Symplocarpus foetidus, to test the hypothesis that the extant species diversity of skunk cabbage is the result of a post-glacial range expansion from southern refugia during the Quaternary Ice Age. A total of 4041 base pairs (bp) of the chloroplast intergenic spacer region (cpDNA) was sequenced from 485 individuals sampled from glaciated  and unglaciated  regions east and west of the Appalachian Mountains. Haplotype number, haplotype diversity, and nucleotide diversity were calculated, and genetic variation within and among populations was assessed by analysis of molecular variance (AMOVA). The geographic pattern of genetic differentiation was further investigated with a spatial analysis of molecular variance (SAMOVA). A total of eight haplotypes and three genetic groups (SAMOVA) were recovered and a much higher haplotype number (eight haplotypes) and haplotype diversity (0.7425) was observed in unglaciated compared to glaciated populations . All haplotypes found in glaciated regions represented a subset of haplotypes found in unglaciated regions. Haplotypes of S. foetidus likely diverged during the Tertiary (mid-Miocene and late Pliocene), predating the last glacial maximum (LGM). Predictions based on ecological niche modeling (ENM) suggested that there was considerably less suitable habitat for skunk cabbage during the LGM, and the habitat range was further south compared to the current distribution. Reduced variation and a subset of haplotypes in glaciated regions suggest a founder effect associated with range expansion via long-distance seed dispersal. Our results do not support the “Driftless Area” scenario for the northern refugium, rather the data suggest a “Northeastern” refugium near the southernmost extent of the LGM.Alternating glacial and interglacial periods during the Quaternary have dramatically affected the distribution and population genetic structure of plant and animal species throughout the northern hemisphere. Surprisingly, little is known about the post-glacial recolonization history of wetland herbaceous perennials that are widely distributed in the understory of deciduous or mixed deciduous-evergreen forests in eastern North America. In this study, we investigated infraspecific variation among 32 populations of skunk cabbage,  During the Quaternary Period, climatic oscillations profoundly affected species distribution as well as population genetic structure across the Northern Hemisphere Hewitt, , 2003. ITrillium grandiflorum (Melanthiaceae) found no significant reduction in genetic diversity in glaciated vs. unglaciated regions. The authors also provide evidence that restricted gene flow shaped population differentiation, and that more pollen flow relative to seed dispersal was important in the occasional long distance dispersal of species during northward migration. Second, Li et al. (Smilax herbacea complex (Smilacaceae) in the eastern U.S. and southeast Canada, found an Appalachian discontinuity, with multiple refugia both east and west of the Appalachians. The authors stressed the importance of a “Driftless Area,” described as a Midwestern northern refugium distinct from southern refugia in the U.S., and postulated contact between the Midwest and the East Coastal lineages during expansion. Although the two plant species from these relative studies share similar life history traits and distribution (eastern U.S. and southeastern Canada), different patterns of genetic structure and phylogeography were observed. Thus, additional studies are necessary in order to explain emerging patterns of the distribution and genetic structure of herbaceous plant species found in the understory of temperate forests in eastern North America.Phylogeography aims to relate evolutionary processes to spatial, temporal and environmental factors in an effort to understand past and present biodiversity  is a relatively recent innovation that has greatly enhanced phylogeographic studies, in particular those of systems that have few physical specimens such as species that diverged in the Pleistocene refugia , only S. foetidus (L.) Salisb. ex W. P. C. Barton, is likely native to North America, with a vast distribution in eastern North America. Like other congeneric species in East Asia, S. foetidus occurs in swamps, wet woods, along streams, and other wet low areas . S. foetidus is common in southeastern Canada  and the eastern U.S., ranging from Minnesota to North Carolina and Tennessee. However, at its southern range limit, S. foetidus is less common and is an endangered species in Tennessee, being highly restricted to the northeastern corner of the state   has been estimated at approximately 10 m, likely facilitated by rodents  in North America, where half of their diet in 1994 and almost the entire diet (99%) in 1996  noncoding regions to elucidate the refugial and recolonization history of herbaceous S. foetidus in eastern North America. Specifically, the aim of this study was to address the following questions: (1) Is there evidence of reduced genetic diversity in recolonized populations in the northern range limit? We would expect a trend of south to north decreasing genetic diversity as a result of repeated founder effect if the current populations of S. foetidus arose from a southern glacial refugium. (2) Is there evidence for the existence of the Driftless Area northern refugium for S. foetidus? If this area has served as a suitable refugium during the LGM, we expect higher genetic diversity, increased relative abundance of endemic and ancestral alleles, and genetic divergence among refugia. In addition, sharing of some unique/ancestral haplotypes between the Driftless Area and the glaciated areas of the Midwest is expected. Finally, (3) are there post-glacial colonization patterns in the present geographical range of S. foetidus?S. foetidus individuals, representing 32 populations in the U.S. and Canada, were sampled  to the southernmost population . The approximate extent of the ice sheets at the Last Glacial Maximum was based on Dyke et al.  based on the normalized PIC  values  of the large single copy region were selected for their relatively high level of variation. Then these four regions were used to survey all 485 accessions. The PCR mixture consisted of the template DNA, 0.5 μM of each primer, 1.25 units of Inclone™ Taq polymerase , 100 mM dNTPs, and 2.5 mM MgCl2. Polymerase chain reaction (PCR) amplification was done using the following program: initial denaturation at 95°C for 5 min followed by 35 cycles of denaturation at 95°C for 1 min, primer annealing at 54–56°C for 1 min, and primer extension at 72°C for 2 min; followed by a final extension step of 10 min at 72°C. PCR products were stained with Loading STAR staining  on a 1% agarose gel and visualized under ultraviolet light. The amplified PCR products were purified with IncloneTM Gel & PCR purification kit  and subsequently sequenced using a BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) at Geno Tech Corporation . The program Sequencher 4.7 program  was used to assemble the contigs and edit the sequences. Haplotype sequences of S. foetidus were deposited in GenBank: accession numbers MH064178-MH064185 for psbJ-petA, MH064186-MH064193 for trnS-trnG, MH064194-MH064201 for rpl32-trnL, and MH064202-MH064209 for trnQ-5′rps16.Total genomic DNA was extracted from silica gel-dried samples using the DNeasy Plant Mini Kit , following the manufacturer's instructions. Given the lack of available nuclear genes and simple sequence repeat markers (SSRs), we sequenced plastid DNA to acquire haplotypes for phylogeographic analyses. Twenty-nine noncoding regions  was used as outgroup based on previous phylogenetic results . Genetic variation within and among populations was assessed by analysis of molecular variance (AMOVA) implemented in Arlequin 3.5  that maximizes the statistic FCT, which is an indicator of the proportion of total genetic variance due to differences between groups of populations, and minimizes FSC, the proportion of total genetic variance shared between spatial groups within groups. Each run began with a new random starting point, and 100 starting points (iterations) as recommended by the manual. For the SAMOVA analysis, 32 populations of S. foetidus with intra population samplings  were analyzed. We repeated the analyses with different numbers of groups (K = 2 to 10 simulated groups) until the FCT value (the proportion of genetic variation among groups) reached a plateau.All cpDNA sequences were aligned with MAFFT version 7  as an outgroup (3 accessions) based on its sister relationship to the Orontioideae and included representative sequences of Orontium (4 accessions), Lysichiton (5 accessions for each species), Symplocarpus nipponicus (10 accessions), and S. renifolius (5 accessions from Japan). All these sequences were deposited in GenBank (accession numbers MH203650-MH203753). We used the GTR+I+G model, Yule process speciation prior, and lognormal relaxed clock. The analyses were run for 40 million generations, sampling every 5000 generations. Tracer v. 1.6.0  and the last inter-glacial . We obtained the occurrences from the Global Biodiversity Information Facility (GBIF) database  and our own field investigations. We manually filtered occurrences data, and sorted location inconsistences based on the expected distribution ranges. The final dataset included 1,524 occurrence records of S. foetidus. We used 19 bioclimatic factors as environmental data for ENM to predict the species' current distribution, then projected the species' probable distribution to the LGM and LIG. The environmental data describing the baseline climate , data for the LGM with spatial resolution of 2.5 arc min resolution and for the LIG , maximum iterations  and 25% of the sites from model training. Habitat stability was calculated as the sum of logistic suitability values of niche projections during present, LGM, and LIG. Habit stability per population was calculated as the average of habitat stability values of each point location. Pearson correlation coefficients (r) were used to estimate the linear associations of habitat stability with haplotype diversity (Hd), nucleotide diversity (π), and private haplotype number. Furthermore, the associations of latitude with haplotype diversity, nucleotide diversity, and private haplotypes were estimated using R  based on the current distribution of psbJ-petA: 1,128 bp; trnS-trnG: 995 bp; rpl32-trnL: 1,041 bp; trnQ-5′rps16: 877 bp) were sequenced from 485 individuals (32 populations) of S. foetidus in eastern North America. The aligned sequences were 4,041 bp long in total and contained 36 variable sites, including 8 nucleotide substitutions, 11 indels, and one inversion (17 bp inversion in psbJ-petA region). We excluded mononucleotide repeat regions and coded the remaining indels as binary character (A or T). We found one substitution (position 207) and one large inversion  in psbJ-petA, two indels  and one non-mononucleotide substitution/indel  in trnG-trnS, four substitutions  in rpl32-trnL, and three substitutions  in trnQ-rps16  were identified from the data set. There were five haplotypes from the glaciated regions , while eight haplotypes were found from the unglaciated regions (H1-H8)  and 29.6% (144 accessions), respectively   for the major lineage 1 and 6.62 mya  for the major lineage 2. Within each intraspecific lineage, haplotypes diverged during the Pleistocene, ranging from 1.90 mya  for H1 and H2 haplotypes to 2.41 mya  for H7 and H8 haplotypes. The divergence between S. foetidus in eastern North America and S. renifolius in East Asia was estimated to be 22. 25 mya .The ML analysis (not shown) and Bayesian dating Figure  supporteS. foetidus  were clearly multimodal , haplotype diversity and latitude , nucleotide diversity and latitude , and private haplotypes and latitude   as the optimal number of genetic “groups” (K) based on spatial locations and cpDNA haplotypes. Although there was very little difference between three and four groups , FCT (among-group variation) reached a plateau when the number of groups (K) was set at 3  in the unglaciated region ranged from 0.0000  to 0.001079 (NC1) Table . Haploty Figures . In term Figures . For thrThe cross validation of the climate envelope models revealed a high mean model fit with AUC = 0.9669 (SD 0.002). For the present Figure , these pTrillium grandiflorum, which has similar life history traits and distribution patterns as S. foetidus, no significant reduction in genetic diversity was reported between unglaciated and glaciated regions on the basis of allozyme diversity  was sampled from western Wisconsin and an adjacent Midwestern state (Illinois), and this area was most likely colonized by populations from the Appalachian Mountains  and east of the Appalachians . Because this same haplotype was shared among all but three populations  from the Midwestern states  this haplotype likely represents colonization by the populations from the Appalachians  found in the glaciated states , this haplotype was recolonized by the east side Appalachian refugium located in Delaware, Maryland, Pennsylvania, and Virginia  appear to be consistent with hypothesized colonization routes in S. foetidus. Another purported refugial area, the mid-Appalachians, the associated colonization routes of select amphibian species  and eastern North America (S. foetidus) within the genus Symplocarpus (ca. 6.88 ± 4.18 mya)   for the origin of S-HK and S-CK designed the experiment, drafted the manuscript. S-HK, M-SC, PL, and S-CK collected samples. S-HK performed the experiments. S-HK and M-SC analyzed the data. All of the authors have read and approved the final 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.


Saccharum officinarum and S. spontaneum accessions. A total of 285 alleles was obtained by a set of 12 SSRs primer pairs that taken together were able to efficiently distinguish and capture the genetic variability of sugarcane commercial cultivars and basic germplasm accessions allowing its application in a fast and cost-effective way for routine cultivar identification and management of sugarcane germplasm banks. Allelic distribution revealed that 97.6% of the cultivar alleles were found in the basic germplasm while 42% of the basic germplasm alleles were absent in cultivars. Of the absent alleles, 3% was exclusive to S. officinarum, 33% to S. spontaneum and 19% to other species/exotic hybrids. We found strong genetic differentiation between the Brazilian commercial cultivars and the two main species (S. officinarum: S. spontaneum: S. officinarum and S. spontaneum. Genetic divergence found between cultivars and S. spontaneum accessions has practical applications for energy cane breeding programs as the choice of more divergent parents will maximize the frequency of transgressive individuals in the progeny.The Brazilian sugarcane industry plays an important role in the worldwide supply of sugar and ethanol. Investigation into the genetic structure of current commercial cultivars and comparisons to the main ancestor species allow sugarcane breeding programs to better manage crosses and germplasm banks as well as to promote its rational use. In the present study, the genetic structure of a group of Brazilian cultivars currently grown by commercial producers was assessed through microsatellite markers and contrasted with a group of basic germplasm mainly composed of  Sugarcane has a worldwide economic importance, with Brazil being the largest producer, harvesting over 657.2 million tons of sugarcane in 2016/17 [Saccharum, related to other genera as Erianthus sect. Ripidium, Miscanthus sect. Diandra Keng Narenga Bor, and Sclerostachya Hack A. Camus, which grouped together, constitute the Saccharum Complex [Sugarcane belongs to the genus  Complex .Saccharum genus occasionally yields viable offspring with superior phenotypes via interspecific crosses [th and 20th century in Asia by Dutch cane breeders [S. officinarum, known as “noble cane”, a decaploid  with thick, juicy stalks rich in sugar but highly susceptible to several biotic and abiotic stresses, and S. spontaneum, an octaploid  with no significant sugar content but highly resistant, vigorous [S. officinarum accessions to recover the sucrose content in a process known as nobilization [S. spontaneum and the remaining 5–10% being recombinant chromosomes [Saccharum species such as S. robustum, S. barberi and S. sinense, although used less extensively in the first artificial interspecific hybridizations, also contributed to the genome of modern sugarcane cultivars [Saccharum germplasm, very few accessions took part in the first interspecific hybrids [A major breakthrough for sugarcane came with the discovery that the  crosses . Artificbreeders  and basivigorous ,7, and eoid 2n = 0, x = 10lization . Therefoomosomes . Other Sultivars . Despite 2n = 40–8, x = 8  hybrids  leading Saccharum species and related genera including those collected in the sugarcane first expeditions, acting on an internationally collaborative level [According to the International Union for Conservation of Nature (IUCN), the conservation of genetic diversity is crucial for the maintenance of biodiversity ,15. For ve level .Saccharum species [Undoubtedly, the genetic variability present in most of the germplasm banks is underexploited. Sugarcane germplasm banks have thousands of accessions, which complicate logistics and planning of future crosses. Organization and classification are key factors for the functionality of germplasm banks. Although morphological descriptors have an important role in accession classification and organization, errors may occur once environmental conditions affect phenotypes. Obtaining the molecular profile of the accession, which is a more reliable way to identify a given individual ,21, is a species ,23. Micr species . SSRs ar species . In addi species . Due to  species . SSRs ar species . For sug species  or from  species –32.S. officinarum and S. spontaneum), permit breeders to better manage their crosses and germplasm banks.The narrow genetic base of sugarcane breeding programs and its implications for future sugar yield and productivity gains are concerns among sugarcane breeders around the world ,34. HoweS. spontaneum has been used as parental in crosses with commercial cultivars to meet the demand for biomass production [The characterization of sugarcane basic germplasm accessions has gained attention because of its utilization by sugarcane breeding programs , mainly oduction .Due to the importance of the sugar and ethanol sector in the Brazilian economy, new sugarcane cultivars are release almost every year by the breeding programs increasing the number of cultivars available for the cane growers. However, the assessment of its genetic structure and hence, the narrow genetic base’s impact on the magnitude of the Brazilian sugarcane genetic breeding pool has not yet been completely investigated.S. officinarum and S. spontaneum accessions. Here, we also report a set of twelve SSR primer pairs that, taken together are capable to distinguish sugarcane commercial cultivars and basic germplasm accessions, showing high power in capturing genetic variability and hence allowing the application of molecular markers in a fast and cost-effective way for routinely cultivar identification and management of sugarcane germplasm banks. We found significant contribution of S. spontaneum in the genetic variability of Brazilian sugarcane cultivars. Nonetheless, our data suggest that there is still potentially exploitable allelic richness  that can be incorporated in the gene pool of Brazilian breeding programs through energy cane cultivar development. This is the first report to investigate the genetic diversity at the molecular level in a huge group of currently planted Brazilian sugarcane cultivars.In the present study, the genetic structure of a panel of Brazilian commercial cultivars currently in use by cane growers  was assessed through microsatellite markers and contrasted against a group of basic germplasm composed mainly of S. officinarum, 26 S. spontaneum and 11 genotypes comprising five Saccharum spp. exotic hybrids and six accessions representing other species—S. barberi, S. robustum and Miscanthus spp.) were collected from the IAC Sugarcane Germplasm Collection maintained at the Sugarcane Center, in Ribeirão Preto, SP, Brazil. For clarity, the last group of 11 genotypes will henceforth be referred to as “other species/exotic hybrids.” Most of the S. spontaneum accessions and other species/exotic hybrids was imported from the USDA’s World Collection of Sugarcane and Related Grasses . The 56 accessions, which compose the basic germplasm group, were previously selected, taking into consideration molecular data, as the most divergent, non-redundant and historically relevant accessions of the IAC Fingerprint SSR-based Data Bank, among over 300 others. Some of the historically relevant individuals [A total of 137 genotypes encompassing 81 Brazilian commercial sugarcane cultivars and 56 basic germplasm accessions . Reactions were amplified on a MyCycler Thermal Cycler (BIO-RAD®) thermocycler. For CV  primer pairs: CV29, CV37, CV38, CV60, CV79, CV94, and CV106 [Total genomic DNA was extracted from young leaves or leaf roll tissue according to the CTAB method . PCR reand CV106 ,30 thermnd CV106 . For thend CV106 ,41 thermnd CV106 , allied nd CV106  and alsoDue to the polyploid nature of sugarcane, the amplified products were scored as presence (1) or absence (0). The 50-350pb molecular weight standard (LI-COR Biosciences®) was used to calibrate the sizes of the amplified alleles on each run. The gel images were manually scored and revised with Saga GT™ software (LI-COR Biosciences®). This software identifies the amplified product size to build the genotyping matrix.PIC), Effective Multiplex Ratio (E), Marker Index (MI), Resolving Power (Rp) and the percentage of different profiles (haplotype assessment) were taken as indexes to evaluate the efficiency/discriminatory potential of the 12 SSRs primer set to capture polymorphism and distinguish sugarcane genotypes. The percentage of different profiles refers to the different molecular patterns (fingerprints) generated by each SSR primer pair considering the total number of genotyped individuals with no missing data point. Furthermore, for each SSR primer pair, the frequency of each type of allele and the presence of exclusive alleles  were also investigated.The Polymorphism Information Content (PIC) was calculated [p is the frequency of the jth pattern for marker i, and n is the number of alleles) using the PIC Calculator program [n) was estimated as the average number of fragments amplified per genotype while the Effective Multiplex Ratio (E) was estimated by the expression E = nβ in which n is the multiplex ratio and β the fraction of polymorphic loci [MI) was obtained by the expression MI = PICxE. The percentage of different profiles refers to the different molecular patterns (fingerprints) generated by each SSR primer pair. The Resolving Power (Rp) for each primer pair was calculated from the genotyping matrix by using the expression Rp = ∑Ib, where Ib is the allele information, i.e., Ib = 1 − (2 × |0.5 − M|), and M the proportion of the total genotypes containing the allele [Polymorphism Information Content  was estimate for each group of species and commercial cultivars according to the expression Pij is the frequency of the jth pattern for marker i, summed across n patterns [The frequency of the different allele types  observed for a SSR primer pair was estimated by dividing the number of times that each type of allele occurs by the total number of alleles amplified by the respective SSR primer pair. Genetic diversity  was performed by the Arlequim v.3.5 software  to quantThe number of sub-populations (k) and the membership proportion (Q) was inferred by using Structure v.2.3.4 software ,51. The The pair-wise dissimilarity was estimated based on the Jaccard similarity coefficient complement (Dissimilarity = 1 –Similarity) ,54. The Aiming to detect the presence of some clustering pattern among the evaluated genotypes, Principal Coordinate Analysis (PCoA) –65 was cThe 12 SSR primer pairs generated 285 alleles (markers) among the 137 genotypes. This set of SSR primers produced high quality fingerprints , minimizPIC estimates varied from 0.776 (CV94) to 0.934 (CV38), with a mean value of 0.871 for all the 137 accessions, and for the cultivars, from 0.720 (CV60) to 0.922 (SCB312), with mean of 0.862. The Effective Multiplex Ratio estimated values varied from 2.956 (CV60) to 7.387 (CV29), with mean of 5.580 for all accessions, and from 2.938 (CV60) to 8.457 (SCB381), with mean of 5.837 for the cultivars. The Marker Index estimate values ranged from 2.262 (CV60) to 6.800 (CV29), with mean of 5.028 for all accessions, and from 2.115 (CV60) to 7.743 (SCB381), with mean of 5.126 for the cultivars.To assess the ability of the primer pair for distinguishing among genotypes, the Resolving Power was calculated for the total of genotypes or for the cultivars only. For total genotypes, the Resolving Power estimates ranged from 3.149 (CV60) to 11.746 (SCB312), while for the cultivars it varied from 1.778 (CV94) to 10.642 (SCB312).The highest index values for PIC, Effective Multiplex Ratio, Marker Index and Resolving Power across all accessions was obtained for primer pairs CV38 (0.934), CV29 (7.387), CV29 (6.800) and SCB312 (11.746) respectively. Considering only the group of cultivars, the highest index values for PIC, Effective Multiplex Ratio, Marker Index and Resolving Power were obtained for primer pairs SCB312 (0.922), SCB381 (8.457), SCB381 (7.743) and SCB312 (10.642) respectively. Primer pair CV38 generated the highest number of different profiles  for the whole 137 accessions .Haplotype assessment revealed that eight primer pairs  were able to produce different SSR profiles in more than 90% of the individuals while CV106 in 64.2% and SCB436 in 59.9%. The lowest percentage of different profiles (38.0% and 29.9%) was obtained for CV94 and CV60 respectively.S. officinarum, S. spontaneum, and other species/exotic hybrids was 166, 186, 246 and 215, respectively  were not found in the Brazilian cultivars , representing 1.4% of the total alleles, were exclusive to the commercial cultivars. One hundred and nineteen alleles (41.8%) spread across the basic germplasm accessions were not present in the Brazilian cultivars. Of these, 55 putative exclusive alleles of the basic germplasm group  showed one putative exclusive allele (SCB436.0186) derived from S. officinarum. In addition, six exclusive alleles  derived from S. spontaneum were spread among twenty cultivars . None of the nineteen other species/exotic hybrids exclusive alleles were observed in cultivars.To find the species origin of the alleles present in the Brazilian commercial cultivars, the presence of putative exclusive alleles from Alleles CV38.0210, CV106.0135, SCB381.0244 and SCC423.0300 were absent in the basic germplasm  and hencS. spontaneum had the highest genetic diversity (D) estimate value (0.900) followed by other species/exotic hybrids (0.898), S. officinarum (0.883), and Brazilian commercial cultivars (0.873).Regarding to the gene diversity values , S. sponS. officinarum accessions (S. spontaneum (S. officinarum and S. spontaneum accessions (The degree of the genetic differentiation estimate (P<0.001) .S. officinarum and S. spontaneum), capturing most of the data structure in three groups . In fact, around 140 markers would be sufficient to obtain a CV average estimate around 10% .S. officinarum, S. spontaneum, and other species/exotic hybrids was 0.176, 0.206, 0.248, and 0.285, respectively. The average dissimilarity values between the cultivars and S. officinarum was 0.247, while for between the cultivars and S. spontaneum it was 0.277, and between the cultivars and accessions from other species/exotic hybrids it was 0.272. In relation to the two main sugarcane species, the average dissimilarity between S. officinarum and S. spontaneum accessions was 0.279. The average dissimilarity between the accessions from other species/exotic hybrids and S. officinarum was 0.282, and with S. spontaneum it was 0.278  was the accession most genetically similar (0.203) to the commercial cultivars and CHUNNEE the most divergent (0.326)  .We observed a low dissimilarity value (0.077) between IAC87-3396 and SP87344, which are full sibs from the cross CO740 and SP701143, for example.S. officinarum and S. spontaneum), with the accessions from other species/exotic hybrids spread among these two groups suggesting the existence of three groups  which agrees with the K = 2 value (two subpopulations) pointed to by the software Structure. In addition, the accessions were mainly clustered according to their species  explained 19.18% of the total variability expressed among genotypes, based on dissimilarity measurement . AccordiUndoubtedly, the availability of an efficient and adequate size set of SSR primer pairs has a great impact for routine management of sugarcane germplasm and cultivar identification in a cost-effective way. Several indexes are available to evaluate primer pair efficiency for polymorphisms at the molecular level ,22,28,47Saccharum spp. molecular database.Microsatellites allow the amplification of different allele types at a locus. Due to the sugarcane’s high ploidy level and consequent heterozygosity, more than two types of alleles per locus in a single individual can be observed and the “banding patterns” reveal the different allele types of a locus defined by one SSR primer pair, although the allele dosage or number of copies cannot be determined ,41,44. TThe high number of alleles obtained by the 12 SSR primer set also positively impacted PIC and Resolving Power (Rp). Most of the PIC values were above 0.7 for either the total accessions or the cultivars. The Resolving Power, which reveals the ability of a given primer pair to distinguish between genotypes , was higS. spontaneum is considered the most diverse species in the Saccharum genus [S. spontaneum (63%), as compared to S. officinarum (6.5%) and other species/exotic hybrids (31%).Due to its large ecogeographical distribution, um genus ,68. ThisS. officinarum, two S. spontaneum and one S. sinense clones were extensively used to form the genetic base of modern sugarcane during the first interspecific hybridizations [S. officinarum contribution [Cultivars encompassed more than half of the total alleles occurring in the basic germplasm showing a considerable representation of its allelic richness. However, a reasonable number of putative exclusive alleles derived from the basic germplasm (41.8%) was not inherited by the Brazilian commercial cultivars. In fact, a very limited number of accessions were used in the early interspecific crosses to develop the first commercial sugarcane cultivars ,69. Onlyizations . This geribution .S. officinarum and S. spontaneum, respectively. This could be a result of the nobilization process, as almost 80% of the commercial cultivar genome is from S. officinarum, indicating that most of the alleles present in the Brazilian commercial cultivars are shared with S. officinarum. On the other hand, the proportion of S. spontaneum exclusive alleles found in the Brazilian commercial cultivars seems to be close to the S. spontaneum genome contribution (around 10–15%) expected for commercial sugarcane cultivars [Of the few putative-species-origin exclusive alleles within the Brazilian commercial cultivars, only one of four (25%) and six of thirty-nine (15.4%) were derived from ultivars .S. spontaneum had a significant contribution to the genetic variability of commercial cultivars used in Brazil. Aitken et al. (2006) [S. spontaneum chromosomes.This result also suggests that . (2006) , assessiWe also reported the presence of putative exclusive alleles derived from commercial cultivars, suggesting that either the basic germplasm sampled in our work did not encompass the whole genetic pool used in the prior breeding programs, or that new alleles in commercial cultivars could have emerged over time as a result of DNA polymerase slippage or unbalanced crossing-over, which are mechanisms that lead to insertions or deletions of one or more repeats ,73.ST. According to Wright (1978) [ST values between 0 to 0.05 signals little population differentiation while values in the 0.05–0.15 and 0.15–0.25 ranges, respectively, indicate moderate and strong genetic differentiation. The genetic differentiation value between the Brazilian commercial cultivars and S. officinarum was slightly lower (21.1%) than that observed between commercial cultivars and S. spontaneum (21.6%), and in turn, these values reflect a strong genetic differentiation between the commercial cultivars and the two main Saccharum species. This strong genetic differentiation between Brazilian commercial cultivars and these main species reflects the previously mentioned founder effect, combined with the genetic drift effect, as a small number of these two progenitor species’ clones contributed to the current genetic base of commercial sugarcane cultivars. Moreover, for the Brazilian cultivars, there is a high number of shared common parents among them, which also contributes to an increase in the genetic differentiation between commercial cultivars and their ancestor species.The genetic differentiation index (. (1992)  is consit (1978) , FST valS. officinarum and S. spontaneum signals that considerable genetic variability can be incorporated in the current genetic breeding pool by exploiting both species, especially S. spontaneum for energy cane development.Despite the limited genotypes employed in the first breeding programs, the sugarcane genetic base has not narrowed as much as had been suspected , especiaS. officinarum and S. spontaneum exposing a genetic relationship between these two species. As pointed out by Aitken and McNeil (2010) [S. spontaneum in the Himalaya foothills of northern India, from which, as a consequence of evolutionary forces such as selection, migration, and introgression, two centers of diversity, respectively for S. spontaneum in India and S. robustum in New Guinea had resulted. However, it is well accepted that S. officinarum has emerged from S. robustum in New Guinea by domestication of this later species [Moderate genetic differentiation was obtained between l (2010) , based ol (2010) , it was  species .S. officinarum, S. spontaneum, and other species/exotic hybrids) was confirmed by the PCoA results. The commercial cultivars were assigned to a separate cluster, as expected, since most of them share the same parents and were bred to meet the high demand for sugar and ethanol production. The S. officinarum accessions have outstanding features such as high sugar production, low fiber content, and thick stalks, among others, which differs from related species. In accordance with the phenotypic differentiation, divergence at the molecular level was observed between S. officinarum accessions and the other species/exotic hybrids, justifying the formation of an isolated cluster composed by genotypes of this species. S. spontaneum, and the other species/exotic hybrids are more similar, and some overlap was shown between these groups. However, there is a slight trend toward separation among them  was observed within the accessions of the other species/exotic hybrids group while the lowest (0.176) within the cultivars group. The average dissimilarity obtained within cultivars was 1.2 and 1.4 times lower than that within  parents  amplifieThe average dissimilarity within cultivars implies that on average the commercial Brazilian cultivars shared 82% of the genomic regions assessed with SSRs. This is the first report in which a very large number of currently planted Brazilian cultivars was investigated at the molecular level, giving a more complete picture of their genetic relationship.The pairwise dissimilarities of Brazilian cultivars ranged from 0.077 (IAC87-3396 x SP87344) to 0.258 CTC2 x SP801836), and was lower than that observed in SSR assays by Pocovi and Mariotti (2015) 1836, and assessedS. spontaneum individuals, GLAGAH and IN8488, were grouped within S. officinarum. In fact, despite its classification as S. spontaneum, GLAGAH is a possible natural hybrid between Saccharum species. Although IN8488 is considered a S. spontaneum clone, the membership proportion showed high identity correlation with S. officinarum, suggesting that it probably is also a hybrid. POJ2878 and KASSOER are tightly clustered in the phylogenetic analysis. In addition, the structure analysis revealed almost the same membership proportion of the three groups in both individuals, revealing an already expected low genetic dissimilarity (0.0947) because POJ2878 is a direct F2 descendant of KASSOER [Clustering and phylogenetic analyses revealed the parenthood among the total genotypes evaluated. They tend to cluster according to their species and genealogy, forming consistent groups in agreement with the pedigree data available. However, some discrepancies were observed. Two  KASSOER .S1 FigPolyacrylamide electrophoresis assays generated high quality images allowing accurate genotyping.(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 TableNames with respective species, female and male genitors, and origin.(DOCX)Click here for additional data file.S2 TableNumber of alleles, exclusive alleles, species exclusive alleles found in commercial cultivars, absent alleles in commercial cultivars and gene diversity.(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.


Salix psammophila, a sandy shrub known as desert willow, is regarded as a potential biomass feedstock and plays an important role in maintaining local ecosystems. However, a lack of genomic data and efficient molecular markers limit the study of its population evolution and genetic breeding. In this study, chromosome counts, flow cytometry and SSR analyses indicated that S. psammophila is tetraploid. A total of 6,346 EST-SSRs were detected based on 71,458 de novo assembled unigenes from transcriptome data. Twenty-seven EST-SSR markers were developed to evaluate the genetic diversity and population structure of S. psammophila from eight natural populations in Northern China. High levels of genetic diversity  were dectected in S. psammophila. The weak population structure and little genetic differentiation (pairwise FST = 0.006–0.016) were found among Population 1-Population 7 , but Pop8 (Ningxia) was clearly separated from Pop1-Pop7 and moderate differentiation (pairwise FST = 0.045–0.055) was detected between them, which may be influenced by local habitat conditions. Molecular variance analyses indicated that most of the genetic variation (94.27%) existed within populations. These results provide valuable genetic informations for natural resource conservation and breeding programme optimisation of S. psammophila. Salix; Salicaceae) are dioecious catkin-bearing plants that are mainly distributed in the northern hemisphere. Worldwide, there are approximately 330–500 species, with various growth habits ranging from tall trees, through shrubs and bushes, to prostrate, dwarf, and rockery plantsSalix genus is 19, and the ploidy levels range from diploid (2n = 2x = 38) to dodecaploid (2n = 12x = 228), with 40% of polyploid species being tetraploid to octoploid1235Salix miyabeana is a tetraploid species that has been hybridised with diploid species in recent breeding to produce triploid progeny with superior performance. An investigation of its interspecific hybrid progeny found that the triploid progeny had higher yields and better overall performance than did the diploid or tetraploid progenyWillows S. reinii on the southeastern slope of Mount FujiS. lanata and S. lapponum at Coirre Sharroch of the Glen Clova Mountains, whereas clonal growth plays an important role in S. herbacea at Meall GhaordieS. purpurea populations are higher than that among native S. eriocephala populationsS. daphnoides at the westernmost foothills of the Carpathian MountainsS. viminalis in the Czech Republic and Europe28S. caprea have been detected in semi-natural woodlands across IrelandS. psammophila has received little attention. Thus, to speed up its marker-assisted selection breeding programmes, developing species-specific SSR markers for examining the genetic diversity and population structure of S. psammophila is essential.To date, some polymorphic SSR markers have been developed in S. psammophila is still at an early stage and the development of SSR markers is limited by the lack of genomic and transcriptomic information. In recent years, Illumina high-throughput sequencing technology has emerged as a powerful tool for obtaining genomic or transcriptomic dataCryptomeria japonica, Coffea arabica, Vigna radiate, Lavandula, Caragana korshinskii and Phaseolus vulgaris), and ample expressed sequence tag (EST)-SSR markers have been developed from the transcript sequences32333435Currently, research into S. psammophila, reported the first transcriptome sequencing from various tissues of S. psammophila, developed EST-SSR markers based on the transcriptome sequencing data, and analysed the genetic diversity and population structure of eight natural populations in Northern China. This work provides fundamental information for natural resource conservation and breeding programme optimisation of S. psammophila in the future.In this study, we performed chromosome counts and genome size estimation of S. psammophila, eight genets from different populations that represented the main distribution areas, including six populations from Inner Mongolia, one population from Shaanxi Province and one population from Ningxia Province  technique was performed to estimate the genome size using the fresh leaves of 27 genets, including the eight genets used for chromosome counts and 19 additional genets. With its genome size of approximately 425–429 Mb, S. suchowensisS. psammophila to that of S. suchowensis was equal to 1.645, indicating that the genome size of S. psammophila is estimated to be approximately 699–706 Mb  were retained. The GC content was 45.28%, and the Q20 and Q30 were 94.73% and 90.55%, respectively. With the aid of the short-reads assembling software Trinity, these clean reads were de novo assembled into 159,737 contigs, and then into 71,458 non-redundant unigenes, with an N50 length of 1,274 bp and a mean length of 713 bp (http://www.ncbi.nlm.nih.gov/Traces/sra) under accession number SRA3189819.Equal quantities of RNA from five tissues including the root, stem, leaf, female catkin and male catkin of f 713 bp . The seqS. psammophila using a bioinformatics approach, a sequence similarity search was conducted in six public databases  were determined to contain 6,346 SSRs. Among these developed SSRs, 424 (7.55%) contained two or more SSRs, and 403 (6.35%) comprised compound SSRs  and class II (n ≥ 20 nucleotides). As shown in All the unigenes were used to detect EST-SSRs. Of the 71,458 (50.97 Mb) unigenes of und SSRs . The freund SSRs . There wund SSRs . The mos S. psammophila genets from eight natural populations in Northern China (S. psammophila based on 27 SSR markers was shown in E) and the Shannon–Wiener index ranged from 0.436 to 0.883 and from 0.696 to 2.133, with averages of 0.689 and 1.345, respectively. The polymorphism information content (PIC) was in the range of 0.386–0.888, with an average of 0.714. Markers c-59 and c-25 shared the highest and lowest genetic diversity, respectively, with values of 0.883 and 0.436 for HE and 0.888 and 0.386 for the PIC.A total of 168 EST-SSR markers containing di- and tri-nucleotide repeats were randomly selected and primers were designed according to their flanking sequences. Of the 168 EST-SSR primers, 125 pairs showed successful amplification. Finally, 27 pairs that showed stable amplification and had multiple alleles were selected to analyse the genetic diversity and population structure of 240rn China . These 2rn China . A summaS. psammophila is tetraploid in the natural population.Using these 27 EST-SSR makers, all 240 genets amplified a maximum of four alleles at some loci. Due to co-dominance, SSR markers present different alleles if they are heterozygous. Thus, SSR analyses also indicate that S. psammophila genets. The optimal number of PCs (45) was assessed using the optim.a.score function. The cluster membership probabilities of each genet based on the discriminant functions of DAPC were performed from K = 2 to K = 8 to K = 8 . Most ge8 , and the greatest genetic differentiation was detected between Pop3 and Pop8 (FST = 0.055) , whilst the genetic differentiation between Pop8 and Pop1-Pop7 was relatively high . The genetic differentiation between Pop8 and Pop6 (FST = 0.045) was slightly lower than that between Pop8 and the six other populations  . The genulations .E, Shannon-Wiener index and PIC, suggesting that the genets from Ningxia have low genetic diversity compared with those from the Inner Mongolia and Shaanxi populations. Obvious differences in genetic diversity between Pop8 and the other seven populations were detected.The genetic diversity of the eight populations was assessed, and the result was shown in S. psammophila. To test the proportion of genetic variance between clusters with the K = 2 suggested by the DAPC analyses, AMOVA was performed and showed that 18.56% of the total molecular variance was attributed to between two clusters  indicated that 5.73% of the total molecular variance resided among eight populations, whilst 94.27% was attributed to variance within populations , suggestclusters . Though Salix genus comprises many species, among which polyploids are common1232S. psammophila was found to be tetraploid by chromosome counts. Second, using FCM technique, the G1 peak means of 19 additional genets were similar to that of the eight tetraploid genets, suggesting that these 19 genets are also tetraploids. The genome size of S. psammophila was estimated to be approximately 699–706 Mb, which is approximately 1.6-fold than S. suchowensis with approximately 425–429 Mb. Previous studies of soybeans have shown that the genome size of the allotetraploid soybean (Glycine dolichocarpa) is approximately 1.9-fold than its diploid progenitor (G. tomentella and G. syndetika) and the tetraploid retains at least approximately 87% of the genes that were initially duplicatedKnautia, the results of ploidy and genome size of 23 species indicated that genome size of tetraploid K. arvernensis and hexaploid K. ressmannii is approximately 1.8-fold and approximately 2.7-fold than diploid K. calycina, respectivelyChenopodiumAvenaS. psammophila genets presented a maximum of four alleles at some loci in the SSR analyses, indicating that these genets are tetraploids. Multiple alleles were also detected in S. reinii using SSR markers, suggesting that S. reinii is polyploid; this inference was confirmed by the FCM technique17S. psammophila genets. Other ploidy levels  of S. psammophila may exist in natural populations, and these materials are more meaningful for the study of genome evolution. Therefore, the intraspecific ploidy variation of S. psammophila requires further research.The S. suchowensis and S. purpurea)S. psammophila was verified to be tetraploid and mainly distributed in the desert regions in Northern China. Therefore, the tetraploid nature of S. psammophila may be associated with strong adaptability to infertile soil and harsh climatic environment. Moreover, polyploids have been reported to affect the gene expression at the transcription level compared with diploids. For example, studies in watermelon have shown that 5,362 and 1,288 genes were up- and down-regulated, respectively, in autotetraploid compared with diploid watermelonS. psammophila.Some hypotheses suggest that polyploids have greater resistance to harsh arctic climates than diploids, or that polyploids are more successful than diploids in colonising after deglaciationS. suchowensis (http://115.29.234.170/willow/)S. purpurea  have been released in public databases. In addition, transcriptome analyses of S. matsudana and S. viminalis have been performed to reveal their stress response mechanisms48S. psammophila. In our study, 71,458 non-redundant unigenes with a mean length of 713 bp were de novo assembled, and 37,465 (52.43%) unigenes were aligned to the sequences of six public databases. Based on these unigenes, 6,346 SSR loci were detected. The AG/CT repeat was the most abundant di-nucleotide motif, which is similar to S. babylonica, S. suchowensis, Populus and Eucalyptus; however, the most abundant tri-nucleotide motif was the GAA/TTC repeat, which differs from these species S. psammophila agrees with previous studies that showed that CG/GC and CCG/CGG are very rare in dicotyledonous plants but common in monocotsS. psammophila.Recently, willows have become a focus of research as promising sources for bioenergy crops because of their short rotation coppice cycle and high biomass yields1S. psammophila genets from eight natural populations in Northern China. The mean number of 10.63 alleles per locus and mean HE 0.689 were determined using 27 developed EST-SSR markers in S. psammophila, which was consistent with SSR-derived genetic diversity in S. viminalis 28S. caprea S. psammophila were sampled within a 220 kilometres radius, it is a dioecious and cross-pollination species, and its pollen can disperse over long distances due to windy weather in the pollination period and few obstacles to airborne pollen flow in desert regions, which may explain its high levels of genetic diversity. The small seeds (thousand seed weight of approximately 0.1 g) of S. psammophila are wrapped in fine hairs that facilitate dispersal by wind. Moreover, the tetraploid nature of this species may be another reason for the high genotype diversity of S. psammophila.As with other willow species, high levels of genetic diversity were found in 240 Salicaceae species, such as S. viminalis (92.3%)Salix caprea (91.7%)Populus simonii (85.8%)P. euphratica (85.5%) and P. pruinosa (78.8%)Salicaceae species are generally dioecious and combine anemophilous and entomophilous pollination with an outcrossing breeding system and high dispersal ability, which could enhance intraspecific diversity and reduce population differentiation2754S. psammophila. However, pollination dynamics and the mating system of S. psammophila remain to be poorly understood, and should be further studied to better understand the distribution of genetic variation.AMOVA indicated that most (94.3%) of the total variance was within-population variance. This agrees with the results obtained for other FST = 0.006–0.016) were found among Pop1-Pop7 (Inner Mongolia and Shaanxi), but Pop8 (Ningxia) was clearly separated from Pop1-Pop7, and moderate differentiation (pairwise FST = 0.045–0.055) was detected between them. Previous studies have demonstrated that the habitat types or local environment conditions have a significant influence on the genetic diversity and population structure in some plant species, such as Ranunculus acrisParis quadrifoliaRhododendron jinggangshanicumTamarix chinensisS. psammophila were selected and appeared to differentiation during the long-term evolutionary process. A germplasm collection base of S. psammophila was established approximately 20 kilometres northeast of Pop1 in Inner Mongolia, with its habitat condition belonging to an arid desert habitat. In recent years, we found that most of Pop8 genets appeared dead due to unadaptability to the arid desert habitats in the germplasm collection base, and this phenomenon did not occur in Pop1-Pop7 genets, supporting above mentioned hypothesis. Moreover, the habitat variability could lead to different phenophases, which may constrain the gene flow between Pop8 and other populations. However, detailed reasons for the population genetic structure need to be further studied.The weak population structure and little genetic differentiation  was established in Ordos Dalad, Inner Mongolia in 2008. In total, 21 S. psammophila populations were collected, including Inner Mongolia (15 populations), Shaanxi (4 populations) and Ningxia (2 populations); each population contained approximately 50 samples. To avoid sampling clones, each sample was randomly selected and separated by at least 50–100 m from the next sample. Two-year-old twigs were collected and cut into 15–20 cm cuttings. Subsequently, these cuttings were planted in the germplasm collection base of S. psammophila. In this study, eight populations covering the main natural distribution areas were selected from the 21 populations in the germplasm collection base for population genetic analyses of S. psammophila, and 30 genets were randomly sampled in each population. Details of the locations of the eight populations used in this study are shown in To protect the germplasm resources and to carry out the molecular biology study of S. psammophila and grown in hydroponic culture in a growth chamber under long-day conditions (16 h light/8 h dark) at 25 °C. When new roots grew out, root tip squashes were used to enumerate the chromosome number according to the method described in a previous study with some modifications612O. The root tips were fixed in Carnoy’s fluid  for 24 h at room temperature, steeped in 90% ethanol for 30 min, and preserved in 70% ethanol at 4 °C. To prepare slides, roots were hydrolysed in 1 M hydrochloric acid for 15–20 min at 60 °C. The slides were prepared according to the squash technique with carbol fuchsin. Images were captured using Zeiss Axio Imager A1 microscope. The chromosome number was determined by counting more than ten well-spread chromosomal cells from each of the eight genets.Eight genets  were used for chromosome counts. Twigs were collected from 1-year-old seedlings of S. psammophila was estimated using flow cytometry  according to a previous protocol−1. After discarding the supernatant, the nuclei suspension was stained with 200 μl propidium iodide (PI) at 4 °C for 30 min in the dark. S. suchowensis (approximately 425–429 Mb)The genome size of S. psammophila. Total RNA was extracted using the RNeasy Plant Mini Kit  according to the manufacturer’s instructions. The quality and quantity of total RNA were determined by the NanoDrop system  and an Agilent2100 Bioanalyser . mRNAs were enriched with a NEBNext Poly (A) mRNA Magnetic Isolation Module . Equal quantities of enriched mRNAs of the five tissues were mixed and used as templates to synthesize cDNA. The cDNA library was constructed using the NEBNext Ultra mRNA Library Prep Master Mix Set for the Illumina  and NEBNext Multiplex Oligos for the Illumina  according to the manufacturer’s instructions. Finally, the cDNA library was sequenced on an Illumina HiSeq2500 instrument by Biomarker Technologies Co. Ltd. , with paired-end sequencing technology and read lengths of 125 bp. As a part of the quality control procedure, adapter sequences and low quality reads were filtered for subsequent analyses. The resulting high quality sequences were assembled using Trinity softwareFive tissues  were obtained from an elite tree of −5) with public databases, including the NCBI non-redundant protein sequences (Nr) database (http://www.ncbi.nlm.nih.gov/), the Swiss-Prot database (http://www.expasy.ch/sprot/), the eukaryotic orthologue groups (KOG) database (http://www.ncbi.nlm.nih.gov/COG), the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/), and the protein family (Pfam) database. Gene Ontology (GO) annotation of these unigenes was produced using the Blast2GO programFor functional annotation and classification, the assembled unigenes were aligned using BLASTx alignment . The parameters were designed to identify di-nucleotide motifs with a minimum of six repeats, and tri-, tetra-, penta-, and hexa-nucleotide motifs with a minimum of five repeats2, 1.0 unit Taq DNA polymerase (Takara), 8 pmol each reverse primer and the M13 universal primer (5′-TGTAAAACGACGGCCAGT-3′) that was fluorescently labelled with Cy5 at the 5′ terminus, and 2 pmol forward primer that was added to the M13 primer sequence to the 5′ terminus. PCR amplification was performed on an ABI Applied Biosystems instrument under following conditions: 94 °C for 5 min; 30 cycles at 94 °C for 30 s, 53 °C for 30 s and 72 °C for 30 s; 8 cycles at 94 °C for 30 s, 57 °C for 30 s and 72 °C for 30 s; and a final extension step at 72 °C for 10 min. The capillary electrophoretic separation of PCR products was performed using GenomeLab GeXP , and the data were analysed by the fragment analyses module of the system.The obtained unigenes were used to identify potential SSRs using the MISA tool (http://www.vub.ac.be/APNA/)et al.et al.FST) was calculated using “polysat” softwareadegenet package version 2.0.1optim.a.score function. The genetic distances between the genets and populations were calculated using Populations version 1.2.31 (http://www.bioinformatics.org/~tryphon/populations/) according to the method described by Nei et al.Using the MAC-PR method, all alleles of each locus were analysed in pairwise combinations to determine their dosages in the genets by calculating the ratios between peak areasHow to cite this article: Jia, H. et al. De novo transcriptome assembly, development of EST-SSR markers and population genetic analyses for the desert biomass willow, Salix psammophila. Sci. Rep.6, 39591; doi: 10.1038/srep39591 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Signal transducer and activator of transcription 3 (STAT3) is phosphorylated by various kinases, several of which have been implicated in aberrant fibroblast activation in fibrotic diseases including systemic sclerosis (SSc). Here we show that profibrotic signals converge on STAT3 and that STAT3 may be an important molecular checkpoint for tissue fibrosis. STAT3 signaling is hyperactivated in SSc in a TGFβ-dependent manner. Expression profiling and functional studies in vitro and in vivo demonstrate that STAT3 activation is mediated by the combined action of JAK, SRC, c-ABL, and JNK kinases. STAT3-deficient fibroblasts are less sensitive to the pro-fibrotic effects of TGFβ. Fibroblast-specific knockout of STAT3, or its pharmacological inhibition, ameliorate skin fibrosis in experimental mouse models. STAT3 thus integrates several profibrotic signals and might be a core mediator of fibrosis. Considering that several STAT3 inhibitors are currently tested in clinical trials, STAT3 might be a candidate for molecular targeted therapies of SSc. STAT3 is a transcription factor that is activated in fibrotic diseases such as systemic sclerosis. Here the authors show that STAT3 is the converging point for multiple pro-fibrotic signalling pathways, and that its genetic ablation or inhibition ameliorate skin fibrosis in mouse models. Their common histopathological feature is an excessive accumulation of extracellular matrix, which disrupts the physiological tissue architecture2. Tissue fibrosis can occur after defined stimuli with a subsequent inflammatory response, but in many fibrotic diseases, no initiating trigger can be identified. These idiopathic fibrotic diseases can manifest on virtually every organ. A prototypical systemic idiopathic fibrotic disease is systemic sclerosis (SSc)3. Failure of the affected organs is common in SSc and results in high morbidity and mortality and no targeted therapies are yet available for the treatment of fibrosis in SSc3. Myofibroblasts are the principle source of extracellular matrix during physiologic tissue repair as well as in fibrotic diseases. However, while tissue remodeling is tightly controlled in normal wound healing and is turned off as soon as the damage is repaired, the fibroblasts escape this regulation1. Myofibroblast differentiation may initially be depended on profibrotic cytokines, but with prolonged external stimulation, myofibroblasts become independent of external stimuli and remain persistently activated in fibrotic diseases.Fibrotic diseases impose a major socioeconomic burden on modern societies and have been estimated to account for 45% of deaths in the developed world5. The levels of TGFβ are increased in fibrotic diseases and fibroblasts display activation of TGFβ signaling with increased transcription of TGFβ target genes6. Moreover, stimulation of resting fibroblasts with TGFβ induces an activated myofibroblast phenotype and a gene expression profile in resting fibroblasts that is reminiscent of SSc fibroblasts7. The key role of TGFβ signaling in the pathogenesis of fibrosis is further demonstrated by the development of systemic fibrosis in mice with fibroblast-specific overexpression of constitutively active TGFβ receptor type I (TBRact)8. The effects of TGFβ are mediated by a complex network of intracellular signaling events. SMAD proteins that mediate canonical TGFβ signaling are considered as major intracellular mediators9. SMAD independent, so-called non-canonical TGFβ pathways, such as mitogen activated involving the mitogen-activated protein kinases (MAPKs), focal adhesion kinase (FAK), the tyrosine kinase c-ABL, and early growth response 1 are also transducing in the pro-fibrotic effects of TGFβ11. However, inhibition of these downstream pathways does not completely abrogate the pro-fibrotic effects of TGFβ14, indicating that additional pathways are important to transduce the stimulatory effects of TGFβ. Identification of these novel downstream mediators of TGFβ might have translational implications and may provide the basis for novel-targeted therapies for fibrotic diseases.Although the molecular mechanisms leading to aberrant activation of fibroblasts are incompletely understood, transforming growth factor-β (TGFβ) has been identified as a core pathway of fibrosis16. Upon binding of these ligands to their receptors, STAT3 is activated by phosphorylation at Tyr-705 in the STAT3 transactivation domain. Phosphorylation of STAT3 at Tyr-705 can be executed by kinases of the JAK and SRC families, but also by other receptor- and non-receptor-associated tyrosine kinases, such as JNK and c-ABL19. Phosphorylation at Tyr-705 is essentially required for STAT3 signaling as it allows STAT3 to dimerize, translocate to the nucleus, and to modulate the transcription of target genes and is thus commonly used to assess the activation of STAT3 signaling. STAT3 regulates fundamental cellular processes including inflammation, cell growth, proliferation, differentiation, migration, and apoptosis20. Given its broad regulatory effects, it may not be surprising that deregulation of STAT3 signaling has been linked to the pathogenesis of various diseases. STAT3 is an oncogenic transcription factor and constitutive activation of STAT3 has been observed in numerous malignancies23. STAT3 is activated by several pro-inflammatory cytokines, including interleukin-6 (IL-6)26, which is a prime target for therapeutic intervention in several inflammatory diseases including rheumatoid arthritis, Still’s disease and giant cell arthritis. The levels of IL-6 are also increased in SSc and treatment with monoclonal antibodies against IL-6 receptor may improve clinical outcomes in an inflammatory subgroup of SSc patients27. Of particular interest, STAT3 has also been linked to mesenchymal tissue responses during development and in cancer. STAT3 is essentially required for mesoderm induction in Xenopus embryos28. STAT3 has very recently been found to promote tumor progression by promoting desmoplastic reactions and tumor invasion30. The identification of STAT3 as a potential therapeutic target in various diseases promoted the development of STAT3 inhibitors and these efforts generated numerous candidates31. First of those inhibitors have already been tested in clinical trials with promising results.Signal transducer and activator of transcription 3 (STAT3) was originally identified as an interleukin-6-activated transcription factor and subsequently reported to also transduce signals from several other stimuli including additional cytokines, hormones, and growth factors32. Given the convergence of those mediators toward STAT3 activation, we hypothesized that STAT3, in addition to its central role in inflammation, may be a crucial checkpoint for fibroblast activation and tissue fibrosis.Several of the upstream kinases that regulate phosphorylation and thus activation of STAT3, such as JAK2, JNK, and SRC, have previously been characterized as mediators of non-canonical TGFβ signalingIn this study, we demonstrate that STAT3 signaling is hyperactive in SSc by the combined action of JAK, SRC, c-ABL, and JNK kinases. Pharmacological or genetic inactivation of STAT3 inhibits TGFβ-induced fibroblast-to-myofibroblast transition and collagen release in cultured fibroblasts and ameliorates skin fibrosis in two mouse models of SSc. We thus demonstrate that STAT3 is a central integrator of multiple profibrotic signals and a candidate for molecular-targeted therapies of fibrosis in SSc.P < 0.0001 by Mann–Whitney U-test) , the common readout for STAT3 activation, in the skin of SSc patients as compared to age- and sex-matched healthy volunteers. P-STAT3 accumulates in the SSc skin with prominent staining in spindle-shaped cells in the dermis, whereas only few cells in the dermis stains positive for P-STAT3 in healthy individuals Fig. . Costainst) Fig. . Consistst) Fig. . The actst) Fig. .Fig. 1Ac33, prevents the upregulation of P-STAT3 in bleomycin-challenged mice , P-JNK (Thr-183/ Tyr-185), P-SRC (Tyr-416), and P-c-ABL (Tyr-412) in fibroblasts of SSc skin as compared to skin from healthy individuals Fig. . StimulaSSc Fig. .Fig. 3AcIn vitro studies demonstrate that not only inhibition of JAK2, but also of JNK, SRC, and c-ABL by both siRNA-mediated knockdown and by respective small molecule inhibitors of JAK2, JNK, SRC, and c-ABL all significantly inhibit the TGFβ-induced phosphorylation of STAT3 Fig. . These rTo confirm those findings in vivo, we selectively inhibited JAK2, JNK, SRC, and c-ABL by selective small molecule inhibitors in the experimental murine models of bleomycin-induced skin fibrosis and TBRact-induced skin fibrosis. Individual inhibition of JAK2, JNK, SRC, or c-ABL signaling all significantly ameliorates the bleomycin-induced accumulation of P-STAT3 Fig. . Consist36 or indirectly by transphosphorylation of JAK237, we decide to further investigate the effects of JAK1 inhibition on TGFβ-induced activation of STAT3. Inhibition of JAK1 by siRNA-mediated knockdown or small molecule inhibitors targeting JAK1 and JAK2 partially reduces STAT3 activation in human dermal fibroblasts , highlighting that STAT3 is considered as a prime target for pharmaceutical intervention.These findings may have direct translational implications. Several approaches to target STAT3 are currently in clinical development with small molecular inhibitors of STAT3 dimerization and oligodeoxynucleotides being most common. Indeed, more than 10 different clinical trials investigating STAT3 inhibitors in various solid tumors, hematologic neoplasms, or psoriasis are currently ongoing or have already been completed  encoding for Cre recombinase at an IFU of 80/cells. AAV encoding for LacZ served as controls.Murine and human dermal fibroblasts were obtained from sterile 3 mm × 3 mm skin punches from murine or patients. The skin fragments were further fragmented using sterile scalpel and digested with Dispase II solution  (0.8 mg/ml in PBS) for 3 h at 37 °C and 800 rpm. The digested sample was filtered using 100 µm nylon filter and centrifuged at 1400 rpm for 5 min. The pellet was resuspended in Dulbecco’s modified Eagle’s medium-Ham’s F-12 (DMEM) containing 10% fetal bovine serum and put in culture flasks. Before an experiment, dermal fibroblasts were serum-starved in DMEM containing 0.1% fetal bovine serum for 24–48 h. In selective experiments, serum-starved cells were incubated with recombinant human TGFβ-1 (10 ng/ml) , the STAT3 inhibitor S3I-201 (15 µM) , the JAK2 inhibitor TG101209 (500 nM) (Selleckchem), the JAK1/2 inhibitor Ruxolitinib (5 µM) , the SRC inhibitor SU6656 (500 nM) , the JNK inhibitor SP600125 (500 nM) , and the c-ABL inhibitor imatinib mesylate (500 nM) . To delete STAT3 from cultured murine fibroblasts isolated from STAT3JAK2, JAK1, SRC, c-ABL, and SMAD3  and 100 nM SignalSilence siRNA I for SAPK/JNK . The transfection efficiency was determined by western blot analysis. Non-targeting siRNAs (nt siRNA)  served as controls. The sequences of the pre-designed sense siRNAs are described in Supplementary Table Human dermal fibroblasts were transfected with reporter plasmid constructs or siRNAs using using the Nucleofection technique and Nucleofector Solution V  using the manufacturer’s recommended protocol. Experiments were conducted 24–48 h after transfection and thereafter cells were harvested. Gene silencing was achieved by nucleofecting 3 μg pre-designed siRNA duplexes against Cignal Lenti STAT3 Reporter (luc) Kit and Cignal SMAD Reporter (luc) Kit from Qiagen  were used to determine STAT3- and SMAD3-dependent reporter activities. Dual-luciferase activities were determined by using Luminoskan™ Ascent Microplate-Luminometer . The constitutively expressed non-inducible Renilla luciferase activity served as internal control for normalizing transfection efficiencies.ACTB) was used to normalize for the amounts of cDNA within each sample. All primer sequences are presented in Suppplementary Table Gene expression was quantified by SYBR Green real-time PCR using the ABI Prism 7300 Sequence Detection System (Life Technologies). Samples without enzyme in the reverse transcription reaction (non-RT controls) were used as negative controls. Unspecific signals caused by primer dimers were excluded by non-template controls and by dissociation curve analysis. Beta-actin , rabbit anti-P-STAT3 , rabbit anti-JAK2 (dilution 1:1000), rabbit anti-P-SRC , rabbit anti-SRC (dilution 1:800), rabbit anti-c-ABL (dilution 1:800), rabbit anti P-c-ABL , rabbit and anti-JNK  from Cell Signaling Technology ; rabbit anti-JAK1 (1:200), goat anti-P-JAK1 , and rabbit anti-P-JAK2  from Santa Cruz Biotechnology ; rabbit anti-SMAD3 (dilution 1:500), rabbit anti-P-SMAD3 (dilution 1:250), and rabbit anti-P-JNK  from Abcam  and rabbit anti-P-SRC (dilution 1:500) from MyBioSource . Horseradish peroxidase-conjugated antibodies  were used as secondary antibodies. Blots were visualized using enhanced chemiluminescence . Beta-actin  or Lamin A/C  antibodies served as controls for equal loading. PageRuler™ Prestained Protein Ladders #26616 and #26619 from ThermoFisher Scientific  were used. Bands were quantified using the ImageJ Software . The original uncropped scans of western blots presented in the main figures are shown in Supplementary Fig. Fibroblasts were collected in lysis buffer composed of 400 mM NaCl, 20 mM HEPES (pH 7.9), and 1 mM EDTA. An aliquot of 20 μg from the lysates was used as input. Cell extracts were incubated with 20 μl Protein A/G Sepharose and 3 μg of SMAD3 or normal IgG antibodies . Unbound proteins were removed by washing with 0.05% NP-40. Sepharose-bound protein complexes were analysed by western blotting.65. Briefly, cell culture supernatant was mixed with sirius red dye for 30 min at room temperature. After centrifugation, the pellet was dissolved in alkali reagent. Measurement was performed using a SpectraMax 190 microplate spectrophotometer  at a wavelength of 540 nm.Total soluble collagen in cell culture supernatants was quantified using the SirCol collagen assay  as described previouslyFormalin-fixed, paraffin-embedded skin sections or 4% PFA-fixed, 0.25% Triton X-100-permeabilized cells were stained with appropriate primary antibodies, including mouse anti-STAT3 (dilution 1:200)  and rabbit anti-P-STAT3 (dilution 1:500) (Abcam). For double-staining experiments, the samples were incubated again with primary antibodies against P-SRC (dilution 1:500), P-JAK2 (dilution 1:500), P-c-ABL (dilution 1:300), and P-JNK (dilution 1:500). Fibroblasts were stained specifically with vimentin (Sigma-Aldrich) overnight at 4 °C. HRP-conjugated or Alexa Fluor antibodies  were used as secondary antibodies. Fibroblasts incubated with isotype control antibodies  were used as controls. Counter staining of cell nuclei was performed using DAPI (Santa Cruz Biotechnology). Stained cells were visualized using a Nikon Eclipse 80i microscope (Nikon).13.For quantification, single, spindle-shaped cells in the dermis positive for the required positive antibody were counted in six randomly chosen high-power fields at 200-fold magnification by two experienced researchers in a blinded manner as describedIntegrated density was analyzed using ImageJ Software .Confocal images of tissue sections were acquired using a Leica SP5 II confocal laser scanning microscope  with ×63 glycerol-immersion objective and scanning resolution of 512 × 512 pixels, zoom factor 6.4. Image stacks consisting of a series of images at 1 μm intervals throughout the entire cell nucleus were taken at randomly selected tissue areas. The images were deconvoluted with the Huygens deconvolution software (Scientific Volume Imaging B.V.). The DAPI channels were then smoothed by convolving with a Gaussian kernel (sigma = 5 pixels) and nuclei were segmented by automatic global thresholding (using ImageJ’s Otsu method). Likewise, YY in the XX channel were segmented using automatic Otsu global thresholding. Afterwards, the ratios of YY between inside and outside of nuclei were calculated for each image. The entire workflow was performed fully automatically to exclude any bias, by a custom ImageJ macro.66. For bleomycin-induced skin fibrosis, 6-week-old mice received repeated subcutaneous injections of bleomycin (100 µl) at a concentration of 0.5 mg/ml in defined areas of 1 cm2 at the upper back every other day for 4 weeks. Mice injected with equal volumes of 0.9 % sodium chloride served as controls. In a subset of experiments, mice were treated with intraperitoneal injection of c-ABL inhibitor imatinib mesylate (150 mg/kg/day) or SRC inhibitor SU6656 (12 mg/kg/day dissolved in 20% DMSO/80% NaCl) or an oral gavage of JAK2 inhibitor TG101209 (200 mg/kg/day) or JNK inhibitor CC-930  for 21 days. Control mice were injected with bleomycin and with vehicle for the respective individual inhibitors. All inhibitors were dissolved in 0.9% NaCl unless otherwise mentioned. For TBRact-induced fibrosis, 4-week-old mice received of 6.67 × 107 pfu/mouse of replication-deficient type 5 adenoviruses encoding for TBRIact into defined areas of 1 cm2 at the upper back four times per 2 months. Mice injected with LacZ-expressing viruses served as controls. To selectively inactivate STAT3 in fibroblasts, mice carrying two conditional alleles of STAT3 (STAT3fl/fl) were crossbred with col1a2-Cre-ER mice to generate col1a2-Cre-ER STAT3fl/fl. Cre-mediated recombination was induced by repeated i.p. injections of tamoxifen over 5 days. Control groups were injected with corn oil. For pharmacological inhibition of STAT3, we used selective STAT3 inhibitor S3I-201 (10 mg/kg/day). All mouse experiments were approved by the governments of Mittelfranken and/or Unterfranken.Two different mouse models were employed: bleomycin-induced skin fibrosis, and fibrosis induced by injections of replication-deficient type 5 adenoviruses encoding for a constitutively active TBRI construct65. For visualization of collagen content, trichome staining was performed (Sigma-Aldrich). Hydroxyproline content and α-smooth muscle actin positive myofibroblasts were analyzed as described previously67.The injected skin areas of all mice were fixed in 4% formalin and embedded in paraffin. Histologic sections were stained with hematoxylin and eosin for the determination of dermal thickness. The dermal and hypodermal thickness was visualized using a Nikon Eclipse 80i microscope (Nikon) and analyzed at four different sites in each mouse in a blinded manner as describedU-test for non-related samples and by the Wilcoxon signed rank tests for related samples. In a subset of experiments, the mean values of the control groups were set to 1. All other values were expressed as x-fold changes compared with the respective controls used as ‘comparison mean values’. P values less than 0.05 were considered significant.All data are presented as median with interquartile range (IQR). Differences between the groups were tested for their statistical significance by Mann–Whitney All data generated or analysed during this study are included in this published article and its  files. ASupplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2


Chrysoperla externa haplotypes and investigate whether it exhibits structure based on genetic composition as opposed to geographic location. The genetic diversity of C. externa, analyzed by AMOVA using the COI and 16S rRNA genes as mitochondrial markers, showed significant haplotype structure arising from genetic differences that was not associated with sampling location. This was reflected in the network grouping. Bayesian inference showed that haplotype distribution may have its origins in C. externa divergence into two distinct clades, which dispersed to various locations, and their subsequent diversification. The evolutionary history of C. externa may include multiple ancestral haplotypes differentiating within the same geographic area to generate the current broad genetic diversity, so that the earlier geographical history has been erased, and now we have highlighted its more recent genetic history.This work aimed to elucidate the distribution of  Chrysoperla Steinmann 1964 comprises 36 lacewing species, four of which are found in Brazil: Chrysoperla defreitasi Brooks 1994, Chrysoperla raimundoi Freitas & Penny 2001, Chrysoperla genanigra Freitas 2003, and Chrysoperla externa (Hagen 1861). The last named is one of the most common lacewing species in the Americas and can be found from the southern USA to Argentina . We sampled 123 localities in Brazil in the states of São Paulo (110), Minas Gerais (7), Paraná (3), Goiás (1), Rio Grande do Norte (1) and Distrito Federal (1) and at a location in Paraguay; all the localities are georeferenced (This study gathers new specimens of ferenced . Collectferenced . The spe® Genomic DNA Purification Kit (Promega), following the manufacturer’s protocol and it is stored at the Laboratório de Biologia Evolutiva (LaBE) do Departamento de Biologia Aplicada à Agropecuária—FCAV-UNESP.DNA was extracted from the thorax, while the head, wings, and abdomen were stored in absolute ethanol also in the Coleção de Referência Entomológica at the Departamento de Fitossanidade of the FCAV-UNESP . The DNACOI and 16S rRNA were amplified by PCR in a Mastercycler thermal cycler (Eppendorf). The reaction was conducted in a final volume of 25 μL consisting of 12.5 μL of GoTaq® Colorless Master Mix (Promega), 0.4 μM of each of the primers, and 2.5 μL total DNA (~40 ng). The COI gene was amplified with primers C1-J-2183 (5’CAACATTTATTTTGATTTTTTGG3’) and TL2-N-3014 (5’TCCATTGCACTAATCTGCCATATTA3’) [16S gene was amplified with primers LR-J-12887 (5’ CCGGTTTGAACTCAGATCATGT 3’) and SR-N-13398b (5’ CRCYTGTTTAWCAAAAACAT 3’) [® SV Gel and PCR Clean-Up System (Promega) according to the manufacturer’s protocol. The products were sequenced using the same primers and amplification conditions. The sequencing reactions were conducted in an ABI 3730 XL DNA Analyzer  automatic sequencer using the BigDye® Terminator v.3.1 Cycle Sequencing Kit (Perkin-Elmer Applied Biosystems), and the sequences were deposited in the Laboratório de Biologia Evolutiva (LaBE) database at the Departamento de Biologia Aplicada à Agropecuária, FCAV. The haplotypes were deposited in the GenBank database under the accession numbers KJ586656 to KJ586673 and KX099401 to KX099607   under thKX099607  Tables.COI gene and 1 to 5 for the 16S gene. For each run, 10,000 iterations were carried out after a burn-in of 10,000 iterations. The assumed parameters for both genes were "no model admixture" because they were haploid mitochondrial genes, and "all frequencies correlated" since the sequences had high similarity [COI gene was GTR+I+G and for 16S gene was HKY+I, obtained using JModelTest v.2.1.3, utilizing the Bayesian Information Criterion [Chrysoperla defreitase , Chrysopodes divisa , Ceraeochrysa cubana , Leucochrysa cruentata , and Hemerobiidae, Neuronema laminatum (NC_028153.1) were used as outgroups. The priors were checked in Tracer v.1.6 [The sequences were read in Chromas Lite v.2.1.1  and aligmilarity . To detemilarity  in the Smilarity . A graphmilarity . Tajima’milarity  and Fu’smilarity  neutralimilarity , to detemilarity  using thmilarity , 27. Themilarity  with conriterion . We perfer v.1.6  and the er v.1.6 , consideer v.1.6 .COI gene and 158 haplotypes , showing that there was not sufficient evidence to prove that there is genetic structure among geographic populations.The genetic structure was used to investigate the species COI gene comprised four groups . The population pairwise FSTs also presented high values, demonstrating that Group 4 did not skew this structure , and the Ftructure .COI gene, individuals were grouped into six clusters . Chrysoperla externa split into two distinct clades with 100% posterior probability, one comprised of individuals appearing only in the TCS Group 4 and a second clade including individuals of other groups  and -17.508 (p = 0.000), respectively, for Group 4, and -2.68706 (p < 0.01) and -315.397 (p = 0.000), respectively, for other groups.Based on the Bayesian inference separation, the neutrality test was conducted for Group 4 separately from the remaining groups. The observed values of Tajima’s D and Fu’s FC. externa has significant haplotype structure arising from genetic differences not shown to be influenced by present geography. Almost all the 158 COI haplotypes and 58 16S haplotypes were found in all sampled locations.Our results demonstrated that C. externa [The AMOVA demonstrated that haplotype differences among locations were not significant, showing that the distances between sampling sites, in Brazil and Paraguay, did not act as a barrier, and produced no geographic differentiation. This would be a necessary premise for the phylogeographic inference test, which could assess the haplotype and geographic association . This di externa –7. HowevPhylogeographic studies use the clades formed in the haplotype network to test the hypotheses of genetic/geographical association and several studies show a correspondence between the geographic distribution and network topology –43. TherC. externa haplotypes into two distinct clades, which diversified posteriorly Click here for additional data file.S2 FigSquare represents the ancestral haplotype. The size of circles reflects the frequency of the haplotypes in the sample. Haplotype names correspond to names in (TIF)Click here for additional data file.S1 Supporting InformationCOI gene, and from 731 to 1218 are for the 16S gene.Positions from 1 to 730 are for the (TXT)Click here for additional data file.S1 TableCOI/16S), and Chrysoperla externa voucher number.Sampling localities, geographic coordinates, number of specimens analyzed ((PDF)Click here for additional data file.S2 TableCOI gene; number of specimens containing each haplotype; GenBank accession number; Chrysoperla externa voucher number.Identification code (ID) of haplotypes for (PDF)Click here for additional data file.S3 Table16S gene; number of specimens containing each haplotype; GenBank accession number; Chrysoperla externa voucher number.Identification code (ID) of haplotypes for (PDF)Click here for additional data file.S4 TableCOI and 16S genes); number of specimens containing each haplotype; Chrysoperla externa voucher number.Identification code (ID) of haplotypes for concatenated data ((PDF)Click here for additional data file.