POij = 0.283) between clones indicated low overlap between female receptivity and pollen shedding. A higher POij value within clones than within outcrossing combinations indicated a high risk of selfing in two seed orchards, particularly for early- and late-flowering clones. The number of female strobili and POij  significantly influence seed production. Overall, fertility and synchronization variation had notable consequences for seed production. Scientific genetic management is indispensable for promoting fertility uniformity and synchronization to obtain maximal genetic gain.Variations in fertility and synchronization information is fundamental to seed orchard management. Our objective was to determine clonal variation and stability in strobili production, phenology, synchronization, and seed production in two generation clonal seed orchards (CSO) of Chinese fir. The number of female and male strobili and the phenology of 42 clones in both the 2.0- and 2.5-generation clonal seed orchards were investigated and recorded to calculate the variation and stability of fertility and synchronization. In both seed orchards, an obvious variation in gamete contribution was found among clones, indicating deviation from random mating. Female receptivity was in the pollen shedding stage, which is favorable to pollination. However, low synchronization (mean  The genetic gain and gene diversity of seeds from seed orchards are expected to be high as a result of continuing improvements to successive generations3. For the progeny of seed orchards to reach the highest possible genetic gain, complete synchronization is necessary4. However, seed orchards often deviate from their “ideal” expectations5. For example, fertility variation has been reported in many tree species8. The difference in female and male strobili production among clones would cause an imbalance in gamete contributions. Commonly, the imbalance of parental contribution will decrease the seed yield in seed orchard1. For instance, a significant difference in female and male strobili caused an unequal contribution to the gamete gene pool in Cedrus libani seed stands1. The deviation of the effective population number from an ideal population was also reported in Picea sitchensis, which indicated the presence of a disproportional contribution by the clones5. In a Pinus sylvestris seed orchard, a differentiation in gene frequencies was exhibited between the parent and the progeny, which mainly resulted from the variation in the imbalance of female and male gamete contributions6. Therefore, knowledge of fertility variation is becoming important to evaluate the maintenance of gene diversity and scientific management in seed orchards.Seed orchards are an important seed source and are essential for global seed production programs2. Phenology affects the frequency of gene exchange among clones and the genetic composition of seeds derived from seed orchards10. Previous studies have documented that phenological variation among clones reduces the effective population size, promotes selfing, and increases the risk of contamination11. Data from P. patula seed orchards suggest that the overlap between female receptivity in orchards and pollen shedding in natural stands produces a risk of pollen contamination12. Conversely, the low synchronization between natural stands and seed orchards of P. patula lowers the risk of pollen contamination and high seed quality failure9. The pronounced variation of gametic contribution and synchronization in P. sylvestris seed orchards causes a substantial drift of gene frequency between the seed orchard and its offspring6. Overall, knowledge of phenology and synchronization is fundamental for making scientific management decisions regarding seed orchard activities and advanced seed orchard design, such as supplemental mass pollination (SMP) and parent configuration, to attain a high level of genetic gain12.Studies on phenology and synchronization have contributed to evaluating the level of effective out-crossing and the condition of genetic diversity in seed orchard seed lots4. Information about fertility and synchronization is important for seed orchard managers, especially with respect to seed production output. Over a six year period, an obvious difference in phenology existed in a Chinese fir seed orchard, but the flowering order of clones remained stable13. Monitoring data in a Korean pine seed orchard for five consecutive years showed that the fertility variation would impact the genetic diversity of seed crops and possible management options14. In some previous reports, the stability of fertility, phenological traits, and cone production in some tree species remained stable, and with little change from year to year, so we could make an early evaluation and selection of these traits16. An inverse report showed unstable cone production over time in a 1.5-generation seed orchard of P. koraiensis, indicating that continuous monitoring was necessary for making wise management proposals17. Therefore, knowledge of the stability or variation in fertility and phenology is important for seed orchard management.A complete and stable synchronization and balance of gamete contamination are required to reach the intended genetic gains and maximize the genotypic diversity of the progeny in seed orchardsCunninghamia laceolata (Lamb.) Hook.) is an important timber tree species for afforestation in southern China due to its fast growth rate and high wood quality19. As the first batch of tree species for which seed orchards were established in China, the Chinese fir provided a large amounts of high-quality seeds for artificial restoration. However, unfavorable conditions, such as low seed production and decreased genetic diversity, also occurred in some seed orchards. Little information exists on fertility and phenology, which limits formulations for seed orchard management. Therefore, the objectives of the current study are to evaluate the variation and stability of fertility and synchronization of Chinese fir seed orchards and their effects on seed production.Chinese fir (Nf) and male (Nm) strobili varied between the two seed orchards and among clones within a clonal seed orchard (CSO)  was significantly higher than that in the 2-generation CSO (CSO2) . The analysis of variance (ANOVA) of Nf and Nm both showed significant differences among clones (P ˂ 0.01), but only Nf showed a significant difference between the two seed orchards (Table Hc2) was low for Nf and Nm. The parental balance curves for female and male strobili production significantly deviated from the ideal situation in CSO2 and CSO2.5  was 0.428 and 0.291 for males and 0.204 and 0.304 for females in CSO2 and CSO2.5, respectively, which indicated an unbalanced gamete contribution in seed orchards. In CSO2.5, the four clones with the highest contribution  produced more than 50% strobili. A change in female and male strobili production rank between seed orchards indicated that clone reproductive capacity differed between CSO2 and CSO2.5  and male (ψm) parents varied between the two seed orchards, especially for ψm, which showed a large difference in fertility between males (Table ψ) in CSO2.5 was higher than that in CSO2. This was inversely mirrored by the effective number of males (Npm) and parents (Np) and the relative effective number of females (Nrf), males (Nrm), and parents (Nr).The numbers of female (SO) Fig. A. The NfF = 1.04, P = 0.319). A regression between male onset date and duration of pollen shedding showed that early flowering clones had a longer period of pollen shedding, such as clone 42 in CSO2 (Julian day 67 onset with 13 days duration) and clone 48 in CSO2.5 (Julian day 67 onset with 27 days duration)  in CSO2 and CSO2.5 was 0.240 and 0.325, indicating 24.0% and 32.5% overlaps in the time of female receptivity and pollen shedding, respectively , indicating that there were more female strobilus available when males were at the peak of pollen shedding than male strobilus available when females were at the peak of receptivity. There was no significant difference in POij  between males and females in CSO2.5. In most clones, there was a similar rank of POij between the two seed orchards, such as clone 12 with a high POij, as females had a lower POij than males in both CSO2 and CSO2.5. The POij within clones was higher than the outcrossing combination in both CSO2 (0.337 vs. 0.238) and CSO2.5 (0.357 vs. 0.325), indicating a greater chance of self-pollination. For both female and male strobilus, the POij of clones in the early, intermediate, and late classes showed significant differences between the two seed orchards (Table POij.The average of the synchronization index (Cp) varied between the two seed orchards and among clones within a clonal seed orchard (CSO) , thousand seed weight (TSW), and seed production of one cone (Psc) among clones; there were also significant differences in Cp and TSW between the two seed orchards (Table H2) of Psc indicated that Psc exhibited higher genetic control than Cp and TSW in Chinese fir seed orchards. The results of linear regression analysis showed that there were significant relationships between Nf and Cp in both seed orchards .The cone production (SO) Fig. A. A chanSO) Fig. B. There ds Table . The higrds Fig. . Additio16. The number of female strobili (Nf) showed significant differences between CSO2 and CSO2.5, and among clones within the seed orchard. The significant age-age correlation of Nf between the two generation seed orchards indicated that early prediction was feasible for female strobili in Chinese fir seed orchards. Differences among clones in female and male strobili were also found in other Chinese fir seed orchards, but clones had a positive or negative depending on the years20. Fertility can be influenced by genetic and environmental factors such as parent selection, management, tree growth, altitude, and seed orchard age21. Tree age and form can affect the number of strobili, pollination, and fruiting by regulating light, nutrient transport, and other factors. In these two seed orchards, difference in tree age and form are the dominant factors inducing the variation in Nf. It should be emphasized that tree form control is important for seed production and seed orchard management. The sibling coefficient (ψ) value was greater than 2 in two seed orchards, indicating that the probability that more than 2 individuals share a parent is more than twice as high as when the parental fertility is equal across the orchards22. The effective number of parents (Np) can estimate the loss of gene diversity between parents and their progeny4. In CSO2, ψ is lower, but Np is higher than that in CSO2.5, indicating that seeds from CSO2 would maintain a relatively high level of genetic diversity.Fertility and synchronization are critical in seed orchard design and management and are key to obtaining the potential maximum genetic gains2. On average, pollen shedding and female receptivity occurred over 14 days in both seed orchards, corresponding closely with observations in the Chinese fir clonal archive in Fujian Province, China23. In 1994, pollen shedding and female receptivity occurred over 34 days, which was a much longer period than that found in our study13. The higher temperature, shorter rainy days, and lower humidity in this study during the flowering period, compared to those in previous study, would induce lower periods of pollen shedding and female receptivity. Female receptivity of clones followed pollen shedding in these two seed orchards, which contributed to crossbreeding in seed orchards as a mean to meet the need for sufficient pollen production. A similar result was also reported for the 1-generation seed orchard of Chinese fir13. The significantly positive relationship between male strobili onset and duration of pollen shedding indicated that early blooming clones shed pollen duration longer than late blooming clones. High synchronization is desirable for successful seed production in seed orchards10. Our analyses showed that the average value of POij was remarkably low but stable across the two seed orchards. Moreover, the synchronization (POij = 0.240 in CSO2 and 0.325 in CSO2.5) was obviously lower than that of other coniferous tree species, such as Pinus patula (POij = 0.49–0.60)9, P. massoniana (POij = 0.513)24, and P. tabuliformis (POij = 0.552)25, probably due to the dichogamy and failure of some strobili lowered the POij value in our study. In this study, POij was mainly distributed from 0.300 to 0.500, implying that there was an imbalance in the gamete contribution and nonrandom cross-fertilization in the seed orchards. Self-pollination is an important factor affecting genetic diversity and genetic gain in seed orchards2. In this study, most clones probably had a higher rate of self-pollination, as indicated by the POij value in the sample clone being higher than its average POij value as a male or female parent. Methods should be considered to dilute this effect when establishing advanced seed orchards, such as deploying clones with similar phenology and increasing the number of ramets per clone. Considered together, pollen contamination seems more important than self-fertilization in affecting the genetic efficiency of the orchard, given the amount of trees releasing pollen in the orchard’s neighborhood12. Low pollen production and synchronization can create scenarios where some clones are more exposed to contamination by external pollen or less represented in the seed orchard’s progeny26. In two seed orchards of Chinese fir, the POij values were lower than 0.4, which made it possible for clones to be confronted with a high risk of pollen contamination and have a negative effect on genetic gain. The absence of a difference between the POij values of two seed orchards indicated the likelihood of pollination due to clone stability from tree growth. This result is similar to the stability in synchronization indices found in Scots pine and radiata pine27.Synchronization of pollen shedding and female receptivity is one of the major factors affecting genetic gain in seed orchards14. The low value of parental gamete contribution (U) (0.204–0.428) indicated an extreme imbalance of female and male gamete contribution. The variation in fertility led to a significant difference in cone production (Cp), thousand seed weight (TSW), and seed production of one cone (Psc) among clones. There were significant positive correlations between Nf and Cp in both CSO2 and CSO2.5, but no correlations between Nm and Cp, indicating that the effect of the number and receptivity of female strobili on seed production is higher than male strobili. Therefore, balancing the number of strobili, especially female strobili, should be carefully considered for management in the main production years to obtain high seed yield and quality. For instance, injections or fertilizations of gibberellic acid (GA4/7) can be used to stimulate or enhance strobili production and further enhance cone production. Information about the different clone contributions to the gamete gene pool is important for the proper estimation of the expected genetic composition and genetic gain11. The Cp and TSW were under low genetic control and genetically different, and they varied widely with age and among clones, which should affect the phenotypic diversity of the resulting seeds, as has been shown in other coniferous species17. These traits may be affected by many factors, such as self-fertilization and synchronization clones4. In our case, wide clonal variation in POij values as females was significantly associated with Cp and TSW. The lack of synchronization in the seed orchard provides a warning regarding self-fertilization and genetic contamination risks, particularly for late-flowering clones. These two factors have negative effects on the genetic quality expected in the seeds produced in the seed orchard. However, this negative effect could be reduced by management practices, such as SMP, parent configuration, and hormone application . Considering that the data were collected in a single year, we should further strengthen the multi-years monitoring of phenology and fertility variation of clones to provide more stability data and improve the genetic gain of seed orchards.The difference in fertility among parents would cause seed orchard clones to contribute differently to the seed cropThe study was carried out in two clonal seed orchards (CSOs) of Chinese fir: the 2-generation clonal seed orchard (CSO2) and the 2.5-generation clonal seed orchard (CSO2.5)  were selected and marked for investigation in both CSO2 and CSO2.5. Four standard twigs  per tree were marked to investigate the strobili, cone, and seed production and phenology data.Nm) and female strobili (Nf) in every tree were surveyed and recorded. The phenological progress of female and male strobili was monitored from March to April 2021. Data were recorded every two days. The phenology of female and male strobili were divided into four stages: the initial stage, peak stage, final stage, and end stage13. Male strobili: 1, initial stage (ISm), staminate strobili whitening with little pollen shedding; 2, peak stage (PSm), a large amount of pollen shedding; 3, final stage (FSm), 90% pollen shedding; and 4, end-stage (ESm), 100% pollen shedding and staminate strobili browning. Female strobili: 1, initial stage (ISf), female strobili bud presenting yellow with invisible ovules; 2, peak stage (PSf), at least two wheels of ovuliferous scale expanding and ovule presenting golden yellow with droplet; 3, final stage (FSf), ovule forfeiting fresh with little droplet; and 4, end-stage (ESf), ovuliferous scale completely closing. The trees were divided into three types  by the start of pollen shedding and female receptivity23. The pollen shedding or receptivity of the early-type tree was two days at the beginning of flowering, the pollen shedding or receptivity of late-type tree referred to the last two days of flowering, all other trees were classified into the intermediate-type group.In 2021, strobili production was investigated by the method described by Chen et al. (1995). The numbers of male (Cp) was calculated. Then, 3–5 cones per tree were randomly selected to measure the seed production of one cone . All cones were dried, and seeds were collected to measure the thousand seed weight .All cones were picked, and the cone production per entire tree  was analyzed by Pearson’s correlation. Afterward, if there was a significant correlation between female and male strobili production then the sibling coefficient (ψ), which means combined fertility variation, was calculated by the following formula29.The variance in fertility, phenology, and production were calculated by Genstat 21st edition software through two-way analysis of variance (ANOVA). Within clonal heritability , the effective number of female (Npf = n/ψf) and male gametic parents (Npm = n/ψm), the relative effective number of parents (Nr = Np/n), and the relative effective numbers of female (Nrf = Npf /n) and male parents (Nrm = Npm /n)31. The parental balance curve was used to estimate the uniformity index of female and male gametic contribution (U)32. The trees were ranked from high to low according to female and male strobili production; then, the cumulative percentage calculations were plotted against the equal cumulative percentage in the seed orchard5.An ANOVA for dichogamy were performed between CSO2 and CSO2.5. The parameters of the effective number of parents in the seed orchard are calculated as follows: the effective number of parents (POij) was calculated through the following formula in R33.POij is the phenological synchronization index between the ith and the jth clones, Mki is the proportion of pollen shedding of the ith clone at day k, Pkj is the ratio of the female receptivity of the jth clone at day k, and n is the days of total strobili duration of i and j.The synchronization index (POij values were performed at the two seed orchards for female and male strobili. A linear regression analysis between flowering onset and duration was performed to determine the initial stage for phenology duration. A linear regression analysis between strobili production, POij, Cp, Psc, and TSW also was performed to determine fertility and phenology synchronization for seed production and quality.Separate ANOVAs for All field studies were performed in accordance with the local legislation in China and complied with the convention on trade in endangered species.


Hungarosaurus  and Mochlodon   were analysed to investigate their dietary ecology, using several complementary methods—orientation patch count, tooth replacement rate, macrowear, tooth wear rate, traditional microwear, and dental microwear texture analysis (DMTA). Tooth formation time is similar in Hungarosaurus and Mochlodon, and traditional and DMTA microwear features suggest low-browsing habits for both taxa, consistent with their inferred stances and body sizes. However, Mochlodon possesses a novel adaptation for increasing dental durability: the dentine on the working side of the crown is double the thickness of that on the balancing side. Moreover, crown morphology, enamel thickness, macrowear orientation, and wear rate differ greatly between the two taxa. Consequently, these sympatric herbivores probably exploited plants of different toughness, implying dietary selectivity and niche partitioning. Hungarosaurus is inferred to have eaten softer vegetation, whereas Mochlodon likely fed on tougher material. Compared to the much heavier, quadrupedal Hungarosaurus, the bipedal Mochlodon wore down more than twice as much of its crown volume during the functional life of the tooth. This heavy tooth wear might correlate with more intensive food processing and, in turn, could reflect differences in the metabolic requirements of these animals.Dentitions of the sympatric herbivorous dinosaurs  Ornithopods exhibit some of the most elaborate dentitions and jaw mechanisms27, but it is now clear that thyreophorans also had varied ways of processing food36. In both groups, complex jaw mechanisms, distinct from the simple orthal jaw closure that was symplesiomorphic for the clade, were present in numerous taxa, and allowed more efficient chewing (and subsequent digestion) of the food consumed. A large body of work has been devoted to reconstructing the evolutionary history of the dental and cranial features associated with feeding in Ornithischia , but only a few studies have investigated possible differences in the feeding behaviours of sympatric taxa (e.g.39).Ornithischian dinosaur feeding traits  displayed substantial variation during the 140 million year history of the clade , exploited the available vegetation in similar or different ways and to use this information to explore their comparative dietary ecology. In order to characterize their feeding habits in as much detail as possible, we compared 15 dental characters  of Iharkút —42. All of the jaw elements and teeth were collected as isolated specimens, except for the dentary (MTM 2007.25.2) of Hungarosaurus tormai, which is part of the fifth skeleton of this species. All of the specimens are housed in the Vertebrate Paleontological Collection of the Hungarian Natural History Museum, Budapest, Hungary (MTM). See Table The specimens used in this study were collected between 2001 and 2021 from the approximately 30 cm thick, Szál-6 bone-bearing bed of the Upper Cretaceous (Santonian) Csehbánya Formation, Iharkút, western Hungary. All specimens are from the same stratigraphic horizon. For reviews of the geology, faunal composition, and taphonomy of this site seeEach specimen was prepared mechanically in the laboratories of either the Department of Paleontology, Eötvös Loránd University or the Hungarian Natural History Museum. Broken specimens were reassembled using cyanoacrylate glue. Great care was taken to avoid damage to the tooth crowns and their wear facets during collection and preparation, and wear facets were not treated with consolidants or coatings where possible.Hungarosaurus tormai and four teeth of Mochlodon vorosi were thin-sectioned  and cutting with a Buehler Isomet 1000, the cut surface was treated with additional resin, sanded with grit abrasives , and then bonded to a glass microscope slide. Subsequently, the surface was treated with resin again, after which it could be sanded down to a final thickness of approximately 70 µm.Seven teeth of ed Table . These t44. We redrew the lines between the von Ebner incremental bands (VEIB) revealed in the thin-sections using Photoshop and measured the thicknesses of the tissue between these lines using ImageJ. The use of the term ’von Ebner incremental line’ in the earlier literature is not always clear as to whether the line is a band composed of both its light and dark parts or the boundary between the daily formed bands. Here, we refer to the unit consisting of one dark and one light part as the von Ebner incremental band (VEIB), which we believe is a better indication of its extent.Images of the thin-sections were taken with a QImaging MP5.0 digital microscope camera using a Nikon LV 100 polarized light microscope, and processed with Image Pro Insight v. 8.0. Measurements from the teeth and thin-sections were taken using ImageJ v. 1.48Hungarosaurus tormai and 25 teeth of Mochlodon vorosi were used for 3D modelling. 3D models from the teeth were created using a Polyga HDI Compact S1 3D Scanner. Before scanning, the specimens were coated with either a Pfinder 871 solvent-based aerosol spray or talcum powder in order to reduce the reflections from their shiny surfaces. The specimens were scanned on a turntable connected to the scanner. The point clouds from the multi-directional scans have been precisely merged and the surface generated in FlexScan3D v. 3.3.21.8. The resulting 3D polygon files had polygon lengths between 30 and 80 µm. Further operations were performed in Geomagic Wrap version 2017.0.2.18 . Measurements of the worn surface area and crown volumetric data were also taken using the latter software  and 25 RPT in mesial-distal direction. Orientation Patch Count Rotated (OPCR) was calculated using eight cardinal directions of 45º, a minimum patch size of three and repeated at eight rotations47.3D models of one unworn Hungarosaurus tormai and five Mochlodon vorosi teeth were analysed using traditional (2D) microwear and DMTA (3D) analysis. Macrowear is defined herein as a wear feature larger than 0.5 mm, whereas microwear features are considered to be smaller than this. Scratches and pits represent most of the wear features seen on the teeth. Following48, pits are defined as having length–width ratios < 4:1. For scratches, this ratio is > 4:1. Alongside the 3D models (see above), light microscopy was used to record gross macrowear patterns. Microwear analysis was performed with a Leica DCM3D confocal microscope . For traditional (2D) microwear analysis four micrographs were saved per tooth as greyscale images using the intensity data from the confocal dataset. Each measured micrograph has a resolution of 768 × 576 pixels, corresponding to a 637 × 477 μm FOV. Measurements were carried out using a Leica HC PL Fluotar EPI 20X lens after a series of sensitivity tests with 20X, 50X and 100X lens. Due to the nature of the investigated samples, no additional information could be gained with higher magnification lens i.e. the increase in level of detail of the 3D dataset with a 100X lens was negligible. On the other hand, using a 20X lens allowed for a quicker acquisition of a comparably larger area with a lateral (X and Y) resolution of 0.83 micron and a vertical resolution of 0.015 micron. It should be noted that the used FOV and spatial resolution is larger than the typical FOV used in DMTA analyses. Nonetheless, 2D analysis based on intensity maps showed a very high level of detail, and therefore should produce comparable results. Images of the microwear facets were analyzed using Microware v. 4.0 following the procedure described by49. The generated 768 × 576 pixel grayscale images were viewed on a 27″ Full HD monitor for 2D analysis, which corresponds to a physical image size of approximately 24 × 18 cm (assuming a pixel density of 81 pixel per inch) when viewed at 1:1 scale. The slides were scaled 1:1 in Microware before conducting the 2D analysis. 2D microwear analysis was conducted by the same operator. Four variables were assessed from the micrographs: (1) the percentage incidence of pitting; (2) mean scratch width; (3) mean pit width; and (4) mean pit length. We also report the number of features measured and the standard deviation of means  per tooth, concentrating on the more resistant mantle dentine located directly under the enamel. In addition,30 identified an average of 44.26 features (scratches and pits) across all of the taxa they studied. By contrast, on the four Hungarosaurus tormai and five Mochlodon vorosi teeth sampled, we identified averages of 281.43 and 404.97 features, respectively. This discrepancy could be due to our inclusion of a high number of small, point-like features on our micrographs that might have not been counted in30 analyses, which could account for the much higher number of pits reported in our sample . In the present study the attributes of SSFA measured are anisotropy , complexity , scale of maximum complexity (Smc), and heterogeneity of area-scale fractal complexity (HAsfc(9 × 9)). The selected parameters are summarized in Supplementary data 3D topographic data from the wear surfaces were collected using the same confocal technique. Raw measurements were post-processed in Mountains v. 8. Datasets were leveled using a least-squares plane method leveling algorithm. Non-measured points were filled with a smooth spline method. No additional data processing was conducted before surface analysis. This approach was chosen to accelerate 3D analysis and to avoid potential misinterpretation of surface features, minimize subjectivity and increase reproductivity. A 500 × 500 pixel area was extracted from each micrograph for evaluation purposes. Each 3D topographic dataset was analyzed by scale-sensitive fractal analysis (SSFA) based on several previous studies . For a multivariate analysis, Principal Components Analysis (PCA) has been used , using a Zeiss Metrotom computer tomograph with interslice distances of 130 µm. In addition, nanoCT with voxel sizes between 50 and 600 nm was used to detect further details of the jaws and teeth, using a Bruker Skyscan 2211 nano-CT cone-beam scanner  at the University of Szeged, Hungary. The images acquired were reconstructed in volumetric NRecon Reconstruction , which allows the correction of typically occurring artifacts . For 3D visualization CTVox 3D Micro-CT Volume Rendering  was used.Micro- and nanoCT-imaging was used to investigate the internal structure of the teeth and jaw elements and to reveal replacement teeth. One dentary of 56, VEIB that can be observed in thin-sections or on the cut, etched surfaces of tooth crowns represent daily tooth formation time. In Hungarosaurus tormai, based on four transverse  and three longitudinal  sections of seven isolated but well preserved specimens, tooth formation time is 63–126 days  and Edmontonia (279 days56). The mean widths of VEIB in the transverse  and longitudinal  sections are 16.97 µm and 18.7 µm, respectively. These values are higher than in Edmontonia (13.9 μm34) or Pinacosaurus grangeri (15.7 μm34). In five of the seven thin-sections, the thickness of VEIB decreases slightly towards the enamel-dentine junction, whereas in the other two taxa the opposite is true. The average enamel thickness in H. tormai is 20–25 μm, with slightly thicker enamel around the marginal denticles.Tooth formation times are summarized in Table Mochlodon vorosi is 77–140 days . In Matheronodon provincialis, based on a coronal section of a tooth57, a tooth formation time of 50–100 days has been calculated. However, counting the number of VEIB on Fig. 57 results in a minimum tooth formation time of 110 days, and the mean width of VEIB is 24.55 µm.Based on four transverse  thin-sections made from four teeth, the tooth formation time in Mochlodon vorosi and Hungarosaurus tormai revealed the number, sizes and positions of the replacement teeth. One right dentary of H. tormai (MTM 2007.25.2) possesses well-preserved functional teeth in the 4th, 6th–10th, 12th, 14th, 16th, and 17th alveoli  represent different stages of eruption through 13 alveoli containing at least one tooth generation .MicroCT scanning of the dentaries of rmai MTM 007.25.2 Mochlodon vorosi dentaries in our sample . The most complete functional tooth is found in the 7th alveolus of the right dentary . For comparison, the minimum tooth replacement rate in Pinacosaurus grangeri is 53 days , which is nearly proportional to that of H. tormai when scaled for tooth size . In Edmontonia, whose teeth are at least four times larger by volume than those of Pinacosaurus grangeri, and 1.5–2 times larger than those of H. tormai, the tooth replacement rate was presumably much longer than in H. tormai based on the small size of replacement teeth of Edmontonia34.Tooth replacement rates could not be calculated using the non-invasive approach oforn Fig. a, and beMochlodon vorosi, although there are no functional teeth in the sampled jaws .The situation may have been similar in aws Fig. b. There Hungarosaurus tormai tooth macrowear were described by32. In dentary teeth, the wear surface extends from the tip of the crown to the base of the cingulum and is bowl-shaped on the labial side of the crown ) and are extremely steep, with an angle of 5–10° relative to the apicobasal axis of the crown. Wear facets on the upper teeth are less extensive , and occur mainly on the apical region and are oriented at shallower angles (25–45°) relative to the apicobasal axis. Calculations of tooth crown volume based on the 3D models of the complete and strongly worn dentary teeth indicate that, on the most highly worn teeth, 28% of the complete crown volume was lost through wear  and steeply inclined . In later stages, the facet is more extensive, covering 60–95% of the crown surface . However, the wear surface undulates slightly mesiodistally reflecting the alternation of some very shallow close-to-vertical grooves and ridges resulting from orthal jaw movement (see38). The volume of the tooth crown lost during wear is much greater in Mochlodon vorosi than in Hungarosaurus tormai. In the most heavily worn crowns, the wear facet reaches the cingulum and we calculate that up to 58% of the original complete crown volume was lost  and, even in strongly worn crowns, this angle does not exceed 30–40°. Double wear facets are rare  microwear and DMTA (3D) analysis in four study areas per tooth  and anisotropy (epLsar) values measured by the 3D analysis fall within similar ranges for both taxa Fig. c. The anHungarosaurus tormai revealed 21.75 patches with 50 RPT (nine with 25 RPT). This relatively high complexity value is related partly to the denticulate mesial and distal craniae, and to the crenulated cingula. While the maxillary and dentary teeth are morphologically identical in H. tormai, they differ slightly in Mochlodon vorosi. Nevertheless, tooth complexity values for the unworn maxillary and dentary teeth of M. vorosi are almost identical, at 20.5 and 20.175 patches per tooth with 50 RPT (8.25 and 9.125 with 25 RPT), respectively  and fall in the middle range of modern saurians, in the insectivore and omnivore range62.OPCR analysis of an unworn, complete tooth crown of ely Fig. . These v20, the two Iharkút taxa, as expected, show similar patterns to those of other ornithischian dinosaurs . They do not reach the highest value seen in Fruitadens haagarorum (36.5) or the average value (33.75) measured in Nanosaurus agilis, but are similar to that for the iguanodontian Camptosaurus dispar (20.15) , which might be related to the presence of a crenulated cingulum in the former.Compared with the complexity values measured in other dinosaursgarorum 3.5 or theHungarosaurus tormai and Mochlodon vorosi reveal both differences and similarities in their dental characteristics. In spite of their markedly different functional tooth numbers and crown morphologies, the reconstructed maximum tooth formation time is quite similar . Tooth replacement was not rapid in either taxon, as the single generation of replacement teeth is not visible under newly erupted, unworn teeth but only becomes evident later, beneath teeth that are already worn. This suggests that new tooth mineralisation in both taxa started only after the functional teeth had begun to wear. However, the interval between the onset of wear and the appearance of a new tooth germ remains unknown. By contrast, the two taxa differ substantially histologically, and in M. vorosi the mean width of VEIB is almost twice that seen in H. tormai. Similarly, the tooth crown volumes in M. vorosi are 2–2.5 times greater than in H. tormai.Comparisons between the sympatric ornithischian dinosaurs Mochlodon vorosi, comparisons between a fully intact (volume [V]: 290.81 mm3) and heavily worn (V: 122.38 mm3)—but originally similarly-sized —maxillary tooth crowns show that up to 58% of the original tooth crown volume was lost as a result of feeding-related wear. In Hungarosaurus tormai, however, only 28% of the crown was lost, based on the volume difference between equally-sized intact (V: 136.28 mm3) and heavily worn (V: 98 mm3) teeth  of the total original tooth volume in a single jaw quadrant was abraded in each tooth generation, compared to 1684.3 mm3 (10 × 168.43 mm3) for M. vorosi. Hence, in spite of similar tooth replacement rates and OPCR complexity in these taxa, M. vorosi wore through more than double the amount of tooth tissue lost in H. tormai, despite possessing much more thickly enamelled teeth (twice the enamel thickness). These differences in the extent of tooth wear, along with the markedly different crown morphologies of these taxa (compressed and phylliform in H. tormai vs more robust and chisel-like in M. vorosi), as well as the differences in orthal (M. vorosi) vs palinal (H. tormai) jaw movements during the powerstroke suggest that these taxa were exploiting the vegetation available to them in very different ways (see below).In Hungarosaurus tormai and other thyreophorans are currently limited to Edmontonia and Pinacosaurus grangeri56. Edmontonia exhibits strong wear on its teeth30, which differs from that present in H. tormai but is also due to occlusion; however, P. grangeri shows only minimal wear on the tips of its marginal denticles, suggesting the absence of occlusion and that the wear present was caused by tooth-food contact33. It is interesting to note that the different wear types and rates of wear seen among these three genera are not reflected in either tooth formation time or tooth replacement rate. Hungarosaurus tormai and Edmontonia had similar rates of wear with different mean widths of VEIB , whereas P. grangeri, with minimal wear, had thicker widths of VEIB (15.7 μm) and a proportionally rapid tooth replacement relative to tooth size34. This suggests that the rate of feeding-induced wear in ankylosaurs (even in forms with occlusion) was not high enough to require the acquisition of rapid tooth formation or higher rates of tooth replacement (by increasing the number of replacement tooth generations), in contrast to the conditions seen in either sauropods59, hadrosaurids63, or ceratopsids64.Unfortunately, comparisons between the dental wear of 36. This small tooth crown (height ~ 4 mm) possesses 95 VEIB with a mean width of 16.24 μm  and suggested a high tooth replacement rate. However, comparing these values with those of the above mentioned ankylosaurs, it seems that the Siberian stegosaur tooth was quite similar with respect to tooth formation. Its tooth size is almost identical to that of Pinacosaurus grangeri34 and the mean width of VEIB is also very similar (15.7 μm in the latter). Due to the more extensive, and perhaps faster, wear compared to that inferred in ankylosaurs36, a faster tooth replacement rate cannot be excluded, but further evidence from replacement teeth of this unnamed stegosaur taxon will be required to test this issue.To our knowledge, tooth formation time has only been reported in one other thyreophoran, a taxonomically indeterminate stegosaur tooth from the Early Cretaceous of eastern Siberia65), there are limited data on tooth replacement in non-hadrosaurid ornithopods, which consist primarily of descriptions of replacement teeth (see e.g.68). For non-hadrosaurids, tooth formation time and replacement rate has only been calculated in the rhabdodontids Matheronodon provincialis57 and Mochlodon vorosi . These studies, along with the results presented here, further support the hypothesis of58 that the process and rate of tooth replacement in these relatively early-diverging ornithopods were quite similar to those of thyreophorans. In rhabdodontids and thyreophorans, the maximum number of replacement teeth per alveolus is one, but the number of replacement teeth in each jaw quadrant varies between groups. For example, while some non-iguanodontian and rhabdodontid ornithopods have replacement teeth in approximately every second alveolus, Iguanodon bernissartensis and Owenodon hoggii, among others, have them in almost every alveolus70 suggesting that tooth replacement rates increased in ankylopollexians.Although ornithopods were diverse, and their varied, often highly sophisticated feeding-related characters have been studied extensively  or in different parts of the crown 71. This may result from the crown shape being narrower labiolingually than mesiodistally (e.g. in Hungarosaurus tormai). In the latter case, however, the difference in mean thickness of VEIB does not exceed 20–25%. The nearly double-thickness of VEIB in the labiolingual sections of M. vorosi teeth are always on the side of the crown involved in occlusion and where most of the resulting wear occurs . It is hypothesised that the reason for this phenomenon is that the occluding side of the crown had to be mechanically more resistant, which required a greater amount of dentine to be formed on a daily basis on the working side of the crown than on the balancing (i.e. non- or less occluding) side, which is consistent with the thicker enamel also present on the working side of the tooth in the majority of cerapodan ornithischians.As far as we are aware, the different lingual and labial mean tissue thicknesses of VEIB in 72, noted that there are two ways to increase the durability of the dentition: 1) increase the wear resistance of the dental tissues; and/or 2) increase the amount of dental tissue available for wear. An example of the first solution is presented by hadrosaurs and ceratopsians, which convergently increased the complexity of their dental tissues64. The second solution can manifest either as increased tooth size, increased functional crown height  or increased tooth number. An increase in tooth size has been documented in the rhabdodontid Matheronodon provincialis57 and the iguanodontian Lanzhousaurus magnidens74. An increase in relative crown height has been observed to some extent in sauropods75, while an increase in the number of teeth, in terms of accelerated tooth replacement, increased numbers of replacement teeth, and increased tooth counts are known in many dinosaur groups . However, it appears that marked asymmetrical thickening of the dentine component of the tooth crown only occurs in some basal iguanodontians, where tooth replacement has not yet accelerated to the same high rates seen in later-branching taxa, but where continual tooth-tooth occlusion and/or less frequently replaced teeth with longer functional lives require more resistant teeth. Further studies are needed to confirm this hypothesis .In mammalsHungarosaurus tormai and Mochlodon vorosi shows pit dominance in both taxa  microwear analysis of Hungarosaurus tormai, as a ground-level feeding ankylosaur, might have habitually consumed plants somewhat closer to ground-level than the bipedal Mochlodon vorosi. Nevertheless, the degree of pitting is similar in both taxa, which is consilient with their maximum vertical feeding ranges of up to ~ 1 m above ground-level .The small difference in the number of pits seen in the two taxa could be due to the fact that 87, we see that they are most similar to herbivorous, algivorous and frugivorous forms. However, for anisotropy, the ranges for herbivorous lizards are lower than those for the two dinosaurs studied. The DMTA ISO values measured in pterosaurs88 and sauropod dinosaurs89 are not directly comparable to those of the two ornithischian dinosaurs.The low anisotropy and widely scattered complexity values obtained from DMTA for both taxa are consistent with the traditional 2D microwear results. Comparing the complexity values of the two dinosaurs with those of modern lepidosaurs52). However, although the comparisons present in Fig. A comparison of the DMTA values of the two dinosaurs with the tooth wear data of modern ungulate mammals shows that whereas they are most similar to browser and frugivore forms in terms of complexity, for anisotropy we see much higher values and no overlap with mammals see Fig. b. Nevert33; ornithopods91; sauropods94; ceratopsians30). Mallon and Anderson30 reported much lower pit ratio counts in North American ankylosaurids and nodosaurids 94, which is identical to the browse height assumed for H. tormai. Whether, this similar high pit count could be indicative of a similar plant diet, despite the marked differences in feeding-related characters between the two groups , requires further research.Dental microwear studies have only been conducted on a few dinosaurs and focused primarily on basic characterisations of the wear features present  using 2D-micrographs . This suggests that, although there might have been some overlap in browsing height, M. vorosi and hadrosaurids consumed different food plants or plant parts. The high proportion of scratches in hadrosaurids suggests that, in contrast to M. vorosi, they consumed a diet obtained from near ground-level, similar to modern grazers . Conversely, M. vorosi appears to have consumed plants of taller stature, up to 1 m above ground-level, which might have either been tougher or processed more intensively, as also suggested for its close-relative Matheronodon provincialis57. Rather than increasing tooth replacement rates to compensate for wear, M. vorosi seems to have adopted a different strategy in which teeth were invested with extremely thick layers of dentine and a longer functional life (see above).For ornithopods, the numbers of microwear features present are known from only a few hadrosauridsHungarosaurus tormai and Mochlodon vorosi  and the relatively lower degree of wear seen in H. tormai are not only interesting in terms of potential differences in diet, but also with respect to their potential metabolic demands. The estimated body mass (Mb) of M. vorosi is ~ 41 kg101, while that of H. tormai is an order of magnitude greater at 650–688 kg101. When these parameters are considered alongside their different feeding adaptations, several more speculative avenues for further investigation emerge. Firstly, there is wide variation in the digestibility and calorific content of the different plant groups available during the Mesozoic (e.g.103), which might have led these taxa to select particular plant species or plant organs preferentially on the basis of their nutritional value. Secondly, due to its lower Mb, M. vorosi should be expected to have higher mass-specific standard metabolic rates than the larger H. tormai . By analogy with extant herbivorous mammals (e.g.105), this might suggest, in turn, that M. vorosi would have selected more easily digested or more nutritious fodder than H. tormai in order to fuel this increased demand. Thirdly, the extensive tooth wear and robust, thickened tooth crowns of M. vorosi are potentially indicative of greater dependency on oral processing than suggested by the less heavily worn teeth of H. tormai , whereas H. tormai might have used a combination of oral processing and fermentative digestion, with the latter consistent with its greater Mb, lower mass specific metabolic requirements (in concert with longer gut passage times) and greater overall food intake . Finally, it is possible that the bipedal M. vorosi was more active than the quadrupedal and heavily-built H. tormai, which is potentially consistent with the need to release energy and nutrients from its food more rapidly.The results presented above suggest that, in spite of similar microwear characteristics and tooth-replacement rates, the other significant differences in tooth wear between Hungarosaurus tormai and Mochlodon vorosi reveals some similarities in their feeding relating characteristics but also highlights key differences indicative of niche partitioning, which might also indicate differences in dietary physiology. Compared to hadrosaurids and ceratopsids, both of these taxa can be characterized by relatively slow tooth replacement rates. The strongly asymmetrical deposition of dentine in M. vorosi tooth crowns appears to represent a novel solution to enable increased functional life for each tooth that has not been reported in any other ornithischian.Using a broad range of morphological, histological, imaging and statistical methods, our comparative study of dentitions from the sympatric herbivorous dinosaurs Hungarosaurus tormai and Mochlodon vorosi strongly support browsing habits for both taxa, which would have consumed plants that were within the first metre above ground-level. However, the markedly different tooth morphology, tooth structure, patterns of tooth macrowear and jaw mechanics of these taxa suggest that they consumed different plant types or organs, providing a basis for ecological niche partitioning. These differences also seem to indicate that M. vorosi consumed tougher and/or more nutritious plant material and/or processed it more intensively than H. tormai. Moreover, when considered alongside the respective body masses of these taxa, we propose that the more extensive wear of M. vorosi might be consistent with a higher mass specific metabolic rate and/or increased activity levels. In addition, this study demonstrates the value of applying a wide range of different methods to dietary reconstruction provides stronger evidence for disentangling complex ecological signals, which would be difficult to detect if only a single proxy were used.We provide new 2D microwear data and DMTA analyses to complement and expand upon the small number of existing studies on non-avian dinosaurs. The high pit count ratios, low anisotropy and scattered complexity observed in both Supplementary Information 1.Supplementary Information 2.


SRY+), 37,X/38,XY mosaicism, and XX/XY leukocyte chimerism. The remaining 14 affected cats were classified as XY DSD (SRY-positive). In this group and 38 normal males, we analyzed a priori selected candidate genes . Only a previously reported nonpathogenic variant was found in SRY. Moreover, SRY gene copy number was determined, and three variants were observed: 6, 5 , and 4 copies in a single DSD case. The known variants in TAC3 and CYP11B1, responsible for testicular hypoplasia, persistent primary dentition or congenital adrenal hyperplasia, were not found in the study group. Nine novel polymorphisms were identified in the LHCGR gene, one of which, a potentially regulatory indel variant in 5′UTR, was significantly associated (p = 0.0467) with XY DSD. Our report confirmed that abnormalities of sex chromosomes are important causes of feline DSDs. We also showed that the indel variant of LHCGR can be considered a promising marker associated with XY DSD phenotype.The genetic background of feline disorders of sex development (DSDs) is poorly understood. We performed comprehensive cytogenetic, molecular, and histological studies of 17 cats with abnormal external genitalia, unusual behavior, or tricolor coats . The DSD phenotype of three cats was associated with sex chromosome abnormalities: X/Y translocation (38,XX It is well known that DSDs have a negative impact on fertility, increase the risk of gonadal cancer, and can affect behavior. The genetic background of these disorders is heterogeneous and three major categories can be distinguished, based on the observed sex chromosome complement: (a) DSD due to abnormal sex chromosome complement, (b) XX DSD: animals with a normal XX set of chromosomes, and (c) XY DSD: animals with a normal XY set of chromosomes3. Primary classification to the appropriate type of DSD is based on the clinical examination of external and internal genitalia, followed by cytogenetic analysis and molecular detection of Y-linked SRY gene, which plays a crucial role in male development1.Disorders of sex development (DSDs) are a serious health issue in mammalian domestic animals, including cats9. However, the molecular background of DSD in cats with a normal set of XX or XY chromosomes has been poorly elucidated. Although a number of candidate genes were evaluated in XY cats with altered external genitalia 13, to date only two causative mutations have been reported. A missense G > A SNP (Arg > Gln) in exon 7 of the CYP11B1 gene, involved in the biosynthesis of adrenal corticoids, has been found to contribute to congenital adrenal hyperplasia and associated gynecomastia, empty scrotum, and aggressive behavior in a tomcat with a normal 38,XY chromosome set14. The second causative variant was located in the TAC3 gene encoding tachykinin 3, which is a precursor of the neurotransmitter neurokinin B. In a domestic shorthair tomcat with testicular hypoplasia, one testicle in the scrotum, primary dentition, and small body size, a homozygous genotype for a missense substitution  was identified13. Genetic screening for this variant in a cohort of 171 randomly selected control cats revealed two carriers. It is known that mutations of this gene are also responsible for DSD phenotype in humans15.There have been number of reports dealing with sex chromosome abnormalities in DSD cats, including X monosomy, XXY trisomy, XX/XY leukocyte chimerism, 37,X/38,XY mosaicism, and two cases of structural rearrangements involving sex chromosomes—namely 37,X/38,X,r(Y) and 38,XX.ish der(X)tSRY gene is a key regulator of male sex determination in mammals and its mutations are well known causes of DSD in males16. So far, only one polymorphism in the coding sequence of this gene has been identified in cats; this is a missense c.389G > C substitution (p.Arg130Thr), but was excluded as a cause of feline DSD based on its distribution17. On the other hand, there is no information about the variability of SRY copy number in cats, while recent study showed that the presence of two copies of the SRY gene, rather than the three copies commonly observed in dogs and wolves, may be associated with XY DSD in Yorkshire terriers18.The Y-linked LHCGR gene, which encodes luteinizing hormone/chorionic gonadotropin receptor, is another candidate gene for DSD in mammals. In XY DSD human patients, mutations that inactivate LHCGR are responsible for female external phenotype  or the incomplete virilization of external genitalia , as well as for a range of penile defects such as micropenis and hypospadias19. DNA variants of LHCGR have not been studied previously in DSD cats.The SRY, TAC3, CYP11B1 and LHCGR).In this study, we aimed to carry out comprehensive cytogenetic and molecular studies of seventeen DSD cats with abnormal external genitalia, unusual tricolor coats in males, and abnormal sexual behavior. All cats were cytogenetically examined in order to establish their sex chromosome complement, while the molecular analysis focused on four a priori selected candidate genes . All experiments were carried out in compliance with the ARRIVE guidelines and all methods were performed in accordance with the relevant guidelines and regulations.Altogether seventeen cats with ambiguous external genitalia, abnormal behavior or tricolor coat selected by veterinarians were included in this study Table . Five DSGonadal samples were fixed in 10% formalin, processed, and embedded into paraffin blocks. The 3 µm serial sections were stained with hematoxylin and eosin. The slides were analyzed on an Axio Lab.A1 microscope  equipped with an Axiocam ERc5s digital camera  or on Olympus BX53 light microscope coupled with an Olympus UC90 camera.in situ hybridization (FISH) with feline X and Y chromosome painting probes  and a locus-specific BAC (RP86-278G21) probe specific to the SRY gene, obtained from the Feline BAC library RPCI-86 (https://bacpac.chori.org/). The probes were labelled with biotin-16-dUTP or digoxigenin-11-dUTP by DOP-PCR and detected using streptavidin‐Cy3   or anti-digoxigenin-fluorescein (Roche Applied Science) . The chromosomes were counterstained with Vectashield containing DAPI (Vector Laboratories). Microscopic evaluation was carried out under a Nikon E600 Eclipse microscope , equipped with a cooled CCD digital camera and Lucia software. For each individual, at least thirty metaphase spreads were analyzed.Chromosome preparations were obtained from lymphocyte culture or skin fibroblast culture using standard procedures. The chromosome complement was analyzed by Giemsa staining and fluorescence SRY and ZFY genes and the X-linked ZFX gene were amplified using the primers shown in Table S220. The SRY gene underwent PCR detection (1022 bp band) and its entire coding sequence was sequenced for the DSD and control cats. The amplicons for ZFY and ZFX (448 bp) were digested with restriction enzyme (BsmI) for all DSD cats. The use of 2% agarose gel electrophoresis allowed us to distinguish ZFY (448 bp band) from ZFX (391 bp and 57 bp bands). Moreover, to verify the size of the translocated region from the Y to the X chromosome, we additionally amplified the Y-linked genes TETY1, TETY2, CUL4BY, CYORF15, HSFY, FLJ36031Y using the primers shown in Table S221. The selected genes are located on short or long arm of the Y chromosome, as it was shown by Wilkerson et al.22 and Li et al.23.Genomic DNA was isolated from peripheral blood using a Blood Mini Kit (A&A Biotechnology) and additionally from fibroblasts for case #7513, with the use of a Genomic Mini Kit (A&A Biotechnology). The Y-linked HpaII restriction enzyme (20U/reaction) at 37 °C for 15 h, followed by heat inactivation at 95 °C for 10 min. The use of HpaII enzyme assured that the active (nonmethylated) X chromosome will be digested, and thus that PCR amplification will be possible only from the inactive (methylated) X chromosome, as described by Mochizuki et al.24 The PCR was performed for the fragment (exon 1) of the X-linked androgen receptor (AR) gene, in which a short tandem repeat (CAG)n polymorphism is present and the study animals were heterozygous in terms of the number of CAG repeats. Standard amplification conditions  were used with the primers shown in Table AR gene) was measured using ImageJ software and calculated for particular peaks as a percentage from a sum for both alleles, which represented 100%. The analysis was performed for the 38,XXSRY+ case (#7318) and for two control healthy females.DNA from blood was isolated with the use of a MasterPure DNA Purification Kit for Blood (Epicentre). The 2 µg of DNA was digested with methylation-sensitive Genotypes for fourteen STR (short tandem repeats) markers  were obtained for cat #7355 and its alleged offspring, in order to verify paternity. The same markers, including CATAMEL and CATZFXY, were used to verify chimerism in the blood leukocytes and hair follicle samples from case #7513. The analysis was performed by an external service .https://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) to amplify exon 3 of the TAC3 gene that harbored the causative SNP c.220G > A13, exon 7 of the CYP11B1 gene that comprised the missense G > A SNP (Arg > Gln substitution)14, all eleven exons of the LHCGR and the entire coding sequence of the SRY gene  and LHCGR with PyroMark PCR kit (Qiagen). The amplicons were purified using FastAP Thermosensitive Alkaline Phosphatase (Thermo Scientific) and Exonuclease I (Thermo Scientific), and the mixture of fragments obtained using BigDye Terminator v3.1 (Applied Biosystems) was filtered on a Sephadex G-50 (Sigma) and separated on 3130 Genetic Analyzer (Applied Biosystems). Chromatograms were aligned to the reference genomic sequences  and analyzed using SeqMan software (DNASTAR). The odds ratio test was used to detect DNA variants associated with an increased risk of DSD.PCR primer pairs were designed using the Primer3 tool  and 5′-regulatory (− 201 to − 300 bp upstream from ATG) regions of the SRY gene. The F2 gene was used as a reference gene. The X and Y copy numbers were estimated for case #7513 through copy number analysis of AMELX (X-linked) and AMELY (Y-linked) in order to detect the XX and XY lines in leukocytes and in the in vitro cultured fibroblasts, following the procedure described elsewhere25 (Table HaeIII (New England BioLabs) and EcoRI (Thermo Scientific) restriction enzymes for SRY and AMELX/AMELY assays, respectively.Droplet digital PCR (ddPCR) was used to estimate 25 Table . Each ddThe PCR reactions were partitioned into approximately 20,000 droplets using a QX200 Droplet Generator (Bio-Rad). PCR was performed on a T100 Thermal Cycler (Bio-Rad) using the following thermal cycle conditions: denaturation at 95 °C for 10 min; 40 cycles at 94 °C for 30 s and 58 °C for 60 s (ramp rate 2 °C/s); 98 °C for 10 min, and 10 °C until reading time. Droplets were detected and analyzed on a QX200 Droplet Reader (Bio-Rad). The number of copies was calculated using the Poisson distribution with QuantaSoft Software (Bio-Rad).26. The potential effect of the detected variants in the 5′UTR region within target sequences for RNA binding proteins was predicted using RBPmap software27. Due to location of the LHCGR gene on the complementary DNA strand in A3 chromosome, all bioinformatical analyses were performed for the DNA strand complementary to the reference genome.Transcription factor- binding sites (TFBS) disturbed by variants in the DNA sequence were designated using PROMOv3.0.2 softwareSRY gene were observed, and they were thus considered as XY DSDs (SRY-positive).Among the seventeen DSD cats we studied, an abnormal sex chromosome complement was found in three cases, which were considered cases of sex chromosome DSDs. In the remaining fourteen cases, a normal male sex chromosome set  and the SRY-specific probe. The signal was visible in all metaphase spreads, so the karyotype was described as 38,XXSRY+. Further molecular analysis confirmed the presence of several genes  located on a short arm of the Y chromosome (Yp), while no genes from the long Yq arm  were detected  gene. It was shown that both X and XSRY+ chromosomes were randomly inactivated. The X-inactivation ratio was 64:36 in the study case and similar in two control females—59:41 and 63:37, respectively  and Y-linked genes (SRY and ZFY) in blood leukocytes  in blood, while the Y-linked genes were not present in the DNA from fibroblasts  were present in blood while absent in DNA from hairs , analyzed using FISH, had two cell lines, 38,XX—41%, [95% confidence interval ] and 38,XY—59%, [95% confidence interval ], in leukocytes and a single cell line  in fibroblasts . The sequencing of the entire SRY coding region revealed the presence of a known missense c.389G > C (p.Arg130Thr) substitution, which occurred in XY DSD and control cats with high frequencies of 0.71 and 0.58, respectively  in exon 7 of the CYP11B114. A single DSD case (#6664) was heterozygous for a synonymous c.1170G > A  substitution but this variant was assumed as not being responsible for DSD due to lack of its effect on the CYP11B1 amino acid sequence.We next searched for the known causative nonsynonymous SNP  in the LHCGR gene, was analyzed. We found a total of nine novel variants in the sequence and their positions in A3 chromosome are shown in Table in silico analysis showed that the g.70455376delC variant in 5′UTR impairs the binding of following RNA-binding proteins (RBPs): RBM4, RBM4B, SRSF10, SRSF2, SRSF8. To test whether these potentially pathogenic variants might be associated with DSD, we compared their distributions in the XY DSD and control cohorts. The odds ratio (OR) test showed that the del variant in the 5′UTR (g.70455376delC) was significantly associated with an increased risk  of DSD  includes over one hundred causative mutations, but only two of these are associated with DSDs. Despite this, abnormalities of sex chromosome complement in DSD male cats are quite frequently reported . This could be due to the genetic background of the orange coat color, which is controlled by a dominant X-linked gene. In females heterozygous at this locus, a tricolor coat is observed due to the random inactivation of one X chromosome. The occurrence of the tricolor coat pattern in males suggests an abnormality in the sex-chromosome complement. In male cats, tortoiseshell and calico coats are frequently seen to cooccur with XXY syndrome8 but the rare phenomenon of whole-body chimerism may also lead to a tricolor coat, as shown in a fertile tortoiseshell male cat with true chimerism 29. It should be mentioned that this type of coat can also be observed in DSD virilized females with leukocyte XX/XY chimerism and in carriers of X/Y translocation leading to the transfer of SRY gene from the Y to the X chromosome  or even in very rare cases of 38,XXSRY− males12.Extension in our knowledge of feline genome organization has facilitated the identification of numerous gene mutations that cause hereditary diseases5 It is worth mentioning, that a similar abnormality  exists in humans and is also rare ; this is called de la Chapelle syndrome30. However, in humans it is caused by unequal crossing over between the X and Y chromosomes, since the SRY is located very close (~ 11 kb) to the pseudoautosomal region (PAR) in the Y chromosome. Feline SRY is, however, located approx. 2 Mb from the PAR as it was presented by Szczerbal and Switonski1.We have here described the second case of X/Y translocation; to best of our knowledge, this type of abnormality in domestic animals has only been found to date in two cats—the present case and a cat reported earlier by Szczerbal et al.9, but the proportion of cell lines was different at 37,X[96%]/38,XY[4%]. This could be responsible for the differing phenotype of our case, which displayed feminine behavior, testes with degenerated spermatogenesis, and rare sperm. Interestingly, this cat has a single offspring, as we confirmed with molecular parentage testing. In the cat described by Balogh et al.9, the testes were not capable of spermatogenesis. Since both cases were recognized as males, it can be supposed that the observed X/XY karyotype was the result of Y chromosome instability during mitotic cell divisions. Such a mechanism was probably also responsible for the disorder of sexual development in a cat with chromosome mosaicism 37,X/38,X,der(Y)6.The second chromosome abnormality described here was a mosaicism  with a predominant 38,XY cell line (90%). Interestingly, the same type of mosaicism has previously been described in a Bengal cat with cryptorchidism and Müllerian duct remnants7 Both cats had a vulva, a rudimentary penis, and abdominally located testicles. The proportion of XX/XY cell lines was also similar , but it is known from studies of such abnormalities in other species (such as cattle and dogs) that there is no correlation between the percentage of XY cells and the extent of the virilization32. We also used ddPCR to quickly and reliably detect the XX and XY cell lines in cats; this approach has a proven usefulness for diagnosis of these type of sex chromosome DSD in cattle and pigs33.The sexual phenotype of the third case, which carried leukocyte XX/XY chimerism, was similar to the previous one reported by Szczerbal et al.34. Of these, mutations of SRY are responsible for at least 15% of cases of GD. In the present study, we found a common missense variant of this gene, which was described earlier by Nowacka-Woszuk et al.10 We also examined the variability of the SRY copy number. It has previously been reported that the feline genome contains a single copy of SRY23, but we have shown here for the first time that the copy number in fact varies between four and six, with a modal number of five. This variability did not show any association with the DSD phenotype in cats. It has recently been reported that a smaller number of SRY copies (two rather than three) may be associated with DSD in Yorkshire terrier dogs18.Of the DSD cats we examined, the majority were XY DSD, and most presented a variable degree of gonadal dysgenesis (GD). This can be caused by or associated with numerous gene variants, as has been observed in humansTAC3, due to the deleterious effect of missense substitution  on testicle development in cats13. We sequenced exon 3 harboring this variant in a cohort of fourteen XY DSD cats, but all of them had normal genotype (GG). Mutations of TAC3 are also quite rare causes of human XY DSD, with the OMIM database containing only a few reports of hypogonadotropic hypogonadism 10, with or without anosmia, caused by mutation of this gene (OMIM #614839). Small testicles and micropenis are observed in such patients36. On the other hand, CYP11B1 mutations may lead to impaired biosynthesis of cortisol and altered level of androgens. In humans they are implicated in 5-8 % of cases of congenital adrenal hyperplasia (CAH), but in males their clinical manifestations usually do not involve external genitalia37. Interestingly, a single cat was described previously with symptoms of CAH, including abnormalities of the genitourinary system, caused by 11β-hydroxylase deficiency due to a missense substitution in exon 7 of CYP11B114. We showed that this variant was not present in the analyzed DSD cats.The next selected candidate gene was LHCGR gene: of nine novel DNA variants, the deletion of G nucleotide, present in 5′UTR, showed an association with XY DSD phenotype. It is worth mentioning that the two DSD cats with the del/del genotype had similarly abnormal sexual phenotypes. Bioinformatic analysis of the sequence harboring this variant revealed that the delG variant impairs binding of the regulatory RNA-binding proteins, which may affect the expression of LHCGR. Studies of knock-out mice with disrupted LHCGR promoter regions showed underdevelopment of internal and external genitalia, including micropenis, abdominal testes, hypertrophy of Leydig cells, and spermatogenic arrest at the round spermatid stage38. The importance of LHCGR mutations has been documented by studies of numerous causative variants, mainly missense substitutions, in humans19.The most interesting molecular observations concerned the 40, or whole genome sequencing (WGS)—would greatly facilitate the search for causal variants for XY DSD in cats.Our study showed that XY DSD cats form a heterogeneous group and that there are no common pathogenic mutations. Studies on large cohorts of affected animals with the same abnormal phenotype should thus be conducted. It can be anticipated that a comprehensive method for analyzing entire genomes—such as dense genotyping array, whole exome sequencing (WES)SRY+, 37,X/38XY, XX/XY leukocyte chimerism). The second group of affected animals consisted of XY DSD cohort, and we were able to exclude known mutations in TAC3 and CYP11B1 as a cause of their DSD phenotype. On the other hand, we showed that a single nucleotide deletion in the regulatory sequence of the LHCGR gene is a promising marker associated with DSD phenotype in some cases. Finally, we detected for the first time an elevated number of SRY copies in normal and DSD cats, but we did not observe an association between the copy number and DSD phenotype. Our study thus extends the knowledge of the genetic background of DSD in cats.In this study, we analyzed a large cohort of cats with complex abnormalities of sex development. Among the studied cases, sex chromosome DSDs were quite frequent, with three different chromosomal abnormalities found (38,XXSupplementary Information.


BRCA1/2 mutations. 63 women completed the Pittsburgh Sleep Quality Index (PSQI) and Godin Shepard Leisure-Time Physical Activity Questionnaire (GSL-TPAQ) and were included in the present cross-sectional analysis. Globally, women showed a PSQI score of 7.0 ± 3.6 and a GSL-TPAQ score of 22.8 ± 18.3. Good sleepers (PSQI score ≤ 5) showed significantly higher PA levels compared to bad sleepers (PSQI score > 5). Women in the higher tertile of GSL-TPAQ total score (≥ 27 METs/week) have a prevalence ratio (PR) of being a good sleeper of 2.85  compared to women in the lower tertile (≤ 11 METs/week). These results were consistent in BRCA1 and BRCA2 women. Considering each single question of PA intensity, the PR of being a good sleeper by unit of increase of MET/week was higher and significant in women engaged in strenuous and moderate intensity PA. These results suggests a direct association between PA and sleep quality in women carriers of BRCA mutations.The aim of the present study is to explore the potential association between sleep quality and physical activity (PA) in women carriers of  BRCA1/2 mutations have a 55% lifetime risk (penetrance) of developing breast cancer (BC)1 and the percentage is even higher at age 80 with 72% (95% CI 65–79%) for BRCA1 and 69% (95% CI 61–77%) for BRCA2 mutation carriers2. In these women the penetrance for ovarian cancer (OC) is also higher, in the order of 16–59%3. Epidemiological studies have suggested that the penetrance of BRCA1/2 genes may be modulated by a number of lifestyle factors. Greater body weight and life-long weight gain have been found associated with an increased BRCA penetrance6 especially for post-menopausal BC8 and for OC9. Consistently, higher fat mass, severe metabolic syndrome (MS) and high serum levels of insulin and insulin-like growth factor-I (IGF-I) affect BRCA penetrance10.Women carrying 14 and are co-morbid with the MS. The association between sleep and BC may be explained by misalignment of the molecular clock circadian rhythm that decreases melatonin secretion, an oncostatic agent and estrogen suppressor17 and by altering the regulation of glucose tolerance and insulin-sensitivity19. Sleep problems or bad sleep quality increase after BC diagnosis and treatment and are more common in BRCA1/2 mutation carriers compared to general population20. In fact, sleep disturbances are very frequent post-surgical symptoms  in the BRCA carriers who chose risk reducing surgery for primary prevention21.In recent years, sleep disorders are emerging environmental factors that influence the risk of BCBRCA1/2 mutations24.Physical activity (PA), especially during adolescence or early adulthood, affects BC risk in women with 28. PA has a protective effect on sleep in both healthy subjects and cancer patients, including sporadic BC women30. PA is a synchronizer of the circadian rhythms by increasing the activity of sympathetic nervous system that modulates melatonin secretion31.PA improves sleep quality and quantity and reduces obesity and MS parameters32.Furthermore, high intensity exercise upregulates the activity of peroxisome proliferator-activated receptor g (PGC-1a) that regulates the expression of the clock gene family, that are involved in sleep behaviour and insulin sensitivityBRCA1/2 mutations. In the present paper we aim to explore the potential relationship between sleep quality and PA in a cohort of BRCA women who joined our dietary intervention trial37.Despite the aforementioned evidence, there are no studies on the association of PA and sleep behaviour in women carriers of BRCA1/2 mutations (reference number: NCT03066856) and its main results have been already described and reported37. In brief, the trial aimed to test if a 6-month dietary intervention based on the Mediterranean diet (MedDiet) with a moderate protein restriction significantly reduces potential modulators of BRCA1/2 penetrance such as body weight, IGF-I, insulin and main factors of MS. Eligible subjects were women aged 18–70 years, carriers of BRCA1/2 mutations, with or without a previous diagnosis of breast and/or ovarian cancer and without clinical evidence of metastases. Unaffected women who underwent bilateral prophylactic mastectomy did not enter the cohort study. All participants signed informed consent, filled questionnaires about their medical history and dietary habits, underwent anthropometric and body composition assessment and donated 20 mL of a blood sample. Anthropometric and body composition measurements, blood samples and dietary data were provided at baseline and the end of the dietary intervention. Among the 502 volunteers randomized into the trial, 63 women recruited between October 2018 and June 2019 also reported at baseline, before starting the dietary intervention, data on sleep and PA habits and were included in the present cross-sectional analysis.The design of the Italian multicenter dietary intervention trial on women carriers of The Ethics Committee of Fondazione IRCCS Istituto Nazionale dei Tumori di Milano approved the study .39 were filled by the volunteers included in the present analysis.Two additional questionnaires for the assessment of sleep and PA habits good sleepers (0–5) from bad sleepers (6–21). In addition, the numbers of hours spent in bed and sleeping and the sleep efficiency can be obtained from the questionnaire40.The PSQI assesses sleep quality and habits in the previous 30 days. This is a retrospective self-report questionnaire, based on 19 items, in order to evaluate seven sleep components: (i) perceived sleep quality, (ii) sleep latency, (iii) sleep duration, (iv) sleep efficacy, (v) sleep disturbances, (vi) use of sleep medications, (vii) daytime dysfunctions. The score for each question ranges between 0  and 3 . The sum of the seven components returns a final score from 0 to 21, with lower scores indicating a better sleep quality. The cut-off value of 5 divides participants into 39 assesses the amount of leasure time PA performed in the last seven days by including three questions about frequency of PA (number of activities/week). Each question refers to PA of different intensity  which corresponds to specific Metabolic Equivalent of Task (MET) amount. Mild PA corresponds to 3 METs, moderate PA to 5 METs and Strenuous PA to 9 METs. The total GSL-TPAQ score is obtained using the following formula: (frequency of mild × 3) + (frequency of moderate × 5) + (frequency of strenuous × 9). If the final score is < 24, the subject is classified as inactive, while in the opposite case (≥ 24) is described as active39.The GSL-TPAQBRCA1/2 using frequencies or means and standard deviation (SD), and compared using χ2 or t tests, as appropriate.General characteristics of the study population were summarized by good (PSQI score ≤ 5) and bad sleepers (PSQI score > 5). Women’s metabolic and anthropometric characteristics were therefore summarized and compared with ANCOVA controlling for age and BMI. Since stratifying the population by disease status (affected and unaffected) or by age  we did not find any significant difference for all the variables under study, we decided to perform the analyses in the whole population and by BRCA mutation type.By using the cut-off of the PSQI score, the study women were stratified into As regards GSL-TPAQ, we considered both the total score and the results for each question of different PA intensity  separately in all the analyses.Spearman coefficient) among all anthropometric, metabolic, sleep and PA data.We conducted a correlation analysis  in the whole population and in BRCA1 and BRCA2 women, controlling for age and BMI. We also repeated the PR analysis by MET/week increase taking into account each question of PA intensity .Since the aim of the present study was to describe potential associations between PA and sleep quality, we performed a binomial regression model to estimate the prevalence ratios (PRs) and 95% confidence intervals (CI) of being a A p-value of < 0.05 was taken as significant. The statistical analyses were carried out using STATA 14  and IBM Statistical Package for the Social Sciences—SPSS Statistics version 27 .The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee of Fondazione IRCCS Istituto Nazionale dei Tumori di Milano .Informed consent was obtained from all subjects involved in the study.BRCA1 and BRCA2 are reported in Table BRCA 1/2 mutations , 39 with BRCA1 and 24 with BRCA2 mutation, were included in the present investigation. The distribution of general characteristics was homogeneous between BRCA1 and BRCA2 women. Taking the whole population, women presented on average a PSQI score of 7.0 ± 3.6 and a GSL-TPAQ score of 22.9 ± 18.3. As regards sleep quality and PA levels we did not found any significance difference due to the BRCA mutations.The general characteristics of the study population by good  and bad sleepers  in the whole population and in BRCA1 and BRCA2 women. Referring to anthropometric and metabolic parameters, bad sleepers showed a slightly worse condition compared to good sleepers without any significant result. In the whole population, we observed that bad sleepers showed a significantly lower total GSL-TPAQ score. These results were consistent also in BRCA1 and BRCA2 mutation carriers. As regards the single question of different PA intensity , we found that bad sleepers showed significantly lower levels of strenuous, moderate and mild PA compared to good sleepers . As regards the stratification by BRCA mutation type we observed consistent results. However, in BRCA1 women the results were significant only for moderate levels of PA while in BRCA2 we observed significant results for strenuous and mild PA.Table Correlation analysis were performed to study the association between sleep quality, PA and anthropometric and metabolic parameters.bad sleep quality, was significantly correlated with higher triglycerides levels . About PA, higher GSL-TPAQ score, which indicates greater PA levels, was significantly correlated with lower values of weight , BMI , waist circumference , hip circumference , insulin , triglycerides levels , fat mass in % , fat mass in kg , and PSQI score . Taking into account the single question of PA intensity, strenuous intensity PA was significantly correlated with lower values of weight , BMI , waist circumference , hip circumference , waist/hip ratio , systolic , diastolic blood pressure , triglycerides , fat mass in %  and fat mass in kg . Even though not significant, strenuous intensity PA showed a tendency to be correlated with lower PSQI score . Moderate intensity PA was significantly correlated with lower PSQI score . Mild intensity PA was significantly correlated with lower waist circumference , but showed no significant correlation with PSQI score .As regard sleep, we observed that a higher PSQI score, which corresponds to good sleeper by considering the increasing tertiles of GSL-TPAQ total score. We observed that women in the higher tertile of GSL-TPAQ total score (≥ 27 METs/week) have a greater and significant PR of being a good sleeper compared to women in the lower tertile (≤ 11 METs/week). These results mean that women who practice strenuous PA for at least 3 times/week (or practice moderate PA 6 times/week) have about three times higher probability to have a better sleep quality. These results were consistent in both BRCA1 and BRCA2 even without any significant association.Table good sleeper by unit of increase of MET/week  have a greater and significant PR of being a good sleeper compared to women in the lower tertile (≤ 11 METs/week). The results were consistent also stratifying the population by BRCA mutation type. Furthermore, taking into account each single question of PA intensity, the PR of being a good sleeper by unit of increase of MET/week was higher and significant in women engaged in strenuous and moderate intensity PA. In contrast, mild intensity PA seemed not to significantly influence sleep behaviours.This cross-sectional analysis suggests a direct association between PA and sleep quality in 63 women carriers of 43.To our knowledge, this is the first investigation assessing the potential influences of PA on sleep behaviour in this population. Previous studies in sporadic BC patients suggested the role of PA on the improvement of sleep behaviour (assessed both with actigraphy and questionnaires) and on the reduction of sleep deficiency at different stages of the BC pathology44. Quality of life usually decreases together with sleep after BC surgery and/or treatments, and their upgrading represents a challenging point for researchers46. The reasons explaining the bad sleep quality could be several: distress and anxiety of developing a very aggressive pathology or for the family's future; pain and hot flashes in consequent to surgery or treatments; fatigue that reduce the possibility to be sufficiently active47.Sleep quality in BC patients is essential for their quality of life. Indeed, BC diagnosis and treatments usually reflect on sleep as a long-term and side-effect symptom48. It also alters cellular signalling associated with mitochondrial respiratory function, insulin/IGF-I signalling32 and diminishes melatonin secretion, enhancing estrogen secretion51. Furthermore, sleep disorders decrease carbohydrate tolerance, alter leptin and cortisol levels53, thus modifying insulin sensitivity and favouring obesity and MS. For these mechanisms, sleep might represent one of the environmental factors involved in the modulation of BRCA penetrance. In this cross sectional analysis we decided not to report results by disease status because affected women showed a slightly worse sleep quality compared to unaffected, but without any significance result. Furthermore, the nature of the analysis and the small numbers of our study would not allow to draw conclusions about a potential effect of sleep on BRCA penetrance.Sleep deficiency has been linked to increased inflammation and impaired immune responseBRCA1/2 women. Increasing PA levels and modifying daily habits in favour of a more active lifestyle has been among the leading research goals in the BC field also in order to improve sleep. The mechanisms on this linkage are not yet fully understood; however, studies from healthy subjects indicated that PA could exert its beneficial action on sleep through body temperature regularization, energy conservation, decrement in anxiety, stress, endocrine function, obesity and immune-inflammation response58. A regular and constant PA practice is recognized to improve body weight, BMI and other anthropometric parameters, insulin and IGF-I levels, adipokines balance, and sexual hormones secretion59. In our study, either total PA or strenuous intensity PA levels were correlated with lower weight, BMI, waist circumference, hip circumference, fat mass and insulin levels, suggesting a positive association also in BRCA1/2 women.This cross-sectional analysis suggests that PA may be a tool for improving sleep quality in 10.The results of the present analysis should be seen in view of their limitations and strengths. Limitations may be attributable to the small sample size and to the use of questionnaires instead of objective assessment instruments for the all population (such as actigraphy). We are aware that is recommended to collect both, subjective and objective evaluations of sleep behavior and PA levels, but this was not foreseen by the MedDiet trial which had different aimsA further intrinsic limitation of this study is the cross-sectional design that does not allow to distinguish the cause from the effect. However, the intention of this paper was mainly to explore the sleep and PA data of the BRCA women who joined our MedDiet trial rather than to give an interpretation to the associations we found.10 demonstrated that a dietary intervention in women carriers is feasible and effective in reducing metabolic and anthropometric parameters. Additional recommendations about PA for a more active lifestyle might be probably useful for the clinical management of this special group of women. These results represent therefore the seed for continuing the recruitment to broaden the sample and to evaluate the association between PA and sleep in a larger population of BRCA mutation carriers.Despite the limitations, the preliminary results of the present analysis suggest new aspects to study in order to improve the quality of life in BRCA mutation carriers. The MedDiet resultsBRCA1/2 mutations, highlighting that higher PA levels are associated with a better quality of sleep behaviour. In this perspective, future studies involving PA interventions are needed to improve sleep and quality of life in women predisposed to hereditary BC.The present study suggests an association between PA and sleep in women carriers of