Data collection followed Arizona State University Dental Anthropology System standards. Genetic correlation estimates were generated using maximum likelihood variance components analysis in SOLAR v.8.1.1. Approximately 23% of deciduous variance components models and 30% of permanent variance components models yielded significant genetic correlation estimates. By comparison, over half (56%) of deciduous–permanent homologues  were significantly genetically correlated. It is generally assumed that the deciduous and permanent molars represent members of a meristic molar field emerging from the primary dental lamina. However, stronger genetic integration among m2–M1/M2 homologues than among paired deciduous traits suggests the m2 represents the anterior-most member of a “true” molar field. The results indicate genetic factors act at distinct points throughout development to generate homologous molar form, starting with the m2, which is later replaced by a permanent premolariform crown.This study aims to expand our understanding of the genetic architecture of crown morphology in the human diphyodont dentition. Here, we present bivariate genetic correlation estimates for deciduous and permanent molar traits and evaluate the patterns of pleiotropy within  and between  dentitions. Morphology was observed and scored from dental models representing participants of an Australian twin and family study (deciduous  Two conditions contribute to this diversity within individuals ,2. Most d molars ,4. For mhyodonty ,6,7. Thehyodonty ,11,12,13heritability traits) or may not (low heritability traits) explain the structure of a corresponding dental data set—e.g., [pleiotropy). With regard to understanding the structure of mammalian dentitions, this work has provided crucial insight into the role of genes in partitioning the tooth row into distinct modules. While many studies have focused on tooth size in human and non-human primates [The field of quantitative genetics has also provided insight into these conditions, outlining the relative contribution of genes to tooth form and the coordination of genetic effects in determining dental phenotypes. In these efforts, pedigreed samples and relatedness coefficients approximate underlying additive genetic variation, which may  dental complex as a cohesive entity. Heritability estimates for crown morphology suggest, on average, a moderately strong and stable contribution of genes to morphological variation across arcades (maxillary–mandibular) and across dentitions (deciduous–permanent), which may explain the high correspondence in crown form between primary and replacement teeth . Recent Recently, the authors reported genetic correlations for anterior crown morphology in a longitudinal dental sample of Australian twins and families . Pleiotrpostcanine tooth row. Although heritability estimates have been reported for crown characters of the deciduous and permanent molars [Yet to be explored is the genetic architecture of morphology of the diphyodont t molars , geneticThe postcanine tooth row is a unique region of the human diphyodont dental complex. In the anterior dentition, primary incisors and canines are shed and replaced by larger permanent elements that are nearly identical in overall shape and morphology. In contrast, the postcanine dentition consists of deciduous molars, which are shed and replaced by permanent premolars ,38,39. TGρ) for deciduous and permanent molar morphology. The analyses test the following null hypotheses:1.Paired deciduous traits are genetically independent of one another:Gρ = 0; dtrait1–dtrait2 Gρ ≠ 1 (no pleiotropy).dtrait1–dtrait2 2.Paired permanent traits are genetically independent of one another:Gρ = 0; PTrait1–PTrait2 Gρ ≠ 1 (no pleiotropy).PTrait1–PTrait2 3.Paired deciduous/permanent traits are genetically independent of one another:Gρ = 0; dtrait1–PTrait1 Gρ ≠ 1 (no pleiotropy).dtrait1–PTrait1 The aim of this study is to examine the structure of additive genetic contributions to postcanine crown variation in the diphyodont dentition, with a focus on crown morphology. Here, our interest is in quantifying the degree to which the same genetic effects contribute to variation in distinct morphological characters within a sample of known relatives. To achieve this goal, we generated a series of quantitative genetic models to estimate bivariate genetic correlations . The UAT represents nearly forty years of data collection with over 1200 twins and 2000 relatives (as of 2014) enrolled as participants from the regions surrounding Adelaide, South Australia and Melbourne, Victoria [Materials and methods follow those outlined in Paul et al. (2021). Molar morphology data were collected for 25 deciduous crown traits and 38 permanent crown traits referencing the University of Adelaide Twin Study (UAT) dental cast collection  ,51. The To test for intraobserver error, a subset (~20%) of the original sample was rescored eight or more weeks after initial data collection—see . Raw graMorphological variants were separated into three distinct variables prior to analysis: (1) left side expression, (2) right side expression, and (3) maximum expression ,36. For Maximum likelihood variance components analysis was used to generate a suite of parameter values, including narrow-sense heritability and bivariate genetic, environmental, and (derived) phenotypic correlations. These methods have been outlined in previous publications ,26,31,32Ω: trait (co)variance matrix; G: genetic (co)variance matrix; Φ: kinship matrix; E: environmental (co)variance matrix; and I: identity matrix. Parameter estimates generated for each model include genetic correlation (Gρ), environmental correlation (Eρ), and derived phenotypic correlation —see  for ρP eGρ = 0; model 2b: |Gρ| = 1). For the genetic correlation parameter, significant difference  all parameter values freely estimated, and (2) parameter of interest held constant  for this sample or because the traits are likely non-homologous with the permanent molar characters upon which scoring standards are based  (Certain deciduous traits were removed from the study because they were monomorphic (m cusp 7) . This is1/M1 metacone, m1/M1 parastyle, M1 anterior fovea, m1/M2 cusp 7 , and m2 /M2 protostylid (maximum error = 3 grades). Of these traits, only m2/M2 protostylid were omitted from the genetic correlation analysis based on error results, as all other listed traits are associated with mean error values less than 0.30. Additionally, as reported in Paul et al. (2020), error data for m1 metacone , M1 anterior fovea , and M1 parastyle  showed observation session biases—either all error session scores were lower or higher than the original scores. Results are presented for associated models but flagged for interpretive purposes  , genetic correlations for morphological isomeres are weaker . Of the significant models, genetic correlations were positive for all but three: m2 metacone–m2 cusp 5, m2 hypocone–m2 anterior fovea, and m2 cusp5–m2 distal trigonid crest  see . While wid crest .1 paracone–M2 cusp 7, M1 protostylid–M2 cusp 7, and M1 cusp 7–M2 cusp 7 . Genetic correlation results for the permanent dentition are presented in 2 cusp 7 . The rem1/M2 hypocone and M1 protostylid account for over 40% of the pleiotropic relationships , and cusp 7. This is a consistent pattern, considering that genetic correlations were not estimated between neighboring teeth for paracone, cusp 6, protostylid, or distal trigonid crest, while deflecting wrinkle and anterior fovea are only scored on a single tooth. For all other significant models, the patterns appear idiosyncratic, although Mionships .Gρ = 0.20; absolute mean Gρ = 0.23)  and mandibular  dentitions, while these values are slightly lower for isomerically paired characters . Significant models yielded positive estimates with one exception: M1 cusp 5–M1 cusp 6  . Again, 1 cusp 6 .The results of the deciduous–permanent genetic correlation analyses are presented in Gρ = 0.36; absolute mean Gρ = 0.43). Interestingly, the results show weaker genetic correlation between maxillary deciduous–permanent homologues  than between their mandibular counterparts . All analyzed trait pairs are positively correlated with two exceptions: m2–M2 cusp 5 and m2–M2 cusp 5; pleiotropy is not indicated for these two models  is similar to that reported for anterior dental morphology (deciduous < 0.21 and permanent < 0.23) in this sample. While this suggests stability in the degree of genetic redundancy occurring in distinct regions of the dentition, these values are markedly lower than reported averages for postcanine crown size in humans (maxillary = 0.73 and mandibular = 0.84) . The samOur results corroborate another reported difference between dental metric and morphological genetic architectures: genetic correlations are, with few exceptions , positiv2 cusp 6, all derived phenotypic correlations for left–right character pairs are positive and exceed 0.30. Together, these results affirm a tendency toward strong morphological symmetry in the molars, which—in nearly all cases—is driven by consistency in genetic effects. Similar results were obtained for anterior (incisor and canine) morphology in this sample [Genetic correlation estimates for left–right paired traits suggest strong bilateral integration for molar morphology. Most values approach 1.0, with complete pleiotropy indicated for all deciduous and most permanent traits . With ths sample  and for s sample ,31 and ns sample . These cHowever, we note that the only other quantitative genetic study of postcanine morphology reported limited symmetry in genetic effects, as interpreted from disparate left–right heritability estimates, limited genetic correlation, and moderate derived phenotypic correlation for paired characters . This ma1 cusp number–m1 cusp 5. This is not surprising, given that cusp 5 variation  directly influences variation in cusp number [Considering deciduous and permanent dentitions separately, molar morphology is minimally integrated. The deciduous molar analyses identified one completely pleiotropic trait pair: mp number ,51,70. Tp number ,72. 2 cusp 7. The genetic redundancy in M1–M2 cusp 7 is expected, given their metameric relationship, but the strong correlations between M2 cusp 7–M1 paracone and M2 cusp 7–M1 protostylid are difficult to interpret. These results may indicate that similar genes are involved in accessory cusp formation and crown elaboration along the buccolingual plane. Yet, if that is the case, it is unclear why similar results were not obtained for M1 cusp 7.In the permanent dentition, all cases of complete pleiotropy involve Mon the same tooth, which suggests a high degree of within-crown morphological integration. Although this pattern reflects a true biological phenomenon—overlapping genes implicated in manifold morphological outcomes—it also underscores the limitations of our methods for quantifying crown variation. By this, we mean that ultimate crown morphology is the result of developmental processes that are highly coordinated within a single tooth: oral epithelial–ectomesenchymal interactions, determination of placode location and size, enamel knot formation and placement, inner-enamel epithelial folding, and tissue (enamel) secretion—as reviewed in [Of the remaining traits that share some degree of genetic redundancy, many occur iewed in . Experimental research has shown these processes are tightly controlled by (epi)genetic mechanisms that act on tooth crowns, and indeed the entire dentition, as cohesive entities ,76,77,78later-forming cusps of the upper molars, especially hypocone, cusp 5, and Carabelli’s trait. Of the deciduous traits that yielded valid model results, m1 hypocone shares an incomplete pleiotropic relationship with ~54% percent of them. This result for m2 hypocone is ~62% and ~32% for Carabelli’s trait. M1 hypocone shows significant genetic correlation with 39% of other permanent traits, while model results for M2 hypocone (56%), M1 cusp 5 (50%), and M2 Carabelli’s trait (50%) indicate incomplete pleiotropy for over half of the characters analyzed. This finding suggests that coordinated additive genetic effects play a major role in the developmental regulation of cusp size and presence in postcanine teeth.Another set of traits that accounts for a large portion of the significant model results includes The pattering cascade model provides a framework for interpreting these findings. It posits that cusp number and size  are determined by: (a) overall size of the tooth germ, and (b) spatial patterning of enamel knots ,90. At tnegative. This suggests the same genes are implicated in lower cusp 5 and cusp 6 expression but their effects are opposing. A similar result was reported by Stojanowski and colleagues in their study of human molar morphology [This pattern is not as strong in the lower molars, however. Interestingly, in the permanent mandibular teeth, the later forming cusps (cusp 5 and cusp 6) are significantly correlated, but the correlation is rphology . Their irphology ,103,104.neighboring. The difference between cusp 5 and cusp 6 in the lower molars and most later-forming cusps of the upper molars is that cusps 5 and 6 are situated next to each other at the distal-most aspect of the crown. Thus, their sizes depend directly on relative enamel organ partitioning and the cellular territory allotted to their neighbor during crown formation [relative to another feature, in this case cusp 5 [Combining this insight with expectations outlined in the patterning cascade model, we might interpret the negative correlation between these neighboring cusps as competition for “cellular real estate” during morphogenesis and enamel secretion ,89,105. ormation . The nate cusp 5 ,51. WhatAs the UAT Cohort 2 sample is longitudinal with most individuals represented by deciduous and permanent dental casts, this study provides a unique opportunity to examine the morphological architecture of the diphyodont molar row. Because m1s are more similar in crown form to permanent premolars, we generated genetic correlations for homologous trait pairs of m2–M1 and m2–M2. Raw and absolute genetic correlations are higher for paired deciduous–permanent molar traits (mean = 0.36 and absolute mean = 0.43) than for paired traits within dentitions , with over half characterized by incomplete pleiotropy. This is a similar pattern to that observed in the anterior dentition for this sample, and it is tempting to draw a similar interpretation: that there is “a stronger genetic mechanism for morphological conservation across the diphyodont dentition than within individual tooth rows” [Unlike the incisors and canines, the permanent molar class is not succedaneous. That is to say, deciduous molars are replaced not by permanent molars but by permanent premolars. Permanent molars do not replace any primary elements and are the only permanent teeth to arise from the primary dental lamina—the same dental lamina from which all deciduous teeth originate . Unlike somewhat premolariform, but, compared to other elements, it does not strongly resemble any other tooth in the diphyodont dental complex. If considered part of a meristic molar field, this unique morphology is unexpected [2, and M3 [That said, the stronger genetic correlation between m2 and the permanent molars corroborates previous research that suggests the m1 is, for lack of a better term, an oddity. The m1 is expected ,108,109.expected ,111,112.expected . While t, and M3 ,42,44. HWhile we note several consistencies between these and previous quantitative genetic findings, sample composition and certain methodological choices may have impacted ultimate results. First, the reported genetic correlations represent a primarily European–Australian sample characterized by its own population-specific morphology profile. Previous research has shown morphological trends in both additive and non-additive genetic factors  that may result in disparate patterns for populations of distinct biogeographic origin . Due to the UAT’s recruitment strategy, this sample also includes a disproportionately large number of MZ twins, which sets it apart from broader pedigree/family study samples. Finally, as in our study of anterior morphology , morphol2 hypocone–M1 hypocone). Further, a greater percentage of deciduous–permanent trait pairs exhibit some degree of redundancy in genetic effects (incomplete pleiotropy). As both the deciduous and permanent molars arise from the primary dental lamina, they likely represent elements of a meristic tooth district. Still, the relative degree of genetic integration noted for m2–M1–M2 morphology as compared to m1–m2 morphology aligns with previous suggestions that the m2 is the anterior-most member of a cohesive primary molar field. This is compatible with the overall distinctiveness of the m1 crown form.Within dentition (deciduous–deciduous and permanent–permanent) correlation values are, on average, lower than those estimated for homologous traits across dentitions (e.g., m


The 250µg-cosyntropin stimulation test (CST) is used to diagnose non-classic congenital adrenal hyperplasia (NCCAH). The current recommendation is to perform CST when follicular 17-hydroxyprogesterone (17OHP) is 6-30 nmol/L, a cutoff derived from radioimmunoassay (RIA). Recently, enzyme-linked immunosorbent assay (ELISA) has replaced RIA.We aimed to (1) determine the RIA and ELISA-based 17OHP cutoffs at which CST should be performed, (2) identify predictors of NCCAH.A retrospective study at an Israeli Health Maintenance Organization. Data were retrieved from women with suspected NCCAH, referred for CST during 2001–2020. NCCAH was defined as a stimulated 17OHP >30 nmol/L. Serum 17OHP levels were assayed by RIA from 1/2000-3/2015, and by ELISA from 4/2015-12/2020. ROC curves were generated and optimal 17OHP thresholds were determined. Multivariate analysis was performed.CST was performed in 2409 women . NCCAH was diagnosed in 4.7% of the RIA group and 7.5% of the ELISA group. The optimal basal 17OHP cutoff values predicting NCCAH were 6.1 nmol/L in RIA  and 8.2 nmol/L in ELISA . In multivariate analysis, higher basal 17OHP, lower LH: FSH ratio, and oligomenorrhea were predictors of NCCAH in RIA. Higher basal 17OHP, androstenedione, and total testosterone were predictors of NCCAH in ELISA. A lower LH: FSH ratio showed similar trend in ELISA.Optimal RIA-based basal 17OHP cutoff was comparable with that recommended in guidelines. The results suggest adopting a higher 17OHP cutoff when using ELISA. LH : FSH ratio improves the negative predictive value of basal 17OHP. The current recommendation is to perform CST when basal serum 17-hydroxyprogesterone (17OHP) levels  are 6-30 nmol/L, and the test is considered positive for NCCAH diagnosis when the 60-minutes post-CST 17OHP level is > 30 nmol/L  has widely replaced RIA in the measurement of serum 17OHP . ELISA iThe 17OHP cutoff of 6 nmol/L for NCCAH screening has been questioned by several studies utilizing RIA assays that suggested lower cutoffs . SeveralSince most of the patients diagnosed with NCCAH are females , 12, we 2This non-interventional, retrospective, cohort study was conducted using the electronic medical database of Maccabi Healthcare Services (MHS), a large health maintenance organization (HMO) in Israel serving over 2 million patients. All medical data were obtained from the MHS automated database. The retrieval of patients’ records was performed using MDClone, a query tool that provides comprehensive patient-level data for a wide range of variables in a defined time frame around an index event , 14. Thi2.1We retrieved data on consecutive women over 16 years of age with suspected NCCAH, referred for 250 µg CST from January 2001 – December 2020. As it is a real-life study, all CSTs for suspected NCCAH were included regardless of pretest 17OHP levels. The time of the CST was set as the index date. Subjects meeting inclusion criteria had clinical data available for at least 12 months before the index date.Subjects using estrogen-containing oral contraceptives (OC), systemic or topical hormone replacement therapy (HRT), or systemic glucocorticoids (GC) were excluded (n=141). Subjects were considered as users of OC (n=109), HRT (n=3), or GC (n=29) if their prescription was filled within three months before the index date.Clinical presentations that led to 17OHP measurements were hirsutism, oligomenorrhea, amenorrhea, acne, alopecia, or infertility that were identified within 12 months before the index date, based on the International Classification of Diseases-9 (ICD-9) or MHS internal diagnostic codes—called “Y” codes.Demographic variables included age and last body mass index (BMI) at the index date. Laboratory values were defined as the last available value during the six months before or at the index date. Laboratory values included pre- and post-CST  serum 17OHP and cortisol, serum TSH, prolactin, total testosterone, dehydroepiandrosterone sulfate (DHEAS), androstenedione, LH, and FSH; LH to FSH ratio was also calculated.NCCAH was defined as a 60-minute post-CST 17OHP serum level >30 nmol/L.2.2Serum cortisol, TSH, prolactin, total testosterone, LH, and FSH levels were measured by chemiluminescent immunoassays (CLIAs) . Serum DHEAS levels were measured by CLIA  from January 2000 through May 2014, and by CLIA  from June 2014 to December 2020. Serum androstenedione levels were measured using RIA (DSL) from January 2000 through December 2011, and by CLIA  from January 2012 through December 2020.From January 2000 through March 2015, serum 17OHP levels were assayed by direct RIA using Wizard gamma counter, Perkin-Elmer , and from April 2015 to December 2020 by ELISA . The intra-assay coefficient of variation was 12.3-8.3% (range 0.345-13.21 nmol/L) for RIA, and 2.8-4.9% (range 7.39-34.57 nmol/L) for ELISA. The inter-assay coefficient of variation was 12-12.8% (range 2.94-22.81 nmol/L) for RIA, and 5.8-9.2% (range 0.78-17.39 nmol/L) for ELISA. The limit of detection was 0.09 nmol/L in both methods. MHS central laboratory performed validation for 17OHP assays, comparing 48 samples from their daily routine. Passing-Bablok regression revealed a good correlation between ELISA and RIA assays . The absolute 17OHP values measured by ELISA were slightly (34% on average) higher than by RIA (ELISA = 1.3462 RIA - 0.04); however, they were still within the allowable limits in accordance with the manufacturer’s package insert reference range.2.3Categorical variables were described as numbers and frequencies. Continuous variables were described as means and standard deviations (SD), or medians and ranges. The Chi-square test or Fisher’s exact test was used to compare categorical variables, and the student’s t-test was used to compare continuous variables, respectively. We allocated the individuals into two groups according to the 17OHP assay method used (RIA vs ELISA). For each group, a receiver-operating characteristic (ROC) curve was generated and an optimal basal 17OHP threshold with the highest sensitivity and specificity was determined. Positive (PPV) and negative (NPV) predictive values were calculated. We compared the sensitivity and specificity between the RIA and the ELISA groups when using the optimal RIA-based basal 17OHP cutoff. For each 17OHP assay group, univariate and multivariate logistic regression analyses were performed to identify variables that predict NCCAH, defined as a post-CST 17OHP serum level of >30 nmol/L. Variables with a p-value <0.1 on the univariate analysis were included in the multivariate analysis, which aimed to determine any independent predictors of NCCAH.A two-sided P-value < 0.05 was considered statistically significant. All analyses were conducted with the statistical software SPSS, version 25 .3CST was performed in 2409 women who satisfied the inclusion criteria (1564 in the RIA group and 845 in the ELISA group). The mean (± SD) age was 24.1 ± 7 years. Symptoms that prompted cosyntropin testing were hirsutism in 45% of subjects, oligomenorrhea in 40%, amenorrhea in 25%, acne in 39.4%, infertility in 13.5%, and alopecia in 12.5%. The two groups were comparable in terms of the indication for testing, except for hirsutism which was more common in the RIA group (p<0.001) Table 1.The mean basal and stimulated 17OHP levels were lower in the RIA group as compared to the ELISA group . Basal serum cortisol, total testosterone, and LH : FSH ratio were higher in the RIA group. Serum androstenedione levels were higher in the ELISA group Table 1.NCCAH was diagnosed in 74 (4.7%) subjects in the RIA group and 63 (7.5%) in the ELISA group (p=0.008) Table 1.Using ROC analysis, the optimal basal 17OHP cutoff values predicting NCCAH were 6.1 nmol/L in the RIA group  Figure 1When applying a post-CST 17OHP diagnostic cutoff of > 40 nmol/L, NCCAH was diagnosed in 69 (4.4%) subjects in the RIA group and 60 (7.1%) in the ELISA group (p=0.007). Using ROC analysis, the optimal basal 17OHP cutoff values predicting NCCAH were 6.1 nmol/L in the RIA group  Figure 3In the multivariate analysis, higher basal 17OHP level (p<0.001), lower LH: FSH ratio (p=0.005), and oligomenorrhea (p=0.021) emerged as independent predictors of NCCAH in the RIA group. Higher basal 17OHP (p<0.001), androstenedione (p=0.004), and total testosterone (p=0.012) levels were identified as independent predictors of NCCAH in the ELISA group. Of note, a lower LH : FSH ratio showed a similar trend in ELISA but did not reach significance (p=0.054). The results of the multivariate analyses are shown in Using ROC analysis, the optimal LH : FSH ratio cutoff values for predicting NCCAH were 1.4 in the RIA group  and 1.2 in the ELISA group .In the RIA group, the combination of a serum basal 17OHP value below 6.1 and an LH : FSH ratio above 1.4 increased the NPV to 100%. In the ELISA group, the combination of a serum basal 17OHP value below 8.2 and LH : FSH ratio above 1.2 increased the NPV to 99.7%. Combining basal serum 17OHP values below the optimal cutoffs with an LH : FSH ratio > 2, yielded an increase in the NPV to 100% in both groups.4This study showed that the optimal RIA-based basal 17OHP cutoff at which to refer a woman suspected of NCCAH to CST was 6.1 nmol/L, comparable with that recommended in the guidelines (6 nmol/L)  who examWhile LC-MS/MS-based 17OHP measurement has been recommended by the Endocrine Society for the screening of NCCAH due to 21-hydroxylase deficiency .Immunoassays are still the assays most frequently used in clinical settings and present good performance in NCCAH diagnosis. The classical basal 17OHP cutoff value of 6 nmol/L for performing CST was established based on immunoassays, mainly RIA . MaffazoNonetheless, in light of the widespread use of immunoassays and the replacement of RIA by the ELISA 17OHP assay, it is pertinent to check whether the guideline-recommended basal 17OHP cutoff applies. This is the first sizable study gleaning data from a large cohort of women with hyperandrogenism and comparing data between the old RIA and the new ELISA method.In the present study, 137/2409 (5.7%) women were diagnosed with NCCAH, defined as a stimulated 17OHP level >30 nmol/L. This figure is similar to previous studies of hyperandrogenic females , 17. ThiNoteworthy, NCCAH was diagnosed more often in the ELISA (7.5%) than the RIA (4.7%) group. This difference could be explained by the upward shift in 17OHP levels in the ELISA group 2 which The validation study, performed by MHS central laboratory, established that when using ELISA, the absolute 17OHP values are higher (34% on average) than that achieved if the same samples were analyzed using RIA. This raises a question of what effect would employing a 34% higher diagnostic 17OHP cutoff (~ 40 nmol/L) have on our results. Interestingly, applying a higher post-CST 17OHP cutoff of > 40 nmol/L did not affect the observed discordance in the diagnosis rates of NCCAH nor the optimal basal 17OHP thresholds in the RIA and the ELISA groups.NCCAH diagnosis might be truly more prevalent in the ELISA group. This might be explained by the introduction of guidelines in recent years that led to more precise referrals and testing. The higher androstenedione levels in the ELISA group may remark a higher degree of clinical suspicion that led to more positive CSTs. However, further studies that incorporate genetic data are needed.We observed that androstenedione levels were higher in the ELISA than the RIA group, while testosterone levels were higher in the RIA. We do not have a clear explanation for these differences; however, as the androstenedione assay was changed in 2012, assays were not identical in the two groups. Consequently, the difference in assays could explain these discrepancies, and the fact that we still have these differences between RIA and ELISA groups in both women with and without NCCAH 3, suppoRemarkably, in the RIA group, lower LH: FSH ratio and oligomenorrhea emerged as independent predictors of NCCAH. In the ELISA group, higher androstenedione and total testosterone levels were independent predictors, while lower LH: FSH ratio showed a marginal trend of significance.Polycystic ovary syndrome (PCOS) is an important differential diagnosis of NCCAH in women and is much more frequent . NCCAH aIn our study, a low LH: FSH ratio emerged as an independent predictor of NCCAH in the RIA group and showed a similar trend in the ELISA group. We found that combining basal serum 17OHP below the suggested cutoff with an LH: FSH ratio > 2 increases the NPV for the diagnosis of NCCAH to 100% in both RIA and ELISA groups. To our knowledge, this is the first study to show that the LH : FSH ratio improves the NPV for the diagnosis of NCCAH.Our study’s main strength is the large number of subjects in each group, which enabled several analyses and allowed drawing important implications for clinical practice. We studied females older than 16 years to avoid 17OHP level differences derived from different pubertal development .This study has some limitations which stem from its retrospective design. An unknown confounding may have influenced our results. The results of this study may not be generalizable to users of other assays. We cannot rule out the inclusion of peri- or post-menopausal women in the study population that may have affected the results, although the median age was 22 years. The lack of genetic profiling is another key limitation, as it may have helped better define the NCCAH populations and draw further distinctions between the 17OHP-assay groups. Further studies that incorporate genetic data are mandatory.In conclusion, our results suggest adopting a higher ELISA-based 17OHP cutoff (8.2 nmol/L) at which patients suspected of NCCAH should be referred for further evaluation. This may help eliminate the number of unnecessary tests. When using RIA, the classical 17OHP cutoff level of 6 nmol/L seems to apply. Utilization of LH : FSH ratio can improve the negative predictive value of the basal 17OHP levels.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Maccabi Healthcare Services (MHS) institutional review board (IRB) and ethics committee. Written informed consent from the participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.AN, LS, and MZ were responsible for the design of the study and data acquisition. AN, LS, and MZ contributed to the analysis and the interpretation of data, drafted the manuscript and revised it critically for important intellectual content. NS, NW, and MS-A contributed to the analysis and the interpretation of data and revised the manuscript critically for important intellectual content. LS-R, SA, and RG contributed to the interpretation of data. All authors read and approved the final manuscript.


The distribution of SNPs markers across the barley genome ranged from 449 markers on chromosome 1H to 1111 markers on chromosome 5H. The average polymorphism information content (PIC) per locus was 0.275 and ranged from 0.094 to 0.375. Bayesian clustering in STRUCTURE and principal coordinate analysis revealed that the populations are differentiated primarily due to the different breeding program origins and ear-row type into five subpopulations. Analysis of molecular variance based on PhiPT values suggested that high values of genetic diversity were observed within populations and accounted for 90% of the total variance. Subpopulation 5 exhibited the most diversity with the highest values of the diversity indices, which represent the breeding program gene pool of AFC, AAFRD, AU, and BARI. With increasing genetic distance, the LD values, expressed as r2, declined to below the critical r2 = 0.18 after 3.91 cM, and the same pattern was observed on each chromosome. Our results identified an important pattern of genetic diversity among the Canadian barley panel that was proposed to be representative of target breeding programs and may have important implications for association mapping in the future. This highlight, that efforts to identify novel variability underlying this diversity may present practical breeding opportunities to develop new barley genotypes.Studying the population structure and genetic diversity of historical datasets is a proposed use for association analysis. This is particularly important when the dataset contains traits that are time-consuming or costly to measure. A set of 96 elite barley genotypes, developed from eight breeding programs of the Western Canadian Cooperative Trials were used in the current study. Genetic diversity, allelic variation, and linkage disequilibrium (LD) were investigated using 5063 high-quality SNP markers  Hordeum vulgare L. ssp.) and is distributed through the Middle East, mostly falling into different classes according to phenology, morphology, and end-use: two-row vs. six-row, winter types vs. spring types, hulled vs. hull-less, feed barley vs. malting barley , Diversity arrays Technology (DArT), and single-nucleotide polymorphisms (SNPs) based on next-generation sequencing (NGS) have been utilized intensively in barley genetic diversity studies . In barlTo build on Canada’s position as a supplier of premium quality barley yield production, the Western Canadian cooperative trials breeding program aims to develop barley varieties with improved traits. For that purpose, a cooperative trial has been formalized in Western Canada in which 25 to 35 lines are evaluated annually in 15 to 20 locations. The result is an extensive collection of historical data demonstrating a wide variety of elite barley genotypes that can be used to discover desirable alleles through association mapping. With such unbalanced historical datasets, association mapping has been constructed for yield in barley . The preA set of 96 elite barley genotypes from Western Canadian breeding programs were used in this study. These panel from eight barley breeding programs and were grown at different times between 1994 and 2016 Table 1.https://bioinf.hutton.ac.uk/iselect/app/index.pl). The markers with unknowing chromosomal positions, monomorphic or had a minor allele frequency of<0.05, and SNPs with missing values of more than 20% of the genotypes were removed. Only, the markers with genetic and physical position information on the 9K SNP array were used. A total of 5063 high-quality SNP markers remained in the dataset and was used for estimating the diversity analysis and population structure.Genomic DNA was collected from the young leaf tissue from each of the 96 genotypes using the DNeasy Plant Mini Kit , then quantified at 230/260 and 260/280 absorption ratios. The barley panel was genotyped using an Illumina 9K barley Infinium iSelect SNP assay at the USDA-ARS Genotyping Laboratory, Fargo, ND, USA. The 9K barley SNP assay can interrogate approximately 9,000 SNP markers, containing the 1,596 SNPs on BOPA1 loci (Genetic diversity statistics for 5063 SNP markers including genetic diversity (GD), major allele frequency (MAF), heterozygosity, and availability were calculated  using Popi and pj are the frequency of the ith and jth alleles of a given locus, respectively.Gene diversity was calculated as, 1−∑pi2 , where pk) given an admixture model with associated allele frequencies. STRUCTURE simulations were performed with the number of presumed clusters from k=1 to k=10. Five independent runs were used for each subpopulation . For each run, simulations were run for 100,000 burn-in and 100,000 Markov Chain Monte Carlo (MCMC) iterations  and the percentage of variations using GenAlEx v6.5 , where AP; diversity among populations, WP; diversity within populations. In addition, the number of loci with private alleles, and the percentage of polymorphic loci were also computed using GenAlEx v6.5 (AMOVA was performed to separate molecular variance at two levels: (a) among subpopulations (genotypes) which were inferred by the analysis of population structure, and (b) within subpopulations. To further verify the genetic differentiation, the number of different (Na), and effective alleles (Ne), expected heterozygosity (He), Fixation index (F), Shannon’s Information Index (I), diversity among and within subpopulations, the unbiased diversity index (uh) and pairwise PhiPT values were calculated for each population according to . The PhilEx v6.5 .2) for each chromosome. To associate the decay of LD among the genotypes, the genetic distance between each pair of SNP markers was plotted against R2 values. The squared allele frequency correlations (R2) for each pair of markers were measured to estimate significant linkage disequilibrium at a p-value<0.001. The density of the filtered SNP distribution on seven chromosomes was drawn using the CMplot package in R.Linkage disequilibrium (LD) was computed using the GAPIT program in R software  as a pai0), Variable fluorescence (FV), Maximum fluorescence intensity (FM), Maximum efficiency of Photosystem II (PSII), (FV/F0), and Maximum yield of primary photochemistry (FV/FM) under low-temperature stress (LTS). In brief, at a three-leaf stage the seedlings were exposed to temperatures raised between 3°C and 5°C, then gradually decreased to -12°C for 4 hours. Chlorophyll fluorescence parameters were measured as an indicator of photosynthetic energy that responds to alters in PSII photochemistry according to . The high-quality SNPs were well distributed across the barley genome and presented in  was plotted against ΔK (The Bayesian model-based method was used to investigate the genetic structure of 96 Canadian barley genotypes using highly informative selected markers (5063 SNPs). To distinguish the optimum number of subpopulations, the number of clusters (ainst ΔK Table 3.st) showed significant divergence within the subpopulations. A high level of genetic variation was observed in subpopulation 3 with the Fst value of 0.600. Subpopulation 5 displayed lower genetic variation with an Fst value of 0.008 among its genotypes (The results of the STRUCTURE estimated that the fixation index (Fenotypes Table 3.P< 0.001) for all the subpopulation-pairwise comparisons  and among (inter) populations by an AMOVA. A two-level AMOVA of 96 barley genotypes, belonging to eight different breeding programs in Canada, revealed that 95% of the total genetic variation exists within populations and 5% among populations based on PhiPT values Table 4.To infer significantly different patterns and levels of population genetic structure, patterns of allele frequency across populations were calculated. The Na and Ne ranged from 30 to 125 and 2.667 to 119.342, respectively. While, the Shannon index (I) and the unbiased diversity index (uh) values ranged from 1.040 to 4.809 and 0.833 to 1.00, respectively  and D′ (the standardized disequilibrium coefficient), we investigated whole-genome patterns of LD using the mixed-model method and population structure as co-factors to understand the predictive value of a marker for the association between its chromosomal region with the phenotype. The r2 between each SNP pair (cM) was calculated to identify the level of co-occurrence of alleles at two loci. The SNP markers with P< 0.001 of R2 on the seven barley chromosomes were selected for use in subsequent analysis. The mean values of R2 over all chromosomes revealed that there was no clear correlation with physical distance. A strong LD was observed among the 5063 marker pairs in the studied population with an approximate average value of 0.021 when the distance was 0.391 cM  . The genetic correlation between chlorophyll fluorescence-related traits is assessed by means of the non-parametric spearman correlations. The correlation coefficient is presented as pairs of scatter plots with an asterisk that have a significant correlation, and the greater the correlation, the more significant. Generally, before the low-temperature stress, an extremely significant and positive correlation (P< 0.001) was observed between F > 0.95) Figure 6V/F0, and FV/FM with all traits under LTS, suggesting relatively high genetic variability among studied genotypes (Ustilago nuda (Jens.) Rostr.] and net-form net blotch [Pyrenophora teres f. teres Drechsler; anamorph: Drechslera teres (Sacc.) were recorded  which have been developed by the US Department of Agriculture. The lines were selected based on their high percentage of winter survival, seed quality, and seed yield. Even though the panel studied does not contain full representations of breeding materials. It reflects the majority of commonly used parental genotypes and suggests that future progress can be achieved from a limited pool of variability if new sources of genotypes are not introduced. Previously,  reportedP< 0.001). A high level of gene flow explains the low level of diversity between subpopulations  was 0.21 , friability, plumpness, β-glucan, and fine extract are some of the traits that influence grain quality. The recent breeding history controlled by the characteristics of the malting productions has had a more dramatic effect on the diversity of barley, entailing a decline in the diversity of the six-row spring types .In the current study, genetic diversity within 96 genotypes of barley was characterized based on 5063 SNP markers. This panel represented elite barley germplasm used to study limit dextrinase and beta-glucanase. These lines have been in the Western Two-Row Cooperative Registration Tests and contain a number of barley check cultivars currently registered in Western Canada. The lines reflect the majority of commonly used parental genotypes, suggesting that further progress can be achieved from a limited pool of variability if the new resource of genotypes is not released subsequently. We have shown that the studied genotypes are genetically diverse, as well as interlinked and structured, indicating a heterogeneous collection. Although an ultimate target of breeders is the genetic diversity based on traits, our main objective was to demonstrate how SNP markers can generate biological variation between barley lines by causing differences in the recipes for proteins that are written in genes. The coverage of markers and population stratification besides the level of LD in our genotypes set was suitable to run different association mapping studies for key economic traits in barley in the future. The results obtained from this study will be useful for future genomic studies, and germplasm management in Western Canada and North American regions to elucidate genetic diversity and structure in their breeding material. Further, the results will assist the selection of parents with complementary allele combinations for future crosses and help identify progeny with the desired alleles is important to develop traits that are expensive and/or time-consuming to measure.https://figshare.com/articles/dataset/Barley9Kplate3Project_96_sample_Raw_data_csv/21760586/1.The datasets generated for this study can be found in FigShare, LC prepared the material for genotyping. AE developed the article concept. AB prepared the individual populations corresponding to each breeding program. AE performed bioinformatics data analysis and interpretation of the results. AE wrote the manuscript. LC, TK, JN, PJ, FC, JS, GR, and AB reviewed the manuscript. All authors contributed to the article and approved the submitted version.


These practices may lead to a reduction in genetic diversity.Genetic diversity is fundamental for the survival of species. In particular, in a climate change scenario, it is crucial that populations maintain genetic diversity so they can adapt to novel environmental conditions. Genetic diversity in wild agaves is usually high, with low genetic differentiation among populations, in part maintained by the agave pollinators such as the nectarivorous bats. In cultivated agaves, patterns of genetic diversity vary according to the intensity of use, management, and domestication stage. In AA. tequilana from three sites of Jalisco, Mexico. For one locality, seeds were collected and germinated in a greenhouse. We compared the genomic diversity, levels of inbreeding, genetic differentiation, and connectivity among studied sites and between adults and juvenile plants.We studied the diversity patterns with genome-wide SNPs, using restriction site associated DNA sequencing in cultivated samples of Agave tequilana presented a genomic diversity of TH = 0.12. The observed heterozygosity was higher than the expected heterozygosity. Adults were more heterozygous than juveniles. This could be a consequence of heterosis or hybrid vigor. We found a shallow genetic structure (average paired STF = 0.0044). In the analysis of recent gene flow, we estimated an average migration rate among the different populations of m = 0.25. In particular, we found a population that was the primary source of gene flow and had greater genomic diversity (EH and OH), so we propose that this population should continue to be monitored as a potential genetic reservoir.A. tequilana. For populations to resist pests, to continue evolving and reduce their risk of extinction under a climate change scenario, it is necessary to maintain genetic diversity. Under this premise we encourage to continue acting in conservation programs for this species and its pollinators.Our results may be the consequence of more traditional management in the studied specific region of Jalisco. Also, the exchange of seeds or propagules by producers and the existence of gene flow due to occasional sexual reproduction may play an important role in maintaining diversity in  Conservation genetics combines evolutionary theory and molecular markers to help biodiversity conservation . An impoIn crop plants, the levels of genetic diversity contained in the managed and in the wild (if still extant) gene pools are relevant to further crop improvement and as a source of resistance to diseases and adaptation to the changing climate . HoweverAgave tequilana Weber var. azul (A. tequilana hereafter). A tequilana is a diploid species (2n = 2x = 60), with a genome size of 3,677.45 Mbp , taro (Colocasia esculenta L.), potato (Solanum tuberosum), grapevines (Vitis vinifera), strawberry (Fragaria spp.) and so on  and, at the same time, mitigating genomic erosion and promoting sustainable practices of the agroecosystems where the main crop is agave used for mezcal, tequila, and other agave distillates production (https://batfriendly.org/).Genetic diversity in cultivated agaves varies according to the intensity of use, management, and domestication . In wildbabuenae . Recent A. tequilana, A. angustifolia  has revolutionized studies on the genetic diversity in plant and animal species . This tystifolia . We beliA. tequilana collected in Jalisco, using SNPs derived from Restriction-site associated DNA sequencing, or RADseq methodology .Plant material was collected from individuals 10 m apart from each other  from three “Bat-friendly” plantations, separated by ~90 to 250 km, of , Mexico . In thesWe analyzed 96 samples collected in two consecutive years (2016 and 2017) from three “Bat-friendly” localities . From thhttps://biotech.wisc.edu/). Each sample was digested using two methylation-sensitive restriction enzymes (PstI and MspI); the choice of enzymes was based on the previous standardization for Agave salmiana and A. lechuguilla . After specific barcodes were ligated to each sample, those were pooled in equimolar concentration and sequenced using the Illumina NovaSeq 2 × 150 platform .For all the samples, genomic DNA was extracted from leaf tissue using a modified “Mini-Prep” CTAB protocol . DNA wasMassive parallel sequencing platforms generate tens of millions of sequences. However, it is essential to verify the quality of these sequences so as not to cause bias in the data analysis. For quality filtering, we first used TRIMMOMATIC ; we remoAgave tequilana .With the paired files generated with TRIMMOMATIC, we used the reference transcriptome—because there is no published agave genome—of thin (100 sites), so that no two sites were within the specified distance from one another, and we also removed SNPs, that significantly deviated from Hardy-Weinberg equilibrium test (—hwe 0.000005). We only retained sites with a mean minimum depth of over 12, and maximum two alleles with no InDels, and also removed sites and individuals with more than 80% missing data and a minor allele frequency (MAF) of <0.01.The final data filtering was performed with VCFtools v.0.1.15 ; we avoimll), and the number of multilocus genotypes (mlg), which are the unique combination of alleles across all loci, estimated using package poppr (Ho), the expected heterozygosity (HE), and the total heterozygosis (HT), for each SNP locus using adegenet V. 2.1.3 (hierfstat (We estimated the multilocus lineages (ge poppr  with thege poppr . We compV. 2.1.3  and hierierfstat . We testierfstat .MLH)—defined as the total number of heterozygous loci in an individual divided by the number of loci typed in the focal individual—and the standardized multilocus heterozygosity (sMLH) for each individual—defined as the number of total heterozygous loci in an individual, divided by the sum of the average observed heterozygosity in the population over the subset of loci successfully typed , a measure of heterozygosity on a per-individual basis and computes observed and expected autosomal homozygous genotype counts for each sample. We used (—ibc) from Plink v1.9 . Nei’s paired genetic distances were visualized using a heatmap. Finally, an analysis of individual ancestry by maximum likelihood was performed using ADMIXTURE v.1.23   from the package , and we  package  using R E v.1.23 , where wi.e., over the last two generations) was inferred using BayesAss V. 3.0.4  was 35.36, and the guanine-cytosine (GC) contents ranged from 49–50%. After demultiplex and removing adapters, the number of reads was 264, 006, 277. Due to the low number of reads in eight samples , they were excluded from further analysis. Therefore, a total of 88 samples were analyzed. Initially, using a reference transcriptome assembly method with Ipyrad, 84, 635 variants were called. After quality control, with depth, allelic number, MAF, and missing data, we retained 979 biallelic SNPs .The RADseq strategy on 96 mll) and genotype (mlg) analyses, we found that the 88 analyzed plants had different genotypes, i.e., each plant presented an unique combination of alleles across all the studied loci. The locality with the highest number of alleles  and the average of expected heterozygosity HE = 0.120 (SD = 0.149) , indicating an excess of heterozygous individuals.Using multilocus lineage  . ObserveHE = 0.129, SD = 0.154), followed by Alteña  and J-Tototlán , while the population with less genetic diversity was Arenal  , Arenal vs Tototlán  and Arenal vs J-Tototlán .Tototlán showed the highest average expected heterozygosity (= 0.150) . We foun= 0.150)  between sMLH = 0.994 (SD = 0.231) . The multi locus heterozygosity (MLH) was , being the highest in Tototlán, followed by Arenal, J-Tototlán, and Alteña .The average standardized multi locus heterozygosity was = 0.231) , with sid Alteña ; Fig. 2AMLH) in all the adults vs juveniles  than in all the adults  .When we compared the levels of genomic multi locus heterozygosity  ; Fig. 2BFIS) in all the analyzed plants of A. tequilana, was slightly negative . On the other hand, the average f index was negative in the adults from the localities of Alteña, and Arenal, indicating an excess of heterozygotes  in relation to what would be expected under random mating. In contrast, the juveniles from Tototlán and the adults from the same locality had a positive and moderate level of f  (The average inbreeding coefficient (ctively) , Fig. 3AFhat3 inbreeding index, we found that A. tequilana individuals have low levels of inbreeding (Fhat3 = 0.012 (SD = 0.182). Interestingly, while in general adults presented negative values, the juveniles from Tototlán (J-Tototlán) had a positive value (average Fhat3 = 0.229). A Wilcoxon test showed that the difference in the inbreeding coefficient (Fhat3) was significant between young and adults   , Fig. S1The UPGMA analysis, based on Edward’s distance , showed FST = 0.0044. The lowest value was found between Arenal and Alteña , followed by Alteña vs J-Tototlán , Tototlán vs J-Tototlán , Tototlán vs Alteña , Arenal vs J-Tototlán , and the maximum value was found between Tototlán and Arenal . Similar results were obtained using Nei’s distance, which ranged from 0.0010 to 0.0089 (average Nei’s genetic distance = 0.0060)  .K (from 1 to 10), the cross-validation error estimates showed that the best model fit was K = 1 (CV = 0.26261)  . Neverth samples . We founm) from the source population of Tototlán (m = 0.309 (SD = 0.013); thus a fraction of individuals in Alteña were migrants derived from Tototlán; followed by Tototlán to Arenal (green), m = 0.289 (SD = 0.023) and Tototlán to J-Tototlán (pink) m = 0.238 (SD = 0.029). In comparison, the lowest migration rate was from Alteña to Arenal m = 0.007 (SD = 0.007).The analysis of recent migration rates using BayesAss software suggested a high migration rate  . These nA. tequilana is higher (>49%) than in other monocots (33–48%) (We found that the GC content in (33–48%) . In theo(33–48%) . It has (33–48%) .Agaves appear to have lower genetic variation in comparison to their wild relatives .Cultivated elatives , 2022, mA. angustifolia in wild and cultivated plants used to produce an alcoholic drink similar to tequila (bacanora), using SNPs derived from restriction site associated DNA sequencing, where HE = 0.25. Similar results were also obtained from other Agavoideae, genotyped with next-RAD strategies, as HE of 0.173 and 0.249 were reported for Yucca valida and Yucca capensis, respectively . In contrast, based on microsatellites (with eight loci), i.e., HE = 0). Similar results were obtained with RAPDs markers, where only 1 of 124 RAPD products (0.8%) was polymorphic, and 39 of 40 plants were completely isogenic (A. tequilana “Sigüin” HE was 0.409 and in A. tequilana “Chato” HE was 0.435 (Previous genetic studies on isogenic . In otheas 0.435 . HoweverAgave tequilana is a species that has been intensively managed since the beginning of the last century (Cucurbita pepo ssp. pepo) HE = 0.185 in populations distributed along Mexico using tunable genotyping by sequencing (tGBS), or for the cultivated runner-red bean (Phaseolus coccineus), HE = 0.167 to 0.221 using genotyping by sequencing (GBS), values similar to what we found in A. tequilana. century . TherefoAgave and Yucca populations, is still maintained in the “Bat-friendly” localities in Jalisco.Genetic diversity is necessary for further evolutionary response to natural selection pressures and to allow for crop improvement , it enhaFIS) and related estimates, measuring the deviation from Hardy-Weinberg equilibrium  a monocot dioecious species, typically clonally propagated (Solanum tuberosum L.), where its high heterozygosity has been explained by the asexual propagation and polyploidy, which provides the potential to display great plasticity that favors adaptation to different environments and challenges (Manihot esculenta ssp.) with a wide tropical distribution, a vegetatively propagated crop (V. vinifera ssp. sativa), cultivars are clonally propagated and highly heterozygous but carry many deleterious recessive mutations  had a positive value, apparently due to some inbreeding in this population. Inbreeding in the juveniles may result of few reproductive events in A. tequilana in this locality, so there may be self-pollination or crosses among relatives.In FIS and heterozygosity excess in the adults may have several potential explanations. It may be due to natural and artificial selection by the farmers, that remove small and weak plants  and select for the most vigorous . A well know case of heterozygote advantage (heterosis) is exhibited in corn, which results from the use of hybrid seeds for agriculture  (Astrocaryum mexicanum (mean for adults FIS = −0.41 and for seeds FIS = −0.19)  , and in = −0.19) . NeverthFST = 0.0044), and alleles shared among all populations. This low differentiation could be due to intensive management of the species, where propagation mainly occurs by propagules and/or clonal. It can also be accounted to the fact that populations have not been separated for so long, and ancestral polymorphisms are still maintained. The juvenile individuals from Tototlán were slightly more divergent than the rest of the populations, however, they did not show significant differences. In our study we found the lowest reported FST value in Agave. For instance, in A. angustifoliaFST = 0.005, while in other Agave species FST from 0.057 (in Agave cocui with isozymes) to 0.76 (in Agave parry cultivated with microsatellites).Genetic structure results from an interaction among ecological factors, historical events, and evolution processes . In natuLeptonycteris yerbabuenae, L. nivalis, and Choeronycteris mexicana  are relatively close to each other geographically (~90 km), while the most distant were Arandas and Arenal (~250 km). Gene flow may affect population structure, as Agaves have long-distance pollen dispersal usually conducted by nectar feeding bats, including mexicana .HE and Ho); thus, we consider important to continue monitoring it for a possible source of variation.Gene flow, therefore, may play an important role in the evolution process of populations because it can increase genetic diversity as new alleles are introduced into the new population . ApparenAgave tequilana is one of the most important economic crop in Mexico (batfriendly.org). Nevertheless, in terms of the program, we consider that it is still too early in the game to show its potential benefits to maintain genetic diversity. However, it is important to highlight that in the present study viable seeds were generated by the naturally pollinated inflorescences, so there can be natural population recruitment. We believe that in future generations, once allowing bat pollination in the agave plantations become mainstream, bats and agaves will be able to continue their millennial association.n Mexico . VariousWe also want to emphasize the importance of bat pollination in agaves in general, since the movement of these mammals is closely related to the reproductive success of the plants . FurtherAgave tequilana besides the var. azul (e.g., the varieties “azul lisado”, “chato”, “bermejo”, “pata de mula”, “sigüin”, “sahuayo”, “moraleño”, “mano larga”, “criollo” and “zopilote”; In a climate change scenario, it is crucial that populations maintain genetic diversity to be able to adapt to the new climatic conditions. It has also been suggested that the inclusion of different varieties of agave in the same field could serve as a germplasm resource and reduce the risk of pests and the loss of diversity . NeverthA. tequilana in Jalisco, Mexico. We found an excess of heterozygotes in the adults, and lower genomic diversity than in the closely related A. angustifolia, but the variation, even if low, was higher than some reports for the species made in more intensive management sites, for example, from around the town of Tequila, Jalisco. We found low genetic differentiation, as reported in most other studies conducted within this genus (The main objective of this study was to evaluate the levels of genomic variation in three traditionally managed areas of is genus , 2021, bA. tequilana found in the adults in our study maybe be explained by more traditional management and clonal propagation. Also, occasional sexual reproduction, and exchange of seeds or propagules by producers may play an important role in maintaining diversity in A. tequilana.The relatively high levels of observed heterozygosity of Our study also demonstrated that massive sequencing related genomic strategies using SNPs, along with the studies of 10.7717/peerj.14398/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.14398/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.14398/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.14398/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.14398/supp-5Supplemental Information 5Click here for additional data file.


According to the written historical sources, the Gepids were a Germanic tribe that settled in the Carpathian Basin during the Migration Period. They were allies of the Huns, and an independent Gepid Kingdom arose after the collapse of the Hun Empire. In this period, the Carpathian Basin was characterized by so-called row-grave cemeteries. Due to the scarcity of historical and archaeological data, we have a poor knowledge of the origin and composition of these barbarian populations, and this is still a subject of debate. To better understand the genetic legacy of migration period societies, we obtained 46 full mitogenome sequences from three Gepid cemeteries located in Transylvania, Romania. The studied samples represent the Classical Gepidic period and illustrate the genetic make-up of this group from the late 5th and early 6th centuries AD, which is characterized by cultural markers associated with the Gepid culture in Transylvania. The genetic structure of the Gepid people is explored for the first time, providing new insights into the genetic makeup of this archaic group. The retrieved genetic data showed mainly the presence of Northwestern European mitochondrial ancient lineages in the Gepid group and all population genetic analyses reiterated the same genetic structure, showing that early ancient mitogenomes from Europe were the major contributors to the Gepid maternal genetic pool. The history of the so-called barbarian populations of the Migration Period is a subject of increasing interest, however, the reconstruction of the population history of these groups has been limited by the low number of reliable written and archaeological sources. From the migration period groups, Gepids had a great impact on the history of Central Europe, yet the investigation of this population so far has been marginal as their legacy is not claimed by any modern nations in Europe .Gepids were one of the East Germanic groups which settled in the Carpathian Basin. Written sources first mention them in the second half of the 3rd century CE, and, based on archaeological data, they originated from the Wielbark Culture, from territory that is today Poland ,2,3; howIn the Transylvanian Basin, the same archaeological tendencies were registered, and two main types of row-grave cemeteries were distinguished . The firThe European migration period is still underrepresented from the genetic point of view, restricted to a few data from populations associated with Avars ,9,10,11,In this study we reveal the first 46 mitogenomes from three Gepid cemeteries located in Transylvania, Romania . All cemThe late 5th century in the Transylvanian Basin was characterized by the appearance of row-grave cemeteries, or Reihengräberfelder, which were strongly connected to the Gepidic expansion . In TranMigration Period cemeteries were often systematically robbed in attempt to recover their rich inventory. Gepid burials were frequently victims of near immediate robbing and disturbing of the deceased, which narrowed our sampling procedure considerably. Another problem regarding the Gepid cemeteries is that there are few cemeteries fully excavated and very limited information is published. In an attempt to carry out a representative sampling from the Classical Gepidic period, we chose 3 cemeteries which were excavated in the 21st century, using modern techniques. One of the cemeteries (Vlaha) is fully excavated and the other two are partially investigated. We collected teeth or petrous bones for DNA extraction from 50 individuals dated to the Classical Gepidic period from three row-grave cemeteries from Transylvania. We selected 7 individuals from the Carei–Bobald cemetery neighboring Northwestern Transylvania, 25 samples from the Șardu cemetery and 18 samples from Vlaha/Magyarfenes-Pad cemetery of which 14 mitogenomes were determined, both located in the North-Western Transylvanian Basin . The selAll methods preceding NGS were performed in the sterile laboratories dedicated for aDNA work at the Department of Archaeogenetics of the Institute of Hungarian Research and Department of Genetics, University of Szeged, Hungary.Sampling was carried out using gloves and facemasks to minimize the risk of contamination with modern human DNA. From each individual, multi-root teeth or petrous bone was collected for the molecular analyses . Teeth wLibrary preparation, mitochondrial DNA capture, sequencing, and sequence analysis was done as described in . LibrariAll libraries were purified using MinElute columns and eluted in 20 µL EB buffer (Qiagen). DNA concentration was measured with Qubit 3.0 Fluorometer using the dsDNA High Sensitivity Assay kit and fragment size was determined using the TapeStation automated electrophoresis system (Agilent).The endogenous human DNA content of each library was estimated by low coverage shotgun sequencing . LibrariThe Cutadapt software [DOI:10.14806/ej.17.1.200] was used to trim the adapters from paired-end reads  and the http://www.ebi.ac.uk/ena, accessed on 21 February 2022) under the accession number: PRJEB50517.The raw nucleotide sequence data of the 46 Gepid mitogenomes were deposited to the European Nucleotide Archive  networks , the selWe used three independent methods for measuring the genetic similarity of Gepids to other ancient populations. In the first approach, we reduced the Hgs number to major Hgs, which decreased our population dataset to 45 dimensions, which is enough for showing the main correlations. The major Hgs frequencies were calculated and the principal component analysis (PCA) was performed using the “prcomp” function in R 3.6.3 .In the second approach, we applied the traditional sequence-based method calculating pair-wise population differentiation values (Fst) with Arlequin 3.5.2.2  from theOur third approach was measuring the so called shared haplogroup distance (SHD) values, as the presence of identical terminal sub-Hgs between populations testifies shared ancestry or past admixture events . Pair-wiHere we report 46 new mitogenome sequences  which weGepid sequences cover almost the entire range of Western Eurasian Major-Hgs with the following frequencies: H 38%, HV 5%, I 8%, J 4%, N 3%, T 20% (T1 5% and T2 15%), U 11% , and V 5% .Phylogenetic relations were illustrated for each Hg using MJ Networks . The phyA few lineages indicated Near Eastern affinity, such as individual Șardu18 (H13a2b), Carei35per1, (N1a1a1a*3) Vlaha0574, and Vlaha1216 (T1) and Vlaha0637 (T1a), which had the closest parallels from Bronze Age Anatolia and Late Chalcolithic Turkey. This Near Eastern affinity is most likely explained as the legacy of early European farmers ,46. The only lineage indicating east Eurasian affinity is the H6a1b Hg of the Carei34 individual  with the same haplotype, belonging to the T2b5 subclade , from neighboring graves, belonging to the T2b4 subclade (We merged all 5–6th century Gepid lineages into a single population and measured their genetic distances from all ancient populations available in the public databases. We compared our data to published mitochondrial data  from 92 To further confirm the genetic relationships of Gepids, an MDS plot was drawn from the linearized Slatkin Fst Values of the same populations, but for better resolution of the European side, 14 Asian populations were left out . The MDSThe SHD approach gave similar results, as the Gepids shared most identical Hg-s with ancient samples from Neolithic–Iron Age Europe . Fst and SHD analysis gave comparable results , as bothUsing NGS technology combined with target enrichment, we described the maternal lineages associated with the Gepid culture in Transylvania. The genetic structure of a Gepid group is explored for the first time, providing new insights into the genetic structures of this archaic people. The retrieved genetic data showed mainly the presence of Northwestern European mitochondrial ancient lineages in the Gepid group, in line with the historically known ancient European geographic origin of these Germanic people. Regarding this, it is interesting to mention the affinities of the analyzed group with the population of the Wielbark culture described in , whose dThe most frequent macrohaplogroup was H, followed by T, U, I, and HV. We detected just a single Asian lineage, indicating that the studied population did not significantly admix with immigrants from Asia. The Central-Northern European ancestry is dominant in the Gepid group and it is closest to Bronze Age populations from nearby regions. Due to the lack of genetic data from other periods in Transylvania we could not assess the relation with other local ancient individuals, but their affinity to the local population of the Carpathian Basin seems significant.Each population genetic analysis reiterated the same genetic structure, showing that early ancient mitogenomes from Europe were the major contributors to the Gepid maternal genetic pool. Population genetic results also imply that the Gepid maternal ancestry may have derived mostly from local residents. This seems to contradict historical data, which describe the Gepids as Germanic immigrants from Northern Europe, however, these data do not exclude the possibility that many of the individuals could be immigrants from Northern Europe, as a comparable European maternal gene pool was present in both regions. The resolution provided by the mitochondrial genomes presented in this study cannot provide a full perspective of the demographic events. In order to depict with better resolution the migration and admixture events, we need to study these groups at the whole genome level, preferably with other contemporary samples from Transylvania and the surrounding regions.


Testudo hermanni in the Italian Peninsula by genotyping 174 individuals at 7 microsatellite loci, with the aim to contribute to planning effective conservation strategies.Hotspots of intraspecific genetic diversity represent invaluable resources for species to cope with environmental changes, and their identification is increasingly recognized as a major goal of conservation ecology research. However, even for iconic and endangered species, conservation strategies are often planned without thorough information on the geographic patterns of genetic variation. Here, we investigated the spatial patterns of genetic variation of the endangered Hermann’s tortoise Ordination-based and Bayesian clustering analyses consistently identified three main genetic clusters, one spread in the central and northern part of the peninsula, and two restricted to southern Italy and Sicily, respectively. The highest levels of genetic diversity were found in populations of the southern cluster and, in particular, at the northern edges of its distribution , that correspond to areas of putative secondary contact and admixture between distinct lineages. Our results clearly identify a hotspot of genetic diversity for the Hermann’s tortoise in southern Italy.We inferred the evolutionary history and the spatial patterns of genetic variation of the Hermann’s tortoise in the Italian Peninsula. We identified three main genetic clusters along the peninsula and a hotspot of intraspecific diversity in southern Italy. Our results underline the urgent need for conservation actions to warrant the long-term persistence of viable tortoise populations in this area. Furthrmore, these data add further evidence to the role of southern Italy as a biodiversity hotspot for temperate fauna, claiming for higher consideration of this area in large scale conservation programs.The online version contains supplementary material available at 10.1186/s12862-022-02075-w. Hotspots of genetic diversity are geographic areas harbouring exceptionally high levels of intraspecific genetic variation and represent appealing resources to investigate the mechanisms shaping the geographic structure of biodiversity –3. StillIn the western Palaearctic region, Mediterranean peninsulas have been identified as major hotspots of intraspecific genetic variation for temperate species , and refTestudo hermanni Gmelin, 1789. The Hermann’s tortoise is a land tortoise inhabiting coastal and sub-mountain regions of the Italian and Balkan peninsulas, some Mediterranean islands, and two small areas of eastern Spain and southern France [T. h. hermanni, which includes populations from the Italian Peninsula, the western Mediterranean islands, and the isolated Spanish and French populations; T. h. boettgeri, which includes populations from the Balkan Peninsula and northern Italy [In this study, we focus on the geographic patterns of genetic variation of an endangered tortoise species, the Hermann’s tortoise n France . Hermannn France –30. Genern Italy . Furtherrn Italy . Howeverrn Italy , 18, 20.rn Italy .Here we employed a set of seven microsatellite loci to analyse the fine-scale population genetic structure of the Hermann’s tortoise in the Italian Peninsula, with a particular effort on the southernmost part of its range. We aimed to (i) providing further insights on the Hermann’s tortoise genetic structure and evolutionary history within the Italian Peninsula, (ii) identifying its hotspot of genetic diversity, (iii) mapping the geographic distribution of its evolutionary lineages. Considering the ongoing reduction of the Hermann’s tortoise habitat, this information will provide fundamental new insights to define effective strategies for the conservation of its populations.Gal236 in populations 8, 11, 13 and 14, and at Test10 in populations 3. However, no significant deviation from the Hardy-Weinberg and linkage equilibria was found after the Bonferroni correction was applied in FSTAT [The dataset consisted of a multi-locus genotype for 174 wild Hermann’s tortoise individuals  performed by the  package  revealedThe Bayesian clustering analyses carried out with TESS 2.3.1 , 38 showFinally, results from the standard Principal Component Analysis (PCA) performed on individual genotypes showed a shallow but clear individual clustering in three main groups, which are consistent with the results from previous analyses see Fig. . Indeed,The analysis of molecular variance (AMOVA) in ARLEQUIN 3.5.1.3  was perfTestudo hermanni in the Italian Peninsula and Sicily showed the existence of three main genetic clusters. Previous studies identified some genetic differentiation between populations from the Italian Peninsula and Sicily, and among populations within the peninsula [Testudo hermanni in southern Italy, in an area spanning from the Pollino massif to the Aspromonte massif, and suggest that the interplay between high topographic complexity and Pleistocene climate changes in this region triggered the formation of this hotspot.The analysis of genetic variation of eninsula , 32. HowThe occurrence of genetic sub-structuring and distinct genetic clusters within the Italian Peninsula has been observed in several temperate species including amphibians , 23, 25,Our data clearly identified two areas of genetic admixture, one located in the northern and one in the southern edge of the Calabrian region. These areas, likely originating from secondary contacts among distinct lineages, closely match with areas of secondary contact and admixture observed in several other taxa , 23, 49.T. hermanni is considered as Near Threatened by the IUCN red list of threatened species at a global scale [Endangered in Italy [Our results have remarkable implications for the management of the Hermann’s tortoise populations. Because of the widespread population decline, al scale  and as Ein Italy . Howeverin Italy , two of in Italy . Indeed,in Italy , 54. At in Italy –57. Thusin Italy .This study emphasizes the importance of integrating a multi-marker approach with a thorough sampling scheme in investigating the geographic structure of intraspecific genetic variation, in order to unveil hidden patterns of fine-scale genetic structuring. By applying this approach, we identified sharp genetic structure in the Hermann’s tortoise populations from southern Italy. Still, our results add further evidence to the role of Calabrian region as a hotspot of biodiversity, and claim for an assessment of the population genetic structure in other supposed well-known taxa inhabiting this region, with special attention on threatened taxa.Testudo h. hermanni within the Italian Peninsula and Sicily, by genotyping 174 wild individuals at seven microsatellite loci , following the protocols described in Biello et al. [We investigated the geographic structure of genetic variation of o et al. .Collecting sites and sample sizes are given in Table adegenet package in R environment [Fragment analysis of PCR products was performed by Macrogen Inc. on an ABI 3730xl Genetic Analyser (Applied Biosystems) with a 400HD size standard. Electropherograms checking and allele calling was performed by using GENEMAPPER® 4.1. Micro-Checker 2.2.3  was usedironment ; allelicironment .adegenet R package [The extent of spatial patterns of genetic variation was investigated by a spatial Principal Components Analysis (sPCA), as implemented in the  package . The anaThe genetic clustering of individuals across the study area was also investigated using the Bayesian clustering algorithm implemented in TESS 2.3.1 and the geographical location of individuals as prior information , 38. Theadegenet R package [Finally, the genetic clustering of individuals was also evaluated without accounting for spatial information by means of a standard PCA of genotypic data, as implemented in the  package .adegenet R package.In order to estimate the amount of variation attributable to differences among the clusters identified by the clustering methods, among populations within clusters, and within populations, a locus by locus analysis of molecular variance (AMOVA) was performed using ARLEQUIN 3.5.1.3 . PopulatBelow is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


Oryza sativa during its evolution process. Natural hybridization-introgression led to the origin of indica, aus, and basmatic subgroups, which adapted to changing cultivated environments, and produced feral weedy rice coexisting and competing with cultivars under production management. Artificial interspecific hybridization-introgression gained several breakthroughs in rice breeding, such as developing three-line hybrid rice, new rice for Africa (NERICA), and some important pest and disease resistance genes in rice genetic improvement, contributing to the stable increase of rice production to meet the expanding human population. We proposed a series to exploit the virtues of hybridization-introgression in the genetic improvement of Asian cultivated rice. But some key issues such as reproductive barriers especially hybrid sterility should be investigated further, which are conducive to gene exchange between cultivated rice and its relatives, and even is beneficial to exploiting interspecific hybrid vigor. New technologies help introduce favorable genes from distant wild species to Asian cultivated rice, such as transgenic and genome editing systems. Rising introgression lines in a wider range with multi-donor benefits allele mining, understanding genetic network of rice growth and development, yield formation, and environmental adaptation. Then, integration of new tools and interspecific hybridization can be a future direction to develop more usable breeding populations which can make Asian cultivated rice more resilient to the changing climate and world.As one of the most important crops, Asian cultivated rice has evolved into a complex group including several subgroups adapting various eco-climate-systems around the globe. Here, we pictured a comprehensive view of its original domestication, divergences, and the origin of different subgroups by integrating agriculture, archeology, genetics, nuclear, and cytoplasm genome results. Then, it was highlighted that interspecific hybridization-introgression has played important role in improving the genetic diversity and adaptation of  The process of plant domestication often leads to genetic bottlenecks. Hybridization supplied chances to increase plant genetic diversity and adaptation ability with its expansion progress. Hybridization has been an important force in generating angiosperm species diversity . At leasOryza sativa, is one of the most important crops to feed the world for thousands of years. It is believed to be domesticated from the wild relative, Oryza rufipogon, in East Asia, then evolved and expanded to form different climatic ecotypes over the world. Regarding the domestication of the Asian cultivated rice, one hypothesis is independent domestication of indica and japonica, the prototype of O. sativa evolved primarily along the foothills of the Himalayas in South Asia and its associated mountain ranges in mainland Southeast Asia and Southwest China, and the genetic differentiation developed parallel to the ecologic diversification process  is responsible for enhanced cold tolerance in the temperate japonica cultivars  chloroplast haplotypes, and encompassing all the chloroplast diversity found in the temperate and tropical japonica  and wild populations  O. rufipogon plant was introduced into O. sativa nuclear background, and successfully bred a series of wild abortive male sterility lines, which led to the famous “green revolution” of hybrid rice. By 2019, more than 7,000 hybrid rice cultivars had been released in China, with a total planting area of about 6 × 108 ha2 and an increased rice grain of nearly 9 × 108 t, which had fed an additional 2.3 billion people. Chinese hybrid rice has been successfully tested or developed in more than 60 countries with planting areas exceeding 6 × 106 ha2  and a new rice type to adapt West Africa upland condition was developed, denoted as NERICA (new rice for Africa), and this led to the naming of many rice cultivars for both rainfed upland and lowland irrigated system of West Africa  is one of the most serious diseases of rice in Asia. A dominant gene Pid3 from O. rufipogon, Pi54rh from O. rhizomatis, Pi54 from O. officinalis, Pi57(t) from O. longistiminata, Pi68(t) from O. glumaepatula, and Pi 69(t) from O. glaberrima were identified (xa-45(t) from O. glaberrima was identified /Boro5//Dianxi1///Hongza135], Yunlu142 [Yundao1/Acc.104613(O. barthii)//Yundao1/3/Yundao1] were bred via interspecific hybridization and released at Yunnan Academy of Agricultural Sciences (YAAS), Yunnan province, China.Some elite cultivars, such as Yundao1 [ or backcross inbred lines (BILs) from a single donor, including O. glaberrima  were generated, and 15 BILs showed >10% yield superiority over the recurrent parents, with yield-related traits . Based on phenotype selection, 6,732 introgression lines (ILs) with agronomic and morphological variation were raised. Using this library, 22 alleles responsible for grain shape were newly found  is greater than inter- and intra-subspecies, which show stronger hybrid vigor  in aus and basmatic rice can also improve this trait of japonica and indica rice  into other subgroups can enrich and improve their diversity in these traits. For instance, ica rice , so as tInter-subgroup hybrids can be another field to utilize heterosis in rice. Heterotic loci existed in wild progenitors of cultivated rice, and cultivated rice was divergently selected among rice subgroups. Divergent selection among the subgroups induced various heterotic alleles in different subgroups. Many heterotic loci in the current commercial hybrids were shaped by genome introgressions from different subgroups . Thus, iIn the past decades, many genes/alleles from wild relative were identified and even cloned, and the corresponding introgression lines (ILs) or near-isogenic lines (NILs) were developed in a specific genetic background. A specific genetic background has the adaptability to a given ecological environment; although distant hybridization can increase genetic diversity, when these ILs or NILs were crossed with parents of distinct genetic backgrounds to develop breeding populations, from the perspective of breeders, the offspring of distant hybridization usually perform poorly under a specific ecological condition. This may be the main reason for the limited utilization of these alleles in rice breeding projects. Yield-related traits usually are quantitative traits, and are often affected by both genetic background and environment, improving them in breeding programs needs to deal with more challenges. Therefore, developing a series of ILs or NILs with these favor gene/alleles in various specific genetic backgrounds will be conductive to reevaluate their genetic effect and breeding value in various genetic backgrounds and environments and to develop adaptive breeding populations.O. rufipogon and evolved thousands of years. During the domestication and evolution progress, when it gained favor mutations or was introduced into changing environments, only a few numbers of individuals could be retained and subsequently expanded, then the genetic bottlenecks occurred and genetic diversity decreased. Fully and long domesticated subgroup japonica showed a higher degree of differentiation and lower genetic diversity than indica (O. sativa, which adapted to changing cultivated environments, and produced feral weedy rice coexisting and competing with cultivars under cultivation management. Artificial interspecific hybridization and introgression gained several breakthroughs in rice breeding, such as hybrid rice, NERICA, and biotic stress resistance genes, which are contributed to the stable increase of rice production to meet the expanding human population. As interspecific hybridization-introgression have played important roles in improving genetic diversity and adaptation of O. sativa, more efforts need to make to exploit its virtues. Some key issues, such as reproductive barriers especially hybrid sterility should be investigated further, which are conducive to gene exchange between cultivated rice and its relatives, and even is beneficial to exploiting interspecific hybrid vigor. New techniques, such as efficient tissue culture, transgenics, genome editing system, and high-quality genome assembly, will help wide distant interspecific introgression and make the crop better. Compared with the huge genetic diversity of wild relatives, the construction of introgression lines in a wider range with more donors is needed, which benefits allele mining, understanding the genetic network of rice growth and development, yield formation, and environmental adaptation. Thus, developing a series of ILs or NILs with these favor gene/alleles in the various specific genetic background will be conductive to reevaluate their genetic effect and breeding value in various genetic backgrounds and environments and helpful to develop more usable breeding populations in various situations. In addition to interspecific hybridization, hybridization between inter-subgroups can make the Asian cultivated rice wider adaptable and stronger heterosis.Asian cultivated rice originated from n indica . InterspDT proposed the concept. JZ conceived and wrote the manuscript. YY, YL, QP, JL, YZ, XD, MW, and JW reviewed and edited the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.