A commercial formulation of poly(tetrafluoroethylene) (PTFE) sheets were surface modified by using non-thermal air at 40 kHz frequency (DC) and 13.56 MHz radiofrequency (RF) at different durations and powers. In order to assess possible changes of PTFE surface properties, zeta potential (ζ), isoelectric points (IEPs) determinations, contact angle measurements as well as Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) imaging were carried out throughout the experimentation. The overall outcome indicated that ζ-potential and surface energy progressively changed after each treatment, the IEP shifting to lower pH values and the implicit differences, which are produced after each distinct treatment, giving new surface topographies and chemistry. The present approach might serve as a feasible and promising method to alter the surface properties of poly(tetrafluoroethylene). Furthermore, plasma treatment is a very versatile technique, since various carrier gases may be employed, giving unique features to the treated material ,2,3,4,5.The strong cohesive force between fluorine and carbon makes poly(tetrafluoroethylene) an inert and nonstick material. Except for other PTFE-like molecules, there is no other molecule that adheres to it ,7,8. OneThe aim of this contribution was to make a comprehensive assessment of the effect of air plasma treatment on PTFE commercial sheets by using 40 kHz (DC) and 13.56 MHz (RF) plasma discharges at different plasma duration and power inputs. This outcome is supported by surface probe techniques, such as Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), zeta potential analysis and contact angle measurements. The extensive and indispensable use of PTFE in our daily life underlies the motivation of choosing this material as the target of this work.® unit, Milipore S.A.S, Molsheim, France, equipped with dual wavelength 185/254 nm UV lamp. The ultra-pure water was used for all experiments and solutions with a water resistivity of 18.2 MΩ at 25 °C and total organic carbon content lower than 5 ppb. Potassium chloride KCl 99%, sodium hydroxide NaOH 98%, anhydrous ethylene glycol C2H6O2 99.8% and diiodomethane CH2I2 99% were purchased from Sigma-Aldrich,  USA. Hydrochloric acid HCl 35% was obtained from Penta, Prague, Czech Republic. The reactants were used as received without any further purification.Poly(tetrafluoroethylene) (PTFE) was obtained from Dow Chemical Company, . Distilled-deionized water was retreated in a Simplicity UV2)  indicating silica gel .Both sides of 5 × 5 × 0.1 cm PTFE foils were exposed to non-thermal air plasma by using the following plasma reactors: Pico  with a cylindrical chamber of 150-mm inner diameter Ø and 320 mm length operated at a frequency of 40 kHz, hereinafter (DC). Pico  Ø 150 mm and 320 mm length operated at a frequency of 13.56 MHz, referred to as (RF). The power inputs were 10, 20 and 50 W. The treatment durations were 0, 1, 2, 5, 10 and 20 min respectively, and the pressure in every experiment was 40 Pa. Once the treatment was completed, the specimens were withdrawn from the plasma reactor and immediately used for the next experiments. The foils were stored in a vacuum desiccator  with stopcock, porcelain plate and cobalt chloride  system equipped with a CCD camera . Deionized water, ethylene glycol and diiodomethane were used as testing liquids at 22 °C and 60% relative humidity. The droplets volume was set to 5 μL for all experiments. Every representative contact angle value was an average of 10 independent measurements. The substrate surface free energy was evaluated by using the acid-basic model.The ζ-potential of the sample surfaces was determined by using a SurPASS electrokinetic analyzer  with a clamping rectangular measuring cell as the one shown in −1 and at resonance frequency of 170 kHz. In order to obtain a reliable result, the average surface roughness was obtained from different spots of the samples. The Scanning Electron Microscope (SEM) was carried out by using a Nova NanoSEM 450  with Schottky field emission electron source operated at acceleration voltage ranging from 200 V to 30 kV and low-vacuum SED (LVD) detector. A coating with a thin layer of gold was performed by a sputter coater SC 7640 .The surface morphology was evaluated by using an Atomic Force Microscope (AFM) Solver PRO . The surfaces were analyzed with standard Si cantilever with a constant force of 10 N·mIn order to estimate the extent of plasma treatment at different treatment times and powers, the PTFE specimens were assessed by contact angle measurements. With regard to the surface energy and surface wettability assessments, the contact angles of water, ethylene glycol and diiodomethane are listed in The surface charge with respect to the plasma duration was appraised by electrokinetic analysis, where the ζ-potential is an indicator for charge formation at the solid-liquid interface and the surface charge is generated by the interaction of the solid surface with the electrolyte solution. Likewise, the isoelectric points (IEPs) are defined as the pH at which a substance has a net charge of zero, or at which it is at its minimum ionization. Concerning the topographical patterning, the scanning electron microscopy (SEM) images along with the AFM ones of the examined specimens are presented in 2. The superhydrophobic character of PTFE plummeted after using air plasma treatment, demonstrating the capability of plasma treatment for surface modification [Solid surfaces may be classified into two basic groups, hydrophilic (wettable with water and high surface energy) and hydrophobic (not wettable with water and low surface energy). The contact angles of the employed liquids on the studied surfaces diminished. For instance, the contact angle of deionized water decreased within the range 74–88°, which indeed indicates a surface wettability change. The typical water contact angle value of poly(tetrafluoroethylene) (PTFE) is ≥105 and its surface energy is typical 20 mJ/mfication ,19,20. Ofication ,22.PTFE has a relatively low surface energy and its total surface energy increased regardless of the treatment. In the case of DC, the highest values correspond to the foils exposed to 20 min treatment with a power input of 50 W; nevertheless, there is no drastic surface energy change if shorter plasma durations are applied, and 1 min of treatment is enough for a cost-efficient surface modification ,24,25. TAs seen from the electrokinetic analysis, which was performed to evaluate the surface charge and to determine the isoelectric points of the studied sample, all the plots have negative values, and these negative charges increased after 20 min RF plasma treatment; conversely, the curve of 20 min RF shifted towards higher pH values. PTFE treated under DC holds the most negative ζ-potential values, which correspond to the highest surface charge. The untreated PTFE showed a value around pH 3.4, which coincides with the IEP found in literature . The stuWith respect to the SEM images, both the untreated sample and the treated ones have wavy areas, and all studied sheets possessed scratches. These anomalies may come from the processing line or an inadequate storage. Nevertheless, the untreated film is relatively smoother and its morphology is more uniform than the treated ones. These alterations may be observed in more detail with the Atomic Force Microscopy (AFM) microphotographs, where the surface topography changes following the PTFE films’ exposure to air plasma treatment are more visible. The treated samples depict relatively rougher morphologies, with the sample treated under DC power being the roughest, with etched features and irregularly shaped textures compared with the untreated film. This may be substantiated in We conclude that the non-thermal plasma sources used in this work are suitable for the surface modification of poly(tetrafluoroethylene). The superhydrophobic character of PTFE is transformed after air plasma treatment. Different treatment times and powers were employed, and as far as this contribution is concerned, plasma treatment at 40 kHz is the most efficacious system and it is in agreement with previous studies. Short duration plasmas are cost-efficient methods to enhance surface properties without causing any negative impact on the treated samples. Surface charge and surface energy have been succinctly characterized by surface probe techniques. All the results demonstrate how the surface charge is gradually changed, and provide the moment that chemical saturation and thermal degradation begin. Surface energy increases with increasing treatment time. Physical and chemical changes are clearly manifested by the rise of surface charge after RF plasma treatment; likewise, the isoelectric point of treated PTFE after DC plasma treatment is lower than the untreated one. The microphotographs illustrate the surface morphology and the etching effects of the treatment, which are corroborated by the Δmass of the treated specimens. This contribution underlines the use of plasma treatment as a reliable tool for surface modification and functionalization.


Peltigera, section Chloropeltigera. We sequenced six loci of the main fungal partner and performed species discovery and validation analyses to establish putative species boundaries. Single locus phylogenies were used to establish the identity of both photobionts, Nostoc (cyanobacterium) and Coccomyxa . Distribution and specificity patterns were compared to the closely related clade, section Peltidea, which includes mainly Peltigera species with trimembered thalli. For section Chloropeltigera, eight fungal species (including five newly delimited putative species) were found in association with nine Nostoc phylogroups and two Coccomyxa species. In contrast, eight fungal species (including three newly delimited putative species) in section Peltidea were found in association with only four Nostoc phylogroups and the same two Coccomyxa species as for section Chloropeltigera. This difference in cyanobiont biodiversity between these two sections can potentially be explained by a significantly higher frequency of sexual reproductive structures in species from section Chloropeltigera compared to section Peltidea. Therefore, horizontal transmission of the cyanobiont might be more prevalent in Chloropeltigera species, while vertical transmission might be more common in Peltidea species. All Peltigera species in section Chloropeltigera are generalists in their association with Nostoc compared to more specialized Peltigera species in section Peltidea. Constrained distributions of Peltigera species that associate strictly with one species of green algae (Coccomyxa subellipsoidea) indicate that the availability of the green alga and the specificity of the interaction might be important factors limiting geographic ranges of trimembered Peltigera, in addition to constraints imposed by their interaction with Nostoc partners and by climatic factors.Species circumscription is key to the characterization of patterns of specificity in symbiotic systems at a macroevolutionary scale. Here, a worldwide phylogenetic framework was used to assess the biodiversity and symbiotic patterns of association among partners in trimembered lichens from the genus  Delimitation of fungal species is paramount to our understanding of the level of specificity among symbiotic partners in lichens and the processes shaping their evolutionary history e.g., . ImprovePeltigera Wild  includes bimembered and trimembered species. The former are associations between mycobionts and cyanobacteria (cyanobionts) from the genus Nostoc, while the latter comprise these two partners and green algae (phycobionts) from the genus Coccomyxa. In trimembered thalli the phycobiont is the main photobiont, whereas Nostoc is restricted to external cephalodia. Molecular systematic studies of Peltigera confirmed and clarified established infrageneric phylogenetic relationships  and species richness seems to be underestimated . While several studies addressed the identity and phylogenetic relationships of lichenized Nostoc , Peltidea  and Phlebia (one known species: P. venosa), include all known trimembered Peltigera species and are composed almost exclusively of trimembered species  includes at least three well-supported monophyletic groups, most likely representing putative cryptic species. To evaluate cyanobiont diversity associated with Peltigera thalli, strongly supported monophyletic groups (Nostoc phylogroups), serving as proxies for species, were circumscribed within a broad phylogenetic context of the genus Nostoc  in association with species of the three trimembered sections of Peltigera, each species showing a different degree of specificity toward Nostoc phylogroups. An unusually high level of reciprocal specificity between Nostoc and P. malacea was detected by Peltidea could potentially represent new species. However, none of them were formally described due to the lack of diagnostic phenotypic features, the need for a more extensive sampling, and the need to assess the level of gene flow among these putative species. Therefore, the limited span of the phylogenetic, geographical, and molecular sampling of past studies, as well as the lack of information about the third partner (Coccomyxa), did not allow a comprehensive understanding of the spatiotemporal interactions among symbionts shaping trimembered Peltigera species.Three  species . Accordievations . Based os Nostoc . O’BrienPeltigera is bimembered (one mycobiont in association with one cyanobiont), which is also the case for the entire order Peltigerales (Nephroma), some evidence suggests that the evolutionary acquisition of a green algal partner by bimembered cyanolichens was accompanied by a change in cyanobiont composition , from section Chloropeltigera (and for selected representatives from section Peltidea), to generate a worldwide phylogeny for this section. We discuss the potential contribution of various biotic and abiotic factors  in shaping the spatial and evolutionary patterns of symbiotic association and levels of specificity among the three main partners of these lichens.In this study we assembled a multilocus dataset for Chloropeltigera were chosen across its global distribution  for this study. These individuals were selected from herbarium material and collected by the authors and collaborators during fieldtrips in China, Russia, Norway, Iceland, Canada, and the United States. Additionally, 32 specimens from section Peltidea used in Ninety-two specimens from section tubulin (β-tub) using primers bt_34F (RPB1) using primers RPB1-AF  was amplified using primers CX and CW . The following PCR settings were used: 94°C for 1 min; 35 cycles of 94°C for 30 s, 55°C for 30 s and 72°C for 1 min; and a final elongation at 72°C for 10 min.The X and CW  followinTM , 2.5 μL of BSA, 15 μL of double-sterilized ultrapure H2O and 1 μL of DNA. All PCR products were cleaned and sequenced as described in All PCR reactions were carried out in a final volume of 25 μL containing 2.5 μL of buffer, 2.5 μL of 10 mM dNTPs, 1.25 μL of 10 mM primers, 0.15 μL of TargeTaq1 with the Q-INS-i strategy. Alignments were then adjusted manually using MacClade v. 4.08  a Nostoc matrix with a total of 425 rbcLX sequences representing a broad sampling of published symbiotic and free-living Nostoc from Nostoc Clade I, and Clade II subclades 1, 2, and 3 (sensuCoccomyxa matrix consisting of 81 new ITS sequences generated as part of this study and 70 sequences from the latest systematic revision of this genus (Coccomyxa found in association with Ascomycota (ascolichens) and Basidiomycota (basidiolichens); and (4) a concatenated matrix for single representatives of each of the 17 species from sections Chloropeltigera, Peltidea and Phlebia for which sequences from the same four loci were available . Perl scripts “compare_and_choose.pl,” “adapt_from_file.pl,” and “exclude_and_adapt.pl”  were used to prepare the concatenated matrices used in this study. All alignments and phylogenies were deposited in TreeBASE2  a mycobiont concatenated matrix with 42 ingroup taxa comprising all specimens from section  3 sensu, includiis genus , includireeBASE2 .tubulin 1st, 2nd, 3rd codon positions and introns, RPB1 1st, 2nd, 3rd codon positions and intron, COR1b, COR3 and COR16 for the concatenated mycobiont matrix; (2) rbcL 1st, 2nd, 3rd codon positions and rbcX 1st, 2nd, 3rd codon positions for the Nostoc matrix; (3) ITS1, 5.8S and ITS2 for the Coccomyxa matrix; and (4) ITS1, 5.8S, ITS2, β-tubulin 1st, 2nd, 3rd codon positions and introns, COR1b and COR3 for the matrix covering three sections of the genus Peltigera. Maximum likelihood (ML) searches for the most likely tree and bootstrap replicates (1000) were performed on each dataset using RAxML HPC2 v. 8  for all known trimembered Peltigera lichens.The most splitting delimitation scheme inferred with the discovery methods was set as prior for species validation in Bayesian Phylogenetics and Phylogeography (BP&P)  using thNostoc phylogroups as a function of the number of samples per section (Peltidea and Chloropeltigera). The R package iNEXT v. 1.0  was computed for each species using equation 1.We examined 253 well-preserved specimens . A linear regression and a statistical correlation test were done in R considering all species with more than 10 available samples. We used the R package iNEXT for the estimation of the rarefaction curves and for comparing the distribution of the number of ITS haplotypes as a function of the sample size per section between Chloropeltigera and Peltidea. Fifty bootstrap replicates were completed to generate a 95% confidence interval.where Chloropeltigera comprised eight well-supported lineages , which might represent species . Newly delimited lineages within P. leucophlebia s. l. were well supported , except for the monophyly of P. leucophlebia 2, which received a bootstrap support of 54%  delimited the same species as the BP&P analysis restricted to section Chloropeltigera (result not shown).Based on the BP&P species validation method, section s Figure . P. leuca Figure . Most an. Figure  into fiv% Figure . The mon. Figure . Four of. Figure . The bGMNostoc rbcLX sequences, obtained from the three sections of the genus Peltigera with trimembered lichens, clustered in 12 monophyletic groups (Nostoc phylogroups) in the ML tree   studied by us was identified as a rare Nostoc phylogroup (XLIV). Phylogroups (III and IV) from Nostoc clade 2 subclade 2 .) Figure . SpeciesCoccomyxa solorinae and C. subellipsoidea were present in all three Peltigera sections without any obvious patterns of specificity toward a single fungus or cyanobiont  and South East Asia (SEA), respectively  was found between the proportion of specimens with apothecia and ITS haplotype diversity (H) in species from sections Chloropeltigera and Peltidea  was nearly twice as large as for section Peltidea  than most species from section Peltidea  of these lineages within this section  . These results can serve as a starting point for a study designed to assess gene flow. The apparent lack of distinguishing phenotypic traits among these putative species could result from gene flow and may be reflected in species population dynamics.Previous studies have already shown the putative cryptic biodiversity within section eltigera . Based on Figure . Togetheunctata) , sectionNostoc phylogroups  consorted with Nostoc phylogroup I, a finding not confirmed by us perhaps due to low sampling. The remaining associations between the mycobiont species and Nostoc phylogroups reported by Chloropeltigera and Peltidea were confirmed and expanded by our study. Phylogenetic branching and very short internodes recovered for most of the phylogroups suggest a predominantly clonal population structure , while C. subellipsoidea, previously reported only from the basidiolichen genus Lichenomphalia  than in Nostoc (12 phylogroups) associated with 17 putative Peltigera species in these three sections , it is likely that additional factors are related to the snow tolerance displayed by P. chionophila. It was previously demonstrated that photobiont switches and associations with a higher number of photobionts  can be associated with the colonization of new niches and geographic regions by the mycobionts, followed by a subsequent increase in specificity   along altitudinal gradients  . Howeverciation) . Within , Canada . Thereforadients  support radients . This caradients  coupled radients .Peltigera species form bimembered thalli with Nostoc as their only photobiont, and they can be found in all biomes (Peltigera species (so far not reported from the Southern Hemisphere) seem limited by the ranges of the green alga Coccomyxa, which appears to be adapted to cold environments  are known to associate with Peltigera species from other sections, which also occur outside of the boreal biome  are highly specific toward each other, confirming, based on a broader geographic sampling, high reciprocal specificity between Nostoc phylogroup III and P. malacea  reported by Collemataceae  by a Nostoc from phylogroup III  have been shown to vary in their specificity depending on macroclimatic variables (Peltigera species studied here generally do not occupy geographic regions with contrasting macroclimates, it is possible that microclimatic differences exist within the distributions of species from different sections, and that such differences might affect symbiont availability and the association patterns. However, a co-occurrence study of the genus Peltigera and their Nostoc partners along a longitudinal and latitudinal transect of nearly 1300 km each, in the boreal biome, revealed that bioclimatic factors at this spatial scale were more limiting for the ranges of Peltigera species than the availability of their Nostoc partners (Several mechanisms have been proposed to explain differences in specificity in lichens. Differences in rate of mycobiont diversification have been shown to affect specificity in dactylon . Howeverdactylon , but as 3 Figure . These pal scale . For comariables . Althougpartners .Peltidea and Chloropeltigera, and their ITS haplotype diversity. Although our molecular sampling was not designed to test directly the prevalence of different types of reproduction, we found a significant positive correlation between the frequency of occurrence of apothecia and ITS haplotype diversity of the mycobiont (Figure Chloropeltigera than in section Peltidea (Figures Nostoc has been shown for a species pair in Degelia (Collema and Leptogium species with contrasting modes of reproduction (Peltigera in section Polydactylon. However, when this hypothesis was tested in bimembered lichen-forming fungi associated with green algae, their reproductive mode was not a good predictor of the level of specificity (Nostoc but not toward Coccomyxa (Figure Modes of reproduction can drive variation in specificity because it translates directly into how photobionts are transmitted from one generation to the next . We lookt Figure . We used Figures . We expe Degelia  as well oduction . Magain cificity . Our fina Figure . It has a Figure ; howeverFL, JM, NM, TG, and CP-D conceived the study. FL, JM, TG, and NM collected the samples. CP-D and NM obtained the molecular data. CP-D performed the phylogenetic and statistical analyses. CP-D, NM, FL, TG, SR, and JM wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


P < 1.54E-06) and 35 suggestive (P < 3.08E-05) single nucleotide polymorphisms (SNPs) were detected. Two pleiotropic quantitative trait loci (QTLs) on SSC 1 and SSC 7 were found to affect more than one trait. Markers WU_10.2_7_18377044 and DRGA0001676 are two key SNPs for these two pleiotropic QTLs. Marker WU_10.2_7_18377044 on SSC 7 contributed 2.16 and 2.37% of the observed phenotypic variance for DFI and RFI, respectively. The other SNP DRGA0001676 on SSC 1 explained 3.22 and 5.46% of the observed phenotypic variance for FCR and RFI, respectively. Finally, functions of candidate genes and gene set enrichment analysis indicate that most of the significant pathways are associated with hormonal and digestive gland secretion during feeding. This study advances our understanding of the genetic mechanisms of feeding behavior and feed efficiency traits and provide an opportunity for increasing feeding efficiency using marker-assisted selection or genomic selection in pigs.Increasing feed efficiency is a major goal of breeders as it can reduce production cost and energy consumption. However, the genetic architecture of feeding behavior and feed efficiency traits remains elusive. To investigate the genetic architecture of feed efficiency in pigs, three feeding behavior traits  and two feed efficiency traits  were considered. We performed genome-wide association studies (GWASs) of the five traits using a population of 1,008 Duroc pigs genotyped with an Illumina Porcine SNP50K BeadChip. A total of 9 genome-wide ( Pork is an important meat source for humans, accounting for nearly 40% of all meat consumed by the world population . The shaIn animal breeding programs, feed efficiency traits are difficult to improve by direct selection because feed efficiency cannot be measured directly . Feed coFeeding behavior is one of the most important factors affecting feed efficiency. Several studies have shown a strong genetic and phenotypic correlation between feeding behavior and feed efficiency traits in swine . For ins1), 639 QTLs have been identified for feeding behavior and feed efficiency traits. Most of these QTL were identified using linkage mapping. Because of the large intervals of QTLs, directly using them for genetic improvement remains difficult  associated with 647 different traits reported in pigs  commercial pigs thanks to its excellent performance on growth traits. In previous studies, several single nucleotide polymorphisms (SNPs) that were significantly associated with FCR were detected on SSC 12 in a Canadian Duroc population by genome-wide association study (GWAS) . HoweverThe experimental procedures used in this study met the guidelines of the Animal Care and Use Committee of the South China Agricultural University (SCAU) . The Animal Care and Use Committee of the SCAU approved all the animal experiments described in this study.n = 1,008) from the Guangdong Wen’s Foodstuffs Group, Co., Ltd.  using the Osborne FIRE Pig Performance Testing System  as previously described , 981 for FCR and 971 for RFI.During the period of 2013–2016, phenotypic data were collected for Duroc pigs  including sex, pig pen, and year-season effects; α is the vector of corresponding coefficients including the intercept; x is the vector of marker genotypes, and β is the corresponding effect size of the marker; u is the vector of random effects, with u ∼ MVNn; 𝜀 is the vector of random residuals with 𝜀 ∼ MVNn; τ-1 is the variance of the residual errors; λ is the ratio between the two variance components; K is a known n × n relatedness matrix; and I is an n × n identity matrix. MVNn denotes the n-dimensional multivariate normal distribution.GEMMA was used to perform GWAS with a univariate linear mixed model , 2014. PGenome-wide significance was determined using the Bonferroni method by dividing the desired type I error level by the number of SNPs tested. To include additional candidate genes and enable gene set enrichment analysis, we also set a more lenient threshold by multiplying the Bonferroni threshold by a constant of 20 .The GCTA tool was used to compute the genomic heritability by dividing the estimated genetic variance by the total variance measured, and phenotypic variances contributed by significant SNPs for each trait , 2011. GA number of SNPs that were significantly associated with the target trait by GWAS were detected based on their strong linkage with highly causal mutants. To demarcate the independence of all significant signals in a putative region, conditional and LD analyses were performed in univariate linear mixed models by fitting the genotypes of peak SNPs as covariates . MoreoveSus scrofa 10.2 genome version were downloaded from Ensembl. The Ensembl annotation of the S. scrofa 10.2 genome version was employed to find genes that were nearest to the significant SNPs1. To annotate significant SNP located in previously mapped QTLs in pigs, all QTL data in pigs were downloaded from http://www.animalgenome.org/cgi-bin/QTLdb/SS/download?file=gbpSS_10.2   . For fun8, 2017) . Fisher’rocesses .Table 1. All phenotypic data conformed to the Gaussian distribution based on the Shapiro test before GWAS analysis (Table 1).We considered five feeding behavior and feed efficiency traits, including DFI, TPD, NVD, FCR, and RFI. Summary statistics for these traits, and their heritabilities are presented in analysis . There eTable 2). Bivariate analysis indicated that the traits are positively correlated with each other, both phenotypically and genetically, except between DFI and NVD. In particular, DFI are strongly positively correlated with FCR and RFI with a genetic correlation of 0.75 and 0.98, respectively (Table 2).We partitioned the phenotypic variance into genetic and environmental components using genetic relationship matrix computed from genotypes. The genomic heritabilities of the traits are moderate, ranging from 0.28 (DFI) to 0.38 (NVD)  to 112.3 kb (SSC1).To understand the genetic architecture of the feeding behavior and feed efficiency traits, we performed GWAS for each trait. After a series of filtering steps for quality control, 32,446 SNPs and 1,008 pigs were available for subsequent GWAS. The number of SNPs on each chromosome and the average distances between pairs of SNPs after QC are provided in P < 1.54E-06 , 9 SNPs on SSC1 were associated with RFI. At a more lenient threshold (P < 3.08E-05) for suggestive associations, 8 SNPs were associated with DFI, 15 with FCR, 5 with NVD, 2 with TPD, and 14 with RFI . QQ plots of P-values and the computed genomic inflation factors (λ) indicated no evidence of population stratification .Single marker tests using mixed model were performed to identify genetic markers associated with the traits. At a stringent genome-wide Bonferroni threshold fication  . To test whether LD caused the associations, we performed conditional analyses with DFI and RFI in which the lead SNP WU_10.2_7_18377044 on SSC7 was fitted in the model as a covariate and the conditional P-values for other SNPs in the vicinity were obtained. While many SNPs in high LD with the lead SNP were significant in the GWAS for DFI , their significance almost diminished completely after the lead SNP was included as a fixed effect in the model . The same pattern was also observed for the same lead SNP for RFI . This is not surprising because the two DFI and RFI were almost perfectly (r = 0.98) correlated genetically (Table 2).Multiple SNPs in close proximity were found to be associated with the same traits, possibly due to their linkage and/or linkage disequilibrium. Indeed, LD block analysis showed that the multiple significant SNPs on SSC1 associated with RFI were located within a haplotype block that spanned 2,169 kb .Similarly, the lead SNP DRGA0001676 significantly associated with RFI and FCR in SSC1 explained the association between multiple SNPs with the traits in the vicinity of DRGA0001676 (Table 3). Eight SNPs associated with DFI and RFI were located on previously reported QTL regions for time spent drinking. One SNP on SSC13 associated with TPD was located on previously reported QTL regions for FCR in pigs. Moreover, 10 SNPs were located in the genomic regions where QTLs were previously detected by GWAS or linkage mapping for average daily gain and growth-related traits in pigs. Given the high genetic correlations (Table 2), overlaps were considered with QTLs for correlated traits as evidence for replication.To evaluate whether SNPs associated with the feeding behavior and feed efficiency trait in this study replicate any previously known QTLs, we search the pigQTLdb based on SNP and QTL locations. A total of 13 SNPs associated with FCR and/or RFI were identified within the genomic regions where QTLs for DFI have been previously mapped in pigs (Sus scrofa 10.2 genome assembly (Table 2). Many candidate genes appear to have biochemical and physiological roles that were are relevant to feeding behavior and feed efficiency traits, including neurensin 1 (NRSN1) and doublecortin domain-containing 2 (DCDC2) for DFI; ADAM metallopeptidase domain 12 (ADAM12) for TPD; phospholipase C beta 1 (PLCB1), GNAS complex locus (GNAS), and ephrin B2 (EFNB2) for FCR; prolactin (PRL) for both FCR and RFI; leucine-rich repeat and fibronectin type III domain-containing 5 (LRFN5), and multiple EGF-like domains 11 (MEGF11) for RFI and FCR.A total of 16 functional genes that were within or near the identified tag SNPs were detected based on annotations of the Supplementary Table S2).To uncover genes and pathways involved in the biology of feed efficiency and feeding behavior traits, we performed gene set enrichment analysis of the 16 candidate genes for all five traits. Interestingly, at an FDR = 0.05, several KEGG pathways and GO terms are significantly enriched for the candidate genes, including pathways related to hormone metabolic process, secretion of digestive enzymes, among others . Indeed, over 87% of SNPs (28/32) significantly associated with feeding behavior and feed efficiency traits were located within genomic regions where QTLs for feeding behavior and feed efficiency were previously reported in pigs (pigQTLdb2).Despite similar design and analytical methods, none of the QTLs identified in this study replicated QTLs in a previous study . There cFigures 1A,B). Signals are detected for both traits on SSC7. And while the strengths of associations differ and may not reach thresholds for both traits, SNPs associated with RFI on SSC1 also tended to have an effect for DFI . However, because of the less than unity correlations, in particular the correlation at the phenotypic level which were used for GWAS mapping, full overlap is not expected.Derivative traits are correlated with each other. For example, RFI and DFI has a highly correlated at 0.81 phenotypically and 0.98 genetically. Indeed, GWAS signals overlapped to some extent for these two traits  is located proximal to the SNP WU_10.2_14_14709705, which is associated with TPD. ADAM12 had been identified as a protease to insulin-like growth factor (IGF)-binding proteins. This gene may have a regulatory function in controlling the amount of free bioactive IGF . Most of the significantly enriched pathways are associated with hormonal and digestive gland secretion during feeding such as thyroid hormone and gastric acid secretion. Given the principal roles of hormone and digestive gland secretion in feeding and metabolism, their involvement in the feeding behavior and feed efficiency traits are conceivable.Functional annotation revealed a number of pathways and biological processes that are significantly overrepresented among the 16 positional candidate genes for feeding behavior and feed efficiency traits (Several GWAS for feeding behavior and feed efficiency traits in Duroc populations with different genetic backgrounds have been reported . We prevZW, JY, and WH: conceived and designed the experiments. RD, MY, XW, JQ, ZZ, SZ, and SL: performed the experiments. RD, JY, and WH: analyzed the data and wrote manuscript. MY, ZX, EZ, GC, and DL: collected the samples and recorded the phenotypes. ZW: contributed materials. All authors reviewed and approved the manuscript.The authors have read the journal’s policy and declare the following conflicts of interest: Wens Foodstuffs Group, Co., Ltd. provided all phenotypic data and ear tissue samples for the research reported in this study. This does not alter our adherence to Frontiers in Genetics policies on sharing data and materials.


Lignin fractions having different molecular weights and varied chemical structures isolated from kraft lignins of both softwood and hardwood via a sequential solvent fractionation technique were incorporated into a tunicate cellulose nanofibers (CNF)—starch mixture to prepare 100% bio-based composite films. The aim was to investigate the impact of lignin structural diversity on film performance. It was confirmed that lignin’s distribution in the films was dependent on the polarity of solvents used for fractionation (acetone > methanol > ethanol > ethyl acetate) and influenced the optical properties of the films. The –OH group content and molecular weight of lignin were positively related to film density. In general, the addition of lignin fractions led to decrease in thermal stability and increase in Young’s modulus of the composite films. The modulus of the films was found to decrease as the molecular weight of lignin increased, and a higher amount of carboxyl and phenolic –OH groups in the lignin fraction resulted in films with higher stiffness. The thermal analysis showed higher char content formation for lignin-containing films in a nitrogen atmosphere with increased molecular weight. In an oxygen atmosphere, the phenol content, saturated side chains and short chain structures of lignin had impacts on the maximum decomposition temperature of the films, confirming the relationship between the chemical structure of lignin and thermo-oxidative stability of the corresponding film. This study addresses the importance of lignin diversities on composite film performance, which could be helpful for tailoring lignin’s applications in bio-based materials based on their specific characteristics. Plastic is a highly useful and convenient material. Although the plastic production benefits the daily life of society, 90% of the plastic products were used once and then discarded, thus creating a global environmental crisis . RecentlLignin is one of such polymers and is the second most abundant biopolymer after cellulose and the most abundant aromatic biopolymers in nature. It is generated in large quantities as the main by-product from the pulp and paper industries. Commercially, 120 Mt of kraft pulp is produced annually, and this corresponds to a total production of 70 Mt of kraft lignin ,14. Recev/v) in a successive manner, and found that the fraction with high molecular weight had high thermal stability [However, the variable nature, chemical functionality and heterogeneity of technical lignin is an obstacle to further develop its material applications ,26. Thistability . A similtability . Recentltability . For thetability .Solvent extraction not only improves lignin’ homogeneity but also results in several lignin fractions with variable physicochemical properties , which wAlthough some interesting results were obtained in previous studies, a better understanding of the impact of the source, molecular weight heterogeneity and chemical structure diversities of lignin on the performance of biocomposites is required. Therefore, the aim of present study is to understand the relationships between lignin structure  and performance in the 100% bio-based composite films . In addition, based on the results obtained from this study, the specific lignin fractions are suggested to fulfil the required properties of the composite films, which is expected to facilitate lignin’s further development in the materials field.Picea abies) and Hardwood  lignin with moisture contents of 6.4% and 5.1% and ash contents of 0.6% and 1.2%, respectively, were obtained using the LignoBoost process [Ciona intestinalis using the prehydrolysis-kraft cooking-bleaching method [w/w), with a high crystallinity index (94%), a charge density of 43 μmol/g, a crystal size of 7.7 nm and an Iβ ratio of 89.94% [Softwood  with a concentration of 0.5%. 4 mL of the 0.5% tunicate CNF suspension prepared above, after blending with 4 mL starch solution and 2 mL lignin solution, was cast on Petri dishes to prepare films after drying at 50 °C overnight. The blank CNF-starch film was fabricated by mixing 4 mL CNF suspension and 4 mL starch solution, followed by the casting and drying conditions mentioned above. To facilitate discussion, the composite films were noted by their corresponding lignin source and the specific solvent used for fractionation, namely, SWEA, SWE, SWM, SWA and SWI for softwood lignin and HWEA, HWE, HWM, HWA and HWI for hardwood lignin, respectively. For example, SW and HW stand for softwood and hardwood, respectively, while EA, E, M, A and I indicate the ethyl acetate, ethanol, methanol, acetone and insoluble fraction, respectively.Starch was dissolved in water (0.5%) under continuous stirring at 90 °C to obtain a clear solution, which was cooled to room temperature. Lignin was dissolved in acetone/water  based on the methods reported by Guerra et al. . The lig31P NMR [3 before performing 31P NMR analysis.The functional groups of lignin were quantified by running 31P NMR . A ~30 m−1. The other operation conditions and data analysis simply followed the ones reported previously [A MicroDouble-shot Pyrolyser Py-2020iD  was connected with a Shimadzu GC/MS-QP 2010 apparatus  to conduct the Py-GC/MS/FID analysis. The separation was achieved by a capillary column RTX-1701  with dimensions of 60 m length, 0.25 mm width and 0.25 μm internal diameter. The pyrolysis was performed at 500 °C with a heating rate of 600 °C sA micrometer  was applied to determine the thickness of the films. The thicknesses at 20 points of each film specimen were measured and the mean value was used for density calculation. The density of the films was calculated based on weight, surface area and thickness and was reported as the mean value of at least three film specimens.A Cressington 208HR high-resolution sputter coater  was used to coat the composite films with gold. The thickness was controlled to be 3–5 nm. Then, a Field Emission SEM  was used to observe the morphological structures of the films.A Shimadzu UV-240  was applied to determine the light transmission (T%) of the composite films. In order to compare different samples, the values were read at 650 nm. Film specimens were cut into a rectangular shape and then were loaded into the test cell. The transmittance values were used to evaluate the transparency of the films.An Instron 4411 mechanical property tester  was applied to analyze the mechanical performance of the films. The measurement was conducted with a 20 mm starting grip gap and a 3 mm/min grip moving rate. The test was performed on at least 3 specimens for each film and the mean values were reported.e device  was applied to conduct thermal analysis. In this study, both nitrogen and oxygen atmospheres were tested under a gas flow rate of 50 mL min−1. The analysis was performed between 20 and 800 °C with a heating rate of 10 °C min−1. The maximum decomposition temperatures were determined by the first derivative TGA curve (DTG).A Metler Toledo Star TOA/SDTA 851When successive solvent fractionation with ethyl acetate, ethanol, methanol and acetone was applied to both softwood and hardwood lignin, five fractions with different yields  were recMn of 1580 and a Mw of 5440, higher than the corresponding values of 910 and 1740 for the initial hardwood lignin. However, softwood lignin showed a much broader molecular weight distribution than hardwood lignin, as indicated by its higher polydispersity index (PDI = 3.5). As seen in Mw) and number average (Mn) molecular weights of each fraction after successive solvent extraction increased along the fractionation procedure, due to the increased dissolving capacity, regardless of lignin source. EtOAc-soluble fractions from both softwood and hardwood lignin had relatively low Mn and Mw, implying that they were mainly composed of oligomeric lignin derivatives [The SEC curves of the lignin fractions are shown in ivatives . The insivatives . All solivatives . In geneivatives ,25. Comp31P NMR technique is used to analyze the contents of the functional groups in the lignin fractions. The obtained NMR spectra and the signal assignment are shown in 31P NMR results, the contents of aliphatic –OH, –COOH and phenolic –OH groups of lignin fractions are calculated and summarized in As an important analytical technique to investigate the complex structures of insoluble polymers, Py-GC/MS/FID method is also applicable to lignin analysis ,54,55,561 + ArC2)/ArC3), can be found in our previous work [To determine the structural diversities in the lignin fractions, the products of lignin after pyrolysis were categorized based on their side-chain and the ortho-substitution in aromatic ring . The detous work . As showThe present study is focused on investigating the impacts of distinct fractions recovered from kraft lignin from different sources (both softwood and hardwood) by sequential solvent extraction on the performance of the corresponding 100% bio-based tunicate cellulose nanofibers (CNF)-starch-lignin composite films. In fact, green composite films prepared from cellulose, starch and lignin were reported by Wu et al. , who use−1 for all the lignin-containing composite films, which generates from the asymmetric aryl ring stretching in lignin [After successive solvent extraction was applied to lignin from different sources (both softwood and hardwood), eight soluble fractions and two insoluble fractions were successfully isolated. All fractions showed great diversities in terms of molecular weight distribution and chemical structures, which is expected to lead to different impacts when they are utilized to fabricate composite materials. To investigate this, we chose tunicate CNF-starch composite film as the blank, and then films containing identical amount of different lignin samples were prepared and characterized. As shown in n lignin . The intAs seen in v/v). However, when lignin solution was mixed with starch and cellulose in water, the acetone/water ratio decreased, and lignin was no longer soluble, resulting in aggregation. In addition, during evaporation, the acetone was easier to remove than water, and the poor solubility of lignin in water also contributed to the lignin particle formation. Irrespective of the source, lignin fractionated with EtOAc formed the biggest particles, with 100–200 nm diameter, followed by EtOH and MeOH-extracted lignin (particles with 50–100 nm in diameter) and then the insoluble fractions (mainly <100 nm). Interestingly, those extracted with acetone showed no visual particle formation. This finding agreed well with the finding mentioned above that SWA and HWA had highest transmittances of 18.22% and 10.47% for softwood and hardwood fractionated lignin, respectively  of the lignin  corresponding to a 5% weight loss and the maximum decomposition temperature (Tmax) were chosen to evaluate the films’ thermal stability. As shown in T5% of 283.3 °C, a Tmax of 355 °C and a residual weight (char) of 10.3% at 800 °C. Lignin addition has been found to influence the thermal stability of the composite films negatively, as indicated by lower T5% (265–278 °C), though the softwood lignin was more thermally stable than hardwood lignin. The addition of both softwood and hardwood lignin did not significantly affect the Tmax of the composite films (354–360 °C). However, the char contents for all composite films (12–19%) were higher than for the blank CNF-starch film (10.3%). The high char formation was due to the lignin which was abundant in aromatic rings with various branches, and these complex and highly condensed structures could result in a high amount of unvolatized residue at 800 °C [All TGA curves of composite films in nitrogen atmosphere were presented in  T5% 265–8 °C, thoTmax) for the former and two peaks of Tmax (Tmax1 and Tmax2) for the latter. This phenomenon suggests a difference between the mechanism of thermal degradation in oxygen and in nitrogen. As shown in T5% of 270.8 °C, a Tmax1 of 325 °C and a Tmax2 of 424 °C, in the oxygen atmosphere. The addition of softwood lignin resulted in higher T5% (271–276 °C) for the composite films. However, the introduction of some hardwood lignin fractions, such as EtOAc-soluble and insoluble lignin, even lowered the T5%. The residue at 800 °C, namely, ashes, weighed 0.8% for blank film. The composite films showed quite similar ash contents at 800 °C to the blank film. In general, the composite films containing softwood lignin showed higher Tmax2 than those with hardwood lignin. Furthermore, the chemical structures of lignin exhibited significant influences on the thermo-oxidative stabilities of the composite films in oxygen. As seen in T5% of the corresponding composite film in oxygen. This finding indicated that more saturated side chains could provide benefit to the thermos-oxidative stability of the lignin-containing films. In addition, as the content of phenols having α-double bonds increased, Tmax2 decreased. However, the Tmax2 increased when increasing (ArC1 + ArC2) per ArC3, until the latter reached approximately 3. Tmax2 was also positively related to the phenolic –OH group content of the lignin, which was also observed in polypropylene-lignin composite films [The thermo-oxidative stability of lignin-containing composite films was further measured in an oxygen atmosphere . All TGAte films .-The lignin aggregated to form particles in the composites, thus affecting the color and transmittance value.-The lignin imparted to the films a lower tensile strain and higher Young’s modulus due to the inherent rigidity of lignin. However, hardwood lignin with lower molecular weight and polydispersity reinforced the film structure, while softwood lignin with a higher molecular weight and lower G+S unit content negatively affected the tensile stress of the films.In this study, the lignin fractions with great diversities have been found to affect the properties of the tunicate CNF-starch composite films significantly:The thermal stability of composite films in a nitrogen atmosphere have a clear positive correlation with the molecular weight of lignin, and the char formation was improved with the addition of the lignin fraction.The direct relationship between the certain lignin structural features and thermo-oxidative stability of lignin-containing composite films was also observed. The same structural features positively influence the antioxidant properties of lignin.The structure–performance relationships found here will help to tune the properties of lignin to fit in specific application in the materials field. For example, as found in this study, the acetone lignin fraction is the best candidate to prepare uniform and highly transparent composite films; insoluble lignin fraction with high molecular weight could render the composite film dense structure; the ethyl acetate lignin fraction with low molecular weight, low PDI, and high content of phenolic –OH group are the best for composite films to have high Young’s modulus and good thermal stability.


The prevalence of pre-diabetes (PD) and type II diabetes (T2D) has risen dramatically in recent years affecting an estimated 422 million adults worldwide. The risk of T2D increases with age, with the sharpest rise in diagnosis occurring after age 40. With age, there is also a progressive decline in muscle mass starting after the age of 30. The decline in muscle mass and function due to aging is termed sarcopenia and immediately precedes the sharp rise in T2D. The purpose of the current review is to discuss the role of protein to attenuate declines in muscle mass and insulin sensitivity to prevent T2D and sarcopenia in aging adults. The current recommended dietary allowance for protein consumption is set at 0.8 g/kg/day and is based on dated studies on young healthy men and may not be sufficient for older adults. Protein consumption upwards of 1.0–1.5 g/kg/day in older adults is able to induce improvements in glycemic control and muscle mass. Obesity, particularly central or visceral obesity is a major risk factor in the development of PD and T2D. However, the tissue composition of weight loss in older adults includes both lean body mass and fat mass and therefore may have adverse metabolic consequences in older adults who are already at a high risk of lean body mass loss. High protein diets have the ability to increase weight loss while preserving lean body mass therefore inducing “high-quality weight loss,” which provides favorable metabolic changes in older adults. High protein diets also induce beneficial outcomes on glycemic markers due to satiety, lowered post-prandial glucose response, increased thermogenesis, and the ability to decrease rates of muscle protein breakdown (MPB). The consumption of dairy specific protein consumption has also been shown to improve insulin sensitivity by improving body composition, enhancing insulin release, accelerating fat oxidation, and stimulating rates of muscle protein synthesis (MPS) in older adults. Exercise, specifically resistance training, also works synergistically to attenuate the progression of PD and T2D by further stimulating rates of MPS thereby increasing muscle mass and inducing favorable changes in glycemic control independent of lean body mass increases. The average lifespan has increased by 20 years, increasing the age of the population worldwide . At the The risk of T2D increases with age, with the sharpest rise in diabetes diagnosis occurring after age 40 . With agLifestyle factors such as diet and exercise play a crucial and central role in glucose handling and IS . While eMuscle mass is important beyond its role to promote movement of the human body. Skeletal muscle also contributes significantly to postprandial glucose disposal, lipid oxidation, resting metabolic rate, and whole-body protein metabolism , 25. MusThe progressive loss of lean body mass that occurs with aging is due to an imbalance between MPS and MPB , 29. WhiMuscle mass loss in aging is worsened in disease states characterized by IR, such as T2D and PD . In healSkeletal muscle is the largest storage depot for glucose in the body, accounting for >75% of glucose disposal , 42. In Currently the recommended dietary allowance (RDA) for protein consumption is 0.8 g/kg/day of protein or 10–35% of total energy intake , 43, 44.Although lifestyle management such as a healthy diet has long been recommended to improve glycemic control, it is not certain what dietary approach is best for individuals with diabetes with most recommendations centered around individualized needs based on glycemic control, age, and co-morbidities . There rHistorically, there were several reasons why higher protein diets were not recommended for older adults and/or individuals with metabolic alterations including T2D, including the thought that dietary protein would adversely raise blood glucose levels and have a detrimental effect on kidney function . The belObesity, particularly central or visceral obesity, is a risk factor for the development of T2D and is common in PD and T2D . Weight Higher protein diets may be especially important to improve IS in older adults with PD or T2D as higher protein intakes during weight loss can help preserve muscle mass , 68, indad libitum caloric intake due to increased leptin sensitivity  increasing muscle mass, which in turn will increase glucose storage capacity, (2) upregulating insulin signaling proteins, and (3) inducing GLUT 4 translocation to the cell membrane to facilitate glucose clearance from circulation during and immediately after exercise . While hWith age there is a loss of muscle mass and the development of IR, increasing the risk of sarcopenia and T2D. When these two conditions coincide, they can create a vicious cycle whereby IR induces greater muscle mass loss, leading to a further reduction in IS and vice versa. Protein has emerged as a potential strategy to combat the decline in muscle mass and IS that occur with increasing age, potentially preventing the development of T2D and sarcopenia. However, protein intake recommendations in older adults are currently insufficient at 0.8 g/kg/day, despite many groups advocating for increased requirements of 1.0–1.2 g/kg/day in older adults and 1.2–1.5 g/kg/day in those who are at risk of malnutrition. While trials in older, IR/PD/T2D populations are lacking, the evidence to date does support a role for higher protein intakes to attenuate declines in muscle mass and IS, particularly when combined with resistance exercise. Further work examining the effectiveness of higher protein intakes, with and without resistance training, in older adults with IR to prevent the development of sarcopenia and T2D are warranted.MD and KB conceived, wrote, and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pleuromamma abdominalis in the poorly known Southeast Pacific region, with samples spanning an ocean gradient from coastal upwelling to the oligotrophic South Pacific Subtropical Gyre. We assessed variation in fragments of the mitochondrial (mt) genes cytochrome c oxidase subunit I (COI) and Cytochrome b as well as in the nuclear internal transcribed spacer (ITS) region and 28 S rRNA. Phylogenetic analyses revealed the presence of 8 divergent lineages occurring across the gradient with genetic distances in the range of 0.036 and 0.44 (mt genes), and GMYC species delimitation methods support their inference as distinct (undescribed) species. Genetic lineages occurring across the zonal gradient showed strong genetic structuring, with the presence of at least two new lineages within the coastal upwelling zone, revealing an unexpectedly high level of endemism within the Humboldt Current System. Multivariate analyses found strong correlation between genetic variation and surface chlorophyll-a and salinity, suggesting an important role for hydrographic gradients in maintaining genetic diversity. However, the presence of cryptic lineages within the upwelling zone cannot be easily accounted for by environmental heterogeneity and poses challenging questions for understanding the speciation process for oceanic zooplankton.Across boundary currents, zooplankton are subject to strong oceanographic gradients and hence strong selective pressures. How such gradients interact with the speciation process of pelagic organisms is still poorly understood in the open ocean realm. Here we report on genetic diversity within the pelagic copepod  This region exhibits high heterogeneity in hydrographic conditions, primarily along a zonal gradient from the coastal zone to the open ocean, with increasing mixed layer depth (40–150 m), sea surface temperature (14–20 °C), sea surface salinity (34.8–35.8) and surface dissolved oxygen concentration (1.5–4.5 mlL−1)6. This high zonal variability is also clearly manifested in productivity of surface waters from the highly productive coastal upwelling zone (Humboldt Current), across to the large ultra-oligotrophic South Pacific Subtropical Gyre (SPSG)10. Moreover, within this zonal gradient there is a changing predominance of different water masses promoting even greater hydrographic variability in this vast ecosystem. The SPSG is the largest subtropical gyre, with an areal extent of 18 × 106 km11, and it has the lowest reported primary production rates (1–2 μg C L−1 day−1)12. The ultra-oligotrophic condition of the eastern SPSG contrasts with the very high productivity of the SEP upwelling system off the coast of Chile, Peru and Ecuador, also known as the Humboldt Current System (HCS), which is one of the most productive eastern boundary current ecosystems in the world13. Primary production in the upwelling zone reaches up to 19.9 g C m−1 d−1 14. Upwelling-driven production extends zonally into a mesotrophic region, also known as the coastal transition zone15, which constitutes an intermediate region between the upwelling system and the oligotrophic region of the SPSG.One of the largest and least explored regions of the open ocean is the Southeast Pacific (SEP). This region is globally important due to its role in controlling regional climate, its atmospheric and oceanic teleconnections at a global scale16. Within the central gyre, small organisms (<500 μm),which numerically represent up to 65% of the zooplankton, are dominated by copepods, whereas other size fractions composed of chaetognaths, euphausiids and siphonophores make up a considerable part of the community16. In the coastal zone in contrast, larger-sized copepods often dominate numerical abundance17. In specific groups of zooplankton, for example siphonophores, tintinnids, and euphausiids, there is an increase in diversity and species richness towards the center of the gyre6. There also are endemic species within the Humboldt Current that are replaced by different species in the oceanic zone20. For example, the endemic copepod Calanus chilensis of the Humboldt Current21 appears to be replaced by the more widely distributed Calanus australis towards open ocean waters19. A similar pattern seems to occur with the endemic Humboldt Current Euphausia mucronata, which may be replaced by other euphausiids in the oligotrophic zone20. How the apparent environmental barriers across the zonal gradient, from the Humboldt Current to the SPSG, have influenced species distributions, population structure and genetic diversity within zooplankton species are poorly known for this ocean basin.Oceanographic variability across the SEP zonal gradient is strongly correlated with changes in zooplankton abundance, biomass and distribution. Zooplankton biomass and abundance can decline by up to 3 orders of magnitude from the coastal zone to the SPSG25. Strong genetic structure among populations within plankton species also has been shown to occur from coastal to oceanic environments26. For example, in the cosmopolitan copepods Eucalanus hyalinus, E. spinifer, Pleuromamma xiphias and Haloptilus longicornis29 ocean regions with strong hydrographic gradients can act as ecological barriers to dispersal, causing strong genetic breaks among populations in distinct subtropical gyres and/or among ocean basins. Because biotic and abiotic characteristics of regions with strong zonal gradients may affect the development or survivorship of populations22, they can act as barriers to dispersal. Responses to these ocean gradients can be species-specific (idiosyncratic responses), due to the variable ecological requirements of each species26. Given that genetic divergence is commonly decoupled from morphological divergence in copepods31, and that broadly-distributed species may include lineages with different evolutionary trajectories25, detection of cryptic diversity in this group is fairly common.Planktonic copepods are the most species-rich taxonomic group of the mesozooplankton, with a large number of cryptic species reportedPleuromamma abdominalis, which has been described to inhabit preferably oceanic environments, has a cosmopolitan distribution in subtropical and tropical waters worldwide, and occurs in high abundance across the mesopelagic and epipelagic zones with active diel migratory behavior37. Within the SEP, seven species of Pleuromamma co-occur, including P. borealis, P. gracilis, P. piseki, P. quadrungulata, P. robusta and P. xiphias35. P. abdominalis can be clearly distinguished from other co-distributed species by the shape of the rostrum, the presence and number of denticles on proximal segments of the A1, body size, and in adult females by the 5th swimming leg segmentation38. In recent studies conducted on this species, large intraspecific variation was reported in both morphological and genetic characters41. Prior work has reported the presence of endemic clades in certain oceanographic provinces, including subtropical waters of the South Pacific41.Within the SEP, there are few copepod species that show broad distribution and high abundance across the zonal gradient. As a result, it is difficult to identify appropriate target species for studies aiming to elucidate the effect of hydrographic variability on genetic structuring within species. One of the few species that meets these requirements is P. abdominalis in the Southeast Pacific. Our objectives were to (1) identify the evolutionary lineages present in the SEP region, (2) evaluate whether they may be distinct, currently undescribed species, using multiple independently inherited markers, and (3) determine the potential effects of hydrographic variability across the SEP region on the distribution of these distinct evolutionary lineages. For this, we used samples collected across a zonal transect of the SEP  and cytochrome b (CytB), as well as nuclear 28 S rRNA and the internal transcribed spacer (ITS) region.In this work, we conducted a molecular study of the evolutionary lineages present within the nominal species  were to  identify were to  identifyP. abdominalis , 100% Bayesian Posterior Probability (BPP)] for both mitochondrial genes  located across the four oceanographic study zones for the two mitochondrial markers. PLAB1 included only the clade 1a_1 Hirai, with haplotypes located mainly in the coastal upwelling zone. Within PLAB2 Hirai, seven clades were observed, which were divided into two large groups with high support values in both gene trees . The first group, including clades 2c Hirai, 2d Hirai, and 2a Hirai, showed variation in tree topology depending on the gene. In the CytB tree, clade 2c Hirai was sister to the pair of clades 2d Hirai and 2a Hirai, while for the COI gene, the clades 2c Hirai and 2d Hirai make up a group with a sister clade 2a Hirai. The second PLAB2 Hirai group, including four clades, also had high nodal support  and variation in topology depending on the gene. In the CytB tree we observed two groups, one formed by the clades 2i Hirai and 2r-NEW and another by the clades 2 f Hirai and 2s-NEW; however, in the COI tree the clade 2r-NEW was placed with clades 2 f Hirai and 2s-NEW.The eight clades were split into two genetically very divergent clades, PLAB2 Hirai and 2a Hirai mainly consisted of haplotypes from the transition and oligotrophic zones, while clade 2d Hirai included only individuals sampled in the ultra-oligotrophic zone. Clade 2 f Hirai consisted of individuals from the coastal upwelling and transition zones, and clade 1a_1 Hirai included specimens from the coastal upwelling zone and a single individual from the oligotrophic zone (COI). Finally, clade 2i Hirai was found in individuals distributed from the upwelling to the oligotrophic zones.Most of the eight primary clades were well supported as reciprocally monophyletic groups in both mitochondrial gene trees . Nodal support values for each clade were high in Bayesian analyses , with 6 or more clades with > 99% BPP support (both genes). ML bootstrap support values were lower, but still supported reciprocal monophyly (>75% ML) in the majority of clades in the CytB gene tree, with lower ML bootstrap values in the COI tree. In addition, placement of animals into clades was entirely congruent across the two mitochondrial trees, with animals present in both trees always occurring in the same mitochondrial clade , covering the equatorial divergence region, oligotrophic zones and even the coastal transition zone off South America 41. This analysis revealed that the sampling performed in this study included all clades reported for the South Pacific with the exception of the clades 2e Hirai, 2k Hirai and 2 m Hirai, which occur in the equatorial region .Two clades primarily sampled in the coastal upwelling and transition zones have not been found or reported in any prior work, and are newly described in this study: They are the clades 2r-NEW and 2s-NEW. Both were well supported as reciprocally monophyletic in Bayesian analyses , with more moderate support in ML results, as was observed in the other 6 previously described clades. The clade 2r-NEW occurred in the coastal upwelling and oligotrophic zones, and clade 2s-NEW included only coastal upwelling zone animals. The eight ion Fig. , and 1b-Hirai and 2r-New (D = 0.4952); however, for the COI gene, the maximum value was observed between the clades 1a_1 Hirai and 2c Hirai (D = 0.1831) (Table Hirai (D < 0.056) and 2a Hirai with 2d Hirai (D < 0.0688), for both genes.Genetic distances (D) calculated using the T92 substitution model for eight mitochondrial clades were higher for the CytB fragment (D = 0.056–0.495), with lower maximum genetic distances among clades observed at COI (D = 0.036–0.183)  showed changing frequency between the haplogroups, in the first clade a lower frequency of Y (5%) with the second one at 58.3%. Finally, clade 2 f Hirai was the only haplogroup with apparently fixed frequencies for all four polymorphic sites. The haplotype network for ITS revealed the presence of two abundant haplotypes separated by two mutational steps  likelihood method and the Poisson Tree Process (PTP) model, using single marker gene trees for both COI and CytB42. GMYC and PTP analyses for both mitochondrial genes supported the inference of several species being present within the nominal species P. abdominalis. In the GMYC results, the number of distinct entities (clusters) associated with the highest likelihood score for the COI gene was 7 , with 9 entities identified for CytB . GMYC support values for most clades were high at both mitochondrial genes  at the COI gene, but was well supported by analyses of CytB. PTP analysis indicated the presence of 11 species within the study sequences for both COI and CytB genes. This analysis showed concordance with the six mitochondrial clades described above with high support values (>0.5 BPP). However, PTP analyses also indicated greater splitting, for example both mitochondrial genes indicated that clade 2d Hirai was composed of 3 species and clade 2c Hirai by 2 species. Because greater congruence was observed between GMYC and phylogenetic results (above), we place greater emphasis on these inferences.Two analytical approaches were used to delimit P. abdominalis mitochondrial clades present in the Southeast Pacific, high variability was found in their relative abundance across the four study zones . In the oligotrophic and transition zones, four clades were observed, including 2a Hirai, 2c Hirai, 2 f Hirai and 2i Hirai. Within these two regions, clade 2c Hirai showed the highest relative abundance (>50%) with a second clade 2a Hirai also occurring at high frequency (>8%). Within the oligotrophic zone, three clades occurred with low relative abundance (<5%) that were not shared with the transition zone, including clade 2d Hirai, with overlapping distribution in the ultra-oligotrophic zone, and 2r-New and 1a_1 Hirai that also were present in the coastal upwelling zone. The coastal upwelling zone was dominated by the mitochondrial clades 2i Hirai (>50%) and the newly discovered 2s-NEW (<30%), the latter being exclusive to this zone. Some haplogroups showed a decrease in their relative abundance from the upwelling to oligotrophic zones as observed in clade 2i Hirai and 1a_1 Hirai; however, others remained relatively constant across zones, as in the rarer clades 2 f Hirai and 2r-NEW , comprised of chlorophyll-a (Chla), sea surface salinity (SSS), wind stress (WT) and photosynthetically active radiation (PAR), explained 94% of the variability . Finally, clade 2d Hirai, the only clade present in the ultra-oligotrophic zone, showed strong association with high SSS environments and had a strong positive correlation with the CCA2 axis   Fig. .44. These distinct lineages commonly exhibit patterns of distribution associated with variable oceanographic conditions22. Our study constitutes a first step to disentangle the genetic diversity within P. abdominalis in the Southeast Pacific. Phylogenetic analysis of mitochondrial and nuclear genes of the single species P. abdominalis showed a complex of genetic entities not described morphologically, suggesting a process of cryptic or pseudocryptic speciation. To assess this possibility, we delimited putative species using a Generalized Mixed Yule Coalescent approach, finding that these 8 clades likely correspond to different species. Subsequently, we investigated the distribution of these cryptic or pseudocryptic species and found them linked to oceanographic variability. Phylogenetic analysis of COI and CytB gene fragments indicated this nominal species as having high diversity over a rather limited geographic range (4000 km), in comparison with some other oceanic copepods of cosmopolitan distribution45. The different mitochondrial lineages observed within P. abdominalis may thus represent distinct species41, or ancient mitochondrial lineages that persist within large populations of oceanic plankton. The P. abdominalis lineages detected and reported here extend the distribution of material examined in Hirai et al.41, and suggest that a number of additional genetic lineages within the nominal species may yet be found across the circumglobal distribution of this species.Molecular analysis in zooplankton species with cosmopolitan distributions has revealed a variable number of genetic lineages in organisms that were ascribed as single species using traditional taxonomyHirai, 2r-NEW, 2i Hirai, 1a_1 Hirai) , while the remaining clades showed an unexpectedly high level of endemism: one in the ultra-oligotrophic zone (clade 2d Hirai), and the other in the coastal upwelling zone (clade 2s-NEW) , and these clades are possibly associated with nutrient-rich waters in the coastal upwelling zone and also in oligotrophic areas subjected to mesoscale eddies that can transport upwelled waters from the coastal zone into the oceanic region48. The clade 2i Hirai was previously described as widely distributed in the upwelling equatorial region Hirai et al.,41 and therefore its presence in the Humboldt Current indicates an extension of the described range towards upwelling areas of the SEP, linked to high levels of Chla.In general terms, the genus its Fig. . The linHirai), and they might also be associated with other oceanographic factors not considered in our study. Weak correlations with the predictors of our study suggest this may be the case. Finally, the lineage located in the ultra-oligotrophic zone showed strong correlation to high salinity (2d Hirai), which is likely a water mass tracer for the subtropical gyre  the productivity in the coastal zone was much higher62, while the subtropical gyre had lower productivity63. This contrast may have acted as an allopatric barrier for dispersal between the zones, explaining the present divergence of the ultra-oligotrophic area (clade 2d Hirai) with respect to the other zones. Later, by the end of the Pleistocene, warming of surface seawater, sea level rise, and lowering of productivity in the coastal zone may have weakened the oceanographic gradient and dispersal barriers between the oligotrophic and upwelling zones, and thus allowing an expansion and possibly secondary contact between zones generating the current parapatric pattern64. In any case, it has been well documented that distributional ranges can change rapidly, as evidenced presently due to climate change (e.g.  range shifts of ~23 km/year)65.The high level of endemism found, in particular in the upwelling and in the ultra-oligotrophic zones, may be a response to both present and historical physical processes. In the pelagic environment there are several physical barriers, such as land masses and oceanic currents, that can influence dispersal and connectivity among populations and act to promote species diversityP. abdominalis is likely a complex of species comprised of genetically divergent clades that are reproductively isolated but difficult to distinguish morphologically. Our results suggest the presence of at least 7 species, within which two appear highly specialized in two particular zones, the upwelling and the ultra-oligotrophic ones. Other studies focused on cosmopolitan species within the same genus, such as P. xiphias, P. gracilis, and/or P. piseki, also have described the presence of several lineages, suggesting that a number of species in this genus are in fact cryptic species complexes44. The prior study by Hirai et al.41 inferred with certainty that P. abdominalis was comprised of at least two species coinciding with the clades PLAB1 and PLAB2, given concordant patterns of genetic variation between mitochondrial and nuclear markers. However, there was an open possibility for many more species, because the observed divergence within both clades was greater than 4.3%41. In the present study, values of divergence greater than 30% were reported among the 8 clades, yet both mitochondrial genes also showed that the greatest genetic distances were observed among the clades PLAB2 and PLAB1. In general terms, many described species of marine copepods exhibit minimum COI divergences  to sister species within the range of 8 to 9%67, while other work suggests that intraspecific variation of copepod lineages occurs within the range of 1 and 4%69.The GMYC analyses of mitochondrial genes clearly indicated that P. abdominalis. In the haplotype networks, both nuclear genes failed to show clear patterns with respect to the pre-established mitochondrial clades , and introgression among closely related species as well as other processes that may obscure species boundaries. Several studies have shown that the use of mitochondrial markers may be more efficient for initially detecting cryptic species45, with subsequent studies using a combination of markers for consistent species delimitation 43. In any case, the high level of genetic diversity and endemism observed here seems unexpected within an ocean basin that is subject to both meridional and zonal advection and water mixing, suggesting that evolution and speciation processes can occur more rapidly than traditionally thought in the open ocean for short-lived species like pelagic copepods.The 28 S and ITS  sequences were less polymorphic and less informative than mitochondrial genes in detecting species limits within des Fig. . Howeverdes Fig. . The lacZooplankton samples were collected during two oceanographic cruises conducted in the Southeast Pacific Fig. , Table 1https://coastwatch.pfeg.noaa.gov/erddap/index.html). We differentiated four zones according to the observed near-surface Chla ranges: a eutrophic zone corresponding to the coastal upwelling region of Chile with Chla > 0.5 mg m−3 (CUP-Z), the mesotrophic coastal transition zone (CTZ) that shows a range of Chla between 0.1–0.5 mg m−3, the oligotrophic zone (OLIGO-Z) with variation between 0.05–0.10 mg m−3, and finally an ultraoligotrophic zone (U-OLIGO-Z) that is located in the central region of the South Pacific gyre with concentrations of Chla < 0.05 mg m−3  on both cruises, and an 8 m2 Tucker trawl net (300 μm mesh size) on CIMAR-22 , with greater abundance of the target species, individuals were chosen randomly. Specimens were collected at a total of 13  stations during the CIMAR-21 and CIMAR-22 cruises: 25 within the CUP-Z, 34 in the CTZ, 24 in OLIGO-Z and 49 in U-OLIGO-Z  following manufacturer protocols. Specimens collected on CIMAR 21 were extracted using the Forensic DNA Kit (Omega) following manufacturer protocols, with the exception of incubating the sample with the lysis buffer and proteinase K for only 1 hour. Extracted DNA was maintained at −20 °C.c oxidase subunit I (COI) and cytochrome b (CytB) as well as nuclear genes encoding the large subunit of the ribosome (28 S) and internal transcribed spacer regions (ITS1-5.8SrDNA-ITS2) were used for phylogenetic analyses. The primers used to amplify each gene fragment, the length of the amplified fragment, and the annealing temperatures used during PCR amplification are specified in Table 2 25, 2 μl of dNTP , 2 μl of each primer (0.35 mM), 0.045 units of the GoTaq G2 Flexi DNA Polymerase (Promega) and 5 μl of the DNA template. PCR products were visualized by 1% agarose gel electrophoresis and sent to Macrogen for sequencing. Sequencing of PCR products was performed primarily along the sense (forward) strand; both strands were sequenced if the forward reads were ambiguous at some nucleotide sites.Fragments of the genes encoding for mitochondrial proteins cytochrome 79. A BLAST search was conducted on confirmed and consensus sequences in order to verify the absence of contamination (correct organism)80. CytB sequence amino acid translations contained no stop codons. Within COI, a total of 6 individuals were found to have a 1-base indel, which would result in a frame-shift mutation. These sequences were inferred to derive from a COI pseudogene and these individuals were removed from all analyses. All included animals have COI sequence translations with no stop codons. Final alignments were created for each gene fragment using MUSCLE within Geneious 7.1.379. A total of 55 sequences were aligned for COI, 75 for CytB, 102 for 28 S rRNA and 80 for ITS. The length of the alignment obtained for each gene fragment is reported in Table Sequences from both strands of the same specimen were aligned, base calls confirmed and edited manually, and consensus sequences produced using Geneious 7.1.381 for COI and CytB, respectively. Bayesian analyses were carried out in MrBayes V.3.182. The Markov Chain Monte Carlo chains were executed in three simultaneous runs with default heating, selected the appropriate models of sequence evolution obtained from MEGA-X V.1081, over 1.0 × 106 generations, and trees were sampled every 1000 generations for both mitochondrial genes. The first 25% of the trees generated were eliminated (as burn-in). Stationarity was estimated by standard deviation of the three runs (>0.001) and by observation of stationarity when the curve of the log-likelihood scores against generation time in Tracer was non-trending83. Maximum likelihood analyses were performed with the GTR + G model and with 5000 bootstrap replicates to calculate the support for each node using RAxML V.8.2.1084. ML and BI trees were inferred for all newly obtained sequences from the SEP for both COI and CytB gene fragments (separately), as well as including representative COI sequences from clades reported in Hirai et al.41. In the latter tree, the analysis included 164 sequences representing all clades of P. abdominalis from the global Hirai et al.,41 study  and Bayesian inference (BI) methods for both mitochondrial genes. The T92 + I and T92 + G models were selected as the best-fit nucleotide substitution models by the Bayesian Information Criterion (BIC), as performed in Mega-X V.1085 to create a graph presentation of a TCS network for the 28 S and ITS fragments, which illustrates the relationship between nuclear haplotypes and mitochondrial clades86. In these TCS networks, nucleotide sites with > 5% ambiguities were masked (not included). In doing this, in the case of 28 S, much of the observed variation was thus masked. Genetic distances between mitochondrial clades were calculated as the net average between clades using final alignments for each gene, with 10,000 bootstrap replicates, using the Tamura 3-parameter (T92) nucleotide substitution model  and uniform rates between sites for COI and gamma-distributed rate variation for CytB (using Mega-X V.1081).We used Popart V 1.7Pleuromamma species in our study region, the Generalized Mixed Yule Coalescent (GMYC) likelihood method and the Poisson Tree Process (PTP) model. The GYMC analysis is a likelihood method that enables delimitation of the number of species based on model fitting the branching patterns expected within and between species87. We inferred ultrameric gene trees for the COI and CytB alignments using BEAST 2.088. Parameters selected for analysis included the HKY nucleotide substitution model, 6 categories for gamma-distributed rate variation, a relaxed lognormal clock, and a Yule model to express the branching pattern of the tree for both genes. Two independent runs were performed to ensure convergence, each run for 50 million generations and with sampling every 10,000 generations. Output files were visualized in Tracer v1.683 to ensure convergence of the chains. The maximum clade credibility (MCC) tree was determined by Tree Annotator v1.889 after removing the first 25% of trees (as a burnin).The number of delimited species was inferred for each MCC gene tree using the “gmyc” function in the splits R package90, with the single threshold option selected, as recommended by Fujisawa and Barraclough87. The GMYC analysis compares a null model (without a threshold) and an alternative hypothesis (with a threshold) in order to evaluate whether all samples constitute a single species (null) against the alternative hypothesis of the existence of more than one species in the data. A likelihood ratio test (LRT) is used to evaluate the model results, with significance calculated using the chi-square test with two degrees of freedom. The analysis also gives the number of genetic entities with their confidence intervals (IC), and the sequences that are associated with each group.Two analytical approaches were used to delimit http://species.h-its.org/ptp/. This model uses coalescence theory and estimates the speciation rate from the number of substitutions observed across the tree. As the input phylogeny, we used the COI and CytB Bayesian phylogenies. We ran the PTP analysis with 5 × 105 generations of MCMC sampling, with a thinning value of 100 and a burnin of 25%. MCMC convergence was visually confirmed as recommended by Zhang et al.91.We also performed a PTP delimitation of species using the bPTP web server 92) was performed to assess the association between hydrographic variables and mitochondrial clades occurring at each station. The model was constructed with eight response variables corresponding to the eight genetic clades, and as predictor variables, we selected sea surface values of chlorophyll-a (Chla), temperature (SST), photosynthetically active radiation (PAR), salinity (SSS), wind stress (WT) at the sea surface, an upwelling index (UI), which is an estimate of water mass being transported offshore by the alongshore wind93, and wind stress curl (WSC) representing the curvature of the tension of the surface wind94. CCA analysis incorporates both ordination and multiple regression techniques for the analysis of relationships among multivariate data, and can be an efficient tool to relate categorical and environmental predictive variables97. Satellite data were used to describe oceanographic conditions during the CIMAR 21 and CIMAR 22 cruises. For each sampling station, the average value was calculated over three days, from one day before to one day after sampling, with a spatial resolution of 0.025 degrees (Table https://podaac.jpl.nasa.gov/datasetlist?search=MODIS),WT was obtained by a sensor aboard the EUMETSAT METOP satellite (https://manati.star.nesdis.noaa.gov/products/ASCAT.php), SSS was downloaded from the SMOS radio telescope , and finally values of UI and WSC were obtained by the instrument Advanced Scatterometer (ASCAT) from NOAA Coast Watch .A canonical correlation analysis  with Chla and salinity, and it was therefore eliminated from the analysis in order to minimize collinearity of hydrographic variables. The CCA was conducted with two contingency tables based on 1000 Monte Carlo permutations: one (X) represented the number of animals within each mitochondrial clade observed at each station and the second (Y) included values of Chla, SSS, WT, IU, WSC and PAR values for each station. The hydrographic variables that explained the largest component of variation in the distribution of the different mitochondrial clades were used to calculate the two most parsimonious canonical components. To evaluate significance of the most parsimonious model the Wilks’ lambda likelihood test was used, which indicates the variance not accounted for by the independent variables, and chi-square tests were used to evaluate the significance of lambda99.The CCA was performed using the CCA package in Rstudio V.3.5Supplementary information.Supplementary information.