Runx1 deficiency results in an anteriorly specific cleft palate at the boundary between the primary and secondary palates and in the first rugae area of the secondary palate in mice. However, the cellular and molecular pathogenesis underlying such regional specificity remain unknown. In this study, Runx1 epithelial-specific deletion led to the failed disintegration of the contacting palatal epithelium and markedly downregulated Tgfb3 expression in the primary palate and nasal septum. In culture, TGFB3 protein rescued the clefting of the mutant. Furthermore, Stat3 phosphorylation was disturbed in the corresponding cleft regions in Runx1 mutants. The Stat3 function was manifested by palatal fusion defects in culture following Stat3 inhibitor treatment with significant downregulation of Tgfb3. Tgfb3 is therefore a critical target of Runx1 signaling, and this signaling axis could be mediated by Stat3 activation. Interestingly, the expression of Socs3, an inhibitor of Stat3, was specific in the primary palate and upregulated by Runx1 deficiency. Thus, the involvement of Socs3 in Runx1-Tgfb3 signaling might explain, at least in part, the anteriorly specific downregulation of Tgfb3 expression and Stat3 activity in Runx1 mutants. This is the first study to show that the novel Runx1-Stat3-Tgfb3 axis is essential in anterior palatogenesis. This condition demonstrates anatomical impairments with various combinations of the defects involving the soft and hard palates, the nasal septum and the alveolar ridge. Palatogenesis requires several developmental steps involving palatal shelf growth and elevation, as well as fusion of the palatal shelves. The palatal fusion begins at the midline of the future secondary palate following bilateral outgrowth of the maxillary process. Thereafter, the anterior secondary palate fuses to the primary palate and the dorsal portions of the secondary palate fuse with the nasal septum. Following contact, the intervening epithelium between the abutted shelves merges to form the epithelium seam that must be removed to provide mesenchymal continuity throughout the fused palate4. Fusion is crucial for the correct formation of the palate, and its defect can lead to CP. Although recent studies using genetically modified mice and human genetics studies have identified numerous genetic and environmental etiology for cleft palate, the etiology that explains the diversity in morphology in cleft palate has remained largely unknown.Cleft palate (CP) represents one of the major congenital craniofacial birth defectsRunx1-null mutants carrying a Gata1 promoter-driven Runx1 transgene (Gata1-Runx1/Runx1−/−)5. Runx1 is a member of the Runx family of genes that encode transcription factors that play various important roles in embryogenesis6 and is a key molecule of hematopoiesis-causative genes in the development of leukemia and other hematopoietic disorders8. Runx1 is also involved in cancer development10. Indeed, families with children diagnosed with acute lymphoblastic leukemia reported a family history of clefts more often than control families11, and adult individuals who survived cancer reported a family history of cleft palate more often than controls12. Microdeletion encompassing RUNX1 causes Braddock–Carey syndrome, which is characterized by distinct patterns of anomalies, including thrombocytopenia and cleft palate13.We previously demonstrated that Runx1 participates in the pathogenesis of cleft palate using Runx1 is expressed in the fusing epithelium of the developing palatal shelf throughout the AP axis, it is of interest that Runx1 mutants develop a localized anterior palatal clefting due to failed fusion between the primary and secondary palates and at the anterior-most part of the secondary palate corresponding to the 1st rugae5. This finding indicates that Runx1 is involved in a different regulatory mechanism for the palatal fusion along the anterior-posterior (AP) axis. Several studies have also revealed such different regulatory mechanism of palatogenesis along the AP axis of the palate15. Some transcription factors and signaling molecules, such as Msx1, Bmp4, Shh, Fgf10, Fgf7, and Shox2 are recognized as anterior-specific15. Spatial distribution of such genes within the palate, together with the palatal phenotypes in their null mutant, clearly highlights the importance of regionally specific regulatory mechanism at the molecular level. However, the majority of experiments have focused on the palatal fusion of the secondary palate, and there is less understanding of the mechanisms in the anterior region between the primary and secondary palate and in the first rugae area of the second palate. Indeed, how Runx1 is involved in anterior-specific clefting has not been investigated.Although 17. The JAK/STAT pathway is the principal signaling mechanism for a wide array of cytokines and growth factors19. Upon activation, phosphorylated Stats dimerize and translocate to the nucleus, where they modulate the expression of target genes. It has been established that Stat3 is activated in a number of epithelial cancers19, in immune response, and also in response to various environmental signals20. Heterozygous loss-of-function mutations in STAT3 lead to the primary immune deficiency Hyper-IgE syndrome and interestingly the cleft palate is observed in this syndrome21. However, little is known about the possible mechanisms underlying involvement of Stat3 in the pathogenesis of the cleft palate.In mouse skin cancer formation and maintenance, signal transducer and activator of transcription 3 (Stat3) is activated by Runx1 signaling as a tumor promoterRunx1 deficiency results in an anteriorly specific cleft palate at the boundary between the primary and secondary palates and in the first rugae area of the secondary palate in mice5. However, the cellular and molecular pathogenesis underlying such regional specific cleft palate remain unknown. In this study, using epithelial-specific Runx1 deletion mice, we demonstrate that Tgfb3 is an essential target of Runx1 signaling in anterior-specific disintegration of the fusing palatal epithelium and that the site-specific downregulation of Stat3 phosphorylation plays a central role in downregulating the Tgfb3 expression and manifesting palatal clefting upon Runx1 loss. We also show that the pharmaceutical inhibition of Stat3 signaling disturbs the expression of Tgfb3 as well as of Runx1, leading to failure of palatal fusion. In addition, we show that the involvement of Socs3 in Runx1-Tgfb3 signaling explain, at least in part, the anteriorly specific downregulation of Tgfb3 expression and decreased Stat3 activity in Runx1 mutants. Together, we demonstrate that Stat3-mediating Runx1-Tgfb3 axis is a novel regulatory pathway that regulates the palatal fusion in the anterior regions.Gata1-Runx1/Runx1−/− mice exhibit anterior clefting between the primary and secondary palates and in the first rugae region of the secondary palate and that Runx1 is specifically expressed in the fusing epithelium, but the mechanisms underlying this phenotype remained largely unknown5. Since Gata1-Runx1/Runx1−/− mice died within a few hours after birth, it was possible that the cleft palate phenotype was simply a consequence of delayed development. To extend the finding and examine how Runx1 regulates the anterior-specific palatal fusion in detail, we used epithelial-specific Runx1 deletion mice (K14-Cre/Runx1fl/fl).We previously reported that K14-Cre recombination using Rosa26R reporter mice at E15.0. β-gal staining was performed in the K14-Cre;R26R mice, and we confirmed that Cre recombination had been successfully performed in embryonic mouse in anterior palatogenesis. β-gal-positive cells were intense at the epithelium overlying the palatal process of the secondary palate and the primary palate  at E15.0 had partial contact between the primary and secondary palate, and that thick K14-immunoreactive epithelial remnants  that is localized at the junction between the secondary palates, the epithelial remnants appeared at the junction between the primary and the secondary palate at the anterior part of the palate , a marker of the periderm26, was sparsely detected in the epithelial remnants in the 2nd rugae area  Fig. . TherefoRunx1 deficiency at E15.0  analysis demonstrated Runx1 binds to the promoters of Socs3, Stat3 inhibitors, to represses their transcription, and ultimately upregulates Stat3 activity by enhancing phosphorylation9. Conversely, Runx1 deficiency results in upregulation of Socs3 expression and Stat3 inactivation. Our whole mount in situ hybridization analyses demonstrated that Socs3 expression was evident specifically in the fusing epithelium of the nasal septum and the primary palate of wild-type and this Socs3 mRNA was further upregulated by Runx1 deficiency. Remarkably, spatial distribution of upregulated Socs3 mRNA overlapped with the regions of downregulated pStat3 immunoreactivity and Tgfb3 mRNA. Hence, involvement of Socs3 on Runx1-Tgfb3 signaling axis could, at least in part, account how Runx1 deficiency leads to spatially-specific downregulation of Stat3 phosphorylation and Tgfb3 mRNA in the primary palate and the nasal septum and at the anterior portion of the secondary palate, and subsequent anterior specific cleft palate.Anterior clefting is a rare palatal phenotype in the genetically modified mice, however, spatial distribution of Runx1 and inhibited Tgfb3 expression, leading to failure in palatal fusion in wild-type mice. Such downregulation of Runx1 by Stat3 inhibitors was also supported by upregulated expression of Socs3, as observed in Runx1 mutants. These findings showed that the Runx1-Tgfb3 signaling axis was affected by extrinsic modification of Stat3. It has been established that Stat3 senses an array of extracellular signals and rapidly responds to them by controlling the target gene expression47. Acute alcohol intake suppresses the Stat3 activity through excessive Socs3 activation in human monocytes48, and nicotine also induces anti-inflammatory action associated with Stat3 phosphorylation in peritoneal macrophages49. Although a previous study did not examine whether or not alcohol intake and/or smoking influence the Stat3 activity in palatal fusion, Stat3 might act as a point of convergence integrating extrinsic environmental input into the genetically defined intrinsic conditions, which might provide a novel understanding of the etiology and pathology of the cleft palate. In the pathogenesis of non-syndromic cleft palate, several genetic susceptibility factors and environmental risk factors have been identified, and a clinically relevant phenotype is identified only when a threshold effect is reached after combining each genetic and environmental susceptibility factor affecting each individual2. Prevention is the ultimate objective with regard to CP. The present findings have identified genetic targets that modify the environmental risk factors, and pharmaceutical modulation of Stat3 signaling may modify the Runx1-Tgfb3 signaling axis, suggesting its utility as a therapeutic procedure for preventing CP in patients with pathologic TGFB3 variant.A striking finding of our study is that the pharmaceutical application of two different types of Stat3 inhibitor disturbed the expression of Stat3 null mutants to directly confirm the function of Stat3. Previous studies using tissue-specific Stat3-deficient mice showed that Stat3 plays a crucial role in a variety of biological functions, including cell growth, anti-apoptosis, apoptosis and cell motility depending on the cell type and stimulus50. However, the cleft palate phenotype has not been reported using such Stat3 mutant mice. In previous studies using epithelial-specific stat3 deletion mice, Keratin 5 (K5) promoter was used for the epithelial-specific Cre-mediated deletion of the Stat3 gene. K14-Cre is another Cre driver for generating epithelial-specific conditional null mutant mice, and interestingly, Dicer-null mutants driven by K14 and K5 do not exhibit similar appendage phenotypes. Indeed, K14-Dicer mutants exhibit more severe phenotypes at birth than K5-Dicer mutants, possibly due to the earlier onset of the K14-Cre driver51. Since palatal fusion occurs in the embryonic stages, it is likely that the K14-Cre driver is appropriate for evaluating the possible function of Stat3 in the fusing epithelium. We intend to investigate the palatal phenotypes using K14-Cre driven Stat3 conditional null mice in future experiments.One limitation associated with this study is that Stat3 signaling was inhibited by pharmacological Stat3 inhibitors. Given the possibility that Stat3 inhibitors also affect other signaling besides Stat3, we used two different Stat3 inhibitors with different mechanisms of action. In future experiments, we will generate epithelial-specific Runx1 epithelial-specific deletion led to the failed disintegration of the contacting palatal epithelium and that Tgfb3 is a critical downstream target in the pathogenesis of anterior cleft palate in the mutants. This Runx1-Tgfb3 signaling axis is independent of previously known signaling systems and is reciprocally mediated by Stat3. In this process, expression of Socs3, an inhibitor of Stat3 signaling, was upregulated specifically in the primary palate by Runx1 deficiency, which could explain, at least in part, how Runx1 deficiency results in anteriorly specific clefting . To generate K14-Cre/Runx1fl/fl mice, we first mated heterozygous K14-Cre mice52 and Runx1fl/fl mice53 to obtain K14-Cre/Runx1fl/+mice. These progenies were subsequently bred with Runx1fl/fl mice. Genotyping was performed by the conventional polymerase chain reaction (PCR) method using each primer set to detect Cre (5′ CTCTGGTGTAGCTGATGATC 3′ and 5′ TAATCGCCATCTTCCAGCAG 3′) and the loxP site of Runx1 (5′ GCGTTCCAAGTCAGTTGTAAGCC 3′ and 5′ CTGCATTTGTCCCTTGGTTGACG 3′). We used littermates that did not carry the K14-Cre/Runx1fl/fl genotype as control animals. We also confirmed the efficiency of the K14-Cre recombination in anterior palatogenesis using Rosa26R reporter mice and X-gal staining, as shown in a previous study54.The mouse embryonic heads were dissected in BGJb medium (Gibco). The palate was evaluated by direct observation and with a dissecting microscope. These tissue were fixed in 4% paraformaldehyde, equilibrated in graded sucrose, and embedded in Tissue-Tek .Immunofluorescence staining was performed on 20-µm sections using polyclonal rabbit-anti-Ki67 , monoclonal rabbit anti-K17 , monoclonal anti-K14 , monoclonal rabbit anti-phospho-Stat3 , monoclonal rabbit anti-Stat3  overnight at 4 °C. Then, Alexa488-conjugated goat-anti-rabbit IgG  or Alexa546-conjugated goat-anti-mouse IgG  was used as secondary antibody. The sections were then counterstained with DAPI  and mounted with fluorescent mounting medium (Dako). At least three embryos of each genotype were used for each analysis.The mice embryonic maxilla were freshly embedded in OCT compound and frozen immediately. Tissues are serially sectioned at −20 °C on a cryostat  at a thickness of 25 μm. The maxilla was sectioned from anterior to posterior throughout anterior palate until the secondary palate appeared. The tissue sections were mounted and thawed on a film-coated slide. In total, there were 12–14 serial sections obtained from the anterior palate at E15.0 (section numbers varied due to the orientation of the frozen block). We stained these slides with cresyl violet dye staining. Anterior palate epithelial and mesenchymal tissue were dissected from the sections using a Leica Micro Laser System  and collected by tube.K14-Cre/Runx1fl/fl mice to extract total RNA. IsogenII (Nippon Gene) was used to extract total RNA according to the manufacturer’s protocol. Total RNA was reverse transcribed to cDNA using an oligo (dT) with avian myeloblastosis virus reverse transcriptase (Takara). For the real-time RT-PCR analysis, the cDNA was amplified with TaqDNA Polymerase (Toyobo) using a light cycler (Roche). The qPCR was carried out with Gapdh used as a housekeeping gene and analyzed as previously described54. Primer sequences are available in the Supplementary Fig. We used the laser-microdissected tissues of the control and in situ hybridization was performed using fixed E12.0, E13.0, E14.0, E14.5 and E15.0 palates. The digoxigenin-labeled RNA probes used in this study were prepared using a DIG RNA labeling kit (Roche) according to the manufacturer’s protocol using each cDNA clone as the template. The probes were synthesized from fragments of Runx1, Shox2, Msx1, Shh, Bmp4, Tgfb3, Socs3 and Stat3 (Allen Institute for Brain Science) and were amplified with T7 and SP6 adaptor primers through PCR. After hybridization, the expression patterns for each mRNA were detected and visualized according to their immunoreactivity with anti-digoxigenin alkaline phosphatase-conjugated Fab fragments (Roche), as previously reported2. At least three embryos of each genotype were used for each analysis.Whole-mount TUNEL assay for apoptosis was conducted according to the manufacturer’s protocol . Frozen sections (10 μm) from samples were prepared. The sections were counterstained with methyl green. At least three embryos of each genotype were used for each analysis.in vitro explants were fixed at each stage in 4% paraformaldehyde overnight and then processed for histological examination and qPCR analyses.The palate was dissected and explanted from the E15.0 embryo and cultured on track-etched polycarbonate membrane filter (Nuclepore) in Trowell type organ culture with serumless, chemically defined BGJb medium (Gibco). In our dissection, the primary palate and the nasal septum were not excluded from our culture system. Affi-Gel beads (Bio-Rad) were incubated in TGFB3 . Bovine serum albumin  was used instead of recombinant protein for the control beads. The beads were immersed in recombinant protein or BSA at 37 °C for 60 min and placed on the primary palate of the explants using a pipette tube. After culture, the Embryo heads from E14.0 ICR mouse embryos were collected in BGJB, and the mandibles, tongues, and brains were removed. The remaining palatal tissue, including both the primary and secondary palate and the nasal septum, were cultured for 24–48 h in a whole-embryo culture incubator (RKI Ikemoto) at 37 °C. Palatal tissues were incubated in BGJb medium with or without AG490  or STAT3 Inhibitor VI, S3I-201 . Tissues were harvested after 24 h of culture and processed for qPCR analysis.The dissected palatal tissues were lysed with RIPA buffer  supplemented with protease and phosphatase inhibitors . The lysates were centrifuged and the supernates were heated in denaturing Laemmli buffer (Bio-rad Laboratories). Proteins were separated by SDS-PAGE and transferred to Polyvinylidene difluoride membranes (Bio-rad Laboratories). The membranes were incubated with either anti-Stat3 , anti-pStat3  and beta-actin . The bound antibodies were detected with HRP-linked antibody  and the ECL detection kit (Bio-rad Laboratories).U test. Differences among the three groups were determined using the analysis of variance (ANOVA) test, and significant effects indicated by the ANOVA were further analyzed with post hoc Bonferroni correction. P values < 0.05 were considered significant. Significance was determined using the statistical analysis software program JMP, version 5 (SAS Institute Inc.).Quantitative variables in two groups were compared using the Mann-Whitney All animal experiments were carried out in strict accordance with the recommendations in the Guide for the Care and Use Committee of the Osaka University Graduate School of Dentistry, Osaka, Japan. The protocol was approved by the Committee on the Ethics of Animal Experiments of Osaka University Graduate School of Dentistry. Mice were housed in the animal facility at the Department of Dentistry, Osaka University. Welfare guidelines and procedures were performed with the approval of the Osaka University Graduate School of Dentistry Animal Committee.Supplementary Figures


Solanum tuberosum L., land cover since 1850 using county‐level agricultural census reports, obtained allele frequency data from 7,408 single‐nucleotide polymorphism loci, and compared effects of historic and contemporary landscape connectivity on genetic differentiation of Colorado potato beetle, Leptinotarsa decemlineata Say, in two agricultural landscapes in the United States. We found that potato land cover peaked in Wisconsin in the early 1900s, followed by rapid decline and spatial concentration, whereas it increased in amount and extent in the Columbia Basin of Oregon and Washington beginning in the 1960s. In both landscapes, we found small effect sizes of landscape resistance on genetic differentiation, but a 20× to 1,000× larger effect of contemporary relative to historic landscape resistances. Demographic analyses suggest population size trajectories were largely consistent among regions and therefore are not likely to have differentially impacted the observed patterns of population structure in each region. Weak landscape genetic associations might instead be related to the coarse resolution of our historical land cover data. Despite rapid changes in agricultural landscapes over the last two centuries, genetic differentiation among L. decemlineata populations appears to reflect ongoing landscape change. The historical landscape genetic framework employed in this study is broadly applicable to other agricultural pests and might reveal general responses of pests to agricultural land‐use change.Changing landscape heterogeneity can influence connectivity and alter genetic variation in local populations, but there can be a lag between ecological change and evolutionary responses. Temporal lag effects might be acute in agroecosystems, where land cover has changed substantially in the last two centuries. Here, we evaluate how patterns of an insect pest’s genetic differentiation are related to past and present agricultural land cover change over a 150‐year period. We quantified change in the amount of potato,  They can impose natural selection, favoring some alleles while eliminating others, can maintain genetic diversity when they are heterogeneous in space and time, and can promote genetic differentiation by constraining gene flow , a specialist leaf beetle of plants in the family Solanaceae, arose when the staple crop of European American pioneers, the potato (Solanum tuberosum L.), reached the Great Plains , to potato was reported in 1859 in central Nebraska , and corn, while shrubland and wheat are the most abundant land cover types in the Columbia Basin  in nonhost crops.Beetle samples from the Central Sands were previously described in Crossley et al. . From ea2.2We isolated DNA from the thoracic muscle tissue of each beetle with the Qiagen DNeasy Blood & Tissue Kit  according to the manufacturer's instructions, with the following modifications: We crushed muscle tissue with a sterile micropestle in 180 µl ATL buffer, added 4 µl of RNase A (Qiagen) prior to overnight incubation at 56°C, and eluted DNA from the spin column with one application of 200 µl AE buffer. We then quantified DNA yield and purity with a NanoDrop spectrophotometer  and by agarose gel electrophoresis. We measured DNA concentrations with a Qubit® fluorometer using the dsDNA High Sensitivity Assay Kit  and used 50 ng template for each sample for GBS library preparation. We prepared GBS libraries as described in Elshire et al. ; in brieprocess_radtags script from STACKS v1.47  using BWA‐MEM  and with greater than 30% missing genotypes among samples . We then imputed missing genotypes using the haplotype clustering method of BEAGLE 3.3.2 . We ran EEMS using default MCMC parameters, with 100 demes for each region.We visualized genetic differentiation over the landscape using estimated effective migration surfaces .We explored relationships between genetic differentiation, genetic diversity, and potato land cover using linear regressions. We calculated average pairwise and Hill  implemen2.4L. decemlineata populations and historic and contemporary potato land cover using county‐level agricultural census data .where L. decemlineata in the early 1860s  of the segment of the shortest path line crossing county i, and LT is the total length of the shortest path line. Values of LRT become large when sites are separated by large spatial extents that lack potato land cover. The assumption that potato acts as a conduit to dispersal is based on the dominance of potato relative to other host plants in agricultural landscapes, and studies documenting up to 75% population reduction when potato fields are rotated as little as 1.5 km away from the previous year's crop  several times initially and adjusted tuning parameters to achieve acceptance rates between 20% and 70%. We then ran 30 independent beta‐binomial Markov chains for four million steps each. We assessed evidence for model convergence by examining trace plots of the posterior probabilities and of the ratios of αE/αD (effect sizes of landscape resistance and geographic distance) and examining scale reduction factors calculated with a Rubin‐Gelman test . We then regressed the residuals on landscape resistance  using linear regression.We visualized the relationship between pairwise 2.6L. decemlineata populations in the Columbia Basin and Central Sands, analyzing each population independently. We generated folded site frequency spectra using minor allele frequencies among the SNPs curated for demographic analysis: 35,985 and 65,535 SNPs among Columbia Basin and Central Sands populations, respectively. We calculated median, 2.5th, and 97.5th percentiles of effective population size among 200 bootstrapped folded site frequency spectra. We defined the sequence length parameter as the number of sites (monomorphic or polymorphic) recovered by Stacks; assumed a mutation rate of 2.1 × 10−9 per site per generation, based on a recent estimate from the insect Chironomus riparius  than in the Central Sands (0.004–0.006) : estimates were generally lower among sites in the Columbia Basin, while most Central Sands sites exhibited high effective migration  in the Columbia Basin and 0.008 (±2 × 10−5) in the Central Sands. Pairwise FST was significantly negatively correlated with nucleotide diversity  was significantly higher (at α = 5% level) in 2012 compared to 1860 and 1910  was negatively correlated with geographic distance in the Central Sands, but not in the Columbia Basin, and was highly correlated between years  and landscape resistance  than in the Columbia Basin (2012). While potato land cover has decreased and become spatially concentrated relative to historic baselines in Wisconsin, it continues to increase and expand in the Columbia Basin. The differences in the history of land cover change in these regions provided an ideal opportunity to detect a legacy of historic land cover on contemporary patterns of genetic differentiation.L. decemlineata populations. While the effects remain weak, importantly this result suggests that land‐use decisions and landscape management could be useful tools to reduce pest movement, alter patterns of genetic diversity and potentially slow adaptation, especially for Colorado potato beetle, whose dispersal and abundance are sensitive to crop land cover composition  for L. decemlineata. Temporal lags vary with migration rate, population size, generation time, and time since landscape change  might be relatively insensitive to agricultural landscape configuration at most scales. Agricultural land use in the United States has changed substantially since the mid‐1800s and will likely continue to do so. Given an uncertain future, it will be important to learn how genetic variation in agricultural pests has been shaped by changing landscapes in the past in order to understand how it might be further shaped in the future. Click here for additional data file.


Neandertals disappeared from the fossil record around 40,000 bp, after a demographic history of small and isolated groups with high but variable levels of inbreeding, and episodes of interbreeding with other Paleolithic hominins. It is reasonable to expect that high levels of endogamy could be expressed in the skeleton of at least some Neandertal groups. Genetic studies indicate that the 13 individuals from the site of El Sidrón, Spain, dated around 49,000 bp, constituted a closely related kin group, making these Neandertals an appropriate case study for the observation of skeletal signs of inbreeding. We present the complete study of the 1674 identified skeletal specimens from El Sidrón. Altogether, 17 congenital anomalies were observed , with at least four individuals presenting congenital conditions . At 49,000 years ago, the Neandertals from El Sidrón, with genetic and skeletal evidence of inbreeding, could be representative of the beginning of the demographic collapse of this hominin phenotype. Some interpretations attribute this extinction to competition with early anatomical modern humans (AMHs), which would present differences expressed for instance through more efficient exploitation of dietary resources, possibly related to differential cognitive, behavioral and cultural abilities, that could rest on life-history and ontogenetic differences5. However, other interpretations have recognized recent findings that support Neandertal dietary flexibility and multiple subsistence strategies7, increasing evidence of symbolic behavior and complex technologies13, and lack of fundamental differences in the overall pace of dental and skeletal growth and maturation in comparison with AMHs14, all of which complicate a scenario of AMHs simply outcompeting Neandertals15. Environmental change also has been considered as a potential important factor in the Neandertal demise, whether acting independently, or in combination with other previously-mentioned differences between the two hominins18. In the context of competition, the very fact of interbreeding within what has been called a hominin metapopulation19, would suggest a complex interaction between Neandertal, AMHs populations and Denisovans that has yet to be defined in detail20. For instance, demographic and ecocultural modeling have included competition models, based on cultural and demographic differences22, and selectively-neutral models, based on migration dynamics and local dispersal and replacement alone 23, with both resulting in the replacement of Neandertals by AMHs. Other models conclude that hunting-prey decline or climatic variations alone was not sufficient to cause the disappearance of Neandertals24. In all cases, most researchers agree that, “whatever the extent to which the eventual replacement of late archaic human morphology involved admixture, absorption, and/or population displacement, the process was ultimately a demographic one”25.The causes of the extinction of the Neandertal populations in western Eurasia by 40,000 BP26, that could have been a critical factor in the Neandertal demise. Although others have recommended caution when making inferences about population size from the archaeological record27, it is not unreasonable to suggest that demographic differences in population size and density, and in group size, could have been an important factor in the disappearance of Neandertals28. In addition, the general demographic structure of Pleistocene Homo, with small effective population sizes (see below), a hunter-gatherer existence and population dispersal into separate small kindred groups, would have favored substantial levels of intragroup, and potentially intrafamily, mating30. Important contributions to Neandertal paleodemography in this direction come from genetic studies, where high levels of inbreeding, or mating among relatives, and a general decrease in heterozygosity have been observed. Specifically, Neandertals from the Altai, Vindija, Mezmaiskaya and El Sidrón sites present low levels of heterozygosity and small estimated effective population sizes averaging around 3000 individuals, both characteristics considered typical of archaic hominins, indicating that they lived in small and isolated populations31. Studies of genetic homozygosity indicate that Neandertals had a long history of high but variable levels of inbreeding. The most extreme values are found in the Altai Neandertal, with long stretches of homozygosity that indicate recent inbreeding consistent with parental relatedness between two half-siblings31. In contrast, Vindija Neandertal homozygosity is comparable to modern human groups like the Karitiana and Pima, suggesting that consanguinity was not ubiquitous among all Neandertal populations31. At El Sidrón, a Neandertal sample (SD1253) had a larger cumulative length of homozygous genomic stretches of 10–100 Kb than samples from Vindija, Altai, Denisova, great apes and modern humans32, indicating a long history of inbreeding. In addition, the mitochondrial DNA (mtDNA) analysis of twelve El Sidrón individuals revealed low mtDNA genetic diversity and close kin relationships within the group33.In this regard, it has been suggested that the archaeological evidence supports substantial demographic differences at the Neandertal-to-AMH transition, with up to a tenfold increase in population density for early AMHs compared with NeandertalsHomo with potentially high levels of intragroup mating would be also phenotypically expressed in the skeleton. For instance, recent analyses of bony labyrinth morphology in the Aroeira 3 cranium suggest a degree of demographic isolation in geographically and chronologically close hominins around the origin of the Neandertal clade34, and as previously suggested35 and recently shown36, there is a high incidence of developmental abnormalities and anomalies in Pleistocene Homo, several of them very rare or with unknown etiology. In past and present modern human populations, dental and skeletal anomalies and low-frequency anatomical variants have been associated with geographical isolation and/or endogamy37. Given the nuclear and mtDNA genetic evidence that indicates that the 13 individuals from El Sidrón constitute a closely related kin group33, El Sidrón is the ideal Pleistocene sample to test for skeletal evidence of inbreeding. Previous morphological analyses of the El Sidrón Neandertals have reported congenital clefts of the first cervical vertebra37 and the retention of a deciduous mandibular canine in two individuals38, but a systematic analysis of the entire sample has not yet been done. Here we present the results of the complete morphological analysis of the 1674 identified skeletal specimens from a total of 2556 remains recovered from El Sidrón.Within this context, it is reasonable to expect that a scenario of small, isolated groups of Pleistocene 42  with other Neandertals (mean 34.13 mm)43 and with modern humans 44, places this maxilla at the smallest extreme of the observed range of variation  preserves several morphological features of interest in its maxilla and mandible Fig. . As desc37 as having another congenital anterior sagittal cleft. In addition, an almost complete atlas from El Sidrón (SD-1643) preserves a congenital posterior sagittal cleft37 and there are two C1 hemi-arches (SD-2045 and SD-1725) from the juvenile skeleton El Sidrón J1 interpreted as a congenital posterior sagittal cleft14. In modern humans, the posterior synchondrosis is fused by six years of age in 95% of individuals and the remaining 5% correspond to cases of congenital posterior sagittal clefts37. Thus the lack of fusion of the posterior synchondrosis PS in El Sidrón J1 is best interpreted as a congenital posterior sagittal cleft of the C137. In total, four out of five atlas specimens with observable anterior or posterior sagittal arches and four out of 13 identified Neandertal individuals at El Sidrón present congenital clefts of the atlas. The frequency of atlas congenital clefts in modern humans ranges from 0.087% to 0.1% for the anterior cleft, and from 0.73% to 3.84% for the posterior cleft37. These clefts have been associated with several different congenital conditions, including Down’s syndrome, Chiari malformation, Klippel-Feil, Goldenhar syndrome, Conradi syndrome, and Loeys-Dietz syndrome, where a higher frequency of anterior (24%) than posterior (16%) C1 clefts have been observed52. It is important to stress, however, that in modern humans these clefts are usually asymptomatic and often only identified in routine examinations37.Several El Sidrón individuals preserve evidence of sagittal clefts of the cervical vertebrae. A first cervical vertebra (C1 or atlas) fragment (SD-636) shows evidence of a congenital anterior sagittal cleft Fig. , similar54  (SD-1601) preserves several morphological features consistent with congenital alterations. First, the right transverse process has not developed and there is bilateral asymmetry in the size of the transverse foramina Fig. . The ver58, clefting of the neural thoracic arch is rare, with few reported dry-bone cases60. A right rib (SD-292) is identified as either a 12th rudimentary or hypoplastic rib or a 13th lumbar rib resulting from a caudal border shifting of the thoracic-lumbar border61  shows cranial displacement of the thoracic transitional vertebra and a sagittal cleft of the arch of the last rib-bearing vertebra with lack of development of the spinous process Fig. . While cr61 Fig. . Rib and63. Three scaphoids  retain a distinctive os centrale projection along the distoulnar border, while another scaphoid (SD-96) is bipartite with a truncated tubercle  most often occur superolaterally or laterally76, rather than inferiorly as in El Sidrón specimen. In modern humans, congenital conditions of the patella are present in more than 35 dysmorphic entities, and patella aplasia or hypoplasia is a hallmark feature of several syndromes 77. This patella also presents other unusual morphological features that suggest a decreased or altered mechanical loading. Specifically, the SD-932 patella lacks the median patellar ridge and a well-developed subchondral bone plate, both of which are typically found in modern humans and Neandertals78, including other patellae from El Sidrón  was recovered from El Sidrón. It presents two articular surfaces inferolaterally and inferomedially for additional ossification centers Fig. . In modein situ articulated bones  and an associated talus and calcaneus were recovered at El Sidrón , and the increased plantar cortical thickness of the lateral tarsals, are consistent with a congenital anomaly of the foot affecting the plantar soft tissue structures  have been documented, and the associations with complex diseases and quantitative traits are inconsistent85. Mild skeletal anomalies or variants have been observed in geographically isolated and/or endogamic human populations. For instance, in Canadian Inuit skeletons a higher frequency and intensity of several spine defects were observed in the smaller and more genetically isolated of the two compared populations93. In a recent study on the impact on patterns of deleterious variation of an extreme and prolonged population bottleneck in Greenlandic Inuit, an increase up to 6% in the genetic load (reduction in mean fitness in a population caused by deleterious mutations relative to a mutation-free population) was observed across all models of dominance94.The health and survival consequences of inbreeding and consanguinity have been studied in humans88, Scandinavian wolf90, and Iberian lynx89 show a range of conditions including heart defects, cryptorchidism and low semen quality91. The Florida panther88 and Scandinavian wolf91 also show mild dental and skeletal  anomalies that have no direct effect on fitness but are indicative of high levels of inbreeding. Mountain gorilla92 genetic analyses indicate a population decline over tens of millennia, recent close inbreeding and increased homozygosity, suggesting that an increased burden of deleterious mutation and low genetic diversity , could have compromised the resilience of the mountain gorillas to environmental change and pathogen evolution92. In addition, genetic analysis of the extinct woolly mammoth reveal low heterozygosity and signs of inbreeding95, including several detrimental mutations96 in one of the last surviving mammoths. Additionally, Late Pleistocene mammoths show a high incidence of cervical ribs, a potential signal of inbreeding and/or harsh environmental conditions97.In a broader biological comparative context, studies on different endangered species that have suffered recent drastic population declines and range fragmentation – similar to conditions potentially experienced by Neandertals – have shown very low genetic diversity and high levels of recent, and in some cases long-term, inbreeding that can result in a reduction in population fitness, or inbreeding depression. For example, the Florida panther98, some studies have observed a larger fraction of putative deleterious alleles in Neandertals than in present-day-humans. Specifically, genes associated to autosomal recessive traits have derived homozygous genotypes with likely deleterious effects, which could be suggestive of an enrichment in recessive disorders99. But when both homozygous and heterozygous alleles in genes associated to autosomal recessive traits are considered, there is no clear difference between Neandertals and modern humans, and the authors conclude that the health significance of the estimated relatively (homozygous) higher genetic load in Neandertals is unclear, with no strong evidence for recessive disorders to have played a significant role in Neandertal extinction99. Other authors have suggested that Neandertals suffered a high load of weakly deleterious mutations, with estimations resulting in at least 40% lower fitness than modern humans on average100. With regard to the Altai Neandertal, it has been estimated that her overall genomic health was worse than 97% of present-day humans, mainly due to high risk for immune-related diseases, cancers, gastrointestinal and liver diseases, metabolic-related disorders, morphological and muscular diseases, and also neurological diseases101. However, these estimates of Neandertal health should not be overinterpreted since the genetic risk scores employed in the study are not deterministic101 and the Altai Neandertal presents greater consanguinity than that of all other Neandertal samples31. Furthermore, within the context of the interbreeding between Neandertals and early AMHs, the interpretation of the genomic landscape of introgression and the functional significance of Neandertal genetic material is complex and would include selection against Neandertal variants but also adaptive introgression106, including potentially adaptive ones related to the immune system108. In this regard, and in the context of the above-mentioned impact of inbreeding and consanguinity on the susceptibility to infectious diseases, it has been suggested that the transfer of pathogens between hominin populations in the Upper Paleolithic could have had negative consequences for Neandertals if these were more susceptible to some novel pathogens brought by early AMHs109. But although differential pathogen resistance might have played a role in the demographic collapse of the Neandertals, an explicit test of this hypothesis looking for an overall decrease in diversity at immune system loci in Neandertals failed to fully support it110.In relation to the Neandertal long-term history of small and isolated populations, where the purging of deleterious alleles is predicted to be less efficient111, supporting the idea that Neandertal variants were progressively purged out. If the findings presented here are indeed related to recessive disorders or syndromes, and this scenario was not infrequent for at least late Neandertals, then this would be compatible with an initial reduction of Neandertal ancestry over time after interbreeding with early AMHs. However, although the morphological anomalies observed in the maxilla, patella and foot could have been clinically relevant, in modern humans several of the described conditions correspond to asymptomatic, incidental findings in routine medical examinations, and a diagnosis of a complex disease based on isolated skeletal elements is not possible. Thus, the possibility that the anomalies found at El Sidrón may reflect an underlying genetic syndrome remains speculative. Even in cases of consanguinity, when assessing the impact of mating between close relatives on any aspect of health, “a clear causal relationship needs to be established, rather than reliance on speculation driven solely by the presence of a close kin union in the family pedigree”29. Thus, whether the findings presented here constitute just a strong skeletal signal of inbreeding and low biological variability, or could be also considered as indicative of recessive disorders remains unsolved.Similar skeletal findings as those presented here for the Neandertals from El Sidrón are observed in some rare syndromes in modern humans, as summarized above. In several of these syndromes, the patient presents congenital anomalies in different parts of the skeleton. At El Sidrón, the maxilla, mandible, spine at different levels C1, C2, T12), ribs, scaphoids, patella and foot are affected , although with varying degrees of intensity and within the frequencies observed in modern human historical samples for the incisors and canines115. Furthermore, previous analysis of the El Sidrón J1 juvenile skeleton indicated that the dental and skeletal growth and maturation values were similar to those of diverse modern human juvenile populations14, while the size and shape studies of the adult postcranial remains from El Sidrón show that these Neandertals fall well within the range of variation documented for this Paleolithic humans119. Together, these analyses offer limited support for unusually harsh environmental conditions impacting the prenatal and/or postnatal growth period as an explanation of the described anomalies in the Neandertals from El Sidrón. This is consistent with Trinkaus’ recent assessment of developmental anomalies and abnormalities in the Pleistocene hominin fossil record, in which he concludes that stress during development could only account for a few of the observed abnormalities36.An alternative or compatible scenario to the interpretation of the described conditions as congenital, genetic and indicative of inbreeding would be the presence of adverse environmental conditions impacting early pregnancy and the growth period. Previous research has shown that Neandertals present nonspecific indicators of stress, such as enamel hypoplasias, at a frequency within the ranges of variation shown by prehistoric samples of modern human foragers92. Therefore, caution has been advised when drawing conclusions about the reasons for Neandertal extinction98, since Neandertals could have evolved diverse genetic and biocultural compensations to cope with a large deleterious genetic load110. The persistence of Neandertals for tens of thousands of years, with increasing evidence for diverse subsistence strategies7, symbolic behavior and complex technologies13 and healthcare120 , demonstrates the resilience of these Paleolithic hominins.Current examples of animal species with a long-term history of low population size and depleted genetic diversity may indicate a resilience to develop strategies to mitigate the effect of inbreeding99 suggest that this individual coped with their dental pathology by alternating left-right hand use and avoiding chewing on the pathological side of the mouth121. Adult 2 also presented the highest incidence of chipping in the dentition, and was the only individual from El Sidrón from whom bitumen or oil shale was recovered from the dental calculus122. Together, these findings suggest that this Neandertal individual could have had a specialized behavior. Furthermore, previous studies have found evidence that this individual probably self-medicated the infected cyst122. Considering the El Sidrón Neandertal group as a whole, and the findings presented here from the perspective of the bioarchaeology of care in Neandertals120, any interpretation of these findings as related to a genetic syndrome or recessive disorder could point to another example of healthcare and resilience in this hominin group.The Adult 2 Neandertal from El Sidrón exemplifies this resilience. This individual had a congenital narrowing of the nasal fossa and a retained deciduous mandibular canine with a subsequent dentigerous cyst secondary to dental trauma to that tooth. Previous analysis of the dental striation orientation123, and the genomes analyzed from the Sunghir site, dated 34,000 years ago, indicate that they were not closely related (third degree or closer)124. Beyond the osteological and paleopathological analysis of the skeletons from Sunghir that could point to inbreeding125, the genetic results of these individuals have been interpreted as already suggesting by 34,000 years ago the presence of the modern human hunter-gatherer social structure “with low levels of within-band relatedness, complex family residence patterns, relatively high individual mobility, and multilevel social networks”124. Currently, the available osteological evidence indicates absence of differences of younger versus adult mortality pattern between Neandertals and early AMHs25, in agreement with biological models that would indicate similar demographic features between both126, and this would suggest that the demographic advantage of early AMHs would have been the result of increased fertility and/or reduced immature mortality25. Fertility and immature mortality would be related to the social structure of Paleolithic hominins, and the study of the impact of a reduced population dispersed in small an isolated groups on that social structure, as well as the study of the reproductive biology of hominins remains an important challenge for the future127.A scenario of small effective population sizes, a hunter-gatherer existence and population dispersal into separate small kindred groups, with probable intragroup mating, would have also affected early AMHs. Even considering the interbreeding episodes with early AMHs, and therefore somehow the permanence of Neandertals, how early AMHs managed to support growing and geographically-expanding populations in the same environment while the Neandertal phenotype disappeared, remains a central question. The genetic analysis of a 45,000 years old modern human male from Siberia indicates lack of recent inbreeding among his ancestors128 indicates that from 48 to 40 ka, the potential niche of Neandertals reduced significantly in size and spatial continuity , while optimal patches of early AMHs remained much better connected. Neandertal habitat reduction and fragmentation suggest that the Neandertal population was sharply decreasing in size and becoming more isolated128, a conclusion that is generally supported by paleogenetic data. The analysis of the genomes of Neandertals (Vindija33.19 and Altai), Denisovans and modern humans by Pairwise Sequential Markovian Coalescent (PSMC) method, indicates that after a reduction of population size that occurred sometime before 1.0 million years ago, the population ancestral to present-day humans increased in size, whereas the demographic history for both Neandertals and the Denisovan shared a recent history of very low effective population sizes129. Recent estimations suggest that in five low-coverage Neandertal genomes who lived around 39,000 to 47,000 years ago 130, the levels of heterozygosity lie below those estimated from the high-coverage Vindija and Altai Neandertals, from around 50,000 years ago or at least predating 44,000 years (Vindija)131. The possibility of particularly low heterozygosity in these late Neandertals could reflect a small number of individuals near the end of its presence in Europe132. At 49,000 years ago, and in the context of a high incidence of skeletal anomalies in Pleistocene Homo36, the Neandertal family group from El Sidrón, with genetic and skeletal evidence of inbreeding, could be representative of the beginning of the demographic collapse of this hominin phenotype.The disappearance of the Neandertals and expansion of modern humans was most probably the result of a process involving several factors, one of them being the low population density of Neandertals. An analysis of the climatic niches of both Neandertals and early AMHsThe bones were inspected using a binocular lens, a Enviromental Scanning Electron Microscope (ESEM Fei-Quanta 200), and were micro-CT scanned with a Nikon XT H 160 at 155-114 kv and 48–85 µA, 1800 projections, reconstructed as 16-bit tiff stacks, voxel size interval from 0.027 to 0.079 mm. The data were loaded into AMIRA 5.4® (Thermo Fisher Scientific) for generating the virtual reconstructions. Photographs were obtained from different views and, when possible, with comparative Neandertal (El Sidrón) and modern human cases. Descriptions of specific procedures followed for the morphological analyses for each skeletal element are presented in the Supplementary Material.SUPPLEMENTARY_INFORMATION


Ciona intestinalis has pelagic eggs and a swimming larval stage lasting for maximum a few days, with the potential for a homogenizing gene flow over relatively large areas. In the eastern North Sea, it is found in a geomorphologically complex archipelago with a mix of fjords and open costal habitats. Here, the coastal waters are also stratified with a marked pycnocline driven by salinity and temperature differences between shallow and deep waters. We investigated the genetic structure of C. intestinalis in this area and compared it with oceanographic barriers to dispersal that would potentially reduce connectivity among local populations. Genetic data from 240 individuals, sampled in 2 shallow, and 4 deep-water sites, showed varying degrees of differentiation among samples (FST = 0.0–0.11). We found no evidence for genetic isolation by distance, but two distant deep-water sites from the open coast were genetically very similar indicating a potential for long-distance gene flow. However, samples from different depths from the same areas were clearly differentiated, and fjord samples were different from open-coast sites. A biophysical model estimating multi-generation, stepping-stone larval connectivity, and empirical data on fjord water mass retention time showed the presence of oceanographic barriers that explained the genetic structure observed. We conclude that the local pattern of oceanographic connectivity will impact on the genetic structure of C. intestinalis in this region.Pelagic larval development has the potential to connect populations over large geographic distances and prevent genetic structuring. The solitary tunicate The online version of this article (10.1007/s00227-018-3388-x) contains supplementary material, which is available to authorized users. Egg and/or larval dispersal are key components of demographic expansion and establishment in sessile and sedentary marine invertebrates. The mode and duration of larval development have been suggested to be important for population genetic structure and gene flow  has a North Atlantic distribution  and, often, a slower southward return current.This part of the North Sea is almost a tidal with spring tides ranging from 24 cm (Vinga) to 37 cm (Väderöarna) and correspondingly negligible tidal currents. The water column in the region is strongly stratified with a pycnocline at 10–15 m . The origin of the surface-water layer is the Baltic Sea, but in the study area, it has a salinity of 15–25 psu and flow northward at ~ 0.5 m sC. intestinalis from each of 6 different sites distributed over three different areas . We used individual Bayesian assignment analysis .Genomic DNA was isolated from ethanol-preserved tissue using NucleoSpin Tissue Kit (Macherey–Nagel). Samples were genotyped at six microsatellite loci Cin-1, Cin-10 following , which should be sufficient to span the ca 200-km model domain. Since the studied geographic area is highly open, there is a significant loss of larvae to areas outside the model domain making the connectivity approach zero as the number of matrix multiplications increases. We thus only interpreted the relative magnitude of multi-generation connectivity between the three study areas  strongly positive, indicating deficiency of heterozygote individuals in all samples. However, individual loci showed very different patterns with two loci (Cin-10 and Cin-15) showing significant FIS values in all samples, whereas the remaining four loci showed few significant FIS values  of only 2–5 individuals per generation , which suggests that none of the loci were affected by strong differential selection, and hence, the Nm estimates are likely to represent gene flow. However, we found no support for isolation by distance , and hence, gene flow was not simply an effect of the geographic distances among sites. These results, instead, suggested a presence of local barriers to gene flow between some of the sites.Under the assumption of selectively neutral genetic variation and a two-dimensional distribution of populations (island model), the range of genetic differentiation found among the latter group of samples corresponded to levels of effective migration  grouped, and the two samples from GUL-deep sampled in different years made a separate group, while both shallow-water samples were different from each other and from all the deep-water samples at the K = 4  was 3. However, one of the loci (Cin-15) had a large number of missing data . Importantly, the overall result of the structure analysis was the same independent of 5 or 6 loci, and the only difference was in the optimal value of K. We show the analysis for 5 loci in Fig. The structure analysis showed support for the presence of local barriers with strong overall support for a similar genetic structure as indicated by the pairwise = 4 Fig. . Note thThe results of the assignment test showed that some of the samples were clearly more discrete than others with high levels of self-assignments, for example, the shallow-water fjord site  and the open-coast shallow-water sample  Fig. . The samThe biophysical model simulated passive dispersal of eggs and larvae, and multi-generational connectivity during 32 consecutive generations under the assumption of a maximum pelagic larval duration of 5 days. The results showed that from the surface down to a depth of 12 m, passive dispersal should be expected in a mainly northward direction with larvae originating in the southernmost site (VIN) spreading north Fig. b. ImportC. intestinalis  on the Atlantic coast of North America  stood out as genetically different from the rest. The authors suggested a mix of anthropogenic and natural causes explaining the complex population genetic structure of C. intestinalis in the English channel.Although the present study was undertaken in a relatively small geographic area, we found FST ranging from 0.054 to 0.093) was similar to what has been observed in marine species with little or no dispersal capacity . In the study region, weak tidal currents and a strong northbound surface current bringing low-salinity water from the Baltic Sea create strong stratification with a low-salinity surface-water layer ~ 10–15-m deep above a more oceanic deep-water layer. The strong density pycnocline that separates these two layers constitutes an effective barrier to passively dispersing particles, and even to actively swimming larvae , genetic differences indicated significant barriers to gene flow between surface and deep populations Supplementary file S2: This file contains 2 figures and 3 tables (PDF 130 kb)Below is the link to the electronic supplementary material.


Fusarium species, is a widespread and destructive fungal disease. In addition to the substantial yield and revenue losses, diseased grains are often contaminated with Fusarium mycotoxins, making them unsuitable for human consumption or use as animal feed. As a vital food and feed ingredient in China, the quality and safety of wheat and its products have gained growing attention from consumers, producers, scientists, and policymakers. This review supplies detailed data about the occurrence of Fusarium toxins and related intoxications from the 1980s to the present. Despite the serious situation of toxin contamination in wheat, the concentration of toxins in flour is usually lower than that in raw materials, and food-poisoning incidents have been considerably reduced. Much work has been conducted on every phase of toxin production and wheat circulation by scientific researchers. Regulations for maximum contamination limits have been established in recent years and play a substantial role in ensuring the stability of the national economy and people’s livelihoods.Wheat Fusarium head blight (FHB), caused by  Triticum aestivum L.), which belongs to the grass family, is widely distributed throughout the world and has a large planting area and total yield. Wheat is not only a vital nutrient-rich food source but also an important industrial raw material and animal feed component, and its distribution and use in China is similar to its global characteristics. With the adjustment of the agricultural structure, wheat has been the third largest food crop after maize and rice. According to the relevant data from the 2018 China Statistical Yearbook, wheat acreage has stabilized at 245,080 km2 over the past ten years, accounting for 14.73% of cereal production in 2017. China’s total production of wheat has reached 134 million tons and has increased 22% from 2007. However, despite the increase, the deficit in the trade balance increased to a billion dollars, which means that China needs to import approximately 4.4 million tons of wheat every year; this importation makes it imperative to breed wheat for increased yield. China is the largest wheat producer, supplying 17% of the total yield globally. More importantly, China is also the largest wheat consumer and accounts for 16% of the total consumption of wheat every year [Wheat (Fusarium graminearum species complex (FGSC), is a destructive fungal disease in most of the world’s major wheat-growing areas [Like other crops, due to the influence of agricultural environments and inputs, there are various safety issues, such as heavy metals and pesticide residues, in wheat-derived products, ,3. Accorng areas . From anng areas . Additiong areas ,10,11,12ng areas . ZEN is ng areas . TherefoFusarium toxins are produced by Fusarium species in suitable environmental conditions, and once they were present, there is no effective way to completely eliminate them from food. Even so, agricultural scientists have developed physical, chemical and biological methods to interfere with Fusarium species infection and toxin accumulation in all phases of wheat production. The present review aims to systematically analyze the occurrence characteristics of the main Fusarium toxins in wheat, the varying trend in human poisoning incidents, and the utility of toxin management measures in the past 40 years to provide a reference for scientific monitoring and effective prevention and control of mycotoxin contamination in China.Concerning the possible substantial effect of toxins on the country’s economy and society, the Chinese government has made many efforts aimed at ensuring cereal safety, such as setting strict limit and rigorous analytical method standards, revising agricultural product quality security laws, and establishing national special projects for agro-product safety risk evaluations. Wheat flour and its products have always accounted for a large proportion of the traditional Chinese diet and have a long history, especially in the north of the country. Due to the widespread occurrence and the complicated toxic effects of poisonous FHB metabolites, their presence in food should be regarded as a potential food safety hazard. Mycotoxicosis outbreaks in humans have been associated with the intake of food contaminated with these toxins and are thus a matter of great concern to consumers.Historically, there were several reports about mycotoxicoses in humans after the consumption of scabby wheat grains, particularly in the 1980s. A total of 21 outbreaks of human intoxication occurred successively from 1984 to 1991 in the main wheat-growing provinces . Some caIn 1985, a severe FHB epidemic occurred in wheat in a suburb of Puyang County, Henan Province. A large proportion of local inhabitants showed typical symptoms including gastrointestinal disorders and nervous system disturbances after eating scabby wheat grains, and the incidence of related diseases was 45% . Most ofAfter a large flood in 1991, extensive outbreaks of human toxicoses affecting a large number of people who consumed moldy wheat grains occurred in parts of Henan, Anhui, and Jiangsu provinces, as appropriate conditions, such as heavy rainfall and low temperature, for an FHB epidemic and mycotoxin production occurred after the flood ,23,24. HFusarium toxins decreased and moved from group outbreaks to individual incidents. For example, in 2000, a whole family in Northeast China developed acute poisoning symptoms twice after eating products derived from flour from home-grown wheat [In the 21st century, human poisoning caused by wn wheat . The latwn wheat .Fusarium toxins are associated with some human chronic diseases  as a species complex with significant genetic diversity, clear divergence of biological species, and an obvious geographical distribution. Currently, FGSC consists of at least 16 phylogenetically distinct species [F. graminearum s. str. is the most widely distributed species and occurs in most FHB areas around the world [F. asiaticum is the main FHB pathogen present in Asia [In recent years, genetic and molecular approaches have led to a new understanding that narrowly defines  species . F. gramhe world , while F in Asia ,52.F. graminearum s. str. isolates are 15ADON producers, while F. asiaticum isolates contain 3ADON and NIV chemotypes. F. graminearum s. str. and F. asiaticum in wheat.FGSC species can produce several mycotoxins; type B trichothecenes are the most common toxic metabolites found in infected cereals . FGSC stF. asiaticum isolates have been collected from warm southern areas [F. graminearum s. str. is mainly distributed in cool northern regions [F. asiaticum strain that produced 3ADON has always dominated [F. asiaticum overwintering, and perithecium production typically favors rice straw under warmer conditions. The better fitness of F. asiaticum on rice and DON producers on wheat has led to the prevalence of 3ADON-producing F. asiaticum in most wheat-rice rotation areas in Southern China. As a result, a 3ADON-producing F. asiaticum population may have been present in this region for a long time, and we suggest that this might have been the main factor underlying the absence of variation in trichothecene genotype frequencies from 1976 to 2014. Recently, based on available data from the literature, we concluded that a cropping system with wheat/maize rotation selects for F. graminearum, while a wheat/rice rotation selects for F. asiaticum [In terms of geographic distribution, the vast majority of rn areas ,57, and  regions ,59. The ominated ,61, and ominated ,64,65,66siaticum .Fusarium, which eventually leads to higher levels of toxins in wheat. A dominant FHB population means that the main toxins have been substantially retained over a long period of time, and the quantity of FHB pathogens is likely to continually increase. Based on this phenomenon, it can be assumed that the epidemic risk of FHB and harmful levels of Fusarium toxins could continue to increase for a long time in the future.Due to the promotion and application of straw-returning methods, more perithecia form and more ascospores are released in the subsequent year. Crop debris in the field feeds and increases the amount of primary FHB inoculum. Climatic or agricultural conditions favor wheat infection by Fusarium toxins in wheat and its products are of great significance. Fusarium toxins in wheat grains and wheat flour, respectively.China is a traditional agricultural country, and wheat is an important food ingredient. FHB severity has exhibited an increasing trend; therefore, the investigation of and surveillance for Fusarium toxin contamination shows various regional differences. The mean content and standard-exceeded rate of toxin was the highest in wheat and flour samples from Anhui and Jiangsu provinces, where FHB occurred severely and frequently. During the wheat heading and flowering period, a high temperature and humidity environment in the middle and lower reaches of the Yangtze River provided favorable conditions for the propagation of Fusarium, the occurrence of the disease and the accumulation of toxins. Although recent trends have indicated that FHB is spreading towards Northeast and Northwest China, the wheat quality in these regions is high. For example, a minimal incidence and concentration of DON and ZEN was detected in wheat from Heilongjiang [ongjiang . A recenongjiang . Especiaongjiang ,78,79. TFusarium toxin contamination shows clear temporal dynamics; the epidemiologic degree of FHB has a clear relationship with the toxin contamination level. The occurrences of Fusarium toxins were studied in detail in Jiangsu, Anhui, and Henan provinces over a long period of time, and there was high concentration of Fusarium toxins over the standard rate in 1985, 1989, 1991, 2010, 2012, and 2015. This is an interesting discovery, as these unusual years were reported to have massive outbreaks of FHB, except for 1991, in which a large flood occurred. In 1985, Henan Province had the worst outbreak in 40 years; almost all the wheat grains were contaminated with DON, NIV, and ZEN in high amounts. The contamination rate for samples containing toxins higher than the tolerance limit of 1000 µg/kg was close to 50%, and the highest number of DON-positive samples reached 40,000.0 µg/kg [.0 µg/kg . In flou.0 µg/kg ,82. Ther.0 µg/kg . In the .0 µg/kg ,85,86. T.0 µg/kg ,88,89. FFusarium toxins are also a potential risk to human health cannot be ignored.Recently, masked DON, a derivative of DON, has become a focus of attention for in-depth research. D3G  is a primary type of masked DON and is the most studied. D3G was for the first time detected in naturally contaminated maize and wheat in 2005 . Until nFusarium toxins in wheat flour remains at a certain level; nevertheless, most flour samples have toxin levels below Chinese regulatory limits and those of wheat grains. During wheat cleaning and flour milling, proper processing removes some toxins and is an effective measure in reducing toxin contamination. Considering the frequency and degree of the FHB epidemic, prolonged, successive, and extensive monitoring of Fusarium toxins in wheat and its products is essential for ensuring food safety and promoting human health.From several national surveys, it was reported that multitoxin, mainly DON and ZEN, were widespread and severe in Chinese wheat grains, especially in the past decade. Although the overall contamination situation varied significantly in different years, wheat quality safety conditions increased in severity with the prevalence of FHB. The detection rate of Fusarium mycotoxins need to be developed. A combination of planting resistant cultivars, adapting agricultural practices, and applying chemical and biological controls for reducing fungi invasion and toxin production may help to control the occurrence of FHB and its associated mycotoxins.Since FHB can lead to economic losses and health concerns, some comprehensive strategies for the reduction in the occurrence of FHB and Fusarium toxins are secondary metabolites produced by Fusarium species during its natural growth; these metabolites are closely associated with cell differentiation, growth, and the response to the external environment. Successful whole genome sequencing of several Fusarium species has provided useful data for researchers to study the biosynthesis pathways and regulatory mechanisms of mycotoxins [2O2 [w), incubation temperatures [Similar to pigments and antibiotics, cotoxins  and has cotoxins ,130 and cotoxins ,132. Envcotoxins ,134, carcotoxins , nitrogecotoxins ,137, H2Oins [2O2 , availaberatures  and regueratures , cyclic eratures ,142, anderatures , can infSeveral studies on detailed species and chemotype identification, population genetic diversity, and biological characteristics of FHB pathogens from wheat in China have been performed ,66,144. Fusarium toxins in wheat samples calls into question the true effects of chemical control on toxin production. In the past, control was measured only by the decrease in the visual disease index and the recovery of economic losses; serious toxin contamination was largely ignored. As such, the efficacy of carbendazim on toxin accumulation is questionable. Poor reductions in toxin accumulation may be due to the increase in Fusarium species that are resistant to carbendazim and the improved synthesis ability of DON in resistant strains compared with that of conventional strains [Spraying carbendazim during the wheat flowering period is a common method for controlling FHB and has played a crucial role in integrated disease control programs since the 1970s. However, the monitoring results of  strains ,146. Hig strains .Fusarium. Field experiments indicated that phenamacril showed better efficacy against FHB than carbendazim, and the yield increase effect was equivalent to that of carbendazim [Phenamacril, a new type of acrylate fungicide, was developed by China; phenamacril provides successful control of a variety of plant diseases caused by bendazim . Moreovebendazim ,148. Cur2O2 production in vitro and further induced DON accumulation in the pathogen [Another kind of fungicide that can significantly reduce the ability of pathogens to infect plants and synthesize toxins is the azole antifungals, including metconazole, propiconazole, prothioconazole, and tebuconazole, which belong to the class of demethylation inhibitors. Several studies have proved that tebuconazole alone at varied concentrations or in combination with other fungicides was effective in inhibiting FHB and DON, and the drug showed optimal performance at the early anthesis stage or later anthesis stage ,149,150.pathogen .Rhizoctonia species, had a remarkable effect on reducing DON synthesis by F. graminearum [F. graminearum was discovered in vitro.According to a new study, validamycin, a type of fungicide for the control of crop diseases caused by minearum ,154. Furminearum . This fiThere are many agricultural practices to combat pathogens, diseases, and contaminants; however, chemical control is still the most powerful and effective measure. The effect of fungicides on mycotoxins is complicated and can be influenced by various factors, such as toxin-producing fungus, environmental conditions, and treatment dosage. More research on the mechanisms of toxin production and compound mode of action is needed to provide theoretical support for the scientific and reasonable use of fungicides. Chinese government is vigorously implementing the strategy of the dosage reduction and efficiency increase of chemical pesticides, as pesticide residue is a threat to food safety and cannot be ignored. More rational and efficient application of these substances is extremely essential.Wheat can experience a serious loss of tissue composition after infection with scab, resulting in a change in the wheat kernel appearance. Diseased grains usually have shriveled surfaces, declined particle diameters, decreased hardness, and decreased weight; therefore, specific gravity separation is the best method to remove infected wheat kernels before storage and processing. Historically, wind force was used to separate diseased and normal grains based on simple devices, and notable results were obtained. Liang et al.  suggesteFusarium, and electric color sorting technology based on color characteristics can separate the contaminated grains to effectively manage toxins. The soft independent modelling of class analogies (SIMCA) model based on near infrared spectrum and hyperspectral imaging technology has been successfully applied in the identification of scabby grains and DON levels, with a recognition accuracy above 90% [It is well known that scabby wheat kernels present different shades of red due to the infection of bove 90% ,160,161.bove 90%  establisbove 90% .Cleaning is the first step in wheat processing and plays a vital role in wheat flour safety. At present, color and gravity sorters have been implemented in most flour milling enterprises and are of great importance to mycotoxin reduction and the quality improvement of wheat flour and its products.Fusarium toxins, many countries and food safety regulators, including China, have introduced maximum or recommended toxin levels for food and feed. Specific regulations at the national level are released by authoritative bodies , harvest control , and postharvest control  should be employed to manage all possible risk factors to prevent mycotoxin contamination. In the future, with the effective implementation of good agricultural practices (GAPs), good manufacturing practices (GMPs), and hazard analysis critical control points (HACCPs), food safety, and consumer health can be improved and guaranteed as much as possible.


Eriphia verrucosa), aiming to unravel potential microevolutionary processes likely involved in shaping its genetic polymorphism. For this purpose, a total of 155 specimens of E. verrucosa from 35 locations across the entire distribution range were analyzed by comparing a 453 basepairs region of the mitochondrial gene cytochrome oxidase subunit 1 (Cox1).The Pleistocene cyclic sea-level fluctuations are thought to have markedly affected the distribution and genetic architecture of Atlanto-Mediterranean biota. Despite the acknowledged key role played by these historical events in shaping population genetic structure of marine species, little is still known about the processes involved in shaping the spatial distribution of genetic variation within intertidal species. We intended in this study to reconstruct the phylogeography of a common and widely distributed coastal species across the East Atlantic and Mediterranean Sea  contains supplementary material, which is available to authorized users. One of the main aims of evolutionary genetics is to discern the evolutionary processes responsible for driving and shaping geographic distribution of genetic variation of populations, as well as disentangling the origin of their genetic structure –4. IntegPalmaria palmata across the English Channel [Sargassum polycystum across the southern Chinese coast [Nassarius nitidus [Carcinus aestuarii [The Pleistocene cyclic sea-level fluctuations are thought to have markedly affected the coastal environment and the evolutionary history of its biota  and rega Channel  and the se coast . The occ nitidus  and the estuarii . In contestuarii . The impestuarii , 15.The Mediterranean Sea and the contiguous northEast Atlantic Ocean represent a suitable area to study biogeographical processes and investigate evolutionary patterns of diversification in marine species , 8, 16. Hippocampus hippocampus [Nassarius nitidus [The above-mentioned biogeographic units could stem from the impact of historical, hydrographic and environmental processes. From a historical point of view, a marked drop of sea level during Pleistocene glaciation peaks  might hapocampus  and the  nitidus . In addi nitidus , 18, 31. nitidus , 32 as w nitidus  responsi nitidus . Besides nitidus  and the  nitidus , 23, 25.Among Atlanto-Mediterranean marine biota, intertidal marine species are still less investigated in terms of their phylogeographic structure and evolutionary history. Furthermore, contemporary spatial distributions of their genetic diversity are thought to have been shaped by the impact of Pleistocene climate oscillations . Hence, Eriphia verrucosa   represents a good model to address the above mentioned issues. This littoral decapod is one of the most ecologically important coastal species, playing a crucial role in structuring intertidal communities, as a highly predatory shore crab [E. verrucosa begins in May or June [E. verrucosa can take 49 days at a temperature of 21 °C. Considering all of these eco-biological aspects, genetic homogeneity and panmixia could be expected among populations of this crustacean species. However, recent phylogeographic investigations, carried out in other Atlanto-Mediterranean decapod species (with similar life-history traits as E. verrucosa), revealed restricted patterns of gene flow, such as in the green crab Carcinus aestuarii [Pachygrapsus marmoratus [Palaemon elegans [The warty crab ore crab , 37. It ore crab , 39. Theore crab . The rep or June . Spawnin or June , dependi or June –43. The  or June . Accordi or June , larval estuarii , the mar elegans . The outE. verrucosa allowing for the occurrence of significant patterning of population genetic structure? (2) Did palaeoclimatic and palaeogeographical evolution of the surveyed geographic area during the Quaternary significantly impact contemporary spatial distribution of genetic polymorphisms and their variations through space and time, and what are the potential evolutionary processes likely involved in shaping genetic diversity and affecting spatial genetic structure within this decapod species? In order to answer these questions, we examined the mitochondrial phylogeography of the species and reconstruct its evolutionary and demographic histories. An intensive sampling of E. verrucosa was conducted from its entire distribution range, stretching from the East Atlantic to the Black Sea  Can the main postulated barriers to gene flow spanning the East Atlantic and the Mediterranean Sea restrict gene flow among populations of  Can the E. verrucosa  and low total nucleotide diversity (π = 0.0034 ± 0.0002). The total mean number of nucleotide differences (K) was 1.575. Details on these parameters of genetic variability in each examined population are reported in Table E. verrucosa were shown to be genetically more variable than their Mediterranean counterparts  in a dataset excluding GenBank sequences, we noticed that East Atlantic specimens of E. verrucosa remain genetically more diversified than their Mediterranean counterparts , after a rarefaction procedure involving 30 replicates, showed that the East Atlantic and western Mediterranean samples were significantly more diversified than their eastern Mediterranean counterparts for all parameters (except for haplotypic richness)  was not significantly higher than the GST value (0.048); P > 0.05, highlighting lack of a significant relationship between genealogy and geographic distribution of Cox1 haplotypes. Within lineage 1, a further separation by two mutational steps was noticed between two sets of haplotypes (or sub-clades), centered around haplotypes 1 and 11. The sub-clade around haplotype 1 was shown to be slightly prevailing in the East Atlantic. However, such observation should be interpreted with caution as this sub-clade is relatively common in a small subset of specimens from the Azores. Besides, the central haplotype 1 and the derived haplotype 3 have been also found in the eastern Mediterranean and may occur in the western Mediterranean but they probably went unnoticed because of the small sample size obtained for this region (only 45 specimens).The TCS statistical parsimony procedure yielded two resolved sub-networks centered around two main and common haplotypes (haplotype 2 and haplotype 11) Fig.  and 4. BE. verrucosa was found to be weak but significant based on both Tajima-Nei distances  and haplotype frequencies . Pairwise comparisons of genetic differentiation showed that only five (based on nucleotide divergence) and six (based on haplotype frequencies) out of the 105 pairwise genetic distances were significant after the B-Y FDR correction  with a 95% high posterior density interval (HPD) of 3.83–5.89%, which is within the range of substitution rates of 1.11–6.58% per Myr, estimated so far for the Cox1 gene in crustaceans , 47–50.E. verrucosa. The first mitochondrial clade (lineage 1) was found to be statistically supported (PP = 0.65); while the more genetically diversified clade (lineage 2) was poorly resolved (PP = 0.42)  . Subsequent diversification within both retrieved mitochondrial lineages started nearly at the same time  Fig. . Within E. verrucosa was in a wide ancestral region encompassing the East Atlantic and Mediterranean regions (biogeographic region AB). The S-DIVA analysis allowed detecting a marked vicariance signal at the most ancestral node  allowed detecting the same patterns at the three most ancestral nodes    could operature , 55. ThiE. verrucosa, as other factors such as large effective population size could also be involved [Alternatively, we can propose that high gene flow and a particular larval behaviour may not be the only causes of the lack of phylogeographic structure discerned within involved . Indeed,involved , 42, it involved .E. verrucosa, they have to be considered with caution as the increase in sample size may yield new insights into the phylogeographic structure of the species across the postulated biogeographic boundaries. The recent study by Fratini et al. [P. marmoratus, previously regarded as panmictic due to geographically and numerically limited datasets.While the above mentioned scenarios may provide possible explanations to the high genetic connectivity within i et al.  showed tOpposed to a background of high regional and and sub-regional genetic connectivity, the East Atlantic peripheral population of Azores was shown to be genetically distinct. Such finding has been already reported for other marine invertebrate and vertebrate species with high dispersal potential –59. ThisE. verrucosa, historical processes might also have exerted significant impact on geographic distribution of genetic variability. This assumption is likely supported by the outcomes of regional genetic diversity, spatio-temporal patterns of genetic diversification, and demographic history. Thereby, a complex series of micro-evolutionary forces encompassing allopatric differentiation, followed by range expansion and secondary contact and admixture among refugial lineages, might have contributed to shaping genetic diversity and structure in this decapod species. Hence, we discuss the evolutionary history of E. verrucosa in the context of the following issues:In addition to the likely involvement of the above-discussed contemporary factors in shaping population genetic structure of E. verrucosa were significantly more diversified than their eastern Mediterranean counterparts for most of the examined parameters. Second, BEAST mitochondrial genealogy highlights potential genetic signatures of late Pleistocene genetic divergence between two lineages. Third, reconstruction of possible past geographic distributions of Cox1 haplotypes, using the S-DIVA method, identified the East Atlantic and the Mediterranean Sea as the most likely ancestral ranges for both retrieved lineages within E. verrucosa. Such integrative evidences suggest that suitable habitats within the East Atlantic and western Mediterranean  might have been important marine biodiversity refugia to retain ancestral relict populations of E. verrucosa during Pleistocene climate changes. This assumption is likely supported by the outcome of recent phylogeographic studies on other Atlanto-Mediterranean marine species, which have also identified potential glacial refugia within the East Atlantic (namely across the Iberian Peninsula and Macaronesia) and the western Mediterranean [As far as can be inferred from the outcome of this study, several lines of evidence support the existence of potential marine glacial refugia in the East Atlantic and western Mediterranean. First, assessment and comparison of regional genetic diversification showed that East Atlantic and western Mediterranean samples of erranean , 63–65. Integrative outcomes of BEAST and RASP analyses allowed retrieving genetic imprints of a late Pleistocene vicariant event across the Gibraltar Strait, which occurred approximately around 66,300 years before present. This historical event corresponds to the glacial period of the late Pleistocene . It haspresent; . The simE. verrucosa. This pattern was shown for all examined diversity parameters except for haplotypic richness. Based on these insights, and referring to the markedly younger demographic and spatial expansion events recorded for the eastern Mediterranean than those retrieved for the western Mediterranean, we hypothesize that areas located to the east of the distribution range of the species might have been recently and rapidly colonized from the nearest refugia, when new habitats became available again.Analysis of regional genetic diversification revealed significantly reduced levels of genetic variability in eastern Mediterranean specimens of However, given the fact that the eastern Mediterranean was shown to be genetically more diversified than the western Mediterranean based on haplotypic richness, the validity of this assumption can be seriously questioned. Two possible explanations can be advanced in order to clarify this discrepancy. First, it has been shown that genetic diversity in re-colonized areas can be either higher or lower depending on the estimator . Second,E. verrucosa: The harsh climatic changes, mainly mediated by significant climate cooling and dropping of sea levels, during the Würm glaciations, might have led to the restriction of biotic exchange across the Gibraltar Strait and consequently to the retreatment of specimens of the warty crab to specific refugia within the East Atlantic and Mediterranean Sea. Subsequent postglacial recolonization of suitable habitats, following the rise of sea level and amelioration of climate conditions, might have led not only to the resumed connectivity across the Gibraltar Strait but also to the recent colonization of the eastern Mediterranean. The resulting gene flow restoration among historically isolated populations across the Gibraltar Strait  is still likely maintained by the impact of contemporary factors including oceanographic features, larval behaviour, and large effective population size.Overall, based on the obtained preliminary results, we propose the following evolutionary history scenario for The results of the present study provide new insights into the phylogeography and evolutionary history of a common but poorly studied Atlanto-Mediterranean decapod species. Specifically, they contribute to the understanding of the impact of historical processes on shaping contemporary population genetic structure and diversity in intertidal marine species. Increase in the sample size and investigation of highly variable nuclear markers are highly required and recommended in future investigation in order to confirm the discerned pattern of phylogeographic structure and provide a more complete and nuanced view of the evolutionary history of this important species.Eriphia verrucosa were obtained from 35 locations across the whole distribution range stretching from the East Atlantic to the Black Sea  Assignment according to geographic origin of the specimens so that only geographically closed locations within the same region (East Atlantic or Mediterranean Sea) or sub-regions (western Mediterranean or eastern Mediterranean) can be combined. 2) Assignment considering the postulated barriers to gene flow . In the latter case, the pooling procedure was not applied on the geographically close locations lying across these biogeographic boundaries even if this pattern of combination may still lead to limited sample size as that obtained in the two defined populations of Northern Tunisia and Alboran Sea (Tables  Assignme AssignmeNh) and polymorphic sites (Nps), haplotype (h) and nucleotide (π) diversities [The nucleotide compositions as well as the number of variable and parsimony-informative nucleotide sites were estimated with MEGA version 7.0.18 . Measureersities , 76, as ersities .h, π, K, Hapr (haplotypic richness), Np (number of private haplotypes), Np/N (proportion of private haplotypes), and Np/Hapr (genetic endemism)). Owing to the difference among the sample sizes of the examined datasets, we applied a rarefaction procedure in order to discard the positive bias of sample size on inferred diversity. For each sample larger than the smallest one with the size N = 42, recorded for the East Atlantic region, a subsample of N individuals was drawn randomly and diversity indices were calculated. In order to infer reliable statistics, the procedure was repeated 30 times for each sample, and then the mean subsample diversity for each sample was calculated. In order to check for the consistency of the obtained results, and see whether the number of replicates could influence the outcome of comparison, analyses were re-carried out with 60 replicates. The unpaired t-test, as implemented in the online software T-Tests-Free Statistics and Forecasting Software  version 1.2.1 [Assessment and comparison of genetic diversification levels among the East Atlantic, western Mediterranean and eastern Mediterranean were carried out by the examination of seven genetic diversity indices  and NST (considering relationship among haplotypes) [Genealogical relationships among Cox1 haplotypes were examined and assessed through construction of the haplotype network with the statistical parsimony procedure included in the software TCS version 1.21 . Correlalotypes) , 81, in P < 0.05). Accurate inference of the significance level was assured by the B-Y FDR correction [P < 0.00954).Patterns of genetic differentiation within the total mitochondrial dataset (by means of one-level AMOVA ), and amrrection   was used to test hierarchical structuring of genetic variation within E. verrucosa, we specifically estimated the mutation rate of the Cox1 gene referring to the already surveyed phylogeny of the genus Eriphia by Lai et al. [Eriphia. We, therefore, implemented a calibration point derived from the biogeographic event of the Pleistocene glacial sea-level low stands, assuming that the vicariant speciation of Indian and Indo-West Pacific species of the genus Eriphia might have been triggered by the impact of dry exposure of the Sunda Shelf at the onset of the Pleistocene glaciation cycles which occurred approximately 2.588 million years ago (± 0.005) [Eriphia by Lai et al. [In order to reliably elucidate the evolutionary history of i et al. , whose f± 0.005) , 88. The± 0.005) . Before ± 0.005) . Cox1 sei et al.  , along i et al. .Eriphia. The Bayesian analysis was accomplished after running the MCMC simulations for 30 million generations (sampled every 1000 generations), checking the generated outputs for convergence  in TRACER version 1.5, and summarizing the resultant trees in TreeAnnotator version 1.7.5 [Temporal patterns of Cox1 haplotypes diversification were estimated in the software BEAST version 1.7.5 with a coalescent tree prior, suggested as an appropriate null model for intraspecific data . A stricon 1.7.5 . The obton 1.7.5 .E. verrucosa was inferred from ancestral area reconstruction based on Cox1 haplotypes using the S-DIVA  method, as implemented in RASP version 3.2 [E. verrucosa Cox1 haplotypes, three main biogeographic regions were defined: (A) Atlantic Ocean, (B) Mediterranean Sea; and (C) Atlantic-Mediterranean. The Most Likely States (MLS) function, as implemented in RASP, was used to highlight the likelihood of possible ancestral range at each generated node of the phylogeny. The MCMC output and condensed tree exported from BEAST and TreeAnnotator, as well as the distribution of the Cox1 haplotypes through all defined biogeographical areas were loaded as ‘trees file’ and ‘distribution file’ respectively in order to perform S-DIVA analysis in RASP.The biogeographic history of sion 3.2 . S-DIVA sion 3.2  and is rE. verrucosa were detected by the analysis of three neutrality tests . A totaindex rg  in ARLEQτ), was inferred for each study case.Both demographic and spatial expansion  models, Eriphia. Two MCMC simulations were run independently for 40 million iterations. After discarding the first 10% iterations (4 millions) as burn-in, LogCombiner version 1.7.5 [Detailed information on the amplitude of historical demographic events that the warty crab populations could have undergone was also retrieved from the outcome of the Bayesian Skyline Plot (BSP) method . BSP anaon 1.7.5  was usedAdditional file 1:Table S1. Estimation of genetic diversity parameters in Atlantic and Mediterranean specimens of Eriphia verrucosa, based on two analyzed datasets (including and excluding retrieved sequences from GenBank). (DOCX 13 kb)Additional file 2:Table S2. Pattern of assignment of the twelve Cox1 sequences of Eriphia verrucosa (retrieved from GenBank) to the detected haplotypes in this study. (DOCX 11 kb)Additional file 3:Table S3. Geographic distribution of the 30 haplotypes of E. verrucosa recorded at the 15 defined populations within the East Atlantic Ocean and Mediterranean Sea. (DOCX 16 kb)Additional file 4:Table S4. Analysis of pairwise genetic differentiation in a total dataset of 143 specimens of Eriphia verrucosa (excluding the twelve sequences from GenBank). (DOCX 17 kb)Additional file 5.Figure S1. Analysis of the historical biogeography of E. verrucosa based on a total dataset of 143 sequences (excluding the GenBank sequences). (DOCX 304 kb)