FANCC), a DNA repair protein. In prior studies, we noted accelerated bone marrow failure in Fancc−/− mice undergoing multiple episodes of emergency granulopoiesis, associated with apoptosis of bone marrow cells with unrepaired DNA damage. Additionally, we found increased expression of Fanconi C and F proteins during emergency granulopoiesis. These findings suggest that Icsbp protects the bone marrow from DNA damage by increasing activity of the Fanconi DNA repair pathway, but the mechanisms for FANCC activation during initiation of emergency granulopoiesis are unclear. In this study, we observed that Stat3 and C/ebpβ activate FANCC transcription and contribute to DNA repair. Our findings indicate that FancC expression is increased during Stat3- and C/ebpβ-induced initiation of emergency granulopoiesis by these transcription factors and is maintained through termination by Icsbp. Our work reveals that Stat3- and C/ebpβ-mediated FancC expression is a critical component for initiating and sustaining key innate immune responses.Interferon consensus sequence–binding protein (Icsbp) is required for terminating emergency granulopoiesis, an episodic event responsible for granulocyte production in response to infections and a key component of the innate immune response. Icsbp inhibits the expression of Stat3 and C/ebpβ, transcription factors essential for initiating and sustaining granulopoiesis, and activates transcription of Fanconi C ( Fancc−/− mice) by repeated stimulation of an emergency (stress) granulopoiesis response ) ) , 17. Thior G-CSF , 17. Alsor G-CSF .Candida albicans or encapsulated bacteria) or an antigen/adjuvant mixture referred to as “Alum”   injection with pathogens (djuvant) , 15, 18.djuvant) , 15. By We found that injecting Alum every 4 weeks repeated this process in WT mice without resulting in morbidity or mortality , 15. We Fancc−/− mice, there was no subsequent expansion of myeloid progenitors or production of mature granulocytes   . We hypoFANCC 5′ flank linked to a firefly luciferase reporter (or an empty control reporter vector) (A). We determined the activity of these constructs in U937 myeloid cells co-transfected with vectors to express Stat3 or C/ebpβ (versus an empty control expression vector). For these studies, we considered two isoforms of C/ebpβ: Lap and Lip. The former is a transcriptional activator, and the shorter Lip isoform is an antagonist of Lap  to normalize for transfection efficiency.To investigate this hypothesis, we assayed the promoter activity of a set of reporter constructs with −1.0 kb to −400 bp of the  vector) A. We dett of Lap . BecauseFANCC promoter  (B). Both Icsbp and C/ebpβ-Lap activated a construct with 450 bp of the FANCC promoter  but Stat3 did not. This suggested the presence of a Stat3-influenced cis element between 450 and 500 bp in the FANCC promoter.We found that Stat3, C/ebpβ-Lap, and Icsbp each significantly increased the reporter activity from constructs with 1.0 kb or 500 bp of the , n = 6) B. Both IB). None of the FANCC promoter constructs were activated or repressed by Stat5 or C/ebpβ-Lip. None of these proteins influenced the activity of an empty reporter control vector, and this was subtracted as background.Further truncation of the promoter to 400 bp abolished activation by C/ebpβ-Lap, identifying a potential cis element between −450 and −400 bp B. None oFANCC promoter activity during differentiation with IL1β  (C). We also found that knockdown of either Stat3 or C/ebpβ significantly decreased FANCC promoter activity , but only in IL1β-treated transfectants. This was consistent with a role for endogenous Stat3 and C/ebpβ in FANCC promoter activation during IL1β-induced differentiation.Stat3 and C/ebpβ are required for emergency granulopoiesis, and IL1β is the essential cytokine for this process. Therefore, we tested the effect of Stat3 and C/ebpβ on ith IL1β . We assa, n = 6) C. We alsFANCC promoter despite interacting with discrete binding sites. For these studies, we co-transfected U937 cells with the 1.0 FANCC promoter/firefly luciferase reporter vector and various combinations of vectors to overexpress Stat3, C/ebpβ-Lap, or Icsbp. Cells were co-transfected with the internal control reporter vector described above. We found that combination of any two of these transcription factors was additive for FANCC promoter activity , and the effect of all three was greater than any two  (C). The total amount of expression vector was maintained at a constant level in these studies . Therefore, these studies indicated cooperation, rather than redundancy, between the three transcription factors.We investigated possible additive or cooperative effects of these transcription factors on the , n = 6) C. The toFANCC promoter (separated by 2 bp). These sites were conserved in mouse and human but slightly divergent from the derived consensus for Stat3 binding N4–5GAA-3′; proximal FANCC site, 5′-TGATTTGAA-3′; distal site, 5′-AGCGTTGAA-3′) (FANCC promoter in the pGL3-promoter vector). Additional constructs were generated with mutation of the proximal, distal, or both putative Stat3 binding sites. Reporter activity was assayed in U937 cells co-transfected with vectors to express Stat3, Stat5, or an empty control vector .To confirm that Stat3 and C/ebpβ activated cis elements within the promoter regions defined above, we performed a sequence analysis (using Vista Tools for Comparative Software) , 21. We TGAA-3′) , 24. We FANCC/minimal promoter construct  (A). Mutation of either consensus significantly decreased this activity , but activation by Stat3 was completely abolished by mutation of both  (A). Stat5 had no effect on the −438 to −474 bp construct, and none of these proteins influenced the empty minimal promoter/luciferase reporter vector (A).We found that Stat3 significantly increased the activity of the −438 to −474 bp , n = 6) A. Mutaticontrol) A. Stat5 r vector A.FANCC promoter  . C/ebpβ-Lap increased the activity of the −385 to −408 bp FANCC sequence significantly  (B). C/ebpβ-Lip did not activate this FANCC sequence, and neither protein activated the minimal promoter/reporter control vector.We identified a potential C/ebp binding site between −392 and −403 in the AATC-3′) . We gene, n = 6) B. C/ebpβFANCC promoter regions by chromatin co-immunoprecipitation. For these studies, lysates of Lin−CD34+ murine bone marrow cells were immunoprecipitated with antibodies to Stat3, C/ebpβ, or an irrelevant control antibody (C) or quantitative real-time PCR (using SYBR Green and the standard curve method) (D).We investigated binding of Stat3 and C/ebpβ to these antibody . Some ceantibody C or quan method) D.C). Co-precipitation of both proteins was significantly increased by IL1β. Neither protein co-precipitated the irrelevant 5′ flank sequence (data not shown).We found specific co-precipitation of the −380 to −410 bp sequence with C/ebpβ and of the −430 to −470 bp sequence by Stat3 C. Co-pre−CD34+ cells were isolated, and some cells were differentiated with IL1β or G-CSF. The amount of G-CSF employed was consistent with serum levels in emergency granulopoiesis  (A). We also found a significant cytokine-induced increase in Stat3 and C/ebpβ mRNA in these cells  (A). The relative increase in expression of Stat3 and C/ebpβ in response to either IL1β or G-CSF was not significantly different .We found significantly increased FancC mRNA in response to treatment with either cytokine  A. We als, n = 4) A. The reB).We next investigated the impact on Stat3 protein. For these studies, cells were analyzed with or without IL1β or G-CSF treatment for total Stat3, Tyr(P)-705–Stat3, or Ser(P)-727–Stat3 by Western blot. We found that either cytokine increased total and phospho-Stat3 protein B.p < 0.001, n = 3) (C). Although phosphotyrosine or phosphoserine Stat3 increased, the relative increase was significantly less than in total Stat3 protein (4-fold increase versus ∼50% increase) (C).To quantify these results, we performed an ELISA for total Stat3, Tyr(P)-705–Stat3, or Ser(P)-727–Stat3. We found that IL1β also significantly increased total Stat3 protein in this assay  C. Althouncrease) C.D). In this experiment, only the C/ebpβ-Lap form was detected (35 kDa), not the smaller Lip form (∼20 kDa).We similarly investigated the impact of emergency granulopoiesis on C/ebpβ protein (by Western blot). We found that IL1β increased C/ebpβ expression, consistent with our mRNA studies D. In thiFANCC cis element. For these experiments, we co-transfected U937 cells with a vector to express Y705F-Stat3 or S727A-Stat3 and the −482 to −517 bp FANCC minimal promoter/reporter vector . Cells were also co-transfected with the internal control reporter vector as described above. Phosphorylation of Tyr-705 enhances transcriptional activation of some target genes by Stat3 .In initial studies, we performed Western blots to verify overexpression of these forms of Stat3 in U937 cells. We found that WT, Y705F, or S727A Stat3 were equivalently expressed under the assay conditions A.FANCC cis element was equivalently activated by tyrosine mutant, serine mutant, or WT Stat3  (B). We found that IL1β increased the activity of the FANCC cis element with or without Stat3 overexpression, consistent with the effect of increased endogenous Stat3 in IL1β-treated cells.We also found that this , n = 6) B. We fouFANCC cis element activity in assays using an inhibitor of Stat3 serine phosphorylation (H7), tyrosine phosphorylation (Stattic), or both. We found that neither inhibitor altered the effect of overexpressed Stat3 on the FANCC cis element, individually or in combination  (B). We verified the effect of these inhibitors on Stat3 in control ELISA experiments (C). None of these forms of overexpressed Stat3, nor treatment with any of the inhibitors, influenced the activity of the control minimal promoter-reporter vector (data not shown).We further investigated the impact of Stat3 phosphorylation on arisons) B. We vereriments C. None o−CD34+ cells were transduced with retroviral vectors to express shRNAs specific to Stat3, C/ebpβ, or both. We tested scrambled shRNAs for each of these proteins as controls, and none influenced the target protein or FancC mRNA, so they were combined for these experiments. Some cells were treated with IL1β, and FancC mRNA was quantified, with quantification of Stat3 and C/ebpβ expression as controls.We also investigated the impact of endogenous Stat3 or C/ebpβ on FancC expression in differentiating murine bone marrow cells. For these studies, Linp < 0.001, n = 3) (A). The combined effect of knocking down both Stat3 and C/ebpβ was greater than either alone  (A). Because the total amount of plasmid was kept constant, the combined amount of shRNA in samples with knockdown of both Stat3 and C/ebpβ was half as much as in experiments with either alone.We found that knockdown of either protein significantly impaired FancC expression in differentiating cells  A. The co, n = 3) A. BecausFANCC transcription at different times during emergency granulopoiesis. Specifically, Stat3 and C/ebpβ are involved in initiating and maintaining emergency granulopoiesis, whereas Icsbp terminates this process (in part by decreasing Stat3 and C/ebpβ expression)  –15. We in = 6 mice per group)  , 15, 18.r group) , 15, 18.p < 0.0001, n = 3 relative to the steady state) and began to decrease at 3 weeks  (B). In contrast, Icsbp mRNA was maximally expressed 3 weeks after Alum injection  and was returning to steady-state levels at 4 weeks (B). FancC mRNA expression was significantly increased 1 week after Alum injection, maximal at 2 weeks , and returning to baseline at 4 weeks (B).We found significantly increased expression of Stat3 and C/ebpβ that was maximal 2 weeks after Alum injection (3 weeks) B. In con 4 weeks B. FancC  4 weeks B.We were interested in determining the impact of Stat3 or C/ebpβ on DNA repair in cells exposed to the stress of emergency granulopoiesis. To investigate this, we used a plasmid-based DNA repair assay we employed previously to study the influence of Icsbp on this process. In this study, a reporter plasmid is treated with mitomycin C to generate DNA cross-links (with an untreated plasmid as a control) , 17. DamRenilla luciferase vector as a control for transfection efficiency). Transfectants were assayed after 24 h of treatment with IL1β.In prior studies, we determined that U937 cells efficiently repaired the MMC-treated plasmid, but treatment of U937 cells with differentiating agents (retinoic acid/dimethyl formamide or Ifnγ) significantly impaired this activity . For thip < 0.001, n = 6) (A). Overexpression of Icsbp, Stat3, or C/ebpβ significantly increased the reporter activity of the MMC-treated plasmid in IL1β transfectants  but had no effect on the activity of reporter vectors that had not been MMC-treated (A). Assays with combinations of the three proteins demonstrated that effects were non-redundant because the total amount of expression plasmid was held constant in these experiments. In the absence of IL1β treatment of the transfectants, we found that the luciferase reporter activity from the MMC-treated plasmid was not significantly different than the activity of the untreated plasmid (data not shown), consistent with our prior studies  A. Overex-treated A. Assays studies , 17.We were interested in determining whether the effects of Icsbp, Stat3, or C/ebpβ on the activity of the MMC-damaged reporter plasmid required FancC expression. To examine this, we co-transfected U937 cells with MMC-treated or an untreated reporter plasmid; vectors to overexpress Icsbp, Stat3, or C/ebpβ; vectors to express shRNAs specific to FancC (or scrambled shRNA control); and a plasmid to control for transfection activity (as above). Transfectants were assayed after 24-h treatment with IL1β.A). These results suggested that these transcription factors were acting through a FancC-dependent mechanism to drive DNA cross-link repair.We found that knockdown of FancC prevented Icsbp, Stat3, or C/ebpβ overexpression from rescuing the reporter activity of the MMC-treated plasmid A. These Emergency granulopoiesis is a high-risk/high-gain response to infectious challenge. During this process, rapid granulocyte production requires increased proliferation and faster differentiation of bone marrow stem and progenitor cells, resulting in genotoxic stress. This risk is further enhanced by the apoptosis resistance and cell cycle shortening that occur during this process , 15. In FANCC promoter and that IL1β increases FANCC promoter activity and enhances the effects of these transcription factors. Also, our study demonstrates that IL1β increases binding of Stat3 and C/ebpβ to their respective FANCC cis elements, perhaps because of the ability of IL1β to increase the expression of Stat3 and C/ebpβ mRNA and protein. We found that IL1β treatment decreases the ability of U937 myeloid cells to repair DNA cross-links and that DNA cross-link repair is rescued by Stat3 or C/ebpβ in a FancC-dependent manner, similar to our prior results with Icsbp and FancC   gene, a calpain inhibitor  antagonized the effects of Lap (liver activating protein) in these cells  , 33. TheFANCC promoter activity and FancC expression in the presence of all three transcription factors compared with Stat3 and C/ebpβ without Icsbp. It would be of interest to determine whether repeated episodes of emergency granulopoiesis enhance drug resistance or progression to blast crisis in CML. Studies are currently being performed in the laboratory to address this issue.It is additionally possible that episodes of emergency granulopoiesis facilitate leukemogenesis under conditions with decreased Icsbp or enhanced expression of Stat3 or C/ebpβ. All three proteins enhance FancC expression, which would be anticipated to protect cells from DNA damage. However, we found significantly more The Icsbp/Irf8 cDNA was obtained from Dr. Ben Zion-Levi  and subcloned into the mammalian expression vector pcDNA , as described previously . WildtypFANCC 5′ flank (1.0 kb from the ATG codon) was generated by PCR from the U937 myeloid cell line. The genomic clone was sequenced to ensure identity with the sequence in the ENSEMBL database  . Some of the −387 to −403 bp FANCC promoter/minimal promoter/reporter constructs had mutation of the proximal, distal, or both Stat consensus binding sequences.The human The human myelomonocytic leukemia cell line U937  was obtaFANCC promoter/luciferase reporter constructs (or empty control reporter vector) and various combinations of vectors to overexpress Stat3, C/ebpβ, or Icsbp (or empty expression vector) or specific shRNAs to knock down Stat3 or C/ebpβ (or scrambled shRNA control vectors). Cells were also co-transfected with an internal control plasmid to normalize for transfection efficiency . Transfectants were assayed for luciferase activity according to the instructions of the manufacturer. Some transfectants were treated with IL1β (50 ng/ml for 24 h) prior to harvesting.U937 cells were transfected with Luciferase activity from control empty reporter vectors was not influenced by IL1β treatment or overexpression or knockdown of any of these proteins and was subtracted as background. All reporter assays were repeated six times in independent experiments (and samples were assayed in duplicate) for each condition.FANCC promoter activity in transfection assays or expression of endogenous mRNA for FancC and gp91phox in cells treated with the cytokine. Some transfectants were studied after treatment for 24 h with H7 (a serine kinase inhibitor for Stat3 (The efficacy of IL1β differentiation of U937 cells was verified for various batches of cytokines used in these studies by determining enhanced or Stat3 ) or Stator Stat3 ).m) for 12 h at room temperature. Plasmid DNA was recovered by phenol:chloroform extraction followed by ethanol precipitation. DNA cross-linking was verified by non-denaturing agarose gel electrophoresis  was incubated with mitomycin C , and vectors expressing specific shRNAs to knock down FancC (or scrambled control). Cells were also transfected with the TK-For Western blots, cells were lysed by boiling in 2× SDS sample buffer. Lysate proteins (50 μg) were separated by SDS-PAGE (10% acrylamide) and transferred to nitrocellulose, and filters were serially probed with antibodies as described previously . Each exA450 (according to the instructions of the manufacturer).In other experiments, total, serine-phosphorylated, or tyrosine-phosphorylated Stat3 proteins in cell lysates were quantified by commercially available ELISA . ELISAs were performed in duplicate on three independent sets of lysates, and the results were graphed as FANCC promoter. The standard curve was generated using total chromatin from murine bone marrow cells. Input chromatin (not precipitated) from each sample was analyzed to normalize data between the samples. At least three independent immunoprecipitation experiments were performed, and the samples were analyzed in triplicate.Cells were incubated briefly in medium supplemented with formaldehyde, and lysates were sonicated to generate chromatin fragments with an average size of 500 bp and immunoprecipitated with Stat3, C/ebpβ, or irrelevant control antibody (Abcam) . ChromatRNA was isolated using TRIzol reagent  and tested for integrity by denaturing gel electrophoresis. Primers were designed with Applied Biosystems software, and real-time PCR was performed using SYBR Green and the standard curve method. The standard curve for these experiments was generated with cDNA from WT cells cultured in GM-CSF, IL3, and stem cell factor. The results were normalized to 18S and actin and presented as mRNA abundance with 1 ng of cDNA set as 1000 in the standard curve. At least three independent samples were evaluated in triplicate.The mice used for this study were C57 Black 6 and maintained in an approved and accredited animal facility at Northwestern University. The mice were housed in a specific pathogen-free, tightly regulated environment that included control of the flow of animals, equipment, and personnel and use of micro-isolator cages and husbandry procedures to minimize pathogen exposure and disease outbreak. All work was reviewed and approved by the Animal Care and Use Committees of Jesse Brown Veterans Affairs Medical Center and Northwestern University.in vitro studies, bone marrow mononuclear cells were harvested by flushing femora repeatedly with Hanks' balanced salt solution until no additional cells were obtained. Washed cells were treated with ammonium–chloride–potassium buffer to lyse red blood cells and then washed extensively. Lin−CD34+ cells were separated using a magnetic bead–based, affinity chromatography–based technique according to the instructions of the manufacturer . Cells were cultured (2 × 105/ml) for 48 h in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 1% penicillin-streptomycin, 10 ng/ml murine GM-CSF , 10 ng/ml murine recombinant IL3 (R&D Systems Inc.), and 100 ng/ml of stem cell factor (R&D Systems Inc.). Cells were maintained in GM-CSF, IL3, and stem cell factor for 24 h or stimulated with 50 ng/ml G-CSF (R&D Systems Inc.) or 20 ng/ml IL1β (R&D Systems Inc.) during this time period. Apoptotic cells were removed before analysis according to the instructions of the manufacturer . Some cells were transduced with retroviral vectors prior to analysis according to techniques described in our prior work (For ior work .WT mice (18–20 weeks of age) were injected i.p. with Alum or saline control (12 mice per group). Mice were randomly assigned to cohorts for injection of Alum or saline control. Alum was prepared as described previously , 15, 18,versus saline injected.Cohorts of mice (6 per group) were sacrificed weekly, and bone marrow was collected from both femora. Successful induction of emergency granulopoiesis in Alum-injected mice  was verified by weekly peripheral blood granulocytes counts . Blood count data were analyzed by an investigator who was blinded to the status of the mice as Alum t test (comparing two conditions) or analysis of variance (for more than two conditions) using SigmaPlot software. p < 0.02 was considered statistically significant. In all graphs, error bars represent ± S.E.Statistical significance was determined by unpaired two-tailed Student's C. A. S., L. Broglie, L. H., L. Bei, W. H., and D. B. D. investigation; C. A. S., L. H., W. H., and E. A. E. methodology; W. H. and E. A. E. conceptualization; E. A. E. formal analysis; E. A. E. supervision; E. A. E. funding acquisition; E. A. E. project administration.


RAD sequencing, we investigated the genomewide effects of urbanization on neutral and adaptive genomic diversity in 140 adult great tits Parus major collected in locations with contrasted urbanization levels . Heterozygosity was slightly lower in the more urbanized sites compared to the more rural ones. Low but significant effect of urbanization on genetic differentiation was found, at the site level but not at the nest level, indicative of the geographic scale of urbanization impact and of the potential for local adaptation despite gene flow. Gene–environment association tests identified numerous SNPs with small association scores to urbanization, distributed across the genome, from which a subset of 97 SNPs explained up to 81% of the variance in urbanization, overall suggesting a polygenic response to selection in the urban environment. These findings open stimulating perspectives for broader applications of high‐resolution genomic tools on other cities and larger sample sizes to investigate the consistency of the effects of urbanization on the spatial distribution of genetic diversity and the polygenic nature of gene–urbanization association.Urbanization is a growing concern challenging the evolutionary potential of wild populations by reducing genetic diversity and imposing new selection regimes affecting many key fitness traits. However, genomic footprints of urbanization have received little attention so far. Using  Great tits are widespread and abundant across Eurasia and can be found in a wide variety of environments, from natural forests to highly urbanized cities. Previous studies based on microsatellite loci showed relatively small genetic differentiation across Europe, yet higher differentiation among southern populations  and in a rural site: the deciduous forest of La Rouvière  where great tits have been monitored since 1991 .From April to May 2012, vegetation cover , and light , air and noise (using car traffic to reflect as a proxy) pollutions were recorded within a 50‐m‐radius disk around each nest box . DNA quality was checked on agarose gels. DNA extracts  were sent to MGX  for libraries preparation using restriction site‐associated DNA sequencing , the SNP model, and α = .05. cstacks was used to build the catalogue of loci using n = 3. With sstacks, loci were searched against the catalog of loci. Finally, individuals were genotyped using the stacks's populations module and filtered using VCFtools  in ROU. We did not filter specifically the other populations for HWE due to smaller sample sizes, and we considered that most of the significant deviations in every site would also be found in ROU given the high gene flow among the populations. This HWE filtering step was mainly applied to remove sequencing or SNP calling errors as well as paralogous sequences  estimate consistency of SNP calling on the two replicated individuals, and (ii) identify highly related individuals. One individual was removed for each pair of highly related individuals  to produce a dataset called “no‐family‐ties.” The rationale for this procedure is that related individuals could artificially increase fst between groups of individuals  on the via identity‐by‐state (IBS), fixation index (fst), a principal component analysis (PCA), and a discriminant principal component analysis (DAPC). We calculated IBS among individuals of the entire dataset to determine whether relatedness among individuals differed between sites and to test whether we could infer dispersal events . Differences in IBS between sites were assessed in R using an ANOVA  using the function snpgdsPCA implemented in the R package SNPRelate to depict genetic structure among the sites, using both the entire dataset and the no‐family‐ties dataset. Finally, we used a discriminant analysis of principal components (DAPC) implemented in the R package adegenet to depict genetic structure among the sites, using the no‐family‐ties dataset. We used the cross‐validation procedure from the adegenet package using the function xvalDapc, to identify the optimal number of principal components, reassigning 30% of the individuals, with a training set based on 70% of the individuals and replicating 100 times the procedure for each set of PCs from 10 to 45 by steps of 5, and then from 5 to 15 by steps of 1.Population structure was investigated n et al.  for moreJombart,  on the n2.6First, we estimated the correlations between urbanization and observed heterozygosity, at the site level and at the nest level.fst (estimated on the no‐family‐ties dataset) and geographic distance, (ii) fst and urbanization difference, (iii) IBS (estimated on entire dataset) and geographic distance, and (iv) IBS and urbanization difference. The correlations of either fst or IBS with geographic distance are used to infer potential isolation by distance, whereby dispersal and accumulation of genetic differentiation increase with geographic distance between populations. The correlations of either fst or IBS with urbanization difference are used to infer potential habitat choice, whereby dispersal and accumulation of genetic differentiation increase with habitat differences. fst is more likely to capture long‐term accumulation of allele frequency differences while IBS captures the present time distribution of relatedness and therefore the dispersal events.Second, to infer the effect of geographic distance and of urbanization difference on dispersal and genetic differentiation, we inferred correlations between (i) Vegan R package , we used a redundancy analysis (RDA), which is a constrained version of PCA . This was motivated by the fact that birds in the Zoo may experience similar selection pressures as ROU birds , while being in the perimeter of the city. Therefore, gene flow among urbanized sites from the city and the zoo could limit the response to locally forestlike environment in the zoo and limit the power of outlier tests. Moreover, this site also represented an opportunity to avoid circularity, first identifying outlier loci excluding ZOO and subsequently inspecting by mean of PCAs (detailed next paragraph) whether ZOO individuals were closer to urban or forest birds. ZOO individuals closer to urban birds would suggest restricted effect of selection relatively to gene flow, at small spatial scale, or absence of habitat choice influenced by outlier loci. In contrast, ZOO individuals closer to forest birds would suggest relatively important response to selection at small spatial scale, or of habitat choice influenced by outlier loci.p‐values, assuming a unique genetic cluster. We displayed the Z‐scores with Manhattan plots. We estimated q‐values from LFMM's p‐values, for each of the six tests separately, using the R package fdrtool  biplots between tests, and (iv) Venn diagrams  per LFMM test were used to obtain average z‐scores, representing the strength of the association between a SNP and an explanatory variable, and associated snpgdsPCA implemented in the R package SNPRelate, followed by linear models in R, to investigate the proportion of variance in individuals’ scores of urbanization at the nest and at the site explained by the genetic distances between individuals when considering several outlier lists of SNPs with q‐values <.05, as well as the entire set of SNPs. This procedure was achieved to inspect the power of gene–urbanization associations. Either the PC1 or PC2 or both were considered to explain individual nest‐ or site‐level urbanization, using a linear model in R. The PCA, realized with the list obtained from tests D, E, and F, was furthermore used to visually inspect the genetic distance of ZOO with other sites.We performed PCAs, using the function To assess to which extent genomic associations with urbanizations were polygenic, we inferred, using linear models, the proportion of variance in nest‐level urbanization that was explained by PCAs using an increasing number of SNPs, from 1 to 49,969, sorted by decreasing Z‐score in test A. Although this is a circular examination, it is primarily an attempt to investigate the cumulative nature of the top outlier SNPs from gene–urbanization association tests. We considered only PC1 as this axis captured most of the variation in urbanization while analyzing outliers from test A (as it will be explained in the result section).Parus major Annotation Release 100). We reported the genes found in the aforementioned lists. Then, gene ontology (GO) enrichment tests, which aim to identify potentially enriched GO terms, were performed in GOrilla . We tested for GO enrichment among genes founds for several lists of outliers . Enrichment tests were corrected with the FDR method.Genes were extracted from the great tit reference annotation . Tukey's HSD test revealed that all pairs of sites had significantly different means in nest‐level urbanization scores (p < .05), except FCO and MOS (p = .99) and ZOO and ROU (p = .52).Average urbanization level per site ranged from −2.19 in ZOO to 2.32 in MNO .Sequencing RAD tags for 140 breeding great tits resulted in a total of 1,815,823,241 raw sequences, with an average of 7,793,233 reads per individual. 107,413 loci were identified in the stacks catalog. After filtering for depth coverage, HWE, and MAF, 49,969 SNPs in 32,756 loci were kept in the final dataset, roughly one locus every 37 Kb. The missing rate per individual ranged from 0.001 to 0.058, with a median of 0.024. The missing rate per locus ranged from 0.000 to 0.100, with a median of 0.014. The average read depth across genotypes  was 72 Appendix , and the3.3F = 11.6, p‐value = .000866, Table There was a general pattern of reduced diversity in individuals from the four most urbanized city sites, in particular MNO, compared to ZOO and to ROU. The observed genomewide observed heterozygosity (Ho) was different among sites  or the no‐family‐ties‐dataset .Pairwise IBS between individuals ranged from 0.73 to 0.89 and was in average of 0.75 Figure a,b. Averfst ranged from 0.004 to 0.009, for an average of 0.007 .At the site level, observed heterozygosity was significantly correlated with urbanization (Genetic differentiation between sites tended (no significant correlation) to increase with geographic distance and with urbanization difference Figure a,b. All,p < .001, Table p < .001) was significant, while nest‐level urbanization and latitude had nonsignificant effects . Using the partial RDA model to test for a nest‐level urbanization effect, after subtracting the effect of longitude and latitude, the proportion of variance explained was not significant (p = .21). Urbanization at the nest explained 0.75% of the total genotypic variance (p = .20).When genetic variance among genotypes was constrained by the full RDA model for latitude, longitude, and nest‐level urbanization, the proportion of variance explained was significant globally . After subtracting the effect of longitude and latitude, the proportion of variance explained using the partial RDA model was still significant (p = .02). Urbanization average per site explained 0.78% of the total genotypic variance (p = .01).When genetic variance among genotypes was constrained by the full RDA model for latitude, longitude, and average urbanization at the site rather than at the nest, the proportion of variance explained was significant globally . In turn, the correlations were relatively small between z‐scores values obtained for the quantitative tests  and for the binary tests  compared to the background list of the 5276 genes  the potential effect of urbanization on genetic diversity and differentiation in great tits, and (ii) the potential existence of genomic footprints of divergent selection driven by urbanization. Observed heterozygosity was only slightly, yet significantly, lower in the most urbanized sites compared to the least urbanized ones. Furthermore, a small but significant proportion of genetic variance was explained by urbanization. These results either suggest that gene flow was only slightly limited along this urbanization gradient or alternatively that relatively large effective population sizes and relatively recent urbanization slow down the rise of genetic differentiation. Although urbanization explained a small proportion of genetic variance, such deviation from panmixia may be sufficient to allow for the rise of local adaptation  populations compared to rural ones  found in this study matches the fst of 0.01 observed earlier, using a SNP chip, between two much more geographically distant populations, respectively, from the Netherlands and the United Kingdom . The absence of strong gene–urbanization associations may be due to relatively large gene flow, suggested by low In turn, our results of numerous small gene–urbanization associations explaining a large proportion of variance in urbanization score (81% explained by a subset of 97 SNPs) may be in line with the hypothesis that selection regimes resulting from urbanization diverge from natural environments in many aspects and act on several potentially complex traits. A formal comparison of the force, shape, and direction of natural selection across the urbanization gradient would be pivotal in understanding how divergent selection really acts. The theory of quantitative genetics and the recent advances in sequencing and quantitative genomics show that variation in adaptive traits often has polygenic origins  great tits usually explore and forage in a relatively vast area around the nest  individual genotypes in a VCF file, (ii) a text file with site, geographic coordinates, and urbanization scores at the nest and site, for each individual.Data available from the Dryad Digital Repository: AG, MS, AC, and CP designed the study; VD, AG, AC, and CP contributed to fieldwork; CP did the bioinformatics; CP and ALDC performed data analysis; CP wrote the paper; all authors contributed to improve the manuscript. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Bromus tectorum L. is an invasive winter annual grass naturalized across the United States. Numerous studies have investigated B. tectorum population structure and genetics in the context of B. tectorum as an ecological invader of natural areas and rangeland. Despite the wealth of information regarding B. tectorum, previous studies have not focused on, or made comparisons to, B. tectorum as it persists in individual agroecosystems. The objectives of this study were to assess the genetic diversity and structure, the occurrence of generalist and specialist genotypes, and the influence of climate on distribution of B. tectorum sourced exclusively from within small grain production regions of the Pacific Northwest. Genetic diversity of B. tectorum sourced from agronomic fields was found to be similar to what has been observed from other land use histories. Six distinct genetic clusters of B. tectorum were identified, with no evidence to indicate that any of the genetic clusters were better adapted to a particular geographical area or climate within the region. Given the apparent random spatial distribution of B. tectorum genetic clusters at the spatial scale of this analysis, unique genotypes may be well mixed within region, similar to what was reported for other inbreeding weedy grass species. Despite the wealth of information regarding the genetic diversity of downy brome, previous studies have not focused on, or made comparisons of downy brome genetic structure in agronomic fields. The lack of downy brome genetic studies within agroecosystems is significant given that downy brome is a widely distributed and a serious pest in small grains and other crops across North America.Downy brome  to assess the genetic variability of downy brome sourced exclusively from within small grain production fields, (2) determine the frequency and occurrence of generalists versus specialist genotypes, and (3) determine the influence of climate on the distribution of genotypes.22.1Downy brome is distributed ubiquitously within agronomic fields of the PNW. No large landscape features, such as mountain ranges, are present that could block gene flow. Climate of the region exists across a longitudinal gradient with annual precipitation in the region ranging from <300 mm to >600 mm, with precipitation increasing from the west to east. Mean annual temperature also varies on an west to east gradient, with the western portion at 11°C and decreasing to 5°C to the east based on a 30‐year average  , was included as a control to determine whether population structure analysis could detect the related species as an outlier. DNA was extracted using a BioSprint 96 Plant Kit and BioSprint 96 workstation . DNA was quantified with the PicoGreen® assay  using a Synergy” HT  microplate reader.In June of 2010 and 2011, a single downy brome plant was collected as either mature panicles or a live plant from each of 130 re‐assigned sampling locations. Each plant was collected at least 10 m from the field border. Live plants were transplanted into a greenhouse and allowed to grow until mature panicles could be collected. Caryopses from collected panicles were later germinated to provide tissue for DNA extraction. On 21 March 2014, as plants were at the two‐ to three‐leaf stage, a single ~4‐cm leaf was collected from 95 (Table 2.2A reduced representation genotype‐by‐sequencing (GBS) approach was employed to identify SNP molecular markers  , based on genetic similarity and upon Bayesian information criterion (BIC), and to initially assign individuals to each cluster. In the second step, synthetic variables called linear discriminants, consisting of linear combinations of alleles, are used to discriminate the cluster membership of each individual. SNPs which are retained by the DAPC, due to their value in discriminating cluster membership of individual accessions, can be considered “more informative SNPs” and will be referred to as such throughout the manuscript.The output from stacks was analyzed in R (R Development Core TeamFST) between each genetic cluster was calculated  were calculated across clusters using the R Package mmod , total diversity (HT), and the ratio of genetic diversity partitioned among clusters (GST and G’ST) using the R Package “mmod”  at individual loci averaged across all accessions ranged from 0.0 to 0.65 with a mean value of 0.006. Across all accessions, expected heterozygosity at each loci (HE) ranged from 0.021 to 0.667 with a mean value of 0.2. Within‐cluster expected heterozygosity ranged from 0.069 to 0.144 with an average of 0.122 (HE)  are likely to be found.Compared to all other genetic clusters, cluster 3 appears to have a higher degree of genetic diversity when described by genetic distance Figure . Both ofFST values and genetic distance. The genetic distinction between other clusters is often slight, but genetic clusters can be separated based upon SNP distribution. Efforts were made to evaluate cluster membership with a different number of retained PCs or with arbitrarily selected k‐values, and those efforts failed to identify ripgut brome as an outlier. The results returned by DAPC may accurately reflect the state of downy brome population structure within the small grain production region of the PNW: an assemblage of inbred individuals with little evidence of outcrossing and varying degrees of shared genetic history, and without strong evidence of adaptation to differing environmental influences.Cluster 6, the ripgut brome outlier, is distinct from all other clusters across all linear discriminants used, and when comparing pairwise While genetic markers linked to neutral gene regions, and SNPs in particular, are well suited to neutral evolutionary process such as genetic drift and gene flow  between farms located in the United Kingdom. Barren brome exists as an assemblage of unique but inbred biotypes within agronomic fields. Similar to what was found from sampling B. tectorum within PNW small grain fields, considerable spatial mixing of genotypes was found across all sampled farm fields  Beauv  P. Beauv. S. geniculata P. Beauv. and S. faberi Herrm. Within the introduced range of the United States, S. geniculata and S. glauca both exhibited lower genetic diversity than what was found within their native range and regional patterns of genetic partitioning, while S. faberi was nearly genetically identical worldwide based upon the isozyme markers used. In summary Wang et al. (Setaria species in the context of a review of genetic diversity of 499 plant species conducted by Godt and Hamrick (Setaria and Bromus species are typical of self‐pollinating, invasive grass species (Green et al., Wang et al.  expandedg et al. ,b descri Hamrick . While sBromus and Setaria species within invaded and agricultural land, high levels of genetic diversity may not be essential for colonizing species. However, the use within this study of neutral markers may have masked genetic diversity conferring local adaption to novel environments (Narum et al., Given the apparently low genetic diversity and the similar genetic structure of Bromus tectorum clusters are highly differentiated and randomly dispersed, suggesting that generalists rather than specialist genotypes predominated across the region. The current structure of diversity within the PNW is likely the result of several independent introductions, constrained by natural selection (Novak et al., Bromus tectorum herbicide susceptibility, germination characteristics, and date of seed production (Ball et al., Within the small grain production region of the PNW, 5Bromus sterilis (Green et al., Analysis of population genetics and genetic structure from downy brome collected within an agronomic region indicates that the heterozygous state of downy brome is similar, if not marginally greater, to what has been reported in previous literature. Downy brome exists within the PNW small grain production region as a series of generalist genotype clusters with limited evidence of spatial adaptation, similar to what was reported Novak et al.  in a broTo expand further upon the current reported findings, future efforts should include more samples of individuals from the same field to increase the spatial resolution of genetic inferences. Additionally, collection of individuals from nearby rangeland and natural areas may allow for the control of climate and the comparison of land use among accessions. Finally, phenotyping of collected individuals in common garden studies across several years or locations would provide traits to be compared across individuals and elucidate the results of DAPC clustering by correlating the separation of genotypes with phenotyping.The authors declare that they have no conflict of interest.https://data.reacchpna.org/resources/reacch-data-library/.Bioinformatic data are archived through the research funding agency website: Nevin Lawrence contributed to study design, collection of plant materials, data analysis, and writing of the manuscript. Amber Hauvermale contributed to writing the manuscript. Amit Dhingra contributed to the data analysis and writing of this manuscript. Ian Burke contributed to the study design, collection of plant materials, data analysis, and manuscript composition and was the principle investigator.


Elodea nuttallii (Hydrocharitaceae), analyses of population structure and genotypic diversity need to be undertaken, for which genetic markers are needed.To enhance the understanding of the recent invasion process of the clonal waterweed DNA enriched for microsatellites was used to develop 24 loci that were characterized in E. nuttallii, 21 of which were polymorphic, with the number of alleles ranging from two to 10. In two populations, expected heterozygosity ranged among loci between zero and 0.796. In the congener E. canadensis, all markers yielded PCR products, 19 of which were polymorphic, with two to nine alleles and expected heterozygosity ranging from zero to 0.690 in two populations.High‐throughput sequencing of E. nuttallii as well as E. canadensis in their native and invasive range.The markers described should be useful for future studies of population structure and clonal diversity of  Elodea nuttallii (Planch.) H. St. John, Hydrocharitaceae) is a tetraploid, dioecious, submerged freshwater macrophyte native to North America that is invasive in Europe and Japan. In its invasive range, almost exclusively female plants have been found and the reproduction is primarily vegetative . Moreover, 60% of these markers only amplified in E. canadensis samples from invasive but not from native populations . Therefore, we developed new markers specifically for E. nuttallii. We demonstrate their utility for native and invasive samples as well as trans‐species amplification in E. canadensis, thus allowing for comparative analyses of clonal variation between native and invasive range and among species.Microsatellite markers are available for E. nuttallii collected in Großer Goitzschesee, Germany , was isolated using the cetyltrimethylammonium bromide (CTAB) extraction procedure of Doyle , medium (140–190 bp), and long (190–250 bp) amplification products. Furthermore, microsatellites with high repeat numbers were favored because they often display a higher mutation rate.These 41 primer pairs were ordered from Eurofins  and were screened using the method of Schuelke , i.e., aE. nuttallii and E. canadensis. For these, we report the number of genotypes (Ngt) and used GenoDive version 2.0b23  and expected heterozygosity (He) correcting for unknown allele dosage of polyploids. To assess allelic variation at the species level, we included additional samples from other native and invasive sites, totaling 262 ramets from 53 sites in E. nuttallii and 92 ramets from 30 sites in E. canadensis , totaling 127 alleles (Table He = 0.350 and 0.376), but genotypic diversity was either very low (mean Ngt = 1.04) or high  and 87 alleles in total  were found in two populations. In both species, the number of alleles detected per individual and locus ranged between one and four, as expected for a tetraploid species (Table Cross‐species amplification in E. nuttallii were also proved to be useful in E. canadensis. Allelic diversity was generally higher in invasive populations of E. nuttallii compared to invasive populations of E. canadensis. This suggests that different processes may drive the invasion of these two morphologically and ecologically highly similar species. The new markers will be useful for further analysis of clonal diversity and genetic structure of E. nuttallii and E. canadensis and, in particular, to investigate their European invasion history.The microsatellite markers developed for


Acacia koa. Admixture and principal components analyses showed that trees from different islands are genetically distinct in general, with the exception of some genotypes that match other islands, likely as the result of recent translocations. Gradient forest and generalized dissimilarity models both revealed a strong association between genetic structure and mean annual rainfall. Utilizing a model for projected future climate on the island of Hawaii, we show that predicted changes in rainfall patterns may result in genetic offset, such that trees no longer may be genetically matched to their environment. These findings indicate that knowledge of current and future rainfall gradients can provide valuable information for the conservation of existing populations and also help refine seed transfer guidelines for reforestation or replanting of koa throughout the state.Identifying and quantifying the importance of environmental variables in structuring population genetic variation can help inform management decisions for conservation, restoration, or reforestation purposes, in both current and future environmental conditions. Landscape genomics offers a powerful approach for understanding the environmental factors that currently associate with genetic variation, and given those associations, where populations may be most vulnerable under future environmental change. Here, we applied genotyping by sequencing to generate over 11,000 single nucleotide polymorphisms from 311 trees and then used nonlinear, multivariate environmental association methods to examine spatial genetic structure and its association with environmental variation in an ecologically and economically important tree species endemic to Hawaii,  For example, knowledge about the association between genotype and environment is important for selecting proper seeds for planting when tree populations are locally adapted . Koa is an endemic outcrossing leguminous hardwood tree that has been under threat due to land use changes, logging, and the introduced fungal pathogen Fusarium oxysporum f. sp. koae , in native Hawaiian forests. It is distributed on all the main Hawaiian Islands, except Niihau and Kahoolawe, and has the greatest densities on Hawaii, Maui, Oahu, and Kauai  in relation to spatial and environmental variables in two predictive nonlinear modeling frameworks: gradient forest and generalized dissimilarity modeling. Our study objectives were to (i) examine genomewide, biogeographic patterns of genetic differentiation in koa across the Hawaiian Islands, (ii) identify the most important geographic and environmental variables structuring genetic variation within the island of Hawaii, and (iii) assess whether koa populations might be vulnerable to potential future environmental changes by applying predicted future climate model data available for the island of Hawaii.To characterize patterns of genetic variation in 22.1A. koaia samples from the island of Hawaii, from November 2012 to September 2013 . For example, adapters were added during the ligation step rather than added to the empty plate and dried down prior to digestion; AMPure XP bead‐based size selection/purification steps were added after the ligation step and repeated after the PCR step to ensure a consistent distribution of fragment sizes between 200 and 500 bp among all preps; and we reduced the number PCR cycles to 16 from 18. Final libraries were checked for the proper size distribution on an Agilent BioAnalyzer  with the High Sensitivity DNA assay and quantified using a Qubit fluorometer . Samples were sent to the UCLA Broad Stem Cell Research Center for single‐end, 100‐bp sequencing on an Illumina HiSeq2000 v3 .Total genomic DNA was extracted from frozen tissue using the NucleoSpin kit . DNA was prepared for sequencing using an efficient restriction‐enzyme‐based approach commonly known as genotyping by sequencing (GBS) , recovered reads whose barcodes had up to one mismatch to the expected barcodes (‐r), removed any read with an uncalled base (‐c), discarded low‐quality reads as defined by default settings (‐q), and trimmed all reads to 87 bases (‐t). Parameters for subsequent steps were optimized based on three samples, one from each of three Hawaiian Islands, that were replicated across four library preparations and lanes of Illumina sequencing. We ran stacks 1.21 repeatedly on these replicates with a variety of parameter values by varying one at a time in each run. Specifically, we evaluated ‐m 3–5, ‐M 1–3, ‐n 1–3, ‐‐max_locus_stacks 3–5, and ‐‐bound_high 0.05 or 0.1  and ‐M 1 (maximum distance between stacks to combine them into a locus) and cstacks parameter ‐n 1 . In comparison with ‐m 4, ‐m 3 also produced similarly small differences among replicates  when in combination with ‐M 1 ‐n 1 but yielded nearly twice as many total SNPs; thus, we preferred –m 3 to –m 4. Little difference was observed among ‐‐bound_high and ‐‐max_locus_stacks alternatives; thus, values of 0.05 and 3 were chosen, respectively. Regardless of the parameter values selected, summary statistics of diversity, such as heterozygosity (0.22 < H < 0.30) and nucleotide diversity (0.0010 < π < 0.0017), did not vary substantially , proportion of “confounded” loci > 0.25 (‐‐conf_filter), and nonbiological haplotypes (‐‐prune_haplo). In each pass of the stacks pipeline through cstacks, the catalog (a reference set of sequences) was built using a geographically representative subset of 14 samples, as is recommended. Only one SNP per “stack” was retained, and SNPs with >30% missing data across all samples (n = 311), lnL < −30 (populations –r 0.7 –‐lnl_lim ‐30 –‐write_single_snp), or minor allele frequency <0.05 were discarded. Another set of SNPs was generated with the same filters considering only samples from the island of Hawaii .SNPs were identified using 0.1 Figs  and S2. 0.1 Figs . The ord0.1 Figs  and prin0.1 Figs . We concly Table . Quality2.4A. koa is autotetraploid because its closest relative, A. heterophylla, which is phylogenetically nested within A. koa, is a putative autotetraploid formed from another diploid Acacia species  to 0.05 as indicated above. Nonetheless, the resulting genotype calls are “coerced” to appear diploid. If koa is an allotetraploid (amphidiploid) formed from two species, then this coercion may be appropriate because divergent homeologous loci are likely to be separated into separate loci in the stacks pipeline under our optimization procedure. If koa is autotetraploid, then we expect allele frequencies to be biased toward intermediate values because allelic dosages of 0.25 and 0.75 will be coerced to 0.5. We expect this coercion to occur equally in both directions (from 0.75 to 0.5 and from 0.25 to 0.5); thus, the primary effect would be to bin all heterozygotes as 0.5. This bias toward intermediate allele frequencies in individuals would not bias the association of these allele frequencies with environmental variables on the landscape (see description of methods below), but the binning may reduce power to detect such associations. Therefore, we are confident that our pipeline is unlikely to lead to erroneous conclusions from downstream analyses.To explore the possibilities that koa is autotetraploid or that it is allotetraploid and our pipeline does not split homeologous loci, we generated histograms of the fraction of reads supporting each allele for each locus with at least 60 × coverage for each individual with at least 1,000 such loci. If either of these issues is present, we expect to find peaks at 0.25, 0.5, and 0.75, rather than just 0.5  and nucleotide diversity (π) were estimated for each island using stacks. Genetic structure was estimated with admixture 1.3.0 . The “optimal” number of clusters was chosen based on the K with the lowest cross‐validation error, as recommended by the developers. For the data set containing all samples, pairwise FST was estimated among clusters using admixture and compared to estimates among the four Hawaiian Islands as calculated in PLINK 1.90b3.29. These FST estimates are useful for relative comparisons and are unlikely to be biased substantially using diploid rather than tetraploid SNP calls, because the effect allele frequency binning averages out over the large number of SNPs. In addition, we performed principal components analyses using PLINK to provide a means for visualizing continuous changes in genetic structure complementary to the discrete clustering approach of admixture.Mean expected heterozygosity (2.6A. koa within the island of Hawaii (n = 201). These approaches are complementary, as GF is a regression tree approach and GDM is a distance‐based approach. All analyses were performed on an individual basis, rather than arbitrary “population” groupings, which are not straightforward with our sampling design and observed patterns of genetic structure and admixture  < .8 with each other. Mean minimum temperature and mean annual rainfall are highly correlated (r > .95) with other mean temperature and rainfall variables, respectively, that were not included  and generalized dissimilarity modeling (GDM), focusing on samples of ture see . In addied Table .http://gradientforest.r-forge.r-project.org/) in R 3.1.2 (R Development Core Team). GF is a nonparametric, machine‐learning regression tree approach , as has been suggested in similar contexts .Generalized dissimilarity modeling  following Fitzpatrick and Keller   Fig. , and mea.9) Fig. . SimilarHistograms of the fraction of reads supporting each allele for 40 individuals that have at least 1,000 loci with at least 60 × coverage demonstrate that heterozygotes are primarily called from situations in which support for each allele is approximately 50:50 (0.5) Fig. . In many3.2H < 0.33 and 0.0016 < π < 0.0018  may be the product of admixture from individuals from Maui and Kauai. Cluster membership is generally not significantly correlated with the amount of missing data nor coverage after accounting for multiple testing , except for cluster K4 which is weakly but significantly associated with missing data percentage . PCA reveals some notable differences among islands and shows similar patterns to those from admixture . Annual rainfall is by far the most important predictor, whereas there is almost no contribution of minimum temperature or rock substrate age, and all other variables contribute modestly Figure . RainfalMapped projections of GDM and GF results onto the landscape are generally concordant and show that eastern part of the island of Hawaii, which is the windward side where rainfall is high, is genetically different from the rest of island Figure . This ea44.1admixture results that show distinct clusters that are unique to each island  of Kauaian populations with a smaller fraction of Mauian populations  or due to natural selection and local adaptation, we expect that climate change will exert pressure on populations in areas where rainfall regimes will change most. These vulnerable areas might be candidates to consider moving genotypes  data.https://doi.org/10.5061/dryad.c014p.Illumina sequence read data are available through NCBI BioProject PRJNA392301, and final SNP data files are available through Dryad  Click here for additional data file. Click here for additional data file. Click here for additional data file.


Braque Français, type Pyrénées is a French hunting-dog breed whose origin is traced back to old pointing gun-dogs used to assist hunters in finding and retrieving game. This breed is popular in France, but seldom seen elsewhere. Despite the ancient background, the literature on its genetic characterization is surprisingly scarce. A recent study looked into the demography and inbreeding using pedigree records, but there is yet no report on the use of molecular markers in this breed. The aim of this work was to genotype a population of Braque Français, type Pyrénées dogs with the high-density SNP array to study the genomic diversity of the breed.The average observed (The online version of this article (10.1186/s13104-017-3112-9) contains supplementary material, which is available to authorized users. Genetic variability and structure in domestic animals largely depend on breeders’ decisions and practices. In selection, breeding within a closed population is common practice, since it allows to fix the desired characteristics and traits of the best representatives of the breed. However, this mating practice can lead to high rates of inbreeding and associated risks , which are a serious threat especially to small populations and to populations originating from a small number of ancestors , 2. FurtBraques Françaises are hunting dogs, originating from a very old type of gun-dog used for pointing the location of game birds to hunters. There are two breeds of Braque Français, both from the south of France: the Braque Français, type Gascogne (larger size) and the Braque Français, type Pyrénées . They are popular hunting dogs in France, but are seldom seen elsewhere. The original Braque Français type of pointing dog has existed since the eighteenth century. The first breed club was formed in 1850, and the standards for both breeds were written in 1880. The demography and inbreeding levels of the Braque Français, type Pyrénées breed were estimated using pedigree records [Blood samples were collected from 48 Braque Français, type Pyrénées unrelated individual dogs , to capture a representative sample of the within-breed genetic diversity. Genomic DNA (gDNA) was extracted from blood samples through a standard ethanol fractionation with concentrated sodium chloride (6 M NaCl) and sodium dodecyl sulphate (10% SDS). The concentration of DNA was adjusted to 50 ng/L is the n. of SNP loci; l; N is the number of individuals (sample size); p and q are the frequencies of, respectively, the A (major) and B (minor) alleles.Basic genetic diversity indices were estimated, including: (i) observed heterozygosity ; and (ihttp://zzz.bwh.harvard.edu/plink/summary.shtml#prune).The contemporary effective population size . To reds) was calculated as proposed by Lencz et al. [N and L are the sample size and number of SNP loci; Runs of homozygosity  were dez et al.  to minimThe inbreeding coefficient based on ROH , the final number of SNPs retained for the analysis was 94,065. All 48 dogs had high quality genotyping and were included in the analysis. The average observed . Table F, [Inbreeding coefficients estimated from SNP marker loci as reported . \documeient  of \docstimated .Fig. 2RuThe results presented in this note constitute the first report on the genomic characterization of the Braque Français, type Pyrénées dog breed using molecular markers. Previously, the demographics and diversity of this breed had been analysed resorting to pedigree data exclusively. The use of molecular data represents an important step towards a more accurate description of the genome of the Braque Français, type Pyrénées dogs. This improved genomic information will prove to be very useful for the management of the breed, both in the perspective of conservation and in that of breeding and selection. However, this is but a preliminary study, that aimed at providing essential initial information to be used subsequently for a larger and more comprehensive studies. Genomic data on a panel of relevant dog breeds are currently being acquired, to be used for a large-scale genome-wide characterization of dog breeds and the genetic distances and phylogenetic relationships between them. Additionally, phenotypes on the hunting ability and morphology of Braque Français, type Pyrénées dogs are being collected: together with the already available genotypic data, these phenotypes will be used in genome-wide association studies (GWAS) and similar approaches (e.g. see ) to dete


Chronic kidney disease is a leading cause of death in the United States. We used cluster analysis to explore patterns of chronic kidney disease in 500 of the largest US cities. After adjusting for socio-demographic characteristics, we found that unhealthy behaviors, prevention measures, and health outcomes related to chronic kidney disease differ between cities in Utah and those in the rest of the United States. Cluster analysis can be useful for identifying geographic regions that may have important policy implications for preventing chronic kidney disease. Chronic kidney disease (CKD) affects 15% of US adults , other uThe Centers for Disease Control and Prevention (CDC) 500 Cities Project provides city-level data for 500 of the largest US cities for year 2014  with theIn our analysis, we included the following prevalence measures related to CKD: 5 unhealthy behaviors ; 3 prevention measures ; and 3 outcome measures .To adjust for socio-demographic characteristics, we regressed each prevalence measure on the following confounding variables simultaneously using a beta regression : age  with Euclidean distance as the distance measure and included the adjusted unhealthy behaviors and the prevention and outcome prevalence measures. Hierarchical clustering, which groups states on the basis of their mutual similarities, is a top-down approach in which all states are initially included in the same cluster, and the algorithm splits the states based on differences down the hierarchy. The algorithm ends when each state is in its own cluster.To identify differences between states, we implemented hierarchical cluster analysis –10 usingWe identified clusters of 50 US states plus Washington, DC, based on the unhealthy behaviors, prevention measures, and CKD-related outcomes among adults living in cities. Among all states, Utah stood out because, compared with the national average among cities, cities in Utah had a higher adjusted prevalence of CKD but a lower adjusted prevalence of unhealthy behaviors. This information may be of interest to public health practitioners and policy makers in Utah, and suggests the need for further research to better understand which factors — beyond socio-demographic characteristics and well-known risk factors — could contribute to the prevalence of CKD. The cluster analysis also showed that several states in the South and Midwest that share geographical borders are clustered together, indicating cities in those states share similar characteristics.This study had several limitations. First, because cluster analysis is a type of exploratory analysis, we could not provide causal inference or prediction based on the results. Second, because data from the 500 Cities Project originate from the Behavioral Risk Factor Surveillance System (BRFSS), which is self-reported, there may be social desirability bias, in which respondents may answer the survey in a way they believe may be viewed positively by others.To our knowledge, this is one of the first articles to analyze data from the 500 Cities Project.


Engraulis encrasicolus), the existence of several populations has been proposed but a global view is missing. Using a multidisciplinary approach, here we assessed the divergence among different ecotypes and its possible causes. SNPs have revealed two functionally distinct ecotypes overlapping in the Central Mediterranean, with one ecotype confined near the river estuaries. The same SNPs outliers also segregated two distinct populations in the near Atlantic, despite their large spatial distance. In addition, while most studies suggested that adaptation to low salinity is key to divergence, here we show that the offshore ecotype has higher environmental tolerance and an opportunistic feeding behaviour, as assessed by the study of environmental conditions, anchovy diet and trophic levels, and passive egg dispersal. These results provide insights into the anchovy evolutionary history, stressing the importance of behaviour in shaping ecotypes.Anchovies represent the largest world’s marine fish catches and the current threats on their populations impose a sustainable exploitment based on sound scientific information. In the European anchovy ( Engraulis encrasicolus) represents one of the most important European fishery resources1. This species is ubiquitous from tropical to temperate areas of Atlantic Ocean, Mediterranean Sea, and Black Sea. Active dispersal of adult individuals plays an important role in species distribution. Moreover, the recruitment of European anchovy is in part influenced by pelagic transport of eggs and larvae from spawning to nursery areas and by diel vertical migration of larvae2.The European anchovy , named wide- (or marine) and narrow-shelf , whose differences reflect ancient dispersal and colonization events of the species9. The current distribution of the marine ecotype is likely attributable to founding events into the Mediterranean Sea, Bay of Biscay and northern European seas by a common ancestral population that fled to the Mediterranean during last glaciation10. In contrast, the current distribution of the coastal ecotype could derive from the colonization of the Atlantic front after the end of the last glaciation6 by an ancestral population probably sheltered in the West African coast10.European anchovy shows high levels of genetic structure attributed to habitat heterogeneity20 could represent specific types of the coastal ecotype. These coastal populations are likely in the process of becoming reproductively isolated from other anchovy populations coexisting together in these basins21as result of parallel genetic differentiation prior to secondary contact17 and to local adaptation20.The estuarine/lagunar populations that have been morphometrically and genetically identified in the Mediterranean Sea, Bay of Biscay and North SeaE. encrasicolus in the Mediterranean Sea, namely coastal and marine , by characterizing anchovy populations from the Italian seas  for a set of 96 gene-associated SNPs22. Additionally, we assessed the evolutionary divergence among the two ecotypes and its possible causes by applying a multi-faceted approach. We simulated current-driven dispersal of eggs and adults and related the presence of the two ecotypes to environmental factors, including zooplankton community, and trophic features. The genetic relationship between the two ecotype pairs in Mediterranean and in the Atlantic Sea was also analysed.In this study, we assessed the existence of two genetic/ecological ecotypes of loci, suggesting high complexity in the evolutionary origin of the two ecotypes. According to fine-scale multidisciplinary analysis, we conclude that the offshore ecotype has higher environmental tolerance and opportunistic feeding behaviour and we stress the importance of behaviour in shaping population and ecotype boundaries.We found that: (i) in the Italian seas, populations of offshore and coastal anchovy ecotypes coexist, occupying partially overlapping niches; (ii) the SNP markers identified as putative outliers in the Mediterranean are among the outlier markers identified in the Bay of Biscay, suggesting allopatric convergent adaptation of the two ecotypes in the two geographic areas,; (iii) a strong differentiation exists also when considering only putatively neutral The number of adult anchovies and eggs successfully genotyped in each sampling site, for the 96 high-resolution SNP panel utilized, is reported in Table et al.20), identified two genetic groups .A Bayesian analysis of population structure, performed after including representative samples from the Atlantic coastal  and offshore (wide-shelf: Bay of Biscay offshore area) ecotypes  values of differentiations  as the most distinct one along the first axis, which explains 21% of the total variance. Al the other samples cluster toward the negative side of the first axis. The two Adriatic samples from PES and CHI were slightly differentiated along the 2nd axis  clustered together with Tyrrhenian samples from river mouths (CVL and CAP) in the positive side of the first axis  . When the other egg samples were included in the PCoA with neutral xis Fig. . Using oxis Fig. .The genetic analyses indicated that, despite the large environmental differences, one of the coastal ecotypes (ecotype-2a) is largely similar between the Mediterranean Sea and the near Atlantic. To test the hypothesis that behaviour and not environmental factors are at the origin of the separation of this group, we carried out supplementary analysis at local scale using a multidisciplinary approach.First, we investigated the presence of mechanisms that may favour a physical separation between sampling sites by evaluating the local physical connectivity, i.e., how dispersal processes connect the different coastal regions of the Tyrrhenian Sea. Excluding the active dispersal of adult anchovies, we simulated egg dispersal only during the months of their occurrence and in the areas where adults were collected. We conducted a series of simulations of passively advected point-like particles released from sampling sites during May-August for three different years . The trajectories were then used to infer the most probable particle density in the sea 30 days  after the release from a given site. Results showed that particles released at CVL/CAP Fig.  and SPL/Second, we performed an analysis of the environmental variables, which showed a clear difference between the northern and southern sectors of the central/southern Tyrrhenian basin. The PCA, carried out separately for the two sectors Table , showed Third, to further investigate how the presence of river mouths can affect population fine-scale patterns, we analysed the oceanographic conditions in a small area north of the Gulf of Naples, including the Volturno river. Acoustic survey showed that the highest anchovy biomasses were recorded in Sperlonga, in the Gulf of Naples and in the Sele River plume Fig. . SatelliEuterpina, Farranula, Oithona, Acartia, and other calanoids and their nauplii, together with other small-sized prey like bivalve larvae. The stable isotope analysis of adult anchovies  revealed values of δ15N and δ13C from 6.61‰ to 11.91‰ and from −17.07‰ to −13.24‰, respectively. The δ15N values showed a clear geographical pattern with an increasing trend northward, although δ15N values in SPL were slightly lower than in CVL and NAP  between the areas GCM/PAO/CAP and NAP/CVL/SPL. Differences of δ13C were not significant, although higher values were detected in NAP samples .Fourth, we assessed that the diet of adult anchovies was almost exclusively based on zooplankton. Results showed that only 0.5% of prey number were represented by phytoplankton cells Table . The cluNAP Fig. . The δ15les Fig. . The remles Fig. , differeles Fig. , as a reEngraulis encrasicolus in the Mediterranean Sea, and we showed the existence of two genetic entities, i.e. offshore and coastal ecotypes, that coexist in most of the sampled sites and strongly resemble the two ecotypes observed on the Atlantic coasts. We therefore confirm and extend the results by Le Moan et al.9 about anchovies in the Central Mediterranean Sea using an higher number of individuals and on a wider geographic scale. Moreover, we found that the evolutionary divergence between the two ecotypes in the Mediterranean Sea is lower than in the near coastal Atlantic.We analysed the genetic diversity among populations of Along the central/southern Tyrrhenian coasts, the coastal anchovy ecotype has been found more abundant at two river mouths where abundance was positively correlated with lower salinity and higher chlorophyll values. In addition, anchovies sampled at the river mouth had a distinct diet characterized by small sized preys such as small copepods and bivalve larvae. The trophic position of adult anchovies showed a clear difference between the northern and the southern sectors of our sampling area in the Tyrrhenian Sea, therefore reflecting differences over a deeper time scale with respect to stomach content analysis.Specifically, a higher trophic level for both the coastal and offshore ecotypes in the northern sector (despite the different diets at the moment of the capture) and a lower trophic level for the offshore ecotype in the southern sector suggest that the offshore ecotype adapts to local food availability, profiting of the richer environments where rivers are presents but able to explore and thrive also in more oligotrophic conditions.loci, suggesting selective divergence between the two ecotypes. Overall, our observations suggest that the two anchovy ecotypes herein identified correspond to the offshore and coastal ecotypes previously described in the Atlantic Ocean for the European anchovy6, despite the remarkable environmental differences between the two regions. Our local-scale analysis showed that the two ecotypes can co-occur in the same localities and that populations with a higher representation of the coastal ecotype have a strong relation with the river mouths, while the other ones have more plasticity, i.e., are able to profit of river-originated productivity though generally live in more oligotrophic conditions. We did not found evidence of a specific adaptation to river environments, in the case of these very small rivers. Two of the outliers genes identified in the analysis were associated with reproductive processes, but this is not sufficient to draw any conclusion. Further analysis assessing traits under selection are necessary.Sampling sites connectivity, governed by marine currents, does not explain the genetic patterns observed, while SNPs neutrality test identified seven putative outlier 20, while that of the Mediterranean Sea is associated to regions with almost complete absence of shelf and with more than 10 °C higher temperatures, much higher salinities, and highly oligotrophic conditions. Given the large differences in environmental conditions, we propose that the common trait is high plasticity that makes this ecotype highly opportunistic when resources are available  while being able to live in highly oligotrophic conditions. It is worth noting that offshore populations from the Bay of Biscay and the Mediterranean Sea have a lower genetic differentiation levels, in respect to the coastal populations. This is likely due to the presence of a common ancestor and to the higher dispersal conferred by higher ecological plasticity.The Atlantic offshore ecotype is associated with wide continental shelves and harbours anchovy populations in the Bay of Biscay and the North SeaThe Atlantic coastal ecotype is associated with narrow continental marine shelves, including populations from Iberian-Atlantic coasts and Canary Islands while in the Mediterranean Sea it seems to be associated to river mouths. However, this narrow-shelf ecotype is not as homogeneous, since Cadiz/Canary samples significantly differ from the coastal group composed by Biscayan and Italian coastal samples Table . Coastalet al.20, stressing that Biscayan and Italian coastal populations are different from Cadiz/Canary narrow-shelf populations, but most probably share a common origin.This result adds relevant information to the observations of Montes et al.9). These ecotypes could have diverged in different refugia, i.e., wide continental and narrow/non-existent continental shelves20, during the last glaciation event (LGE). Then, they could have extended their distribution in different directions, once temperature started to arise. The coastal ancestor migrated from the Atlantic and Mediterranean refugia to estuaries and areas with river inputs, in the two basins, even when these areas were already inhabited by wide-shelf populations. If genomes of the two different ecotypes evolved to incompatibility during allopatry, no (or very little) gene flow would have been possible upon secondary overlap and ecotypes of the same area would have remained genetically distinct24.Atlantic and Mediterranean populations of coastal and offshore ecotypes may have become geographically isolated from each other, resulting in an allopatric divergence followed by secondary overlap  could influence the growth of individuals and development of stocks on the basis of their genetic adaptability. Barriers to gene flow between the two ecotypes could have evolved in a short time because divergence driven by ecological separation requires minor genetic alterations only in few loci among the whole panel of SNPs utilized. Surprisingly and interestingly, 6 of them are among the 16 outlier loci that are involved in differentiation of the Bay of Biscay coastal anchovy20. This finding that the 37.5% of outlier loci separating the two ecotypes in the Bay of Biscay also separate the two ecotypes in the Mediterranean, is higher than the value of 24.1% found by Le Moan et al.9. Nevertheless, it strongly confirms the complexity of the evolutionary origin of the two ecotypes and the possibility that the distinction by functional loci has arisen when the two common ancestors started to separate. The fact that the basigin gene, involved in the fusion of egg and sperm, appears as a common outlier differentiating coastal and offshore populations in both Mediterranean Sea and Bay of Biscay, suggests a selection pressure on reproduction-related genes that could reduce gene flow between these ecotypes on a wider scale. On the other side, the separation observed between ecotypes in the two basins, also when outlier loci are excluded from the analysis, suggests that neutral genetic drift cannot be neglected.Searching for putative outliers in separating coastal and offshore Mediterranean populations, we identified 7 31. The position of spawning areas is genetically fixed and those occur in optimal environmental conditions for survival of offspring. However, distribution and abundance of eggs and larvae are primarily affected by passive transport32, and our data suggest high rate of eggs dispersion along the Italian Tyrrhenian coasts. Patterns of marine currents affect the distribution of small pelagic fishes also by shaping the distribution of their prey, most of which are unable to move for long distance34. The results of the Lagrangian analysis showed that in Central Tyrrhenian Sea the geomorphological conformation of gulfs favours retention of the nutrient-rich river waters, thus amplifying the impact of rivers that are actually very small (flow < 100 m3/s), especially if compared with the rivers along the Bay of Biscay.The coexistence of parental populations and hybrid hauls in the same areas, and the apparent patchiness in hybrids distribution could be an effect of larval transport to areas distant from the areas of fertilizationIn conclusion, we shed light on the existence of two anchovy ecotypes co-occurring along the European coasts: the offshore ecotype, with wide distribution in the North Eastern Atlantic Ocean and in the Mediterranean Sea, and the coastal ecotype with preferences for river plumes. These ecotypes show signs of evolutionary divergence at pan European scale, although their separation in the Mediterranean Sea is lower than in the near Atlantic Ocean. Their co-existence in the same populations and the presence of many hybrids make difficult their inclusion in the stock management plans. Further studies are necessary to address the question whether such genetic differences could lead to differences in reproductive and recruitment success or in other related factors influencing the abundance and spatial distribution of the two groups.A total of 443 anchovies from 15 sites were collected from the Tyrrhenian, the Adriatic and the Ionian Sea . All individuals were genotyped for 96 SNPs22 using Fluidigm Biomark platform 96.96 chips , following the user guide. The panel of 96 highly informative SNP markers was selected in a previous study where it showed optimal resolution in assessing differentiation among E. encrasicolus populations within the Mediterranean and between Atlantic and Mediterranean Sea22. SNP genotyping was performed in the Genomics Facilities-Sgiker (UPV/EHU).Total genomic DNA was isolated from 30 mg of tissue of adult anchovies using Nucleospin Tissue kit , following manufacturer’s protocol. Eggs genomic DNA was isolated by a Hotshot protocol , Canary (CAN), Tarragona (TAR) and Biscay , analyzed in Montes is Table . This wa35 and a principal component analysis (PCoA) was conducted with GENALEX36 and the package ADEGENET v.1.3-1 for R37. STRUCTURE version 2.3.438software was employed to infer main Bayesian genetic clustering. Runs in STRUCTURE were made assuming K = 1– 5, imposing an admixture model with correlated allele frequencies for estimating the ancestral populations and indicating the sampling location information. Each K value was replicated with ten independent runs of 100,000 MCMC iterations, after a burn-in of 10,000 iterations. The most likely number of clusters based on delta K was identified using the method of Evanno et al.39, performed in the software STRUCTURE HARVESTER40. Individuals with assignment probability > 90% were considered as belonging to a given cluster, while individuals with assignment probability between 50 and 90% were considered as putative hybrids.Genetic differentiation was assessed using the program Genepop 4.1.0FST and genetic distances) were calculated by the program ARLEQUIN v.3.541. P-values of FST values were computed using a permutation approach . BARRIER v.2.223 was used with pairwise estimates of FST mapped onto a matrix of geographic coordinates (latitude and longitude), and the Monmonier’s maximum difference algorithm identified breaks in gene flow patterns among geographical sites. BAYESCAN v.2.142 and LOSITAN43 were used to test whether any of the used SNP markers do not behave according to expectations under neutrality. In BAYESCAN, we calculated Q-values as posterior probabilities to estimate the difference in numbers of putative neutral and selected loci, performing 20 pilot runs with 5,000 iterations and 500,000 iterations MCMC with an additional 50,000 iterations as burn-in. LOSITAN is based on a coalescent approach, detecting outlier loci from the joint distribution of FST and expected heterozygosity (He) under the island model of migration. Runs were made with both the Infinite Allele Mutation Model (IAM) and Stepwise Mutation Model (SMM), each with 100,000 simulations. Only the loci that were detected by both methods were considered as true outliers.Patterns of population differentiation among all samples  from the large scale geostrophic vortices (~100kms) down to the turbulent (sub) mesoscale and wind-induced perturbations (<10 kms) and guarantees a smooth nudging with the lower resolution operational model output used to define the open boundary conditions. Initial and boundary conditions are derived from the Mediterranean Monitoring and Forecasting Centre (MED-MFC) physical reanalysis product. The MED-MFC model data are presently being produced and distributed on a weekly basis by the European Copernicus Marine Environment Monitoring Service . They are obtained through a specific implementation of the hydrodynamic model supplied by the Nucleous for European Modelling of the Ocean (NEMO), including a variational data assimilation scheme (OceanVAR) for in situ temperature and salinity vertical profiles and satellite Sea Level Anomaly along-track data47. The MED-MFC data are available on a 1/16° × 1/16° horizontal resolution grid (6–7 kms here) and 72 vertical levels. Hence, since such resolution differs from that of our domain (2 kms), we project the sea surface height, the seawater velocity fields, the temperature and salinity MED-MFC data at the boundaries, and as initial condition, using a trilinear interpolation. We nudge temperature and salt in the regions close to the boundary to allow for a smooth transition from the boundary data, with a nudging linearly decreasing from a time scale of 0.25 days at the boundary to a null value at about 100 kms from the boundary.The Regional Ocean Modeling System (ROMS) is a free-surface, terrain-following, primitive equation ocean model, with online point-particles tracking abilitiesCampania  rivers have been monitored by the Protezione Civile  of the Campania Region since 2001. Observed daily discharge data were incorporated in the model as a point source term.et al.48. It was adapted from the Coupled Ocean-Atmosphere Response Experiment (COARE) algorithm for the computation of surface fluxes of momentum, sensible heat, and latent heat. The air-sea boundary layer is used as a one way coupling with atmospheric models. Here, we use the atmospheric data of the nonhydrostatic version of the SKIRON/Eta modelling system, that was implemented in the Mediterranean and Black sea in order to produce high-resolution (10km) weather hindcasts and forecasts. The surface fluxes, obtained through bulk parameterization, and the SKIRON data have been validated against the NCEP Reanalysis Data. The NCEP/NCAR Reanalysis 1 project is using a state-of-the-art analysis/forecast system to perform data assimilation using past data from 1948 to the present49.The air-sea interaction in ROMS is modelled using a bulk parametrization of Fairall in situ sampling was carried out. Results obtained with ROMS appear as a smooth extension of the MED-MFC data. Figure 52. However, the nudging strongly conditions the results of the simulation and prevents the development of small scales non-linearities. Finally, in order to conduct Lagrangian analyses and infer the physical connectivity along the coasts of the Tyrrhenian Sea, we released and tracked a set of point-like particles being passively advected by currents. Particles are released on the surface along the coast around each sampling sites in a disk-like area with a radius of Rsite = 4 km. Starting in May and during 90 days, a batch of 550 particles is released from every site with an occurrence of 5 days for a total number of around 10,000 particles per site.In order to assess the physical connectivity among sampling sites, we ran simulations of 120 days from May to August for the years 2009, 2012 and 2013, therefore covering the late-spring/summer periods of the year when 53. Then, we inferred from the Lagrangian PDFs the physical connectivity matrix which gives the probability that a particle leaving a given sampling site reaches another sampling site. The combined analysis of LPDFs and connectivity matrix allows for a complete drawing of the seascape and its related transport dynamic.From the particles trajectories computed with ROMS, we inferred the fine-grained Lagrangian PDFs which give the probability density function of a particle leaving its initial location and reaching a given destination location of interest (here any point in the Tyrrhenian Sea), and this after a given advection time scalein situ observations collected during the EVATYR surveys. This analysis included both the comparison of observed tracer distribution mesoscale very clearly mark the small scale circulation features along the Tyrrhenian coasts. This qualitative comparison was carried out using the 1 km resolution MODIS-Aqua chlorophyll-a daily data distributed by CMEMS. The analysis revealed that the surface current variability, mainly driven by rapid changes in the wind-stress, was generally well reproduced in our simulations. The second approach consisted in comparing model data with the see Fig.  and a qu54. Obtained data were post-processed using Myriax Echoview software and adopting an Elementary Distance Sampling Unit (EDSU) of one nautical mile . During the survey, midwater pelagic trawl sampling was carried out in order to characterize the observed echoes in terms of species compositions and to investigate the length frequency distribution of collected species. Anchovy density (t/nmi2) for each EDSU was evaluated by merging the biological and acoustic data, based on the nearest haul method55.The echosurvey was carried out in the period 17 May–9 June 2013. A zig-zag sampling strategy was adopted in the southern part of the study area due to the very narrow continental shelf characterizing this sector, while in the northern part a parallel transects design was adopted. At each transect vertex, vertical profiles of salinity and temperature were also collected by means of SBE911 plus CTD probe. During the cruise, acoustic data were collected at vessel speed of about 8-10 knots by means of Simrad EK60 scientific echosounder working with a split-beam transducer at 38 kHz; the system was calibrated following standard techniquessat); (iii) distance from the nearest river mouth and (iv) depth.To characterize the environmental processes driving differences in ecotype proportions between the Northern and Southern sectors of the Southern/Central Tyrrhenian Sea, a Principal Component Analysis was carried out. Further, the link between anchovy biomass and environmental drivers was investigated by means of Quotient Index analysis in order to verify if identified processes had some influence on anchovy biomass distribution. To this aim, the environmental parameters considered were (i) salinity and temperature at 5 meters depth ; (ii) satellite derived Chl-a values . In this study the PCA scores values were used as environmental factor since they represent identified processes as linear combination of all or some of the original environmental variables. The significance of association/avoidance was tested using randomization procedure57. In order to avoid the presence of few observations in the histogram tails (strongly affetting the resampling procedure), the histogram intervals were computed by balancing the number of observations in each bin.Principal Component Analysis (PCA) was carried out separately in each sector to better discriminate environmental drivers acting in the two areas. The association between anchovy biomass and identified environmental processes was investigated by means of Quotient Index analysis13C values of fishes were no normalized for lipid concentration, being the C:N ratio of anchovies <3.558. Stable isotope measurements were carried out by ThermoFisher Flash EA 1112 elemental analyzer coupled to a Thermo Electron Delta Plus XP isotope ratio mass spectrometer (IRMS). Samples were run against blank cups and known urea standards of certificated isotopic composition. Three capsules of urea were analysed at the beginning of each sequence and one every six samples as a quality control measure. Experimental precision  was < 0.3‰ for δ15N and < 0.4‰ for δ13C. The δ15N and δ13C values were obtained in parts per thousand (‰) relative to Vienna Pee Dee Belemnite (VPDB) and atmospheric N2 standards respectively, according to the following formula:A portion of white muscle was extracted from each fish individual, oven-dried (60 °C for 24 h), powdered and weighted (0.5 mg) into tin capsules for isotopic analysis. The δ59. The Bray-Curtis coefficient of similarity60 and the complete linkage were applied to square root transformed data.In order to describe the diet composition, 60 anchovies were selected from 6 sampling areas Table , with prSupplementary Informations