Studies showed that upregulation of Nav1.6 increased the neuronal excitability and participated in neuropathic pain in the dorsal root ganglion (DRG). However, the molecular mechanisms underlying Nav1.6 upregulation were not reported yet.The paw withdrawal threshold was measured in the rodents following lumbar 5 ventral root transection (L5-VRT). Then qPCR, western blotting, immunoprecipitation, immunohistochemistry, and chromatin immunoprecipitation assays were performed to explore the molecular mechanisms in vivo and in vitro.Scn8a (encoding Nav1.6) promoter and the interaction between STAT3 and p300, which contributed to the enhanced transcription of Scn8a by increasing histone H4 acetylation in Scn8a promoter in DRG. Importantly, intraperitoneal injection of the TNF-α inhibitor thalidomide reduced the phosphorylation of STAT3 and decreased the recruitment of STAT3 and histone H4 hyperacetylation in the Scn8a promoter, thus subsequently attenuating Nav1.6 upregulation in DRG neurons and mechanical allodynia induced by L5-VRT.We found that the levels of Nav1.6 and phosphorylated STAT3 were significantly increased in DRG neurons following L5-VRT, and TNF-α incubation also upregulated the Nav1.6 expression in cultured DRG neurons. Furthermore, immunoprecipitation and chromatin immunoprecipitation assays demonstrated that L5-VRT increased the binding of STAT3 to the Scn8a promoter region in DRG, which contributed to L5-VRT-induced neuropathic pain.These results suggested a new mechanism for Nav1.6 upregulation involving TNF-α/STAT3 pathway activation and subsequent STAT3-mediated histone H4 hyperacetylation in the  Chronic pain is a major health issue worldwide, especially in the patients with nerve injury , 2. HoweVoltage-gated sodium channel (VGSC) is a kind of multi-subunit transmembrane glycoprotein, which consists of nine members, named from Nav1.1 to Nav1.9 . It is wEvidence show that the inflammatory cytokines play an important role in the establishment and maintenance of chronic pain . TNF-α iflox/flox mice (ID: 016923) were obtained from the Jackson Laboratory. Animals were housed in separated cages and provided with food and water ad libitum, and the room was kept at 24 °C temperature and 50 to 60% humidity under a 12/12-h light/dark cycle. All experimental protocols were approved by the Local Animal Care Committee and were performed in accordance with the guidelines of the National Institutes of Health on animal care and the ethical guidelines. All efforts were made to minimize the number of animals used and their suffering.Male Sprague-Dawley rats (200–220 g) and male wild-type (WT) C57 mice (20–30 g) were purchased from the Institute of Experimental Animals of Sun Yat-sen University. STAT3The L5-VRT model was performed as described previously . BrieflyRecombinant rat TNF-α was obtained from R&D Systems . Intraperitoneal injection of TNF-α synthesis inhibitor thalidomide  or intrathecal injection of STAT3 inhibitor S3I-201  and TNF-α neutralizing antibody  were performed 30 min before L5-VRT and for additional 9 days . Intrathecal injection was in accordance with the previously described method . After tVon Frey hairs were used to assess the 50% withdrawal threshold as described previously . In brieflox/flox mice. pAAV-CAG-eGFP-U6-shRNA (Nav1.6) was commercially obtained from OBiO . The sequences of these shRNAs were presented as follows:shRNA1 5′-GGAAGACGCCATTGAAGAAGA-3′shRNA2 5′-GGAAGAATGTCAAGATCAACT-3′shRNA3 5′-GCAGATGGAGAACATTCTTTA-3′Recombinant adeno-associated virus encoding Cre and green fluorescent protein (GFP) marker (AAV8-Cre-GFP) and AAV encoding GFP (AAV8-GFP) were purchased from Beijing Vector Gene Technology Company Ltd. Four microliters (4 μL) of AAV8-Cre-GFP or AAV8-GFP was intrathecally injected in STAT3These reagents were intrathecally delivered according to methods we previously described . The expThe L4 to L6 DRGs were immediately removed at different time points after application of sodium pentobarbital at 50 mg/kg dose (i.p.) and were homogenized on ice in 15 mM tris buffer containing the inhibitors of proteinase (Roche) and phosphatase (Roche) on ice. Protein samples were separated by SDS-PAGE and then transferred onto a PVDF membrane. The PVDF membrane was incubated with primary antibodies against Nav1.6 , TNF-α , phosphorylated STAT3 , STAT3 , acetylated histone H3 (K9) , acetylated histone H4 , p300 , or β-actin , after blocking with the block buffer for 1 h at room temperature (RT). Enhanced chemiluminescence (ECL) (Pierce) was used to detect the immunocomplex after the blots were incubated with the secondary antibodies. The intensity of the band was examined by a computer-assisted imaging analysis system .Perfusion was immediately performed through the ascending aorta with 4% paraformaldehyde after being anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg). The DRG segments were removed and placed into the 4% paraformaldehyde for post-fixing overnight. Cryostat sections (16 μm) were cut, and immunohistochemistry was performed with primary antibody for Nav1.6 , TNF-α , STAT3 , phosphorylated STAT3 (Tyr705) , IB4 , GFAP , and NF-200 . After incubation overnight at 4 °C, the sections were incubated with secondary antibodies, which conjugated with cy3 or FITC for 1 h at RT. To test the specificity of the TNF-α antibody, preincubation with TNF-α peptide was used . Briefly2, 37 °C) and used for western blot within 24 h.The ipsilateral DRG neurons of the procedure were dissociated by enzyme digestion as described previously with slight modifications . In shor−ΔΔCT method. The primer sequences of Nav1.6 were as follows: Forward 5′-TGGACGATACCAGCTCCTCA-3′, and Reverse 5′-ACGCAACCCTCTGTAAAGCA-3′.A TRIzol reagent (Invitrogen) was used to extract total RNA from L4 to L6 DRGs. The reverse transcription was performed using Prime Script RT Master Mix (Takara). Real-time qPCR was processed with SYBR Premix Ex TaqII (Takara). The relative ratio of mRNA expression was quantified through the 2Scn8a (encoding Nav1.6) promoter (which containing the STAT3 binding site) in rats. Finally, the relative ratio of CHIP/input was calculated.The ChIP assays were performed by a ChIP Assay Kit (Thermo). The L4 to L6 DRGs of rats were broken into pieces and placed in 1% formaldehyde for 2 min, then terminated with glycine. The DNA was subsequently digested by micrococcal nuclease after being broken via sonication. After the addition of ChIP dilution buffer, 100-μL samples were saved as input. The antibody of STAT3 (8 μL) or Ac-H4 (8 μL) was added to 500 μL for preclearing, and then the samples were incubated overnight at 4 °C. Immunoprecipitation with control rabbit IgG (Sigma) was performed as negative control. The DNA/protein/antibody complexes are purified into DNA by elution and reversion. QPCR was applied on 5 μL of precipitated DNA samples. Primers 5′-CTAGGCTGGGGACTTCTGTG-3′ and 5′-GCTAGTTTCCAGCAGGCAGT-3′ were projected to amplify a − 1530/− 1625 sequence, which localized on the Coimmunoprecipitation was performed using the Co-Immunoprecipitation Kit (Pierce). In brief, DRG tissues were excised quickly and homogenized in lysis buffer. The p300 antibody or STAT3 antibody, which was immobilized with resin, was used to collect the immune complexes. The eluted complexes from the resin were incubated and washed following the Kit manual and then analyzed by western blot using the STAT3 antibody or p300 antibody.t test and one-way analysis of variance (ANOVA). For the data of behavioral tests, one-way or two-way repeated-measures ANOVA were employed. The threshold for statistical significance was P < 0.05. Although no power analysis was performed, the sample size was determined according to previous publications in pain-associated behavior and molecular studies.Data were expressed as mean ± SEM and analyzed with SPSS 13.0. Western blot and qPCR were analyzed by independent Student’s Consistent with our previous study , 21, theNext, we found that L5-VRT also markedly induced the increase of TNF-α in DRG Fig. a. Then, Our previous study demonstrated that activation of STAT3 in the spinal dorsal horn was involved in paclitaxel-induced neuropathic pain . Here, wflox/flox mice  of the Scn8a promoter which contains the STAT3 binding site with the designed primers. The results of qPCR analysis revealed that the binding of STAT3 to the Scn8a promoter was enhanced in DRG on day 5 and day 15 following L5-VRT compared to the sham group, which can be reversed by the STAT3 activity inhibitor S3I-201 in the modeled rats (Fig. Scn8a promoter region in DRG. Firstly, western blot analysis showed that the total acetylation of histone H4 was increased significantly (Fig. Scn8a promoter region which contains the STAT3 binding site in DRG on day 15, and this H4 hyperacetylation can be reversed by S3I-201 in the modeled rats (Fig. flox/flox mice with L5-VRT (Fig. Scn8a promoter region on day 15 induced by L5-VRT (Fig. Scn8a promoter region, which increased the histone H4 acetylation and facilitated the Nav1.6 expression in the rodents with L5-VRT.As a member of the STAT family of transcription factors, STAT3 is now known to regulate the expression of many genes such as cytokine, anti-apoptotic, and pro-survival genes , 24. HerScn8a promoter in DRG. Suppression of STAT3 activation with local knockdown or inhibitor S3I-201 significantly attenuated H4 acetylation, and Nav1.6 upregulation in DRG, and relieved mechanical allodynia induced by L5-VRT. Importantly, inhibition of the TNF-α activity by thalidomide inhibited the STAT3 activation, H4 acetylation, and Nav1.6 upregulation in DRG following L5-VRT. Taken together, these findings, for the first time, proved that the activation of the TNF-α/STAT3 pathway may be a novel mechanism for Nav1.6 upregulation in neuropathic pain induced by nerve injury.In the present study, we elucidated a novel molecular mechanism underlying the epigenetic upregulation of Nav1.6 by an enhanced TNF-α/STAT3 signaling pathway in DRG, which contributed to neuropathic pain induced by L5-VRT in the rodent models. The results showed that the expression of Nav1.6 and p-STAT3 was significantly increased in DRG neurons following L5-VRT. Incubation of TNF-α also upregulated the expression of Nav1.6 and p-STAT3 in cultured DRG neurons. Moreover, IP and ChIP assays showed that L5-VRT enhanced the interaction between p-STAT3 and p300 and increased the recruitment of p-STAT3 and histone H4 acetylation on the Scn8a promoter increased the histone H4 hyperacetylation and facilitated Nav1.6 expression in DRG induced by L5-VRT.Nav1.6, as an isoform of sodium channel, contributes to the neuron excitability and plays a critical role in the development of CNS diseases. For example, Nav1.6 upregulation is involved in neuropathic pain induced by SNL or diabetes in mice , 26. TheAs a crucial inflammatory factor, TNF-α has the capability to increase the neuronal excitability and mediate the behavioral hypersensitivity in neuropathic pain . Here, wAltogether, our study showed that TNF-α, via activating STAT3, mediated epigenetic upregulation of Nav1.6 in DRG and contributed to L5-VRT-induced mechanical allodynia, which provided a novel potential target for the treatment of nerve injury-induced neuropathic pain.


Nectomys squamipes (Cricetidae: Sigmodontinae) is a semiaquatic rodent from eastern South America that shows shallow genetic structure across space, according to some studies. We tested the influence of hydrography and climatic changes on the genetic and phylogeographic structure of this semiaquatic small mammal.The water rat DNA sequences of two mitochondrial genetic markers (Cyt b and D-loop) and six microsatellite loci from water rats were collected at 50 localities in five river basins in the Atlantic Forest along the eastern coast of South America. We evaluated the genetic structure within and among river basins, and we estimated divergence dates. Species distribution models for the present and past were built to identify possible gene flow paths.N. squamipes haplotypes occurred during the Pleistocene and the river basin cannot explain most of the genetic structure. We found evidence of population expansion during the last glacial maximum, and gene flow paths indicate historical connections among rivers in the Atlantic Forest.Mitochondrial data and species distribution models showed coherent results. Microsatellite loci showed a more complex pattern of genetic differentiation. The diversification of N. squamipes to disperse farther across and within basins, leading to shallow genetic structure. Population expansions and gene flow through the emerged continental shelf during glacial period support the Atlantis forest hypothesis, thus challenging the forest refuge hypothesis.Historical connections among rivers in the Atlantic Forest may have allowed  In recent years, many phylogeographic studies have tested different hypotheses to explain the diversity and genetic structuring patterns found in vertebrates in the Atlantic Forest, eastern South America . QuaternNectomys squamipes (Brants), in the Atlantic Forest of eastern Brazil. The genetic structure of semiaquatic mammals may have been shaped differently from terrestrial mammals because the displacement through rivers may allow semiaquatic mammals to disperse further, leading to less genetic differentiation among populations  evaluate evolutionary hypotheses to explain the biogeographic history of the water rat; (2) test if the genetic structure is strongly influenced by hydrography, leading to low gene flow and high divergence among basins, or if rivers allow N. squamipes to disperse across basins, leading to homogeneity and low genetic structure.Here we integrated analyses of mitochondrial and nuclear molecular markers with species distributions modelling to comprehend the phylogeographic structure of N. squamipes (http://www.ncbi.nlm.nih.gov/genbank/), five were donated by researchers, and 154 liver and muscle samples preserved in ethanol were supplied by three mammal collections: CTA-UFES , MBML , and MCN-M   see . The PCRhttps://code.google.com/archive/p/garli/) and in PhyML 3.0  were above 200 in Tracer 1.6 , Oligoryzomys flavescens (Waterhouse), Oligoryzomys chacoensis (Myers & Carleton) and Oligoryzomys nigripes (Olfers) were used as outgroups.Sequences were used to obtain phylogenies through maximum parsimony (MP) in MEGA 5.2 , BayesiahyML 3.0 . The modhyML 3.0  for Cyt acer 1.6  and thenPseudoryzomys and Holochilus at 2.58 million years ago (Ma) (0.43–5.38 Ma), and the divergence between Cerradomys and Sooretamys at 2.35 Ma (0.42–5.02 Ma), are from O. nigripes, O. flavescens, and O. chacoensis at 3.71 ± 0.035 Ma (Amphinectomys and Nectomys at 2.64 Ma (2.00–3.37 Ma) are from Beast 1.8.2  was used0.035 Ma , and thehttp://www.snirh.gov.br/hidroweb/) and GeoNetwork (http://metadados.ana.gov.br/geonetwork/srv/pt/main.home). We performed Bayesian Analysis of Population Structure (BAPS 6.0) , and genetic divergence (FST)   to estimwork 4.6  using thce (FST)  among clce (FST) , and Arlce (FST) . The gence (FST)  using thRST values were calculated in Arlequin 3.5 . Structure Harvester (K) based on the second order rate of change of the likelihood (ΔK) (K. CLUMPAK (K.BOTTLENECK 1.2.02  was usedarvester  was usedood (ΔK) , and for CLUMPAK  was alsoFST (mtDNA), and RST (microsatellites) with 10,000 replications.For all genetic markers we performed analysis of molecular variance (AMOVA), grouping our samples according to the river basins, and to localities inside the basins. Localities with only one sample were grouped with the nearest locality within the same river basin. We used only sequences without ambiguities, and genotyped samples with one missing locus at most. We conducted AMOVA in Arlequin 3.5 , using F2). For the LGM, we used the MIROC-ESM model (We used MaxEnt 3.3.3k  and modeSM model  and for http://splink.cria.org.br/), Global Biodiversity Information Facility (GBIF: http://www.gbif.org), and Arctos Collaborative Collection Management Solution (http://arctos.database.museum/), or requested from curators. The points of occurrence were reviewed, and only data with precise specific locations were used. The locations of the points of occurrence were verified using Google Maps (http://maps.google.com.br), and two tools from SpeciesLink (http://splink.cria.org.br/) were used when compiling our locality database: (1) “geoLoc”, which searches for place names in gazetteers, was used to georeference localities with no coordinates; (2) “infoXY”, was used to fill out administrative data  for a given lat-long coordinate. The coordinates found in “geoLoc” were not used when building the models, only in the external validation test.A database with 463 georeferenced locations was compiled based on data from museums (242 points), scientific articles (95), theses and dissertations (126). The museum data were obtained from SpeciesLink  and those with correlation values less than 0.7 among them. Theses tests were perfomed in R 3.0.1  using “raster”  and “vegL = linear, Q = quadratic, H = hinge, P = product and T = threshold); with regularization values (RM) from 0.5 to 3.0 (increments of 0.5), and we used block method for partitioning occurrence data, which resulted in 48 individual model runs. The best model was one with minimum Akaike information criterion value (i.e. ΔAICc =0), FC = LQH and RM = 2.0. We evaluated this model with indices in MaxEnt validation and external validation step. We analyzed the area under the curve (test AUC), default values and the p-value for the three thresholds frequently used in MaxEnt: minimum training presence logistic threshold, 10 percentile training presence logistic threshold, and maximum test sensitivity plus specificity logistic threshold. Indices analyzed in the external validation were based on the 10% threshold.To estimate optimal model complexity for the SDM, we used “ENMeval” package in R 3.0.1 . We testWe built scatter plots for the three environmental variables that contributed most to the SDM. The models were based on the 10% threshold and the environmentally appropriate area was calculated for each scenario  to evaluate whether there was variation in the size of the area over time.N. squamipes. We modelled gene pathways under LGM because recovered gene flow may reflect older demographic connections. The most recent Cyt b population expansion may have taken place during the LGM as suggested by the Bayesian skyline plot (see ‘Results’ below). The SDM was converted into a dispersal cost layer by inverting the model’s suitability values ; sequences for both markers were obtained from 70 samples . Five D-loop sequences were discarded due to double peaks. For microsatellites, 152 individuals were genotyped, representing 43 localities, 120 samples with one missing locus at most  were those from Cyt b sequences, recovering three main clades: North, Central and South, in reference to their latitudinal ranges, regardless of the optimality criteria used . The Cened trees . Samplesed trees  showed tN. squamipes were also recovered in BAPS cluster analyses  for N. squamipes that are not related to the river basins , and the three intraspecific clades also display small genetic distances, with little intraclade  and interclade divergences for Cyt b (1.8–1.9%); interclade divergences for D-loop were higher (4.0–4.9%). In addition, samples from the São Francisco basin have greater intraregional diversity  than those from the other basins . The most recent common ancestor of all Cyt b haplotypes from Nectomys spp. dates back to the Plio-Pleistocene period 2.7–1.3 Ma, and Cyt b haplotypes from N. squamipes diverged 1.64–0.51 Ma . Other river basins presented moderate values for FST/RST . Among the genetic markers, Cyt b showed the highest values of population differentiation (FST = 0.16–0.56), while the microsatellite showed the lowest values (RST = 0.01–0.13). River basins explain only 11.08% of the genetic variation found in Cyt b, and were irrelevant for D-loop and microsatellite loci . The projection for the LGM shows a 62% increment , with the most suitable areas on the coast and inland near the headwaters of the São Francisco river, on the Espinhaço range, and along the Paraná river , a 51% decrease compared to the present, and a 70% decrease from the LGM. Highly suitable areas are restricted to the coast, with a break between Bahia and Rio de Janeiro . We therefore believe that the number of microsatellites loci were adequate infer the genetic structure in our dataset.The only results indicating strong genetic structure by river basin were  markers . Indeed,an males , which man males  also fouan males  found siThe phylogeographic break at Serra da Bocaina is concordant with the boundaries of two freshwater ecoregions, Ribeira do Iguape and Paraíba do Sul, delimited by the Freshwater Ecoregions of the World (FEOW) system provided by Arvicola sapidus Miller  (Castor fiber Linnaeus) and otters [Lutra lutra (Linnaeus)], also display a shallow genetic structuring among populations in different river basins (Hydrochoerus hydrochaeris (Linnaeus)), the importance of river basins is variable according to river system  few statistically supported clades; (ii) clades with short branches in the phylogenetic trees; and (iii) few isolated haplotypes in the haplotype networks. These same factors were found in the small semiaquatic rodent, s Miller . In addintiation . Geneticinnaeus)  corroborr basins . These cr basins . Even for system . These rr system . Some spr system , which uy season , and theThere has been a recent population expansion in the Southeast Atlantic and Central clade/cluster according to the neutrality tests. Moreover, there are haplotypes in the Southeast Atlantic shared by other river basins, which indicates gene flow from adjacent basins is taking place now or in the recent past because the shared haplotypes concentrate at the borders. The microsatellite data, on the other hand, suggest stability rather than expansion in the Atlantic Southeast. This incongruence might be the result of the distinct temporal scales documented by these markers. Mitochondrial loci reflect older events than microsatellite loci  and, in N. squamipes. For example, Hoplias malabaricus (Bloch) nearly identical to presented here for N. squamipes. They explained this structure by recent dispersal events congruent with historical connections among river basins caused by sea level variations and stream piracy. The same factors may also explain the connections among river basins found here for N. squamipes populations  was the most important variable for constructing the distribution models. This variable is directly related to the biology of this water rat because it lives near water bodies where the temperature is more stable during the day, and therefore coastal areas with narrow temperature range are highly suitable in the SDM. Population fluctuations over the past 120,000 years followed the temperature variations documented by the stable isotope oxygen-18 (δ18O)  , with anNectomys squamipes populations may have been favoured during glacial periods despite possible decrease in plant cover and increase in rainfall seasonality (N. squamipes, a more generalist species that prefers small marshy streams over larger rivers with faster-running water (N. squamipes during the LGM is the decrease in ocean levels, which were on average 68 m lower than their current level (N. squamipes (N. squamipes as well.sonality . This scng water . Anothernt level . This innt level , allowinquamipes . Therefoquamipes . Pleistoquamipes , and theN. squamipes and for the three main clades. This result indicates that climate oscillations in the period may have been important both for the speciation of the genus and for the genetic structuring of N. squamipes. The three main geographic regions resulting from modelling the LIG are similar to the clades found in the phylogenetic trees, indicating correspondence between the modelled ecology data and the genetic data, as described in other studies (N. squamipes populations may have undergone recurring processes of population reduction and expansion (Dating revealed a Pleistocene origin for the Cyt b haplotypes of  studies . Taking xpansion .N. squamipes. The presence of suitable areas and gene flow paths through the continental shelf, and population expansions during the glacial period, give support to the Atlantis forest hypothesis. Rivers and historical connections between rivers allowed N. squamipes to disperse farther across and inside basins, leading to a shallow genetic structure.Our results showed a shallow genetic structure in the water rat, slightly influenced by river basins. The pattern of genetic diversity is best explained by latitude with three main clades structured from north to south. There is evidence of recent population and suitable area expansions during glacial period for 10.7717/peerj.5333/supp-1Appendix S1Click here for additional data file.10.7717/peerj.5333/supp-2Appendix S2Click here for additional data file.10.7717/peerj.5333/supp-3Appendix S3Click here for additional data file.10.7717/peerj.5333/supp-4Supplemental Information 1Click here for additional data file.10.7717/peerj.5333/supp-2Supplemental Information 2Click here for additional data file.


Vitis vinifera L cv Monastrell is the main red grape variety grown for vinification in the Denomination of Origin Jumilla (southeast Spain). Different strategies are still being tested to optimize available resources both in terms of the environment and to achieve wines with better organoleptic and functional characteristics. The objective of this work was to combine two strategies: the application of methyl jasmonate (MeJ), benzothiadiazole (BTH), and methyl jasmonate + benzothiadiazole (MeJ + BTH) elicitors to Monastrell leaves, and pre-fermentative cold maceration. During two seasons, the experiment was carried out to improve the extraction of the phenolic compounds, whose levels may have increased following the application of elicitors in the field, and to assess the effect of both strategies on the wine quality. Discriminant analysis revealed that independently of the meteorological conditions during both years, the pre-harvest response to the application of elicitors MeJ, BTH, and MeJ + BTH, induced increases in total anthocyanin concentration of the treated grapes, allowing the distinction of the treatments. This analysis also allowed the distinction between the types of maceration used, showing greater extraction of phenolic compounds by the application of low temperature, giving wines with a higher index of total phenols, a greater intensity of color, and a lower luminosity. Vitis vinifera L. cv Monastrell is the fourth most cultivated red wine grape variety in Spain, where Murcia and Alicante are its main production areas [on areas . This vaon areas , and is on areas . Analysion areas , which con areas , the useon areas . Among ton areas ,7.Different strategies are still being tested in this region in an attempt to optimize the use of available resources with respect to the environment and to improve the polyphenolic quality of the main vine varieties cultivated in order to obtain wines with better organoleptic and functional characteristics since the organoleptic properties of red wines depend on the phenolic compounds , and theAmong these strategies are clonal and sanitary selection programs of the Monastrell , deficitHowever, the increases achieved in the fresh skins of Monastrell have not been reflected in any great extent in the wines produced, as observed in a previous study . For thiVitis vinifera (syn. Mourvedre) red wine grapevines grafted onto 1103-Paulsen (clone 249) rootstock and trained to a three-vine vertical trellis system. Vine rows were arranged from N–NW to S–SE with the between-row and within-row spacing of 3 x 1.25 m .The experiment was carried out over two consecutive years (2016–2017) on the Monastrell variety located in Jumilla, Murcia (southeastern Spain). The study was performed on 14 year old v/v). Control plants were sprayed with aqueous solution of Tween 80 alone. The treatments were carried out twice, at veraison and 1 week later. When grapes reached technological maturity , they were harvested and transported in boxes to the winery for physicochemical analysis and vinification.All treatments were applied to three replicates and were arranged in a complete randomized block design, with 10 vines for each replication. The protocol used to apply the different treatments, as well as the doses used, have been described previously [Total soluble solids (°Brix) were measured using an Abbé-type refractometer (Atago RX-5000). Titratable acidity was measured using an automatic titrator  with 0.1 N NaOH. Tartaric and malic acids were measured using enzymatic kits from Boehringer 146 Mannheim GmbH . The methodology used to carry out these analyses is described in OIV  (2018) .2/100 kg). Total acidity was corrected to 5.5 g/L with tartaric acid, and selected yeasts were added . Two types of vinification were carried out: a pre-fermentative cold maceration (PFCM) followed by traditional maceration (TM), and another using only a traditional maceration.All vinifications were made in triplicate in 50-L stainless steel tanks using 50 kg of grapes, which were destemmed, crushed, and sulfited (8 g SOPre-fermentative cold maceration (PFCM): Tanks containing crushed grapes were placed in a cold room at 4 °C for 10 days, after which the tanks were returned to the winery to continue the traditional maceration.Traditional maceration (TM): All vinifications were conducted at 25±1 °C. The fermentative pomace contact period was 10 days. Throughout the fermentation pomace contact period, the cap was punched down twice a day, and the temperature and must density were recorded. At the end of this period, wines were pressed at 1.5 bars in a 75 L tank membrane press. Free-run and press wines were combined and stored at room temperature. The analyses were carried out at the end of alcoholic fermentation (AF) in triplicate.−1. Elution was performed with a gradient starting with 2% B to reach 32% B in 40 min, isocratic for 15 min, 50% B at 70 min, 60% B at 75 min and then isocratic for 5 min. Chromatograms were recorded at 280 nm (phenolic acids and flavan-3-ols), 320 nm (hydroxycinnamic acids and derivatives), 360 nm (flavonols), and 520 nm (anthocyanins). Anthocyanins were quantified at 520 nm as malvidin-3-glucoside, using malvidin-3-glucoside chloride as external standard . Phenolic acids and flavan-3-ols were quantified at 280 nm, and flavonols at 360 nm using the pure compounds as external standards. For the quantification of hydroxycinnamic derivatives, caftaric acid was quantified as caffeic acid and coutaric acid as coumaric acid.Grapes were peeled with a scalpel, and the skins were stored at –20 °C until analysis. Samples (2 g) were immersed in methanol (40 mL) in hermetically closed tubes and placed on a stirring plate at 150 rpm and 25 °C. After 4 h, the methanolic extracts were filtered through the 0.45 μm nylon filters and directly analyzed by HPLC, according to Gil-Muñoz et al. . The HPLWine was previously centrifuged and color intensity (CI), CIELab parameters, total phenols (TP) and total anthocyanins (TA) were analyzed by a Shimadzu UV–vis spectrophotometer, model 1600PC , as described in Paladines-Quezada et al. . CI was p < 0.05) when the ANOVA test was significant; and a multivariate discriminant analysis was applied to identify the most discriminant variables.Significant differences among wines and grapes and for each variable were assessed by analysis of variance (ANOVA) using the Statgraphics 5.0 Plus package . The Duncan test was used to separate the means  stimulated the polyphenol biosynthetic route, increasing the concentration of almost all the anthocyanins present in Monastrell , such asIn 2017, the rainfall between July and August A was greTo the greater precipitation of 2017 must be added a greater number of days with maximum temperatures above 30 °C 33 days; C compareHowever, during 2017, the grapes treated with MeJ + BTH were the only ones that presented a higher concentration of anthocyanins/gram of skin with respect to the control grapes , especiaAn analysis of flavonols present in grapes was carried out , since tIndependently of the treatments carried out, the results obtained in 2017 showed considerable increases in the concentration of isorhamnetin, as well as an important decrease in quercetin. As was mentioned above, the high temperatures that the grapes were exposed to this year C, togethThe treatments carried out in the field during 2016 did not influence the concentration of any flavonol with respect to the values of control grapes. However, in 2017, treatments with MeJ and MeJ + BTH halved the concentrations of myricetin, and the concentration of quercetin in grapes treated with MeJ + BTH was also substantially reduced, so that these grapes were the only ones that presented a lower concentration of total flavonols compared to the control. However in the same year, the treatments with MeJ, BTH and MeJ + BTH produced a substantial increase in the concentration of kampferol with respect to the control grapes.The results for the 2016 vinification with traditional maceration  were preThus, in 2016, wines made from grapes treated with MeJ and BTH using traditional maceration (TM) showed higher concentrations of total anthocyanins A, resultIn the 2017 season, only wines made with grapes treated with BTH and PFCM + TM showed increases in the concentration of total anthocyanins compared to the control A, providThe wine anthocyanin concentration obtained by HPLC  showed nThe wines elaborated using grapes treated with MeJ and TM during 2016  showed aThe wines in 2017 made from grapes treated with MeJ and MeJ + BTH, and elaborated with TM , presentThe wines produced by TM in 2016 using grapes treated with elicitors  did not In the 2017 vinifications using TM , wines mAs occurred in the comparison of anthocyanin profiles in grapes and wines, differences were found between the grape and wine profiles of flavonols, the grapes were produced using different agricultural techniques (the application of elicitors in our case) and react differently, depending on the climatic conditions, while the different processes of maceration may also have influenced the concentration of flavonols analyzed. It is therefore difficult to talk of a characteristic flavonols profile in Monastrell, such as that detected by Makris et al.  and MartTo improve the visualization and interpretation of all the results obtained, and to check whether the measured variables could distinguish between pre-established groups (vine treatments with elicitors and types of maceration), a multivariate discriminant analysis was made. This analysis revealed that, independently of the meteorological conditions during both campaigns, treatment with elicitors increased the concentrations of phenolic compounds in the treated grapes A; achievThe discriminant analysis between the two types of maceration used, allowed to distinguish between traditional maceration and that in which cold was applied, especially in 2016 B; showinThe higher precipitation, together with a greater number of days with temperatures above 30 °C between July and August 2017, induced variations in the processes of Monastrell maturation, affecting to a greater extent the response to the treatments in the vineyard, compared with 2016. These changes included an earlier beginning to veraison, increases in berry size (generating the dilution of phenolic compounds), the degradation or inhibition of anthocyanin synthesis, as well as an increase in some minor flavonols. Spectrophotometric measurements in wine samples showed that the application of the PFCM + TM technique allowed the phenolic compounds to be extracted to a greater extent in those grapes that increased their phenolic composition through the use of elicitors, giving wines with a higher index of total phenols, a greater intensity of color, and a lower luminosity. Finally, the discriminant analysis revealed that independently of the meteorological conditions during both years, the different treatments with elicitors induced increases in the concentrations of phenolic compounds in the treated grapes, allowing the distinction of the treatments. This analysis allowed the distinction between the types of maceration used, showing a higher proportion of phenolic compounds by the application of low temperature.


Ochotona princeps), representing the northern and southern Rocky Mountain subspecies, within Rocky Mountain National Park. The limited spatial extent of gene flow observed may be the result of geographic barriers to dispersal, selection against hybrid individuals, or both. Our fine-scale population genetic analysis suggests gene flow is limited but not completely obstructed by extreme topography such as glacial valleys, as well as streams including the Colorado River. The discovery of two subspecies within this single protected area has implications for monitoring and management, particularly in the light of recent analyses suggesting that the pikas in this park are vulnerable to fragmentation and local extinction under future projected climates. Future research should focus on the fitness consequences of introgression among distinct genetic lineages in this location and elsewhere, as well as within the context of genetic rescue as a conservation and management strategy for a climate sensitive species.Genetic variation is the basis upon which natural selection acts to yield evolutionary change. In a rapidly changing environment, increasing genetic variation should increase evolutionary potential, particularly for small, isolated populations. However, the introduction of new alleles, either through natural or human-mediated processes, may have unpredictable consequences such as outbreeding depression. In this study, we identified a contact zone and limited gene flow between historically separated genetic lineages of American pikas ( When tinction . Therefotinction , 6. Locatinction –7.Anthropogenic manipulation of genetic structure, whether intentional or accidental, also may influence adaptive potential. Translocations and augmentations, whereby individuals from one locality are moved to another location to found a new population or to supplement an existing population , 9 have Naturally-occurring hybrid zones among distinct and formerly isolated genetic lineages provide opportunities to learn more about introgression, such as how genes spread through admixed populations and the subsequent effects on physiology and behavior , 18. AmoOchotona princeps, within Rocky Mountain National Park (ROMO) and discuss the implications for conservation of the species within this management unit and elsewhere. American pikas are often considered a sentinel species with respect to climate change due to numerous observations of local extinctions, particularly in lower, drier, and hotter habitats [In this study, we describe a previously undocumented hybrid zone between two distinct genetic lineages of American pikas, habitats –30. Recehabitats . Pikas ahabitats . They arhabitats –36. Theyhabitats –40. Predhabitats . More rehabitats . Some pohabitats . Americahabitats  and werehabitats . Thus, uO. p. princeps) and Southern Rocky Mountain (O. p. saxatilis) lineages [Previous work based on morphology, dialect, and both mtDNA and nuclear coding sequence data identified a potential historic contact zone between the northern and southern Rocky Mountain lineages in the vicinity of ROMO –47. Estalineages . Given tlineages , the Amelineages , 51, 52,lineages , we expeIn this study, we focus on ROMO as the context for contact between the two Rocky Mountain subspecies, in comparison with data from two additional study sites, Grand Teton National Park (GRTE) and Great Sand Dunes National Park (GRSA). GRTE and GRSA fall within the geographic range of the northern and southern Rocky Mountain lineages, respectively  Fig 2)..Fig 2). et al. [et al. [mtDNA region for a subset of our samples from ROMO, plus one from GRSA and GRTE. Due to the low quality and quantity of fecal DNA template as compared to fresh tissue, we designed an additional five primers to amplify smaller regions ranging from 483–566 bp  initial denaturation; 39 cycles of 30 sec. denaturation (95°C), 45 sec annealing (60°C), and 30 sec (72°C) extension; and a final 5 minute (72°C) extension. We sequenced all DNA fragments in both directions and visually inspected sequence alignments using Geneious 6.1.2 .We extracted genomic DNA from fecal samples using a modified AquaGenomic DNA extraction protocol . We genotyped individuals at 24 microsatellite loci in four multiplex polymerase chain reactions (PCR) using a Qiagen Multiplex PCR kit . Detailed PCR protocol, primer sequences, and methods for calling and screening microsatellite genotypes are provided in Castillo et al. . In orde [et al.  to ampliStructure  to infer population structure within ROMO. We ran 10 replicates for each inferred number of populations (K = 1 to 10) totaling 100 replicates, with 100,000 MCMC steps each of burnin and run steps. We used eight regions within the national park, identified by geographic features, as sampling localities for location prior information in the admixture model, as well as correlated allele frequencies among populations as run parameters. We analyzed the model output using Structure Harvester  according to the ΔK method proposed by Evanno et al. [Clumpp  to determine the optimal assignment of individuals across the ten runs for the best supported value of K. Finally, we visualized the output from Clumpp spatially using ArcMap 10.0 .We performed a Bayesian clustering analysis in program o et al. . We compStructure and methods to interpret such analyses, particularly in the case of two genetic clusters [find.clusters function, to identify the optimal number of groups based on Bayesian Information Criterion (BIC).Given the limitations of clusters , we alsoclusters  using thclusters  in R [67clusters . We perfOchotona princeps individuals, plus one O. collaris, from GenBank (Table B in http://tree.bio.ed.ac.uk/software/figtree/).We obtained previously published sequence data covering the Cyt-b and D-loop region of the mitochondrial genome (c. 1650 bp) for 112 FST [D [h) and nucleotide diversity (π) for the ROMO mtDNA dataset using DnaSP [Once we identified two genetic clusters within ROMO see , we quanFST  using thFST  in R. AdFST [D , 74 usinFST [D  package FST [D  in R. Fing DnaSP .Structure analysis for K = 2 to equilibrium cline models using the Metropolis-Hastings Markov chain Monte Carlo algorithm implemented in the R package HZAR . We fit 15 candidate models that varied in the number of cline shape parameters estimated and selected the best model according to AIC corrected for small sample size. We were thus able to estimate the geographic center and width of the cline. Finally, we performed a hybrid detection analysis to estimate the probability that individual genotypes reflect genotype frequency categories corresponding to pure individuals, F1 or F2 hybrids, or backcrosses. We categorized individuals with Q > 0.99 in either northern or southern cluster as “pure” individuals and categorized all others as of unknown origin. We performed the hybrid detection analysis using the program newhybrids , implemented in the R package parallelnewhybrid , with 100,000 burnin reps and 500,000 sweeps. We performed ten replicate runs and averaged the posterior probabilities for each individual across all ten runs.We fit the admixture proportion values from the After removing individual samples that either failed to amplify, were contaminated (contained more than 2 microsatellite peaks at any locus), or gave inconsistent genotypes, our final dataset included 230 genotyped individuals from ROMO, 194 from GRTE, and 54 from GRSA. After screening loci for significant deviations from expected Hardy Weinberg proportions and removing loci that failed to amplify consistently across sites, we included 22 microsatellite loci. Number of alleles per locus across all four sites ranged from 5 to 28 (mean = 14).Structure analysis supported two genetic clusters within ROMO  had more intermediate values along the first principal component axis compared to individuals with higher Q values . Geograclusters . This waQ values .Structure analyses for the individuals assigned to the northern and southern clusters with Q ≥ 0.6. Within the northern group, there was support for three clusters, with one primarily in the northeast and another mostly restricted to the west of the Colorado River  . Likewise, microsatellite heterozygosity was similar among the study sites (h) and nucleotide diversity (π) calculated from mtDNA sequences was similar between ROMO N and ROMO S. We identified 7 haplotypes in each site. Genetic diversity was slightly higher in ROMO N  than ROMO S .Pairwise lineages  and incrlineages . FST was pattern . Microsady sites  and relaQ ≥ 0.8  and therefore robust to the K = 2 conundrum common in STRUCTURE analyses . Moreoveet al.  identifiet al. . In theiet al.  describeet al. –82, as cStructure analyses. However, admixture appears geographically limited to within a <10 km zone  to populations of Florida panthers (P. c. coryi) [Oncorhynchus clarki lewisi) be protected under the ESA and not hybrids with Yellowstone cutthroat trout (O. c. bouvieri) or rainbow trout (O. mykiss). While the ESA is a powerful management tool, it is often challenging to reconcile complex ecological and evolutionary processes with structured legal decisions.The presence of two distinct genetic lineages as well a hybrid zone within a single park boundary presents some interesting, and potentially complicated, implications for management of this species within ROMO. Previous research demonstrated that there are five major American pika genetic lineages, each with independent evolutionary trajectories . Galbrea. coryi) . In cont. coryi)  recommenS1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(CSV)Click here for additional data file.


Pinus kesiya Royle ex Gordon var. langbianensis (A. Chev.) Gaussen) is one of the most important tree species in the production of timber and resin in China. However, the genetic diversity of the natural populations has not been assessed to date. In this study, sequence related amplified polymorphism (SRAP) markers were used to investigate the genetic composition of natural Simao pine populations.Simao pine , the unweighted pair group method with arithmetic mean (UPGMA), and Principal coordinate analysis (PCoA).stG = 0.1701), and a slightly high level of gene flow (mN = 2.4403) were observed among populations using AMOVA. Eleven populations of Simao pine were gathered into four distinct clusters based on molecular data, and the results of UPGMA and PCoA also illustrated that assignment of populations is not completely consistent with geographic origin. The Mantel test revealed there was no significant correlation between geographic and genetic distance .The 11 populations revealed a high level of genetic diversity  at the species level. A moderately low level of genetic differentiation (The SRAP markers were very effective in the assessment of genetic diversity in Simao pine. Simao pine populations display high levels of genetic diversity and low or moderate levels of genetic differentiation due to frequent gene exchange among populations. The low genetic differentiation among populations implied that conservation efforts should aim to preserve all remaining natural populations of this species. The information derived from this study is useful when identifying populations and categorizing their population origins, making possible the design of long term management program such as genetic improvement by selective breeding. Pinus kesiya Royle ex Gordon var. langbianensis (A. Chev.) Gaussen), a geographic variant of Pinus kesiya, is distributed naturally in the humid and sub-humid areas of the tropical and subtropical zone within Yunnan Province  is a kind of molecular marker technology based on polymerase chain reaction (PCR). The method is convenient, exhibits high co-dominance, is uncomplicated in the separation of strips and sequencing, and doesn’t require knowledge of the sequence information of species in advance . The SRAIn this study, SRAP markers were used to investigate the genetic composition of natural Simao pine populations, which are limitedly distributed in Southwest Yunnan, China. The study’s aims were to evaluate genetic diversity at population and species levels in the Simao pine; assess the distribution of the genetic variation within and among populations, and construct a dendrogram demonstrating the genetic relationships among them. The genetic information can then be used as a tool for assessing the current conservation management plan for this species and for designing conservation strategies.n >  100), 30 adult individuals were selected within a distance of >  100 m. In small populations (n <30), all available adult individuals were sampled. For each sampled individual, young fresh pine needles (∼10 g) were collected, dried in silica gel, and stored at −80 °C until subsequent DNA extraction.Eleven natural populations of Simao pine with a total of 290 individuals were sampled throughout the species distribution range . Field eGenomic DNA was isolated from the pine needles of each individual tree according to the cetyltrimethyl ammonium bromide (CTAB) method . All gen2, primer 1 µM, dNTPs 0.2 mM, and 1U Taq DNA polymerase . The following cycling parameters were used for amplification: 4 min denaturing at 94 °C, five cycles of 94 °C for 1 min; 35 °C for 45 s and 72 °C for 1 min, 30 cycles of 94 °C for 1 min, annealing for 1 min, 72 °C for 1 min, and 5 min 72 °C for final extension. The PCR products were stored at −20 °C. Finally, amplification products were separated on 6% denaturing polyacrylamide gel and visualized by silver nitrate staining.Fifteen primer combinations , selecteP, %), Nei’s gene diversity analysis (H), Shannon’s information index (I), the observed number of alleles (Na), and the effective number of alleles (Ne). Total gene diversity (Ht), genetic diversity within populations (Hs) as well as the relative magnitude of genetic differentiation among populations (Gst), gene flow (Nm), and Nei’s genetic distance were also evaluated. Polymorphic information content (PIC) was calculated using a simplified formula  or absence (0) and transformed into a 1/0 binary character matrix. The binary data matrix was analyzed using the program POPGEN v.1.32  to estim formula .K values) was run from 1 to 20, with 10 independent runs for each K. The contribution to the genotypes of the accessions was calculated based on 105 iteration burn-in and 105 iteration sampling periods. Then, the optimal number of clusters K was identified following the procedure of Genetic differentiation within and among populations was estimated by the analysis molecular of variance (AMOVA) software package in GenAIEx 6.5 . To analmax), minimum monthly precipitation (Prmin), annual mean temperature (Tmean), maximum temperature of the warmest month (Tmax), and minimum temperature of the coldest month (Tmin) were extracted from Worldclim-Global Climate Data1 with ArcGIS 9.3 , maximum monthly precipitation  ranged from 77.27% (A) to 100% (C), with an average of 93.46%, and 100.0% at species level. The mean observed number of alleles (Na) ranged from 1.7727 to 2.0000, while the mean effective number of alleles (Ne) varied from 1.5469 to 1.8181. Nei’s genetic diversity (H) varied from 0.3089 to 0.4418, with an average of 0.3801, and Shannon’s information indices (I) ranged from 0.4511 to 0.6310, with an average of 0.5529. At species level, H and I were 0.4567 and 0.6484, respectively  of the species and genetic diversity within populations (Hs) were 0.4580 and 0.3801, respectively (Gst) was 0.1701, thus leaving 82.99% of the total genetic variation kept within the populations. The average Nm obtained was 2.4403, suggesting the existence of a certain degree of gene flow among natural distribution populations. This finding was consistent with the results of AMOVA, which detected the highest genetic variation within populations (86%), whereas the variance among populations was only 14% .The applied measure of genetic similarity was used to construct UPGMA dendrograms . The 11 Principal coordinate analysis (PCoA) was performed to determine the genetic relationships among the accessions with minimum distortion. The first two principal components displayed 37.69% and 60.12% of total variation, respectively, and the 72.74% was expressed by the first three components . CorrespK = 2, Q scores of 0.2 < Q < 0.8. The individuals in NE, JD, JG, ZY populations with Q values of >0.8 belonged to cluster I, and the ML, JH, Mla, SM populations with Q values of <0.2 belonged to cluster II. Individuals with admixed population assignments were from MJ, JC and LC populations.The results of the Bayesian clustering analysis of genetic structure showed that the populations analyzed of Simao pine, best fit two genetic groups K = 2, . The perBased on the ecological factors and genetic diversity parameters data, a Spearman nonparametric correlation coefficient matrix had been constructed by SPSS. The result showed no significant correlations between the genetic diversity parameters and geoclimatic variables.In this study, SRAP markers were used to evaluate levels of genetic variation within and among 11 natural populations of Simao pine in China. Previously, three natural populations of Simao pine genetic variation were investigated using nine enzyme systems . The repP) were used to measure genetic diversity. A high level of polymorphism was observed in numerous amplification products generated in the course of the analysis. In this study, PIC ranged from 0.43 to 0.77, with an average of 0.63, indicating that the SRAP markers could develop high loci polymorphism useful for genetic variation of accessions studied in this research (P) of Simao pine was 93.46%, revealing a considerable level of genetic diversity, similar to that of its relatives Pinus yunnanensis  was 0.4567 and Shannon’s information indices (I) was 0.6484, indicating that Simao pine had a high basis of genetic diversity. Previous studies also showed that narrowly distributed species could have higher genetic diversity (The PIC and polymorphism rate (research . The periversity .Ht) of the species and genetic diversity within populations (Hs ) were estimated to be 0.4580 and 0.3801 respectively. Compared to previous SRAP-based studies of pine plants, Simao pine showed higher diversity than Pinus taxa  was the ratio between the additive genetic variance among populations and the total additive genetic variance (Gst = 0.1701) among populations of Simao pine. This value was higher than the mean value (Gst = 0.073) recorded for 121 woody species examined using allozyme markers , which suggested no significant geographic restriction to gene flow among the populations. The reason for slightly higher Nm may be due to the morphological character of Simao pine, as tall plants benefit from reduced resistance to pollen movement in the air. A long-distance pollen flow also enhanced the gene recombination  was performed to provide spatial representation of the relative genetic distances among populations defined by cluster analysis. The results of UPGMA and PCoA also showed that the assignment of populations was not completely consistent with their geographic origin, a result that has been observed in previous studies . This fiK = 2) for Simao pine. Cluster I was composed of the NE, JD, and ZY populations from the northwest of the Simao pine natural range, collected from regions that had higher altitude  and latitude. The ML, JH, Mla, and SM populations which belonged to cluster II were collected from southern regions that had lower altitude  and latitude. These results possibly reflected an adaptational difference. It implied that geographical and environmental factors together created stronger and more discrete genetic differentiation than isolation by distance alone.A Bayesian cluster analysis performed with STRUCTURE, showed that the most probable number of genetic groups in the data was two (In the study, there were no significant correlations between the ecological factors and genetic diversity parameters according to Spearman nonparametric correlation analysis. It was suggested that the ecological factors had less effect on genetic diversity. This may be due to the limited distribution, and the small geographical and ecological differences of the studied populations. The high level of genetic diversity in Simao pine is mainly caused by the traits of long-life, generation overlap, wind pollination, and wind-seed dispersal, which lay a broad genetic base for the species .Retaining genetic diversity was the key to multi-generation improvement of forest trees and fine provenances should have an extensive genetic base and significant genetic gain . ThorougThe present study is understood to be the first genetic investigation of Simao pine using SRAP markers to understand distribution and genetic variation. The results indicated that SRAP markers can be efficiently used in the study of genetic diversity and genetic variability of Simao pine. The study revealed high levels of genetic diversity and low or moderate levels of genetic differentiation. Slightly frequent gene flow existed within Simao pine populations, weakening genetic differentiation among these populations. Based on these results, conservation strategies for Simao pine have been formulated.10.7717/peerj.6529/supp-1Data S1Click here for additional data file.10.7717/peerj.6529/supp-2Data S1Click here for additional data file.10.7717/peerj.6529/supp-3Data S1Click here for additional data file.10.7717/peerj.6529/supp-4Supplemental Information 1Click here for additional data file.10.7717/peerj.6529/supp-5Supplemental Information 2Click here for additional data file.10.7717/peerj.6529/supp-6Supplemental Information 3Click here for additional data file.10.7717/peerj.6529/supp-7Supplemental Information 4Click here for additional data file.


Since domestication, chickens did not only disperse into the different parts of the world but they have also undergone significant genomic changes in this process. Many breeds, strains or lines have been formed and those represent the diversity of the species. However, other than the natural evolutionary forces, management practices (including those that threaten the persistence of genetic diversity) following domestication have shaped the genetic make-up of and diversity between today’s chicken breeds. As part of the SYNBREED project, samples from a wide variety of chicken populations have been collected across the globe and were genotyped with a high density SNP array. The panel consists of the wild type, commercial layers and broilers, indigenous village/local type and fancy chicken breeds. The SYNBREED chicken diversity panel (SCDP) is made available to serve as a public basis to study the genetic structure of chicken diversity. In the current study we analyzed the genetic diversity between and within the populations in the SCDP, which is important for making informed decisions for effective management of farm animal genetic resources.Many of the fancy breeds cover a wide spectrum and clustered with other breeds of similar supposed origin as shown by the phylogenetic tree and principal component analysis. However, the fancy breeds as well as the highly selected commercial layer lines have reduced genetic diversity within the population, with the average observed heterozygosity estimates lower than 0.205 across their breeds’ categories and the average proportion of polymorphic loci lower than 0.680. We show that there is still a lot of genetic diversity preserved within the wild and less selected African, South American and some local Asian and European breeds with the average observed heterozygosity greater than 0.225 and the average proportion of polymorphic loci larger than 0.720 within their breeds’ categories.It is important that such highly diverse breeds are maintained for the sustainability and flexibility of future chicken breeding. This diversity panel provides opportunities for exploitation for further chicken molecular genetic studies. With the possibility to further expand, it constitutes a very useful community resource for chicken genetic diversity research.The online version of this article (10.1186/s12864-019-5727-9) contains supplementary material, which is available to authorized users. Chickens are of major and increasing importance for agricultural production as an efficient source of high quality protein. There have been concerns about loss of animal genetic resources and erosion of many genotypes due to crossbreeding or replacement by the high performing commercial hybrids resulting from highly efficient selection programs , 2. SuchDomestication history of chickens is still a matter of scientific debate, and has enjoyed the interest of researchers and scholars, from tracing the centers of domestication to exploring the archeology and dispersion of the chickens across different parts of the world –10. One In Europe, many local type breeds were developed mostly by intense selection and crossbreeding for desired phenotypic traits. In the nineteenth century, with an increasing popularity local strains maintained for centuries in Europe have been developed into standardized chicken breeds. At the same time, Asian breeds such as Cochin and Langshan were imported to Europe. In addition to keeping them as purebred populations, many new breeds evolved from crossing European breeds and newly imported Asian breeds following the European Poultry Standards . Fancy cwww.synbreed.tum.de) project. The SYNBREED chicken diversity panel used here consists of 174 chicken populations, representing four continents . The SCDP also includes broiler and layer purebred lines, as well as two wild populations . We have included some commercial lines in our analyses as representatives of the most favored stocks in breeding programs whose end products are distributed globally. They are not at risk for extinction, but may threaten local breeds by crossbreeding. We show their share of genetic diversity with a much wider spectrum of chicken breeds in SCDP set which these commercial lines do not cover. We have analyzed the genetic diversity within and between the populations and report here the current status of global genetic diversity based on this panel.The history of the origin of chickens together with management practices following domestication provides an important backbone to assess the genetic make-up and diversity between today’s chicken breeds. Low resolution studies of chicken biodiversity using microsatellites have shown that genetic diversity has been greatly affected by management practices. Highly selected layer lines, in particular white layers, showed reduced genetic variability while the wild type and less improved indigenous village chickens retained high genetic diversity , 26. In Gallus gallus gallus and Gallus gallus spadiceus, as well as samples of nine local breeds and four broiler lines taken from the previous EU project AVIANDIV  samples were collected from a wide range of chicken populations across the globe under the umbrella of the SYNBREED project (project lifetime 2009–2014). First, samples were collected from 80 fancy chicken breeds in Germany between 2010 and 2012. Fancy breeds are chickens which have been developed following hobbyists’ selection programs to create phenotypes which meet the requirements of the poultry standards (i.e. the European Poultry Standards). The German Association of Poultry Breeders  maintains a wide spectrum of traditional and fancy poultry breeds. They reflect various types of breeds of very different origins and breed histories according to the European Poultry Standards. Additional samples were collected from chicken breeds kept by farmers organized in “The Society for the Conservation of Old and Endangered Livestock Breeds ”. Blood samples were collected from the wing vein using EDTA as anticoagulant. Sampling was carried out in strict accordance to the German Animal Welfare regulations, and notice was given to the authorities of Lower Saxony according to § 8 of the German Animal Welfare Act  and with the written consent of the animal owners. The collection was completed by samples of two Red Jungle Fowl populations, see also ). In addAfter 2012, the panel was complemented with DNA samples of 71 populations from 22 countries provided by partners (see Table The populations were classified into twelve categories based on their continent of origin and/or type as shown in Table DNA samples were genotyped with the Affymetrix® Axiom™ Genome-Wide Chicken Genotyping Array encompassing over 580 K SNPs . Genotyp2 (pruning of markers with a pairwise r2 of greater than 0.2), respectively, leaving 123,273 SNPs for further analysis. Furthermore, imputation was performed on the remaining SNPs using Beagle 3.3 [In total, genotype information for 580,961 SNPs was obtained from the array. 579,621 of the SNPs were annotated using the genome assembly Gallus_gallus-5.0 . We deleagle 3.3  to recovWe estimated Reynolds’ genetic distances  between We performed a principal components analysis (PCA) using SVS. Because of the large number of 3235 individuals, we calculated the average principal component (PC) scores for each population to make their positions in the PCA plot presentable. We then plotted the average scores of each population for PC 1 & 2 with different colors to highlight the breeds’ categories.We evaluated the relatedness of the populations through admixture analysis using ADMIXTURE 1.3 software . ADMIXTUHo) and expected (He) heterozygosity were used to evaluate the genetic diversity within populations. The observed heterozygosity was calculated directly, while the expected heterozygosity was estimated as: He = 2q(1 − q) where q was the frequency of one of the alleles [He and Ho estimates for all individuals within each population were averaged over all SNPs. Because of low sample sizes and the fact that a number of the populations did not form mating groups, the calculated expected heterozygosity values should be treated with caution. Consequently, for many of the breeds we avoided making conclusions based on the Hardy–Weinberg principles.Genetic variability measures such as proportion of polymorphic SNP loci, levels of observed . Cluster 3 encompasses mainly breeds from the Middle East and geographically nearby areas, sampled in Saudi Arabia, Egypt, Pakistan, Israel, Sudan, Ethiopia, Turkey, and Italy. The close relationship of the breeds in this cluster is likely due to their neighboring geographic distribution and distribution routes of chickens in these regions. The NJ tree further shows a very small cluster (cluster 4) which consists of two populations of Vorwerkhuhn (VWco and VWcoE) and Lakenfelder (LAco). Vorwerkhuhn was recognized as a standardized breed in Germany in 1919 and one of the founder breeds was the Lakenfelder breed. Cluster 5 consists of European bantam breeds as well as some Asian game birds which were sampled in Germany. Cluster 6 consists exclusively of chickens sampled in Finland. Following this group, several populations of European background were arranged in the middle of the tree, but were not forming a visually distinct cluster. They were found between cluster 6 and 7. Among these breeds there were three populations of the Araucanas. Cluster 7 branches into two sub-clusters with African populations on the one side and South American Mapuche populations on the other. Among the African populations, there were two Tanzanian ecotypes (MOxx and CWxx). Though five ecotypes from Tanzania were included in this study, they did not cluster together in the NJ tree. The remaining three breeds from Tanzania clustered with populations in cluster 9. The second sub-cluster including the South American sub-cluster also contained some populations from Eastern Europe .In cluster 8 commercial brown layers and broilers are found. Close to the four commercial purebred brown layer lines (BL_A-D), there were also two lines of New Hampshire (NHL68 and NHbr) and the fancy breed of Rhode Island Red (ROro). Two of the brown layer lines (BL_A and BL_B) originated from the breed Rhode Island Red while the other two lines (BL_C and BL_D) are based on White Plymouth Rock. New Hampshires may have formed a part of the dam lines used in the development of brown layer lines . The PlyAverage scores of each population for PC1 versus PC2 are shown in Fig. It is noteworthy that the second PC could be related to the breed’s body size. This is because the PC2 shows a transitioning of mainly the small sized (mostly bantams) birds at the lower part of the PCA plot and the normal sized birds towards the upper part. However, the separation is much clearer for the European type breeds than for the Asian types.Admixture analysis results for K = 2 and K = 5 are displayed in Fig. In agreement with the PCA results, the admixture exhibited a gradually increasing separation of breeds from European (green) background from breeds of Asian (red) background with K = 2, with the African and South American breeds situated in the middle of the spectrum. The commercial white layers were completely homogeneous in the European gene pool (green) at K = 2 while the brown layers and broilers were admixed, however, with more proportion of the Asian ancestry cluster. In the NJ tree, cluster 3 is made up of populations from Asia  and Africa  clustering in the middle of European clusters. On the admixture plot  which is not shared among many breeds. Thereby, the White Leghorn line LER11) was more affiliated to this white layers’ cluster, while the other populations which clustered with the commercial white layer populations in NJ cluster 1  than those of European background which constitute a large proportion of the European (green) cluster. This suggests a higher diversity within the Asian breeds than within the European breeds.p), observed (Ho) and expected (He) heterozygosity for each population are shown in Table S1.In Fig. p within category) = 0.394  of polymorphic loci, i.e. the Leghorn line (LER11), and the Hamburger silver spangled (HAsl) and the Jaerhoens (JAExx) breeds.The proportion of polymorphic loci was lowest in commercial white layers with p= 0.483, Table S1) while the remaining populations of this group displayed average values above 0.600. The Totenko black breasted red (TOgh) and Koeyoshi (KYswi), both breeds of Japanese origin, were outliers among the Asian breeds sampled in Germany with a very low proportion of polymorphic loci . They formed a homogeneous cluster in the admixture analysis plot  had a higher proportion of heterozygous SNPs , with some contribution from Gallus sonneratii in Southwest India [Gallus lafayettii in Sri Lanka  or sampled in Germany from fancy breeders grouped together in the NJ tree  as well as in the PCA plot, but separated from the majority of the European breeds which segregate in NJ clusters 1 and 2. The wild populations fitted well into the Asian cluster. They display high levels of genetic diversity as shown by the levels of heterozygosity, SNP polymorphism and their high admixture. This finding is in agreement with the widely accepted opinion that chickens were first domesticated in Asia, predominantly from the Red Jungle Fowl , aniewed by , 38. TogThe Asian breeds covered a huge spectrum of genetic diversity. Despite some of the breeds being sampled in Germany (see ‘http://www.fao.org/dad-is/browse-by-country-and-species/en/). Indeed the Swiss chicken, Transylvanian Naked Neck and Hungarian Yellow do show slightly higher levels of observed heterozygosity than expected which may suggest possible crossbreeding.The European breeds sampled in Germany clustered very well with the rest of the European breeds. Consistent with that, breeds of European origin are represented close together in the PCA plot, distinct from the breeds of Asian origin (PC1). The second PC distinguishes bantam breeds from large chicken breeds in the European gene pool. The Iron Age is assumed to be the main period for dispersion of chickens through Europe. Our results suggest that the majority of breeds categorized as typical European breeds according to the European Poultry Standards (those categorized as DE_Europe and DE_Europe_Ban) have been little or not exposed to crossbreeding with Asian breeds as they do not overlap with Asian breeds. However, there are some exceptions for local breeds sampled in Europe. In the PCA plot Figure , a few bChickens in Africa and South America originated from both Asian and European chickens , 9. NoneThe split of African breeds between both Asian and European clusters supports the reports on their origin from both an Asian and a European origin . MitochoSouth American breeds were exclusively represented by the Mapuche chickens in this study. These populations showed a good share of affiliation with the Chinese populations at K = 5 Figure  of the aGallus gallus species and also had the lowest genetic diversity among the local Asian populations of its respective NJ cluster 9. This is probably the result of a limited number of breeding individuals and the absence of a continuous gene flow from other breeds while trying to keep the breed pure (as it is for many fancy breeds in Germany) for its interesting fibromelanosis trait. Another examples are the Leghorn line (LER11) and Hamburger breeds which have the lowest genetic diversity within the European categories. The LER11 is a White Leghorn line which has been kept as a closed population at least since 1965 when it came to the Institute for Small Animal Breeding in Celle (Germany) and was most likely based on a narrow gene pool . The Hamburger breed is a fancy breed with a small effective population size [The highest genetic diversity was observed within populations sampled in Africa, South America and Asia, some of which exhibited even higher diversity than the wild populations. Generally, the local type breeds from the four continents exhibit more genetic diversity than those from German fancy breeders and commercial layer lines Figure . The locion size . AdditioWith respect to commercial purebred lines, white layers are clearly distinct from brown egg layers, while broiler lines cluster more in the center Figure  and closMeasures are needed to preserve genetic diversity, between and within breeds. There is a large spectrum of between breed diversity preserved in the local breeds from different origins and the fancy breeds from Germany. Additionally, the local chickens have proven to be great reservoirs of within breed genetic diversity. However, the low genetic diversity within the fancy breeds, and the non-structured, non-monitored breeding programs of local breeds raised concerns about their vulnerability to go extinct , 42, 53.In this study we assessed genetic diversity between and within breeds from chickens collected across the world, from various backgrounds. It is very evident that the origin, geographic expansion, selection and different management practices have had a major impact on the global pattern of chicken diversity. Overall, the commercial white layers had the lowest variation among the commercial lines, the bantams displayed lower genetic diversity than the normal sized breeds in the respective category of origin ; and breeds that were sampled in Germany (both European and Asian breeds) had lower genetic diversity than those sampled in various places in the respective continent of origin. At the current state, the commercial breeding lines seem to have not yet reached selection plateau in the current breeding programs and are still responding to the breeders’ objectives. However, the limited diversity they cover (as shown by PCA and NJ tree), and the very low within breed diversity, in particular within the white layer purebred lines, might limit the flexibility to respond to unforeseen future needs. There is still more genetic diversity within the less selected African, South American and some local Asian and European breeds. Therefore, it is required that genetic diversity in these chickens be maintained in order to have the opportunity to respond to future challenges.http://www.imageh2020.eu/index.php), with collaborative effort to characterize and manage genetic diversity in livestock and poultry species. Not only is this panel the biggest gene pool of chicken data by far; it also has the potential to expand as new breeds and other sources of genetic materials will be added from other parts of the world. The SCDP data set presents ample opportunities for exploitation for further chicken molecular genetic studies and is made available for public access .As conservation measures are costly, it was stressed by  that “coAdditional file 1:Table S1. Population information, population diversity measures and origin of DNA samples. (DOCX 88 kb)Additional file 2:Document S1. Description of clusters. (DOCX 46 kb)Additional file 3:Figure S1. Admixture Analysis. (PDF 366 kb)


Curculio spp.) predating in hazel and oak mixed forests; and (ii) the consequences that trophic breadth and host distribution have in the genetic structure of the weevil populations. DNA analyses were used to establish unequivocally host specificity and to determine the population genetic structure. We identified 4 species with different specificity, namely Curculio nucum females matured earlier and infested a unique host  while 3 species  predated upon the acorns of the 2 oaks (Quercus ilex and Quercus pubescens). The high specificity of C. nucum coupled with a more discontinuous distribution of hazel trees resulted in a significant genetic structure among sites. In addition, the presence of an excess of local rare haplotypes indicated that C. nucum populations went through genetic expansion after recent bottlenecks. Conversely, these effects were not observed in the more generalist Curculio glandium predating upon oaks. Ultimately, co‐existence of weevil species in this multi‐host‐parasite system is influenced by both resource and time partitioning. To what extent the restriction in gene flow among C. nucum populations may have negative consequences for their persistence in a time of increasing disturbances (e.g. drought in Mediterranean areas) deserves further research.Synchrony between seed growth and oogenesis is suggested to largely shape trophic breadth of seed‐feeding insects and ultimately to contribute to their co‐existence by means of resource partitioning or in the time when infestation occurs. Here we investigated: (i) the role of seed phenology and sexual maturation of females in the host specificity of seed‐feeding weevils ( Curculio spp.) in mixed forests. Integrative Zoology13, 267–79.Arias‐LeClaire H, Bonal R, García‐López D, Espelta JM (2018). Role of seed size, phenology, oogenesis and host distribution in the specificity and genetic structure of seed weevils .Supporting InformationClick here for additional data file.