1.5 was also applied as described by Excoffier , where tGeographical distances of pairs of populations were calculated using the latitude, longitude, and elevation of each population. The Mantel Z-statistic  was used to test the correlation between geographical distances and genetic distances . As one 8 A amplified only 6 bands. The average polymorphic information content varied from 0.28 (AG)8CC) to 0.74 (AC)8AG), whereas the marker index ranged from 2.48 (AC)8TC) to 5.84 (AG)8TA). The mean polymorphic information content and marker index of the 14 primers was 0.55 and 4.63, respectively. The ISSRs that exhibited a high polymorphic information content value, together with a higher multiplex ratio, were likely to be efficient for the analysis of intra-specific genetic variation in a species like S. furcifera for which no prior sequence information was available.Fourteen ISSR primers were selected from a total of 67 on the basis of clarity, usability, and reproducibility of their banding patterns; the data are shown in FST of population differentiation, which is equivalent to an FST statistics when the degree of relatedness among the genetic variants is evaluated  among regions, indicating a high level of genetic differentiation.To assess the overall distribution of genetic diversity, the AMOVA program was used to analyze the distance matrix, the data are shown in valuated . AMOVA aThe similarity matrices calculated from the polymorphic ISSR bands showed highly variable genetic distances among the different populations. The genetic distance was the highest (1.1052) between the northeast population  and Red River Delta population  in Yunnan Province, and the lowest (0.1475) between southern population  in Yunnan Province and Maten population in Vietnam.S. furcifera populations; the data are shown in The dendrogram generated from the NJ cluster analysis showed the genetic relationship among 47 S. furcifera in the Greater Mekong Subregion. However, there were a few populations that did not fit into any group , and the Red River Delta populations were split into Group 2 and Group 3 in the PCOA while belong to cluster I in NJ tree. The lack of fit was also reflected in the results of the Mantel test, where the geographical and genetic matrices did not have an overall correlation , indicating the role of geographic isolation did not shape the present population genetic structure of S. furcifera.The results of PCOA showed three main groups in the two dimensional PCOA; the data are show in S. furcifera in Yunnan Province, China and the potential original areas of this migratory pest in the adjoining south and southwest countries. ISSRs can be informative at various levels of genetic variation (Drosophila melanogaster) where the poly (AC) and poly (AG) repeats are common repeat motifs across animal groups . The PCOA analysis proved relatively regional distribution of  to July . So mostBased on AMOVA analysis, 79.84% of the total variation was found among regions, while 20.16% variance was attributable to population divergence within the regions. The population genetic structure of a species is affected by a number of evolutionary factors including its mating system, gene flow, and its mode of reproduction as well as its natural selection , and theChrysoperla cornea species complex, and demonstrated the migratory capabilities of the individual species comprising the C. cornea group of lacewings, and indicated that understanding the population ecology of an insect species is necessary to investigate the complete migration syndrome. Xian et al.  not only due to the special geographic environment and meteorologic condition of Yunnan Province , but also as their small size, short lifespan, large population sizes, rapid aerial population dilution and the very long distances over which these insects may fly (S. furcifera are of particular interest in this regard. In addition to demonstrating the usefulness of ISSR markers for DNA profiling, the genetic structures among populations of S. furcifera analyzed in this study enable us to infer its evolutionary relationship. Based on the results of the study, it can be speculated that S. furcifera migrates to Yunnan Province primarily by two routes, one from northern Vietnam and Laos to the Red River Delta which is in the southeastern Yunnan Province, China; and the second one from Myanmar to the southwestern areas of Yunnan Province such as Lincang and Baoshan prefectures. After that, these two migrators disperse, spreading and finally causing outbreaks in the whole rice area of the Province, even to the whole country. This speculation was supported by Wu et al. (S. furcifera migrated to China via two routes: one was to southwestern Yunnan by southwest monsoon from the northern Thailand and Myanmar; and the other one was to the Red River Delta in Yunnan and Guangxi, Guangdong Province due to the southwest monsoon from Indochina. The present data does not clearly indicate the migration pattern of S. furcifera as there still is a dearth of information on genetic architecture. Phenotypic variation in migratory propensity has long been known, but the genetic basis of such variation is still relatively unexplored. Even more important, although it is recognized that migration is not a single trait but a suite of traits that include both larval and adult components, more data are needed on the functional and genetic relationships among traits (The present study is the first attempt of assessing the genetic diversity of  may fly , the appu et al. , as theyg traits . Therefo


Passer domesticus) in a metapopulation at Helgeland, Norway. Genomic (gDNA) and transcribed (cDNA) alleles of functional MHC class I and IIB loci, along with neutral noncoding microsatellite markers, were analyzed to obtain relevant estimates of genetic variation. We found lower allelic richness in microsatellites in the inbred population, but high genetic variation in MHC class I and IIB loci in both populations. This suggests that also the inbred population could be under balancing selection to maintain genetic variation for pathogen resistance.Small populations are likely to have a low genetic ability for disease resistance due to loss of genetic variation through inbreeding and genetic drift. In vertebrates, the highest genetic diversity of the immune system is located at genes within the major histocompatibility complex (MHC). Interestingly, parasite-mediated selection is thought to potentially maintain variation at MHC loci even in populations that are monomorphic at other loci. Therefore, general loss of genetic variation in the genome may not necessarily be associated with low variation at MHC loci. We evaluated inter- and intrapopulation variation in MHC genotypes between an inbred (Aldra) and a relatively outbred population (Hestmannøy) of house sparrows ( Genetic variation may determine the ability of a population to respond to environmental changes and hence significantly contribute to the persistence of a population . TherefoIn vertebrates, the major histocompatibility complex (MHC) is one of the most variable regions of the genome . The MHCLow genetic variation at MHC has been associated with, for example, small population size  and potePasser domesticus) is a small passerine bird with a wide natural distribution  and Hestmannøy  located approximately 13 km apart in an insular metapopulation off the coast of Helgeland in northern Norway . From each sampled bird, we collected ca 25-µl blood, which was stored in 100% ethanol for later extraction of genomic DNA. In addition, ca 25-µl blood was collected and stored in TRIzol LS , which maintains RNA in the sample without degradation. Samples were kept at –18°C until back in the laboratory, 0–2 days later, and were then stored at –80°C until analysis. All laboratory analyses were carried out at the Department of Biology, Norwegian University of Science and Technology, Norway.Genomic DNA was isolated from whole blood samples according to the phenol–chloroform protocol described in To determine the intrapopulation levels of neutral genetic variation, we typed individuals from the two islands on 15 polymorphic microsatellite loci; Ase18 , Pdoµ1 aThe forward primers were fluorescently labeled with either 6-FAM (Invitrogen), or VIC, NED, or PET , and a “pigtail” -) was added to the 5′ end of the reverse primers. Seven and eight loci were multiplexed, respectively, in 10-µl polymerase chain reactions (PCRs) containing 5-µl QIAGEN Multiplex PCR Master Mix , 2 µl of a mix of all primers , and 3-µl genomic DNA. The PCR profile consisted of an initial denaturation step at 94°C (15 min), followed by 12 cycles where the temperature was decreased by 1°C each cycle starting at 62°C annealing temperature: 94°C (30 sec), 62–50°C (1.5 min), 72°C (1 min). These cycles were then followed by 25 cycles at 50°C annealing temperature: 94°C (30 sec), 50°C (1.5 min), 72°C (1 min). The PCR was ended by a final extension step at 60°C (5 min). After PCR, 0.5 µl of the multiplexed PCR products were mixed with 0.5-µl GeneScan LIZ600 (Applied Biosystems) and 10-µl HiDi Formamide (Applied Biosystems). Alleles were resolved in a 16 capillary ABI 3130xl Genetic Analyzer (Applied Biosystems) and scored using GeneMapper 4.0 (Applied Biosystems).2 (2.5 mM), primers (0.2 µM) , MgClnd A23H3  for clasnd A23H3  for clasP > 0.160) or in the Aldra population (P > 0.119), except for Pdoµ3, which had a level of significance of P= 0.024 in the Aldra population. After correcting for number of tests within the population , this deviation from Hardy–Weinberg was however not significant. Tests for linkage disequilibrium (LD) carried out in GenePop v. 4.0.10 showed that none of the loci with enough information to carry out the test were in significant LD either within (P > 0.087) or across the two populations (P > 0.056). Moreover, microsatellite genotype data from a more extensive study that included the same loci except Pdo33 and Pdo40, but more sampled individuals from the two populations (Aldra n= 23 and Hestmannøy n= 43 (P < 0.001) that deviated from Hardy–Weinberg expectation in either population . Furthermore, this extended microsatellite dataset showed that only three pairs of loci  in the Hestmannøy population and one pair of loci (Pdo10-Pdo17) in the Aldra population were in significant linkage disequilibrium with each other after adjusting the significance level for the large number of tests carried out within each population (Bonferroni-adjusted significance level: P < 0.00047). We used t-tests to examine whether the neutral genetic variation  differed between Aldra and Hestmannøy. Means are presented as ±1 standard error, and all statistical tests are two-tailed.Mean expected heterozygosity of microsatellite loci within each population was estimated using the observed allele frequencies and assuming unlinked loci and that the populations were in Hardy–Weinberg equilibrium (eq 8.4 in øy n= 43 ), showedhttp://align.bmr.kyushu-u.ac.jp/mafft/online/server/). Sequences were considered verified if they occurred in two independent PCRs. We further performed a BLAST search on GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi), using our verified sequences, to look for published nucleotide sequences of MHC class I and IIB in house sparrows. These were used in the alignments and construction of trees. Because our primers are nested within those used by N/dS). For trees and distances, rates were assumed to be uniform among sites, and patterns among lineages homogenous, and bootstrapped 1000 times to obtain SE values. We tested for positive selection (dN > dS) on PBR and non-PBR regions of both MHC classes with the Z-test in MEGA v. 4.1, computing overall averages using the Nei–Gojobori (Jukes–Cantor) method. We also calculated codon-wise dN/dS ratios and tested for positively selected amino acid sites with CODEML in the PAML 4 package (http://www.datamonkey.org/) using the REL method for site-by-site selection, the REV nucleotide substitution bias model, and the default significance level  were assembled and edited using BioEdit v. 7.0.0, MEGA v. 4.1, and aligned with MAFFT v. 6 online . In fact, Aldra had a lower number of alleles in all but four microsatellite loci tested, the exceptions being Pdo16, Pdo27, Pdo32, and Pdo40, for which the numbers of alleles were the same for both populations. This resulted in a significant overall decrease in the number of alleles across loci on Aldra compared to Hestmannøy . In general, half or more of the alleles for each microsatellite locus were shared between the two populations, with one notable exception. For Pdo47, eight alleles were found in total, of which only one was shared, two were private to Aldra, and five to Hestmannøy. On average, Hestmannøy had significantly more private alleles than Aldra . There were however no significant differences between the two populations in mean observed heterozygosity , or mean expected heterozygosity , although the mean expected heterozygosity was slightly higher on Hestmannøy (0.83 ± 0.053) than on Aldra (0.74 ± 0.033). The estimated neutral genetic differentiation (FST) between the two populations based on microsatellite genotypes was 0.046.The mean allelic richness  of the 15 microsatellite loci was significantly higher on Hestmannøy compared to Aldra . In addition, the average (mean ± SE) number of alleles per individual did not differ between the two populations in either MHC class I  or MHC class IIB , although being slightly higher for MHC class IIB in Aldra. Consequently, for MHC class IIB, there was no difference in APD between Hestmannøy and Aldra . However, APD did differ between the populations for MHC class I, being higher in Hestmannøy . Nucleotide diversities for both MHC classes were similar for the two populations .In contrast to the results on microsatellites, variation at MHC loci, measured as total number of alleles, did not differ between the two populations  were found in Hestmannøy and 17 (10 short and seven long) in Aldra . The twoFor MHC class IIB exon 2, 25 verified sequences were found in total, distributed with 14 in Hestmannøy and 19 in Aldra . HestmanNot all verified sequences were found as both genomic and transcribed sequences. Ten MHC class I sequences were found both in gDNA and cDNA samples, while eight were found only in gDNA and nine only in cDNA . HoweverThe number of verified sequences found per individual varied between four and eight for MHC class I, and between three and seven for MHC class IIB, indicating a minimum of four loci for both MHC classes  and 2. AAs in previous studies on house sparrow MHC , the MHCN/dS ratios than regions outside the PBRs . However, eight (PAML) and six (HyPhy REL) sites were under positive selection for MHC class I long sequences, and 10 (PAML) and three (HyPhy REL) sites were under positive selection for MHC class IIB  and adaptive (MHC) loci in two house sparrow populations differing in recent population history. One population (Aldra) was established 10 years prior to sampling by a handful of individuals, while the other population (Hestmannøy) had been a comparably large population for decades. As predicted, we found a lower level of genetic variation in neutral markers in Aldra compared to Hestmannøy . In contThe results on microsatellite variation are in accordance with Petroica traversi), for example, drift seems to have resulted in monomorphism in at least one class of MHC loci (Sphenodon guntheri) had a substantially lower level of MHC variation than a nearby large population of a related species , which is extraordinarily monomorphic in hypervariable neutral genetic markers, but shows unexpectedly high level of heterozygosity in four MHC class II loci , as well as a lower total demographic variance compared to Hestmannøy. Thus, a larger proportion of the adult population on Aldra reproduce compared to on Hestmannøy. This could reduce the rate of allele loss on Aldra. In addition, the most likely mating pattern on Aldra seems to be random mating . Because Aldra was founded by only four individuals, among them one female, and the population size thereafter has been relatively low, the potential for mate choice could have been limited. The high MHC variation in this population may also be a result of gain rather than reduced loss of alleles. New alleles could have been introduced by immigration, after the founder event, if the immigrants reproduced successfully. In the first 10 years, after the Aldra population was established, 15 immigrants were recorded  and these individuals are likely to have carried previously lost or novel alleles. Analyses have shown that these immigrants also contributed to the population's gene pool . There may also be selection against inbred individuals on Aldra, since lifetime reproductive success was negatively related to individual inbreeding level in this population , although the mechanisms behind this selection are not known yet.One could speculate about the mechanisms behind the selection for maintaining MHC diversity in a population such as Aldra. Balancing selection could come about through interactions with pathogens, or possibly inbreeding avoidance mechanisms such as mate choice. A study by Phylloscopus trochilus) in Sweden showed both temporal and geographical variation between three different sampling years , feather mites , Isospora spp. and Capillaria spp. , as well as the parasitic gapeworm Syngamus trachea . House sparrows infected with S. trachea have been found on both Aldra and Hestmannøy, and individuals showing symptoms of gapeworm infection have a reduced probability of survival until the next breeding season . Thus, similar selection pressure, such as exposure to the same pathogens, is one possible mechanism that should be explored in relation to maintenance of MHC variation in future studies.Finally, selection pressure caused by exposure to a variable community of pathogens may have helped to maintain the MHC variation gained or restored by immigration. For example, in insular avian populations of the West Indies, avian malaria communities have been shown to be relatively stable over short time periods (1 year) but to have lineage turnover during the course of a decade . Infecting years . In partVariation at neutral loci may not reflect variation at adaptive loci. This was clearly demonstrated in the current study where we found high levels of MHC variation in an inbred house sparrow population with a low level of variation at neutral loci. The level of MHC variation in the inbred population was also comparable to that of a much larger outbred house sparrow population in the same area. The mechanisms behind such maintained functional genetic variation need to be further investigated, but immigration and selection on MHC variation through pathogens are possible candidates in this system.


Styela clava in the northeastern Pacific. We utilized microsatellite data to estimate gene flow between samples collected throughout the known range of S. clava in the region, and assessed correlation of these estimates with patterns of intracoastal commercial vessel traffic. Our results suggest that recent shipping patterns have contributed to the contemporary distribution of genetic variation. However, the analysis also indicates that other factors—including a complex invasion history and the influence of other vectors—have partially obscured genetic patterns associated with intracoastal population expansion.Understanding the factors contributing to expansion of nonnative populations is a critical step toward accurate risk assessment and effective management of biological invasions. Nevertheless, few studies have attempted explicitly to test hypotheses regarding factors driving invasive spread by seeking correlations between patterns of vector movement and patterns of genetic connectivity. Herein, we describe such an attempt for the invasive tunicate  The risks posed by invasive populations depend in large part on their ability to expand their nonnative ranges through colonization of new habitats. Many authors have recognized the importance of factors driving movement of invasive propagules (“propagule pressure”) not only to sites of initial colonization outside a taxon's native range, but also to sites of secondary colonization following establishment  and the Canadian Marine Communications and Traffic Services database (BC). These data represented >96% of all vessel arrivals from a 2-year period (2003–2005) for US ports and for 1-year in BC. For all regions, data included arrival port, date-of-arrival, and previous port. The only suspected minor under-reporting of vessels may have occurred for barges because regulatory changes affecting barges occurred at the time. The database comprised more than 32,000 vessel arrivals, and patterns of connectivity among ports (and from overseas ports) were derived from a common denominator of one annual cycle of vessel flux .Styela clava were collected in 2006 as part of a large-scale effort to assess the distribution of genetic variation within multiple established and widely distributed coastal marine invasive species in the northeastern Pacific. Collections were conducted at a total of 36 sites along the Pacific US coast, including all known sites where S. clava populations had been previously reported. Styela clava specimens were successfully collected from eight of those sites  following the manufacturer's protocol.se sites . Subsequ2, and 10–100 ng of template DNA. Amplified products were sized on an ABI 3730xl DNA Analyzer using GeneScan-500 LIZ size standard  and raw data were analyzed using GENEMARKER v. 1.60 .Polymerase chain reaction amplification of 10 species-specific microsatellite loci  was conducted using the PCR cycling parameters described by Dupont et al. . ReactioK) best supported by the genetic data, we assessed models with K ranging from 1 to the total number of samples (13). For each value of K, we conducted five independent Markov–Chain Monte Carlo (MCMC) runs with 105 generations discarded as burn-in followed by an additional 106 generations of data collection. The value of K best representing the population structure present in the genetic data was inferred using the procedure described by Evanno et al. ; Dana Point, Newport Bay, and Los Angeles ; Port Hueneme and Channel Islands ; and Pleasant Harbor and Blaine Marina  ; and for shipping connectivity based on directional vessel visits  were observed between the Puget Sound cluster and all other clusters, whereas migration rates were highest among southern clusters between San Francisco and San Diego  ranging from 3.62 to 4.14 alleles and expected heterozygosity (HE) ranging from 0.53 to 0.59 , nor was migration . However, when Puget Sound was excluded from the dataset, the correlation between migration and shipping became significant . The correlation between FST and distance remained significant  in this case.When considering the full dataset, the only significant correlation observed was that between S. clava as a component of fouling communities on maritime vessels, including commercial ships, is widely recognized as a prominent mechanism for both initial introduction and post establishment spread . That hypothesis would also explain why the correlation between intracoastal shipping and distribution of genetic variation is only apparent when PS is excluded from the analysis, as the introduction of diversity from outside the region would obscure patterns driven by vectors within the region.This significant correlation was only observed when the cluster at Puget Sound (PS) was excluded from the analysis . The PS S. clava populations in the eastern Pacific, including a population in Puget Sound . This failure of FST to capture the affect of shipping on colonization patterns may reflect violations of equilibrium assumptions. The expected inverse relationship between genetic distance as measured by FST and dispersal between populations assumes equilibrium between mutation and genetic drift , even in the case, where that correlation is statistically significant. First, it is possible that the correlation is obscured by the activity of alternate dispersal vectors. There is reason to believe that at least two other vectors, recreational boating and the movement of aquaculture stocks, contribute substantially to the expansion of S. clava populations worldwide (Minchin and Duggan S. clava in other parts of its nonnative range (Dupont et al. S. clava in the northeast Pacific, and possible that these vectors may be stronger predictors of the observed distribution of genetic variation. Unfortunately, data on the vessel flux and movement patterns for these vectors remain unavailable, at present, precluding direct tests of those relationships, such as we have performed herein for commercial vessels.There are several potential explanations for the relatively weak correlations observed between genetic and shipping patterns (Styela clava has been present on the US Pacific coast since at least the early 1930s (Lambert and Lambert S. clava's residence in the region, and heavier traffic from faster and larger ships over time may well have played a role in redistributing S. clava northward. Unfortunately, data on directional patterns of historical vessel flux could not be readily assembled. The trend of apparent northward spread of invaders on the coast is well documented for many species and ships are strongly implicated (Ruiz et al. It is also possible that there may be temporal mismatch of the genetic and shipping datasets that would confound our analysis. Gemma gemma. In none of these cases, was it possible to develop connectivity networks that quantify directional movement of vectors. The movements of recreational vessels have proven particularly challenging to assess given the lack of reporting requirements in most contexts, although researchers have developed various methods for overcoming the lack of empirical data (Padilla et al. Botryllus schlosseri populations in Nova Scotia. However, even that study was limited by an inability to register directionality of recreational boat traffic. Detailed databases that track directional vector intensities (e.g., the National Ballast Information Clearinghouse, http://invasions.si.edu/nbic; Verling et al. Our study demonstrates the feasibility of using genetic data to quantitatively test hypotheses regarding the mechanisms driving spread of invasive populations. In particular, it suggests that the combination of nonequilibrium genetic methods and vector datasets revealing true source-to-destination connectivity may provide a powerful means to assess correlations between anthropogenic drivers of dispersal and genetic structure. However, it also exposes difficulties likely to be associated with such attempts. First, the assembly of useful datasets on the movement of potential vectors may prove a limitation for many analyses. Previous studies focusing on aquatic bioinvasions have been forced to adopt various estimates of vector transport that are incapable of registering precise source-to-destination linkages between sites (Herborg et al. Equally problematic is the fact that, for many invasive species, there will be multiple vectors potentially contributing to population expansion. Invasive spread is frequently determined by both natural and anthropogenic processes, and a large number of recent studies have revealed stratified dispersal patterns among invasive populations, with both local diffusive spread driven by natural dispersal mechanisms and regional spread driven by long-distance anthropogenic vectors (Wilson et al. S. clava, that continue to expand their global ranges through the activity of multiple anthropogenic dispersal vectors (Davis and Davis More generally, our results suggest that complex invasion histories, particularly those that involve multiple independent introductions, may make it problematic to infer the most important drivers of regional population expansion. By introducing external influences on contemporary distributions of genetic variation, multiple introductions will likely obscure the expected relationships between patterns of population genetic structure and patterns of regional vector intensity. Unfortunately, the past decade of invasion genetics research suggests that multiple introductions may be an all too common characteristic of successful biological invasions (Roman and Darling