ST = 0.048, ΦST = 0.109, p < 0.01) and microsatellite allele frequencies , consistent with long-term fidelity to calving areas. However, most genetic comparisons of calving grounds and migratory corridors were not significant, supporting the idea that whales from different calving grounds mix in migratory corridors. Furthermore, we find a significant relationship between δ13C stable isotope profiles of 66 Australian southern right whales, a proxy for feeding ground location, and both mtDNA haplotypes and kinship inferred from microsatellite-based estimators of relatedness. This indicates migratory culture may influence genetic structure on feeding grounds. This fidelity to migratory destinations is likely to influence population recovery, as long-term estimates of historical abundance derived from estimates of genetic diversity indicate the South Pacific calving grounds remain at <10% of pre-whaling abundance.Fidelity to migratory destinations is an important driver of connectivity in marine and avian species. Here we assess the role of maternally directed learning of migratory habitats, or migratory culture, on the population structure of the endangered Australian and New Zealand southern right whale. Using DNA profiles, comprising mitochondrial DNA (mtDNA) haplotypes (500 bp), microsatellite genotypes (17 loci) and sex from 128 individually-identified whales, we find significant differentiation among winter calving grounds based on both mtDNA haplotype (F Dermochelys coriaceaCarcharhinus leucasMegaptera novaeangliaeFidelity to migratory routes or destinations is a common trait across many taxa, including fishMigratory traditions between two sets of habitat patches, breeding and non-breeding , are often a driver of population structure across an entire migratory networkEubalaena australis) have migratory networks spanning thousands of kilometres, from sheltered coastal wintering grounds to offshore summer feeding grounds. The species was the target of a prolonged hunt across the southern hemisphere, with up to 150,000 southern right whales killed between 1790 and 198016Southern right whales , including key calving grounds, show a high degree of interchange based on the movement of photo-identified individualsThe delineation of southeast and southwest Australia into distinct management units was proposed based on the difference in recovery rate and photo-identification data suggesting movement within each region is greater than between the two regions20n = 13), is consistent with the hypothesis of maternally directed fidelity to wintering grounds. Female, and to a lesser extent male, southern right whales show long-term fidelity to their natal wintering ground, based on long-term photo-identification and paternity studies20242523This finding, although preliminary as it was based on a small sample size from southeast Australia  New Zealand and Australian calving grounds, (2) previously defined southwest and southeast Australian management units , with New Zealand as a comparator, and (3) between migratory corridors and calving ground samples. In addition, we use a combination of genetic and stable isotope data to investigate the influence of migratory fidelity to feeding grounds on population structure by testing the hypotheses (1) there is maternally directed fidelity to feeding grounds in Australian southern right whales and (2) that there is a correlation between genetic estimates of relatedness and feeding ground preferences. Finally, we use mtDNA data to provide long-term estimates of genetic diversity and historical population size of the right whale populations in the South Pacific. In doing so, we provide an understanding of the role of migratory culture as a driver of genetic structure in the contemporary population and provide targets to inform our understanding of the recovery of the species in the New Zealand and Australia.n = 51) were used here as a comparison. Sampling was carried out following protocols that were approved by the Macquarie University Animal Ethics Committee, Department of Primary Industries Parks Water and Environment Animal Ethics Committee and the University of Auckland Animal Ethics Committee. Sampling was conducted in accordance with and under permits from: Department of Environment, ; Department of Environment and Heritage ; Department of Primary Industries, Parks, Water and Environment (Tasmania); Department of Natural Resources and Environment (Victoria); Office of Environment and Heritage ; Department of Conservation and Land Management  and the New Zealand Department of Conservation .Small samples of skin were collected from southern right whales across New Zealand and Australia using a stainless-steel biopsy dart fired from a modified veterinary capture rifleWe broadly categorise sampling areas into calving/nursery areas  and migratory corridors . Calving grounds are areas used regularly by females with calves who show long-term fidelity to such sites6It should be noted that the majority of New Zealand samples come from the sub-Antarctic Auckland Islands, which is a key wintering ground for all demographic classes and so represents both a key calving area and management unit. Samples comprise those used in previous analyses13C and δ15N) for southern right whales skin biopsy samples collected in Australia  and microsatellite genotype (up to 17 loci), for southern right whale samples collected around New Zealand and Australia. This was a mixture of previously published data and newly generated data  and 2. Iustralia .h) and nucleotide (π) diversity for each sampling location and putative management unit. Differentiation between (1) Western Australian, Victorian and New Zealand calving grounds and (2) the southwest Australia, southeast Australia and NZ management units, was estimated using pairwise F-statistics (FST), ФST and a hierarchical analysis of molecular variance  calving grounds and (2) management units. We also estimated Jost’s D statisticFor the microsatellite data, we estimated the mean number of alleles per locus and observed and expected heterozygosity for each sampling location and management unit using CERVUS v3.0ST and ФST and microsatellite-based FST and Jost’s D, between the calving grounds and migratory corridors, as described above . We used GenetixWe also investigated the hypothesis that individuals from different calving grounds were mixing on the same migratory corridor. This was done by calculating pairwise mtDNA-based F13C and δ15N by mtDNA haplotype to visually identify any patterns. We then tested for significant differences in δ13C and δ15N values between individuals with different mtDNA haplotypes using the Kruskal-Wallis test.The hypothesis of maternally directed fidelity to feeding grounds leads to the expectation that whales that share mtDNA haplotypes are more likely to have similar stable isotope profiles than those with different mtDNA haplotypes. We conducted several analyses to investigate the relationship of stable isotopes with both the mtDNA and microsatellite data, using the Australian samples only. First we tested the data for normality and plotted δ13C or δ15N values. Model fit was assessed using AIC and estimated the size of the effect of mtDNA on stable isotope values by assessing the weight of models that included mtDNA as a explanatory factoret al.et al.We used generalised linear modelling in R13C and δ15N values. Pairwise genetic similarity between individuals was calculated in the program COANCESTRY13C and δ15N values and the relatedness estimators using the R package apeTo further investigate the relationship of the genetic and stable isotope data, we tested the hypothesis that individuals that show a higher level of relatedness, based on their microsatellite genotypes, have more similar isotopic profiles. To do this, we estimated the Euclidean distance between individuals based on their δth genealogy, discarding the first 20,000 of each search.We used program LAMARCNC) following Roman and Palumbi’se(f)μ, where μ is the substitution rate per generation and Ne(f) is effective female population size. We took 2,000 samples of θ from the LAMARC output  and for each we estimated Ne(f) using: (1) the mutation rate of 2.0 ×10−8 bp−1 year−152, estimated from the closely-related bowhead whale Balaena mysticetus and previously used in similar analyses for right whalesNe(f) to NC using two correction factors. Firstly, we adjust Ne(f) to number of mature females, NT(f), by multiplying by twoNT(f)to NC by multiplying NT(f)by a value sampled from a uniform distribution between 2.5 and 4.71. This distribution represents the ratio of the proportion of reproductively mature females in a population to the total population size, in pre-disturbance, equilibrium conditions  and NC. By accounting for uncertainty in generation span and proportion of mature females in the population, we attempt to account for fluctuations in the population size over time, due to whaling or other historical bottlenecks.We converted θ into census population size : see  × 10−14 . Only onThere were between 5 and 6 mtDNA haplotypes per region, and each had reasonably high levels of haplotype diversity, with the exception of New Zealand as previously characterised . BakHapAWe tested for population structure on wintering grounds in two ways: testing first between calving grounds and then between management units, the latter comprise different habitat types including calving grounds and migratory corridors.ST = 0.0086, p < 0.01) and mtDNA data, based on both the haplotype frequencies  and haplotype distances . There was a significant difference between New Zealand and Western Australia based on both mtDNA and microsatellite data , but not microsatellite data . As found previously, there was significant differentiation between southwest Australia and New Zealand based on both microsatellite allele and mtDNA haplotype frequencies. There was some differentiation between southeast Australia and New Zealand based on mtDNA data but not microsatellite allele frequencies  and the Western Australian calving ground . There was a significant difference between the South Australia migratory corridor and the New Zealand calving ground . However, New Zealand was not significantly different to either the Tasmanian or the New South Wales migratory corridors (at p < 0.05).There was no significant differentiation between the Australian calving grounds and migratory corridors, based on either mtDNA haplotype or microsatellite allele frequencies . The excInspection of the FCA results using the calving ground samples only show individuals from each calving ground group together, but with some overlap between calving grounds . Further13C and δ15N for individual whales were normally distributed based on the Shapiro-Wilk normality test. We found a significant difference in δ13C values , but not δ15N values (p = 0.771 p = 0.760) associated with mtDNA haplotypes. Given the latter, we only report the comparison of genetic data with δ13C values (see 15N data).The measurements of δ13C against two-way combinations of factors revealed interactions, so we considered general linear models with interaction terms. For the δ13C data, the best-supported model according to AIC indicated that mtDNA, sex and state were all influential in determining δ13C  between two whales from the same matrilines was on average smaller than the distance between two whales from different matrilines. There was also a significant relationship between the microsatellite-based relatedness estimates of Ritland13C, based on the results of the Mantel and permutation tests.The randomisation test of Valenzuela Ne(f)and NC in NCwere similar between New Zealand and Western Australia, with historical abundances of 30–35,000 whales. In contrast, θ and NC for Victoria was lower with historical abundance estimated at 22,000 whales.We used LAMARC to estimate θ for each calving ground while accounting for the effect of migration and changes in population sizes. From these estimates we derive estimates of historical population size. Inspection of the LAMARC output in the program TRACERHere we demonstrate genetic structure at both ends of a migratory network, winter calving grounds and summer feeding grounds, in the Australian southern right whale. This population structure appears to be driven by migratory culture, which is consistent with long-term behavioural studies that show migratory fidelity appears in general to persist across a lifetime in females . The inclusion of whales ‘from’ southwest Australian calving grounds in the southeast Australian management unit via migratory corridor samples could contribute to the increase in differentiation seen between the southeast Australian and New Zealand management units, compared with the calving ground samples.If present, such migration patterns could lead to a decrease in the signature of genetic differentiation between the two Australian management units when migratory and calving area samples are pooled. This may have contributed to the decrease in the estimate of differentiation between the two putative Australian management units in this study compared with previous research , derived from a logistic growth model incorporating a regional catch series and constrained by genetic dataGlobal estimates of pre-whaling abundance, estimated using logistic growth modelling and a global catch series, had previously suggested that the historical circumpolar population size was substantially smaller, at 55,000–100,000 whalesn = 39) took 20 years to collect. The dataset therefore contains samples from whales of different ages, sex, reproductive state and migratory status, all of which may also influence the stable isotope profile13C than when it is not relying on fat stores13C is moderately strong, suggesting that maternally directed fidelity to feeding grounds is very likely and overrides other factors. We suggest that the implications of this finding may be important in other stable isotope analysis studies. Stable isotope analyses are often conducted in the absence of concurrent genetic analyses, and for rare and uncommon animals frequently opportunistic rather than truly random or systematic. These findings suggest caution in both sampling design and in interpretation when underlying drivers such as those found here cannot be attributed.The low abundance and wide range of the previously heavily exploited southern right whale off southeast Australia meant that the sample size used here .


Cytokine or growth factor activated STAT3 undergoes multiple post-translational modifications, dimerization and translocation into nuclei, where it binds to serum-inducible element GAA’)-bearing promoters to activate transcription. The STAT3 DNA binding domain  mutation in hyper immunoglobulin E syndrome (HIES), called the HIES mutation , which elevates IgE synthesis, inhibits SIE binding activity and sensitizes genes such as TNF-α for expression. However, the mechanism by which the HIES mutation sensitizes STAT3 in gene induction remains elusive. Here, we report that STAT3 binds directly to the AGG-element with the consensus sequence ‘AGG(N3)AGG’. Surprisingly, the helical N-terminal region (1–355), rather than the canonical STAT3 DBD, is responsible for AGG-element binding. The HIES mutation markedly enhances STAT3 AGG-element binding and AGG-promoter activation activity. Thus, STAT3 is a dual specificity transcription factor that promotes gene expression not only via SIE- but also AGG-promoter activity. STAT3, known as the acute-phase response element, was first described in hepatocytes ,2 and caSTAT3 C-terminal K685 acetylation and Y705/S727 phosphorylation are involved in C-terminal dimerization and enhance some formation. The repeating β-sheets of the DBD (320–494) recognize and bind the serum-induced element (SIE) with the consensus sequence of ‘TTC(N3)GAA’  are a major cause of hyperimmunoglobulin E syndrome (HIES) and unexpected hyper-TNF-α promoter activity . Mice wiIn this study, we applied ChIP-cloning and ChIP-on-ChIP approaches to identify other STAT3 binding elements. While ChIP-on-ChIP analysis is based on hybridization to identify the peaks of tagged DNA sequences, ChIP-cloning approach is based upon transcription factor DNA binding sites of variable affinities provides information regarding the genome-wide distribution ,22. We n2 atmosphere.HepG2, 293T and PC3 cells were obtained from the Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences . Cells were cultured in high glucose DMEM  containing 10% fetal bovine serum (FBS)  with 100 units/ml penicillin and 10 μg/ml streptomycin  at 37°C in a 5% CO® 680RD (926–68071) and goat anti-mouse RDye® 800CW (926–32210), were from LICOR. Anti-pY45-STAT3 and anti-acetyl-K78-STAT3 polyclonal antibodies were prepared by AB-land, Inc. .The polyclonal antibody against STAT3 C-20 (sc-482) and monoclonal antibodies for Myc (sc-40) and pY20 (sc-508) were from Santa Cruz Biotechnology, Inc. The GFP monoclonal antibody (11814460001) was from Roche and the polyclonal antibody for acetyl-lysine (#9441s) was from Cell Signaling Technology. The secondary antibodies, including goat anti-rabbit RDyeRecombinant human IL-6 was from Life Technologies, LPS was from Sigma and recombinant human LIF was from Millipore. The dual-luciferase reporter assay system kit (E1910) was obtained from Promega.7 HepG2 cells in a 6-well plate were treated with IL-6 for 30 min and fixed in 1% formaldehyde. Chromatin was sheared into 600-bp fragments with sonication. Nuclear lysates prepared from these cells were incubated with anti-STAT3 (5 μg) for immune-precipitation and ChIP analysis.ChIP experiment: chromatin preparation and immunoprecipitation experiments were carried out as described previously . In brie®18-T Vector  and transformed into competent bacteria. For sequencing, 200 colonies were selected. ChIP-on-ChIP experiments were performed following the KangChen Biotech protocol (Shanghai).ChIP-cloning: The ChIP precipitates were amplified by polymerase chain reaction (PCR) using six base random primers according to the Whole Genome Amplification Kit . The PCR-amplified gene products were then inserted into a pMDChIP-PCR analysis: IL-6-treated HepG2 cells were used for the ChIP experiment. ChIP precipitates were used as PCR templates with nuclear lysates as input. PTK6, IL-6R, NTKR2 and EP300 were selected for ChIP-PCR. The nucleic acid sequences represented by the red boxes in Figure PTK6-F,5-CCAGCACTTTGGGAAGC-3;PTK6-R, 5-GGAGTCTCGCTCTGTCG-3;NTRK-F,5-TGCTTTCTGCTGGAAT-3;NTRK-R, 5-TACAGGTGCGTGCTAC-3;EP300-F,5-CTCACGCCTGTCATCCC-3;EP300-R, 5-GCAACCTCTGCCTCCTG-3;IL-6R-F,5-TGGCTGACACGGTGAAAC-3;IL-6R-R, 5-AGGCTGGAGTGCAGTGGT-35 cells per well) were cotransfected with 0.5 μg of firefly luciferase reporter, 0.02 μg of pRL-SV40 renilla luciferase reporter (Promega) and wild-type or mutant cMyc-STAT3 using Lipofectamine 2000 according to the manufacturer's instruction (Life Technologies) for 24 h followed by treatment with IL-6 (20 ng/ml), LPS (10 μg/ml), LIF (20 ng/ml) or no treatment. Twenty-four hours later, whole cell lysates were prepared for the luciferase reporter activity analysis using a Dual-Luciferase® Reporter Assay System (Promega). The pRL-SV40 plasmid was used to normalize transfection efficiency. Data are presented as the mean ± SD of three experiments.293T cells in 24-well plates (1 × 10™ II DNA Transfect Reagent (PuFei 2012–100) for 36 h followed by IL-6 treatment for another 30 min. Whole cell lysates were prepared and Myc-STAT3 proteins were purified with extensive washes by using Pierce Mammalian C-Myc Tag IP/Co-IP Kit . Such Myc-affinity purified STAT3 proteins were applied for electrophoretic mobility shift assay (EMSA) and DNA affinity binding assay.293T cells were transiently transfected with Myc-STAT3 or Myc-STAT3 mutants using SuperFectinNuclear lysates were prepared from HepG2 cells treated with or without IL-6 according to the instructions of a Nuclear and Cytoplasmic Protein Extraction Kit . The 5′-biotin-labeled oligonucleotides (AGG-element) synthesized by Sangon Biotech Company were incubated with HepG2 cell nuclear protein or purified protein and DNA binding proteins were separated in the gel according to a Light Shift Chemiluminescent EMSA Kit .® M-280 Streptavidin (#11205D Life Technologies) at room temperature for 30 min, washed three times with 1× B&W buffer  and resuspended in DNA binding buffer . An equal volume of cell lysate was then added and the sample was incubated for 60 min at room temperature, washed three times with DNA binding buffer and boiled for 10 min in sodium dodecyl sulphate (SDS) sample loading buffer . The elution was then separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotted with anti-STAT3 or anti-Myc.The DNA affinity binding assay was performed according to our previous publication . Briefly293T transfectants were harvested in ice-cold RIPA lysis buffer containing protease inhibitors and incubated for 60 min at 4°C with spinning. Cell lysates were cleared by centrifugation at 14 000 rpm for 5 min at 4°C. The supernatants were incubated with 1 μg of primary antibody overnight at 4°C followed by the addition of protein A/G beads  and rotation for 3 h. The beads were collected and washed three times with 1 ml of RIPA lysis buffer and then denatured by boiling in SDS sample loading buffer. The supernatants were analyzed by western blot assay with different primary antibodies  or anti-acetyl-Lys). Proteins were visualized using the Odyssey Infrared Imaging System (LI-COR Biosciences) with the appropriate secondary antibodies as per the manufacturer's instructions.Nucleic acid sequences containing the 5′ ends of the nucleic acid fragment plus a HindIII recognition site and the 3′ end of sequences plus a SalI recognition site were generated, and three protective bases were added to both ends. The nucleic acid sequence and its complementary sequence were diluted with STE buffer  and annealed. Double-stranded nucleic acid fragments were then digested with HindIII/SalI and subcloned into a luciferase reporter vector. The sequences used to generate luciferase vectors are shown in Supplementary Figure S1. Two deletion mutants based on the AGG-Luc vector (AGGΔ1-Luc and AGGΔ2-Luc) were constructed. Sequences used to generate the deletion mutation are as follows:AGGΔ1-Luc F: TGAAAGGCTGTGCGCGCGAGGGAGGG;AGGΔ1-Luc R: CCCTCCCTCGCGCGCACAGCCTTTCA;AGGΔ2-Luc F: TGCGCGGAGGGCGGGGGCCGTCGACC;AGGΔ2-Luc R: GGTCGACGGCCCCCGCCCTCCGCGCA® XL site-directed mutagenesis kit . The following primers were employed:GFP-tagged STAT3 and truncated STAT3 were constructed using the corresponding Myc-STAT3 expression vector. Myc-STAT3 and truncated STAT3 expression vectors were digested with EcoRI/ApaI, and the target fragments were subcloned into the pEGFP-C1 vector. The deletion mutation (170–180Δ) and other mutants  were constructed usinga QuikChange170–180ΔF, GTGGAGAACCTCCAGGACAGCCAAGGAGACATGCAG170–180ΔR, CTGCATGTCTCCTTGGCTGTCCTGGAGGTTCTCCACE434/435A-F, TCCTTGATCGTGACTGCCGCCCTGCACCTGE434/435A-R, CAGGTGCAGGGCGGCAGTCACGATCAAGGAV461/462/463A-F, TCCTTGCCAGCCGCCGCCATCTCCAACATCV461/462/463A-R, GATGTTGGAGATGGCGGCGGCTGGCAAGGAR382Q-F, CAGAGGGTCTCAGAAATTTAACATR382Q-R, ATGTTAAATTTCTGAGACCCTCTGR382W-F, CAGAGGGTCTTGGAAATTTAACATR382W-R, ATGTTAAATTTCCAAGACCCTCTGR423Q-F, GGGAATGGAGGCCAGGCCAATTGTR423Q-R, ACAATTGGCCTGGCCTCCATTCCCV463Δ-F, TTGCCAGTTGTGATCTCCAACATCTV463Δ-R, AGATGTTGGAGATCACAACTGGCAAS465A-F, TTGTGGTGATCGCCAACATCTGTCAS465A-R, TGACAGATGTTGGCGATCACCACAAGenomic DNA was extracted, and the corresponding primers were synthesized. We then amplified the TNF-α promoter region. The nucleic acid amplification sequences are shown in Supplementary Figure S2A. The PCR products were digested with HindIII/SalI and inserted into the luciferase plasmid as TNF-Luc. The following primers were utilized:TNFα-promoter F, CCCAAGCTTCCAGGGCTATGGAAGTCGTNFα-promoter R, ACGCGTCGACGAGAAGAGGCTGAGGAACAAGTwo deletion mutants based on the TNF-Luc vector were constructed(TNFΔ1-Luc and TNFΔ2-Luc). The following primers were employed:TNFΔ1-Luc -F: GGAGGCAATAGGTTTTCAGTTGTTGGCACACCTNFΔ1-Luc -R: GGTGTGCCAACAACTGAAAACCTATTGCCTCCTNFΔ2-Luc -F: AACTTTCCAAATCCCCCTCATGGGTTTCTCCATNFΔ2-Luc -R: TGGAGAAACCCATGAGGGGGATTTGGAAAGTT® (DRR002A), Prime STAR® Max DNA Polymerase (DR045S), Prime Script RT-PCR kit (DRR014S) and DNA mate (D605A) were from TaKaRa Bio.All DNA sequencing was performed by the Sangon Biotech Company (Shanghai). The PCR cleanup kit (AP-PCR-50), plasmid miniprep kit (AP-MN-P-50) and DNA gel extraction kit (AP-GX-50) were from AxyGen Biosciences. A ligation high kit , high pure maxi plasmid kit  and TRIzol  were used. Dpn I (1233A), EcoRI (1040A), ApaI(1005A), HindIII (1060A), SalI (1080A), Takara Taq™ (DR001A), Takara LA TaqHepG2 cells were transiently transfected with Myc-STAT3, wild-type STAT3 or HIES mutant STAT3 as previously described, followed by LPS or IL-6 treatment. Total RNA was extracted with TRIzol (Life Technologies) according to the manufacturer's instructions. Reverse transcription was performed with oligodT primers and reverse transcriptase M-MLV . Real-time PCR was carried out in a Bio-RAD-IQ5 Multicolor-Real-Time PCR system with SYBR Green PCR Master Mix (TaKaRa #DRR041A). Relative levels of gene expression were determined with β-actin as the control. RT-PCR was carried out using a Bio-Rad T100 Thermal Cycler PCR system with β-actin as the control. The following primers were used for gene amplification:TNF-α-F, GTCTGGGCAGGTCTACTTTTNF-α-R, TGGGCTCCGTGTCTCAPTK6-F, CGGGAGACTGACACGAAGCPTK6-R, CCAGCGACAAAGGTGAAGGEP300-F, GCCAGTGTCGGAATGCCAATTEP300-R, GCTGAGCAGTCTGAGGAGGGIL-6R-F, CTGGGTGCTCAGGAATCAGGIL-6R-R, GTTGGCGACGCATAGGGNTRK2-F, ATGGCTTTCTAGTCTTGTGGNTRK2-R, GCATACTCGGCTCTTTGAβ-actin-F, ATGCCATCCTGCGTCTGβ-actin-R, ATGCCATCCTGCGTCTGTo uncover novel STAT3-promoter binding activities, we performed ChIP cloning and ChIP-on-ChIP experiments in HepG2 cells. In addition to non-specific binding sequences, many DNA samples recovered from the STAT3 immuno-precipitates contained the so-called AGG-motif with the consensus sequence ‘AGG(N3)AGG’. From a careful analysis of 194 sequences obtained from STAT3-ChIP cloning results, 20.21% of the DNA sequences contained the AGG motif, whereas only 12.44% DNA sequences contained the SIE motif Figure . SimilarTo investigate STAT3 binding capacity to the AGG-element, we performed DNA oligonucleotide affinity precipitation and mass spectrometry analysis. STAT3 was identified among the proteins pulled down by both the AGG-oligonucleotide beads and the SIE-oligonucleotide beads as the positive control. Mass spectrometric analysis clearly revealed that STAT3 was efficiently pulled down by both the SIE-oligonucleotide beads and the AGG-oligonucleotide beads Figure . To confTo provide additional evidence of the specificity of STAT3-AGG-element binding, we mutated the flanking AGG sequences of the probes. The three mutations, designated M1, M2 and M3, are listed in the right panel of Figure To confirm the direct binding activity of STAT3 for AGG element, we performed EMSA by using purified STAT3 protein. Myc tagged STAT3 proteins were affinity purified from 293T transfectants treated with IL-6. After extensive washes, Myc affinity purified STAT3 protein was fairly pure with no visible contamination of other proteins Figure . We thenThe central region of STAT3 contains the DBD responsible for SIE binding . To confSTAT3 1–355 responded to IL-6 for the AGG-element binding, suggesting that the STAT3 N-terminal region might undergo post-translational modification upon IL-6 treatment. Post-translational modifications within the N-terminal domain of STAT3 have been reported ,10,13. TTo investigate STAT3 N-terminal region transcription activity, we performed a luciferase reporter activity assay by adopting three luciferase reporter systems: SIE-luciferase, STAT6 SIE-luciferase and AGG-luciferase. The AGG-luciferase reporters were constructed using the promoter sequences obtained from the ChIP-on-ChIP results . STAT3 activation was monitored by including c-Src or CBP for cotransfection. As expected, STAT3 transcriptional activity was clearly detected by cotransfecting STAT3 with c-Src or CBP and the SIE-element reporter but not with the STAT6 SIE reporter, which contains one additional base between ‘TTC’ and ‘GAA’ (TTC(N4)GAA) Figure . When thTo identify the motif critical for STAT3 1–355 transcriptional activity, we tested a series of STAT3 variants. Compared to STAT3 full length or 1–355, the deletion of STAT3 170–180 markedly abolished AGG-luciferase reporter activation in response to CBP cotransfection Figure . In contWe analyzed the STAT3 N-terminal region for nuclear translocation. Punctate STAT3 nuclear distribution accompanied by CBP and CBP-mediated histone acetylation was previously reported as an indication of transcriptionally active chromatin. These punctate STAT3 nuclear distributions were defined as the STAT3-specific nuclear body by Herrman . RecentlThe central and C-terminal regions of STAT3 contain the DBD and the SH2 domain, respectively. STAT3 mutations of these two domains, including R382Q, R382W, F384S, R423Q, V463Δ, S465A, T662I, E652K, V637M, were reported to lead to HIES ,17. All Among the HIES mutations, R382 is the most commonly mutated residue. Surprisingly, while STAT3 binding to the SIE-element was disrupted, STAT3 with the R382Q mutation markedly enhanced its AGG-element binding activity in response to IL-6 treatment Figure . PurifieWe then compared STAT3-R382Q mutant SIE- and AGG-luciferase reporter activation. The R382Q mutation dramatically increased AGG-luciferase reporter activation Figure .Double Glu E434, E435) and triple Val V461-V463) were previously reported as the key STAT3 DBD motifs for SIE binding . Two AGG-deletion mutations were introduced into the AGG-luciferase reporter vector . Deletion of the AGG-element disrupted both STAT3 wild-type and STAT3-R382Q mutant activity in response to CBP cotransfection and IL-6 treatment Figure . These rThe TNF-α gene promoter, either human or mouse, does not contain any conserved SIE sequence, even though STAT3 activation of this gene in B cells was reported . An AGG-PTK6, IL6R, EP300 and NTRK promoters also contain AGG-element(s)  NF-κB binding sites ,28. LPS ) Figure . IL-6 ac) Figure . Express) Figure . CollectThe STAT3 HIES mutation is a loss-of-function or dominant negative mutation in terms of SIE DNA binding and gene regulation in cells. However, in cells expressing the STAT3 HIES mutant, the TNF-α level was unexpectedly high ,16, suggThe N-terminal region of STAT3 is sufficient for STAT3 nuclear translocation and punctate distribution, as this region alone is capable of manipulating gene expression. This helical region likely evolved from the ancient coiled-coil domain with some similarity to the helix-turn-helix. While the helix-turn-helix is the major DNA binding structure –32, the STAT3 exists in multiple isoforms in mammalian cells. The three major isoforms are STAT3α, STAT3β and STAT3γ. They have very different DNA binding activities and gene regulation activities –36. STATLPS induces gene expression either directly via NF-κB or IRF3 activation or indirectly via their gene expression products  . AlthougIn summary, STAT3 is a dual specificity transcription factor that can regulate both the canonical SIE- and the non-canonical AGG-containing promoters. The HIES mutation can dramatically increase STAT3 intrinsic activity for AGG-element DNA binding and gene regulation activities.


Canis aureus), we characterised its population genetic structure and attempted to identify the origin of European populations. This provided a unique insight into genetic characteristics of a native carnivore population with rapid large-scale expansion. We analysed 15 microsatellite markers and a 406 base-pair fragment of the mitochondrial control region. Bayesian-based and principal components methods were applied to evaluate whether the geographical grouping of samples corresponded with genetic groups. Our analysis revealed low levels of genetic diversity, reflecting the unique history of the golden jackal among Europe’s native carnivores. The results suggest ongoing gene flow between south-eastern Europe and the Caucasus, with both contributing to the Baltic population, which appeared only recently. The population from the Peloponnese Peninsula in southern Greece forms a common genetic cluster with samples from south-eastern Europe , although the results based on BAPS and the estimated likelihood in STRUCTURE indicate that Peloponnesian jackals may represent a distinct population. Moreover, analyses of population structure also suggest either genetic distinctiveness of the island population from Samos near the coast of Asia Minor , or possibly its connection with the Caucasus population . We speculate from our results that ancient Mediterranean jackal populations have persisted to the present day, and have merged with jackals colonising from Asia. These data also suggest that new populations of the golden jackal may be founded by long-distance dispersal, and thus should not be treated as an invasive alien species, i.e. an organism that is “non-native to an ecosystem, and which may cause economic or environmental harm or adversely affect human health”. These insights into the genetic structure and ancestry of Baltic jackals have important implications for management and conservation of jackals in Europe. The golden jackal is listed as an Annex V species in the EU Habitats Directive and as such, considering also the results presented here, should be legally protected in all EU member states.In the first continent-wide study of the golden jackal ( An implementation of molecular techniques to study population genetics has broadened our knowledge about several aspects of wildlife biology and ecology, including breeding characteristics , 2, popuChanges in the geographical range are recognized as natural processes and have occurred in the history of most species –23. TheoUrsus arctos) population in Finland revealed disappearance of initial structuring and homogenization, as well as gradual increase of genetic diversity )nx lynx) –47 or thnx lynx) , 48–50).nx lynx)  suggestenx lynx) ). Low genx lynx) . A signinx lynx)  also disnx lynx) , 54.Genetic relationships of the European golden jackals with jackals from the Asiatic part of the species’ range, were not yet determined. Moreover, none of the studies so far analysed genetic structure of the population on the larger scale . Consequently, the understanding of historic development of jackal populations in Europe is lacking. One of the hypotheses suggested that the European population goes back to the introduction of jackals from northern Africa in the 15th century . This waThe aim of the present work is to characterise for the first time the population genetic structure of European golden jackals on the continental scale, with the incorporation of samples from hitherto unstudied regions. Therefore, we included samples from the Peloponnesus Peninsula (southern Greece), which could possibly originate from the Neolithic population ; the insn = 5), Croatia , Slovenia , Ukraine , Serbia , Hungary , and northern Greece ; ii) the Caucasus (CAU)—comprising samples from Mountainous Karabakh , Armenia , and Georgia ; iii) the Baltic States (BAL)—comprising samples from Lithuania  and Estonia ; iv) southern Greece (GRE-P) —comprising samples from the Peloponnese ; and v) the island of Samos .Tissue samples used in this study were obtained from 97 individuals originating from five geographical regions , i.e. i)Genomic DNA was extracted using NucleoSpin Tissue kit (MACHEREY-NAGEL) with standard protocol. We amplified 15 microsatellite loci: CPH4, CPH5, CPH8, CPH12, CPH6, CPH9 , CPH22 , FH2004,5’-TCCCTGACACCCCTACATTC-3’) and H576 (5-CGTTGCGGTCATAGGTGAG-3’) . To. ToK = 1K . The number of iterations used to estimate the admixture coefficient for individuals, and the number of reference individuals from each location were 50 and 200, respectively. The number of iterations applied to estimate the admixture for reference individuals was set at 15.The Bayesian-based method implemented in the Bayesian Analysis of Population Structure software  was We also obtained an additional representation of the genetic structure using Principal Components Analysis (PCA). This multivariate descriptive method is not dependent on any model assumption and can thus provide a useful validation of the Bayesian clustering output –84. We uH) in the total sample, as well as in particular geographical regions and genetic groups, haplotype diversity (h), nucleotide diversity (π) and mean number of nucleotide differences among haplotypes (k) were all calculated using DNAsp 5.10 [θST among regions using haplotype frequencies. The test for significance was performed with 1,000 permutations. The overall genetic structure, based on haplotype frequencies, was estimated in DNAsp, using HST  was discovered at locus FH2137, the lowest (A = 3) at CPH5 . In south-eastern Europe (SEE)—the region represented by the highest number of the samples studied—there were three loci manifesting deviation from the HWE on account of heterozygote deficiency and one, FH2096, indicative of heterozygote excess . Allelic richness was similar in SEE, CAU and BAL, though slightly lower in two groups from southern Greece, i.e. from the Peloponnese (GRE-P) and Samos (GRE-S). Observed heterozygosity (HO) was highest in SEE. The lowest HO was found in the insular GRE-S population.When samples were grouped by geographical distribution, a significant overall K = 6), but also of individuals of mixed ancestry. Jackals from CAU and BAL were assigned to two other clusters, with more or less equal probability of ancestry from each of them. The ΔK statistic (K = 2). On the basis of the K = 3 value, BAL and CAU formed the first genetic group, SEE and GRE-P the second, and GRE-S the third (K = 3). In both of these cases, certain individuals from SEE had the highest probability of ancestry from the CAU/BAL group. These were four individuals from Ukraine  and two individuals from Serbia  was assigned to the CAU/BAL cluster, one sample from SEE  was assigned to the GRE-S cluster, and one sample from BAL  was assigned to SEE  suggested the presence of four genetic groups, with a very limited admixture among them . In gened to SEE . A simild to SEE , althougd to SEE . The remFST = 0.199, 95% CI = 0.147–0.258). Pairwise FST ranged from 0.05 to 0.39 (FST > 0.20). Similarly, marked genetic differentiation was found between GRE-P and CAU and GRE-P and BAL, while moderate genetic differentiation characterised the pairwise comparisons of data for SEE, as set against GRE-P, BAL or CAU.Genetic differentiation among the geographical regions was high . Apart from BAL, we identified two haplotypes per region. The highest level of haplotype diversity was found in GRE-P, while the most marked nucleotide diversity and highest average number of pairwise nucleotide differences was found in CAU  control region fragment, we identified four unique haplotypes in 93 samples. Both haplotype diversity and nucleotide diversity were low , as was d in CAU .Haplotype H1 proved to be most frequent, being absent only from GRE-S. In BAL this was the only haplotype found. Haplotype H2 proved to be unique to GRE-S, while H3 was shared between CAU and GRE-S, and H4 between SEE and GRE-P , Fig 5.Comparing haplotypes identified in this study with those deposited in GenBank , we found that H1 corresponds with a haplotype identified previously in Italy, Croatia, Serbia, Bulgaria, Ukraine, NW Poland, and the Caucasus, while differing by just a single mutation from another haplotype from the Caucasus (H5). A haplotype observed previously in Indian jackals (H6) differed from H4 by just four mutations .HST = 0.486 for geographical groups, P < 0.001). Pairwise θST was highest for the comparison of SEE with GRE-S. No genetic differentiation was noted between BAL and CAU or between BAL and SEE , Slavonia  and Serbia (A = 4.0), Dalmatia (A = 2.8), and Italy (A = 3.7). Moreover, the analysis of the mitochondrial control region revealed four mitochondrial haplotypes  [Canis anthus) [In the present continent-wide study we supported previous findings of Zachos et al.  and Fabb vulpes) , 102. Th vulpes) , which s anthus) . For exa anthus) , 105. ThH = 0.34) with just four haplotypes despite the large sampling area. In jackals from the mainland sites , it was the haplotype recorded in previous studies (H1) that was found to occur most frequently. However, it was absent from the island population (Samos), where the unique H2 haplotype is prevalent. Higher mtDNA diversity compared with previous studies is mainly connected with the larger sampling area including the Caucasus (one ‘new’ haplotype H3) and Greece . The highest level of haplotype diversity was found in the Peloponnese. The greater number of haplotypes in the Aegean region could suggest that the present population in Greece may, at least partially, descend from the ancient Greek population. However, confirmation of this hypothesis requires further study, preferably including fossil material. Moreover, in the south-eastern European population, alongside the haplotype discovered by Zachos et al. [C. aureus is uniform in regard to control-region polymorphisms, as suggested by earlier studies [In contrast with the authors of previous studies , 52, we s et al.  and Fabb studies , 52. Hen studies , 107. HoA = 2.8) and more limited heterozygosity (HO = 0.37) in Dalmatian jackals and suggested a long-term isolation of this population. In respect to this, we also call to attention that golden jackals were present in southern Dalmatia already in the Middle Ages [Previous studies of golden jackals in Europe emphasized the limited degree of genetic structuring, with only the coastal population from Dalmatia clearly differentiated from other Balkan samples , 52. A gdle Ages  and possdle Ages .Our analysis extending to the whole of Europe has pointed to the existence of a pronounced genetic structure in relation to both nuclear and mitochondrial markers. Individuals from an extensive area of south-eastern Europe generally form a uniform genetic group, as already noted by Zachos et al. . Fabbri STRUCTURE and BAPS suggested ongoing gene flow between the Caucasus and Europe as well—some individuals from SEE had the highest probability of ancestry from the CAU/BAL cluster. Interestingly, when the microsatellite genotypes are concerned, an individual from south-eastern Europe . Unfortunately, there was no access to samples from the Turkish mainland, so it remains unclear whether the geographical barrier of water restrics gene flow between the island and that mainland. However, our genetic data indicate that there are or were some connections between Samos and northern Greece , and from the population expanding out of south-eastern Europe (Lithuanian case). This dual origin does not support the idea that jackals were introduced by humans, as it is unlikely that someone would capture jackals in different regions and smuggle them to the Baltics. Additionally, recent records of jackal occurrence from Slovakia, Ukraine, Belarus, and north-western and eastern Poland , 113–116The dynamics of species’ range expansions depend on habitat connectivity, but also on dispersal ability  and habiAnother case resembling the sudden occurrence of several jackals in the Baltics, refers to the first colonization of Slovenia in the mid-20th century. In winter 1952/3 several jackals suddenly appeared in Central Slovenia near Ljubljana, with reported observations of groups of up to six animals  and lateMore recent records that can be considered potential cases of long-distance dispersal of jackals include:-a male observed several times from 1996 and then shot in 1998 in Südbrandenburg in Germany  and an i-five photo-records of a jackal in 2011 in the Northwestern Alps of Switzerland , with th-an individual shot in 2014 near Olevsk in Northern Ukraine , 430 km -an individual shot in 2012 near Tomašovka in Belarus , 410 km -a young male found dead in April 2015 on a road in NW Poland, close to the German border , ca. 610-GPS-GSM collared 1.5 year old female, which travelled 220 km during 12 days in Hungary in 2014 (J. Lanszki unpubl. data).Based on this review of jackal occurrences and our genetic data, we suggest that it is not uncommon for golden jackals to disperse over several hundred kilometers in human-dominated landscapes. This could explain the speed of jackal expansion in Europe that has been observed in the last decades . We alsoper se, but rather with individuals of admixed origin between several canid species. Furthemore, the parasite load in the European golden jackal is similar to or lower than that in other carnivores  in the region [The golden jackal has already been declared an alien, potentially invasive species in all Baltic States (e.g. ). Howevee region –143, andNevertheless, results presented here have several management and conservation implications. The existence of long-distance dispersal in the golden jackal would seem to warrant the initiation of international coordination in management of the species in Europe and more focus on management at the population, rather than at the national level, especially considering considerable differences that currently exist among countries . We therS1 FileTable A, List of examined specimens including specimen number, sex, locality information, date, geographical coordinates, and mtDNA haplotype designation. Table B, Microsatellite genotypes. Table C,Per locus genetic diversity in 97 samples of golden jackal. Table D,Per region genetic diversity estimated based on polymorphisms in 15 microsatellite loci.(DOC)Click here for additional data file.


Lithobates sevosus, is a critically endangered species found only in coastal Mississippi, with low genetic variability as a consequence of isolation and population size reduction. Conservation management practices have been implemented, but their efficacy has not been addressed. We genotyped individuals collected 1997–2014 to determine temporal trends in population genetic variation, structure, and effective size. Observed and expected heterozygosity and allelic richness revealed temporally stable, but low, levels of genetic variation. Positive levels of inbreeding were found in each year. There was weak genetic structure among years, which can be attributed to increased effects of genetic drift and inbreeding in small populations. L. sevosus exhibited an increase in effective population size, and currently has an estimated effective size of 33.0–58.6 individuals, which is approximately half the census size. This large ratio could possibly be explained by genetic compensation. We found that management practices have been effective at maintaining and improving effective size and genetic diversity, but that additional strategies need to be implemented to enhance viability of the species.Monitoring temporal changes in population genetic diversity and effective population size can provide vital information on future viability. The dusky gopher frog,  The maintenance of genetic variation within populations and species is a primary concern in conservation biology because this variation allows species and populations to adapt to their changing environments , which can be estimated through the use of microsatellite markers. Effective population size is defined as the size of the ideal population that experiences genetic drift at the same rate as the population of interest  can be used together to calculate a Ne/Nc ratio. This ratio is useful because census size is easier to measure than effective population size , as opposed to Ne  Waples . Of these Waples . The rele Waples .Lithobates sevosus) is a prime study species in determining the effects of population isolation on genetic diversity, population structure, and effective population size and in evaluating the efficacy of management practices. The species is listed as Endangered by the United States Fish and Wildlife Service  forests  appears to have fewer than 20 adults and could possibly be extinct based on egg counts  determine temporal trends in population genetic variation and implications for future population persistence for L. sevosus based on these data and (2) estimate effective population size (or effective number of breeders). This information will allow for an evaluation of the effectiveness of current management practices in terms of preserving and enhancing genetic variability. Furthermore, this study hopes to inform broadly applicable questions in the field of conservation biology, including (1) Is there temporal genetic structuring in isolated populations? (2) How do conservation management practices, like tadpole headstarting, impact genetic diversity over time? and (3) Are Ne/N ratios stable overtime in small, isolated populations?Extensive genetic and demographic research has been conducted . This population was selected because Glen's Pond represents the largest, if not only remaining, population and has the most stable breeding activity. Specifically, one egg was collected from each clutch deposited in 1997 (n = 58), 2005 (n = 20), 2008 (n = 50), 2013 (n = 31), and 2014 (n = 51). These years were selected to address the efficacy of the headstarting program, with 1997–2005 largely representing preconservation management practices, and 2006–2014 representing postconservation management practices. Individuals were genotyped for nine previously developed microsatellite loci, four loci from Richter and Broughton  and expected heterozygosity (He) were calculated using GenAlEx Version 6.501, and allelic richness using rarefaction were calculated using FSTAT Version 2.9.3.2 (Goudet FIS) was calculated using GENETIX 4.05 with 95% CIs generated via bootstrapping 10,000 times within each year  on temporal distance was computed, and a Mantel test was performed using FSTAT Version 2.9.3.2 to assess significance levels  on heterozygosity, allelic richness, and inbreeding, a two‐way repeated measures ANOVA was performed such that year 1997 represents “early” preconservation efforts, year 2005 represents “late” preconservation efforts, year 2008 represents “early” postconservation efforts, and year 2014 represents “late” postconservation efforts. Year 2013 was omitted from these analyses to create a 2 × 2 level factor design.Nb limits were set to 4 > Nb > 500. The Nb of simulated populations with summary statistics resembling that of the test population were used to infer Nb of the target population  values were estimated from demographic data based on the number of adults captured during each breeding season (Richter and Seigel N(−1)). However, amphibians have overlapping generations, and recognized bias and inaccuracies could be introduced into these estimates.Census size . The Wilcoxon signed‐rank test indicated a significant pairwise difference in observed heterozygosity values for 2013 compared to all other years, with uncorrected P‐values ranging 0.01–0.02. Allelic richness ranged from 3.20 to 3.99, with a nonsignificantly decreasing, then increasing over the sample period . Wright's inbreeding coefficient values ranged from 0.01 to 0.28, with significant differences over the sample period . Sample year 2013 was different from all years except 2008, with P‐values ranging 0.02–0.04. A heterozygote deficiency was detected in 2013, with FIS values significantly >0 (95% CI 0.08–0.41). The overall FST value was 0.023 (95% CI 0.01–0.04), indicating weak genetic differentiation among years. Significant differentiation existed between all pairs of sample years, with the exception of years 2008 versus 2014, 2005 versus 2008, and 2005 versus 2014 .Null alleles were suggested to be present in 3 years of the sampling period. Data were reanalyzed upon removal of loci with null alleles, and the results were similar. Additionally, there was no consistent pattern across years or loci, so all loci were included in final analyses. Deviations from Hardy–Weinberg equilibrium occurred at locus Lica 41 in the year 2013. No pairs of loci exhibited linkage disequilibrium. When analyzing the data year‐by‐year the amount of genetic variation within F1 = 3.03, P = 0.12; expected heterozygosity: F1 = 5.02, P = 0.06; allelic richness: F1 = 2.16, P = 0.18; Wright's inbreeding coefficient: F1 = 0.42, P = 0.54).No statistical relationship was found between conservation period or stage on heterozygosity, allelic richness, or inbreeding. Graphically, however, a nonsignificant antagonistic interaction is revealed between stage x period and heterozygosity, allelic richness, and inbreeding Fig.   than in the preheadstarting period (1997–2014)  within L. sevosus followed an increasing then decreasing pattern over the sample period. This trend can be strengthened by demographic data, which reveals an inverse pattern of a decreasing then increasing number of breeding individuals  mating  and conservation stage (early or late) reveals that the conservation practices have had a positive effect on heterozygosity, allelic richness, and inbreeding. Both observed and expected heterozygosity and allelic richness show values decreasing from early to late stages in the preconservation period, and then increasing from early to late stages in the postconservation period. Similarly, inbreeding values increase from early to late stages in the preconservation period and then decrease from early to late stages in the postconservation period. These slope changes suggest that the management practices in place have reversed the previous trajectory of decreasing genetic variation and increasing inbreeding and have replaced it with a more promising outlook.L. sevosus exists as one population, Ne (or Nb) estimates can be interpreted at both scales.Effective population size is an important parameter that influences population viability and conservation management decisions. This estimate can be interpreted both at the global and the local scale, depending on the scope of the project. At the global scale, information such as long‐term viability, adaptive potential, and maintenance of genetic variation can be gained. At the local scale, immediate threats to population persistence can be revealed  are in line with the theoretical values of Nunney  with small population sizes. Similarly, species of conservation concern tend to have larger Ne/Nc (or Nb/Nc) ratios than stable populations  ratios at low population sizes. Genetic compensation suggests that when population sizes are low, most individuals participate in breeding activities. In an evolutionary context, this compensation would lessen genetic variation reduction in times with low population numbers  by reducing inbreeding. These results suggest that L. sevosus might be experiencing genetic compensation in concert with effective conservation management practices that have stabilized Ne/Nc (and Nb/Nc) ratios and genetic variation over the sample period.Frankham , in a ref Nunney . Jehle ef Nunney  found siIn the 2015 breeding season, 92 egg masses were laid in Glen's Pond—a greater amount than any year in our study . Also, 18 egg masses were laid in Pony Ranch Pond via natural migration and recruitment. This large reproductive output is consistent with the trend seen in previous years, which show an increasing amount of egg masses laid following the implementation of conservation management practices. The findings of this study support and strengthen the large demographic dataset, and show that as reproductive output continues to increase, so does genetic variation and effective size.L. sevosus appear to have been successful at preventing further genetic erosion. The addition of headstarting tadpoles appears to be an effective way of maintaining genetic variation in study systems subject to variable reproductive output due to environmental stochasticity or disease. However, further efforts are still needed to continue restoring genetic variation to higher levels. Second, L. sevosus exhibits weak temporal genetic structuring. This differentiation is likely due to increased effects of genetic drift and inbreeding in small, isolated populations. Future studies should continue to focus on temporal genetic structuring, as it provides genetic information on microevolutionary changes. Third, the effective population size and effective number of breeders estimates can be variable, but overall, effective population size of L. sevosus is around half that of the estimated census size. These Ne/Nc and Nb/Nc estimates contribute to the literature on anuran effective population size—helping to aid in the search for a common ratio among taxa, and suggest that genetic compensation might be acting within this system, reducing the loss of genetic diversity. Temporal estimates demonstrated that Ne increased, potentially because of headstarting efforts, indicating further success of the conservation plans and that the decrease in heterozygosity in year 2013 does not appear to be affecting the number of genetically contributing individuals each breeding season. Despite the success of multiple management practices in place, L. sevosus still has a reduced amount of genetic variation, and new, interconnected populations must be established in order to improve population persistence and ensure future viability of the species. The recent breeding events at Pony Ranch Pond provide encouraging possibilities for the establishment of population connectivity. Additional wetland restorations could benefit from the use of head‐started tadpoles from Glen's Pond and captive zoo populations, although careful consideration must be taken to eliminate the risk of transferring pathogens from captive to natural populations and between natural populations.Overall, this study illustrates three major findings. First, the current management practices in place for None declared.


Greece  is located in southeastern Europe and is bordered on the north by Albania, the Former Yugoslav Republic of Macedonia (F.Y.R.O.M.) and Bulgaria; on the east by the Aegean Sea and Turkey; and on the west and south by the Ionian and Mediterranean seas, respectively. The country consists of the mainland, the Peloponnese peninsula and more than 3000 islands, of which about 170 are inhabited, including Crete, Rhodes, and Corfu, and the Ionian, Dodecanese, and Cyclades groups , with the vast majority being of Greek descent (98%) and the remainder belonging to other ethnicities. Over 95% of Greeks are Orthodox Christians. Muslims comprise 1.3% of the population and the remaining 0.7% includes Catholics, Jews, Old Calendar Orthodox, Jehovah Witnesses, Protestants, and other religions. The majority of the Muslim minority is concentrated in the area of northern Greece called Thrace. They are the only officially recognized minority in Greece and received legal status through the Treaty of Lausanne in 1923. Nearly half of the population lives in the two major cities, Athens and Thessaloniki, after a significant urbanization trend that was observed in the 20th and 21st centuries.According to the most recent official consensus in 2011, the population was 10,816,286 .Studies, theories, and observations on the inheritance of physical traits in humans can be traced back in ancient Greek literature from the eighth to the fourth century BC (Bazopoulou-Kyrkanidou Hippocrates (460–377 BC), the father of medicine, believed in the inheritance of acquired characteristics so he devised the hypothesis known as “pangenesis.” He proposed that all organs of a parent’s body gave off invisible seeds, which were like small building components transmitted during procreation to help the formation of the unborn child.“Euploidy,” “euchromatin,” and “eugenics” begin with the Greek preface “eu” which means good/well. Every word in the ancient and modern Greek language which begins with “eu” implies something good Galton . Plato (Aristotle (384–322 BC), who was a student of Plato, developed a theory of inheritance, according to which each sex contributes to heredity, so that daughters resemble their mothers and sons their fathers. This concept was expounded through many generations. He also emphasized the importance of blood in heredity. One of his theories was that blood supplied genetic material for building all parts of the adult body and was the basis for passing on the power to the next generation. Today, people still speak of certain traits as being “in the blood” or “of blood lines” or “blood ties.” Furthermore, Aristotle was a central figure in our knowledge of ancient theories of reproduction/conception in animals, which were also applicable to humans. Democritus (460–370 BC) introduced the notion of nature–nurture when he wrote: “more people become capable by exercise than by their natural predisposition.”Cyclopia is one of the severe forebrain lesions. Cyclops appears in Homer’s Odyssey (eighth to seventh century BC). The next time Cyclops appears in literature is in “Theogonia” written by Hesiodus (seventh century BC) and a satyric play of Euripides which is titled “Cyclops” (fifth century BC)  founded in 1983, which is financed by the state budget, social insurance contributions, and private payments Economou . VariousSince July 2011, increasing austerity measures resulted in citizens being forced to contribute more toward the cost of their healthcare services and medication. Insufficient public funding has forced households to look for care in the private sector  at the Choremion Research Laboratory of the First Department of Pediatrics of the University of Athens has the leading role in introducing modern molecular genetics methods for the diagnosis, carrier screening, and prenatal diagnosis of most genetic disorders in Greece. As the only major public, academic medical genetics center for the whole of the Hellenic area, DMG is also active in teaching and research. Located within the largest pediatric hospital in Greece  it receives a large number of referrals from the hospital clinics, as well as outside referrals from Greece and other Balkan countries. Annually these include (approximately):Clinical genetics and dysmorphology as an outpatient clinic offers:Patient evaluation Genetic counselingOur well-equipped Genetics Laboratory provides:Molecular geneticsCytogenetics Molecular cytogenetics Prenatal diagnosis (monogenic diseases and karyotype)Preimplantation genetic diagnosisNoninvasive prenatal diagnosis (sex and rhesus)Microarrays technologies for CGH (Comperative Genomic Hybridization) and expression studiesThe genetic services include:Concurrent to the major role that the DMG plays in clinical services, it has also made substantial contributions in several areas of applied research in the field of genetics. Public funding for genetic testing outside a standard karyotype is limited and the patient’s family usually pays the remainder of the genetic, metabolic, and enzymatic testing privately.A number of other public genetic centers appeared in the 1990s at the Universities of Thessaloniki, Ioannina, Patras, Larissa, Alexandroupoli, and Crete Fig.. CurrentThere are official referral centers for hemoglobinopathies across the country offering patient treatment, carrier screening, and prenatal diagnosis.There is no official recognition of laboratories to perform diagnostic genetic tests, quality assessments, or accreditation for genetics services. In recent years though, several molecular genetics laboratories have joined the quality assessment programs of EMQN and one cytogenetics laboratory has joined the UK NEQAS in clinical cytogenetics. Also, the DMG at the Choremion Research Laboratory is a member of the UK NEQAS for Molecular Genetics, Special Advisory Group for external quality assessment (EQA) for Preimplantation Genetic Diagnosis. There are over 24 public and private institutions currently offering a variety of genetic services in Greece, with ∼17 entries for molecular DNA diagnostic laboratories in the European Directory of DNA Diagnostic Laboratories (EDDNAL) website.The number of trained physicians in Clinical and Biochemical Genetics is extremely limited. Contacts and scientific collaboration with various foreign countries have provided an avenue for further exploration of genetic disorders. Clinical Genetics has not as yet been recognized as a separate specialty by the Hellenic Ministry of Health to date. Clinical dysmorphology services exist as a consulting and outpatient service at the “Aghia Sophia” Children’s Hospital in Athens, Choremion Research Laboratory, but are almost nonexistent elsewhere in Greece  syndromes in Greece.β-Thalassemia  carrier frequency is ∼8%, while 1.5% of the population are carriers of the HbS [β6(A3)Glu→Val] mutation, sickle cell disease (SCD). The rate of β-thal carriers can be as high as 15–20% in some areas, especially in the low-altitude areas of Thessaly, Western Peloponnese, and Western Epirus. The distribution of HbS shows a similar pattern with certain regions, such as Viotia, western Peloponnese, and the Chalkidiki peninsula reaching a carrier frequency of up to 20%.Thalassemias are the most frequent genetic disorders in Greece. This resulted in the interest of the international scientific community regarding the clinical symptoms, the pathophysiological findings, and the inheritance of these diseases. For many decades, these served as models for the study of monogenic diseases on a clinical, biochemical, and later at a molecular level.The Greek program for thalassemia started in 1974 as a nationwide government-sponsored and prospectively organized program, the National Program for the Prevention of Haemoglobinopathies, and is implemented free of charge through a network of 23 Prevention Units distributed across the country. All procedures are coordinated and supervised by the Central Prevention Unit at Laikon Hospital in Athens.β-globin chains, which had to exceed an empirically set level. This was a delicate step as in Greece most fetuses with thalassemia major are β+/β+ and therefore displayed values that could overlap with those of carriers. The procedure has been performed since then in thousands of cases  annual reports  or Crete that represent genetic isolates and have allowed studies on genetic drift of certain disease-associated variants. Mutation analysis was performed in 110 and nine Wilson disease families originating from two isolated populations in Sardinia and the Greek Island of Kalymnos  trial,” 2012–present.The TREAT-NMD rare disease registries for DMD (Duchenne muscular dystrophy) .The DMG at the Choremion Research Laboratory of the University of Athens organized in collaboration with the MDA Hellas (Muscular Dystrophy Association), a Greek-wide registry of patients diagnosed with a neuromuscular disease : Praksis, Doctors of the World, UNICEF, Médecins Sans Frontières, the Hellenic Society for the Protection and Rehabilitation of Disabled Children (ELEPAP), and the Red Cross are the most well known.There are centers for Social Support for People with Disabilities such as the K.A.A.K.Y.A.MEA and the National Institution for the Rehabilitation of Disabled People (E.K.A.).www.tomellon.com.gr) is one of these, which provides social and financial support to parents with children suffering from genetic syndrome with or without intellectual disabilities. It also organizes seminars and meetings in order to deliver scientific information to professionals, members, and the general public.Various support groups for all different genetic diseases exist. “To Mellon”  which is the representative of Eurordis in Greece and many others. All these groups cooperate with professional medical staff.In 1982, the Hellenic Association of Medical Geneticists (Σ.Ι.Γ.Ε.) was founded and consisted of professionals working in the field of medical genetics. This was a historical event in the development of Genetics in Greece. In January 2011, the Hellenic Society of Medical Genetics (HSMG) was established. Its aim was to unite all the medical doctors involved with genetic diseases and help the recognition of the specialty of Clinical Genetics in Greece.In the last 10 years, Greece has gone forward in leaps and bounds in the fields of clinical genetics/dysmorphology, laboratory testing, and research. It has now introduced “state-of-the-art” technologies in diagnostic and prenatal testing and has success in the prevention of hemoglobinopathies. Some areas have difficulties in accessing highly specialized centers of clinical genetic services. Participation in extended newborn screening, expensive molecular genetic testing, especially whole exome sequencing, and costly therapies, such as enzyme replacement or gene and stem cell therapies, are difficult to cover by health insurances. The financial crisis has posed a burden in the services and support programs provided by the National Health System, with charities and the private sector trying to fill the void.Academic institutions will have to play a critical role in public education in genetic disorders that will help improve their quality of life and social integration. Multidisciplinary care for the long-term needs of patients with complex genetic syndromes is difficult in urban areas, due to small numbers of genetically trained physicians. Access to information and genetic care is particularly challenging in rural areas, such as mountainous regions and smaller islands. Telemedicine has gained support and limited funding and could provide an avenue of sharing expertise in remote areas of the country.There are patients with genetic diseases in all fields of Medicine. The contribution of Clinical Genetics is significant and essential for the diagnosis, prognosis, prevention, and risk assessment of genetic disorders. The correct evaluation of the clinical symptoms and of the morphological abnormalities constitutes the basis for accurate laboratory genetic testing and treatment in daily clinical practice. In Greece, the availability of genetic testing has expanded dramatically. Recently, whole exome or whole genome sequencing has begun to help solve diagnostic problems.A major aim of the Greek genetics community is to have Clinical Genetics recognized as an independent Medical Specialty in Greece. In this way, we hope to (1) encourage young doctors to be trained and gain experience for the benefit of the patients; (2) promote scientific research in the field of medical genetics; (3) develop and specify what is necessary for the foundation of certified medical genetic centers; (4) organize quality control of the genetic services; and finally (5) gain recognition of medical geneticists as scientific professionals.


Psammodromus, including the Western Sand Racer , the Edward’s Sand Racer (P. edwardsianus) and the Spanish Sand Racer (P. hispanicus). In this article, the genetic variability and population structure of Psammodromus edwardsianus, P. hispanicus, and P. occidentalis were studied in the Iberian Peninsula covering their natural geographic distribution. Mitochondrial DNA showed genetically different units in all species with higher genetic variability in their southern populations . Genetic differentiation was different among species and contrasted to those of species with similar characteristics. Our results therefore highlight the importance of species-specific studies analysing population structure.The knowledge of a species’ population structure is essential for the development of adequate conservation actions as well as for the understanding of its evolution. The population structure is unknown in all species of the Genus  The knowledge of the population structure of a species is basic for its efficient conservation and management as well as for the understanding of subjacent evolutionary processes implicated on it . DifferePsammodromus P. occidentalis, P. edwardsianus and P. hispanicus  have been recently described as new lizard species given their old divergence and their genetic, phenotypic, and ecological niche differences  Mya from the ancestor of P. edwardsianus and P. hispanicus, and the latter diverged 4.8 (1.5–8.7) Mya  Mya . The natlineages . PhylogePsammodromus species  was studied, using mitochondrial DNA and a sampling covering the biggest area of their natural geographic distribution. Results will be very useful, for conservation and management issues, among other reasons.Some phylogenetic and phylogeographic studies have been previously done in different  species . Neverthsolation  and woulPsammodromus hispanicus (n = 69), P. edwardsianus (n = 135) and P. occidentalis (n = 43) were used in this study. The study employs all of the currently available genetic data and it includes three of the six described species of the Genus (note: for the other species of this genus no intensive population sampling has been conducted to date). Cytochrome B (cytB) sequences of the studied species (the only marker with enough sample sizes for this study) were obtained from GenBank , where two populations from neighbouring provinces have been joined  because unfortunately not any of the sampled southern P. occidentalis populations consisted of more than 8 captured individuals.All populations with a sample size of at least nine individuals were included in the sequence datasets . The onlπ) diversity, as well as genetic differentiation among populations, measured as FST , were calculated with Arlequin v3.11 software  and nucleotide  are linearly linked with the logarithm of the distance (in ln[km]) among the sampled populations, as predicted by a two-dimensional migration model  was studied with GENEPOP  employinon model . In P. oPsammodromus edwardsianus, from 0 to 86% in P. occidentalis and from 17 to 56% in P. hispanicus. P. hispanicus was the only species without monomorphic populations. Nucleotide diversity ranged from 0.000 to 0.566 in P. edwardsianus, from 0.067 to 0.848 in P. hispanicus and from 0.000 to 0.113 in P. occidentalis. Neither tests . FST values (P < 0.05) among almost all populations of P. edwardsianus except between the two northernmost populations (population pairs PrBa-Zr and Zr-Gir). In P. occidentalis three of the six population differences were significant  in P. edwardsianus. In P. hispanicus and P. occidentalis, no significant IBD was found.Isolation by distance (IBD) tests resulted highly significant  phylogeographic studies exist  had lower genetic variability than southern populations , nor by anthropogenic infrastructures such as roads. Big highways or heavy traffic roads determine the movement of other lizard species and small and big mammals . Our results suggest that roads do not determine their population structure, at least at global scale, suggesting that road networks affecting species gene flow might be species-specific . The subset rendered drastic differences since IBD was not longer significant (P = 0.192). This suggests that in P. hispanicus and P. occidentalis IBD results might be affected by the sampling. Thus, the here observed absence of IBD in the two species requires careful interpretation and its presence cannot be discarded.As commented above, IBD was only significant in Psammodromus species are distributed along the Iberian Peninsula in fragmented units with higher genetic variability in southern populations . In conservation terms, isolated populations  as well as those with low genetic variability  require specific measures and have to be specially managed together with the more variable populations, because a balanced and dynamic conservation strategy is needed (In sum, mitochondrial DNA showed that all three studied s needed . In addis needed . This fis needed  and high10.7717/peerj.994/supp-1Supplemental Information 1Psammodromus edwarsianus Cytochrome B sequences dataset in fasta format.Click here for additional data file.10.7717/peerj.994/supp-2Supplemental Information 2Psammodromus occidentalis Cytochrome B sequences dataset in fasta format.Click here for additional data file.10.7717/peerj.994/supp-3Supplemental Information 3Psammodromus hispanicus Cytochrome B sequences dataset in fasta format.Click here for additional data file.


Anopheles culicifacies, is currently increasing. To predict malaria vector dynamics, knowledge of population genetics and gene flow is required, but this information is unavailable for Sri Lanka. This study was carried out to determine the population structure of An. culicifacies E in Sri Lanka.The disease re-emergence threat from the major malaria vector in Sri Lanka, An. culicifacies E collected from six sites in Sri Lanka during 2010-2012. Standard population genetic tests and analyses, genetic differentiation, Hardy-Weinberg equilibrium, linkage disequilibrium, Bayesian cluster analysis, AMOVA, SAMOVA and isolation-by-distance were conducted using five polymorphic loci.Eight microsatellite markers were used to examine FIS values in the pooled population (p < 0.0100). Three loci showed high deviations in all sites except Kataragama, which was in agreement with HWE for all loci except one locus (p < 0.0016). Observed heterozygosity was less than the expected values for all sites except Kataragama, where reported negative FIS values indicated a heterozygosity excess. Genetic differentiation was observed for all sampling site pairs and was not supported by the isolation by distance model. Bayesian clustering analysis identified the presence of three sympatric clusters (gene pools) in the studied population. Significant genetic differentiation was detected in cluster pairs with low gene flow and isolation by distance was not detected between clusters. Furthermore, the results suggested the presence of a barrier to gene flow that divided the populations into two parts with the central hill region of Sri Lanka as the dividing line.Five microsatellite loci were highly polymorphic with high allelic richness. Hardy-Weinberg Equilibrium (HWE) was significantly rejected for four loci with positive An. culicifacies E specimens isolated in Sri Lanka. There was no effect of geographic distance on genetic differentiation and the central mountain ranges in Sri Lanka appeared to be a barrier to gene flow.Three sympatric clusters were detected among  Anopheles culicifacies Giles sensu lato, the major malaria vector in Sri Lanka, is widely distributed across the dry and intermediate zones of the country , Kandy , Nikaweratiya , Thanamalwila , Monaragala  and Kataragama  .Genomic DNA was extracted from mosquitoes using a phenol:chloroform extraction method . Out of FIS) using FSTAT software v2.9.3.2 2-1}/8 2-1}/8 , 31, whe gambiae  were use gambiae . Ne estiion rate . The effT values . The anaT values  in the IT values , with 10distance . Spatialdistance  to invesK) within the studied population using STRUCTURE 2.3.4 software [K (K =1 to 10) with a burn-in period of 100,000 iterations and 10,000 steps for Monte Carlo Markov Chain (MCMC) replications. The Evanno method [ad hoc quantity, DK, which is based on the second order rate of change of the likelihood function between successive values of K.Bayesian analysis was carried out to identify possible clusters  and the AcAIIB5 locus had the fewest (6). Locus AcA59 was also the most polymorphic, with allele frequencies ranging from 0.19 to 0.36, and a maximum value at all test sites. Allele richness based on a minimum sample size of 26 diploid individuals per locus was between 4.963 and 9.830, with locus AcA59 showing the highest richness for all sites . A marked deviation in AcA59, AcAIIB5 and AcAVb93 was observed at the sampling sites. Locus AcA59 significantly deviated at all six sampling sites. Meanwhile, AcAIIB5 deviated at five sampling sites and confirmed HWE in Kataragama. The AcAVb93 locus deviated at all sites except for Kataragama and Monaragala. Kataragama specimens showed HWE for all loci except AcA59 (p < 0.0016). Observed heterozygosity was less than the expected heterozygosity at all test sites except for Kataragama, which had negative FIS values that indicted a heterozygosity excess.Hardy-Weinberg Equilibrium was significantly rejected for four loci: AcA59, AcAVIB213, AcAIIB5 and AcAVb93, with positive p < 0.005). Among the collection sites, only one pair of loci (AcAVb93 - AcA59) in Anuradhapura showed significant linkage disequilibrium.Two pairs of loci in the pooled population (AcAVb93 - AcA59 and AcAVb93 - AcAIIB5) were significant in the exact test for linkage disequilibrium   and the longest distance was between Kataragama and Anuradhapura (239 km). The highest levels of genetic differentiation were observed between Kataragama and Anuradhapura (239 km), Kataragama and Kandy (124 km) and Kataragama and Nikaweratiya (198 km) (Table Genetic variability between populations was estimated using pairwise 3) Table . SignifiFST values, showed greater variation within population pairs (86.44 %) than that among the populations (13.56 %), thus confirming genetic differentiation between population pairs. The Mantel test revealed that there is no significant correlation between pairwise genetic distance (FST/(1- FST) and the natural logarithm of pairwise geographical distance , which supports the lack of correlation between the genetic differentiation of populations and geographic distances between population pairs , which is calculated based on irs Fig. .Fig. 2aNm values (number of migrants per population per generation) were calculated based on FST values as t/M = FST/(1-FST) for all population pairs  was observed between Kataragama and Anuradhapura populations (Nm = 0.98), Kataragama and Kandy populations (Nm = 1.76), and Kataragama and Nikaweratiya (Nm = 1.85) populations, which were separated by 239 km, 124 km and 198 km, respectively. The highest degree of genetic differentiation in terms of FST comparisons was also observed for these three population pairs, suggesting that a barrier to gene flow may exist within these populations. Monaragala and Thanamalwila, which are separated by 53 km, showed the highest Nm value (50.35) and the least genetic differentiation. According to the Nm values and the geographic distances between the populations, there is no correlation between the measure of gene flow (Nm) value and the distance  ranged between 4,124 and 12,034 for collecting sites while 12,626 for the pooled population  likelihood score optimized for K = 3 clusters) . The number of alleles seen in this study varied according to the microsatellite loci studied. Locus AcA59 had the highest number of alleles (14) compared to 8 alleles for the same locus of species A in India. Meanwhile, locus AcAVIB213 had 12 alleles, while the Indian sibling species A had 17. The fewest alleles were seen for locus AcAVB93A in both Sri Lankan E and Indian A . FurtherAn. culicifacies complex, given that a study of spatial or Bayesian genetic structure has not been undertaken for any sibling species in the complex. These microsatellite loci have not been physically mapped to An. culicifacies polytene chromosomes, and therefore the location of these loci with respect to the polymorphic chromosome forms is unknown. Linkage disequilibrium was detected only within two pairs of loci in the entire population out of ten comparisons as well as in a single pair of loci at all collecting sites, suggesting the absence of significant linkage between loci, which may have a random distribution in the genome.The genetic structure of a sibling species in the Samples isolated from Kataragama site agreed with HWE with excess heterozygosity while all other localities showed significant deviations. The heterozygote deficiency observed at all other sites could be due to the population substructure (Wahlund effect), inbreeding, selection or null alleles. The heterozygote deficiency observed at multiple loci likely was not due to selection, which generally engages only one locus . If inbrIn this study a possible barrier to gene flow was observed in the East-west direction across the central area of the country that was close to the Kandy sampling site, which has one of Sri Lanka’s highest altitudes and is surrounded by a number of hills Fig. . These hAn. culicifacies E for the Kataragama site relative to the other collection sites could arise from influences by the coastal environment.The Kataragama site has the lowest altitude and Fig.  is near An. culicifacies E are concerned, although environmental factors such as temperature and humidity might vary among these sites. Therefore, the availability of breeding sites was not expected to influence the gene flow between collection sites. Most of the areas that An. culicifacies inhabits experience high amounts of rainfall only in the South West Monsoon season (December to February). During this period, the abundance of mosquitoes falls drastically due to monsoon-driven flushing of egg clutches and larval breeding sites. Thus, mosquito population abundance undergoes seasonal changes with high densities reported only during the dry seasons. The high level of genetic diversity observed in this study suggests that An. culicifacies E can maintain a relatively high effective population size despite large population fluctuations.All sample collecting sites had similar topologies as far as breeding sites for Finally, studying insecticide susceptibility and performing parasite susceptibility tests to determine insecticide resistance levels and vectorial capacity of species E in two regions separated by a physical barrier is recommended to provide baseline genetic information about the vector. Such knowledge is useful for implementing new vector control strategies or to revise ongoing national malaria control practices, as well as to drive a re-emergence of malaria prevention programs.An. culicifacies E specimens collected in Sri Lanka. There was no effect of geographic distance on genetic differentiation in a pairwise population analysis. The central mountain ranges in Sri Lanka appeared to act as a barrier to gene flow.Three sympatric clusters were detected among