Na), allelic richness (Rs), Nei’s genetic diversity index (He), observed heterozygosity (Ho) and polymorphism information content (PIC) revealed increase of diversity over the periods in year of releasewise and longevitywise classification of rice varieties. Analysis of molecular variance (AMOVA) suggested more variation in within the decadal periods than among the decades. Pairwise comparison of population differentiation (Fst) among decadal periods showed significant difference between all the pairs except a few. Analysis of trends of appearing and disappearing alleles over decadal periods showed an increase in the appearance of alleles and decrease in disappearance in both the categories of varieties. It was obvious from the present findings, that genetic diversity was progressively on the rise in the varieties released during the decadal periods, between 1970s and 2000s.Genetic diversity in representative sets of high yielding varieties of rice released in India between 1970 and 2010 was studied at molecular level employing hypervariable microsatellite markers. Of 64 rice SSR primer pairs studied, 52 showed polymorphism, when screened in 100 rice genotypes. A total of 184 alleles was identified averaging 3.63 alleles per locus. Cluster analysis clearly grouped the 100 genotypes into their respective decadal periods i.e., 1970s, 1980s, 1990s and 2000s. The trend of diversity over the decadal periods estimated based on the number of alleles ( In rice, like many crops, selection preference has been for improvement of yield enhancing traits like compact panicle with more grains/panicle, large seed size, non-shattering habit etc. The selection process continued for centuries result in cultivars far different from the wild/weedy progenitor species in their habit and potential. Since the advent of the short statured high yielding varieties in the mid-sixties, the selection priority of breeders has been for higher stability or performance, need based crop duration, tolerance to various stresses and consumer preferred grain quality. Excessive breeding emphasis in this direction given during the last 50 years knowingly or unknowingly has led to some sort of genetic uniformity among the currently cultivated high yielding varieties. The improved germplasm is being excessively depended on for needed variability for progressive improvement of the crop with no precise knowledge of extent of exploitable variability beyond simply inherited traits. Also it is not clear to what extent breeding strategies Precise knowledge of exploitable genetic diversity and genetic relatedness among the constituents of the improved gene pool is vital for meticulously planning and executing target directed breeding especially when genetic relatedness among the improved parental sources appears to become increasingly narrow. Unlike morpho-physiological traits used earlier to estimate genetic variability/relatedness, molecular markers have become quite handy in precisely understanding the extent of genetic divergence among varieties being chosen these days as parental sources in breeding programs. Many recent scientific papers tracing the trend of genetic diversity in crop varieties released over the years reveal the diversity levels to often fluctuate strongly from one time period to the successive periods Since the introduction of semidwarf high yielding varieties in the mid 1960s, simultaneously launch of the All India Coordinated Rice Improvement Programmes (AICRIP) and active involvement of International Rice Research Institute (IRRI), Philippines facilitated rapid exchange of germplasm among breeders, institutes and between countries culminating in the release of around 1000 high yielding varieties in the country. Despite such large number of varieties developed using diverse germplasm, molecular marker based diversity analysis has shown the genetic base of Indian rice gene pool to be surprisingly narrow viz., 1970s, 1980s, 1990s and 2000s. They were as well classified into “longevitywise” as differences in genetic diversity could be observed between them, especially when a variety is popular within and across decades.The experimental material comprised of 100 rice cultivars, which included 89 high yielding varieties released between 1970 and 2010 in different states of India and 11 traditional varieties . They weHind III DNA as standard. Based on the intensity and thickness of genomic DNA bands, as compared to lambda (λ) Hind III DNA, the concentration and quality of DNA in individual samples were determined.Genomic DNA was isolated from 20 day-old seedlings germinated in sterile petri dishes lined with moist filter paper using the CTAB (Cetyl Try methyl Ammonim Bromide) method described 2, 0.2 mM each dNTPs, 5 pmol of each forward and reverse primer, 0.5U of Taq DNA polymerase (NEB), and 5 ng of genomic DNA. Reactions were carried out in GenAmp PCR system 9700  thermal cycler using the following temperature profile: an initial denaturation of 5 min at 94°C followed by 35 cycles of 45 s at 94°C, 45 s at 55°C and 1 min at 72°C, then a final extension of 5 min at 72°C. Amplification products were resolved on 3% metaphor agarose gels using a horizontal gel electrophoresis unit . The DNA fragments were then visualized under UV-transilluminator and documented using ALPHA IMAGER gel documentation system  which was stored for further scoring and permanent records.In all, 64 hypervariable microsatellite markers distributed on all the 12 chromosomes of rice were picked up from our previous study 2 - ΣΣPi2 Pj2 where ‘i’ is the total number of alleles detected for SSR marker and ‘Pi’ is the frequency of the ith allele in the set of hundred genotypes investigated and j = i+1 Only clear and unambiguous bands of SSR markers were scored. The sizes of the amplified fragments were estimated with the help of Alpha image software by Gel documentation system using 100 bp DNA ladder (NEB) as size standard. Markers were scored for the presence (1) or absence (0) of the corresponding band among the genotypes. To measure the informativeness of the markers, polymorphism information content (PIC) for each of the SSR markers was computed according to the formula: PIC = 1-ΣPiviz., number of alleles (Na), observed heterozygosity (Ho), Shannon Index (I) and Nei’s genetic diversity index (He) http://www.ualberta.ca/~fyeh ). The allelic richness (Rs), is a measure of the number of alleles independent of the sample size, was measured using FSTAT t-test (http://www.stattools.net/Wilcoxon_Pgm.php). In addition, number of rare alleles  and high frequency or common alleles  were estimated using Microsoft Excel 2007. The F-statistics (Fst) were used to analyze genetic differentiation of the varieties in all possible pairs of decades based on allelic discrepancy at each locus by the procedure of AMOVA , besides the genetic variation within and among the populations using the software ARLEQUIN v 2.0 http://darwin.cirad.fr/). The dendrogram based on unbiased genetic distances among decadal periods was constructed by UPGMA (Unweighted pair-group method with arithmetic average) employing POPGENE v 1.31.Genetic diversity parameters et al (7.8) et al. (7.2) et al. (4.37) et al.et al.et al.et al.He values ranged from 0.15 (RM12031) to 0.76 (RM14735) with an average of 0.59 and more than 40% of the loci were in the frequency of 0.7 to 0.8  markers distributed evenly on all the 12 chromosomes were chosen to assess the genetic relatedness among the 100 genotypes. Fifty-two of them were found to show polymorphism (81.25%). In all,7 to 0.8 . The aveviz., landraces, 1970s, 1980s, 1990s, and 2000s  in improved Samba mahsuri, remaining in the improved versions of the varieties even after many backcrosses. Longevitywise clustering of the 100 genotypes too was in total agreement with the decadewise clustering. Even though some varieties were popular beyond the decade(s) of their release, they do not affect the clustering pattern of the genotypes as well as the genetic diversity of successive decadal periods. For instance, Tella Hamsa and Jyoti remain popular even as late as in the 2000s, albeit released decades back in the states of Andhra Pradesh and Kerala, respectively.The unweighted neighbour-joining (UNJ) dendrogram constructed on the basis of genetic similarity matrix grouped the 100 genotypes into five clusters nd 2000s . The phyGenetic distance between each pair of decadal periods suggest that it was the highest between the landraces and the high yielding varieties of the 1990s (0.2715), while the lowest between the decades 1990s and 2000s (0.0905) in the year of releasewise group. In case of longevitywise group also genetic distance was highest between the landraces and the high yielding varieties of the 1990s (0.210) but lowest between those of the 1980s and 2000s (0.0237) . A dendr(Na), Nei’s genetic diversity index (He) and polymorphism information content (PIC)  along with sets of landraces and Basmati accessions was studied based on number of alleles nt (PIC) . As wellNa) was observed from landraces to varieties of 2000s in the categories of both year of releasewise and longevitywise classified rice varieties. Na values were increased by 13.38% and 13.87% from landraces to 2000s while it was 12.79% and 9.83% from 1970s to the 2000s, respectively for both the categories. On an average, landraces to total modern cultivars percentage of Na increase has been observed to be 9.02 for year of releasewise and 10.91 for longevitywise varieties. This is in contrast to the observation of Wei et al.Na score of 7.8% from the 1950s to the 1990s.Overall increasing trend of number of alleles (He) also reveals that the genetic diversity has been increased approximately 10% from landraces to 2000s and landraces to modern cultivars in the year of releasewise category while it was 16.02% and 13.39% for longevitywise category, respectively. However, from 1970s to 2000s the trend of increase was 4.71% and 8.92% for year of releasewise and longevitywise varieties, respectively. The observed trends in the present study are in agreement with Mantegazza et al.,et al.,et al.,Genetic diversity index  also shows increasing trend over decadal periods, though not to the level of He = 0.54) as estimated by the Nei’s genetic diversity index in the present study is nearly equal to that of the representative world collection of indica varieties (He = 0.55). It is, however, overwhelmingly high as compared to the representative world collection of temperate japonica (He = 0.39) indica varietal group to be much more genetically diverse than japonica group which is in agreement with many earlier reports The genetic diversity in Indian cultivars (Na), polymorphism information content (PIC) and genetic diversity index (He), to increase progressively in the advancing decadal periods from 1970s to 2000s and interestingly, it was higher as compared to even landraces. Use of genotypes, which cover longer periods always offer added advantage in temporal studies. In the present study only a few landraces which are being cultivated for their certain features by local farmers has been included to compare genetic diversity in varieties released before and after 1970s. The analysis of genetic diversity using various parameters clearly reveals the increasing trend from landraces to the decadal period 2000s. While this observation is in agreement with those of Mantegazza et alet alet alet alindica subspecies of Oryza sativa, we could not compare with the japonica varieties in terms of allele appearance and disappearance. However, earlier indica varieties than in japonica. This phenomenon can be attributed to the higher diversity nature of indica varieties compared to japonica.Overall, the present investigation reveals a genetic diversity as estimated using different parameters such as allele number -1 and IR8 and varieties derived from crosses involving them with traditional Indian varieties could have introduced large variability for yield, earliness, photo-insensitivity, seed non-dormancy etc. In the subsequent decades, the focus has been shifted towards the development of polygenic durable resistance to multiple pathogens, adaptation to diverse rice ecologies and consumer preference driven quality improvement. For instance, high yielding varieties of the initial decades were more susceptible to indigenous as well as introduced diseases and insect pests like bacterial blight, tungro virus, hoppers etc. warranting to exploit exotic cultivar and wild/weedy sources. Convergent breeding efforts have made most of the high yielding varieties of today multiple pest resistant. Given the challenges ahead which have all the potential to impede further progress in crop improvement, there is need to enrich the variability employing multiparent breeding and exploitation of hidden variability from progenitor species and primitive cultivars.et al.Fst) among decadal periods reveals significant genetic differentiation among all except between the landraces and the decadal period of 1970s as estimated by both year of releasewise and longevitywise analysis. Also there was no significant differentiation between the 1970s and rest of the decades 1980s to 2000s in longevitywise analysis  has revealed most of the variation was existed within decadal periods i.e., 92.12% and 96.66%, respectively for year of releasewise and longevitywise as against very low variation between the decadal periods i.e., 3.34% for longevitywise classified varieties and 7.88% for year of releasewise classification . These ranalysis .viz., RM11356, RM12031, RM13131, RM14735, RM15004, RM15580, RM5693, RM5907, RM18384, RM22554, RM22565, RM22688, RM23741, and RM8207. Interestingly, three loci viz., RM11313, RM12353, and RM6965 have been found to explain more than 20% of the variation among the decades (Locus-by-locus AMOVA showed significant (p<0.01) differences in genetic variation among the decadal periods as exhibited by 14 loci  decades .Analysis for identification of unique population specific alleles has revealed all the decadal periods found to show specific alleles with the exception of the 1970s as estimated by year of releasewise as well as longevitywise classification. As expected, the landraces exhibited as many as six  and seven  specific alleles compared to the decadal periods in both year of rleasewise and longevitywise categories, respectively . While tQualitative analysis of genetic diversity revealed that the landraces were comprising of more number of population specific alleles as compared to the modern high yielding varieties suggesting loss of some of the landrace specific alleles on account of intensive human selection over the decades. The present findings are in agreement with those of earlier workers . In contrast, Qi etalEver since the domestication of rice, man’s breeding priority has been yield enhancement and “breeding out” the wild progenitor traits like lax panicle, shattering, awns, etc., and later the focus had been shifted towards “breeding in” the traits of plant architecture and resistance to biotic and abiotic stresses and acceptable cooking quality. In that long breeding process, except the genomic regions, which govern domestication related traits, a major part of the rice genome remains unchanged and hence large unfolded genetic diversity. To estimate genetic variability in crop improvement research, increasingly crop specific microsatellite markers are used. Since most of the microsatellites are non-coding, estimates made and inferences drawn based on them may not reflect true genomic diversity of a crop. To understand precisely the changing trends of genetic diversity in breeding material over periods, it is necessary to consider markers located both in genic and non-genic regions. In the present study, thus, microsatellite markers located in both genic and non-genic regions have been chosen to understand the level and trend of genetic diversity in varieties developed over the last four decades in comparison to the traditional varieties. The study using two marker types separately and together interestingly showed an increasing trend of genetic diversity . AnalysiNa), number of effective alleles (Ne), Shannon index (I) and Nei’s genetic diversity index (He) estimated according to their region of release in India , ecology (irrigated and rainfed), grain size (long and medium) and days to 50% flowering  of a representative set of Indian rice varieties, observed variation was not correlating with their classification , genetic diversity (He) over decadal periods using genic and non-genic SSRs along with gene-specific markers(previous studies).Changes in number of alleles (TIF)Click here for additional data file.Figure S2(Na), number of effective alleles (Ne), Shannon index (I) and Nei’s genetic diversity (He) estimated in different groups of rice varieties i.e., region, ecology, grain size and days to 50% floweringwise.Number of alleles (TIF)Click here for additional data file.Table S1Details of the rice cultiavars used in the study.(XLS)Click here for additional data file.Table S2Summary statistics of genetic diversity parameters of major Indian rice cultivars.(DOCX)Click here for additional data file.Table S3Locus by locus AMOVA among populations.(DOCX)Click here for additional data file.Table S4Population specific alleles and their corresponding markers.(DOC)Click here for additional data file.Table S5List of genic and non-genic SSRs and their functions.(DOC)Click here for additional data file.Table S6Summary statistics of genetic diversity parameters of Indian rice cultivars using different microsatellite classes.(DOCX)Click here for additional data file.Table S7Summary statistics for genetic diversity parameters of Indian rice varieties classified according to their region, ecology, grain size and days to 50% flowering.(DOCX)Click here for additional data file.


ST = −0.00335, p > 0.05), corroborating our previous study. Three variable sites, however, showed significant base composition heterogeneity between samples from the northern and southern waters . Nucleotide substitutions at the three sites converged on an apical clade, which consisted of the five southern individuals, whereas its sister clade consisted only of the three northern individuals. This phylogenetic information corroborates previous ecological studies indicating the presence of separate stocks in the northern and southern waters.This study deals with mitochondrial phylogenetic information of Japanese flounder in the Pacific coast of Tohoku Japan to estimate the genetic population subdivision that was undetectable by conventional population statistics. We determined complete sequences of mitochondrial NADH dehydrogenase subunit-2 (ND2) and subunit-5 (ND5) genes for 151 individuals from northern  and southern  waters. Samples from both waters showed high genetic diversity, including 126 haplotypes. These haplotypes were located at mixed and nested positions on an inferred phylogenetic tree, and traditional F-statistics indicated no significant population divergence (ϕ Many marine organisms spawn pelagic eggs, and their larvae are capable of dispersal over long distances . SignifiParalichthys olivaceus is widely distributed along the coastal waters off Japan, Korea and China [Japanese flounder nd China ,15. Thisnd China ,17. Ecolnd China . These snd China –27. Elevnd China ,27, inclIn the Pacific coast off northern Japan, Tohoku district, previous tagging data and life history research suggested two diverged populations, each comprising northern  or southern  waters –24. TherST or even ϕST that was suggested to be a better index for population diversification [ST ≈ 0). However, if migration between subpopulations is limited for a while during which new clades appear by additional nucleotide substitutions, migrants do not carry members of some of these clades by chance. The limited migration thus leaves genetic footprints, i.e., biased distribution of some clades and base composition heterogeneity at sites that define these clades.The mitochondrial genetic difference between samples from the northern and southern waters, if any, would also be small or undetectable by conventional population statistics, e.g., Ffication . In an iIn this study, to explore the possibility of genetic divergence of Japanese flounder in the Pacific coast of Tohoku Japan, we tried to reveal genetic footprints of the incipient state of population subdivision with mitochondrial nucleotide variation in relation to the character state along phylogenetic tree.We determined the whole sequences of NADH dehydrogenase subunit-2 (ND2: 1046 bp) and subunit-5 (ND5: 1839 bp) genes for all 151 individuals. There was no gap in these regions among the material fish examined. The haplotype diversity values (h) of samples from the northern and southern waters were 0.9973 ± 0.0022 and 0.9966 ± 0.0040, respectively. The nucleotide diversity values (π) of the samples from northern and southern waters were 0.0079 ± 0.0039 and 0.0075 ± 0.0037, respectively. The neighbor-joining tree showed the two major mitochondrial clades (lineage-A and -B) with deep branching , as in op < 0.01) .We found three nucleotide sites , which show base composition heterogeneity between samples from the northern and southern waters  . All of  < 0.01) . A “C” tWild Japanese flounder show high mitochondrial haplotype diversity with two major lineages sympatrically distributed along the coast of Japan . SamplesST = −0.00335, p > 0.05) that is better to display divergence between highly variable populations than FST [A value of fixation index  and 60 in Fukushima and Miyagi Prefectures  in 2005 . BecauseExtractions of genomic DNAs from muscle tissues followed . We sequI + G distance, whose substitution model was selected by AIC comparison, using MEGA5 [ST) between the two samples was calculated using Alrequin version 3.11 [Calculations of haplotype and nucleotide diversities for each samples followed . We counng MEGA5 . Then, tng MEGA5  under thion 3.11 .The present study showed that upon phylogenetic and base composition analyses, even neutral or near neutral genetic markers could indicate small and incipient population subdivision, which is undetectable with conventional population statistics. The phylogenetic information of Japanese flounder corroborates previous ecological studies indicating the presence of separate stocks in the northern and southern waters of the Pacific coast of Tohoku Japan.


Zootoca vivipara (= Lacerta vivipara) in the Iberian Peninsula and test the role of the Pyrenees and the Cantabrian Mountains in restricting gene flow and driving lineage isolation and divergence. We also assess patterns of introgression among lineages upon secondary contact, and test for the role of high-elevation trans-mountain colonisations in explaining spatial patterns of genetic diversity. We use mtDNA sequence data and genome-wide AFLP loci to reconstruct phylogenetic relationships among lineages, and measure genetic structure.The geographic distribution of evolutionary lineages and the patterns of gene flow upon secondary contact provide insight into the process of divergence and speciation. We explore the evolutionary history of the common lizard The main genetic split in mtDNA corresponds generally to the French and Spanish sides of the Pyrenees as previously reported, in contrast to genome-wide AFLP data, which show a major division between NW Spain and the rest. Both types of markers support the existence of four distinct and geographically congruent genetic groups, which are consistent with major topographic barriers. Both datasets reveal the presence of three independent contact zones between lineages in the Pyrenean region, one in the Basque lowlands, one in the low-elevation mountains of the western Pyrenees, and one in the French side of the central Pyrenees. The latter shows genetic evidence of a recent, high-altitude trans-Pyrenean incursion from Spain into France.Z. vivipara lineages at large geographic scales. However, mountain ranges are not always effective barriers to dispersal, and have not prevented a recent high-elevation trans-Pyrenean incursion that has led to asymmetrical introgression among divergent lineages. Cytonuclear discordance in patterns of genetic structure and introgression at contact zones suggests selection may be involved at various scales. Suture zones are important areas for the study of lineage formation and speciation, and our results show that biogeographic barriers can yield markedly different phylogeographic patterns in different vertebrate and invertebrate taxa.The distribution and age of major lineages is consistent with a Pleistocene origin and a role for both the Pyrenees and the Cantabrian Mountains in driving isolation and differentiation of  The geographical distribution of intraspecific lineages and the pattern of gene flow among them provides valuable insight into the process of lineage divergence and speciation . Of partZootoca vivipara (= Lacerta vivipara) in the Ibero-Pyrenean region. The species has a broad Eurasian distribution composed largely of viviparous lineages, yet individuals in this region belong to an oviparous lineage isolated from the nearest viviparous populations in the French Massif Central by a gap of unsuitable habitat [Z. vivipara in this region have documented a sharp contact zone between two divergent maternal lineages corresponding generally to the French and Spanish sides of the Pyrenees, and showing consistent results with mtDNA 16S and cytochrome b gene sequences [One of these regions of secondary contact is the Ibero-Pyrenean suture zone, an important biogeographic region where both inter and intra-specific lineages are known to come into contact in a number of taxa -6. In th habitat . Recent equences ,9 and a equences . HoweverInferring the demographic and evolutionary history of lineages at various spatial and temporal scales often requires the use of molecular markers from genomic regions with different modes of inheritance and rates of evolution . MitochoZ. vivipara. We also use this large-scale phylogeographic context to assess patterns of introgression and test hypotheses related to the history of contact zones.Here we undertake a multilocus phylogeographic survey across the region using both mtDNA sequences and genome-wide AFLP loci and an improved geographic sampling in order to document the presence and distribution of major genetic lineages and test the role of the Pyrenees and the Cantabrian Mountains in driving lineage divergence in Z. vivipara from 48 localities .Sequencing of 200 individuals of es Table  revealedPhylogenetic analysis revealed two major clades corresponding generally to southern France and northern Spain, respectively Figures  &2A. Thes, consistent with a recent burst of population growth , Φ41 Table .BayScan 2.0 analysis across all comparisons, although one of them (locus “15c194”) was identified as an outlier in the K = 2 comparison, and another one (locus “19c84”) in the K = 3 and K = 4 comparisons  and thus most markers contributing to the pattern of genetic structure were selectively neutral.None of the 34 loci showed significant departures from neutrality in the m locus “5c194” waUsing the geographic distribution of the main four mtDNA clades and the comparable AFLP-defined genetic units corresponding to K = 4, we detected localities with mixed haplotypes (for mtDNA) or assignment probability values (for ALFPs) in two main areas within the region, one corresponding to the contact zone between blue and green lineages across the Pyrenees, and one between blue and red lineages in Navarre, just east of the Basque Country Figure .The contact zone in southern France between divergent blue and green mtDNA lineages, apparent in Figures At a second mtDNA contact zone detected between the red and blue clades at the Ibañeta site in the Spanish side of the western Pyrenees Figure A, the AFA final discrepancy between the two datasets pertains to the assignment of the Beret site (BER), near the border in the central Pyrenees, where the six individuals sampled carry southern France mtDNA haplotypes (green clade), yet their AFLP profiles cluster with the NE Spain group (blue cluster) Figure , a situaZootoca vivipara lineages. The high variation found in the ND2 gene relative to cyt-b, together with a more thorough geographic sampling allowed us to identify several distinct and geographically segregated clades in northern Spain, which help explain their historical interactions with the divergent clade found in southern France and clarify the phylogeographic history of the region.Our mtDNA dataset revealed four major clades and a remarkable degree of geographic structuring among Z. vivipara at the Ibero-Pyrenean zone do not show an actual “suture zone” between Iberian and northern European lineages [Chorthippus parallelus[Phylloscopus collybita[Z. vivipara on opposite sides of the Pyrenees form a monophyletic clade with respect to all other European lineages, indicating a relatively long time in isolation in this region. However, our mtDNA data do support a major role for the Pyrenees in isolating populations, and the two main divergent lineages  and the Pyrenean taxa , suggesting similar patterns of isolation as in Z. vivipara albeit at longer time scales [Lacerta schreiberi along northern Spain are genetically uniform and expanded from a refuge in central Portugal since the last glacial maximum [Podarcis bocagei[Chioglossa lusitanica[Overall, the general pattern of allopatric differentiation in isolated highlands is consistent with the species ecology, as in the southern part of its distribution the common lizard is found mainly in humid bogs and associated habitats ,32, whice scales ,34. In c maximum , a pattes bocagei and the usitanica.Z. vivipara lineages associated with different mountain areas along the northern Iberian peninsula is consistent with a pattern of “refugia-within-refugia” documented for a number of other organisms [Z. vivipara lineages are younger than those reported in other taxa, such as Psammodromus hispanicus[Alytes obstetricans[Lissotriton boscai[Alytes cisternasii[The pattern of distinct rganisms ,38,39, atetricans, or Lisson boscai, and morsternasii.Z. vivipara in the region provides some support for this hypothesis, and analysis of morphological traits has revealed marked phenotypic differentiation of Cantabrian populations with respect to those in the Basque Country and Pyrenees [Z. vivipara. A closer association between traits under selection and nuclear instead of mitochondrial neutral makers has been documented in other studies. For example, in a contact zone between central European newts, Babik et al. [Ensatina salamander complex of North America shows that divergence in neutral nuclear markers is better correlated with reproductive isolation than mtDNA [The pattern of spatial variation in genome-wide AFLP markers revealed both important similarities and differences compared to that of mtDNA sequence. Both datasets revealed the presence of four genetic units that are geographically concordant, yet relative divergence among groups differs in both datasets. AFLP variation forms two main genetic groups that separate NW Spain from the rest, while the two main mtDNA clades separate Spain from France. This difference suggests that at a large geographic scale different neutral and/or selective factors have shaped diversity and structure in both types of markers, yet identifying the specific cause is beyond the scope of our dataset. Potential causes range from neutral factors such as random sorting of loci or chromosomal rearrangements, to the role of natural selection. Even though the AFLP loci we used appear to be largely neutral, these markers are likely to be spread randomly across the nuclear genome, and are therefore more likely to be associated with regions under selection than mtDNA markers. If this were the case, we would expect to find a stronger association between traits under selection and AFLP structure. Available phenotypic data on Pyrenees . This paPyrenees ,45 are yk et al.  documentan mtDNA . AdditioCorthippus grasshoppers [Phylloscopus birds [Z. vivipara. Another potential contact zone not detected in the present study corresponds to that found in north-central Spain, between the yellow and red lineages, and further sampling would be necessary to confirm its existence.Our phylogeographic results reveal the presence of two independent contact zones between lineages: one in southern France across the high Pyrenees , although recent evidence from the same study area in Gabas-piet  indicates that further replication across years will be necessary to obtain conclusive evidence. Several studies in diverse organisms have shown that mtDNA can introgress further than nDNA [Relatively good sampling in the central Pyrenees allows us to infer the demographic and introgression history of lineages there. Different lines of evidence  support the hypothesis that the presence of NE Spanish (blue) haplotypes in southern France is due to a recent population expansion from the Spanish side of the Pyrenees, which led to the colonization of southern France across the high Pyrenees. The marked predominance of haplotype “AA” there suggests that this incursion took place recently, probably during or since the last glacial maximum, as indicated by time estimates from the mismatch distribution. The relative geographic congruence among mtDNA and AFLP genetic groups allows us to compare patterns of introgression of maternal and bi-parentally inherited loci. Genome-wide AFLP data revealed limited introgression of nDNA into France, with individuals of mixed origin being restricted to the upper Ossau river drainage and the Spain-France border, suggesting that the contact zone among lineages is located within 10 or 15 km of the border. In contrast, Spanish mtDNA haplotypes are found at least tens of kilometres further than nDNA. Interestingly, it appears that Spanish haplotypes expanded into France (mostly towards the W and NW), they were then trapped at river valleys towards the east , giving rise to a sharp contact zone there. This apparent barrier to gene flow in mtDNA but not nDNA at river valleys could be due indirectly to Haldane’s rule , which pn et al. 11, althohan nDNA ,51-54, ahan nDNA ,56. DistAdditional fine-scale sampling at contact zones among lineages will also be needed to understand introgression dynamics at other areas, such as the Beret site, where all individuals carry French mtDNA and Spanish nDNA, or the contact zone in the western Pyrenees between the blue and red clades detected at the Ibañeta site, where individuals show blue central-Pyrenean nDNA, yet half of them carry red mtDNA haplotypes. Patterns at these individual sites are similar to those found at some sites in southern France, and a more thorough geographic context provided by finer-scale sampling will be needed to help determine whether they belong to active contact zones with ongoing introgression or instead represent populations left in the genetic wake of a contact zone that shifted away.Z. vivipara in northern Iberia and the Ibero-Pyrenean suture zone show marked levels of differentiation that is congruent with major topographical features in the region. Our data reveal that mountain ranges do not necessarily represent impassable barriers for this species, as demonstrated by a recent high-elevation colonization event across the Pyrenees that has led to secondary contact and partial introgression among divergent lineages. In contrast, we find indirect evidence of contact zones in lowland areas, in the absence of obvious barriers to dispersal. Our results underscore the importance of multilocus datasets in reconstructing the evolutionary history of independent intraspecific lineages [Z. vivipara lineages in secondary contact is similar to that found among different lizard species [Z. vivipara lineages in the region may in fact represent separate species. Further research using both molecular and phenotypic data will help determine the extent to which reproductive isolation is underway despite the presence of gene flow [Z. vivipara show that even major biogeographic barriers can yield markedly different phylogeographic patterns in different vertebrate and invertebrate taxa depending on their demographic histories and ecological traits. Combining intraspecific studies of genetic variation at the lineage level with ecological data will be essential to better understand phylogeographic differences among taxa and advance our understanding of diversification mechanisms.Our results show that lineages of lineages , and und species , suggestene flow . Suture Z. vivipara were captured at 49 localities across the species range in Northern Spain and Southern France (Table Specimens of ce Table . The taib gene (including 21 bp from the adjacent Glu-tRNA) using primers MVZ04 and MVZ05 [Genomic DNA was extracted from tail clippings using a Qiagen™ DNeasy Kit and following the manufacturer’s protocol. The following regions of the mitochondrial DNA were amplified: a 427 bp fragment of the cytochrome nd MVZ05 ; and 964nd MVZ05 . DetaileSequencher 4.1.  and variable sites were checked visually for accuracy. Coding gene sequences were unambiguously translated into their amino acid sequence and no double peaks were observed in the chromatographs, suggesting sequences were of mitochondrial origin and not nuclear copies. For most analysis, the two gene fragments (cyt-b and ND2) were concatenated into a single sequence of 1370 bp. Haplotype and nucleotide diversity indices were calculated in DnaSP v5[Sequences were automatically aligned using  DnaSP v5.jModelTest[b) and obtained an optimal partitioning scheme for the dataset (by gene and by codon position) using the sotware package PartitionFinder [MrBayes 3.2.2 (http://mrbayes.csit.fsu.edu/) [Tracer v1.5 [We constructed a haplotype network using a maximum parsimony algorithm as implemented in the program TCS 1.21 , settingModelTest to deteronFinder . We thensu.edu/) , and rancer v1.5 , and outBeast[Tracer v1.5 [b, a rate estimated from a comprehensive phylogenetic study of several squamate groups [To estimate divergence times among lineages we used a coalescence approach that uses Bayesian inference and MCMC simulations to generate posterior probability values for divergence times as implemented in the program Beast. We partcer v1.5 , and alle groups  that usee groups ,71. We se groups ,35,38,72Fs (generated with Arlequin 3.5) indicate an excess of recent mutations and reject population stasis . To estimate the time elapsed since such a sudden population expansion, we used the parameter τ obtained from the distribution of pairwise differences among Cyt b haplotypes, as τ = 2ut, where t is the time elapsed between initial and current population sizes and u = 2μk, where μ is the mutation rate (0.01 substitutions/site/My per lineage) and k is the length of the sequence [Arlequin 3.5 [We tested for past sudden changes in effective population size using Fu’s test of neutrality , which dsequence . We estisequence  and compquin 3.5 .et al.[EcoRI and MseI (Tru9) and fragments were ligated to oligonucleotide adapters with T4 DNA ligase. A random sub-sample of fragments was obtained through a pre-selective amplification reaction using primers ET and MC, followed by three selective amplifications using primer pairs ETAG/MCGA, ETAG/MCGT, and ETAG/MCTA, with the E primer fluorescently labelled. Ten pairs of selective amplification primers were tested, but only those producing sufficient loci and repeatable and unambiguously scorable profiles were selected for the analysis. Selectively amplified fragments were run in an ABI 3730XL genetic analyser with a LIZ500 size standard. Peaks were visualized using Genemapper 3.7 and scored manually by a single observer (BM). Only unambiguously scorable loci and individuals were included in the analysis and peaks found in less than 2% of individuals were excluded. Methodological error rate was assessed by running a subset of 10 individuals twice from the pre-selective amplification step. The average per-locus genotyping error rate for the AFLP loci selected, measured as recommended by Bonin et al. [Amplified fragment length polymorphism (AFLP) profiles were generated using the laboratory protocol described in Milá et al. , which wet al.. In brien et al. , was lowBayeScan 2.0, which uses differences in allele frequencies between populations and a reversible-jump MCMC algorithm to estimate the locus-specific posterior probability for a model including selection vs. a neutral model [To determine whether AFLP markers were selectively neutral, we conducted an outlier analysis using the program al model . We usedal model .He) was based on allele frequencies calculated using the method by Lynch and Milligan [Aflp-surv v. 1.0 [FST values using the FST analogue ΦPT was calculated with Genalex 6.0 [PT is calculated as VAP/(VAP + VWP) where VAP is the variance among populations and VWP is the variance within populations. Probability values of pairwise ΦPT were based on 10,000 permutations.We estimated allelic frequencies using Zhivotovsky’s  BayesianMilligan  as implev v. 1.0 . A matrialex 6.0 . ΦPT is Genalex 6.0. [Structure 3.1 [Locprior setting, which incorporates sampling locality as a prior without affecting the value of the optimal K [Structure Harvester[To assess genetic structure among samples we conducted a principal coordinate analysis  on a gelex 6.0. .We furthture 3.1 . This prture 3.1 , and useptimal K . We run ptimal K  as impleHarvester.b (KF593892-KF593905 and KF593907-KF593908) haplotypes.Sequences used in this study have been deposited in GenBank under accessions for ND2 ( KF593862-KF593891) and Cyt The authors declare that they have no competing interests.BM, YSG, BH and PF designed the study; all authors carried out the field sampling; BM and YSG generated the molecular data; BM conducted all genetic analyses; BM wrote the manuscript with contributions from all authors, who read and approved the final manuscript.Zootoca vivipara mtDNA haplotype used in the study. Figure S1. Plots generated by the software Structure Harvester to determine the optimal K in the program Structure 3.1. (A) Delta K values from the method by Evanno et al. (2005). (B) Mean values of the estimated Ln probability of different K values. Figure S2. Results from an outlier analysis to detect loci under selection among 34 AFLP loci using the program BayeScan 2.0. Plots show Fst vs. posterior odds for selection for each locus in three population comparisons: (A) K = 2, (B) K = 3, and (C) K = 4. The vertical line in each plot indicates the threshold leading to a false discovery rate (FDR) of no more than 5%. Loci to the right of the line deviate from neutrality for the comparison shown. The loci represented by the dots sitting on the vertical lines are locus “15c194” in (A) and locus “19c84” in (B) and (C).GenBank accessions for each Click here for file


Triturus (i.e. the marbled and crested newts) as the climate shifted from the previous glacial period  to the current interglacial.The major climatic oscillations during the Quaternary Ice Age heavily influenced the distribution of species and left their mark on intraspecific genetic diversity. Past range shifts can be reconstructed with the aid of species distribution modeling and phylogeographical analyses. We test the responses of the different members of the genus Triturus species on composite maps.We present the results of a dense mitochondrial DNA phylogeography  and species distribution modeling (using two different climate simulations) for the nine Triturus. The combined use of the two independent techniques yields a more complete understanding of the historical biogeography of Triturus than both approaches would on their own. Triturus newts generally conform to the ‘southern richness and northern purity’ paradigm, but we also find more intricate patterns, such as the absence of genetic variation and suitable area at the Last Glacial Maximum (T. dobrogicus), an ‘extra-Mediterranean’ refugium in the Carpathian Basin (T. cristatus), and areas where species displaced one another postglacially (e.g. T. macedonicus and western T. karelinii). We provide a biogeographical scenario for Triturus, showing the positions of glacial refugia, the regions that were postglacially colonized and the areas where species displaced one another as they shifted their ranges.The combined use of species distribution modeling and mitochondrial phylogeography provides insight in the glacial contraction and postglacial expansion of  The null model approach prevents interpreting model quality based on an arbitrary AUC threshold and precludes the requirement to set aside part of the localities for model testing [The species distribution models were tested for statistical significance against a null model derived from random localities . For eacools 1.3 ; [http:/ testing .The species distribution models were projected on the current and Last Glacial Maximum climate layers. Composite maps were created for each set of climate layers (in the same way as explained for the genetic approach). Maxent provides predicted probability values between zero and one and we used a color scheme running from blue to red to reflect these values.Triturus sequenced newts comprise 315 haplotypes .The 2470 Zi) populations was 44 and 48 out of 52 populations for T. carnifex, 60 and 88 out of 104 for T. cristatus, 29 and 40 out of 44 for T. dobrogicus, 55 and 79 out of 79 for western T. karelinii, 13 and 16 out of 24 for central T. karelinii, 27 and 31 out of 31 for eastern T. karelinii, 32 and 37 out of 72 for T. macedonicus, 34 and 36 out of 36 for T. marmoratus and 49 and 50 out of 51 for T. pygmaeus . Measures of genetic diversity within and genetic divergence among populations can be found in Additional file Triturus species generally show considerable spatial variation in their genetic composition  and genetic divergence among (Triturus) are provided in Additional file Triturus species are predicted to have been restricted at the Last Glacial Maximum. A composite map showing species distribution models projected on present day climate layers is provided in Figure Species distribution models perform significantly better than random  [T. cristatus (SE Poland) likely reflects the comingling of haplotypes belonging to the two distinct clades present in this species  likely represents standing genetic variation, given the lack of phylogeographic structure among the haplotypes involved. Species distribution modeling can further help to distinguish both processes by establishing whether an area became suitable only recently or whether it has continuously been so on the long term.A high value for genetic diversity within populations Figure  can resuronment) . To sepas Figure . On the T. macedonicus) show many hotspots. Such areas would be expected to have been habitable on the long term. On the other hand, genetically homogenous areas, due to high levels of gene flow and/or demographic expansion stand out as cold areas (e.g. both of these processes likely determine the absence of genetic structuring among T. dobrogicus populations). These two processes are more difficult to disentangle: whereas demographic expansion would particularly be expected in areas that became suitable postglacially, gene flow could reduce genetic structuring both inside and outside of glacial refugia. In some cases geographical barriers to gene flow stand out, such as the Greater Caucasus mountain range separating eastern T. karelinii on its northern and southern side. Such barriers reflect climatically unsuitable regions and can be identified as such in the species distribution models.Maps showing genetic divergence among populations Figure  reflect The reliability of genetic diversity values for populations depends on sample size, and some populations are relatively poorly sampled in our study. The genetic turnover between populations provides additional insight that counters the adverse effects of low sample sizes. If populations within an area are genetically diverse, but sample sizes per population are low, genetic diversity per population might be underestimated due to chance effects in sampling. However, these effects are random per population. Therefore, the more populations that are being compared within that area, the less likely it is that actual genetic diversity remains hidden by chance. For this same reason determining the genetic turnover between populations is less susceptible to sample size to begin with: low sample size per population adds up to a big sample sizes within a region. So, in short, if multiple populations within a region are found to be genetically similar, even though samples sizes per population are low, together they provide a good indication that genetic diversity in the region really is low. We thus recommend exploring genetic diversity within populations and genetic divergence among populations together to improve the estimate of spatial genetic variation, especially when sample sizes are low.Triturus, the similarity of the introgressed mitochondrial DNA to that present in the ‘donor’ species suggests a recent  transference across the species boundary. Given the climatic oscillations during the Quaternary Ice Age, the Triturus newt species are presumed to have been in periodic contact through time. However, we did not identify ancient mitochondrial DNA introgression events  genetic marker. Although we consider our results indicative (and use the species distribution modeling results as an independent check), it should be taken into account that a single marker may not accurately reflect species history . In fact in e.g. -58). A pT. cristatus but only the latter shows the high genetic variation expected to be present in a former glacial refugium). Another issue is that species distribution models are based on the climatic conditions currently experienced by a species, but the actual conditions under which they could occur might be broader .A disadvantage of species distribution models is that they cannot distinguish between potential and realized distribution: areas that had suitable conditions at the Last Glacial Maximum may not have actually been inhabited. Genetic information can narrow down the position of glacial refugia  was present in the northern part of the area currently occupied by T. pygmaeus. Striking is that both T. marmoratus and T. pygmaeus mitochondrial DNA occurs throughout this area .T. cristatus was unsuitable at the Last Glacial Maximum  was colonized post-glacially, as the species is genetically depleted outside of the Carpathian Basin. Although T. cristatus shows asymmetrically introgressed western T. karelinii mitochondrial DNA south of the Danube river, relatively distinct T. cristatus haplotypes also occur here  is low in T. dobrogicus . Another option would be that the niche of T. dobrogicus has evolved in a brief time interval to adapt to the environmental shift [T. dobrogicus must have survived multiple glacial-interglacial cycles in situ.Compared to the other s Figure . Triturue Figure . This ise Figure . Evidentns (e.g. ) this wallozymes ). This sal shift . We consT. macedonicus is predicted to have been suitable during the Last Glacial Maximum  are suggested to have harbored suitable area at the Last Glacial Maximum according to species distribution models . The. TheTriton  ‘eastern T. karelinii’ extended their ranges into temperate Europe from their southerly positioned refugia. However, postglacial expansion is best exemplified by T. cristatus. The contemporary range of T. cristatus outside its glacial refugium in the Carpathian Basin encompasses a huge (c. 4.75 million km2) postglacially acquired area, stretching all the way from western Europe to Scandinavia and central Russia. This gives an indication of the speed with which postglacial colonization can be accomplished.Similarly, areas typically identified as having been postglacially colonized from glacial refugia are predicted as such for Triturus  within each Triturus species.Click here for fileFull genetic similarity maps The genetic (dis)similarity among populations within the different Triturus species, not cut according to the current species ranges.Click here for fileModel performance. The AUC values for the species distribution models of each Triturus species, tested against null models.Click here for fileFull species distribution models. The species distribution model of each Triturus species projected for Last Glacial Maximum and current climate conditions, not cut according to the current species ranges.Click here for file


The liver tissue was analyzed macroscopically, histologically and immunohistochemically, at protein level by Proteome Profiler Arrays and Western blot analysis. -/-Matn2 mice exhibited higher susceptibility to hepatocarcinogenesis compared to wild-type mice. In the liver of -/-Matn2 mice, spontaneous microscopic tumor foci were detected without DEN treatment. After 15 μg/g body weight DEN treatment, the liver of -/-Matn2 mice contained macroscopic tumors of both larger number and size than the WT liver. In contrast with the WT liver, spontaneous phosphorylation of EGFR, Erk1/2 GSK-3α/β and retinoblastoma protein (p-Rb), decrease in p21/CIP1 level, and increase in β-Catenin protein expression were detected in -/-Matn2 livers. Focal Ki-67 positivity of these samples provided additional support to our presumption that the lack of Matn2 drives the liver into a pro-proliferatory state, making it prone to tumor development. This enhanced proliferative capacity was further increased in the tumor nodules of DEN-treated -/-Matn2 livers. Our study suggests that Matn2 functions as a tumor suppressor in hepatocarcinogenesis, and in this process activation of EGFR together with that of Erk1/2, as well as inactivation of GSK-3β, play strategic roles.Matrilin-2 (Matn2) is a multidomain adaptor protein which plays a role in the assembly of extracellular matrix (ECM). It is produced by oval cells during stem cell-driven liver regeneration. In our study, the impact of Matn2 on hepatocarcinogenesis was investigated in  Matrilins are non-collagenous glycoproteins implicated in the organization of extracellular matrix (ECM) Matn2) is expressed in a great variety of tissues, but at highly variable levels The matrilin-2 gene  treatment. Furthermore, we uncovered several alterations in signaling pathways likely to be involved in the pathomechanism of tumor formation.Targeted disruption of Matn2 in embryonic stem cells and generation of inbred (129/SV) mouse strains have been described elsewhere +/−Matn2 heterozygotes were mated to generate +/+Matn2  and -/-Matn2 homozygotes for tumorigenesis. Fifteen-day old male WT and -/-Matn2 mice were injected intraperitoneally with DEN at a dose of 15 μg/g body weight. Each group, including WT and -/-Matn2 untreated controls as well as DEN-treated mice, consisted of ≥10 animals. Mice were terminated 10 months after DEN exposure by cervical dislocation in ether anesthesia. At termination, body weight and liver weight of the animals were measured and the number of macroscopically detectable tumors was counted.Inactivation of All animal experiments were conducted according to the ethical standards of the Animal Health Care and Control Institute Csongrád County, Hungary. The protocol was approved by the Committee of the Animal Health Care and Control Institute Csongrád County, Hungary (permit No. XVI/03047-2/2008).RNA was isolated from frozen livers. After homogenization in liquid nitrogen the total RNA was isolated using the RNeasy Mini Kit , according to the protocol provided by the manufacturer. The yield and purity of the isolated RNA was estimated by the ND-1000 spectrophotometer . The integrity and size distribution of the total RNA was analyzed using Experion Automated Electrophoresis Station .−ΔCT method.cDNAs were generated from 1 μg of total RNA by M-MLV Reverse Transcriptase kit  following the instructions of the supplier. Real-time PCR (RT-PCR) was performed by ABI Prism 7000 Sequence Detection System , using ABI Taqman Gene Expression Assays for β-actin (assay ID: Mm00607939_s1), Matn2 (assay ID: Mm01166023_m1) according to the manufacturer's protocol, mouse β-actin was used as endogenous control. All samples were run in duplicates in a 20 μl reaction volume with 50 ng of cDNA. Results were obtained as threshold cycle (CT) values. Expression levels were calculated by using the 2For phosphoprotein antibody arrays and Western blot analysis proteins were isolated from frozen liver tissues. After homogenization in liquid nitrogen 1 ml of lysis buffer ) was added to the samples. After incubation for 30 min on ice, samples were centrifuged at 13000 rpm for 20 min. Supernatants were saved and protein concentrations were measured according to Bradford et al The activities of phospho-mitogen-activated protein kinase (phospho-MAPK) and phospho-receptor tyrosine kinase (phospho-RTK) were assessed by their relative levels of phosphorylation using the Proteome Profiler Array  according to the manufacturer's instructions. The same liver protein samples were used for Western blot. Pooled samples of three livers from the same experimental group were homogenized in lysis buffer (described above) and adjusted to 300 μg protein/250 μl lysate. Signals were developed by incubating the membrane in SuperSignal West Pico Chemiluminescent Substrate Kit , and visualized on a Kodak Image Station 4000MM Digital Imaging System.Thirty μg of total proteins were mixed with loading buffer containing β-mercaptoethanol and were incubated at 95°C for 5 min. Pooled samples of three from the same experimental group were loaded onto a 10% polyacrylamide gel and were run for 30 min at 200 V on a Mini Protean vertical electrophoresis equipment . Proteins were transferred to polyvinylidene fluoride (PVDF) membrane  by blotting overnight at 100 mA. Ponceau staining was applied to determine the efficiency of blotting. Membranes were blocked with 3% (w/v) non-fat dry milk (Bio-Rad) in TBS for 1 hour followed by incubation with the primary antibodies , p-GSK-3α/β, p-c-Myc, extracellular signal-regulated kinases 1/2 (Erk1/2), p-Erk1/2, cyclin-dependent kinase inhibitor 1 (p21/CIP1), phospho-retinoblastoma (p-Rb), diluted 1∶500-1∶1000) at 4°C for 16 hours. Beta-actin served as loading control. Membranes were washed 5 times with TBS containing 0.5% (v/v) Tween20, then were incubated with appropriate secondary antibodies conjugated with HRP enzyme for 1 hour. Afterwards membranes were washed as before and signals were visualized with SuperSignal West Pico Chemiluminescent Substrate Kit (Thermo Fisher Scientific Inc.). Band densities were determined by Kodak Image Station 4000MM Digital Imaging System. Antibody data are provided in Supporting Information .Liver samples were either frozen for further processing or fixed in 10% formaldehyde and embedded in paraffin for histological analysis. Paraffin sections were dewaxed in xylene and stained with hematoxylin and eosin (HE). Stained sections were used for histological diagnosis.Immunofluorescence staining was performed on methanol-acetone-fixed 10-μm cryosections. Nonspecific binding was blocked first with 10% (w/v) normal donkey or goat serum in phosphate buffered saline (PBS) for 1 h. The specimens were incubated at 4°C overnight with primary antibodies . After washing in PBS, sections stained for β-Catenin, GSK-3β, p-GSK-3α/β and p-c-Myc were incubated with Alexa Fluor 555 donkey anti-rabbit IgG secondary antibody (Invitrogen by Life Technologies) for 30 min at room temperature in a dark chamber. Nuclei were stained with 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI) in PBS for 5 min. Sections stained for Matn2 and laminin were washed and incubated with a combination of Cy3-conjugated donkey anti-goat IgG and Cy2-conjugated donkey anti-rabbit IgG antibodies, respectively . Pictures were taken by Nikon Eclipse E600 microscope with the help of Lucia Cytogenetics version 1.5.6 program or Bio-Rad MRC 1024 confocal laser microscope.2O2 in methanol for 30 minutes at room temperature. Normal serum was applied to block any nonspecific binding, and the slides were incubated with primary antibodies raised against specific proteins  at 4°C overnight. Subsequently, the slides were incubated for 30 minutes with biotinylated goat anti-rabbit IgG secondary antibody at room temperature. A detailed list of the antibodies used in this study is presented in Supporting Information  in a pressure cooker under maximal pressure for 3 minutes, followed by proteinase K (0.5 mg/ml) digestion for 10 minutes. To inhibit endogenous peroxidases, samples were treated with 10% Hormation . Signal After immunostaining of Ki-67 in liver sections of 5-5 animals, the number of immunopositive cells was counted in 10 fields of each slide at a magnification of 200× by Olympus BX50  microscope.2*b*π)/6 where ‘a’ refers to width (mm) and ‘b’ stands for length (mm).To determine the tumor volume of livers, HE stained sections were scanned by Panoramic Scan . The length and width of tumors in each section were determined with the help of Panoramic viewer program (3D Histech Ltd.) Tumor volume was calculated as V  =   and Students' t-tests depending on the distribution of the data. The independent experimental sets were then compared for reproducibility. Only reproducible significant changes were considered as „significant”. Significance was declared at the standard p<0.05 level.All statistical analyses were made with Graphpad Prism 4.03 software (Graphpad Software Inc.). Data were tested for normal distribution by D'Agostino & Pearson's omnibus normality test. Significance of changes in WT control vs. WT DEN, WT control vs. -/-Matn2 mice, confirming the knockout phenotype of these animals   .-/-Matn2 livers compared to WT samples (p<0.001) (-/-Matn2 samples (p<0.001)  protein, two key regulatory molecule of the G1 cell cycle checkpoint were examined in whole liver homogenates. Western blot analysis revealed a ∼50% decrease in p21/CIP1 protein levels in p<0.001) . In parap<0.001) . In a gon the WT . MoreoveT livers .-/-Matn2 control livers as revealed by Western blot   /1, 3B, p-/-Matn2 livers GSK-3α/β becomes inactivated by phosphorylation and hence fails to phosphorylate c-Myc and β-catenin, thereby rescuing them from degradation. As a consequence, β-catenin translocates and accumulates in the cytoplasm, and c-Myc is allowed to act as a transcription factor in the nucleus  was slightly higher in  samples .-/-Matn2 and WT control groups with regard to signaling are summarized in Differences between the -/-Matn2 and WT mice was induced by administering a single dose of DEN. DEN-exposed WT and -/-Matn2 mice were compared to mock-injected littermates 10 months after treatment. DEN treatment induced macroscopic tumors in 78% of WT mice, whereas tumor frequency was 100% in the liver of -/-Matn2 animals. A higher number of macroscopic tumors developed in -/-Matn2 livers, with an average number of 23 tumors/liver in contrast with 4 counted in WT livers   .In WT animals, DEN treatment initiated HCC formation with molecular changes already known from earlier studies of hepatocarcinogenesis. In DEN-provoked livers 15% higher matrilin-2 mRNA level was detected than that of untreated control livers (p<0.05) .Whereas no immunopositivity for Ki-67 was detected in untreated WT liver sections, the reaction in DEN-exposed WT livers revealed a high number of immunopositive cells with an average count of 49/field (p<0.001 vs. WT control) see . These cDEN treatment caused a ∼1.5-fold elevation of total Erk1/2 proteins compared to the untreated WT animals. Moreover, phosphorylation of these proteins was also significantly higher after DEN treatment than in controls, as seen both by immunostaining and Western blot /1, 3B.The inactive phosphorylated forms of GSK-3α/β were increased in tumor-bearing livers. Compared to the normal livers a 60% elevation for α and 80% for β was measured by densitometry of Western blots . In paraCompared to WT controls, inhibition of GSK activity resulted in 1.5× higher amount of β-catenin and 70% less c-Myc phosphorylation in WT DEN liver homogenates, as revealed by Western blots . ImmunosBy phospho-RTK array, significantly higher phospho-EGFR levels were detected in the homogenates from DEN-treated WT wild-type animals compared to control ones . No chanDifferences between WT control and WT DEN groups are summarized in -/-Matn2 animals; -/-Matn2 control and WT control vs. WT DEN groups. Our data indicate that -/-Matn2 hepatocytes harbor alterations that make them prone to malignant transformation even without hepatocarcinogenic challenge; indeed, -/-Matn2 mice developed spontaneous foci in the liver. Only a single protein, p21/CIP1, behaved differently in these comparisons. p21/CIP1 was induced upon DEN treatment in WT livers; however, the lack of Matn2 seemed to interfere with the induction of p21/CIP1.-/-Matn2 livers had an activated, pro-proliferatory phenotype without any experimental challenge.As mentioned previously, Matn2-deficient mice were more sensitive to DEN treatment, displaying higher number of tumors with significantly larger volume in their livers compared to the WT DEN group see . We also-/-Matn2 control, 24.4 times more Ki-67 immunopositive cells were counted in sections of matrilin-2 deficient livers after DEN treatment (-/-Matn2 DEN homogenates relative to -/-Matn2 controls (p<0.01) (-/-Matn2 DEN samples as compared to -/-Matn2 controls. These molecular changes are summarized in When compared to reatment . In para(p<0.01) . Among t(p<0.01) /1, 3B. F(p<0.01)  in Matn2-/-Matn2 livers. The elevation of Ki-67 was the most prominent, as the tumors developed upon HCC induction had a high rate of cell division. In conclusion, since the lack of Matn2 had already driven hepatocytes into an activated state, DEN treatment caused only modest intensification of pro-proliferative signaling.As seen in -/-Matn2 mice both treated with DEN, higher proliferation rate was seen in -/-Matn2 DEN livers, as the average Ki-67-positive cell count was 49 in WT and 100 in -/-Matn2 livers after DEN exposure (-/-Matn2 livers as compared to DEN-treated WT (p<0.001) (-/-Matn2 vs. WT upon DEN treatment (-/-Matn2 DEN the levels of p-c-Myc were only 37% of those measured in WT DEN (-/-Matn2 DEN relative to WT DEN (-/-Matn2 DEN was as low as 45% of that measured in WT DEN (When comparing groups of WT and exposure . In parap<0.001) . With rereatment /2, 3B ann WT DEN /1, 3B. Co WT DEN /1, 3B. Hn WT DEN .-/-Matn2 DEN groups are summarized in -/-Matn2 livers may involve enhanced GSK-3β inactivation and hindered p21/CIP1 induction, leading to elevated proliferation rate indicated by Ki-67.Differences between WT DEN and -/-Matn2 mice. In matrilin-2-deficient animals, however, significantly more, and more voluminous tumor foci have developed. Furthermore, quite surprisingly, sporadic microscopic tumor foci spontaneously appeared in the liver of -/-Matn2 mice without DEN treatment at age of 10 months. This result evidenced that the lack of Matn2 triggers an activated state in the liver, making it prone to tumor development. Our observations suggest a protective role for Matn2 against liver carcinogenesis. To explore the underlying mechanisms, activation of key signal transduction proteins in -/-Matn2 animals has been investigated in more detail.Despite the relatively detailed data available on matrilin-2 structure and tissue expression, its function has not yet been elucidated -/-Matn2 livers was increased phosphorylation of Erk1/2. Activation of the MAPK pathway most likely was a critical event related to the absence of Matn2 from the ECM, and this might be one of the key alterations responsible for the increased susceptibility of hepatocytes to cancer development. In addition, inactivation of GSK-3α/β proteins was revealed by Western blot. This implies that beside Erk1/2 activation the signal generated in the absence of Matn2 utilizes the Wnt pathway and inactivates GSK-3β and -3α by phosphorylation at Ser9 and Ser21 residues. Although it is mostly protein kinase B (Akt protein) that inactivates GSK-3β The most conspicuous finding in -/-Matn2 mice was confirmed by immunostaining for Ki-67. Whereas β-catenin in the WT control livers was confined to the cell membrane, the protein accumulated in the cytoplasm in -/-Matn2, but no nuclear localization was ascertained. Currently we cannot explain why β-catenin failed to translocate to the nucleus, since its localization is thought to be solely determined by the availability and phosphorylation of its binding partners. This aspect of the mechanism needs further investigation In accordance with the inactivation of GSK-3β we were able to demonstrate an increase in β-catenin protein levels, and a decrease in the inactivating phosphorylation of c-Myc at the Thr58 residue. Suspension of GSK-3β-mediated inhibitory effects brings hepatocytes into a proliferative state Beside the β-catenin-related mechanism, Jin et al. reported that GSK-3β has a critical role in the phosphorylation-mediated inactivation of the Cyclin-dependent kinase 4/CyclinD3 complex (Cdk4/CyclinD3). This offers another approach how the inactivation of GSK-3β may result in increased proliferation Our results regarding p21/CIP1 fit very well into the model outlined. p21/CIP1 levels increased upon DEN-treatment in WT mouse liver, which is in line with the available reports -/-Matn2 animals; however, this induction failed to compensate for the initially lower p21/CIP1 levels in -/-Matn2. Hence, the absolute amount of p21/CIP1 remained lower in the DEN-treated -/-Matn2 mice, too. In -/-Matn2 animals, activation of GSK-3β/c-Myc/p21/CIP1 axis may result in transcriptional inhibition of p21/CIP1 synthesis. The above-mentioned Erk1/2-mediated stabilization of p21/CIP1 is also likely to be Matn2-dependent Erk1/2 activation in the context of DEN treatment has been reported to increase the expression and stability of p21/CIP1 -/-Matn2 indicates that, as a consequence to p21/CIP1 downregulation, Rb becomes inactivated by a Cdk4/CyclinD3-mediated process -/-Matn2 mice, since the ablation of Matn2 was shown to be associated with elevated Erk1/2 activity.Increased phosphorylation of Rb protein at Ser780 in To find the link between altered ECM composition and modified signaling, tyrosine kinase receptor phosphorylation was assessed. This revealed increased phosphorylation of EGFR only. Considering that Matn2 contains several EGF-like domains it can be hypothesized that Matn2 as a decoy molecule may interfere with the binding of the real EGF ligand to its receptor. This could imply that the lack of Matn2 increases the binding capacity of EGFR to its real ligand, resulting in increased activation. However, this hypothesis requires further studies to prove. Also, despite our efforts, integrin receptors possibly involved in mediating the effects of Matn2 could not so far be identified.-/-Matn2 animals showed increased susceptibility to cancer induction, and spontaneously developed atypical microscopic foci in the liver. Ablation of Matn2 is thought to contribute to hepatocarcinogenesis through 1) activation of the MAPK signaling pathway; 2) inactivation of GSK-3β with consequent increase in the amount of β-catenin and activation of c-Myc; and 3) lowered levels of p21/CIP1 and consequent Rb phosphorylation by Cdk4/CyclinD complexes  and WT DEN-treated (white bars), compared to control (black bars) and -/-Matn2 DEN-treated samples (light grey bars).(TIF)Click here for additional data file.Figure S2Matn2 mRNA expression in wild type control and DEN-exposed livers detected by real-time RT-PCR. As seen, 1.15-times more Matn2 mRNA was detected in tumorous samples (WT DEN) compared to control ones (WT control). Data are expressed as mean ± SD, n = 10; *p<0.05.(TIF)Click here for additional data file.Table S1Antibodies used in the present study.(DOC)Click here for additional data file.


Stylidium hispidum, a species endemic to Southwestern Australia. Multivariate ordination of 134 AFLP markers analyzed genetic structure and detected two clusters of paired sites that diverged significantly for marker variation along a latitudinal boundary. To test for outbreeding depression and to determine the consequences of molecular population divergence for hybrid fitness, we conducted controlled pollinations and studied germination and survival for three cross categories  for paired sites distributed within and between the two genetically differentiated regions. We found evidence of outbreeding depression in long-distance hybrids (111–124 km), and inbreeding depression among progeny of within-population crosses, relative to short-distance (3–10 km) hybrids, suggesting an intermediate optimal outcrossing distance in this species. These results are discussed in light of the evolutionary consequences of mixing seed sources for biodiversity restoration.The benefits of composite rather than local seed provenances for ecological restoration have recently been argued, largely on the basis of maximizing evolutionary potential. However, these arguments have downplayed the potentially negative consequences of outbreeding depression once mixed provenances interbreed. In this study, we compared intraspecific F1 hybrid performance and molecular marker differentiation among four populations of  The spatial distribution of genetic diversity and the genetic distance between interbreeding individuals can have significant consequences for the ecological restoration of native plant communities  fitness in a controlled environment. As a result, we were able to (1) test for significant population genetic divergence among sites, (2) contrast the performance of hybrid progeny relative to parental population fitness and compare performance with estimates of genetic divergence, and (3) predict the potential for outbreeding depression to occur in restored populations of this species established from mixed provenance seed.While the avoidance of inbreeding in reintroduction programs is a significant concern, and multiple publications argue in favor of genetic diversity of introduced sources, few studies have tested genetic structure and the consequences of seed mixing in restoration relative to the potential impacts of outbreeding depression  forest alone, more than 550 hectares of post-mined land are restored each year  occur in a landscape where greater than 70% of the natural vegetation has been altered or lost due to land development (Hopper and Gioia Stylidium hispidum Lindl. (Stylidiaceae), or white butterfly triggerplant, is a perennial species endemic to the northern E. marginata forests of Southwest Western Australia. Plants have a basal rosette growth form and produce one or more flowering racemes each spring. The style and filaments of S. hispidum flowers are fused to form a column that is “triggered” when an insect, such as a native bee lands on the petals . The two southern sites are located near two bauxite mines operated by Alcoa World Alumina Australia .Fresh leaf or bud samples were preserved on ice in the field and stored at −80°C prior to DNA extraction. Total genomic DNA was extracted using the methods of Wagner et al.  with modEcoRI for Pst1 as well as EcoRI/MseI adaptors in the restriction-ligation mix, and the direct substitution of EcoRI primers for Pst1 primers in subsequent PCR reactions. The restriction-ligation mix and PCR products were diluted 1:20 prior to pre-selective and selective amplification using an ABI Veriti 96-well thermal cycler . Selective PCR reactions were pooled and fragments detected on a Beckman CEQ 8000 DNA sequencer using the CEQ Size Standard 400 . Two primer combinations, EcoRI-AGG/MseI-CTG and EcoRI-ACC/MseI-CTG, resulted in 134 markers scored between 60 and 400 bp using CEQ 8000 Genetic Analysis System software. DNA markers were double checked manually and replicate samples were run to test the reproducibility of AFLP fingerprints. Individual markers were selected to minimize scoring errors, and final error rates were calculated at ≤3%. Fragment analyses were performed on a total of 135 individuals and an average of 34.5 individuals per site (Amplified Fragment Length Polymorphism (AFLP) marker analysis was performed using a modified version of Vos et al.  describeper site . Data fiper site .j). Population structure (FST) was also calculated in AFLP-SURV using the method of Lynch and Milligan  in PRIMER-E 6.1.13 software . Collections were subsequently transplanted into forestry pots (90 mm × 90 mm × 180 mm) using native soils that remained around plant roots and a standard greenhouse mix , and placed in the UWA greenhouses. Each population was represented by a minimum of 20 adult plants. Mortality rates were low and individuals were replaced when necessary prior to experimental crosses.Whole Greenhouse collections flowered in spring 2008 over a period of 4 months. Plants from northern sites flowered earlier by 2 or more weeks, but continued to flower as plants from southern populations began to bloom. During that time, triggers of male-phase flowers were removed to hand-pollinate female-phase flowers both within and between populations. Cross-pollinations were conducted following the methods of Burbidge and James  in a cloEarly fitness was investigated for 764 seeds representing three cross categories: within-population (or intrasite) crosses, short-distance hybrids (3–10 km), and long-distance hybrids (111–124 km). Specifically, 100 seeds were selected from within-population crosses for each northern (AV1 and AV2) and southern (CPC and TOR) population for a total of 400 seeds. A total of 100 seeds corresponded to short-distance hybrids between the two northern Avon populations or the two southern Dwellingup populations. Lastly, 200 seeds were selected to represent long-distance hybrids divided among the four cross combinations between northern and southern populations. Once the original 700 seeds were planted, we increased the sample size based on the availability of selected crosses and greenhouse space. This resulted in an additional 19 seeds for within-population crosses, 29 seeds for short-distance crosses, and 16 seeds for long-distance crosses. Each cross combination  in the germination study represented a minimum of 10 maternal plants, and seeds were divided into approximately equal numbers of reciprocal hybrids where each site alternated as the pollen source.3) and planted into 50 mm forestry tubes containing the standard greenhouse mix in February 2009. Pots were watered daily in the UWA greenhouses, and germination was monitored weekly over a 4-month period until newly germinated seedlings were no longer visible. The number of surviving seedlings was recorded approximately 6 months later. Prior to planting, average seed weight was recorded for each diallel cross to detect possible maternal effects as well as effects of reciprocal parental combinations. Air-dried seeds were weighed in lots to the nearest 0.01 mg and average seed weight was calculated per cross by dividing the number of seeds in each lot by their total weight. This resulted in a mean of 442 seeds weighed and averaged for each parental combination. Analysis of variance (ANOVA) was used to test the effects of maternal population  and experimental cross type on seed mass using JMP version 9.0 . The effects of reciprocal pollinations were also investigated. Seed mass was log transformed to meet the assumption of normality.Seeds were soaked overnight in a solution of 100 ppm gibberellic acid . Given the potential for maternal effects to influence the survival of progeny, mean seed mass for each reciprocal cross was included as an explanatory variable. Observations in the study had a larger variance than expected given a binomial distribution, and we used the William's method to correct for overdispersion Allison . The likS. hispidum individuals were genotyped for analysis. All but two of the 134 AFLP markers were polymorphic, and values for percent polymorphism and gene diversity were relatively high . Nested AMOVA partitioned 74% of the total genetic variation within sites (or populations), 5% between sites within regions, and 21% between regions .A total of 135 ely high . EstimatRANOSIM = 0.623, P = 0.001), but ANOSIM did not detect evidence for divergence between populations within either region.Non-metric multidimensional scaling separated populations from northern and southern regions, with large overlap among populations within regions . One outS. hispidum failed, and the crosses that developed seeds set a single seed per fruit. Although greenhouse flowers that were not experimentally pollinated failed to set seeds in almost all cases, it is possible that the rare cases where flowers did set seeds were due to inadvertently transferred pollen. Overall, results support the hypothesis that S. hispidum is self-incompatible, but further study may be required to determine the breeding system of this species.All but two of the experimental pollinations to test self-incompatibility in F3,760 = 43.25, P < 0.0001). The average seed mass for experimental crosses with maternal plants derived from southern sites (CPC and TOR) was 19% greater than seed mass resulting from crosses of maternal plants derived from northern sites (AV1 and AV2). Seed mass did not differ significantly for maternal plants within each set of paired sites. ANOVA also detected an effect of cross type on seed mass . The average mass of seeds resulting from long-distance crosses was significantly lower than seed mass for either short-distance hybrids or within-population crosses . Reciprocal cross effects were also apparent for progeny resulting from short-distance pollinations and separate t-tests indicated significant effects of maternal parent on seed mass for both northern and southern sites (P < 0.0001).Results of ANOVA for seed mass detected a significant effect of maternal population regardless of cross type  and among regions (21%). However, MDS ordination, with support from ANOSIM, detected only two significant clusters split between northern and southern sites. This division corresponded to the greatest cross distance for hybrid comparisons, and predicted two regions or zones for seed transfer among experimental locations. Nested AMOVA results for S. hispidum reflect moderate levels of genetic subdivision detected in other endemic, outcrossing plant species from the region  where early seedling survival of long-distance and short-distance hybrids was equally likely. Thus, our molecular marker data predicted the scale of outbreeding depression among these four sites, providing support for marker use in defining optimally outcrossing seed provenance zones for ecological restoration. Other findings of a significant correlation between genetic diversity and population fitness  that was not detected in seed mass of within-population (inbred) crosses. Reciprocal cross effects on seed mass were also apparent, and cytogenetic factors may contribute to outbreeding depression.S. hispidum suggest that there would be a detrimental effect of inbreeding depression if seed collections were limited to few, neighboring plants from single sites. However, inbreeding depression was alleviated via crossing at distances as short as 3 km (range: 3–10 km). This distance falls well within the ∼20 km range for provenance collections currently in practice for post-mining Jarrah forest restoration (Koch Generally accepted guidelines (e.g., Mortlock ion Koch . Thus, wS. hispidum, composite collections at long distances would likely result in a significant decline in hybrid and mean population fitness. At a practical level, data for gene dispersal curves among species are often no more available than data for adaptive differentiation, and applications of this composite seed sourcing method may also limit genetic diversity if collections at long distances are made from few source plants or sites. The selection of a seed collection protocol should consider measures to avoid both inbreeding and outbreeding depression, and be based on the management of risk for individual species and plant communities (Byrne et al. Sgrò et al.  support With mounting evidence for outbreeding depression and an optimal outcrossing distance beyond the local population in plant species, we recommend that composite seed mixes for restoration be restricted to a local provenance zone. In doing so, we advise against highly restrictive collection areas, and instead recommend that zones be established with flexible boundaries to represent a mobile collection radius or patch size similar to that in use in Western Australia mine site restoration Koch . While v


AbstractAnguis veronensis Pollini, 1818) was performed and their genetic differentiation at the mitochondrial 16S rRNA gene fragment from a Spanish sample has been assessed. The Italian populations were karyologically uniform, all showing 2n=44 elements, of which 20 were macrochromosomes and 24 microchromosomes. Comparison with literature data on Central European populations showed a difference on the morphology of the 10th chromosome pair: submetacentric in Italian populations and telocentric in the Central European ones. Our analysis showed the presence of a fragile site on chromosomes of this pair, suggesting its propensity for structural rearrangements. Analysis of the 16S rRNA gene fragment showed uniformity among Italian populations (uncorrected genetic distance of 0.4%), and their genetic distinctness from the Spanish individual (uncorrected genetic distance of 4.2%). Our results confirm the existence of two different Anguis fragilis Linnaeus, 1758 lineages, each one characterized by a different cytotype.A karyological analysis on six Italian populations the slow worm ( Anguis Linnaeus, 1758 in the Palaeartic region: Anguis cephallonica Werner, 1894 and Anguis fragilis Linnaeus, 1758, commonly known as slow worms. The first species was considered a Mediterranean endemic restricted to the Peloponnese and some Ionian islands, while the second (Anguis fragilis) was considered a widespread taxon distributed from Western Europe to NW Iran and from the Mediterranean coast to Scandinavia, with a broad altitudinal distribution, ranging from the sea level up to 2300 m above sea level , Anguis graeca Bedriaga, 1881 , Anguis cephallonica (limited to the Peloponnese), and Anguis colchica  (widely distributed from Eastern Europe to Iran) and subdivided into three main lineages  each with a geographically distinct distribution; and more recently Anguis veronensis Pollini, 1818 . Both the mitochondrial (mtDNA) and the nuclear (nuDNA) analyses analyses identified Anguis cephallonica as the sister lineage of a wider clade comprising Anguis fragilis, Anguis colchica and Anguis graeca  and 24 microchromosomes, arm number (A.N.)=48. The populations of the Matese Regional Park differed in showing as submetacentric the elements of the tenth macrochromosome pair (A.N.=50). On the basis of the recent finding of Anguis species were found  1x, and incubation in hypotonic solution  per 30 min, cells were fixed in 3:1 methanol-acetic acid. This non-invasive method, useful for both karyologycal and molecular investigations, allows the avoidance of specimen sacrifice and animals were released at capture sites immediately after the sampling. In addition to standard staining method (5% Giemsa solution at pH 7) we performed various chromosome banding techniques: Ag-Nor banding (3+DAPI staining (3-methyl green staining (Late Replicating banding pattern (LR), bromedeoxyuridine  was added to blood cultures during the last six hours and differential staining was revealed by staining chromosomes with 4% Giemsa solution in 2% 4Na-EDTA for two minutes , one male; Ancona , two males; Valle Agricola , one male and one female; Giffoni Valle Piana , one male and one female; Ruvo del Monte , two males and one females; Monte Cocuzzo , one males. As already successfully performed , adult i banding , C-bandi banding , C-bandistaining , Chromomstaining ; G bandistaining . For Lat minutes .PageBreakGenBank (NC012431). A homologous sequence of Ophisaurus attenuatus Baird, 1880 (EU747729) from HG003678-HG003683). Uncorrected pairwise distances (p-distances transformed into percent) within individuals of Anguis veronensis and between species were computed using MEGA, version 5.05 . All individuals showed a karyotype of 2n = 44 elements, comprising ten pairs of macrochromosomes and twelve pairs of microchromosomes. The first macrochromosomes pair was metacentric, the fourth and fifth were submetacentric and the others were telocentric with the exception of the tenth pair  and 2 insertion/deletions between samples from Italy and Spain, corresponding to an average uncorrected genetic distance of 4.2%, thus confirming the genetic distinctiveness of the Italian populations. On the contrary, the analyzed Italian specimens were genetically very uniform and showed an intraspecific uncorrected divergence of 0.4%, whereas their uncorrected genetic distance from the outgroup, n a tree  with larnth pair ; Table 1nth pair , arrows.nth pair . The C-bnth pair . Both G nth pair . Howevernth pair  (Fig. 3Bnth pair . The prenth pair , as it inth pair . Thus, tanalysis .Anguis veronensis.To conclude, our karyological study is consistent and provides further support for the recently discovered molecular and morphological differentiation of the Italian slow worm lineage , who asc