ST is among the most widely used measures for genetic differentiation and plays a central role in ecological and evolutionary genetic studies. It is commonly thought that large sample sizes are required in order to precisely infer FST and that small sample sizes lead to overestimation of genetic differentiation. Until recently, studies in ecological model organisms incorporated a limited number of genetic markers, but since the emergence of next generation sequencing, the panel size of genetic markers available even in non-reference organisms has rapidly increased. In this study we examine whether a large number of genetic markers can substitute for small sample sizes when estimating FST. We tested the behavior of three different estimators that infer FST and that are commonly used in population genetic studies. By simulating populations, we assessed the effects of sample size and the number of markers on the various estimates of genetic differentiation. Furthermore, we tested the effect of ascertainment bias on these estimates. We show that the population sample size can be significantly reduced  when using an appropriate estimator and a large number of bi-allelic genetic markers . Therefore, conservation genetic studies can now obtain almost the same statistical power as studies performed on model organisms using markers developed with next-generation sequencing.Population genetic studies provide insights into the evolutionary processes that influence the distribution of sequence variants within and among wild populations. F ST, first defined by Wright (1951).Studies on wild populations give important insights into population dynamics leading to genetic differentiation. One important goal of population genetic studies is to estimate the amount of genetic differentiation among populations in order to draw conclusions on the demographic history. A common measure for the degree of genetic differentiation is the fixation index FST outlier methods n>20) are required to yield reliable estimates of differentiation ST. Reducing the sample size per population would make it possible to analyze a larger number of different populations at the same cost, and it offers an important advantage in conservation genetic studies on rare organisms. Furthermore, understanding the statistics of different FST estimators is especially important in the context of detecting regions under selection.Until recently, most studies on wild population of non-reference species used moderately large numbers of samples per population (>20), but only a small number of genetic markers (<20), preferentially microsatellites, for which more than two alleles can often be distinguished. Studies on human populations were among the first using thousands of markers, with single nucleotide polymorphisms (SNPs) as markers of choice. SNPs are typically the most abundant sequence variants in genomes. Their distribution throughout the entire genome at high density, combined with well-established models for handling mutation rates and error rates, and inexpensive methods for high throughput genotyping make them appealing for population genetic studies ST becomes inflated with small sample sizes. We concentrated on three different estimators. The first one was proposed by Wright (1951), which by definition lies between zero (no genetic differentiation) and one . However, Wright assumed infinite sample sizes in his definition, but population size is finite in real datasets. The absence of negative FST values in Wright's (1951) definition can lead to an overestimation of FST, particularly when the populations are only weakly or not differentiated. Cockerham and Weir (1984) proposed an unbiased estimator that can also have negative FST estimates and that has been widely used n = 4–6), and the behavior of this new estimator will be evaluated as well. Here we compare all three estimators for their performance on the same bi-allelic data set. We addressed the following four questions. First, what is the effect of small sample size on the type I error rate, i.e. falsely detecting genetic differentiation in a panmictic population? Second, does a small sample size result in an overestimation of the FST in cases where populations are genetically differentiated? Third, if estimates are imprecise due to small sample sizes, does precision increase with the number of loci genotyped? Fourth, what is the effect of different allele frequency distributions on the FST statistics, in particular, does a relative excess of common or rare alleles lead to a bias in FST estimates?In this study we used simulations to examine whether the estimation of genetic differentiation measured by Ft generations of random mating, we estimated FST on the complete simulated dataset comprising two populations with 1,000 individuals each that were genotyped at 21,000 loci  will cause additional drift and differentiation over and beyond that expected from the theoretical distribution. Because of the absorption state of SNP loci , such random chance events are not completely offset by an overly equal reproductive distribution (i.e. runs in which too many families with exactly 2 offspring). As a consequence, the simulated FST, like a ratchet, tends to increase slightly faster than the theoretically expected value.After l and ST . The reaSTF = 0, 0.01, 0.05, 0.1, 0.2, 0.4). k = 100 and k = 1,000 at varying sample sizes. n = 4 and n = 20 at increasing number of loci genotyped. Since combining all the different parameters  resulted in a large number of estimates, we chose these four combinations in order to illustrate our general findings . STWF severely overestimated genetic differentiation in small sample sizes (n = 2–6) (e.g. n = 10–50), STWF slightly overestimates the level of genetic differentiation. Since these observations were consistent for all datasets, we will in the following concentrate on the behavior of the two other estimators.We tested the influence of increasing the sample size on the estimate by taking 2, 4, 6, 10, 20 and 50 individuals from each population at different levels of genetic differentiation (–6) e.g. . MoreoveSTW&CF and STRF gave on average similar, fairly good estimates at all sample sizes (ST≥0.1) the FSTW&C estimator tended to slightly overestimate genetic differentiation with small sample sizes (n≤6), whereas the estimator FSTR showed the same average estimate irrespective of samples size. Though, with increasing sample sizes the size of 95% CIs decreased. The 95% CIs were large and included zero at low genetic differentiation (FST<0.05), when using a small sample size (n = 2–6) and a moderate number of loci (k = 100). Increasing the number of loci had no impact on the average estimate of FST  differences between sample sizes did not have an impact on the average estimates of FST of either estimator (FSTW&C and FSTR). If genetic differentiation was moderate to high (FST≥0.1), the FSTW&C overestimated genetic differentiation when the sample size of population 2 was small , this will lead to ascertainment bias and an overestimation of genetic differentiation.In order to test the influence of biases of allele frequency distribution in the analysed marker set, we generated two datasets with 10,000 loci each, where in one set MAF>0.25 and in the other set MAF≤0.25. The simulations show that a shift towards common polymorphisms in the marker set leads to overestimation of genetic differentiation, whereas a shift towards rare polymorphisms leads to underestimation. These erroneous estimates were observed in both the yped see  and S3. ST estimators has been analyzed before, this study is the first to evaluate different FST estimators in population genetic studies of thousands of loci and very small sample sizes. This is a timely matter, given that Next Generation Sequencing (NGS) methods have revolutionized the field of marker development. Furthermore, we have included a new estimator employed by Reich et al. (2009). Our simulations show that even when sample sizes are small , accurate and unbiased estimates of FST can still be obtained when a large number of bi-allelic markers such as SNPs are used, as long as the appropriate FST estimator is chosen. The original FSTW estimator severely overestimates the level of genetic differentiation when using small sample sizes. Since this estimator cannot have negative values, these results were expected for values of FST<0.5, because overestimates that are higher than 0.5 over the actual FST cannot be compensated by a negative value at another locus. The two other estimators we tested showed similar performance on large sample sizes (n≥20), and consistent with a previous study STW&C overestimates FST when analyzing small samples. Importantly, the estimator proposed by Reich et al. (2009) showed better performance in cases where sample sizes were small (n≤6). Our simulations suggest that genetic differentiation is not falsely detected due to small sample sizes when using this unbiased estimator. Furthermore, we showed that increasing the number of genetic markers has no impact on the mean FST estimates, but that it considerably reduces the 95% CIs. Although this is not unexpected, given that increasing the number of loci decreases the coefficient of variation of the estimates, we quantify this effect here for the first time. Finally, the precision of a pairwise estimate depends on the smaller sample size taken from one of the populations and not on the number of loci.Although the statistical behavior of different FST rather than the sample size taken. A bias towards common polymorphisms in the marker set leads to overestimation of genetic differentiation, whereas, visa versa, FST becomes underestimated when there is a bias towards rare polymorphisms. The practical implication is that if the panel is developed based on the screening of a small number of individuals, this will lead to ascertainment bias and an overestimation of genetic differentiation of the study populations.A previous study suggested that increasing the sample size might be more beneficial than increasing the number of markers genotyped random individuals. However, in practice, random sampling cannot always be achieved in nature. Estimates on extremely small samples, as suggested here, might be severely affected by non-random sampling, e.g. due to population structuring. Furthermore, our simulated dataset contained only fully unlinked markers. If subsets of markers are linked they represent only one informative marker. Hence, the number of fully unlinked markers depends on the genome size of the organism analyzed. For the same reason, detecting regions under selection with FST outlier methods require larger sample sizes (n>10). In such cases, FST estimates are based on small sliding windows in which markers are  linked, and hence, they represent a smaller number of independent loci. Baird and colleagues (2008) proposed a method that has been proven particularly useful to develop a large number of genetic markers in non-reference organisms with less ascertainment bias Theoretically, precise estimates of genetic differentiation can be obtained based on very small sample sizes of t) of generations according to the Wright-Fisher model without mutations . Consequently, the population sizes were kept constant, the generations were non-overlapping and the frequencies in the next generation were a binomial random sample based on the frequencies in the current generation. Random dioecious mating was simulated by randomly drawing one female and one male with replacement. Thus all males and females were equally likely to be chosen and could mate multiple times, and the draw was independent of the number of times an individual has been chosen before. The two individuals drawn became the parents of one member of the next generation. Since we assumed all loci to be completely unlinked, we simulated gametogenesis by simply selecting at random one allele from each parent. This process was repeated until all members of the next generation have been created. We simulated different degrees of genetic differentiation . The number t of generations was determined by n equals the number of mating individuals  and t equals the number of generations needed to achieve the required amount of differentiation We simulated an ancestral population with 1,000 individuals  and 21,000 bi-allelic loci. The genotypes at the loci were generated by randomly drawing from eight allele frequency classes . The two starting populations consisted of the same 1,000 individuals, which were genotyped at 10,000 loci randomly taken from the 21,000 loci of the ancestral population. We assumed an isolated island model (i.e. no migration between the two populations after separation). Genetic drift was simulated for a certain number (ST was introduced by Wright (1951) as a measure of correlation of gene frequencies und suggested the first and simplest estimator, STWF. For one allele at locus k,ip among the sampled populations i  and STWF for multiple loci is calculated by taking the mean across k loci.FSTW&CF, preserves Wright's definition of FST in terms of correlation of gene frequencies and is the most widely used estimators  ST at moderate population sample size  and small number of loci (k = 10). The estimates can also have negative values which do not have a biological meaning k, STW&CF is defined as2s is the observed variance of allele frequencies, n is the number of individuals per population, r is the number of sampled populations and k loci is derived byThe second estimator tested, STRF, for bi-allelic loci and pairwise population comparison. In their study they used a very high number of loci, but small sample sizes per population. Therefore, we decided to test this estimator as well. Again STRF is calculated as followsu is the allele count for population 1, v is the allele count for population 2, t and s are the total number of individuals for population 1 and 2, respectively Recently, Reich and colleagues (2009) proposed a new unbiased estimator, t generations of random mating among 1,000 individuals, 10,000 of the 21,000 loci were randomly chosen to test the STF estimates. In order to test the influence of ascertainment bias in marker design, we generated three different datasets. The first set contained loci with equally distributed allele frequencies, the second set contained only loci with minimum allele frequency MAF>0.25, because SNP marker sets are often biased in the direction of more common polymorphisms. However, we also generated a dataset of the other extreme containing only markers with MAF≤0.25.After ST estimate and derived the 95% confidence interval. We used a custom Java program to perform the simulations and estimations of FST that will be made available upon request.We used sample sizes of 2, 4, 6, 10, 20 and 50 individuals. For each sample size we sampled 10, 20, …, 100, 200, …, 1000, 2000, …, 5000 loci. For each number of individuals and genotyped loci we sampled from each population 1,000 times. We took the average FFigure S1Effect of increasing sample sizes and increasing marker numbers with uniform allele frequency distribution.ST = 0, 0.01, 0.05, 0.1, 0.2, 0.4). The results (average FST and 95% CI) of each estimator are depicted in the different graphs: FSTW (blue circles), FSTC&W (purple squares) and FSTR (green triangles). The dashed red line indicates the actual FST for the simulated population.(PDF)Click here for additional data file.Figure S2Effect of increasing sample sizes and increasing marker numbers for MAF>0.25.ST = 0, 0.01, 0.05, 0.1, 0.2, 0.4). The results (average FST and 95% CI) of each estimator are depicted in the different graphs: FSTW (blue circles), FSTC&W (purple squares) and FSTR (green triangles). The dashed red line indicates the actual FST for the simulated population.(PDF)Click here for additional data file.Figure S3Effect of increasing sample sizes and increasing marker numbers for MAF<0.25.ST = 0, 0.01, 0.05, 0.1, 0.2, 0.4). The results (average FST and 95% CI) of each estimator are depicted in the different graphs: FSTW (blue circles), FSTC&W (purple squares) and FSTR (green triangles). The dashed red line indicates the actual FST for the simulated population.(PDF)Click here for additional data file.Table S1ST estimates according to Wright (1951).F(XLSX)Click here for additional data file.Table S2ST estimates according to Weir & Cockerham (1984).F(XLSX)Click here for additional data file.Table S3ST estimates according to Reich et al. (2009).F(XLSX)Click here for additional data file.


Since pulmonary hypertension (PH) in patients with chronic obstructive pulmonary diseases (COPD) causes poor prognosis and inflammatory process involved in PH, it is supposed that Statins with anti-inflammatory effects might be useful in the treatment of PH.The aim of this study was to evaluate the influence of Atorvastatin on the treatment of pulmonary hypertension in patients with COPD.A registered (IRCT201108257411N1), triple-blind, randomized controlled trial was performed in Rasoule Akram hospital, Tehran, from 2009 to 2011. Forty five patients with secondary pulmonary hypertension due to COPD were recruited and randomized to two groups receiving either Atorvastatin 40 mg/d or placebo in addition to their current treatment for 6 months. The outcomes including systolic pulmonary arterial hypertension (SPAH), cardiac output (CO), right ventricular size (RVS), CRP, 6 min walk distance test (6MWD), and spirometry parameters were measured after 6 months.Baseline characteristics were similar in both groups. After 6 months, pulmonary hypertension changed from 48.5 ± 6.9 to 42.9 ± 9.3 mmHg for Atorvastatin users and from 49.7 ± 11.4 to 48.2 ± 14.6 mmHg for Placebo users , 6MWD after 6 months was 339 ± 155 meters in case group versus 340 ± 106 meters in control group . There were no significant changes in other outcomes including CRP, RVS, CO and spirometry parameters.Although we found a trend towards decreasing SPAH and improving 6MWD, no statistically significant shift were detected in our outcomes due to inadequate sample size. COPD is a systemic inflammatory disease, not limited to the pulmonary system . The effPulmonary hypertension (PH) is one of the most important functional derangements in COPD and leads poor prognosis. The mechanisms leading to pulmonary hypertension in COPD have been ascribed to chronic hypoxia and loss of vascular bed from emphysematous destruction . In thisIn addition of lowering cholesterol levels and improving peripheral vascular perfusion, statins have a multiple biological effects including alteration of vascular wall structure; modulation of endothelial function , 5, 7 doSince the majority of evidence endorsing potential therapeutic benefit of statin drug class comes from a series of animal studies and there are a few data from studies in patients especially in the area of PH due to COPD, the necessity of studying such gray topic in a well-designed RCT on human seems so profitable in treating COPD patients. In our literature review, we found just one randomized clinical trial about the effect of statins on PH due to COPD . TherefoWe performed a triple-blind, parallel-group, randomized controlled trial in Rasoule-Akram hospital that was conducted from 2009 to 2011. The trial protocol was approved by the institutional ethical review board at Tehran University of Medical Sciences (TUMS). The trial was registered at Iranian Registry of Clinical Trials with registration number (IRCT201108257411N1). We initially planned to recruit 60 subjects with secondary pulmonary hypertension due to COPD over approximately two years with convenience sampling method. Due to slower anticipation than recruitment, the target sample size was decreased to 45 patients. We included patients > 18 years old with SPAH and without risk factors for adverse events from this medication. Inclusion criteria were considered as Systolic pulmonary hypertension > 40mmhg, no drug history of prostanoids, statins, endothelin antagonists and phosphodiesterase, able to doing 6-min walk distance test, obstructive pattern in PFT and functional class 2,3 (NYHA). Exclusion criteria include PAH with underlying cause other than COPD; LDL < 70 mg/dl, ALT or AST > 3x upper limit normal. All participants provided written informed consent.The criteria for diagnosis of COPD was based on American Thoracic Society standards , with a The primary outcome was SPAH at 6 months after medication consumption. For evaluating of SPAH we used Two-dimensional and Doppler echocardiography using standard techniques on commercially available equipment with a predefined imaging protocol. To estimate systolic pulmonary artery pressure, the maximal tricuspid regurgitated jet velocity (m/s), derived from the continuous wave signal, was added to estimated right atrial pressure (RAP) (based on inferior vena cava size and its changes with inspiration) in the modified Bernoulli equation . One speThe primary analysis proceeded according to the intention-to-treat principle. Hypothesis testing for the primary and secondary end points was conducted with 2-sided α = 0.05. Continuous variables are presented as mean ± SD or median (interquartile range) and categorical variables as n (%). Baseline characteristics of the two treatment groups were compared with independent T tests. Response to Atorvastatin on SPAH, right ventricular size, cardiac output, lung function versus placebo was assessed by analysis covariance, where the response was change from baseline and the baseline value was the covariate. For non-parametric variables, we used mann-withney test. All data were analysed using SPSS software.Of the 45 patients who were randomized to either the Atorvastatin treatment (n = 24) or the placebo (n = 21) 36 patients completed treatment (19 in Atorvastatin group and 17 in placebo), 7 patients had incomplete data, two patients died because of deterioration of their diseases . BaselinThe two groups were well balanced with respect to these characteristics. Changes in clinical outcomes after Atorvastatin treatment are listed in There is a lot of interest in the use of statins for the treatment of pulmonary arterial hypertension (PAH). Statins have recently come under evaluation for the treatment of PAH. The potential role of statins in treating COPD is controversial. The current evidence shows that statins have an effective influence on outcomes in patients with COPD. Despite these hopeful results, in all researches up to this time, there are many limitations that would be in mind. The effective dose, interval and duration of administration and effect of various statins will be needed to appear the advantage of them, in target population is unclear. Although these are new evidence but insufficient to vindicate a clinical indication for statin therapy in patients with COPD regardless of protection of them on cardiovascular system. This information support that new RCT should be designed to approved these advantages. A positive effect on outcomes, such as exacerbation, hospitalization and mortality rates in patients with COPD, potentially could have a large beneficial effect on the individual, social, and economic consequences of this disease.To our knowledge the present study has been one of the first randomized controlled trials to evaluate the effects of statin treatment on SPAH due to COPD. In this study we found no statistically improvement in the atorvastatin group compared to the placebo group in SPAH, lung function, CRP, 6MWT in COPD patients with pulmonary hypertension. There are several limitations to this study  The MajoThe only RCT of statin use in patients with COPD and PAH was done by Lee et al. that evaluated the hypothesis whether statins improve hemodynamic in COPD patients with PH by affecting ET-1 synthesis. A total of 53 patients with stable COPD [FEV1 (forced expiratory volume in 1 s) 56% predicted] and PH (defined as a systolic PAP > 35 mmHg by echocardiography) were randomized to receive either pravastatin or placebo in a double-blind group for 6 months. The primary outcome was exercise capacity, tested using a Naughton stress test treadmill protocol, and was significantly improved in the pravastatin-treated group in comparison with placebo group. Systolic PAP decreased significantly from 47 to 40 mmHg, and the Borg dyspnoea index was also improved. The main conclusion of the authors was that “pravastatin significantly improved exercise tolerance, and decreased PH and dyspnoea during exercise, probably by inhibiting ET-1 synthesis in COPD patients with PH . The desPeter N. Kao evaluated the safety and efficacy of simvastatin for treatment of patients with PAH in an open-label observational study performed on sixteen patients with primary and secondary causes of PAH. Simvastatin was prescribed at 20 to 80 mg/d and continued in the absence of adverse effects. Serial measurements of 6-min walk (6MW) performance, hemodynamics, and echocardiographic estimates of right ventricular systolic pressures (RVSPs) were recorded on each patient. Improvements in 6MW performance, cardiac output, or decreases in RVSP that may be attributable to simvastatin treatment, were demonstrated . Martin Przemysław K and his colleagues evaluated the influence of simvastatin on selected inflammatory markers in patients with COPD. The study shows that a 3-month treatment with simvastatin does not reduce circulating inflammatory markers in the patients . BlamounIn another study, Reed et al. evaluated 112 patients with severe COPD regarding clinical characteristics, pulmonary function, cardiac catheterization findings, and medical co morbidities in a cross-sectional study. They compared these outcomes between 30% statin users and 70% non-statin users. They found that statin use was associated with a 4.2 mmHg  lower PAWP and a 2.6 mmHg  reduction in mean PAP independent of PAWP . In a su


The role of dispersal potential on phylogeographic structure, evidenced by the degree of genetic structure and the presence of coincident genetic and biogeographic breaks, was evaluated in a macrogeographic comparative approach along the north-central coast of Chile, across the biogeographic transition zone at 30°S. Using 2,217 partial sequences of the mitochondrial Cytochrome Oxidase I gene of eight benthic invertebrate species along ca. 2,600 km of coast, we contrasted dispersal potential with genetic structure and determined the concordance between genetic divergence between biogeographic regions and the biogeographic transition zone at 30°S. Genetic diversity and differentiation highly differed between species with high and low dispersal potential. Dispersal potential, sometimes together with biogeographic region, was the factor that best explained the genetic structure of the eight species. The three low dispersal species, and one species assigned to the high dispersal category, had a phylogeographic discontinuity coincident with the biogeographic transition zone at 30°S. Furthermore, coalescent analyses based on the isolation-with-migration model validate that the split between biogeographic regions north and south of 30°S has a historic origin. The signatures of the historic break in high dispersers is parsimoniously explained by the homogenizing effects of gene flow that have erased the genetic signatures, if ever existed, in high dispersers. Of the four species with structure across the break, only two had significant albeit very low levels of asymmetric migration across the transition zone. Historic processes have led to the current biogeographic and phylogeographic structure of marine species with limited dispersal along the north-central coast of Chile, with a strong lasting impact in their genetic structure. It has long been established that the geographical structure of the genetic diversity of benthic species is strongly shaped by life history traits. Among the main life history attributes, mode of larval development, duration and characteristics of larval life, have been highlighted as features that influence the maximum distance of dispersal in many benthic marine species In the recent years however, the correlation between pelagic larval duration (PLD) and the spatial scale of genetic differentiation has been repeatedly questioned by meta-analyses and comparative studies of benthic and coastal marine species Among the different alternatives, biogeographic transitions have been considered as ideal situations to study the historical and contemporary processes that affect the distribution and abundance of species. Indeed, depending on the species ability to disperse and survive in different environmental conditions, biogeographic breaks often reflect species range limits Lessonia nigrescensMazzaella laminarioidesAcanthina monodonConcholepas concholepas, and the squat lobster Pleuroncondes monodon and the cephalopod Dosidicus gigas, did not show genetic structure at a macrogeographic scale across 30°S Limnoria by rafting on macroalgae Pyura chilensis on boat and ship hulls Lessonia nigrescens and A. monodon have planktonic dispersive stages that last a few hours to a few days, whereas C. concholepas, J. cirratus, P. monodon and D. gigas can spend weeks to several months in the water column. Thus, these studies strongly suggest that the biogeographic break is the result of a past discontinuity that is currently not perceivable in the macrogeographic genetic structure of species with high dispersal potential.This study further tests these predictions by undertaking a comparative phylogeographic analysis across a biogeographic boundary along the coast of Chile. The lineal coast of Chile, north of 42°S, encompasses approximately 2,600 km with a natural temperature gradient associated to latitude, but with neither apparent geographic barriers to dispersal nor any sharp environmental discontinuity. The cold and rich nutrient Humboldt Current influences the coastal area between 18 and 42°S, with upwelling as the dominant feature influencing coastal communities The current study aims at contributing to the knowledge of phylogeographic patterns of marine benthic organisms along the Chilean coast under the influence of the Humboldt Current focusing on eight species with high and low dispersal potential. More broadly, the role of dispersal potential on the population genetic diversity and differentiation is addressed. We predict that species with low dispersal potential will have greater genetic structure, lower genetic diversity than high dispersers and the presence of a historic phylogeographic break around 30°S, coincident with the biogeographic transition zone.Emerita analoga (Stimpson) (Hippidae) and Petrolisthes violaceus Stimpson (Porcellanidae); two starfishes of the class Asteroidea (order Forcipulatida), Heliaster helianthus (Lamarck) (Heliasteridae) and Stichaster striatus Müller and Troschel (Asteriidae); and the sea urchin Tetrapygus niger (Molina) . The remaining three species were considered as having low dispersal potential: the peracarid Orchestoidea tuberculata Nicolet  that broods its embryos, and the gastropods Scurria scurra (Lesson)  and Tegula atra (Lesson)   with reflects an upper bound of dispersal rather than a mean dispersal time Emerita analoga JX130229 - JX130344; Heliaster helianthus JX130054 - JX130098; Orchestoidea tuberculata JX129929 - JX129953; Petrolisthes violaceus KC543356 - KC543409; Scurria scurra JX130193 - JX130212; Stichaster striatus JX129954 - JX130053; Tegula atra JX130156 - JX130192; Tetrapygus niger JX130099 - JX130155.COI gene sequences were obtained from 2,217 individuals of eight species , coverinT. niger to 672 in S. scurra. Variable sites varied between 2.7% in S. scurra to 23.2% in O. tuberculata, with an average of 10.5% . The genetic structure detected for S. striatus was weaker than on the three low dispersers.St values . The lowerculata , and oveerculata . Mantel’ST values were significantly associated with the interaction of dispersal potential and biogeographic differentiation , suggesting that differences detected between low and high dispersers were not biased by the inclusion of O. tuberculata and S. striatus.Overall, GLMMs indicated that pairwise Φrsal see  on the rTegula atra and S. striatus showed a very similar pattern; although there was greater structure in T. atra both species had a significant and high portion of the total variance within populations , and a considerable amount explained by biogeographic region . Overall, GLMMs revealed significant differences between high and low dispersers for each of the AMOVA’s Φ-statistics Fig. 4), erculata , revealiad k = 4 . For botS. striatus  and divergence times were >0, indicating that not all shared haplotypes are a consequence of gene flow but there is also ancestral polymorphism  . In genestriatus ; Fig. 5Bt recent , confirmmorphism ; Table 7plitting . ScurriaOur results suggest that the phylogeographic patterns of marine benthic organisms of the Chilean intertidal have been strongly influenced by their dispersal potential. Compared to other regions of the world, such as the northeast Pacific et al.E. analoga and O. tuberculata that inhabit sandy beaches. All the rest are all found in the mid-intertidal rocky shore. The species with the most divergent habitat is O. tuberculata that inhabits the supralittoral (STF) Several studies have explored the determinants of the geographic structure of the genetic diversity of marine species. Among them, Weersing & Toonen littoral , howeverS. striatus, was assumed to have high dispersal potential based on the estimated PLD of its congener S. australisS. striatus did not change the implications of results, i.e. dispersal potential was still a strong predictor of genetic diversity, genetic differentiation, and of the presence of a phylogeographic break at 30°S. Of the four species with structure across 30°S, S. striatus had the least structure. For example, ΦCT (among groups) was non-significant for S. striatus while it was for all low dispersers. When analyzing genetic differentiation and isolation by distance of S. striatus at each side of the break, only the northern biogeographic region had significant differentiation and isolation by distance. The southern region showed non-significant differentiation and lack of an isolation by distance pattern. Contrastingly, all low dispersers had significant genetic differentiation and isolation by distance in both biogeographic regions. There was a tremendous difference between the genetic diversity (2 Ne) of the northern and southern biogeographic regions for S. striatus. Presumably the genus Stichaster has cold-water affinities S. striatus: (1) S. striatus is a true high disperser and its spatial genetic structure is the result of the effects of strong local genetic drift, or (2) it is a medium to low disperser, in which case observed results would be consistent with the marine connectivity paradigm. The effective size inferred from isolation-with-migration model is comparable to the two most structured species (O. tuberculata and S. scurra) and much smaller than the other low dispersal species, T. atra, suggesting that genetic drift may indeed have been strong in S. striatus. On the other hand, PLD is highly variable within the order Forcipulatidae, with a minimum of zero days . The main way in which populations and species loose genetic diversity at neutral markers is by genetic drift. However, the actual influence of genetic drift depends on its interaction with other evolutionary forces, such as gene flow: reduced dispersal may allow local populations to demographically fluctuate and increase extinction-recolonization rates Finally, because low dispersers have subdivided populations they are prone to metapopulation dynamics, i.e. extinction and recolonization S. scurra, T. atra and S. striatus) showed similar and relatively recent divergence times, suggesting that the genetic discontinuity originated either during the last glacial period (based on t 2%) or just after (when based in t 10%). However, O. tuberculata had a divergence time between biogeographic regions of an order of magnitude higher and overlaps those estimated for other reported genetic discontinuities in the same region, such as the kelp Lessonia nigrescens with estimated divergence at 3095 ky ±1200 ky Bayesian analyses based on the isolation-with-migration model take full advantage of the data by using a coalescent approach to jointly estimate migration, time of divergence and effective population size. Ideally, when using coalescent approaches such as those implemented in IMa2, several polymorphic and unlinked loci should be used for a robust estimation of parameters and to deal with phylogenetic uncertainty S. striatus and T. atra. Both species showed a greater contribution from the southern to the northern region. Likely these two species have been able to achieve a low yet significant connectivity between biogeographic regions (probably enhanced by the Humboldt Current), slightly eroding the historic signature, which is anyhow strong in both species. Concordantly, shared haplotypes of S. striatus and T. atra between biogeographic regions are best explained by a combination of migration, shared ancestral polymorphism and drift. For the northeast Pacific coast, Wares et al.Perumytilus purpuratusMazzaella laminarioidesMigration estimations suggested low yet significant northward migration across the 30°S break in two of the four species that had genetic structure among biogeographic regions, O. tuberculata and S. scurra) and biogeographic regions (the north for S. striatus and the south for T. atra) may have stronger effects of genetic drift. Concomitantly, biogeographic regions carry within their structure the signatures of historic effective population size, low migration and historic isolation, which probably leads to the significant interaction of dispersal potential and biogeographic region in models associated to nucleotide diversity and genetic differentiation between low and high dispersers.Consistently dispersal potential was the variable that always best explained the contrasting genetic structure of low and high dispersers. Genetic structuring measures that are more associated with genetic divergence , not only were determined by dispersal potential but also significantly by its interaction with biogeographic region. Based on isolation-with-migration analyses, the biogeographic regions have been completely or partially isolated in recent times. Isolation has led to differentiation, which in some cases is likely to have been enhanced by low effective population size. Genetic diversity was estimated to be unequal between biogeographic regions and species, such that some species  is based on reports that show that PLD is short and very conserved in the Patellogastropoda Tegula have conserved PLD’s of 6 to 7 days T. atra.Amphipods, as all peracarids, brood their embryos up to a crawl-away stage. Thus, E. analogaT. nigerH. helianthus can persist for at least 2 months without reaching metamorphosis. The New Zealand starfish Stichaster australis, the only congener of S. striatus from the southeast Pacific coast, has larvae that spend 38 days in the water column S. striatus; albeit it is possible this extrapolation is wrong given that PLD is highly variable in asteroids  to Chiloé (42°S) along the coast of Chile. The intertidal areas are not privately owned in Chile, however, there are areas of restricted access (management or protected areas) where we did not sample. All sampling was performed in free access areas for which no permit was required. Also no permits were required for the sampling as species studied did not involve commercially important species or species endangered or protected.H. helianthus and S. striatus) and the urchin (T. niger), ambulacral feet and gonad tissue were sampled, respectively. Tissues were placed in absolute ethanol and stored at −20°C. Total DNA was extracted using standard phenol/chloroform protocol Organisms were processed a few hours after being extracted from nature, obtaining from them a piece of tissue. In most cases tissues corresponded to skeletal muscle. From starfishes  and ArbaR (5′TATACTTCAGGGTGTCCAAAGAATCA′3). These latter primers were designed for this study in GENEIOUS 5.5.4 2, 0.4 µM of each primer, 0.22 mM of each dNTP, 1.5 mg ml−1 of bovine serum albumin, and 1.5 U of Taq polymerase. Cycling conditions consisted of an initial denaturation at 94°C for 10 min followed by 35 cycles of 1 min denaturing, 1 min annealing at 50°C, and a 2 min extension at 72°C.A partial sequence of COI was obtained using the universal HCO and LCO primers nn statistic nn or the nearest neighbor statistic For each species, sequences were aligned and then truncated on both extremes using GENEIOUS. DnaSP 5.0 ST) were calculated in ARLEQUIN 3.5 nn and ΦST.Pairwise and global population genetic differentiation values based on sequence data (ΦST among and within regions). All models were constructed nesting the species as a random factor. The syntax of the diversity models was: Genetic Diversity = Dispersal X Biogeographic Region+Species . For the differentiation model the syntax was: Genetic Differentiation = Dispersal X Biogeographic Differentiation+Species .To test the influence of dispersal capacity and the 30°S transition zone on the genetic diversity and differentiation Generalized Lineal Mixed Models (GLMMs) were performed using the package lme4 in R. These models are appropriate for data influenced both fixed and random effects. In the models population-specific data of genetic diversity were grouped into biogeographic regions (north and south of 30°S). For models of genetic differentiation data was grouped according to biogeographic differentiation . To evaluate the presence of a genetic discontinuity coincident with the biogeographic break at 30°S, ARLEQUIN software was used to perform AMOVA by grouping individuals CT). The number of groups was denoted as k. In each species, statistical variance was evaluated with 1000 random permutations running repeatedly SAMOVA from 2 to 1-n populations, where n was the total number of sampled localities. The k value that maximized FCT was interpreted as the most likely geographic subdivision. A genetic break coincident with the biogeographic transition was inferred if when k = 2 the clustering FCT was significant and the groups of individuals were located north and south of 30°S.Two further analyses were conducted for species that displayed genetic structure with AMOVA, i.e., those that had less than 95% of the variation within populations. The clustering method implemented by SAMOVA 1.0 t (splitting time), effective population sizes (Θ = 2 Ne), and migration rates in each direction across the biogeographic break. For each analyzed species, we generated a random balanced subsample of individuals per biogeographic region with a double goal of having equal number of individuals per biogeographic region and reducing the number of sequences analyzed, which is recommended when using the model of isolation-with-migration with information from a single locus 3) algorithm The second analytical approach applied only on species with structure across 30°S, consisted in the evaluation of asymmetric migration and time of divergence across 30°S using the isolation-with-migration demographic model


Rhinella marina) within their invasive Australian range. Metamorph body size is highly plastic in this species, and our laboratory studies showed that larger metamorphs had better locomotor performance (both on land and in the water), and were more adept at catching and consuming prey. In mark-recapture trials in outdoor enclosures, larger body size enhanced metamorph survival and growth rate under some seasonal conditions. Larger metamorphs maintained their size advantage over smaller siblings for at least a month. Our data support the critical but rarely-tested assumption that all else being equal, larger body size at metamorphosis is likely to enhance an individual’s long term viability. Thus, manipulations to reduce body size at metamorphosis in cane toads may help to reduce the ecological impact of this invasive species.Body size at metamorphosis is a key trait in species (such as many anurans) with biphasic life-histories. Experimental studies have shown that metamorph size is highly plastic, depending upon larval density and environmental conditions . To test the hypothesis that this developmental plasticity is adaptive, or to determine if inducing plasticity can be used to control an invasive species, we need to know whether or not a metamorphosing anuran’s body size influences its subsequent viability. For logistical reasons, there are few data on this topic under field conditions. We studied cane toads ( An individual’s body size and mass can affect its viability in many ways; for example, a smaller body often may increase an individual’s vulnerability to mortality sources such as desiccation, predation, competition, starvation or infection Pseudacris triseriata by Smith Rana sylvatica by Berven Bufo terrestris by Beck and Congdon Rana lessonae and R. esculenta by Altwegg and Reyer Some of the best examples of developmental plasticity in body size come from studies on anuran amphibians with aquatic larval stages. The size at which a tadpole metamorphoses not only can trade-off against the duration of the larval period The fitness consequences of within-cohort variation in metamorph size are of interest from applied perspectives also. Many anuran species have been widely translocated by humans, and some have become invasive within their new ranges, with substantial ecological impacts on native taxa Although intuition suggests that larger body size at metamorphosis should be beneficial, that assumption may not always hold true and it has been rarely documented with field-based data, e.g. Rhinella marina) in the Australian wet-dry tropics. Toad invasion has had major ecological impacts on native predators, stimulating a search for novel methods of toad control In the current study, we build upon extensive research on invasive cane toads (Rhinella marina) and with native frog species that are not endangered. Two egg clutches of cane toads (R. marina) and egg clutches of the native frogs were collected by hand from natural ponds. Another six cane toad egg clutches were obtained by injecting hand-collected adult toads  with 0.25 mg/mL of leuprorelin acetate. No animals were killed. All procedures were approved by the University of Sydney Animal Ethics Committee (Protocol L04/6-2010/3/5333).The study was performed at the Tropical Ecology Research Facility (University of Sydney) located at Middle Point, Northern Territory, Australia (see below for further details). The Facility does not belong to a National Park or other protected area of land. Permission for research on amphibian species at this location was granted by the Parks and Wildlife Commission of the Northern Territory (permit numbers 36597 and 40463) and the University of Sydney Animal Ethics Committee (Protocol L04/6-2010/3/5333). The study was conducted with an invasive species , native to a wide area of the Americas , was introduced to north-eastern Australia in 1935 as a biological control agent of insect pests of sugar cane The cane toad  was first colonized by cane toads in 2005 R. marina and one of the native ornate burrowing frog . We reared cane toad tadpoles at two densities: (1) 10 R. marina tadpoles per container, (2) 10 R. marina tadpoles plus 10 O. ornatus tadpoles per container. For the second trial (December 2011), we obtained one cane toad clutch by injecting local field-collected adults  with 0.25 mg/mL of leuprorelin acetate , to induce amplexus and spawning. The resulting tadpoles were allocated to two density treatments: 10 or 20 R. marina per tub. Each rearing trial was a randomized block design with four replicates per treatment.We reared toad tadpoles at different densities in two trials to produce toad metamorphs of different body sizes. Tadpoles were reared in plastic bins (60×40×40 cm), each filled with 65 L of bore water and a substrate of dirt collected from natural temporary ponds. We initially added 1–1.5 g of crushed algae wafers  to each bin as a food source for tadpoles. For the first trial (November 2010), we obtained two naturally-spawned clutches from local waterbodies: one of We checked the containers daily and collected individuals with emerged forelimbs over a two-week period after the first metamorph had emerged. We tested a total of 60 metamorphs: 36 from the first larval trial and 24 from the second one. These animals were kept in 1 L rectangular plastic containers with a small amount of water. On the day metamorphosis was complete , we conducted performance trials between 1000 and 1400 h  in a shaded building at the University of Sydney Tropical Ecology Research Facility, Middle Point . For each individual, we tested jumping performance on the concrete floor by touching the urostyle of the metamorph with a wooden stick (50 cm length) to induce it to jump. We recorded the distance of each of the first three jumps (measured with a ruler from the damp “prints” produced by the metamorph). After 5 minutes rest in its container, we tested the individual’s swimming performance in a water-filled trough . We recorded the total distance swum by the metamorph in 2 minutes. After the swimming trial, we waited another 5 minutes before evaluating feeding performance. We placed the metamorph plus 30 live termites in a plastic container (34×22×10 cm) on a layer of wet dirt, and recorded the number of termites eaten during the next 5 minutes. We measured the snout-urostyle length (SUL) and weight of each metamorph with electronic digital calipers and an electronic balance  at the completion of the performance and feeding trials. We did not measure metamorphs prior to these trials because pilot studies showed that handling can induce abnormal behaviour. These pilot studies also showed that metamorphs that ate up to six termites increased in mass by <0.05%; therefore, the prey consumed during a trial  had a negligible effect on their mass.R. marina per container, or 20 R. marina per container, or 10 R. marina plus 10 tadpoles of a native species. We used eight species of native tadpoles in these trials . Most of the smaller metamorphs were from treatments where toad tadpoles were exposed to native tadpoles or to high densities of conspecific tadpoles, whereas the larger metamorphs came from treatments with less larval competition. The metamorphs used in the growth and survival trials emerged from rearing tubs over an eight-day period. As metamorphs emerged, they were collected and kept in 1 L rectangular plastic containers with a little water and fed with termites three times weekly for one or two weeks depending on their dates of emergence.We performed three mark-recapture trials to assess the consequences of body size at metamorphosis on growth and survival. Metamorphs were obtained from laboratory experiments on larval competition. The experiments were performed in 65 L or 15 L plastic bins, with natural substrata and crushed vegetable wafers as larval food. One experiment was conducted in May 2009 (“Experiment 1”) and one in January 2010 (“Experiment 3”). We used a total of four and eight treatments respectively, with either 10 larval Crinia sp., Limnodynastes convexiusculus, Litoria australis, L. bicolor, L. dahlii, L. nasuta, L. rothii, L. rubella), as well as by other potential prey, competitors, and predators . Piles of dry grass 10 cm from the water of each pond provided pondside refuges for the metamorphs.The three mark-recapture trials were performed from June to August 2009 , October to November 2009 , and February to August 2010 . In each trial, metamorphs were released into three large (15×4 m) adjacent open-topped outdoor enclosures with similar conditions and resources to those present in and around nearby natural waterbodies. The walls of these enclosures were formed by metal sheets (0.8 m high) and were located within the grounds of the Tropical Ecology Research Facility. Each enclosure housed a pond (2.5×1.8×0.6 m) that contained, and was surrounded by, natural vegetation. The ponds and enclosures had been constructed 4 years prior to our trials, and had already been colonized by native anurans  and mass of each metamorph. Body size was recorded with electronic digital calipers and mass was measured with an electronic balance . The range of body sizes was 7.4–13.5 mm SUL, and 0.03–0.25 g body mass. For Trial 1, we individually marked 63 toads by toe clipping  and these individuals were released around the ponds into the field enclosures the following day. Each of the enclosures was allocated one-third of the animals . We followed the same methodology for Trials 2 and 3, with 94 and 148 metamorph toads respectively. Every 2 weeks we exhaustively searched for the metamorph and juvenile toads, during both day and night and collected them to record individual identity, SUL, and mass before returning them to their enclosures the following day. The duration of each of the three trials differed, dependent upon mortality rates. The number of recaptured metamorphs declined rapidly during the first two trials, but not in Trial 3; thus the duration in the last trial was greater . Records of air temperature and rainfall during the mark-recapture period were obtained from an automated weather station located <5 km from the enclosures.P>0.05), and a marginally significant effect of trial # for jumping performance . Thus we combined the results obtained in both trials and used linear regression to compare the performance of metamorphs of different sizes, with metamorph mass as the independent variable and mean distance jumped, total distance swam, and total number of termites consumed as response variables. We compared the average rainfall (ln –transformed) and air temperature among trials data using ANOVA.In a preliminary covariance analysis with metamorph mass as a covariate and trial # as the factor, we found no significant effects of trial # on swimming or feeding performance . We used multiple regression to assess the effects of initial mass, climate variables and Trial # on growth rates and included toad ID (identity) as a random effect. We also repeated the analysis using larval treatment group as an independent variable, instead of initial body mass, to determine which variable better explained growth rate variation. Models containing both initial mass and larval treatment groups were over-parameterized or too unbalanced to run. To assess whether the growth advantage conferred by larger initial size was retained over time, for each Trial we carried out an ANCOVA with growth rate as the independent variable and with initial mass as the covariate and recapture period as a factor. For all three trials the first recapture period occurred after 16–20 days, the second after 30–34 days, the third after 40–51 days, and, for Trial 3, a final recapture took place after 185 days. Because some individuals were captured during more than one period, we included ID as a random effect in these ANCOVAs. All analyses were performed using JMP 9 software .Survival was estimated using the Cormack-Jolly-Seber model for mark-recapture experiments with MARK 6.0 software R2 = 0.737, F1,58 = 163.33, P<0.0001; R2 = 0.608, F1,58 = 90.25, P<0.0001; R2 = 0.467, F1,58 = 50.88, P<0.0001; Larger metamorph toads jumped further than did their smaller siblings , with 30 of them recaptured on Day 15, 17 on Day 30, 12 on Day 51 and 8 on Day 65. Of the 94 metamorphs released in Trial 2, 35 were recaptured on Day 16, 14 on Day 30 and 4 on Day 40. In Trial 3, 148 metamorphs were released, 33 were recaptured on Day 10, 19 on Day 20, 19 on Day 34, 13 on Day 48, 30 on Day 134, and 28 on Day 185. Some of the individuals in this last trial were recaptured as juveniles (from 30–90 mm SUL), based on the classification of F2,285 = 68.16, P<0.0001). Trial 2 was the warmest (30.1°C) followed by Trial 3 (27.1°C), and Trial 1 was the coolest (24.4°C). Rainfall also varied significantly among trials . Trial 1 was conducted during the dry season and experienced only 0.2 mm of cumulative rainfall. Trial 2 (conducted immediately prior to the wet season) experienced a total of 83.6 mm rainfall while during Trial 3 (conducted on the wet-season), the study site experienced 338.2 mm rainfall.Average air temperatures differed significantly among the three trials  than toads in Trial 1  and Trial 2 . In these latter two trials, some young toads in the outdoor enclosures failed to grow during their first month post-release, and a few decreased in mass over this period. At Day 30, toads in Trial 1 had, on average, not gained any mass, toads in Trial 2 were twice their initial mass, and toads in Trial 3 were 20 times heavier than at the time of release .F2,193 = 4.21, P = 0.016). This interaction indicated that the effects of temperature on growth differed among the three trials; therefore we carried out subsequent analyses on each trial separately. In Trial 1, there were no significant effects of initial mass, air temperate or rainfall on growth rate . When larval treatment group was substituted into the model in place of initial mass, it was non-significant also .An initial model, testing the effects of Trial #, initial mass, air temperature and rainfall on growth rates revealed a significant interaction between Trial # and air temperature  and air temperature  but the effect of rainfall was marginally non-significant . When larval treatment group was substituted into this analysis in place of initial mass, it was significant , but had much less explanatory power than did initial mass.During Trial 2, growth rate was affected by initial mass , temperature  and rainfall . The effect of larval treatment group was non-significant  when it replaced initial mass in the analysis.In Trial 3, growth rate increased with initial mass , being lowest during the first period, intermediate during the second, and highest during the third period. However, growth rate was not significantly related to initial mass  nor was there an interaction between initial mass and period   indicating that the slope of the relationship between initial mass and growth rate varied among the recapture periods. Inspection of the interaction plot . Inspection of the interaction plot indicated that the relationship between growth rate and initial mass was steep during the first two periods, but levelled off during the third and fourth recapture periods  . During ion plot  revealed periods .We fitted data separately for each trial. For Trial 1, the survival probability of a juvenile toad increased with its initial mass, as indicated by the top three models in the mark-recapture analysis . Daily sIn parallel analyses using larval treatment as a grouping variable instead of initial mass, the best fitting models in which larval group affected survival had much higher AIC values than the best models constrained by initial mass .Our data support a commonly-made but rarely-tested assumption: that increased body size at metamorphosis in anurans is associated with traits that definitely  or plausibly  enhance individual fitness. Smaller body size also renders a young cane toad more vulnerable to desiccation Limnodynastes convexiusculus can actually decrease rather than increase survival, because larger maximum gape size increases the ability of a metamorph frog to ingest a  metamorph cane toad Our laboratory-based results showing performance advantages to larger body size at metamorphosis are unsurprising: an abundant literature demonstrates that larger size usually  confers performance advantages in a diverse array of functions in anurans such as metabolic rates and endurance, jumping ability , feeding  and escape from predators, e.g. Rhabdias.Our mark-recapture results provide the first robust evidence of survival and growth advantages to larger metamorph size in cane toads under semi-natural conditions. Although we monitored toad viability in outdoor enclosures rather than around natural ponds, the large size of our enclosures, and the diverse native fauna they contained (reflecting easy ingress and egress by most native fauna) facilitate extrapolation to the wild. The abiotic and biotic conditions inside our enclosures resembled those around nearby waterbodies in which cane toads frequently spawn, although natural ponds are likely to be inhabited by more predators including a wider size range of toads, snakes, wading birds and parasites such as lungworms in the genus Our results highlight the sensitivity of toad growth rates to seasonally varying conditions. During the wet-season  when food and water were abundant, the young toads grew very quickly . In contrast, growth was negligible over the same period during the dry-season . Growth rate was positively related to rainfall and air temperature during Trial 3. Moist substrates during the wet-season not only remove hydration stress, but also allow the young toads to forage widely instead of remaining close to the pond edge where the density of conspecifics  is greater Under dry-season conditions when growth is slow or negligible, there is little opportunity for growth rates to be affected by other factors such as body size at metamorphosis. However, the more rapid growth seen during the wet-season allows abundant opportunity for us to detect other influences on rates of growth. In our field enclosures, low body mass at metamorphosis  continued to affect toad metamorph body size for at least a month post-metamorphosis in Trials 2 and 3. This advantage weakened through time, with no detectable effect of initial body mass on growth rates of larger juvenile toads. The reduction in effect may have several causes. For example, the effects of individually variable phenotypic traits that influence growth rate may become increasingly important relative to initial size. Also, high mortality rates of the smallest animals increasingly remove the lower tail of the body-size distribution, leaving only a subset of individuals that were all relatively large at metamorphosis. Within that relatively restricted size range, initial mass may cease to be a strong predictor of an individual’s subsequent growth trajectory.Scaphiopus bombifrons and salamanders Ambystoma macrodactylum, A. tigrinum nebulosum and Hynobius retardatus, where low food supply and thus hunger (or inducible defences in heterospecific prey) stimulates some individuals to switch their diets towards cannibalism Scaphiopus bombifrons and the salamanders noted above).Low growth rates under stressful dry-season conditions reduce the overall range of body sizes within a cohort of young toads, whereas favourable conditions result in a much wider size range. For example, at the end of one month of growth, young toads in Trial 1 weighed from 0.12–0.57 g (a range of 0.45 g) and from 0.13–1.22 g (range 1.09 g) in Trial 2, whereas young toads in Trial 3 weighed from 0.66–4.41 g (a range of 3.75 g). Asymmetry in the sizes of conspecifics increases the probability of cannibalism Litoria dahlii) around the ponds, strongly suggesting that they had ingested toxic metamorphic toads Odontomachus turneri) on marked toad metamorphs. Under some conditions ants can decimate cane toad populations, with smaller metamorph toads being more vulnerable to predatory ants than are larger conspecifics The link between growth rates and opportunities for cannibalism (above) is not the only mechanism creating a relationship between rates of survival and growth. A more direct relationship is that low feeding rates reduce both survival and growth. In Trials 1 and 3, we found that the smallest individuals at metamorphosis were unlikely to survive. Our data do not identify the factors causing mortality, but these likely involve interactions between predation, cannibalism, parasitism, desiccation and thermal variation Lastly, we consider the implications of our results for the idea that reducing toad size at metamorphosis may help to reduce rates of recruitment of this troublesome invasive species. Size reduction could be achieved by pheromonal means


Occupational exposure to wood dust is a strong risk factor for the development of intestinal-type sinonasal adenocarcinoma (ITAC); however, knowledge on possible precursor lesions or biomarkers is limited. Fifty-one samples of tumor-adjacent mucosa and 19 control samples of mucosa from the unaffected fossa of ITAC patients were evaluated for histological changes and p53 protein expression. Mild dysplasia was observed in 14%, cuboidal metaplasia in 57%, intestinal metaplasia in 8%, squamous metaplasia in 24%, and cylindrocellular hyperplasia in 53% of cases. P53 immunopositivity was generally weak occurring most frequently in squamous metaplasia. Wood dust etiology did not appear of influence on the histological changes, but p53 showed a tendency for higher positivity. Dysplasia adjacent to tumor was indicative of subsequent development of recurrence. In conclusion, precursor lesions do occur in mucosa adjacent to ITAC. This is clinically important, because it may justify the screening of high-risk individuals such as woodworkers. Intestinal-type sinonasal adenocarcinomas (ITACs) are epithelial tumors of the nasal cavities and paranasal sinuses, often related to professional exposure to wood dust. It is a rare tumor representing 8–25% of all malignant sinonasal tumors , 2. AccoIn the northern part of Spain, the incidence is 0.19 cases/100.000 inhabitants per year . It is lIn the clinic, ITAC often appear as indolent, slow-growing tumors with unspecific unilateral symptoms  normal to this site of origin, such as nasal obstruction, epistaxis, or rhinorrhea . FrequenPrevious reports have focussed on finding precursor lesions in series of sinonasal mucosa samples from persons at high risk of developing ITAC, that is, woodworkers. Aiming to find stronger indications, in this study we analyzed normal sinonasal mucosa of patients who have already developed ITAC. In addition to the histological evaluation, we studied p53 protein expression as a possible marker for early neoplastic transformation.Fifty-one paraffin tissue samples were taken from mucosa adjacent to the tumor, with presence of both tumor and normal tissues in the same tissue block. Nineteen control samples were obtained from the healthy, unaffected fossa of patients with ITAC. In 9 cases we obtained a sample from both adjacent tissue and the other fossa. All samples were collected from previously untreated patients seen between 1990 and 2009. Informed consent was obtained from all patients, and the study was approved by the ethical committee of our institute. Of the 51 cases, 1 patient was female and 50 male, with a mean age of 65 years (45–92). Forty-five patients had occupational exposure to wood dust with a median of 35 years (range: 4–55 years), and 27 were tobacco and alcohol users. Fifteen tumors were stage I, five stage II, seventeen stage III, eight stage IVa, and six stage IVb. No patient had metastases at the time of diagnosis. According to the WHO histological classification [Of the 19 controls, 2 patients were female and 17 male, with a mean age of 69 years (45–78). Seventeen had occupational exposure to wood dust with a median of 35 years (range: 1–50 years), and 9 were tobacco and alcohol users.H&E stained paraffin sections were grouped as: (1) mucosa adjacent to tumor and (2) control mucosa from the other unaffected fossa of a patients with ITAC. The surface epithelium of all samples was evaluated for the presence of respiratory epithelium, dysplasia, cuboid metaplasia, squamous metaplasia, and cylindrocellular hyperplasia . Intestinal metaplasia was evaluated with the help of cytokeratin 20 immunostaining. The seromucinous glands were analyzed for signs of dysplasia and mucosecretory hyperplasia.μm paraffin embedded sections with the antibodies anti-p53 clone DO-7 and cytokeratin 20 clone K.208  using an automatic staining workstation  with the Envision system and diaminobenzidine chromogen as substrate. P53 expression was evaluated both in surface epithelium and in seromucinous glands and scored in 4 categories: 0–10%, 10–25%, 25–50%, and 50–100%.Immunohistochemistry was performed on 4 P  values below  .05 were considered significant.Possible correlations were statistically analyzed by SPSS 12.0 software for Windows , using the Fisher Exact and Pearson Chi2 test. Kaplan-Meier analysis was performed for estimation of survival, comparing distributions of survival through the logarithmic range test (log-rank test). In 6 of 51 (12%) samples, no histological abnormality was observed in the mucosa adjacent to ITAC. Of these 6 cases, 3 ITACs were stage T1, 1 T2, 1 T3, and 1 T4b and concerned 3 colonic and 3 mucinous type tumors. Over half of the cases (24/45) showed more than one histological abnormality. P = .029). The 4 cases with CK20 immunopositivity in the surface mucosa also showed positivity in the seromucous glands , cuboid metaplasia in 29 (64%), squamous metaplasia in 12 (27%), and cylindrocellular hyperplasia in 27 (60%) cases . CK20 ims glands . Two pubIn normal mucosa of the unaffected fossa of ITAC patients, we found 9 of 19 (47%) cases without abnormalities and in general much lower frequencies of histological changes than in the mucosa adjacent to tumor . This isP = .261). In addition, all 4 patients with CK20 immunopositivity  in the mucosa adjacent to tumor had a tumor recurrence. Neither of the histological aberrations were related to overall or disease-free survival. To our knowledge, the relation between tumor and clinical characteristics and histological changes in mucosa adjacent to the tumor has not been studied previously in the literature.Although the number of cases without wood dust etiology was very low, our data suggest that wood dust exposure does not cause specific histological changes, except perhaps for cylindrocellular hyperplasia. This is confirmed by Wolf et al. who studied a large series of samples . On the P = .923). It may be speculated that the relatively low level of p53 positivity in the mucosa adjacent to tumor does not indicate TP53 gene mutation, but rather an upregulation of functional p53 in response to inflammatory signals in the wood dust-exposed sinonasal epithelium.In general, p53 positivity was weak and occurred in a low percentage of cells , in the Our data, summarized in When analyzing the p53 results in relation to the adjacent histological tumor type, we noted a higher expression in mucosa adjacent mucinous type ITAC . Howeverμm become trapped in the mucosa of the middle turbinate and ethmoid [ITAC represents an important occupational health problem with serious consequences, needing better ways of prevention, early diagnosis, and treatment. Despite a clear etiology, it is still unknown how they develop. The present model suggests that inhaled wood dust particles larger than 5  ethmoid , 7 and w ethmoid  showed t ethmoid . It willEpithelial cancers are frequently preceded and accompanied by precursor changes in the tissue histology, of which dysplasia is generally believed to be a true premalignant lesion. In previous studies on normal mucosa of woodworkers, dysplasia was found in around 10% of cases –16. In oWhatever the tumor-initiating causes, cancer arises through a multistep sequence in which histological changes are accompanied by the accumulation of genetic aberrations. The analysis of genetic or epigenetic aberrations in these precursor lesions will give a more definitive insight in their possible role in ITAC development.


Aim. To evaluate the effect on physical properties of Mineral Trioxide Aggregate (MTA) of using direct hand compaction during placement and when using hand compaction with indirect ultrasonic activation with different application times. Methods. One hundred acrylic canals were obturated in 3 increments with MTA in sample sizes of 10. One group was obturated by hand with an endodontic plugger and the remainder obturated with indirect ultrasonic application, with times ranging from 2 seconds to 18 seconds per increment. Microhardness values, dye penetration depths, and radiographs of the samples were evaluated. Results. As ultrasonic application time per increment increased, microhardness values fell significantly (P < 0.001) while dye penetration values increased (P < 0.001). Microhardness of MTA ultrasonicated for 2 seconds was significantly higher than hand compaction (P = 0.03). Most radiographic voids were visible in the hand-compacted group (P < 0.001), which also had higher dye penetration depths than the 2-second ultrasonicated samples. Ultrasonication of MTA for 10–18 seconds resulted in significantly more voids than 2–8 seconds of ultrasonication (P = 0.02). Conclusion. The use of ultrasonics with MTA improved the compaction and flow of MTA, but excessive ultrasonication adversely affected MTA properties. A time of 2 seconds of ultrasonication per increment presented the best compromise between microhardness values, dye penetration depths, and lack of radiographic voids. MTA has grown in popularity as a dental material because of its largely favourable properties, including tissue biocompatibility, superior sealing ability, and its ability to promote dental pulp and periradicular tissue healing . HoweverUltrasonics in dentistry has a wide range of applications and has arguably improved treatment outcomes and predictability . UltrasoThese different and contradictory findings may be the result of different times used to ultrasonically compact MTA. Therefore, the aim of this study was to evaluate the effect of different ultrasonic application times on the density, compressive strength, and radiopacity of MTA.n = 10) were obturated with MTA without ultrasonic activation. ProRoot MTA  was mixed according to manufacturer's instructions with the supplied sterile water in a 3 : 1 powder/liquid ratio [Twenty uniform acrylic blocks of five canals each were prepared, creating 100 samples. Each simulated canal measured 1 mm in diameter by 6 mm in length. The canals in two of these blocks (id ratio  and handn = 90) were obturated with indirect ultrasonic activation against the endodontic plugger. Samples 11–20 were compacted with 2 seconds of ultrasonic activation, samples 21–30 for 4 seconds, samples 31–40 for 6 seconds, and so forth until samples 91–100, which were ultrasonically activated for 18 seconds. These samples were also filled in three increments, with an ultrasonic tip held lightly against the plugger at each increment for the desired time period. The Cavi 3D tip of the VDW Ultrasonic unit  was used at the middle setting. The MTA fillings were flush with the surface of the acrylic block, and all blocks were placed into petri dishes under cotton pellets soaked with room temperature distilled water. All blocks were then left in an incubator at 37°C for 24 hours at 95% humidity.All of the other samples  was set at 70 kV, a current of 7 mA, and an exposure time of 0.25 seconds. These radiographs were then examined for voids, which presented as clearly demarcated radiolucent areas within the specimens. The number of voids, when present, was counted in each specimen.The Vickers microhardness was evaluated for the 100 samples. After 24 hours, all the blocks were lightly lapped with 400-grit fine sandpaper to produce a smooth surface, and one cement surface per canal was loaded with one Newton for six seconds, with a slope of 0.99. This resulted in a stamp indent on the MTA surface, with an impression of two orthogonal diagonals. An image of this was brought into focus using the computer and captured immediately after discharge of the diamond indenter. The microhardness of each sample was evaluated using standard calculations .The samples were left in a sealed container for an additional 24 hours after the microhardness testing and immersed in a 0.2% Rhodamine Blue solution  for 72 hours in the incubator and then visually assessed for dye penetration. Each of the 100 specimens was examined under a light microscope with 4x magnification and a Michigan “O” cc periodontal probe  was used to measure the depth of dye penetration. The periodontal probe was held against each specimen and measured to the nearest 0.5 mm.t-test, Regression analysis, and Fisher's exact test with significance set at P < 0.05.Statistical analysis included Pearson Correlations, two-sample r = −0.707, where r is the correlation coefficient; P < 0.001). A two-sample t-test indicated a significant difference (P = 0.03) between hand compaction and two-second ultrasonication. As ultrasonication time increased, the dye penetration of the MTA samples . As dye penetration increased (P = 0.001).Evaluation of the microhardness  showed a samples  also incncreased , a correP < 0.001) of visible voids were noted in the hand-compacted group (90%) than in the ultrasonicated groups overall (22%). Grouping and dichotomising of the ultrasonication groups only indicated a significantly lower proportion (P = 0.02) of groups with voids when ultrasonicated for 2–8 seconds (10%) than 10–18 (54%) seconds.When viewing the radiographs taken of the acrylic blocks, a significantly higher proportion (Hydration of the MTA powder results in a colloidal gel that solidifies to a hard structure in less than three hours, which then continues to set . The basIn another comparison of hand and ultrasonic compaction  ultrasonThe Vickers microhardness test used in this study aimed to quantify the resistance of MTA to plastic deformation. While the compressive strength helps to indicate the setting and hydration reaction progress , 20, micThe method used for the dye penetration study was adapted from that of Bortoluzzi et al.  and aimeOverall, the results indicated that an ultrasonication time of 2 seconds was better than hand compaction. The higher depth of dye penetration and the higher incidence of radiographic voids amongst the hand-compacted samples indicate that the use of indirect ultrasonic activation is beneficial. It is difficult to directly compare the results of the present study with the available literature because they differ in too many aspects, including type of MTA, the assessment method, length of MTA fill, type of canal used, canal dimensions, ultrasonication time, and ultrasonic unit used. Parameters measured also differ and include bacterial leakage , push-ouThe use of ultrasonics with MTA was useful in improving flow and compaction of MTA, but excessive ultrasonication can adversely impact MTA properties. A suggested time of 2 seconds of ultrasonication per increment presented the best compromise between microhardness values, dye penetration depths, and lack of radiographic voids.


Micrasterias. We applied flow-cytometric techniques of DNA quantification to microalgae and mapped the estimated DNA content along the phylogenetic tree. Correlations between DNA content and cell morphometric parameters were also tested using geometric morphometrics. In total, DNA content was successfully determined for 34 strains of the genus Micrasterias. The estimated absolute 2C nuclear DNA amount ranged from 2.1 to 64.7 pg; intraspecific variation being 17.4–30.7 pg in M. truncata and 32.0–64.7 pg in M. rotata. There were significant differences between DNA contents of related species. We found strong correlation between the absolute nuclear DNA content and chromosome numbers and significant positive correlation between the DNA content and both cell size and number of terminal lobes. Moreover, the results showed the importance of cell/life cycle studies for interpretation of DNA content measurements in microalgae.It is now clear that whole genome duplications have occurred in all eukaryotic evolutionary lineages, and that the vast majority of flowering plants have experienced polyploidisation in their evolutionary history. However, study of genome size variation in microalgae lags behind that of higher plants and seaweeds. In this study, we have addressed the question whether microalgal phylogeny is associated with DNA content variation in order to evaluate the evolutionary significance of polyploidy in the model genus  Closterium ehrenbergiiC. ehrenbergii producing 2 zygotes per pairing cells, other genera  are producing 1 zygote per pair of sexualized cells Streptophycean green algae are a sister group to land plants Cyperaceae and Juncaceae) and in the genera Drosera, Myristica, Cuscuta and ChionographisCaenorhabditis elegans. By their nature, holocentric chromosomes have high potential for rapid chromosome rearrangements as chromosome fragments can undergo both normal meiosis and mitosis. For this reason, holocentric organisms are characterised by enormous diversity in both number and size of chromosomes. Chromosome number in desmids ranges from 14 to 592 Cytogenetic research on Desmids is intriguing due to the presence of holocentric (holokinetic) chromosomes Micrasterias species have been produced by Waris and Kallio Micrasterias species in which these conditions were induced Polyploidization, i.e. whole genome duplication (WGD), is considered one of a major evolutionary process in higher plants In higher plants, detection of polyploid taxa within polyploid series became a routine procedure with the development of DNA flow-cytometry (FC) using several different protocols Micrasterias, a model genus of desmids , includes about 60 morphospecies and its cryptic diversity, phylogeny and biogeography have recently been reported Micrasterias taxa, the genus comprises at least eight lineages Recent taxonomic research on microalgae suggests that traditional species/genera boundaries based largely on cell morphology have underestimated the real species diversity Micrasterias species based on chromosome morphology. The first group have small chromosomes with a tendency to aggregation and includes mostly representatives of clade A  assessment of overall DNA content variation; 2) recognition of the significance of DNA content in the evolution at generic and species levels, and 3) the correlations between DNA content and selected cell morphometric parameters.In this study, we asked whether the phylogeny of the genus No specific permits were required for the described field studies. No specific permission was required for any locations and activity. The locations are not privately owned or protected in any way. No activity during field study involved any endangered species or protected species.−2. s−1 with 12∶12 light:dark cycle (cooling box Helkama C5G). Subsequently, two weeks before planned flow cytometric measurements, a rich inoculum of each strain (ca 1 ml) was transferred to fresh medium in a 100 mm Petri dishes and kept at a higher irradiation (40 ìmol. m−2. s−1) with 16∶8 light:dark cycle. All cultures were regularly examined under inverted microscope (Zeiss Axiovert), to check their fitness, cell density and estimation of cell division intensity. Well grown cultures were used for cytometric measurements in exponential phase of growth (14–18 days after subculturing), few slow growing cultures were maintained longer to obtain sufficient cell density. Although the cell density was priority, intensively dividing cultures were preferred because their cell walls were more sensitive to enzymatic disintegration The strains we used were obtained from five public culture collections: Sammlung von Conjugaten-Kulturen, University of Hamburg (SVCK); Culture Collection of Algae, Charles University in Prague (CAUP); Culture Collection of Algae, University of Vienna (ASW), currently deposited in the Culture Collection of Algae at the University of Cologne (CCAC); Culture Collection of Algae, University of Göttingen (SAG); and Microbial Culture collection, National Institute for Environmental Studies, Tsukuba (NIES). Some strains come from the personal collection of Jiří Neustupa Raphanus sativus cv. Saxa , 0.5% Macerozyme R–10  dissolved in modified rinsing solution PGly Micrasterias are haploid in vegetative stage is based on the fact, that all published and observed Micrasterias species are producing 1 zygote per two pairing cells Closterium ehrenbergiiAll desmid strains were measured at least twice, usually three times, except of strain CAUP K604 with unsufficient amount of desmid cells (extremely slow growth). Altogether 3000 particles were measured . Each of the repetitive measurements of the same strain was independent . Thus the possibilities of errors in DNA measurements were minimized. As the majority of cultures represented proliferating cultures with sufficient amount of dividing cells (synchronized by photoperiod), the histogram of relative nuclear DNA content usually shows 2 peaks – first peak represents vegetative haploid cells (1C), second peak (2C) represent cells in G2 mitosis phase . The preM. ‘pusilla’ (NIES 783) and M. radians var. evoluta (SVCK 519) strains with those molecularly characterized by Škaloud et al. M. rotata and M. truncata strains having identical SSU rDNA sequences with those included in the alignment (The phylogenetic tree used to map the character of the evolution was created using Bayesian inference (BI), MrBayes, version 3.1 lignment  were addhttp://www.r-project.org/. Accessed 2013 Dec 19). The output from R was mapped onto the Bayesian phylogenetic tree with TreeExtender v1.03 The existence of a phylogenetic signal in DNA content variation was tested by calculating Pagel's lambda M. rotata strains. The relationship between DNA content and selected cell morphometric parameters  was evaluated by linear correlation analyses in PAST, ver. 2.01 M. rotata subset precisely, we determined the centroid size (CS) of the cells per each strain by a landmark-based shape analysis.Correlation analyses were conducted separately on 1) a dataset of all taxa for which the cytometric data were obtained, and 2) a subset of five Centroid size, a widely used dimension-free size measure is linearly correlated with traditional univariate cell size measures of desmids, such as cell length or width M. rotata strain, 46–51 randomly chosen cells were photographed. In total, we defined 40 landmarks spanning the outline shape of the cells .For each Micrasterias species, including the phylogenetically nested species Triploceras gracile , suggesting a strong correlation between absolute nuclear DNA content and chromosome number.To test the correlation between the estimated DNA content values and real number of chromosomes, we compared our measured values with the chromosome data published by Kasprik  studies  and 3. A studies , a lineaMicrasterias, we mapped the estimated DNA content values along the phylogenetic tree. A phylogram was constructed by the Bayesian inference method, based on the concatenated SSU rDNA, psaA, and coxIII alignment published by Škaloud et al. Micrasterias species with known sequence data. The topology of the resulting phylogenetic tree was highly congruent with the phylogram constructed by Škaloud et al. To better understand the evolutionary history of DNA content changeover during the diversification of the genus M. rotata strains forming the clade C tended to have much higher amounts of DNA. On the other hand, clear differences were found in some closely related strains, indicating potential rapid evolutionary dynamics in the genome size. For example, two Triploceras gracile strains differed six times in their 2C nuclear DNA content . Similarly, M. crux-melitensis (14.3 pg) and M. radians var. bogoriensis (6.6 pg) had 2C DNA content lower than the closely related M. ceratofera (39.2 pg).The existence of a phylogenetic signal in nuclear DNA content was tested using Pagel's lambda calculations. The related species were significantly different in their DNA content . However, the existence of a phylogenetic signal was revealed by the maximum likelihood reconstruction of the ancestral states . WhereasIn addition to analysis of the evolutionary significance of the DNA content variation, correlations between the genome size and selected morphometric characteristics were determined. Statistical relationships, described by correlation coefficients between the estimated DNA content and cell length, cell complexity and number of terminal lobes, are presented in M. rotata strains having identical SSU rDNA sequences. Strong positive correlation was found  with 1C = 0.065 pg equalling ca. 63.4 Mbp Arabidopsis thaliana (L.) Heynhold with five chromosomes only, is approximately only 1C = 157 bp large (0.16 pg) For Streptophyta, 2C nuclear DNA content range from 0.2 to 6.4 pg, excluding the highly polyploid Charales and Desmidiales, which have genome sizes of up to 14.8 and 46.8 pg, respectively. In general, nuclear DNA content is smaller in Zygnematales than in Desmidiales Micrasterias rotata, M. truncata, and Triploceras gracile  depend on stage of cell cycle Chlamydomonas represent different microalgal group and type of cell/life cycle, authors found that G1 peak is always observable and never represents less than 29%, because in each stage exist some cells, which are not dividing. The percentage of both peaks depend on degree of synchrony and division rate which both depend on complex of factors including culture conditions and cell size Histograms of well growing cultures in exponential phase of growth showed two peaks, first representing vegetative cells (G1), second representing dividing cells (G2). We believe, that for desmid genus The exact knowledge of cell cycle and degree of synchrony of each species/sample should lead to exact interpretation, but represent another complication which makes flow cytometry in microalgae extremely time consuming. Moreover, the lack of information on reproduction of the majority of microalgal species, complicate our interpretation of ploidy level. Vegetative cells (G1) can be haploid (some desmids) or diploid (e.g. diatoms), depending on their life histories.Genome size is an important species-specific characteristic in organisms with centromeric chromosomes and was extensively studied in past decade in order to describe its changes during the evolution of angiosperms Micrasterias. For example, clade G consists of strains with a narrow range of genome size variability. However, within other clades, clear differences were found in some closely related strains, indicating the potentially rapid evolutionary dynamics in the genome size. Even more, great variability in genome size was detected in different strains of a single species .Some studies on holocentric angiosperms Small genome size in angiosperms appears to be correlated with phenotypic characteristics such as rapid seedling establishment, short minimum generation times, reduced cost of reproduction and increased reproductive rate Micrasterias were found to be usually larger than that of haploids. Waris and Kallio Closterium, Cosmarium and Staurastrum were polyploid.Ploidy level in conjugating green algae may be of taxonomic significance as cell dimensions are considered to be diagnostic Micrasterias thomasiana. The basic chromosome number of this desmid is n = 39, but morphologically irregular variants contained n = 40, n = 46 or even n = 70 and 75. Some of the cells of this latter clone were inclined to develop more or less typical morphologies, except that they were significantly larger Cosmarium turpinii. Starr considered the production of large forms to be a response to increase in nuclear quantity; whereas the change in shape  he explained as a response to the increase in cell volume.Kasprik We can support partially this hypothesis by our data. In contrast to the number of terminal lobes variation, cell complexity was not significantly correlated with DNA content.Micrasterias and between DNA content and cell size and morphology in the species Micrasterias rotata.This is the first flow cytometric analysis of both interspecific and intraspecific DNA content variation within one microalgal genus. There was strong correlation between nuclear DNA content and chromosome number in strains of the genus Moreover, the results showed the importance of cell/life cycle studies for interpretation of DNA content measurements in microalgae.