ST between breeds was 22%. However, we failed to detect deeper levels of structure, probably consequence of a recent and single geographic origin of domestication together with a non-bifurcating process of breed formation, which were often derived from crosses between two or more breeds. Finally, we found evidence for intrabreed stratification that is associated with demographic and selective causes such as formation of strains, colour morphs within the same breed, or country/breeder of origin. These additional layers of population structure within breeds should be taken into account in future mapping studies.Over thousands of years humans changed the genetic and phenotypic composition of several organisms and in the process transformed wild species into domesticated forms. From this close association, domestic animals emerged as important models in biomedical and fundamental research, in addition to their intrinsic economical and cultural value. The domestic rabbit is no exception but few studies have investigated the impact of domestication on its genetic variability. In order to study patterns of genetic structure in domestic rabbits and to quantify the genetic diversity lost with the domestication process, we genotyped 45 microsatellites for 471 individuals belonging to 16 breeds and 13 wild localities. We found that both the initial domestication and the subsequent process of breed formation, when averaged across breeds, culminated in losses of ~20% of genetic diversity present in the ancestral wild population and domestic rabbits as a whole, respectively. Despite the short time elapsed since breed diversification we uncovered a well-defined structure in domestic rabbits where the F Oryctolagus cuniculus) is the sole progenitor of domestic rabbits and both the wild and domestic forms occur worldwide.The European rabbit  [e.g. hypertension, epilepsy, osteoporosis), making them valuable models in biomedical research [Genetic data supports a domestication origin in France and shows that domestic rabbits display a subset of the genetic variability found in the ulations ,7,11–15.ulations ,16. Howeanimals) ,17. Thesresearch .The genetic studies so far have focused on a small number of breeds –15 or geHere, by taking advantage of the availability of wild rabbit populations, we investigated the domestication history of rabbits and quantified the amount of genetic diversity lost with the initial domestication process as well as with the more recent process of breed formation. In addition, we assessed genetic relatedness and substructure among rabbit breeds.No ethical approval was deemed necessary for this study. The European rabbit is a game animal in all locations sampled and all tissue samples for the wild animals were donated by local associations of hunters, who held appropriate permits for hunting wild rabbit in season. The fieldwork did not involve any endangered or protected species. No experimental studies in live animals were performed in this study. The sampling of blood from domestic animals for DNA extraction was approved and performed at INRA  by specialised veterinaries, and in accordance with the national regulations for the use of animals in agriculture. No animals were purposely killed for this study.To obtain a representative sample of the genetic diversity captured with rabbit domestication we followed three criteria. First, given that historical records indicate that most modern breeds were derived from crosses among ancient breeds , we sampO. c. cuniculus from four localities in the northeastern part of the Iberian Peninsula and nine localities in France  and their genomic location is given on To determine levels and patterns of genetic variation we used a total of 45 microsatellites distributed throughout the rabbit genome . We choshttp://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) , water and genomic DNA. PCR products were separated by capillary electrophoresis using an ABI Prism 3130xl, automated sequencer and Genescan-500 LIZ as size standard . Genotypes were scored with the software GENEMAPPER 4.0 , followed by visual inspection and manual corrections whenever required. Microsatellite genotypes per marker and for each individual sample are available in Primer pairs were designed using the web-based version of Primer3plus (lus.cgi)  and screFor each microsatellite, programs MICRO-CHECKER version 2.2.3  and ARLEHe) and Fixation Index (FIS) were calculated using the program GENALEX 6.4.1 [Ar) and Private Allelic Richness (PAr) were estimated using the program HP-RARE 1.0 [ST [i.e. among breeds/localities, among individuals within breeds/localities, and within breeds/localities). Statistical significance of results was evaluated with 10,000 permutations.Sample size for each locus (n), number of alleles (Na), expected heterozygosity . The software MICROSATELLITE ANALYSER (MSA) version 4.05 [http://tree.bio.ed.ac.uk/software/figtree/).Phylogenetic trees of individuals were constructed using Chord-distances  and the ion 4.05  was usedion 4.05 ,38. Phylion 4.05 . Final tK  ranging from 1 to 30 were performed for a dataset composed of all domestic rabbits. We chose a broad interval of K to i) infer potential ancestral relations among breeds by exploring clustering patterns resulting from lower values of K, and ii) investigate the existence of potential substructure within breeds by focusing on higher values of K. Each run was carried out with 100,000 iterations following a “burn-in” period of 50,000 iterations. The analysis was performed using the admixture model and assuming that allele frequencies are correlated among populations. After the first analysis including all domestic animals, a second analysis was conducted independently for each breed in order to evaluate the existence of additional substructure within breeds. The most probable K was inferred using the method proposed by Evanno et al [K, using the LargeKGreedy algorithm and 10000 random input sequences. Consistency among runs assuming the same value of K was evaluated using a similarity coefficient measure (C) as described by Rosenberg [Genetic structure within the domesticated population was investigated using two distinct approaches. First, we applied the Bayesian clustering procedure implemented in STRUCTURE, version 2.3.3 . Five inno et al . This esno et al  The grapno et al . CLUMPP no et al  was usedosenberg  and calce.g. Principal Component Analysis–PCA, or Factorial Correspondence Analysis–FCA), DAPC maximizes the separation between groups while minimizing variation within a group, providing a better discrimination of pre-defined genetic groups. Moreover, it allows obtaining a graphical representation of the relationships between the inferred clusters. This method has also the advantage of not relying on a particular population genetics model, such as Hardy-Weinberg equilibrium and linkage disequilibrium assumptions [Second, we conducted a Discriminant Analysis of Principal Components–DAPC . In contumptions , a key aumptions  implemenumptions . For theet al [Finally, we conducted a population assignment test among the 16 domestic breeds with GENALEX according to the approach of Paetkau et al .ST values averaged across all markers that indicated less differentiation between these two populations (FST = 12%) than between domestic rabbits and wild rabbits from the Iberian Peninsula (FST = 16%). An alternative phylogeny based on a different genetic distance  produced qualitatively similar results with the exception of the population of Alicante that did not group together with the remaining Iberian populations , wild rabbits from the Iberian Peninsula exhibited the highest levels of genetic diversity averaged across loci (He = 0.825), followed by French wild rabbits (He = 0.723), and finally domestic rabbits (He = 0.581). Allelic and private allelic richness followed similar trends. These reductions in genetic diversity were statistically significant for all summary statistics . This pattern is consistent with the occurrence of population bottlenecks in the colonization of France and in the initial domestication process. We did not find significant differences between levels of genetic diversity for loci residing on the autosomes vs. X-chromosome .Summary statistics of genetic diversity are presented in ST among breeds was 22%, with values ranging from 9% to 41% for the Flemish Giant/Hungarian Giant and Belgian Hare/English Spot comparisons, respectively. We found statistical support for genetic differentiation in all pairwise FST comparisons among breeds  averaged 2.39. Values of private allelic richness were also low and averaged 0.06. An analysis of molecular variance (AMOVA) was conducted for three distinct groups: 1) four sampling localities from Iberian Peninsula; 2) nine sampling localities from France; and 3) 16 domestic breeds . Domestiet al [He. The amount of diversity lost with the colonization of France was estimated by sampling one chromosome from each of the 9 wild French populations and 4 wild Iberian populations localities and calculated as 1 –(He in wild French) / (He in wild Iberian). The loss of diversity caused by the initial domestication process was estimated by sampling one chromosome from each of the 16 rabbit breeds and 9 wild French populations localities and calculated as 1 –(He in domestic) / (He in wild French). Finally, genetic diversity lost in the process of breed formation was estimated by sampling one chromosome from each individual in each breed and one chromosome for each individual across all domestic animals and calculated as 1 –(He in breed) /(He in domestic). We resampled 1,000 times per comparison. It should be noted that the levels of genetic diversity found in current wild populations from Iberian Peninsula and France were used as proxies for the amount of genetic diversity historically available in the wild populations involved in the colonization of France and initial domestication process, respectively.To more formally estimate the amount of genetic diversity lost by the i) colonization of France, ii) initial domestication process, and iii) the process of breed formation, we conducted a resampling procedure following Gray et al . This prConsistent with serial population contractions, we inferred a 12% (95% CI 4–20) decrease in genetic diversity with the colonization of France from the native range in Iberia, a decrease of 21% (95% CI 14–27) in the initial domestication process, and a decrease of 23% (95% CI 19–27) in the process of breed formation when averaged across breeds. . For indUsing chord and allele-sharing distances between individuals, respectively, we constructed two different trees of individuals using only the domestic dataset . The treTo analyse in greater detail the relationships among breeds and to obtain support values, we constructed two additional consensus trees using both chord and allele-sharing distances among breeds instead of individuals . FollowiK interval allowed us to investigate not only ancestral relationships between breeds  but also to analyse eventual substructure within breeds .Next, we investigated patterns of population structure within domestic rabbits by means of the clustering software STRUCTURE . With thK exhibited very low consistency among runs, which may be indicative of a lack of deep structure within domestic rabbits. This observation was also supported by the low values of the similarity coefficient among the different runs, which we had to restrict to values of K below 9 due to computational limitations , and when assuming a number of populations matching the total number of breeds sampled (K = 16), the structure plot shows a clear separation between most breeds   . A populK (K > 19) but in some cases preceded the separation of all breeds  and 19.05% for the New Zealand rabbits. As expected, the breeds more deviated from Hardy-Weinberg equilibrium markers are consistently those with high levels of intrabreed stratification. Even though the population structure patterns found are independently confirmed by a non-model approach (see DAPC below), one should take these results with caution, in particular due to the risk of overestimation of K [One of the STRUCTURE assumptions is that within populations the loci are at Hardy-Weinberg equilibrium (HW). Therefore, in addition to searching for microsatellites that showed significant deviations across all samples in wild populations see  we used ion of K .The DAPC also provides membership probabilities of each individual belonging to a given cluster . SimilarDuring most of its existence, the European rabbit was restricted to the Iberian Peninsula. Today, wild rabbits are distributed across Europe and new populations emerged worldwide as a result of human-mediated introductions ,51. The et al [et al [e.g maize, wheat, sunflower) for which the microsatellite estimates of diversity loss are lower values than those derived from sequence data [One of the main goals of our study was to investigate how the domestication process affected levels of genetic diversity in domestic rabbits . We founet al  with sixl [et al , which ence data –54.i.e. O. c. cuniculus) and exclusively from the genetic pool available in France. Concurrently, the fact that rabbit domestication occurred in an historical time where many other domestic species already existed may have contributed to a more focused and incisive process that might in many ways have resembled the process of breed formation. Historical records suggest that the early rabbit domestication occurred in closed environments such as monasteries [e.g. dogs and pigs), a practice that can increase genetic diversity levels in the domesticated population [The similar reduction of genetic diversity with the domestication and breed formation is surprising given that the later is normally seen as a more direct and extreme process, where founder effects, population contraction and artificial selection should be stronger. Several non-mutually exclusive explanations may account for this observation. First and perhaps most importantly, rabbits seem to have a single origin of domestication. In fact, while many domestic animals emerged from multiple domestication events or originated from different wild species or subspecies , domasteries –10. Thispulation . Our resST values among breeds (FST = 22%). This well-defined breed structure is also evident in the phylogenetic tree in which individuals of the same breed consistently grouped together  or betw chicken ,73). It K = 8. Second, the Rex breed, which consists of individuals belonging to three different colour morphs, began to show a subdivision signature between the strain Castor and the other two strains beyond K = 5, and later between Chinchilla and White strains  highlighting the Pyrenees mountain range that separates the Iberian Peninsula from France.(PDF)Click here for additional data file.S2 Fig(A) Neighbour-joining tree for 471 individuals rooted with wild rabbits from the Iberian Peninsula. (B) Unrooted Neighbour-joining tree for 340 domestic individuals from 16 different breeds. Branches are coloured according to individual’s origin.(PDF)Click here for additional data file.S3 FigBars show values of genetic diversity lost for the colonization of France (green), domestication process (red), breed formation process (blue) and for each one of the 16 breeds (grey). Values were estimated using a resampling methodology (described in Methods). Error bars represent 95% confidence limit.(PDF)Click here for additional data file.S4 FigTrees include domestic rabbits, wild French rabbits and is rooted with wild Iberian rabbits. (A) Tree based on chord genetic distance B) Tree based on allele-sharing genetic distance. Domestic rabbits include 16 breeds, two of which composed by two different strains. The node values correspond to the support values obtained from the consensus of 1000 trees and the branch length is proportional to support values.(PDF)Click here for additional data file.S5 FigK value, and K varied from 2 to 30.Five independent runs were performed for each (PDF)Click here for additional data file.S6 Fig(PDF)Click here for additional data file.S7 FigThe scatterplot shows the first two principal components  using breeds as prior for genetic clusters. Each dot represents an individual, and genetic clusters are depicted by colours and 95% inertia ellipses. The eigenvalue components are show in the lower right pane with relative magnitude.(PDF)Click here for additional data file.S1 FileThe table contains individual rabbit genotypes for each microsatellite. Microsatellites are ordered according to the multiplex number order and group code represents breed/population.(XLSX)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.S5 Table(PDF)Click here for additional data file.S6 Table(PDF)Click here for additional data file.S7 Table(PDF)Click here for additional data file.S8 TableValues for domestic rabbits (DOM), wild rabbits from France (WF), and wild rabbits from the Iberian Peninsula (WIP).(PDF)Click here for additional data file.S9 Table(PDF)Click here for additional data file.S10 TableValues for breed formation process were averaged across breeds and for each individual breed. Values were estimated using a resampling methodology (described in the Methods section).(PDF)Click here for additional data file.S11 Table(PDF)Click here for additional data file.S12 Table(PDF)Click here for additional data file.


Dalechampia scandens (Euphorbiaceae). These measures were compared against rates of evolution and patterns of divergence among 24 populations in two species in the D. scandens species complex. We found clear evidence for genetic constraints, particularly among traits that were tightly phenotypically integrated. This relationship between evolvability and evolutionary divergence is puzzling, because the estimated evolvabilities seem too large to constitute real constraints. We suggest that this paradox can be explained by a combination of weak stabilizing selection around moving adaptive optima and small realized evolvabilities relative to the observed additive genetic variance.If genetic constraints are important, then rates and direction of evolution should be related to trait evolvability. Here we use recently developed measures of evolvability to test the genetic constraint hypothesis with quantitative genetic data on floral morphology from the Neotropical vine  Population and quantitative genetics provide precise predictions for the short-term dynamics of allele frequencies and phenotypes, but how far can these predictions be extrapolated? It is customary to distinguish two extreme positions. The first is the extrapolationist view that macroevolution is microevolution writ large, or simply that macroevolution can be fully understood by use of concepts and parameters from quantitative genetic theory e.g. –6). The . The 6])The research paradigm in evolutionary quantitative genetics initiated by Lande and Arnold e.g. ,13) is a is a13])A key test of the macroevolutionary relevance of evolutionary quantitative genetics is to see whether macroevolutionary divergence is influenced by patterns of genetic variation as measured in contemporary populations. If there is no such relationship, then either genetic constraints are not important or they are not captured by the observed patterns of genetic variation. Many studies have asked this question, most concluding qualitatively in support of constraint (see Discussion). However, such studies face substantial conceptual and methodological challenges –20.The most important determinant of phenotypic divergence among populations is likely to be the dynamics of local peaks in the adaptive landscape ,6,21–23.Dalechampia scandens species complex, which we then compare with among-population divergence in 24 populations (12 each from the two species).Here, we use recently developed theory on the measurement of evolutionary potential in a multivariate context , and con2.(a)e, and from the standard equations of quantitative genetics, we get e ≡ Δz/β = IA, where Δz is the mean-scaled selection response, β is the mean-scaled selection gradient and IA is the mean-standardized additive genetic variance .,37.Dalecpulation . Larger pulation , and blopulation ,42,43. Tpulation ,42,44.FDalechampia scandens contains at least two reproductively isolated groups with overlapping geographical distributions The plants used in the quantitative genetics analyses were derived from seeds collected in two distinct populations, a large-glanded population near Tulum, Mexico  and a small-glanded population near Tovar, Venezuela . Fruits with seeds were collected from separate individuals in these two populations in 1998. We germinated one seed per maternal family and conducted two separate block diallels in which 12 and nine sets of five parents, in Tulum and Tovar, respectively, were combined into complete 5 × 5 diallels with both reciprocals and selfed crosses. Two individuals were raised from each cross and subsequently measured. The first diallel (Tulum) was conducted between 1999 and 2000 and results from this have been published ,47,48. T(c)In total, we obtained data on trait means from 24 populations , including the two populations on which the quantitative genetics experiments were conducted (see above). The measured plants in the remaining 22 populations were from fruits with seeds sampled from roadsides in Mexico  during the autumn of 2007. All plants were grown in the same greenhouse in Trondheim (Norway) during the same time period. Sample sizes ranged from one to 33 (median 12) plants per population .DA [D. scandens [We constructed a neighbour-joining tree  based onDA  using 70scandens . The gen(d)All analyses were conducted on two sets of traits . The fir(i)MCMCglmm [z is the trait value, u is the trait mean, a is the breeding value, b is the non-genetic plant-level effect, d is the measurement-date effect and q is the residual within-plant effect. The subscripts, i, j, k and l represent trait type, plant, day and blossom, respectively. The random effects are assumed to be distributed as a ∼ N, b ∼ N, d ∼ N and q ∼ N, where A is the additive relatedness matrix, I is the identity matrix and ⊗ is the Kronecker product. The model estimates the additive genetic variance matrix G, the among-plant environmental variance matrix B, the among-date variance matrix F and the residual variance matrix E. The traits were mean standardized before the analyses to obtain mean-standardized variance matrices. The complete posterior distributions of all G-matrices are reported in the electronic supplementary material, tables S4–S7.For the quantitative genetic experiments, we fitted mixed models with the R package MCMCglmm where z iMCMCglmm), we used zero-mean normal distributions with very large variances (108) for the fixed effects, half-Cauchy distributions with scale parameter 20 [V and n for the residuals. The matrix parameter V was a crude guess based on the phenotypic variance matrix, and the value of n was set to x − 0.998, where x is number of traits in the analysis [As priors for the Bayesian mixed models evolvability . The measures e(β), r(β) and c(β) are explained in the theory section. We also use a measure of evolutionary integration, i(β) = 1 − c(β)/e(β), that measures the fraction of additive genetic variance bound up in the other traits. This integration index varies between zero, no integration, and one complete integration. To calculate the average, the minimum and the maximum evolvability  for each G-matrix, we used the average, the minimum and the maximum of the eigenvalues. The minimum and maximum evolvability correspond to the evolvability in the directions of the smallest and largest eigenvector of G, while the average evolvability corresponds to the expected evolvability in a random direction. To calculate the average respondability, conditional evolvability and integration  we took an average over 1000 random selection gradients uniformly distributed on the unit sphere for r(β), c(β) and i(β) instead of using the analytical approximations in Hansen & Houle  independently from a zero-mean Gaussian distribution with unit variance, and subsequently normalizes each vector to unit length.To generate the set of random selection gradients uniformly distributed in R2, between evolvabilities, e(β), r(β) and c(β), computed along 1000 random selection gradients. These estimates of R2 describe the amount of variance across directions in the evolvability parameter of one G-matrix that can be explained by the same parameter from another G-matrix. All measures were calculated at each iteration of the posterior distribution of the G-matrices to include uncertainty.For direct comparison of G-matrices, we calculated the squared correlation coefficient, (iii)z is the trait value, u is the trait mean, a is the phylogenetic effect, p is the residual population effect, b is the plant-level effect, d is the measurement-date effect and q is the residual within-plant effect. The subscripts, i, j, k and l represent population, plant, day and blossom, respectively. Random effects were assumed to be identically independently normally distributed, with the exception of the phylogenetic effects for which we allowed phylogentic correlations as A is the phylogenetic relatedness matrix composed of shared branch lengths between populations. We also fitted models in which the residual population effects (p) were excluded, which gave us an estimate of the evolutionary rates MCMCglmm with the same specifications as in the genetic models.We estimated the among-population variance using phylogenetic mixed models ,55. Beca(iv)e, r and c) for all traits and for 1000 random directions (unit-length selection gradients). The different evolutionary models predict different scaling relationships between evolvability and evolutionary divergence (βspecies) was compared to the range of evolvabilities of the G-matrices to see whether this direction had high or low evolvability. Each element of βspecies was calculated by subtracting the average trait values  of the small species from the average trait values of the large species estimated in the phylogenetic mixed models, and dividing by the norm of this vector for standardizing to unit length. Uncertainty was assessed by evaluating the complete posterior distributions.To understand whether population differentiation had happened along lines of high evolvability, we compared evolutionary rates vergence . These si(β) with the fraction of independent among-population variance . Because of statistical power, we were only able to estimate two-dimensional among-population variance matrices using the phylogenetic mixed model described above . We therefore only compared the pairwise combinations of all traits for autonomy and fraction of independent among-population variance. All analyses were done in R v. 2.15.2 [Morphological integration may constrain the independent evolution of individual traits . We inve. 2.15.2 .3.(a)e = 0.55% and 0.23% for the small-glanded and large-glanded species, respectively (e = 0.09% for linear traits reported in a recent compilation [e = 0.85%) in the small-glanded species, but the least evolvable in the large-glanded species . The bract-trait matrices were more consistent .Although the small-glanded species was roughly twice as evolvable as the large-glanded species, their G-matrices were qualitatively similar in the general patterns and levels of integration. There were a lot of particular differences, however, and, for the functional traits, the evolvability measures along different directions were poorly correlated between the matrices . As for non-genetic variance components, we note that there were large components of temporal variance (‘day’) and that most of the residual variance was among individual blossoms and not among plants.(b)rate) because they scale isometrically with the evolvabilities under linear selection (see equations (2.1) and (2.2)). The mean-scaled variance accumulated over the length of the phylogeny was around 0.02 (ln mm)2 for all traits; this equals a CVrate of around 14%. The exception was bract traits in the small-glanded species, which had rates of evolution an order of magnitude lower than the other traits G can change over time, and thereby changing the predictions of among-population divergence. In general, divergence is best predicted by evolvability, less well by conditional evolvability and hardly at all by respondability .Note first that the rates of evolution were very small relative to the estimated evolvabilities. With our estimated evolvabilities, changes of this magnitude could be produced by natural selection over just a few generations. We do not have direct information about the absolute age of the phylogeny, but because the deepest split in the phylogeny is between species, we regard it as likely that the populations within each species have been separated by hundreds of thousands of years. Hence, there is no obvious reason to expect an influence of genetic constraints. However, i = 1−conditional evolvability/evolvability) between pairs of traits predicts the independent evolution of the traits. There was a strong negative relationship between integration and independent evolution for the large-glanded species, but a less clear relationship for the small-glanded species Dalechampia blossom morphology is under direct selection from both pollinators and seed predators [Dalechampia blossom traits are influenced by several factors, including bee community composition, availability of other resin sources for the bees, presence of other Dalechampia species and energetic constraints [D. scandens [Dalechampia in general [redators ,58–60. Cstraints ,42,44,61scandens ,62 and i general . This isα < 2es, The scaling exponent between evolvability and evolutionary rates for the functional traits was clearly below one for the large-glanded populations and closer to one for the small-glanded populations . Such scAmong the functional traits, bract size in the small-glanded populations stands out. This trait has a similar amount of additive genetic variance as the other functional traits, but much less among-population variance. This suggests that there is less dispersion of optima for this trait. This pattern is consistent across all bract traits in the small-glanded populations.α ≈ 2es, s = 1), the value of α will be too large to be consistent with the observation of phylogenetic signal (α ≈ 2es = 0.001 gives t½ ≈ 700 generations). For very weak selection (e.g. s = 0.01), however, this model may be plausible (α ≈ 2es = 0.00001 gives t½ ≈ 70 000 generations). The tight isometric relationship observed for the large-glanded populations G. The field is also marred by fundamental measurement/theoretical problems such as use of inappropriate or incommensurate scales, use of theory-free indices and use of statistical significance testing in place of estimation [Taken at face value our evolvability estimates and even most of our conditional-evolvability estimates predict very rapid evolution at macroevolutionary timescales. Yet, we also find clear evidence for a relationship between evolvabilities and patterns of evolution indicating that genetic constraints may be important. How can these two results be reconciled? It is not that this finding is unique to our study. We know of many studies reporting a relationship between patterns of genetic variation and population divergence or evolutionary rates ,24,64–80timation . Hence, A common problem, especially with the early studies, is the use of correlation matrices ).The third and last alternative is that realized evolvabilities are much smaller than measured evolvabilities, yet correlated with them. For example, it is possible that a conditional evolvability relative to a set of unmeasured traits under stabilizing selection could be quite small, due to a high degree of pleiotropy, and also correlated with the unconditional evolvabilities, since they both depend on the total variation. Similarly, for macroevolutionary changes, standing genetic variation may be less relevant for long-term response than the supply of ‘mutational evolvability’ based on how much genetic variation is generated each generation, and the mutational and standing evolvabilities are likely correlated. This last alternative is in line with several recent reviews concluding that there is good evidence that genetic constraints are important in evolution ,112,113.Distinguishing between these and other explanations is an empirical question, but in our opinion, a combination of relatively weak stabilizing selection and small realized evolvabilities is tentatively the most plausible. This can explain why some populations evolve fast on microevolutionary timescales ,114,115,


Aedes (Finlaya) koreicus was first identified in north-eastern Italy in 2011, during the ongoing surveillance activity of Aedes albopictus. Following this finding, a more intensive monitoring was carried out to assess the distribution of the species and to collect biological data. Herein, we report the new records obtained by four years of surveillance.The invasive species Ae. koreicus was checked using ovitraps, adults traps and by larval collections in all possible breeding sites from May 2011 to July 2015. The monitoring started in the site of the first detection (Province of Belluno) and was then extended in the neighbouring Provinces belonging to four Regions. Aedes koreicus was found in 73 municipalities out of 155 monitored (47.1 %), including 23 municipalities (14.8 %) previously not infested. The area of first detection of Ae. koreicus (Province of Belluno) was also the most infested (68 %). However the mosquito has also been found to the west (Province of Trento) and to the south and south-west (Provinces of Vicenza and Treviso) of the initially infested area.The presence of Ae. koreicus is directed towards south and west from the original infested area, likely due to the dense road connections and the habitat suitability of the new areas. According to these records, northern Italy has a high probability to be invaded by Ae. koreicus in the next decade. These data can be useful to validate predictive models of potential distribution and dispersal of this species in Italy or in Europe.The spread of The online version of this article (doi:10.1186/s13071-015-1208-4) contains supplementary material, which is available to authorized users. Aedes albopictus (Skuse) or Asian tiger mosquito in north-eastern Italy in 1991 [Ae. albopictus. Larvae and adults were then morphologically and molecularly identified as Aedes (Finlaya) koreicus (Edwards) [Ae. koreicus in Italy represents the second incursion in Europe [Ae. koreicus and to collect biological data on its life cycle.Since the discovery of the invasive mosquito species (IMS) Edwards) . The finn Europe , after an Europe . After tAe. koreicus in Italy. The survey started in 2011 from Valbelluna , where the first Ae. koreicus mosquitoes were found. A previous study reported that this species was well established in an area of 2600 km2 [2 and lure) were used in some Provinces . The collection sites were georeferenced. Mosquitoes were sampled from public areas according to the official guidelines of the Regional Health Authorities. Mosquitoes sampled from private areas were collected after verbal consensus of the owners.In this work we update the results of four years of surveillance on the presence and spread of 2600 km2 . Since 22600 km2 . All posThe specimens were morphologically identified , 7. In cAe. koreicus in northern Italy is reported in Fig. The current distribution July 2015) of 5 of Ae. Aedes koreicus was found in 73 municipalities out of 155 monitored (47.1 %) located in four Regions . Aedes koreicus was found mainly in hilly and mountainous areas in a range of altitude between 42 m.a.s.l.  and 1250 m.a.s.l. .The location of the first detection in Italy (Province of Belluno) was also the most infested.Ae.koreicus is now present were not infested before 2012, such as seven municipalities in province of Belluno, three in province of Trento  and six both in province of Vicenza and Treviso . Moreover, unexpected new records scattered in a wide area of northern Italy were found in the last two years . The spread of Ae. koreicus seems to be faster towards south and west from its original and more infested area (Province of Belluno), likely due to the dense road connections and the habitat suitability of new areas. On the contrary, the distribution of Ae. koreicus in Belgium is still limited in a small area of 6 km2 [Several areas where of 6 km2 .Ae. koreicus in this part of Italy. This species probably arrived in the province of Belluno where it was found for the first time and where the current density of population is the highest in the country. During the last years, the new records of Ae. koreicus in Italy could originate either from new introductions from its native range or dispersion from colonized areas, supported by human activities through private vehicles, public transport and/or freight. Previous introductions of IMS in USA and Europe suggest that the trade of used tires and plants is the main cause of invasion in a new area [Aedes species can be frequent as reported in The Netherlands [It is not possible to track back the route of entry and the time of arrival of new area . Throughherlands .Ae. koreicus. The probability of establishment and spread of IMS depends on environmental parameters, host availability and suitable climatic conditions [There is little information on the factors that are driving the dispersal of nditions . The knonditions .Aedes koreicus is native to Korea, Japan, China and eastern Russia. Little information is available on this species, but recent studies are clarifying some aspect of its biology and ecology in the new-colonized areas [Aedes koreicus is reported to feed on humans and domestic animals, and it seems to be well adapted to urban environment [Aedes, Ae. koreicus females lay eggs that can survive during the winter and hatch in the spring [ed areas , 4, 13. ironment . Like ote spring , 16. In Ae. koreicus and Ae. albopictus co-habitation . In nature in our study, Ae. albopictus let a spatio-temporal gap to Ae. koreicus, avoiding larval competition as observed for Ae. japonicus [Ae. koreicus larvae are found early in the spring  and in areas too cold for Ae. albopictus where Ae. koreicus can occupy an empty niche.The results of our monitoring shows a large area of ion Fig. . Interspsquitoes . Prelimiaponicus . Indeed,Ae. koreicus in an area where Ae. albopictus is already present has complicated the current entomological monitoring system, because larvae and adults are difficult to recognize and their eggs are practically indistinguishable.The establishment of Aedes koreicus has been suggested as a potential vector of arboviruses [Ae. koreicus can transmit the dog heartworm, Dirofilaria immitis [oviruses . The speoviruses . More ev immitis , 22.Ae. koreicus in the next decade. These data can be useful to validate predictive models of potential distribution and dispersal of this IMS in Italy or in other parts of Europe.According to these records, northern Italy has a high probability to be invaded by Therefore, early detection of IMS distribution is needed to prevent new establishments and to plan control actions. Unfortunately the eradication of an establish species is very unlikely .


Charadrius pecuarius), white-fronted plover (C. marginatus), and Madagascar plover (C. thoracicus). Bayesian clustering revealed strong population structure in the rare and endemic Madagascar plover, intermediate population structure in the white-fronted plover, and no detectable population structure in the geographically widespread Kittlitz's plover. In contrast, allelic richness and heterozygosity were highest for the Kittlitz's plover, intermediate for the white-fronted plover and lowest for the Madagascar plover. No evidence was found in support of the “watershed mechanism” proposed to facilitate vicariant divergence of Madagascan lemurs and reptiles, which we attribute to the vagility of birds. However, we found a significant pattern of genetic isolation by distance among populations of the Madagascar plover, but not for the other two species. These findings suggest that interspecific variation in rarity, endemism, and dispersal propensity may influence genetic structure and diversity, even in highly vagile species.Understanding the relative contributions of intrinsic and extrinsic factors to population structure and genetic diversity is a central goal of conservation and evolutionary genetics. One way to achieve this is through comparative population genetic analysis of sympatric sister taxa, which allows evaluation of intrinsic factors such as population demography and life history while controlling for phylogenetic relatedness and geography. We used ten conserved microsatellites to explore the population structure and genetic diversity of three sympatric and closely related plover species in southwestern Madagascar: Kittlitz's plover ( It is well established that environmental barriers can restrict gene flow, facilitating genetic isolation by distance , the white-fronted plover (C. marginatus), and the endemic Madagascar plover  amplified polymorphic and clearly interpretable PCR products in at least one of the species, seven of which amplified in all three species. These markers were subsequently screened across all 362 individuals in two multiplexes using the PCR conditions described by Küpper et al. , and the expected (HE) and observed (HO) heterozygosity of each locus. Unbiased estimates of expected heterozygosity (uHE) were calculated to compensate for variable, and in some cases, small sample sizes  equalized to a sample size of four genes  with rarefaction. Significant differences among species in each of the above measures of genetic diversity were identified using two-way analyses of variance (ANOVA) with locus and population specified as random factors. Species comparisons were further evaluated for significance with post hoc Tukey's tests. All analyses were tested at α = 0.05.To account for variable sample sizes, we employed HP-RARE 1.0  in the sample by probabilistically assigning individuals to one or more cluster in a manner that minimizes each cluster's deviation from the Hardy–Weinberg and linkage equilibrium. Membership coefficients represent the assignment probability of each individual's genome to the K inferred clusters. We conducted these analyses using the LOCPRIOR model in STRUCTURE, for which we provided a priori information about the sampling locations of individuals across the study area  of each K over the five independent runs and used both the maximum Ln P[D] and ΔK statistics to infer the most likely number of distinct clusters given our data. ΔK is an ad hoc statistic that uses the second-order rate of change of the likelihood function to reveal the relative amount of inference gained between successive K values  as independent variables. We extracted these location specific climate data from interpolated bioclimatic surfaces provided by WorldClim  that were based on proximity to suitable habitat described in detailed species accounts  suggesting that the overall pattern is not driven by one or a small number of outliers. When we employed cost-weighted dispersal distance as a predictor of genetic differentiation, the pattern strengthened  and center of the study area , revealing north to south gradients in the population structure of two of the three species. We also controlled for any potential biases resulting from variation in sample sizes by incorporating established statistical methods, such as unbiased estimations of heterozygosity Nei , rarefieOur findings also have important implications for plover conservation in Madagascar. The strong population structure of the Madagascar plover suggests that this species in particular may be vulnerable to inbreeding depression and the loss of genetic diversity owing to its low abundance and restricted distribution. Therefore, we advocate continued conservation of critical habitats of this vulnerable species to maintain sufficient genetic diversity needed to promote population viability.Using a comparative approach, we show that the Madagascar plover, an endangered endemic species with low abundance and a restricted range, is strongly structured and has low genetic diversity across its range. In contrast, the Kittlitz's plover, a widespread and abundant species with high dispersal propensity, is panmictic and has high genetic diversity over the same geographic area. The white-fronted plover, which is intermediate in many respects, exhibits moderate population structure and levels of genetic diversity. This pattern is consistent with what we know about these species' life histories, dispersal propensities, and endemic statuses. Thus, species traits may profoundly influence population structure and genetic diversity, with important implications for population, evolutionary and conservation biology.


Chthamalus fragilis is found along the US Atlantic seaboard historically from the Chesapeake Bay southward, and in the Gulf of Mexico. It appeared in New England circa 1900 coincident with warming temperatures, and is now a conspicuous member of rocky intertidal communities extending through the northern shore of Cape Cod, Massachusetts. The origin of northern C. fragilis is debated. It may have spread to New England from the northern end of its historic range through larval transport by ocean currents, possibly mediated by the construction of piers, marinas, and other anthropogenic structures that provided new hard substrate habitat. Alternatively, it may have been introduced by fouling on ships originating farther south in its historic distribution. Here we examine mitochondrial cytochrome c oxidase I sequence diversity and the distribution of mitochondrial haplotypes of C. fragilis from 11 localities ranging from Cape Cod, to Tampa Bay, Florida. We found significant genetic structure between northern and southern populations. Phylogenetic analysis revealed three well-supported reciprocally monophyletic haplogroups, including one haplogroup that is restricted to New England and Virginia populations. While the distances between clades do not suggest cryptic speciation, selection and dispersal barriers may be driving the observed structure. Our data are consistent with an expansion of C. fragilis from the northern end of its mid-19th century range into Massachusetts.The barnacle  Chthamalus fragilis is currently found along the eastern United States, extending from the Gulf of Mexico to the Atlantic coast northward up to Massachusetts  in 1898, and subsequently was observed in other locations south of Cape Cod, in Buzzards Bay and Vineyard Sound  or anthropogenic vectors , or both. C. fragilis possesses a typical biphasic life cycle, with the potential for long distance dispersal. Adults are hermaphroditic with internal fertilization and are capable of self-fertilization  haplotypes from several locations in Massachusetts and Rhode Island with those obtained from locations farther south, in Virginia, South Carolina, Georgia, and Florida. Thus, sampling covered a ∼2,000 km range (minimum linear separation). While confirming the source of populations that are cryptogenic  can be difficult, the existence of private haplotypes shared between the northern populations and a subset of southern populations may indicate the colonization pathway We assembled chromatograms and confirmed sequence quality using Geneious v. 7.1.7 . Sequences were aligned using ClustalW  with defNucleotide diversity, haplotype diversity, Tajima’s D and Fu’s Fs were calculated using DnaSP . An AnalC. proteus, a cryptic sibling species of C. fragilis , and 110 polymorphic sites, of which 58 were parsimony informative. In the amino acid alignment , there were three amino acid substitutions: a valine for an alanine in position 6 in a Charleston, South Carolina sequence; a valine for an isoleucine in position 55 for a Woods Hole, Massachusetts sequence, and an alanine for a threonine in position 157 for a Summerland Key, Florida sequence.ST and AMOVA results showed significant genetic structure particularly between distant sites .Haplogroups differed in their geographic distribution ; Fig. 3.Balanus glandula along the California coast , and one in Virginia and northward locations only. Additionally, we observed significant genetic structure between northern and southern populations. This pattern—a cline between divergent clades—is similar to that observed for other barnacles, including ia coast , Notochtan coast  and Chthern Asia .C. fragilis belonging to haplotype group 2 have characteristics that are less suited to southern locations. Additional research on the physiology and ecology of C. fragilis are necessary to elucidate possible adaptive differences between northern and southern populations.A deep phylogeographic break for species like barnacles with high planktonic dispersal potential may be due to several non mutually exclusive factors, including selection, cryptic speciation, and the presence of dispersal barriers . It is pThe pattern of reciprocal monophyly and large between-clade relative to within-clade divergences can sometimes be used to infer the existence of cryptic species . MitochoChthamalus proteus as a cryptic species distinguishable only through molecular techniques from C. fragilis using enzyme electrophoresis, and these results were supported by Chthamalus moro, Euraphia rhizophorae and E. eastropacensis  is detected in our data, we are cautious about interpretation. The strict interpretation of IBD is an equilibrium pattern between genetic drift and gene flow when migration is limiting, and so allele frequencies become divergent over spatial distance. However, similar statistical patterns emerge by non-trivial disjunct distributions of divergent lineages , and mayC. fragilis . Understanding how the population genetic composition is shifting, and how these changes may impact the overall community structure, is critical for understanding the consequence of climate change on coastal communities.As temperatures continue to increase, ts range . Likely dynamics . Our gen


Tfap2c and its downstream target Cdx2. Our findings provide a novel concept in the realm of Stat3, self-renewal signaling, and pluripotent stem cell biology. Ultimately, this finding may facilitate the development of conditions for the establishment of authentic non-rodent ESCs.Stat3 is essential for mouse embryonic stem cell (mESC) self-renewal mediated by LIF/gp130 receptor signaling. Current understanding of Stat3-mediated ESC self-renewal mechanisms is very limited, and has heretofore been dominated by the view that Stat3 signaling functions in a binary “on/off” manner. Here, in contrast to this binary viewpoint, we demonstrate a contextual, rheostat-like mechanism for Stat3's function in mESCs. Activation and expression levels determine whether Stat3 functions in a self-renewal or a differentiation role in mESCs. We also show that Stat3 induces rapid differentiation of mESCs toward the trophectoderm (TE) lineage when its activation level exceeds certain thresholds. Stat3 induces this differentiation phenotype via induction of  Embryonic stem cells (ESCs) were originally derived by explanting mouse blastocysts onto a layer of mitotically inactivated fibroblasts (‘feeders’) in medium containing fetal calf serum (FCS) . Under tOnce LIF binds to the LIF receptor (LIFR), the receptor forms a heterodimer with the membrane protein, gp130. The heterodimerization of LIFR and gp130 triggers the membrane-proximal cytosolic docking and activation of the associated Janus kinases (JAKs) . The actCurrent understanding of Stat3-mediated mESC self-renewal has been dominated by the view that LIF/Stat3 signaling functions in a binary “on/off” manner. It has not been clear whether Stat3 activation level is critical to ESC self-renewal and whether non-129 strain mESCs can be maintained under feeder-free conditions through modulation of Stat3 activity. Here, we use chemical and genetic approaches to modulate Stat3 activity in mESCs and determined the corresponding effect on self-renewal and differentiation. We report that Stat3 exhibits a dose-dependent effect in mESC self-renewal and differentiation.supplementary material Fig. S1A,B)  . Therefo. S1A,B) . As a re. S1A,B) , likely . S1A,B) . Both B6. S1A,B) , suggestTo further define how Stat3 activation level can affect ESC fate, we introduced both Stat3 and gp130-Y118F transgenes into B6 mESCs  so that we could broadly manipulate Stat3 activation level. B6-S3Y118F mESCs remained undifferentiated without feeders in the presence of LIF . SurprisTo further confirm that Stat3 activity is correlated with the rapid differentiation phenotype, we modulated Stat3 activity by applying different concentrations of GCSF and JAK1i. Indeed, B6-S3Y118F mESCs self-renewal could be maintained at a lower concentration of GCSF or at a higher concentration of GCSF co-administered with an adequate dosage of JAK1i . We examsupplementary material Fig. S2A). However, when treated with 50 ng/ml of GCSF, E14-S3Y118F ESCs underwent rapid differentiation whereas E14-Y118F ESCs remained undifferentiated . Western blot analysis confirmed that the level of Stat3 phosphorylation in E14-S3Y118F mESCs treated with GCSF for 6 hours was significantly higher than those treated with LIF . The differentiation phenotype of E14-S3Y118F mESCs induced by GCSF could also be rescued by co-administration of JAK1i .To determine whether hyperactivated Stat3 inducing rapid differentiation is a general phenomenon in mESCs, we introduced Stat3 and chimeric gp130-Y118F transgenes into E14 mESCs, a widely-used feeder-independent ESC line derived from the 129 strain of mouse. Both E14-Y118F and E14-S3Y118F mESCs could be maintained in LIF condition , indicating rapid differentiation of B6-S3Y118F mESCs induced by GCSF.B6-S3Y118F mESCs underwent rapid differentiation when Stat3 was hyperactivated via GCSF-mediated Stat3 activation . To dete24 hours , confirmsupplementary material Fig. S4). Additionally, the differentiated cells induced by GCSF cannot be maintained or passaged in the trophoblast stem cell culture condition, suggesting that GCSF induces B6-S3Y118F mESC differentiation towards terminally differentiated TE cells .Next, we examined the expression levels of various germ layer markers in B6-S3Y118F mESCs treated with GCSF. Surprisingly, GCSF treatment did not significantly upregulate any of the three somatic germ layer markers examined ; insteadCdx2 and Gata3 were significantly upregulated within 24 hours of GCSF treatment in B6-S3Y118F mESCs , to verify whether Tfap2c is directly regulated by Stat3. Despite CHX application, Tfap2c expression level increased significantly following a one-hour GCSF application, suggesting that GCSF directly regulates Tfap2c expression , we found that TE cells . Forced TE cells . We obsemulation . To furtpression . We nextpression . Additiop2c loci . Taken tTfap2c expression by shRNA  mESC strains through modulation of Stat3 activity. LIF-induced Stat3 activity is not sufficient to maintain non-129 ESC self-renewal under feeder-free conditions, and enhancing Stat3 activity through overexpression of a Stat3 transgene or a gp130 chimeric receptor is necessary and sufficient to obviate the requirement of the feeder layer and maintain an undifferentiated ESC state. However, elevating Stat3 activity over a certain threshold will switch Stat3's self-renewal promoting effect into one that induces ESC differentiation towards the TE lineage.Tfcp2l1, Gbx2, Klf4, Klf5, Pim1, Pim3, Pramel7, and c-Myc  domain, and then phosphorylated on tyrosine 705, leading to dimerization and translocation to the nucleus, where it binds specific DNA sequences and activates target gene transcription. We hypothesize that Stat3 recruits distinct co-activators and activates distinct gene programs depending on its activation level. Stat3 can not only regulate the expression of pluripotency-related genes such as nd c-Myc , but alsnd c-Myc . The outnd c-Myc . When Stnd c-Myc . The plund c-Myc   containing 10% FBS (HyClone), 1 mM Sodium Pyruvate (Gibco), 100 µM Non-Essential Amino Acids (Gibco), 2 mM GLUTAMAX (Gibco), 1 µM β-mercaptoethanol (a formulation hereinafter referred to as “mESC medium”), and 1000 U/ml LIF (Stemgent). The concentration of GCSF used in this study was 50 ng/ml unless specifically indicated. 46C ESCs were routinely maintained on gelatin-coated plates in mESC medium. Trophoblast stem cell medium was prepared according to the previous report . Alkalin5 cells per well into a 24-well plate and cultured in 600 µl of medium per well composed of 300 µl filtered virus-containing supernatant and 300 µl mESC medium with 4 µg/ml Polybrene (Millipore).Stat3 and grgp130-Y118F transgenes were cloned into the pCAG-IRES-IP expression vector or the pSIN-EF2 lentiviral vector. The details on how to generate grgp130-Y118F transgene have been described in a previous study . pCAG-StGapdh. The primers used for qRT-PCR are listed in supplementary material Table S1.Total RNA was extracted with the Quick-RNA MiniPrep Kit (Zymo). cDNA was synthesized with 0.5 µg of total RNA, using the QuantiTech Rev. Transcription Kit (Qiagen). qRT-PCR was performed with Power SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer's instructions. Signals were detected with an ABI7900HT real-time PCR System (Applied Biosystems). The relative expression level was determined by the 2-ΔCT method and normalized against shRNA-expressing plasmids were generated according to Addgene PLKO.1 protocol. Certain genes which have no validated shRNA were knocked down by introduction of multiple shRNA sequences into cells to achieve better efficiency. The target-specific shRNA sequences used in this study are as follows: Control shRNA: AATTCTCCGAACGTGTCACGT; Cdx2 shRNA: GGACAGAAGATGAGTGGAATT; Gata3 shRNA#1: GCCTGCGGACTCTACCATAA A; Gata3 shRNA#2: ATTGCTGAACATTGCATATAA; Gata3 shRNA#3: CAGTTGTTTGATG CATTTAAA; Tead4 shRNA#1: CCGCCAAATCTATGACAAGTT; Tead4 shRNA#2: GC TGAAACACTTACCCGAGAA; Tead4 shRNA#3: CCCTCT CTGTGAGTACATGAT; Tfap2c shRNA: AGCCGCTCTGCAAGTCTAATA. After lentiviral infection, the cells were incubated with 1 µg/ml puromycin, 10 µg/ml Blasticidin S deaminase, or 100 µg/ml hygromycin for 48–72 hours. The surviving ESCs were pulled together to examine knockdown efficiency and to investigate their roles in GCSF induced TE differentiation.Immunostaining was performed according to a standard protocol. Primary antibodies used were the following: Cdx2 . Alexa Flour fluorescent secondary antibodies (Invitrogen) were used at a 1:2000 dilution. Nuclei were visualized with DAPI.


Crassostrea gigas has been introduced into a large number of countries for aquaculture purpose. Following introduction, the Pacific oyster has turned into an invasive species in an increasing number of coastal areas, notably recently in Northern Europe.Originating from Northeast Asia, the Pacific oyster n = 60).To explore potential adaptation of reproductive traits in populations with different histories, we set up a common garden experiment based on the comparison of progenies from two populations of Pacific oyster sampled in France and Denmark and their hybrids. Sex ratio, condition index and microarray gene expression in gonads, were analyzed in each progeny . Candidate transcripts included mRNA coding for sperm quality and insulin metabolism, known to be implicated in coordinated control and success of reproduction.Observed differences suggest that adaptation of invasive populations might have occurred during expansion acting on reproductive traits, and in particular on a female-biased sex-ratio, gamete quality and fertility.The online version of this article (doi:10.1186/s12864-015-1972-8) contains supplementary material, which is available to authorized users. In these organisms, high gene flow between populations can be ensured by high larval dispersal, high fecundity and huge population sizes , identified as outlier ePST both in males and females and more expressed in Danish progeny, is a A-kinase anchoring protein (AKAPs) promoting the selective sequestration of intracellular cAMP-dependent protein kinase (PKA) involved in epithelial sodium channel regulation , more expressed in Danish progeny, is an Actin-binding protein that participates in the anchoring of membrane proteins for the actin cytoskeleton and is involved in sperm morphogenesis in mammals [C. gigas, an Insulin-related peptide receptor has previously been identified in oysters by Gricourt et al. [In males, functional annotation of outlier ePgulation .This progulation  found hi mammals . Finally mammals , Alterna mammals  and in fST, to divergence in phenotypic traits, QST, has widely been used as a method to assess the relative strength of genetic drift and selection [ST levels typically exceed that observed in FST suggesting an important role of natural selection on quantitative traits [ST values but we restricted PST values to additively behaving transcript with intermediate expression levels in hybrid. We found that mean ePST (0.04) was similar to the estimated FST values [ST had values greater than 0.15. Our results therefore suggest that diversifying selection has most probably acted on outlier gene expressions. Finally, gene expression can also be controlled by epigenetic mechanisms, potentially in a transgenerational manner, meaning in a way, by genetic mechanisms non-DNA dependents. There are yet only a few papers studying epigenetics in oysters [A comparison of divergence in neutral markers, Felection . The QSTe traits , 69. In T values  on the s oysters , 71 and  oysters .Palaemon macrodactylus [Pacifastacus leniusculus [C. gigas, an alternative and irregular protandrous hermaphrodite, is complex, it seems to be determined both by environmental and genetic factors [C. gigas. The first suggests the presence of 2-genotypes, a dominant male M allele and a protandric recessive F allele [C. gigas, having an “r” demographical strategy, characterized by high fecundity, reproductive success is greatly dependent on the quantity of gametes produced, especially oocytes, as well as their quality. Furthermore, Cardoso et al. [C. gigas could favour a rapid colonization of new habitats.In our study, we observed a female biased sex-ratio in the Danish progeny. Sex-ratio biased to female in invasive species has also been observed in an estuarine shrimp, dactylus  and was iusculus . Althoug factors . In our  factors . Two genF allele . The secF allele . FurtherF allele , in whicF allele . In speco et al.  found thC. gigas Southern Europe populations and population north to the Wadden Sea is corroborated by phenotypic differentiation based on transcriptomic data, biased sex-ratio and condition index. Overall, our results suggest a population expansion strategy of the studied Pacific oyster Danish population, potentially relying on a females-biased sex-ratio, a greater reproduction effort and gamete quality, noticeable on molecular signatures.To conclude, genetic differentiation previously reported between http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE66103).Microarray data are deposited in the gene expression omnibus (GEO) repository with the accession number GSE66103 (All the experiments were conducted according to the regulations of local and central government, and the study protocol was conducted in accordance with institutional guidelines


Lepidonotothen nudifrons. Starting from a species-specific spleen transcriptome library, we aimed at isolating polymorphic microsatellites  suitable to quantify the genetic variability in this species, and additionally to assess the population genetic structure and demography in nototheniids.Loss of genetic variability due to environmental changes, limitation of gene flow between pools of individuals or putative selective pressure at specific markers, were previously documented for Antarctic notothenioid fish species. However, so far no studies were performed for the Gaudy notothen L.nudifrons. Eleven microsatellites were polymorphic and allele numbers per locus ranged from 2 to 17. In addition, we amplified loci identified from L. nudifrons in two other congeneric species (L. squamifrons and L. larseni). Thirteen loci were highly transferable to the two congeneric species. Differences in polymorphism among species were detected.We selected 43,269 transcripts resulting from a MiSeq sequencer run, out of which we developed 19 primer pairs for sequences containing microsatellite repeats. Sixteen loci were successfully amplified in Lepidonotothen species if genotyped in larger population samples. When compared with anonymous loci isolated in other notothenioids, i.e. Type II (isolated from genomic libraries), they offer the possibility to test how the effects of occurring environmental change influence the population genetic structure in each species and subsequently the composition of the entire ecosystem.Starting from a transcriptome of a non-model organism, we were able to identify promising polymorphic nuclear markers that are easily transferable to other closely related species. These markers can be a key instrument to monitor the genetic structure of the three  Lepidonotothen nudifrons (Lönnberg 1905), the Gaudy notothen [L. nudifrons is especially abundant along the Islands of the Scotia Arc until the Bransfield Strait (Antarctic Peninsula) [L.nudifrons is experiencing any loss of genetic variability or if gene flow is limited among pools of individuals. Genetic differentiation among populations has already been demonstrated for another ecologically important notothenioid (Pleuragramma antarctica) in the same area [Lönnberg 905, the notothen ). L. nudLönnberg 905, the ninsula) . This spninsula) , howeverninsula) , it is eThese investigations require microsatellites, which have proven useful and reliable genetic markers in similar studies , 6. In tDue to their position inside or their flanking coding gene sequence and due to functional constraints, EST-linked SSRs are expected to have higher probability to be under selective pressure . Gene-asLepidonotothen spp. in order to compare their variability with anonymous Type II markers isolated in other notothenioid species.The primary goal of this study is to expand the panel of available EST-linked loci in notothenioids for future studies on the evolution and demographic history of Antarctic fish. Moreover, we aim to isolate specific markers for L.nudifrons [L. nudifrons. Loci identified from L.nudifrons were also tested in two congeneric species: L. squamifrons and L. larseni.We therefore mined microsatellite sequences from the publicly available spleen transcriptome of udifrons  and screL.nudifrons [de novo assembly was performed using the trinity genome-independent transcriptome assembler [release 17 July 2014, Gene-associated SSR markers were identified among 112,477 spleen transcripts of udifrons . The rawudifrons  with folL. nudifrons collected in March/April 2012 near Elephant Island at 70–322 m depth during the RV Polarstern expedition ANT-XXVIII/4. The fish are not considered endangered at the sampling site. Sampling of the animals has been performed in accordance with the Antarctic treaty, and permission was given by the respective national authority (Umweltbundesamt: permit number I 3.5-94003-3/274). All treatments with live animals were in accordance with German law and approved by the competent national authority ).Primer validation was carried out on genomic DNA extracted from 7 specimens of Total DNA was purified from 10 to 20 mg of muscle tissue following the standard protocol of the DNeasy Blood and Tissue Kit . Quality and quantity of DNA extractions were assessed using a NanoDrop™ 2000c spectrophotometer  before samples were stored at −20 °C.L. nudifrons individuals . To this purpose, forward primers were labelled with fluorescent dyes FAM, HEX, and TAMRA  and loci were combined in multiplex PCRs designed with Multiplex Manager v. 1.2 [Initially, PCR primer pairs were tested as single-locus PCR in 20 μl volume, containing 1X reaction buffer , 70 μM dNTPs, 0.25 μM of each primer, 1 unit Taq polymerase  and ~30 ng of genomic DNA. PCR conditions were: initial denaturation at 94 °C for 1 min, followed by 30 cycles of 94 °C for 30 s (denaturation), 54 °C for 40 s , 72 °C for 40 s (extension) and a final single extension step at 72 °C for 5 min. Electrophoresis was carried out at 100 V on 1.5 % agarose gels containing GelRed Nucleic Acid Gel Stain  for a preliminary qualitative polymorphism detection. Only loci that provided a clear PCR product were retained  and their polymorphism was verified on an Applied Biosystems 3130 XL automated sequencer  using a larger sample of 21 r v. 1.2 . AmplifiL. squamifrons (n = 20) and L.larseni (n = 18) were tested to assess loci variability in congeneric species (DNA extraction and amplification conditions as described above). Individuals of these two species were collected around Elephant Island in March/April 2012 (RV Polarstern expedition ANT-XXVIII/4).Specimens of Allele sizes were assigned using GeneMarker v. 2.6.3 . Binning was automated with Flexibin v. 2 . All inp−9.To annotate the transcripts corresponding to the final panel of markers, homology searches were performed using Blastx  against L. nudifrons with no missing genotypes. The allele number per locus ranged from 2 to 17 (Table o) and expected heterozygosities (He) were 0.43 (±0.34 SD) and 0.43 (±0.31 SD). Ten loci out of eleven were in HWE after correction for multiple testing , but it did not provide a consistent amplification and genotyping result for L. larseni. The remaining 13 markers were successfully genotyped (Table L. squamifrons, while in L. larseni we scored multiple alleles in ten loci (Tables L. nudifrons, turned out to be polymorphic in L. squamifrons and L. larseni (Tables In the congeneric species, Lepidonotothen spp. these markers are also easily transferable among different, but phylogenetically closely related species.Our results confirm that polymorphic SSR markers can be effectively isolated from transcriptomes of non-model organisms , 33. ForST values to search for loci showing a significantly high level of genetic differentiation between pairs of species as applied for the Chionodraco genus [Additional tests should be implemented to verify whether these loci could be considered candidates for being influenced by selection. An effective method to verify this, is to compare Fco genus . Agostinco genus  indicateLepidonotothen species, profiling demographic events occurred in the past, and identifying signatures of local adaptation in genetically different populations. To understand the significance of these events and the climate challenges that marine organisms in polar regions are facing, is essential to model and project future population viability for species management and conservation [Moreover, we aim to use our microsatellites in conjunction with other markers such as mitochondrial and nuclear sequences and on a larger sample size. This would effectively help monitoring the genetic structure of the three ervation .


Medicago sativa subsp. sativa L.) was genotyped using 85 genome-wide distributed SSR markers to reveal the genetic diversity and population structure in the alfalfa. Genetic diversity analysis identified a total of 1056 alleles across 85 marker loci. The average expected heterozygosity and polymorphism information content values were 0.677 and 0.638, respectively, showing high levels of genetic diversity in the cultivated tetraploid alfalfa germplasm. Comparison of genetic characteristics across chromosomes indicated regions of chromosomes 2 and 3 had the highest genetic diversity. A higher genetic diversity was detected in alfalfa landraces than that of wild materials and cultivars. Two populations were identified by the model-based population structure, principal coordinate and neighbor-joining analyses, corresponding to China and other parts of the world. However, lack of strictly correlation between clustering and geographic origins suggested extensive germplasm exchanges of alfalfa germplasm across diverse geographic regions. The quantitative analysis of the genetic diversity and population structure in this study could be useful for genetic and genomic analysis and utilization of the genetic variation in alfalfa breeding.Information on genetic diversity and population structure of a tetraploid alfalfa collection might be valuable in effective use of the genetic resources. A set of 336 worldwide genotypes of tetraploid alfalfa ( Medicago sativa subsp. sativa L.), a perennial autotetraploid (2n = 4x = 32) and cross-pollinated forage legume, is the most important cultivated forage plant in the world [Triticum aestivum L.) [Oryza sativa L.) [Lolium perenne L.) [Setaria italica (L.) Beauv.]  –10, cucuivus L.) , and cotutum L.) . The genutum L.) –16. Tucautum L.)  assessedutum L.)  estimateutum L.)  investigutum L.) .Although several studies have employed marker-based estimation of genetic diversity and population structure in alfalfa, most of these studies are limited in the number of accessions included or the number of markers used to characterize genetic diversity in alfalfa. Some of these studies have been conducted using germplasm specific for a breeding program , or diplM. sativa subsp. sativa accessions were analyzed in the study. Each accession was represented by four genotypes, except for the Chinese accessions that were represented by eight genotypes for each accession. Nine accessions from China were obtained from National Herbage Germplasm Bank of China; two accessions from Syria, one from Libya and one accession from Sudan were provided by the Institute of Animal Science, Chinese Academy of Agricultural Science ; the other 62 accessions were provided by the USDA National Plant Germplasm System (NPGS). Detailed information about the 336 genotypes used in this study is provided in A total of 336 cultivated tetraploid genotypes from 75 2, 1μL template DNA, 17.8μL ddH2O. The following PCR program was used: an initial denaturing for 3min at 95°C, followed by 10 cycles of 95°C for 30sec, 60°C for 30sec, 72°C for 30sec; and 20 cycles of 95°C for 30sec, 55°C for 30sec, 72°C for 30sec; a final extension at 72°C for 6min. PCR products were separated on an ABI3730xl DNA Analyzer . Fluorescence-labeled primers were synthesized at Applied Biosystems Company. Fragment sizes were determined using an internal size standard , and the outputs were analyzed using GeneMapper software (http://www.appliedbiosystems.com.cn/).Young leaves of each of all 336 genotypes were freeze-dried and ground to a fine powder, separately. Genomic DNAs were extracted from each powder sample, following the CTAB method . DNA quahttp://hi.baidu.com/luansheng1229/item/306815126d58e3a4feded5a4) described by Botstein et al. [http://www.pge.cnrs-gif.fr/bioinfo/populations/index.php) was used to calculate pairwise genetic distances between the genotypes using Nei et al.’s [Individual tetraploid genotypes were scored from microsatellite banding patterns in the electropherograms following the Microsatellite DNA Allele Counting-Peak Ratios (MAC-PR) method of Esselink et al . For eacn et al. . The sign et al.  with then et al. . The POPet al.’s  and the et al.’s . We usedet al.’s .Out of the 159 SSR primer pairs used for genotyping, 74 could not be scored with confidence. The remaining 85 markers amplified a total of 1056 alleles, confirming that most of the selected markers were highly informative. The average allele number per locus was 12.4, ranging from 4 to 40. The expected heterozygosity (He) ranged from 0.283 to 0.901 with the average of 0.677 and the polymorphism information content (PIC) ranged from 0.273 to 0.893 with an average of 0.638. A summary of genetic characteristics of 336 alfalfa genotypes based on 85 SSR markers are listed in A comparative analysis of genetic characteristics was performed at the chromosome level . The meaK  was evaluated by two methods , PopA-2 (96 genotypes), PopA-3 (49 genotypes), and two subpopulations in PopB, namely PopB-1 (36 genotypes), and PopB-2 (59 genotypes)  . ClearlyThe principal coordinate analysis (PCoA) was conducted to further assess the population subdivisions identified using STRUCTURE. The first three principal coordinates explained 24.2%, 16.4% and 16.0%, respectively, and 56.6% of the total variation. The PCoA was largely consistent with the STRUCTURE analysis, the first principal coordinate (PCo1) clearly separated 336 alfalfa genotypes into two populations as identified by the STRUCTURE analysis (PopA and PopB). The PopB could be divided into two subpopulations (PopB-1 and PopB-2) by the PCo 2, whereas the PopA -1, PopA-2 and PopA-3 were clustered together .The values of genetic distance (GD) were calculated based on all markers, ranging from 0.09 to 0.59 with the average 0.39 while most of the GD values ranged from 0.35 to 0.45 (ca 76%). Overall, the lowest average GD was observed between wild accessions, followed by landrace (0.391), cultivar (0.387) and cultivated accessions (0.368). No significant difference for the average GD values for different improved types of alfalfa accessions was found. A Neighbor-joining tree of 336 alfalfa genotypes was constructed using Nei’s genetic distance and five clusters I, II, III, IX, and V were identified . ClusterThe genetic diversity of each subpopulation identified by the STRUCTURE was assessed . PopB-2 We also determined the genetic diversity for the accessions from cultivars, landraces, cultivated materials, and wild germplasms with different improvement statuses . The meaM. falcata germplasm using eight SSR markers and identified 16.7 alleles per locus and a He value of 0.77. Flajoulot et al. [M. sativa—falcata complex, it was slightly lower than that of the diploid germplasm  [The study worked on the genetic diversity and population structure of worldwide collection of tetraploid cultivated alfalfa germplasm as accessed by SSR markers. In China, alfalfa has been identified a major forage species for genetic improvement. The genetic diversity revealed by 336 genotypes from a worldwide collection of alfalfa germplasm in the study was high as reflected in average allele number per locus (12.4), He (0.677) and PIC (0.638). Bagavathiannan et al.  evaluatet et al.  investigt et al.  which det et al.  versus 2 = 0.71) . The sim = 0.71)  that minIn the present study, alfalfa landraces represented the highest genetic diversity followed by wild materials while cultivars materials had the lowest values . One posM. sativa—falcata complex [Population structure analysis enables to understand genetic diversity in a given collection and identify the appropriate population for association mapping . Many pr complex , 33. In It is necessary to use a large number of accessions from each geographical location to confirm whether the clustering pattern of accessions from closer geographical origins agrees with the available pattern identified in this study. However, different relationships may be obtained using different numbers of markers, genome coverage, degree of linkage disequilibrium, the type of DNA sequence variation detected with different marker systems and/or the type of sequence assays (low/high copy).S1 FigViolin plots show density distribution of PIC values, horizontal bar indicates average value, median is shown as white circle, top and bottom of vertical bar represent the first and third quartile.(TIF)Click here for additional data file.S2 FigB Estimating number of subpopulations using delta K values for K ranging from 1 to 10 using method proposed by Evanno et al. (2005)(TIF)Click here for additional data file.S3 FigViolin plots show density distribution of PIC values, median is shown as white circle, top and bottom of vertical bar represent the first and third quartile.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.


The recent availability of sheep genome-wide SNP panels allows providing background information concerning genome structure in domestic animals. The aim of this work was to investigate the patterns of linkage disequilibrium (LD), the genetic diversity and population structure in Valle del Belice, Comisana, and Pinzirita dairy sheep breeds using the Illumina Ovine SNP50K Genotyping array.r2 between adjacent SNPs across all chromosomes was 0.155 ± 0.204 for Valle del Belice, 0.156 ± 0.208 for Comisana, and 0.128 ± 0.188 for Pinzirita breeds, and some variations in LD value across chromosomes were observed, in particular for Valle del Belice and Comisana breeds. Average values of r2 estimated for all pairwise combinations of SNPs pooled over all autosomes were 0.058 ± 0.023 for Valle del Belice, 0.056 ± 0.021 for Comisana, and 0.037 ± 0.017 for Pinzirita breeds. The LD declined as a function of distance and average r2 was lower than the values observed in other sheep breeds. Consistency of results among the several used approaches  showed that while Valle del Belice and Pinzirita breeds formed a unique cluster, Comisana breed showed the presence of substructure. In Valle del Belice breed, the high level of genetic differentiation within breed, the heterogeneous cluster in Admixture analysis, but at the same time the highest inbreeding coefficient, suggested that the breed had a wide genetic base with inbred individuals belonging to the same flock. The Sicilian breeds were characterized by low genetic differentiation and high level of admixture. Pinzirita breed displayed the highest genetic diversity  whereas the lowest value was found in Valle del Belice breed.Average This study has reported for the first time estimates of LD and genetic diversity from a genome-wide perspective in Sicilian dairy sheep breeds. Our results indicate that breeds formed non-overlapping clusters and are clearly separated populations and that Comisana sheep breed does not constitute a homogenous population. The information generated from this study has important implications for the design and applications of association studies as well as for development of conservation and/or selection breeding programs.The online version of this article (doi:10.1186/s12863-014-0108-5) contains supplementary material, which is available to authorized users. If pedigree information is not available, LD analysis might offer an alternative perspective for the estimation of Ne [The application of recently developed genomic technology, such as high-density single nucleotide polymorphism (SNP) arrays, has great potential to increase our understanding on the genetic architecture of complex traits, to improve selection efficiency in domestic animals through genomic selection , and to on of Ne . In locaon of Ne . Currenton of Ne .In Sicily, dairy sheep production represents an important resource for the economy of hilly and mountain areas, in which other economic activities are limited . NowadayIn the present study, the Illumina OvineSNP50K Genotyping BeadChip was used to characterize LD and to analyze genetic diversity and population structure in Sicilian dairy sheep breeds.Out of a total of 54,241 SNPs genotyped, 378 were unmapped and 1,450 were located on sex chromosomes. Thus, 52,413 SNPs mapped onto 26 sheep autosomes were used, and after filtering see , the finr2 was used as measure of LD, because is the most suitable measure of LD for biallelic markers [r2 ranged from 0.133 ± 0.187 for OAR14 to 0.182 ± 0.229 for OAR2 in Valle del Belice, from 0.134 ± 0.188 for OAR14 to 0.189 ± 0.237 for OAR2 in Comisana, whereas the lowest values among chromosomes were observed in Pinzirita, where r2 ranged from 0.107 ± 0.164 for OAR23 to 0.148 ± 0.208 for OAR2 , marker types (microsatellite or SNP), marker density and distribution, and population demography [r2 estimated for all pairwise combinations per chromosomes that ranged from 0.006 in OAR1 to 0.015 in OAR20, and an average r2 of 0.061 for SNPs separated by 200–500 kb, in agreement with our results. Usai et al. [r2 over 1,000 kb of 0.072. Previous studies in five populations of domestic sheep based on microsatellite markers [r2 = 0.192 ± 0.131 for markers separated by 5–10 cM, and high levels of LD observed over 4 Mb, respectively). Levels of r2 for adjacent SNP pairs and all pairwise combinations of SNPs in Sicilian sheep breeds were lower than values observed in other livestock species such as pig [r2 level of 0.2. Qanbari et al. [r2 threshold of 0.25 as a useful LD value for association studies. In fact, species with extensive LD will require fewer markers than those with low levels of LD. Therefore, these results support the need to use more dense SNP panels for high power association mapping and genomic selection efficiency in future breeding programs for Sicilian dairy sheep breeds, and in particular for Pinzirita breed.The extent of LD was first evaluated for each adjacent SNP pairs. The  markers  and to a markers . The aveFST and Neighbor network were used to visualize and explore the genetic relationships among breeds. Genotypes from seven additional sheep breeds belonging to International Sheep Genomics Consortium , Bayesian model-based clustering algorithm, calculation of After data editing, a total of 42,422 SNPs common to 486 individuals from 10 sheep breeds were analyzed. The PCA showed that most sheep breeds formed non-overlapping clusters and are clearly separated populations, except for Sarda white and Sarda black sheep breeds  clustered animals from the same breed together, the Comisana breed showed two groups Figure . In factMoreover, some individuals of Comisana breed were positioned near the cluster of Pinzirita breed. The genetic closeness between these two breeds might be explained considering that they are characterized by a common breeding system and geographical husbandry area, which might have led to genetic exchange between them . The relFST among all populations, Chios was the most divergent breed. The highest values were observed between Chios and both Sarda breeds , while the lowest one was observed between Comisana and Pinzirita breeds (FST = 0.025). These results may be explained considering the geographic origin of the breeds: Chios and the two Sarda breeds belonging to different countries, whereas Comisana and Pinzirita were two breeds reared in the same island. The FST value between pairs of breeds was also used to reconstruct the NeighborNet graph , pointed out that the best fitting number of populations present in the total sample was K = 12. A graphic representation of the estimated membership coefficients to the 12 clusters is shown in Figure FST, Neighbor networks) supported the robustness of our conclusions.Understanding the relationships among and within populations is an important step to establish conservation priorities and strategies. Consistency of results among the several used approaches , whereas the lowest value in Pinzirita breed (0.016 ± 0.042). In Valle del Belice breed, the high level of genetic differentiation within breed, with individuals spread out on PC1 . In fact, LD is affected by population history and demography, representing an important tool to be applied to genetic population. Effective population size (Ne) is a general indicator of the risk of genetic erosion, contains relevant information for the monitoring of the genetic diversity, and helps to explain how populations evolved [e, estimated 50 generations ago, was observed for Pinzirita breed (Ne = 685), whereas the lowest one was observed for Valle del Belice breed (Ne = 369)  used to assess relationships among breeds showed high degree of congruence and supported the robustness of conclusions. Analysis of genetic diversity indicated high genetic variability and low inbreeding. The identification of genetic relationship and gene flow among livestock breeds/populations is important for breeders and conservationists. In order to maintain the existing genetic diversity, breeding strategies aiming at maintaining effective population size, minimizing inbreeding and genetic drift should be implemented for the Sicilian breeds. The information generated from this study can have important implications for the design and applications of association studies as well as for the development of conservation and/or selection breeding programs.This study reported for the first time estimates of linkage disequilibrium, genetic diversity and population structure from a genome-wide perspective in Sicilian dairy sheep breeds. Knowledge concerning the behavior of LD is important for performing genomic selection and genome wide association analysis. LD declined as a function of distance and average A total of 221 unrelated animals were collected from several farms in different areas of Sicily, to capture a representative sample of within breed genetic diversity, and were used for the analysis.The procedures involving animal sample collection followed the recommendation of directive 2010/63/EU. Number of animal sampled per flock ranged from 5 to 10. Samples consisted of 72 Valle del Belice, 72 Comisana and 77 Pinzirita individuals. For these sheep breeds, pedigree data were not available. About 10 ml of blood was collected from jugular vein using tubes with EDTA as anticoagulant. Genomic DNA was extracted from buffy coats of nucleated cells using salting out method . The conP-value > 0.001. SNPs that did not satisfy these quality criteria were discarded.All animals were genotyped for 54,241 SNPs, using the Illumina OvineSNP50K Genotyping BeadChip following standard operating procedures recommended by the manufacturer. Genotyping was performed by Dipartimento Scienze Agrarie e Forestali, University of Palermo. Raw signal intensities were converted into genotype calls using the Illumina GenomeStudio Genotyping Module v1.0 software  by applying a no-call threshold of 0.15. Genotyping data were initially tested for quality using the same software. Markers in each breed were filtered to exclude loci assigned to unmapped contigs. Therefore, only SNPs located on autosomes were considered in further analyses. Moreover, quality control included: Call Frequency (proportion of samples with genotype at each locus) ≥ 0.95, minor allele frequency (MAF) ≥ 0.05, and Hardy-Weinberg Equilibrium (HWE) r2), was used as measure. Pairwise LD between adjacent SNPs was calculated on each chromosome using PLINK [r2 was estimated for all pairwise combinations of SNPs using LD plot function in Haploview v4.2 software [r2 was calculated for SNPs between 0 and 50 Mb apart. To visualize the LD pattern per chromosome, r2 values were stacked and plotted as a function of inter-marker distance categories. Average r2 for SNP pairs in each interval was estimated as the arithmetic mean of all r2.A standard descriptive Linkage Disequilibrium (LD) parameter, the squared correlation coefficient of allele frequencies at pair of loci  was calculated as IBS2 + 0.5*IBS1/N, where IBS1 and IBS2 are the number of loci that share either one or two alleles identical by state (IBS), respectively, and N is the number of loci tested. Genetic distance (D) was calculated as 1-As. These values were calculated using PLINK [-cluster and -distance-matrix. PCA of D matrix was performed using the multidimensional scaling (MDS) algorithm of pairwise genetic distance implemented in PLINK [D matrix, it is numerically identical to PCA [R software (R Development Core Team) with RColorBrewer package.First, the average proportion of alleles shared between animals (ng PLINK  through in PLINK . It shoul to PCA . The graR software (R Development Core Team).Extents of population substructure were evaluated through the model-based clustering algorithm implemented in the software Admixture . The mosFST among all breeds. Neighbor networks were constructed from pair-wise estimates of FST using Splitstree [Genepop  softwarelitstree .F) for Sicilian sheep breeds. Files used for basic diversity indices  were pruned in PLINK considering 50 SNPs per windows, a shift of 10 SNPs between windows and variation inflation factor’s threshold of 1.5. Moreover, effective population sizes (Ne) were calculated as Ne = (1/4c)*(1/ r2-1) [r2 is the value of LD and c is the marker distance in Morgans between SNPs.PLINK  was also1/ r2-1) , where r


Oncorhynchus spp.), seasonal declines in reproductive life span have been attributed to adaptation-by-time, in which divergent selection for different traits occurs among reproductively isolated temporal components of a population. We evaluated this hypothesis in kokanee (freshwater obligate Oncorhynchus nerka) by testing for temporal genetic structure in neutral and circadian-linked loci. We detected no genetic differences in presumably neutral loci among kokanee with different arrival and maturation dates within a spawning season. Similarly, we detected no temporal genetic structure in OtsClock1b, Omy1009uw, or OmyFbxw11, candidate loci associated with circadian function. The genetic evidence from this study and others indicates a lack of support for adaptation-by-time as an important evolutionary mechanism underlying seasonal declines in reproductive life span and a need for greater consideration of other mechanisms such as time-dependent, adaptive adjustment of reproductive effort.Seasonal declines of fitness-related traits are often attributed to environmental effects or individual-level decisions about reproductive timing and effort, but genetic variation may also play a role. In populations of Pacific salmon ( Oncorhynchus, fish arriving later for spawning often have short reproductive life spans (RLS), resulting in less time available for reproductive activities and parental care prior to their rapid physiological decline and death. These declines in RLS are apparent over short arrival periods of 12–35 days in multiple populations and species and are quite steep .In seasonal breeders, traits related to reproductive effort and parental care often decline with breeding date. In temperate-breeding birds, for example, later breeders may have smaller clutch sizes, slower nestling growth rates, or lower fledging masses Brinkhof . In PaciWhile adaptation-by-time can apply to any seasonally varying trait, seasonal declines in RLS are an obvious contender because of seasonally varying selective pressures, highly heritable breeding dates , and evidence of reproductive isolation-by-time (Hendry and Day O. nerka) entering a creek 21–25 day apart or 13–15 day apart, FST was 0.006 (significant) and 0.003 (nonsignificant), respectively  , RL = red late , SE = silver early , and SR = silver that arrived early (5–8 September) but matured in a pen . With these groups, we were able to compare genetic variation on the basis of arrival date or maturation date. All procedures performed on live animals were approved by Western University’s Animal Use Subcommittee (AUP 2013-033).Over 18 days in September 2013, we used dip nets to capture females at the enumeration fence. A short breeding season is typical of this population, and previous research shows steep seasonal declines in the period between arrival and death over a 21-day arrival period , and all allele calls were manually verified.DNA was extracted from each sample using a NucleoSpin Tissue Kit (Macherey-Nagel) following manufacturer’s protocols. Nuclear genotypic data were collected from nine neutral microsatellite loci and three circadian-linked microsatellites or genes with fragment length polymorphisms Table . All forFST outlier detection tests as implemented in LOSITAN , and expected heterozygosity (He) were calculated at each locus for each group using GenAlEx  using 1000 simulations and both the chi-squared and Fisher’s testing approaches. Power was estimated as the proportion of significant outcomes. Levels of genetic differentiation among groups under various grouping strategies were estimated by pairwise comparisons of θ  from 1 to 5 with 20 iterations per value of K. The most likely number of clusters was determined by plotting the log probability of the data (ln Pr(X|K)) across the range of K values tested and selecting the K where the value of ln Pr(X|K) plateaued as suggested in the STRUCTURE manual. We also calculated ΔK  were performed using ARLEQUIN  not previously detected in kokanee (Lemay and Russello The β > 0.8) to detect small levels of divergence (FST = 0.005), with the proportion of significant outcomes at this level equaling 0.958 and 0.934 for the chi-squared and Fisher’s exact tests, respectively  and was longer in 1999 than in 1998 . Using RMA regression, the estimated slope of the decline was −0.593 in 1998 and −0.592 in 1999. Over an 18-day period, a decline of this magnitude would correspond to a total shortening of RLS by 11 days.In 1998 and 1999, female RLS declined with spawning date  and their timing of maturation (early or late), there was no genetic differentiation Table , with anOtsClock1b or other circadian-linked loci. This result may not be surprising given only three OtsClock1b PolyQ length variants in our study. Moreover, no other study has reported temporal genetic structure of circadian-linked loci at such a short time scale. Only at time scales beyond a month do genetic differences begin to emerge. For example, in one of five comparisons, OtsClock1b showed significant divergence (FST = 0.042) between seasonal runs of Chinook salmon (O. tshawytscha) , pink salmon (O. gorbuscha), and chum salmon  maturing over a 2-month period, Abadía-Cardoso et al. (h2 = 0.50) and females (h2 = 0.56).Overall, phenological traits such as migration timing and maturation timing show high heritability in salmon, with median o et al.  estimateIf adaptive seasonal declines in RLS are not the outcome of adaptation-by-time, what other mechanism could be responsible? An alternative explanation is condition-dependent arrival timing  that affect the timing of maturation. In salmonid fishes, reproductive development seems to be controlled by developmental decisions far in advance of breeding, although the specific factors influencing variation in rate or onset of sexual maturation are not well understood (Thorpe et al. One important limitation of our study is the reliance on a limited number of neutral loci to detect population structure and only three targeted gene regions to detect loci under selection. The possibility remains that a full genomic scan would reveal loci under selection in association with early- versus late-arriving or with early- versus late-maturing fish. Russello et al.  applied