Ficus sp., Moraceae) pollen over enormous distances (> 100 km). Because of their extensive breeding areas, Neotropical figs are expected to exhibit weak patterns of genetic structure at local and regional scales. We evaluated genetic structure at the regional to continental scale  for the free-standing fig species Ficus insipida. Genetic differentiation was detected only at distances > 300 km (Jost´s estD = 0.68 ± 0.07 & STF = 0.30 ± 0.03 between Mesoamerican and Amazonian sites) and evidence for phylogeographic structure (RST>>permuted RST) was only significant in comparisons between Central and South America. Further, we assessed local scale spatial genetic structure  in Panama and developed an agent-based model parameterized with data from F. insipida to estimate minimum pollination distances, which determine the contribution of pollen dispersal on SGS. The local scale data for F. insipida was compared to SGS data collected for an additional free-standing fig, F. yoponensis (subgenus Pharmacosycea), and two species of strangler figs, F. citrifolia and F. obtusifolia (subgenus Urostigma) sampled in Panama. All four species displayed significant SGS (mean Sp = 0.014 ± 0.012). Model simulations indicated that most pollination events likely occur at distances > > 1 km, largely ruling out spatially limited pollen dispersal as the determinant of SGS in F. insipida and, by extension, the other fig species. Our results are consistent with the view that Ficus develops fine-scale SGS primarily as a result of localized seed dispersal and/or clumped seedling establishment despite extensive long-distance pollen dispersal. We discuss several ecological and life history factors that could have species- or subgenus-specific impacts on the genetic structure of Neotropical figs.Wind-borne pollinating wasps (Agaonidae) can transport fig ( Ficus spp., Moraceae). Fig trees have specialized, often species-specific relationships with minute (1–2 mm) wasps in the family Agaonidae [In contrast with high latitude forests, where wind is often the primary vector of pollen- and seed-mediated gene flow in trees, animals are the predominant pollinators and dispersers of tropical trees ,2. Biotigaonidae ,5,6). Algaonidae ,7. Figs,gaonidae . Thus, lgaonidae ,9.Ficus life forms and subgenera [Ficus more generally. Nazareno et al. [Urostigma) in Brazil and indeed found evidence of significantly lower SGS as measured by the Sp statistic, which measures degree of SGS [There is some debate about how patterns of gene dispersal distances, and therefore spatial genetic structure (SGS), are likely to differ among ubgenera ,11,12. Fubgenera ,11,13, te of SGS .Ficus, the two subgenera exhibit contrasting life histories that could further influence genetic structure (see Pharmacosycea) require high light intensities mostly encountered in large light gaps [Urostigma) establish as hemi-epiphytes on host trees, rendering effective seed dispersal a more spatially random process.Within the Neotropical ture see . For exaght gaps , which cFicus insipida subs. insipida showed significant cpDNA haplotype differentiation between Mesoamerican and western Amazon samples [At the biogeographic scale, genetic structure has been shown for several fig species ,17. A re samples . Using aFicus insipida, and compares local scale spatial genetic structure (SGS) in an additional free-standing fig, F. yoponensis (subgenus Pharmacosycea), and two species of strangler figs, F. citrifolia and F. obtusifolia (subgenus Urostigma) within Panama. Using data from F. insipida, we developed and parameterized an agent-based model to estimate minimum pollen dispersal distances. We then evaluated the extent to which pollen dispersal is expected to drive SGS in our study species. Finally, in order to determine whether our microsatellite data concurs with results from plastid and ITS sequences in showing population differentiation for F. insipida across its distribution range [F. insipida (subgenus Pharmacosycea) broadly within the Panama Canal Watershed, and in Costa Rica and Peru and tested for phylogeographic structure at these nuclear loci.Here we present an analysis of genetic structure in Neotropical figs based on nuclear microsatellite data. Our study spans local, regional, and continental spatial scales for on range , we sampFicus species (family Moraceae) worldwide [Pharmacosycea is phylogenetically distant from Americana [Pharmacosycea are pollinated by fig wasps of the genus Tetrapus (Agaonidae) and grow as free-standing trees that mostly establish in early successional stages due to their initial dependency on high light intensity [Urostigma (section Americana) grow mostly as hemi-epiphytes or strangler figs on host trees (hereafter we will refer to Urostigma as “strangler figs”). Americana figs are pollinated by fig wasps of the genus Pegoscapus [There are more than 750 orldwide ,20 from orldwide . Moleculmericana . Trees ontensity . Speciesgoscapus .F. insipida. At the local scale we included a second free-standing fig species F. yoponensis, and two species of strangler figs, F. citrifolia and F. obtusifolia, all of which are mainly dispersed by fruit-eating bats. Ficus obtusifolia usually grows as a strangler fig or hemi-epiphyte, and seldom as free-standing tree. Large individuals are most frequently found in mature forests. In contrast, F. citrifolia mostly grows as a small hemi-epiphyte or bush and is frequently found on palm trees in the study area  where adult figs have been censused repeatedly since 1973 [F. insipida (n = 190) is by far the most common fig species in the vicinity of the Panama Canal and relatively abundant along the shoreline as well as on the North-Eastern part of BCI which is covered by secondary forest of about 90–150 years [F. yoponensis (n = 37) is relatively rare along most shorelines and thus sampling was limited to the secondary forest on BCI. The strangler figs F. citrifolia (n = 62) and F. obtusifolia (n = 59) were mostly sampled along the shoreline, as few individuals are known from the core region of the main island , within in Soberanía National Park, for more intensive studies of spatial genetic structure, including a comparison of SGS in adults  and saplings . While mostly situated within 20 m from the dirt road, some individuals were located up to 600 m in the forest interior , Ministerio del Ambiente, Energía y Telecomunicaciones, Costa Rica  and Instituto Nacional de Recursos Naturales, Peru .F. citrifolia and F. popenoei [Microsatellite data are within Supporting Information files , expected heterozygosity (He), global estimates of FIS over alleles [For each species and sampling site we calculated total numbers of alleles , the natural logarithm of the spatial distance between individuals, and F1 being the mean pairwise kinship coefficient of the first distance class that includes nearest neighbors . To determine the significance of b we conducted 9999 permutations of the spatial positions of all individuals. Standard errors (SE) of b were approximated by jackknifing. We restricted the range for regression to dij = 0–3.0 km, with 3.0 km being the maximum distance of the smallest sampling area (for F. yoponensis on BCI). For F. insipida, we calculated Sp for all samples collected in the BCNM and for samples exclusively from BCI to compare them to the results of F. yoponensis. We always refer to the Sp statistics when discussing SGS in this manuscript. When comparing SGS among sampling sites or species we classified differences of Sp as significant if standard errors (± 2 SE) of b did not overlap.For the local scale SGS analyses, we calculated the kinship coefficient e et al.  that meaquencies . With thnd Hardy . This stFij for n = 5–14 distance classes such that the number of pairwise comparisons within each distance interval was approximately constant. This way, the values for the first distance class do not correspond to the F1 values calculated for the Sp statistics. We obtained 95% confidence intervals (CI) by permuting Fij of all pairs of individuals per distance class 9999 times. Standard errors were assessed by jackknifing for each distance class. All calculations were performed with the program SPAGeDi version 1.3d [Fij over ln(dij) in R [In order to visualize the data for each species, we plotted mean ion 1.3d . In addiij) in R  and geneDest and FST to determine whether there is genetic differentiation among sampling sites of F. insipida [Dest among the sampling sites and SPAGeDI to calculate FST. In order to test whether Dest increases significantly with distance we conducted a regression of Dest over distance in R version 3.0.1. To this end, we calculated a mean value across all pairwise comparisons for the Panamanian sampling sites that were not genetically differentiated (one data point) and used the pairwise comparisons among Panama, Costa Rica, Eastern and Western Peru (six data points). Further, we performed a STRUCTURE analysis [K, we tested K values from 1–10 and carried out 10 runs for each K. Afterwards, we followed the procedure suggested by Evanno et al. [K. With the same settings, we analyzed all samples from Panama, Costa Rica and Peru with 10 repetitive runs for each K = 1–7.At larger spatial scales, we used Jost´s insipida . Under ainsipida ,41. We uinsipida  to calcuanalysis  to test analysis . We usedo et al.  to selecRST with its value after permuting allele sizes within loci (“permuted RST”—similar to FST) using SPAGeDI with 10,000 permutations. A significant one-sided test establishes the alternative hypothesis of RST > “permuted RST”. Sequential Bonferroni corrections [Phylogeographic structure can be inferred from the microsatellite DNA data set if allele sizes within a populations or geographic region are more similar than in the overall sample . This warections  were appR programming language [F. insipida trees in the BCNM. Based on the available census data we included all F. insipida trees on BCI that were still alive in 2007 and all trees mapped along the shoreline of the islands and peninsulas  of 7.0–10.9 and total allele numbers of 56–109 alleles per species  and low inbreeding coefficients (mean FIS = -0.056–0.054), except for F. obtusifolia, which exhibited a relatively high FIS of 0.146. Ficus obtusifolia had an excess of homozygosity in 4 (of 7) loci. Because FIS is not uniformly high across all loci, we interpret deviations from HWE in the 4 loci as the result of allelic drop-out rather than inbreeding, which would affect all loci. Allele dropout is commonly encountered in microsatellite markers that were not developed for the focal species.Each sampled individual had a unique multi-locus genotype. All of the species exhibited high levels of heterozygosity . Along PLR, SGS was significant in adults , but only marginally significant in saplings .For Sp statistics revealed strongest SGS for the strangler fig F. obtusifolia  compared to F. citrifolia , F. yoponensis  and F. insipida. When comparing Sp based on the overlap of 2SE, only F. obtusifolia and F. insipida showed significant differences with much stronger SGS in F. obtusifolia. When we restricted analysis for F. insipida to trees growing on BCI to better compare it to F. yoponensis, we found no significant SGS . To exclude the possible of null alleles affecting these results, we repeated the analysis by omitting markers with null alleles  for distance classes up to 1 km in insipida . For botF. insipida trees had no potential mate within a radius of 500 m (mean number of mating partners < 500 m: 1.7 ± 1.0). On average, 4.0 ± 2.1 potential pollen donors could be encountered within a radius of 1 km in our simulations  and did not increase with distance. The STRUCTURE analysis detected only one cluster in this entire area. In contrast, at the biogeographic scale (Costa Rica to Peru) we found a strong linear correlation of Dest over linear Euclidian distance  and Peru (P < 0.01)  . There wF. insipida. Significant genetic differentiation among Mesoamerican and Amazonian sites indicates that pollen dispersal is restricted over biogeographic scales. At the local scale we detected weak but significant SGS in F. insipida as well as in three other sympatric fig species from both New World subgenera Pharmacosycea (free-standing figs) and Urostigma section Americana (strangler figs). Our model results were consistent with previous genetic studies [We collected nuclear microsatellite data to estimate genetic structure at different spatial scales in the common free-standing fig  studies  that shoHO and low FIS values in our study which indicate high levels of random mating. Our model results using data from F. insipida are consistent with Nason et al.´s [F. insipida yet have similar asynchronous flowering patterns.Figs exhibit the highest pollination distances known in insect-pollinated plants . In Panaet al.´s  findingsFicus species for other reasons, including life history differences associated with fruit size, and differences in habitat requirements for seedling establishment, both of which we explore in the following paragraphs. As any measure of SGS will only provide a temporally and spatially restricted snapshot, we also discuss the potential influences of colonization history and fluctuations in population density on SGS of the studied fig populations.While long distance pollen dispersal is a common feature in Neotropical figs, SGS patterns may vary predictably among Neotropical F. insipida in our study, coupled to the fact that SGS is not significant even in saplings, suggests that light gaps are being seeded by multiple maternal sources. As free-standing figs are most dominant in early successional stages of forests, colonization history has to be considered as well. The northwestern part of BCI experienced a long history of agricultural use and the forest only began to regenerate about 150 years ago. Almost all F. insipida and F. yoponensis trees on BCI are restricted to this secondary forest [Ficus trees established in early successional stages and appear to be the same age on BCI. As the forest matured, further recruitment was impeded by low light conditions under the canopy cover and, currently, sapling Ficus trees are extremely rare in BCI´s closed canopy forest [F. insipida on BCI  indicata on BCI  could beF. insipida and F. obtusifolia should be dispersed over larger distances and exhibit lower SGS . We . We F. c schemes , we reco schemes  that mayF. insipida despite the spatial limitations of seed dispersal that likely generated SGS at the local scale. This is consistent with prior estimates of breeding area based on pollen dispersal studies for strangler figs [FST, RST and Dest only permit a post hoc interpretation, the phylogeographic structure of F. insipida detected between our Central and South American samples is consistent with the previously published cpDNA phylogeography [Ne (1/4 the Ne of nuclear markers) and genetic drift therefore plays a much stronger role in cpDNA markers. Chloroplast capture from congeneric species is also possible, and has been demonstrated in Ficus and other tree taxa [Ficus [F. insipida, and perhaps other fig species, to overcome the geographic barriers that have caused vicariance in many other tree species.At the regional scale within the Panama Canal watershed we detected no population substructure for ler figs . At the ler figs  . S1 Dataset(XLSX)Click here for additional data file.S1 FigF. insipida, F. yoponensis, F. citrifolia and F. obtusifolia.Shown are all sampled individuals of (PNG)Click here for additional data file.S2 FigK = 5 where samples from Peru and Costa Rica were assigned to separate clusters while samples from Panama were jointly assigned to three clusters. The graph was based on the run that had the highest Ln P(D) value with K = 5.The most likely number of cluster was (PNG)Click here for additional data file.S3 FigHere, tree 54 (blue dot) is in its receptive phase and all trees highlighted in red release fig wasps at a given day. Blue circles around tree 54 indicate the distance classes .(PNG)Click here for additional data file.S4 FigBoxplots are based on 10 runs where the release and receptive phase last for five (A) and seven (B) days each.(TIF)Click here for additional data file.S5 FigFij). Dotted lines represent 95% confidence interval. Depending on the spatial scale the x axes vary between graphs. .Pairwise kinship is determined by Loiselle´s kinship coefficient ((TIF)Click here for additional data file.S6 FigRegression in based on all pairwise comparisons within a radius of 3 km. Regression was performed in R using a linear model. Within each graph, r2, the regression slope b and p are indicated .(TIFF)Click here for additional data file.S1 MethodsWith Table A: Allele length for the tested cpSSRs from Weising & Gardner (1999).(DOCX)Click here for additional data file.S2 Methods(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Tablea), the expected heterozygosity (He) and the inbreeding coefficient (FIS).For each marker, we provide the percentage of missing data, the number of alleles (N(DOCX)Click here for additional data file.


Mustela nigripes) as a case study for evaluating this emerging biomedical technology as a tool for genetic restoration. The black-footed ferret has depleted genetic variation yet genome resource banks contain genetic material of individuals not currently represented in the extant lineage. Thus, genetic restoration of the species is in theory possible and could help reduce the persistent erosion of genetic diversity from drift. Extensive genetic, genomic, and reproductive science tools have previously been developed in black-footed ferrets and would aid in the process of developing an iSCNT protocol for this species. Nonetheless, developing reproductive cloning will require years of experiments and a coordinated effort among recovery partners. The information gained from a well-planned research effort with the goal of genetic restoration via reproductive cloning could establish a 21st century model for evaluating and implementing conservation breeding that would be applicable to other genetically impoverished species.Interspecies somatic cell nuclear transfer (iSCNT) could benefit recovery programs of critically endangered species but must be weighed with the risks of failure. To weigh the risks and benefits, a decision-making process that evaluates progress is needed. Experiments that evaluate the efficiency and efficacy of blastocyst, fetal, and post-parturition development are necessary to determine the success or failure or species-specific iSCNT programs. Here, we use the black-footed ferret ( Puma concolor coryi) provides an example of the power of genetic restoration as a management tool for in situ conservation. A population on the verge of extinction, Florida panthers showed classic symptoms of inbreeding depression: cryptorchidism, heart defects, and declining reproduction (Puma concolor stanleyana) into the remaining isolated population of 20–25 Florida panthers in 1995 led to positive population response  Peltophryne lemur) Aphanius baeticus) Ailuropoda melanoleuca; In parallel to the management of One technology which has emerged as a useful tool for biomedical research is somatic cell nuclear transfer SCNT;  which enNearly all of the recent reviews of genetic restoration by reproductive cloning have been published in reproductive biology or cloning journals , and thuin vitro and then implanted surgically or by nonsurgical methods into surrogate females that carry the developing clone through to parturition. The resulting animal is considered a clone of the donor of the somatic cell.Reproductive cloning via SCNT allows for the genetic duplication of an individual. What made SCNT revolutionary was that it eliminated the need for the desired genetic material to come from a germ cell; genomes of interest could, in principle, come from any somatic cell. In practice, these cells are often fibroblasts in cell culture. A donor oocyte, whose nucleus has been removed, becomes the cellular vessel that holds the diploid genome from a different organism. Depending on the method of fusion used, the donor ooplast may retain the cytoplasmic architecture of its donor including mitochondrial DNA, RNA, and other organelles in the cytoplasm or a mix of cytoplasmic material from the egg and the nucleus donors . Donor oThe process of reproductive cloning is inefficient. Many enucleated oocytes must be merged with many nuclei to produce viable clonal embryos, and not all embryos develop to term . As conshttp://www.frozenark.org/), and regional biological resource banks have been created for southern African wildlife  the opportunity no longer exists to add genetically unique immigrants from the wild, because wild populations are extinct. Curated, frozen repositories of somatic and germ cells  have been created for the purpose of both assisted breeding and reproductive cloning. These collections provide unique genetic resources to these critically endangered species with the goal of increasing the effective population size of the species. For example, cryopreservation of sperm has successfully been used to genetically augment many captive breeding programs , differences in gestation length prohibited the use of domestic goats as foster mothers, and F1 hybrids were ultimately used  and totipotency  . BecauseExisting reproductive and developmental resources for black-footed ferrets greatly surpass what is available for the vast majority of currently endangered species. The species was used to pioneer assisted breeding in endangered species, and thus, many details are known about the reproductive biology of this species . SCNT haCynomys spp.), and ultimately disease reduced the species to one population. A century of local population extirpation resulted in a 30–50% loss of genetic diversity in the species . Indeed, the black-footed ferret has previously been suggested as a candidate for conservation cloning  which diverged approximately 800000 years ago from black-footed ferrets  and M. putorius (2n = 40) show strong evidence of F1 hybridization and backcrossing in the wild, which suggests that F1 animals are fertile (Mustela nigripes (2n = 38) have previously been bred with domestic ferrets , and M. nigripes × M. eversmanni (2n = 38) crosses produced healthy offspring  incompatibilities of black-footed ferrets with either Steppe polecats or domestic ferrets would inhibit the production of a viable, full-term animal. Indirect evidence suggests that the likelihood of creating a viable interspecies blastocyst is high, given that incomplete reproductive isolation is a feature of this clade. Other mustelids in the ferret clade hybridize in captivity  and the  fertile . Mustelaffspring . Mustelarrogates  and M. nin vitro until the blastocyst stage and then implanted into a surrogate mother. Finding the appropriate species  would require embryo transfers to determine if healthy animals of one species could be carried to term and born to another species. For SCNT cloning of domestic ferrets, the transfer of the embryos to the surrogate requires surgery in order to place the embryos in the oviduct. The risk of surgery may make highly endangered black-footed ferrets a less desirable surrogate mother.For the sake of further discussion of iSCNT in ferrets, we will assume the domestic ferret to be the oocyte donor species, with the understanding that other ferret species may ultimately be found to be more suitable for iSCNT. To clone black-footed ferrets, domestic ferret oocytes would need to be harvested, enucleated, and fused with the fibroblast of black-footed ferrets . The resIn vitro experiments whose goal was to reduce improper genetic reprogramming to increase healthy blastocyst and ultimately fetal and juvenile development would need to be undertaken. As discussed earlier, promising use of treatments to erase methylation patterns seem to mimic the process of genetic reprogramming and hold promise for endangered species iSCNT . Fibroblasts are regularly used as sources of nuclear material for SCNT and thus have a high possibility of success compared to other sources of somatic material. In addition to their feasibility as source genomic material, these cell lines were collected from individuals that did not directly contribute to the foundation of the captive breeding population, and thus inclusion of their genetic material in the current gene pool would represent an increase to standing genetic diversity in the black-footed ferret gene pool. The relatedness of these 2 cell donors to each other or to the genetic founders of the extant population is unknown, and thus, the magnitude of their potential contribution to genetic diversity is currently unknown. Nonetheless, as has been demonstrated repeatedly in other cases of genetic rescue, the addition of variation from even 1 individual can change the demographic trajectory of a population  population for 5000–8000 years as intermontane grasslands became separated from Great Plains grasslands by high elevation sagebrush habitat . The genNew genomic resources that may eventually benefit conservation cloning have been discussed in scientific forums e.g.  but havehttp://www.ncbi.nlm.nih.gov/sra/?term=SRP044096) and could be used as a scaffold to understand and reconstruct genomic variation across the geographic breadth of this species. These endeavors unto themselves will be valuable to understanding the nature of loss of genetic variation.For black-footed ferrets, short fragments of mtDNA from museum specimens have been successfully extracted  and utilex situ populations of endangered species, positive or negative fitness consequences may occur. Gene pool enrichment has been observed to increase population viability through increased fecundity, juvenile survival, recruitment, and immunocompetence in both facilitated translocation and natural migration events .University of Florida’s Department of Wildlife Ecology and Conservation, Lincoln Park Zoo, San Diego Zoo Institute for Conservation Research, and supporters of Revive and Restore (


Stat3 or IL-6 signalling in a Pten-deficient PCa mouse model accelerates cancer progression leading to metastasis. Mechanistically, we identify p19ARF as a direct Stat3 target. Loss of Stat3 signalling disrupts the ARF–Mdm2–p53 tumour suppressor axis bypassing senescence. Strikingly, we also identify STAT3 and CDKN2A mutations in primary human PCa. STAT3 and CDKN2A deletions co-occurred with high frequency in PCa metastases. In accordance, loss of STAT3 and p14ARF expression in patient tumours correlates with increased risk of disease recurrence and metastatic PCa. Thus, STAT3 and ARF may be prognostic markers to stratify high from low risk PCa patients. Our findings challenge the current discussion on therapeutic benefit or risk of IL-6/STAT3 inhibition.Prostate cancer (PCa) is the most prevalent cancer in men. Hyperactive STAT3 is thought to be oncogenic in PCa. However, targeting of the IL-6/STAT3 axis in PCa patients has failed to provide therapeutic benefit. Here we show that genetic inactivation of  IL6-STAT3 signaling is activated in prostate cancer, however inhibiting this pathway has not lead to a survival advantage in patients. Here, Pencik et al. show that loss of the IL6-STAT3 axis in mice and humans leads to metastasis due to loss of ARF, unravelling STAT3 and ARF as potential prognostic markers in prostate cancer. ARF was shown to augment p53 stability by promoting the degradation of Mdm2, a negative regulator of p53. Concomitant inactivation of Pten and p53 leads to bypass of senescence and as a consequence to a malignant PCa phenotypePten-deficient PCa model accelerates cancer progression leading to metastasis. Loss of IL-6/Stat3 signalling in PCa bypasses senescence via disrupting the ARF–Mdm2–p53 tumour suppressor axis. We identify ARF as a novel direct Stat3 target. Notably, loss of STAT3 and p14ARF expression correlates with increased risk of recurrence in PCa patients. In addition, STAT3 and p14ARF expression was lost in metastasis compared with the primary tumours. We identified STAT3 and CDKN2A mutations in primary PCa patients. Furthermore, PCa metastases show a high frequency of STAT3 and CDKN2A deletions. We propose STAT3 and ARF as prognostic markers for high versus low risk PCa patient stratification.In this study, we show that loss of IL-6/Stat3 signalling in a Pten in the prostate epithelium (Pb-Cre4 Ptenfl/fl) hereafter referred to as Ptenpc−/− (ref. Ptenpc−/− compared with wild-type (WT) prostate epithelium  proliferating cells and reduced numbers of cleaved caspase 3 positive (CC3+) apoptotic cells compared with Ptenpc−/− prostates  PCa with liver and lung metastases  at 19 weeks of age, whereas Ptenpc+/−mice developed only prostatic intraepithelial neoplasiaPten-deficient PCa. We found reduced p53 protein expression in Ptenpc+/−Stat3pc−/− prostates (Stat3 (Stat3pc−/−) led to development of PIN lesions in prostates at 19 weeks of age  mediated knockdown of Stat3. Western blot and IHC analyses confirmed efficient Stat3 knockdown in these cells   . To corrl shRNA)  increasel shRNA) . Notablyl shRNA) , led to l shRNA) . These dPten triggers senescence thereby restricting cancer progression and metastasisPtenpc−/−PCa cellsPtenpc−/−Stat3−/− tumours lacked p21 expression, displayed reduced numbers of PML nuclear bodies and decreased SA-β-Gal activity compared with Ptenpc−/− tumours  and WT mouse embryonic fibroblasts (MEFs). Both p19ARF protein and mRNA levels were significantly reduced in the absence of Stat3 (Stat3C/+ and Stat3C/C) from the endogenous Stat3 locusARF protein and mRNA z-scores of IL-6 or STAT3 expression. Specifically for IL-6 expression, samples that had z-scores<−2 were defined as low IL-6 and all others (z-scores>−2) were used as a comparator for prognostic significance. Conversely, samples that had STAT3 z-scores>2 were defined as STAT3 high and all other samples (z-scores<2) were used as comparators. Time to biochemical recurrence (BCR) was assessed as an indicator of individual prognosis. BCR was defined by an increase to >0.2 ng ml−1 PSA in serum. Of note, patients with low IL-6 expression levels showed a significantly higher risk of BCR compared with patients with high IL-6 expression  using validated antibodies  and low p14ARF  expression were associated with poor outcome   . Using mwith GSC . In accoth GSC 25. IHC anath GSC 25, suggestpatients . Moreove disease . This is disease , where iSTAT3 (2.5%) and CDKN2A (2.3%) in a cohort of 529 patients with primary PCa .ARF , p14ARF , p21 , PML , Ki-67  and Cleaved Caspase 3  and ISG15 , AR . Staining for SA-ß-Gal activity was performed according to the manufacturer's protocol . We used PTENARF antibody was validated using the human melanoma cell linesARF) and VM-7 (deletion at the p14ARF locus).Immunohistochemistry and haematoxilin/eosin staining was performed with formalin-fixed paraffin-embedded (FFPE) tissue using standard protocols using consecutive sections. The following antibodies were used for immunohistochemistry: pY-Stat3 , pS-Stat3 , Stat3 , p-Akt , Akt , p53 , p19www.tissuegnostics.com) as described in detail in , p53 , p21 (1:500 dilution), β-actin , p-Akt , Akt , AR , GAPDH , Pan-Cytokeratin , Mdm2 , PML  and GAPDH . For protein expression analysis by western blot, frozen tissue samples and cell lysates were prepared as describedTotal RNA was isolated using Trizol (Invitrogen) according to the manufacturer's instructions. For quantitative reverse transcription PCR (qRT–PCR) analysis, 1 μg of total RNA was reverse transcribed to cDNA using the Transcriptor First-Strand cDNA Synthesis kit (Fermentas). qRT–PCR was performed in triplicate with aa MxPro3000 and SYBR GreenERqPCR mix (Invitrogen). Real-time monitoring of PCR amplification was performed using the LightCycler 480 detection system (Roche). The relative amount of specific mRNA was normalized to β-actin in each sample. Primer pairs are listed in Stat3-null MEF were isolated by trypsin treatment of individual littermate E13.5 embryos from a cross of Stat3+/− heterozygous mice. Stat3+/− mice were generated from conditional Stat3+/fl micein vivo using Mox2-Cre. Cells were amplified and used in experiments starting at passage 2. Stat3−/− MEFs were grown in DMEM supplemented with 10% FBS, 2 mM L-Glutamine, 0.1 mM NEAA, 20 mM HEPES and pen/strep using standard techniques. Stat3C/+ and Stat3C/C MEFs were generously provided by Valeria Poliin vitro cultures LNCaP, RWPE-1 and PC3 were cultured under standard conditions.Primary WT and Ptenpc−/− mice at 19 weeks of age was cut into pieces of 0.2 mm, plated in tissue culture dishes coated with type I collagen (BD Pharmingen) and supplied with a minimal amount of epithelial growth medium containing 1% FBS serum. After 24 and 48 h, more medium was added. After an additional 24–72 h, cells began to grow out from the prostate tissue. Clean epithelial cell lines were obtained by removing the unwanted cell type with a cell scraper or pipette tip. Cells were passaged using Accutase . For the generation of cell lines, cells were passaged until they survived crisis and only cells with typical epithelial morphology were maintained. WT and Ptenpc−/− prostate cell lines were established and maintained in DMEM/F12  plus 10% foetal bovine serum , 25 mg ml−1 bovine pituitary extract , 5 mg ml−1 EGF , insulin/transferrin/selenite , pen/strep , cholera toxin  and ciprofloxacin . Prostate tumour epithelial cell lines expressed the epithelial marker Pan-Cytokeratin detected by immunofluorescence and western blot analysis. Pten deletion was confirmed by PCR and western blot analysis.Dissected prostate tissue from WT and −1 PSA in serum, a log-rank (Mantel–Cox) test was used to test significance. The hazard ratio and its confidence interval were calculated using the Mantel–Haenszel method. All the human samples for TMA and their use in this study were approved by the Research Ethics Committee of the Medical University Vienna, Austria (1753/2014) and by the Research Ethics Committee for Germany (395/2008BO1) .The human tissue arrays representing a panel of 204 patient samples were obtained from the Institute of Pathology, Tuebingen, Germany and from the Clinical Institute of Pathology (CIP) of the Medical University of Vienna (MUW), Vienna, Austria. The cohort contains tumour material from PCa patients who consecutively underwent radical prostatectomy at the University Hospital of Tuebingen or the University Hospital of the MUW. Each PCa specimen was represented by two cores on tissue microarrays (TMA). From the CIP of the MUW, we furthermore obtained 41 PCa and 23 metastasis FFPE patient samples. In addition, we include FFPE material from 67 matched patient samples with PIN and PCa (GSC≤7), as well as 7 matched patient samples with primary PCa and PCa metastases, all from the CIP, MUW. The FFPE tissue blocks were sectioned as 3-μm-thick sections mounted on slides and stained with haematoxylin and eosin. Subsequently, the area of cancer was marked by a pathologist (S.P.). Cores, each of up to 0.6 mm in diameter, were taken from the corresponding donor block and placed into a TMA recipient block using a semiautomatic tissue arrayer (Beecher Instruments). Tissue sections (3-μm thick) were placed onto superfrost slides. IBM SPSS Statistics 20 (IBM) was used for statistical analysis. For determination of time to BCR, which is defined by an increase to >0.2 ng mlt-test and for comparing more than two groups Tukeýs post hoc test was used. Fisher's exact test was employed when differences in distributions within groups were monitored. For Kaplan–Meier analysis and log-rank statistical evaluation of time to BCR, as well as evaluation of prognostic power in univariate and multivariate analysis, we used the IBM SPSS version 22 programme. To analyse Affymetrix SNP Chip 6.0 primary data, we used the AGCCytoScan Software together with the Chromosome Analysis Suite 3.0 (both Affymetrix), and deletions >200 kb were considered significant. For analysis of mutations in primary PCa, we used the ICGC (www.icgc.org).Data were analysed using GraphPad Prism 6 software. For comparing two groups unpaired Students −1; R&D Systems) in PBS. After blocking, 100 μl aliquots of cell lysates or culture supernatants were added. IL-6R bound to the plate was detected by biotinylated goat anti-mouse IL-6-R pAB BAF1830  followed by streptavidin–horseradish peroxidase (R&D Systems). The enzymatic reaction was performed with soluble peroxidase substrate (BM blue POD from Roche) at 37 °C and the absorbance was read at 450 nm on a SLT Rainbow plate reader (Tecan). For the detection of murine IL-6 in sera of mice, the samples were diluted 1:3 with 1% BSA–PBS.For the detection of soluble sIL-6R in sera of mice, the samples were diluted 1:15 in 1% BSA–PBS. The ELISA was performed as describedHow to cite this article: Pencik, J. et al. STAT3 regulated ARF expression suppresses prostate cancer metastasis. Nat. Commun. 6:7736 doi: 10.1038/ncomms8736 (2015).Figures 1-19, Supplementary Tables 1-3, Supplementary Methods and Supplementary References


For example, close genetic relationships are seen in cortical regions within the same lobes and, the frontal lobe, a region showing great evolutionary expansion and functional complexity, has the most distant genetic relationship with other lobes. The frontal lobe also exhibits the most distinct expression pattern relative to the other regions, implicating a number of genes with known functions mediating immune and related processes. Our analyses reflect one of the first attempts to provide an assessment of the biological consistency of a genetic phenomenon involving the brain that leverages very different types of data, and therefore is not just statistical replication which purposefully use very similar data sets.The many subcomponents of the human cortex are known to follow an anatomical pattern and functional relationship that appears to be highly conserved between individuals. This suggests that this pattern and the relationship among cortical regions are important for cortical function and likely shaped by genetic factors, although the degree to which genetic factors contribute to this pattern is unknown. We assessed the genetic relationships among 12 cortical surface areas using brain images and genotype information on 2,364 unrelated individuals, brain images on 466 twin pairs, and transcriptome data on 6 postmortem brains in order to determine whether a consistent and biologically meaningful pattern could be identified from these very different data sets. We find that the patterns revealed by each data set are highly consistent (p<10 Although functional and anatomical connections among cortical regions have been intensively explored, genetically-mediated relationships between cortical regions have not been pursued to the same degree. Identifying genetic factors that mediate these relationships among different brain subcomponents can provide insight into how the human brain is organized and functions. We have assessed the genetic relationships among cortical regions using an integrated approach that considers twin data, genotype information among a large set of unrelated individuals, and gene expression measurements from postmortem neural tissues. We looked for evidence that subsets of cortical brain regions are under common or unique genetic control. We found that the patterns of genetic relationships are highly consistent across three independent data sets and multiple lines of evidence, suggesting that the patterning of cortical surface area is strongly mediated by genetic factors and, furthermore, likely reflects underlying anatomical and possibly functional relationships among cortical brain regions. The human cerebral cortex is known to be composed of functionally and anatomically specialized regions based on lesion, neurophysiological and neuroimaging studies . DespiteWe assessed the consistency of cortical patterns likely attributable to polygenic factors in humans by comparing genetic correlations between every pair of 12 pre-defined cortical regions among 2,364 unrelated individuals, 466 twin pairs, and postmortem brain samples from 6 individuals. Each of these data sets has unique features and requires a different set of statistical modeling and data analysis techniques. As such, if the results of each reveal a consistent genetically mediated pattern among the 12 human cortical regions, it would suggest that the pattern exists. Note that most replication studies of a particular phenomenon, especially in the context of genetic association studies and GWAS, focus on replicating the actual study designs in detail and not on the biological consistency of the findings in different contexts. Thus, a complementary way of validating a particular phenomenon is to assess it using different approaches altogether, looking for independent evidence of the phenomenon of interest across all of them. A recent paper by Richiardi et al. does attempt to look for consistency of results from fMRI studies and post-mortem brain gene expression studies and is thus similar in orientation to our approach . Howeverrg, which is simply the genetic covariance divided by the product of genetic standard deviations of each region ). The most highly correlated pairs of regions (correlation coefficient r > 0.25 in at least one data set) involved neighboring regions within the conventional lobar divisions. This pattern was consistent between the two data sets. The most anti-correlated pairs of regions, again consistent between the two data sets, were mostly between regions on the frontal lobe and regions on the other three lobes.The phenotypic correlation matrices derived from the VETSA and C5C data had entries that were highly correlated  model . The magThe Mantel test results for correlation matrix comparisons are shown in To further investigate the phenotypic and genetic relationships of cortical surface areas, we compared the inter-regional genetic correlations with the gene co-expression profiles using a third independent data set: data from the publicly available Allen Human Brain Atlas , 18. We As exhibited in Figs In addition to visually comparing the correlation matrices and rigorously testing the similarities between them with Mantel tests, we further examined how the genetic relationships between regions cluster those regions. We took an average over the twin genetic correlations of VETSA , genotyp-4). Half of the associated genes were from the set of transcripts distinctively found in the frontal lobe. Our integrated analyses resulted in three main findings: (1) We observed consistency of a genetically-based cortical parcellation scheme among our twin sample and C5C sample of unrelated individuals. The effective number of independent phenotypes from the 12 cortical regions was estimated to be between 11 and 12 for both sample sets, suggesting that the parcellation did identify phenotypically and genetically distinct cortical subdivisions within the spatial resolution of our brain images. (2) Genetic correlations of surface area among cortical regions estimated from twin modeling were highly consistent with those estimated from genome-wide genetic markers using an independent sample of 2,364 unrelated individuals. Similarly, the gene co-expression pattern among cortical regions in six postmortem brains was also highly consistent with genetic correlations among the surface areas of the cortical regions estimated from twin or genotype-based analysis. (3) We found that the majority of genes (71.4%) were ubiquitously expressed in the cortex, whereas the minority of genes showed region-specific expression patterns. The frontal lobe exhibited the highest number of distinctively expressed genes whose level of expression was not as pronounced in the other brain regions. These genes included some immune related genes, and a larger proportion of expressed intergenic transcripts, which we consider in more detail below.How different brain structures are genetically related to each other is still something of a mystery. Here we explored the genetic relationships between cortical brain structures, asking if there is any evidence that subsets of cortical brain regions are under common or unique genetic control, and how individual cortical regions are organized genetically. To address this question, we examined evidence for pleiotropy on a whole genome basis  among various cortical structures in the human brain. The genetic correlation patterns we found are highly consistent across samples . For the genetic patterning to converge from different sample sets across different study designs using different analysis methods, several conditions intrinsic to all the data sets have to be met, including: (1) The boundaries of genetic cortical regions must be generally aligned; (2) the relative positions of genetic cortical regions must be preserved; (3) all pairwise genetic correlations between genetic cortical regions are similar; and (4) consistent genetic relationship estimates exist in genotype polymorphisms, genetic information based on twin data and gene expression data, regardless of data type, ethnicity, gender and age disparities across samples, because we have controlled for all these factors. Our first main finding was to test the requirements (1) and (2), and the second main finding was for the requirements (3) and (4). It is not trivial to meet all these requirements and derive convergent results from different computational models see ‘’ below. The highly conserved genetic correlation patterns across individuals suggest that, despite substantial structural and functional variability among individuals, the rudimentary genetic patterning of the human cortex in terms of these 12 large cortical regions is fundamentally similar. Our findings are consistent with the notion of the “protomap” hypothesis, which states that regional layout of the cortex is established at early stages of development by intrinsic genetic mechanisms. The cortex is initially patterned by gradients of signaling molecules and transcription factors within cortical progenitors –31. ThesA notable feature of cortical genetic patterning is that the spatial patterns of gene expression recapitulate the spatial topography of the cortex . We founWe examined the number of genes distinctively expressed in each of the four lobes. A majority of the genes, 71.4%, were ubiquitously expressed in cortical surfaces of all four lobes. Approximately 2.1% of, or 602, genes were expressed in the cortical surfaces of at least one but not all four lobes, suggesting that subtle differences in gene expression profile in terms of spatial locations in the brain may have significant consequences for cortical functional divergence. The frontal lobe, a region showing great evolutionary expansion, showed the highest number of distinctively expressed genes. This finding was consistent with the observed negative genetic correlations between the frontal regions and the regions of the other lobes in our genotype-based and twin-based analyses . The negThese selectively expressed genes in the frontal lobe are associated with immunity, cell cycle regulation and transport. The most significantly associated pathways are related to interferon-gamma, which is critical for immune response. A recent study has found that certain psychiatric disorders have involvement of immune-related gene loci . The genThe frontal lobe also has a larger proportion of the expressed intergenic transcripts compared to the distribution of all the analyzed transcripts, suggesting stronger regulatory involvements. Intergenic transcripts have been suggested to contribute to functional differences between humans and chimpanzees in an evolutionary comparison study .The genotype-based approach implemented in the GCTA program presumably captures the additive genetic effect contributed by all common single nucleotide polymorphisms (SNPs), but may not capture all the factors contributing to the heritability of a trait or phenotype , which are reflected in twin heritability. Hence, there is usually a difference between the overall heritability of a trait estimated from twin or pedigree data and that estimated from genotype data. Genetic correlations between two traits obtained by the twin and genotype-based analyses are likely to exhibit similar differences. Furthermore, although dominance effects at multiple loci and higher-order epistatic interactions were not explicitly considered, the consistency of the correlations across the data sets suggests that the exclusion of dominance and epistatic effects did not confound our analyses in substantive ways.Subjects making up the unrelated individuals in the C5C sample were limited to those with European ancestry to avoid stratification and genetic background effects. The analysis method for the classic twin design and for computing gene co-expression profiles were not susceptible to mixture of genetic ancestry; therefore, those analyses included Caucasians, African Americans and Hispanics. In addition to ancestry heterogeneity, our samples contained differences in gender and age. The twin sample only had male twins between 50–59 years of age; the C5C sample had both genders between 3–90 years of age; and the postmortem brain samples also had both genders between ages 24–57. We have adjusted age and gender in the analyses. The observed consistent genetic relationships were still evident and not affected by demographic heterogeneity.While our genetic analysis of MRI data focused on genetic correlations on one particular aspect of cortical morphology , and thus implicates only those genetic elements related to cortical surface area, the transcriptome analysis we pursued examined transcriptional variation across the cortex based on all genes. Therefore, although we found consistent patterns between our genetic and transcriptome analyses at the gross anatomical level, some level of discrepancy was expected.We found a consistent pattern of genetically-mediated relationships among cortical brain regions across different data sets and different analytical techniques. These cortical brain regions are genetically defined and largely correspond to known functional specialized regions. Thus, our results suggest that the overall cortical patterning, as reflected in the relationships among cortical regions, is shaped by genetic factors and, further, that this conserved spatial pattern may be important to organize functional modules of the cortex. This robust and consistent configuration might originate from a common evolutionary and developmental pattern of cortical regionalization. Although we know that several transcription factors are key players in intrinsic genetic mechanisms of cortical regionalization, especially based on animal data, there is a large knowledge gap regarding our understanding of polygenic contribution by common genetic polymorphisms to human cortical regions. Our work sheds light on the genetically-mediated organization of cortical regionalization. Identifying the specific variants underlying the likely polygenic pleiotropic effects we observed, however, will require further, likely very large-scale, studies.UCSD IRB approved this study as part of Project #131068X: "The above-referenced project was reviewed and approved by one of this institution's Institutional Review Boards in accordance with the requirements of the Code of Federal Regulations on the Protection of Human Subjects (45 CFR 46 and 21 CFR 50 and 56), including its relevant Subparts." Each study was approved by the local Institutional Review Board: South East Norway (TOP and NCNG) and Mid Norway (HUNT) Regional Ethical Committee (HUNT), and UC San Diego (PING and VETSA).A combined sample of five sub-study cohorts (C5C) is made of 605 subjects from the Thematically Organized Psychosis (TOP) study, 842 Health Study of Nord-Trøndelag (HUNT) subjects, 325 Norwegian Cognitive Neuro-Genetics (NCNG) subjects, 726 Alzheimer’s Disease Neuroimaging Initiative (ADNI) subjects, and 1198 Pediatric Imaging Neurocognition and Genetics (PING) subjects. The samples for the twin analysis was part of the Vietnam Era Twin Study of Aging (VETSA) study . There wEach study was approved by the local Institutional Review Board (IRB): South East Norway (TOP and NCHG) and Mid Norway (HUNT) Regional Ethical Committee (HUNT), and UC San Diego (PING and VETSA). The current study was approved by the IRB of UC San Diego.Magnetic resonance imaging (MRI) data of the brains were collected for all subjects with various scanners. The imaging data were analyzed using FreeSurfer software and the cortical surface was reconstructed to measure surface areas at 160k surface locations for each hemisphere. To account for global effects, we divided the area measure of each location by the total surface area in each subject. The surface locations were then parceled into 12 regions and surface areas of each cortical region computed. The regions were previously defined using a data driven clustering technique that identified parcels of the human cortex maximizing their genetic correlations based on twin modeling [All subjects from C5C were genotyped with different commercial arrays. Genotypes from each sub-study were imputed separately with European panels from the 1000 Genome Project. After quality control and removal of related individuals and individuals of non-European ancestry, 2364 subjects with 2,480,482 genome-wide imputed variants from the C5C remained for subsequent analyses.In this final combined cohort, 52% of the individuals were female; the subjects were aged 47 ± 24 y ; and 273, 128, 131, 147, and 66 subjects were diagnosed with mild cognitive impairment (MCI), Alzheimer’s disease (AD), schizophrenia (SCZ), bipolar disorder (BIP), and other psychosis (OP), respectively.The genetic correlations of surface area between cortical regions were derived using the classical twin modeling for the VETSA samples . A standA standard bivariate variance component model  similar Transcriptomic data of six human brains at hundreds of anatomical locations was obtained from Allen Human Brain Atlas , 18. TheNetwork analysis for frontal lobe genes were performed using GeneMANIA  and visuTo account for errors in both twin-based and genotype-based estimates of genetic correlations, a linear regression with errors-in-both-variables (EIV) model , 43, insMantel test , 44 was Full details are given in S1 Figrg estimates, clipped at 1. The genetic correlations shown in the graph were original values without standardization. Where an overall correlation between the two sets of genetic correlations was observed, some pairs of regions exhibited strong correlation (or anti-correlation) consistently. For instance, the pairs opercularis and subcentral region and the superior temporal region (clusters 5 & 6) had high rg from both sample sets, and the surface areas of the two regions were also highly correlated, suggesting that the genetic correlation is likely underlying the phenotypic correlation, which is also consistent with both regions belonging to the same human-specific subdivision involved in language. The occipital region (cluster 12) was consistently anti-correlated with regions in frontal lobe (clusters 1–5), while correlated with superior parietal region (cluster 10) and likely with precuneus region , both genetically and phenotypically, across both datasets Click here for additional data file.S1 TableSee also (DOCX)Click here for additional data file.S2 TableSee also Figs (DOCX)Click here for additional data file.S3 TableSee also (DOCX)Click here for additional data file.S4 TableSee also (DOCX)Click here for additional data file.S5 TableA small percentage of the genes were either distinctively expressed in the cortical surface areas of one lobe or co-expressed in multiple but not all four lobes of the brain, which are listed below. See also (DOCX)Click here for additional data file.S6 TableSee also (XLSX)Click here for additional data file.S7 TableSee (DOCX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.


Munroa argentina, an annual grass distributed in the biogeographic provinces of Puna, Prepuna and Monte. We collected 152 individuals from 20 localities throughout the species’ range, ran genetic and demographic analyses, and applied ecological niche modeling. Phylogenetic and population genetic analyses based on cpDNA and AFLP data identified three phylogroups that correspond to the previously identified subregions within the SATZ. Molecular dating suggests that M. argentina has inhabited the SATZ since approximately 3.4 (4.2–1.2) Ma and paleomodels predict suitable climate in these areas during the Interglacial period and the Last Glacial Maximum. We conclude that the current distribution of M. argentina resulted from the fragmentation of its once continuous range and that climate oscillations promoted ecological differences that favored isolation by creating habitat discontinuity.The South American Transition Zone (SATZ) is a biogeographic area in which not only orogeny (Andes uplift) and climate events (aridification) since the mid-Miocene, but also Quaternary glaciation cycles had an important impact on the evolutionary history of the local flora. To study this effect, we selected  The uplift of the Andes in the Neogene had a strong impact on the evolutionary history of South American biota –3. The rLate Neogene and Quaternary climate oscillations that involved several ice age cycles influenced species and community distribution patterns on Earth, and although aridification occurred mainly during the Pliocene, fluctuations in climate during the Pleistocene also contributed to the expansion of arid and semiarid regions in South America ,8,26–33.Hordeum [Recent studies on the evolutionary history of animal and plant taxa in the SATZ provinces ,33,42–49Hordeum ,39,42, aHordeum ,42. TherHordeum ,42.Munroa argentina (Poaceae), an annual [Hordeum and with a wide distribution in three SATZ provinces  [M. andina, with a common ancestor that inhabited the southern area of the SATZ [M. argentina is an ideal subject for studying the effects of these climate oscillations and the Andean uplift because it was settled in this biogeographic region before these events occurred. This species inhabits the open areas of plains and mountains from mid- (1000 m a.s.l.) to high elevations (4200 m a.s.l) on alluvial and sandy-stony soils, along seasonally dry creeks and rivers [M. argentina’s distribution and the structure of its genetic variation. Analyzing these two aspects can help reconstructing the evolutionary history of a species and identify putative Pleistocene refugia [To better understand the effect of not only the Quaternary climate oscillations but also of the Late Neogene climate oscillations and the uplift of the Andes in grassland taxa, we selected n annual  grass whd Monte) ,51. We hthe SATZ . Thus M.d rivers –52. In t refugia ,37,39, r refugia ,39,42,44 refugia ,42,44. BMaterial was collected and deposited in CORD the Herbarium of the Universidad Nacional de Cordoba, Argentina. The herbarium is recognized by the "Secretaría de Ambiente y Desarrollo Sustentable de la Nación" for doing that. Dr. Ana M Anton is the curator of Poaceae and participated in the collection of material. Collections were made under three scientific collecting permits granted by the following government agencies in Argentina “Secretaría de Medio Ambiente y Desarrollo Sustentable” in Salta ”, by the “Secretaría de Ambiente y Desarrollo Sustentable, Subsecretaría de Conservación y Áreas Protegidas, Dirección de Conservación y Áreas Protegidas” in San Juan (Res. 056)” and by the “Administración de Parques Nacionales” .M. argentina is a predominantly cross-pollinating annual grass with flowers with exserted anthers [M. argentina has been reported in Argentina, Bolivia and Peru [ anthers ,51. Plan anthers ,51. Sing anthers . M. argeand Peru ,51.M. argentina were sampled from 20 localities that cover its entire distribution range and its elevation gradient from 1000 to 4200 m a.s.l.  were assayed following the protocol of Lachmuth & al. . After tA statistical parsimony network of haplotypes was constructed using TCS ver. 1.2.1  with theM. argentina haplotypes during Pliocene, Pleistocene, and Holocene events, we performed molecular dating under a Bayesian approach as implemented in BEAST ver. 2.1.3 [M. argentina’s haplotypes was estimated using the age obtained from the previous dating for Scleropogoninae and Swallenia alexandrae [Sohnsia filifolia , Scleropogon brevifolius, Swallenia alexandrae, Erioneuron avenaceum, Munroa pulchella, and Munroa andina were outgroups. The node of the Scleropogoninae clade was constrained using a lognormal prior distribution : 0; Log (Stdev): 0.5, range: 19.5–17.5). The node of the Swallenia alexandrae was constrained using a lognormal prior distribution : 0; Log (Stdev): 0.5, range: 17.0–15.5). Analyses were run using a molecular clock model with uncorrelated rates, assuming a lognormal distribution of rates. Models of sequence evolution were the same as for the MrBayes analyses (GTR+I) and a coalescent model assuming logistic population growth was selected. Two MCMC analyses were run each with 100 million generations, and sampling every 10 000th generation. The time series plots of all parameters were analyzed in Tracer v.1.5 [http://tree.bio.ed.ac.uk/software/tracer/) to check for adequate effective sample sizes (ESS > 200) and convergence of the model’s likelihood and parameters between each run. Trees were combined in Log Combiner v.1.6.1 [To estimate genetic differentiation among r. 2.1.3 . The divexandrae –64. Sohner v.1.5  , haplotype diversity  and nuch; [π; )—were cah; [π; . Genetich; [π; . This anh; [π;  and inveh; [π; .D [Fs [R2 [−9 substitutions per site per year (s/s/y) for chloroplast-wide, synonymous substitution rates described for most angiosperms [−9 s/s/y calculated for the cpDNA loci in Munroa  and expected heterozygosity (HE) were calculated for each population groups using Arlequin and TFPGA v. 1.3  [For nuclear data (AFLP), SplitsTree v. 4.10  was utilquencies . The optno & al. . Geneticnalyses)  respectiM. argentina. A total of 200 occurrence points were compiled both during the fieldwork of this project and also using specimen records from the following herbaria: CORD, CASTELAR, BAA, BAB, IBODA, LIL, and LPB (Abbreviations for herbaria follow "Index Herbariorum" http://sweetgum.nybg.org/ih/). Environmental scenarios in the present and in the past were represented by a series of 19 variables summarizing aspects of climate [2. Four estimates of ecological niche models were made to identify: 1) the potential current distribution, 2) the potential past distribution during the Last Interglacial, and 3) the potential past distribution during the extreme conditions during the Last Glacial Maximum (LGM). For the first analysis, we used the current layer, followed by the last interglacial period layer , and the LGM  layer. For the LGM analysis we used general circulation model simulations from two models: the Community Climate System Model  and thel . All mol  was 1704 bp with 43 variable sites. Forty-one haplotypes were identified for the 152 individuals sampled from 20 localities . Haplotype H5 is shared by three localities BY, BP, BT (Bolivian Puna), and haplotype H13 is common in JT, JH, and JS (Argentinean Puna). Thirteen haplotypes were private to seven localities in both Argentinean and Bolivian Puna . HaplotyM. argentina haplotypes in a single and well supported clade (PP = 1.00), corroborating the monophyly of M. argentina. The topology of the recovered phylogram (not shown) is congruent with the result of the statistical parsimony network and identical to the chronogram obtained with BEAST  grouped all M. argentina began in the Middle Pliocene–Late Pleistocene, approximately 3.4 (4.2–1.2) Ma separating the Puna phylogroup from the ancestor of the Prepuna–Monte phylogroups  ranged 0.78–0.85, while nucleotide diversity (π) ranged 0.0008–0.0012 . AMOVA rFs, Tajima’s D, R2 test, and the SSD values suggest a historical population expansion by three phylogroups defined by TCS and phylogenetic analyses (Under a model of sudden population expansion (P>0.05), the Puna and Monte phylogroups have a strongly unimodal mismatch distribution indicating a historical population expansion event, while the Prepuna phylogroup have a bimodal mismatch distribution, indicating a historical population contraction/expansion . The samanalyses . The Bayanalyses . The PunΔK indicated that the most likely number of population clusters was three (K = 3) and four (K = 4)  were polymorphic. The neighbor-net diagram showed a genetic split between three groups with a high degree of support (Bootstrap support = 87–100). The three groups were identical to groups recognized by the haplotype network and the phylogenetic analyses  . BayesiaM. argentina where it has never been recorded  Ma from the Late Miocene to the Late Pliocene when climate conditions were suitable, which agrees with our previous results [M. argentina. The split among these phylogroups occurred in the Late Pliocene (4.2–1.2 Ma) and mainly in the Pleistocene. These results suggest that although the early stages of the evolutionary history of M. argentina (i.e. its divergence) are linked to the (semi)aridification processes that gave rise to the SATZ, and to the final uplift of the Southern Andes (5 Ma), the following stages and conformation of its range are rather linked to Quaternary glaciations (1 Ma– 15 Ka) [M. argentina, through the fragmentation of an ancient, more widespread distribution. The three phylogroups  do not share any haplotypes; moreover, the phylogroups identified by cpDNA and AFLP data were congruent. These results are consistent with a hypothesis of ancient habitat fragmentation, as previously posited by Templeton [Geological, climate, and genetic studies suggest that the SATZ arose from the Middle Miocene to the Upper Pliocene ,20,29,33 results . Analyse– 15 Ka) ,89,90. Cempleton  and repoempleton ,44.M. argentina, although the highest within-population genetic variation was found in the southern area of the Prepuna, genetic variation was also high in Prepuna and Monte. Prepuna is the southern limit of this species’ distribution, suggesting that it was probably the habitat occupied by the ancestors of M. argentina. Demographic parameters, the negative value of Fu’s Fs and Tajima’s D, and the results of the mismatch distribution analysis all suggest a growing population size in M. argentina. Based on this evidence, we suggest that populations extended their range northward and westward into the Puna and Monte provinces, respectively. From there, the once continuous distribution underwent fragmentation due to the climatic oscillations in the Pliocene-Pleistocene.It is commonly inferred that areas exhibiting high levels of genetic variation were glacial refugia for relatively large populations , while aM. argentina appears to be continuous, its populations are actually isolated. From our field observations, we noticed that while the populations are often found within short distances of each other, they inhabit areas with different ecological conditions and their flowering periods differ considerably , given that large suitable areas are necessary for population subdivisions to occur without any marked reduction in genetic diversity; similar results were found for Hordeum [The predicted suitable range of  Hordeum .M. argentina within its extant distribution for at least the last ice age cycle. The ecological niche modeling, revealed that the climate conditions were suitable for M. argentina in the SATZ, and that the niche for this species has not changed since at least the LGM.Our study utilized two independent approaches to reconstruct the present and past population distribution. The phylogeographic approach detected survival of Recent studies are finding complex phylogeographic patterns in the biota of South America –42,95–99Munroa argentina has been able to persist in the SATZ despite orogenic changes, climate changes and ice age cycles, and that its apparently once continuous range has undergone fragmentation. Our analyses detected a deep intraspecific phylogeographic structure with multiple lineages for this species. We identified three phylogroups  with low historical gene flow among them and a strong genetic structure that matches geographic distributions in known biogeographic provinces with distinct histories.Our study has revealed that, since approximately 4 Ma, the grass species S1 FigA-D, Mismatch distributions of pairwise nucleotide differences for population clusters of M. argentina. Dashed lines show the observed frequency distributions and solid lines show the distribution expected under the sudden-expansion model. E-I, Bayesian skyline semilog plots showing medians for the historical demographic trends for total populations of M. argentina.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


Mounting evidence of climatic effects on riverine environments and adaptive responses of fishes have elicited growing conservation concerns. Measures to rectify population declines include assessment of local extinction risk, population ecology, viability, and genetic differentiation. While conservation planning has been largely informed by neutral genetic structure, there has been a dearth of critical information regarding the role of non-neutral or functional genetic variation. We evaluated genetic variation among steelhead trout of the Columbia River Basin, which supports diverse populations distributed among dynamic landscapes. We categorized 188 SNP loci as either putatively neutral or candidates for divergent selection  using a multitest association approach. Neutral variation distinguished lineages and defined broad-scale population structure consistent with previous studies, but fine-scale resolution was also detected at levels not previously observed. Within distinct coastal and inland lineages, we identified nine and 22 candidate loci commonly associated with precipitation or temperature variables and putatively under divergent selection. Observed patterns of non-neutral variation suggest overall climate is likely to shape local adaptation  of steelhead trout in the Columbia River region. Broad geographic patterns of neutral and non-neutral variation demonstrated here can be used to accommodate priorities for regional management and inform long-term conservation of this species. Cathaya argyrophylla pineceae, Ge et al. Naja atra, Lin et al. Gopherus polyphemus, Clostio et al. Management strategies implemented for species conservation are highly contingent on a host of correlated life history and demographic information. In concert with these data, genetic structure is vital for characterizing population distinctions and limitations on productivity related to the decline of many species , are established based on a core set of criteria including population ecology and viability, ancestry and descent, reproductive isolation, and local adaptation  loci  occur as two evolutionarily divergent lineages, delineated east (inland) and west  of the Cascade Mountain Crest. The inland redband trout (O. m. gairdneri) are typically a stream maturing, summer-run type, while coastal rainbow trout (O. m. irideus) are dominated by an ocean maturing, winter-run type , steelhead trout , but some hatchery exceptions are included (n = 12). Both the coastal and inland lineages were represented but in skewed numbers of populations: coastal (n = 24), and inland , followed by sequencing via primers developed for EST sequence flanking regions. Generally, the coding or noncoding nature of specific ESTs, and associated functions were unknown  were used to screen for species ID and hybridization between O. mykiss and O. clarkii subspecies  were subsequently removed from the data set prior to analyses. Following this screening exercise, the three species-diagnostic loci were removed from the data set.A total of 191 SNP loci were genotyped with Taqman assays . Our locus panels were comprised of SNPs developed by multiple sources . All loca priori candidate loci for selection related to environmental variables. Specifically, two loci are putatively associated with thermal stress-induced mortality  have been previously identified as associated with temperature variation in desert versus montane environments, and one locus (Omy_hsf2-146) was putatively associated with precipitation in the same study  and FST were generated with the program genalex version 6.2 , available at: http://www.critfc.org/fish-and-watersheds/fishery-science/data-resources-for-scientists/critfc-data-download/. Mantel tests of isolation by distance (IBD) were evaluated in genalex v.6.2 using matrices of pairwise FST and pairwise stream distance. The Markov Chain Monte Carlo (MCMC) approximation of Fisher's exact test implemented in genepop v. 3.3 (1000 batches with 1000 iterations) was used to test for deviations from Hardy–Weinberg equilibrium (HWE) expectations, evaluated across 188 SNP loci and 145 populations. Linkage disequilibrium was tested for all pairs of loci among populations using a simulated exact test in genepop. For all pairs of loci with significant nonrandom association (linkage), the locus with the lower MAF was excluded from further analyses. Statistical significance (α) was adjusted for the number of simultaneous tests  for both HWE and linkage tests via the B-Y FDR method , observed heterozygosity , or fractional group fidelity among individuals in each of 145 populations, setting k = 2 . Default settings were used for the ancestry model  and frequency model , with a burn-in of 50 000 and 250 000 MCMC repetitions. Locus-specific and global pairwise FST  was performed in genalex version 6.2 based on matrices of pairwise FST to quantify amounts of variation.Throughout our methods and analytical approach, the coastal and inland lineages were evaluated separately. First, we verified the resolving power of our SNP markers to discretely differentiate the two steelhead lineages that have been characterized in previous studies based on other genetic markers . Due to the potential high error rate of the hierarchical island model  – unaffected by selection , (ii) – directly under selection (affecting one or more traits under selection), or (iii) – indirectly affected by the process of selection (selection at a linked locus). In all subsequent discussion, the term ‘non-neutral’ variation will be used to represent the latter two distinctions. We used an outlier approach based on simulation methods initially proposed by Beaumont and Nichols  to identFST  and were used to gather all physical variable measurements  were generated using parameter-elevation regressions on independent slopes model (PRISM) of the Oregon Climate Service; http://www.prism.oregonstate.edu/. Monthly average maximum and minimum air temperatures were simulated at 800-m cell resolution from a model based on climate normals from a 30-year period (1971–2000) in PRISM. Water temperature readings were unavailable, and therefore, we used air temperature readings as a proxy for in-stream temperature. In the Pacific Northwest region, stream temperature trends closely with air temperature, particularly on temporal scales  was selected based on five iterations for each potential k value. We used program default settings for the Monte Carlo Markov Chain procedure, with 30 000 burn-in followed by 150 000 MCMC repetitions. The (Δk) statistic derived from the second order rate of change of the likelihood function , and the mean Q values for each population were established using the full search algorithm in CLUMPP  inferred admixture proportions from structure and the first three Eigen vectors (EV) from PCoA analysis were used to control for underlying neutral population differentiation in subsequent association tests.The highly dependent nature of Beaumont . When isBeaumont , it can P-values for the correlation coefficient were calculated in Microsoft Excel. Statistical significance (α = 0.05) was adjusted for the number of simultaneous tests as a conservative measure using a Bonferroni correction to exclude false-positive results  is established, the remaining variables are fit to the model, while those selected in previous steps are held as constants. Specific associations were determined on the basis of highest rank (P < 5%) variable for a given locus, and the method accounts for correlations that are likely present between the variables used to characterize climates (Q and EV) variables were evaluated in respective ‘sets’ as demonstrated by Anderson et al.  loci with precedence of association , which occurred at k = 6 (Q1–Q6) within each lineage independently , two groups in the upper Willamette DPS (Q2 = 58% and Q4 = 61%), the Washington coast population from the Quinault River (Q1 = 89%), and the Big White Salmon population on the Cascade Crest (Q6 = 81%). Most of the remaining lower Columbia populations had greatest proportions in Q3, ranging from 34% to 77%. For the inland lineage, the six inferred populations partially distinguished major subbasins, where the middle and south forks of the Salmon River were both dominant in Q1 (74%), the south and middle forks of the Clearwater River in Q4 (53%) and Q3 (76%) respectively, the Klickitat River in Q2 (68%), and the upper Salmon River in Q5 (56%). Regions with highest mean admixture proportion in Q6 included the majority from the upper and middle Columbia, and the Grande Ronde and Imnaha rivers in the Snake River Basin. Most individual populations within these regions did not exhibit definitive or strong fidelity in Q6 , and the highest admixture proportion by watershed occurred in the Yakima and John Day Rivers, with mean Q6 of 59% and 54%, respectively  compared to values within the coastal lineage . Results of PCoA based on pairwise FST corroborate primary distinctions revealed from structure analyses. Among the inland lineage, observed distinctions coincide well with several of the MPGs within the Snake River DPS.For the coastal lineage, mean ectively . DiffereP < 0.00029. On the basis of linear regression alone, we identified 12 loci in the coastal lineage that were significantly correlated with one or more environmental variables  were candidates for climate association in both lineages. Additional novel candidate loci were detected that have not previously been identified as loci putatively under selection.Of the eight SNP loci showing precedence as candidates for local climate from previous studies, seven exhibited significant association with climate variables in at least one lineage in our broad-scale evaluation across many populations. Two loci previously identified as associated with survival under thermal stress  with precedence of putative association for climate or life history had no confirmed associations for any habitat variable in our analyses and was deemed ambiguous as it may be a candidate at smaller, more local scales.The two loci that were previously determined to be associated with differentiating resident from anadromous life-history forms  may have ranked highest for a given locus, the corresponding variable sets  may have ranked low for that same locus  were equally the most commonly correlated environmental factors. To clarify, the common point of origin for all measured migration distances was the Columbia River estuary, and therefore, distance associations were not necessarily an example of IBD gene flow which relates to direct distance between populations. In the inland lineage, environmental correlations were dominated by precipitation, then temperature. Specifically, spring, summer and total annual precipitation were the physical variables most often associated with genetic variation, and several significantly correlated loci spanned both lineages Fig. . In someme locus . An explThe NJ tree topologies based on neutral SNP panels generally showed genetic distance relationships among populations that accurately aligned with the five DPS delineated under the ESA Figs A and 5A.n = 9 loci) and inland (n = 22 loci) lineages. Trees presented population clustering patterns that often did not correspond with delineations of DPS or MPG , mean spring precipitation , and mean summer precipitation . The panel of 158 neutral loci for the coastal lineage was also tested to account for potentially confounding neutral patterns of IBP, but none were observed. Mantel tests of IBP based on 15 precipitation-associated loci in the inland lineage (pairwise comparisons between 121 populations) indicated significant IBP for mean spring precipitation , and for mean summer precipitation ; no correlations were observed between seasonal precipitation and the panel of inland lineage neutral SNPs (n = 146).Allele frequencies were most commonly correlated with the precipitation variables in association tests. This was further verified with Mantel tests of isolation by precipitation (IBP). Tests were based on a five-locus subset of nine candidate loci in the coastal lineage and a 15-locus subset of 22 candidate loci in the inland lineage; subsets were chosen based on significant associated with precipitation specifically Table . For theO. mykiss at a broad geographic scale that will be a valuable contribution to improved conservation management of this species in the CRB. Neutral structure was complementary to preceding studies .Additionally, we show clear evidence for non-neutral (putatively adaptive) variation that is significantly associated with climate in the region. Specifically, several candidate markers were primarily associated with precipitation and temperature. This study demonstrates that candidate markers can be applied at broad geographic scales to describe the extent of potential local adaptations across highly variable climates. To evaluate non-neutral differentiation, our designation of candidate SNP loci was applied conservatively to reduce false-positive results. Conclusions of climate association were based on a large and diverse number of populations and supported by a framework of multiple test criteria and strict likelihood thresholds , it is challenging to decipher the biological relevance of those correlations. Climate variables such as temperature may affect behaviors and phenotypes alike . In contrast, population-clustering patterns identified using candidate loci  were presumably based upon environmental variation and frequently did not align with current steelhead trout DPS delineations. Moreover, clustering similarities based on non-neutral variation did not necessarily coincide with geographic proximity, nor were patterns among populations always transparent in regard to biology . Thus, the juxtaposition of genetic signals show that neutrally dissimilar populations may exhibit non-neutral similarity (and vice versa) related to environment, and irrespective of geographic distance. Compared with neutral variation, candidate loci revealed some novel distinctions between populations, presumably reflective of environmental differences between regions and locales, particularly for the inland lineage. For the coastal lineage, less variable environments may produce moderate selective pressure for local adaptation among surveyed portions of the species range. More extreme environments in the southern portion of the coastal lineage range  might be expected to provide stronger selective pressure.The relative influences of climate or environment on genetic variation  may occur during particular life-history stages of an organism, which can be difficult to discern. Temperature for example has been shown to have variable effect on different life stages of fish (Fowler et al. Omy_stat3-273 was previously identified among desert and montane resident O. mykiss populations as being associated with temperature (Narum et al. If correlations between loci and physical variables are indicative of an adaptive influence (Bonin et al. Global climate change and effects of climate on ecosystems has earned the attention of the scientific community and freshwater fish are expected to be negatively impacted (McCullough et al. Therefore, conservation strategies based heavily on neutral genetic variation, with an under-emphasis on the distribution of non-neutral variation, risk detrimental impacts to locally adapted population segments and should be scrutinized Pearman . We concThere is a palpable consensus among researchers and managers cautioning against broad scale, rigid approaches to conservation, and acknowledging the need to address productivity limitations of myriad organisms in need of protection (Clostio et al.