Panthera pardus) persist outside protected areas throughout most of their range, occupying diverse habitat types and land uses, including peri-urban and rural areas. Understanding of leopard population dynamics in mixed-use landscapes is limited, especially in South Africa, where the majority of leopard research has focused on protected areas. We use spatially explicit capture-recapture models to estimate leopard density across a mixed-use landscape of protected areas, farmland, and urban areas in the Overberg region of the Western Cape, South Africa. Data from 86 paired camera stations provided 221 independent captures of 25 leopards at 50 camera trap stations with a population density estimate of 0.64 leopards per 100 km2 (95% CI: 0.55–0.73). Elevation, terrain ruggedness, and vegetation productivity were important drivers of leopard density in the landscape, being highest on elevated remnants of natural land outside of protected areas. These results are similar to previous research findings in other parts of the Western Cape, where high-lying natural vegetation was shown to serve as both a refuge and a corridor for leopard movement in otherwise transformed landscapes. Given the low leopard density and the prevalence of transformed land intermixed with patches of more suitable leopard habitat, prioritising and preserving connectivity for leopards is vital in this shared landscape. Ecological corridors should be developed in partnership with private landowners through an inclusive and multifaceted conservation strategy which also incorporates monitoring of and rapid mitigation of emerging threats to leopards.Large carnivores face numerous threats, including habitat loss and fragmentation, direct killing, and prey depletion, leading to significant global range and population declines. Despite such threats, leopards ( Housing and food requirements to support the global human population of over 8 billion people place extreme pressure on land and natural resources. Over half of the planet’s land is shared by both wildlife and people . Large cPanthera pardus) are the most widespread large carnivore and can survive across varied land use types outside of protected areas, attributed to their adaptability and opportunistic hunting strategy [Leopards . The topography varies from 1,300 m (Cape Fold Mountains) to sea level along the southern coast of South Africa. There are 17 types of vegetation in the study area, of which 10 are threatened  , 52 S2 .2 and Hermanus mixed landscape = 86.7 km2 [A camera trap survey consisting of 86 paired camera stations was active for 160 days (19 August 2021 to 25 January 2022). Sufficient spatial recaptures of individuals are essential for precision and accuracy in density estimates , but attCamera stations consisted of paired Cuddeback C1 Strobe Flash 1279 trail cameras set at a height of 30–40 cm on opposite sides of a pathway to obtain an image of both the left and right flanks of animals. Camera locations were chosen to maximise leopard detections, and were placed on least resistance pathways , ‘natural funnels’ created by the local topography, and where there was signs of leopard presence including scat, spoor, and tree markings. At six-week intervals the camera traps were serviced to maintain functionality, and vegetation was cleared in front of each camera.Only adult leopards were included in the analysis. Individual leopards were identified by their unique pelage pattern on the left and right flanks using the program Hotspotter , 57. An http://mcmc-jags.sourceforge.net). In data augmentation, we set M to 200 –larger than the largest possible population size . Buffer sizes around the trapping grid were plotted to test for density stabilisation [2) cells, and we included a buffer of 12 km beyond the outermost camera stations and excluded cells with centroids located in pixels classified as water  approach. Leopards exhibit sex-specific differences in space-use and behaviour, with the home range of a single adult territorial male overlapping with smaller home ranges of several females . Locatiolisation . The habas water . The 12 We used the centroids of capture locations of individual animals caught as the starting values for activity centres, ensuring these occurred in suitable habitat as defined in the habitat mask. We implemented three Markov Chain Monte Carlo (MCMC) chains with 60,000 iterations, a burn-in of 1,000, and a thinning rate of 10 to ensure an adequate number of iterations to characterise the posterior distributions. We checked chain convergence using the Gelman-Rubin statistic , R-hat, For leopard density surface modelling, we used maximum-likelihood SCR density models with covariates in the package ‘secr’  in R, ve0  and σ . To avoid models converging on local maxima, models were rerun with the starting values as the output of previous runs. Relative statistical support was selected using Akaike’s Information Criterion corrected for small sample sizes (AICc). Models that had AICc scores below seven relative to the best performing model were considered to show adequate support [Leopard density was also estimated using a maximum likelihood approach in the package ‘secr’  in R, ve support The 86 camera stations were active for 13,500 camera trap nights. Leopards were detected at 50 of the 86 camera stations (58%). Independent captures (a sampling occasion is 24 hours) of leopards totalled 221 occasions and included 25 individual adult leopards . Seven juveniles and three unidentified individuals were captured on cameras but excluded from further analysis. In the first 100 days of the survey, 92% of individual adult leopards were detected  was comprised of patches of protected land. Natural land made up most of the study area (63%) followed by agricultural (35%) and urban (2%). Camera traps were placed on eight different vegetation types  and ranged across the study area with the lowest densities near the coast (The Bayesian p0(sex+trap)sigma(sex) model fitted well to the data , and R-hat values for all model parameters were below 1.1. Leopard population density was estimated as 0.64 leopards per 100 kmhe coast . Female he coast .0. AICc scores revealed that MD7, MD5 and MD1 all have support  was 0.0140 (95% CI: 0.0088–0.0222) and 0.0245 (95% CI: 0.0148–0.0404), respectively. MD7 showed adequate support and therefore, sex was used as a covariate on σ and λ0 for inhomogeneous models. MD7 was chosen over MD6, as the model contains fewer parameters, and therefore the inhomogeneous density models performed better with only sex as a covariate on σ and λ0.Using a maximum likelihood approach, the best performing model, MD6, includes the covariate sex on the detection parameter σ, and a combination of sex, vegetation type and the interaction on λAll individual covariate density models showed some support. Elevation, terrain ruggedness, and NDVI showed high support (dAICc < 2); with human density and protected status showing intermediate support (dAICc < 7) and land-use category and vegetation bioregion the least support . The besThe coefficient of elevation (β = 0.58) indicates a positive relationship between elevation and leopard density . TerrainThis study’s estimate of 0.64 leopards per 100 km2 falls in the middle of density estimates for the Western Cape region  and caracal  in a mixed land-use region of the Western Cape revealed that both species prefer low lying land but are displaced to higher elevations by anthropogenic land-uses [Elevation, terrain ruggedness, and vegetation productivity most strongly influenced leopard density in the Overberg. Elevation, which was correlated to the terrain ruggedness index, may serve as a proxy for leopard refugia with high relative elevation invariably precluding urban and agricultural land-use , 15. Leoand-uses , 82. In Both NDVI  and terrBottom-up factors (habitat suitability and catchable prey) are the principal drivers of apex predators’ movements in natural systems . HoweverThe importance of protected areas for leopards is well established , 27, 48,However, this study did indicate that leopard density was higher in low intensity use agricultural land than urban land—showing a clear negative cascade from natural to urban land-use. Although leopards can be attracted to agricultural areas due to the potential availability of both domestic and synanthropic food sources –96, resuWith large home ranges which span up to 910 km2 in the Western Cape  and the The large spatial requirements of leopard in the Cape emphasise the role leopard can play in conservation as an umbrella species but also places extra pressure on protecting and expanding connectivity for the species, especially across tracts of modified land such as in the Overberg. Connectivity, particularly in small populations, is important for finding mates, allowing dispersal, and ensuring genetic diversity in the population –40. The Improved connectivity and the persistence of leopards outside of protected areas provide a critical buffer for stochastic events such as extensive fires and droughts that may both be exacerbated by climate change –108. TheProtected areas are becoming increasingly fragmented from each other with a hardening of their edges and do not fully shield species from anthropogenic risks , 112; th2; [High leopard densities outside of protected areas is a phenomenon more commonly associated with countries like India  where t2; . There a2; , it is uS1 File(DOCX)Click here for additional data file.


This study aims to determine the hard and soft tissue shape variation and its changes in Class II division 1 malocclusion before and after orthodontic treatment using Geometric Morphometric Analysis.o). 32 landmarks in Cartesian coordinates were created and identified using MorphoJ software to establish a shape analysis.This retrospective study included 141 pre-treatment and near-end treatment lateral cephalometric radiographs of Class II division 1 malocclusion patients aged 16–40 years with a skeletal II pattern (ANB > 4p < 0.0001). T2 showed significant uprighting of upper incisors (17.5o) and lower incisors (3.7o), improved NLA (8o), an increase in upper lip thickness (1.5 mm), and a reduction in lower lip thickness (0.7 mm) (p < 0.05).The vertical dimensions  showed the largest variation in the general shape of hard and soft tissue, followed by the anteroposterior dimensions . Variations of lip shape (long to short), lip protuberance (everted to inverted), and nasolabial angle (obtuse to acute) were present. Orthodontic treatment affected the shape of the hard and soft tissue significantly (Vertical and anteroposterior shape variations were found. Orthodontic treatment had an impact on both hard and soft tissue shapes. Hence, understanding both the hard and soft tissue shape variations and the orthodontic treatment changes is crucial for an accurate diagnosis and treatment plan to achieve a successful outcome and excellent patient satisfaction.The online version contains supplementary material available at 10.1186/s12903-023-03684-7. The prevalence of Class II division 1 malocclusion is the highest after Class I malocclusion and is one of the most common malocclusion seeking orthodontic treatment in an orthodontic clinic . AlthougConventional lateral cephalometric radiographs can quantitatively describe and analyse facial forms using linear distances, angles, and ratios. However, it is unable to adequately describe the craniofacial morphology, distinguish changes in curvature, and describe size, shape, and growth –13. MultRecently, GMA has been increasingly utilised in the orthodontics field. Knigge et al.  studied Nevertheless, some studies using lollipop and wireframe graphs for shape illustration may not accurately replicate shape differences. Based on current updates, no studies have ever been done on hard and soft tissue shape changes during orthodontic treatment. The hypothesis has been that no hard and soft tissue shape variations or significant shape changes exist after orthodontic treatments. Therefore, this study aims to investigate the shape variations and changes of both hard and soft tissue in a Class II division 1 malocclusion before and after orthodontic treatment. This study could provide information on the shape variations of Class II division 1 malocclusion and provide graphical illustrations of the orthodontic treatment effects on the maxilla, mandible, and soft tissues, including their relationships with each other.o). This study excluded patients with poor LCR, craniofacial or dental anomalies, clefts, a history of trauma or surgery to the mandible, a history of periodontitis, a history of previous orthodontic treatment, and missing incisors.This retrospective study looked at the lateral cephalometric radiographs (LCR) and hard and soft tissue shapes and sizes in Class II division 1 malocclusion pre- and near-end-orthodontic treatment. All patients have completed orthodontic treatment in the Department of Family Oral Health, Universiti Kebangsaan Malaysia. The inclusion criteria for subjects were as follows: Age 16–40 years old, Class II division 1 malocclusion, medically fit and healthy, presence of all incisors, excellent LCR quality with all hard and soft tissue landmarks identifiable, and a skeletal II pattern  and near-end-(T2) treatment LCR were retrieved from the Department of Radiology at Universiti Kebangsaan Malaysia. Using Planmeca Romexis software , 141 LCR were obtained from both pre- and near-end treatment. The exposure parameters were 60–90 kV, 1–14 mA, an exposure time between 9 and 37 s, and a distance of 1.5 m between the source and the midsagittal plane. The LCR were digitally traced using VistaDent OC (2D) . To ensure accurate and consistent LCR tracing, all radiographs were rotated in such a way that Frankfort horizontal plane is used as the true horizontal plane, as a reference line to define the precise location of all other landmarks.Thirty-two 2D landmarks were identified on the LCR. The chosen landmarks are commonly used in the traditional metrical and geometric morphometric systems, are familiar to most orthodontists in lateral cephalogram tracing for malocclusion determination, and show significant biological relevance Fig. ; Table 1Digital copies of LCR in JPG format were converted into TPS files via TPSUtil software . A dataset containing the landmark coordinates  of all samples was created using TPSDig2 software. This dataset was then exported into MorphoJ software  for shape analysis.For GMA, all shape and statistical analyses were done using MorphoJ software. The hard and soft tissue shape variations were analysed using Generalised Procrustes Analysis (GPA). LCR were superimposed, translated, scaled, and rotated until the minimal sum of squared distances between corresponding landmarks was detected . This prThe primary outcome of the study was the hard and soft tissue shape variation (T1), obtained by calculating the most significant variation in the dimension of the hard and soft tissue shapes via Principal Component Analysis (PCA). This technique reduces the shape data dimensionality and identifies variation patterns. The covariance matrix is calculated from the shape data, to compute the eigenvectors of the matrix, also known as Principal Components (PC). Each PC represents the directions of the maximum shape variance in descending order .Another primary outcome compared the shapes and changes before (T1) and after (T2) orthodontic treatment. Discriminant Analysis (DA) was done, where a permutation test (1000 random permutations) was used to assess the significant shape differences between repeated measurements of the hard and soft tissues. This is critical to prevent digitizing errors and subsequently introduce variance in the subsequent analysis. DA was also done separately for the maxilla, mandible, and soft tissue for better visualisation. Procrustes distance was used to evaluate the statistical differences at T2.p < 0.05 and a 95% confidence level to appraise the mean changes before and after orthodontic treatment. In the presence of normally distributed data, descriptive statistics were calculated for each measurement before and after orthodontic treatment while significant differences between them were tested with the dependent t-test at T1 and T2.In addition to GMA, angular and linear measurements were obtained to confirm the changes in DA as per Fig. p < 0.05). In addition, outlier identification was also done in MorphoJ to ensure the robustness of the samples; no outlier was found.Inter-examiner and intra-examiner reliability tests were carried out on 20 randomly selected cases 1 month apart. The tests were performed using the Interclass Correlation Coefficients (ICC) test. The correlation coefficients for intra-examiner reliability and inter-examiner reliability were both above 0.97 and 0.94, respectively, demonstrating excellent agreement between measurements. For GMA, measurement error from landmarking was checked with Procrustes ANOVA using MorphoJ software. The measurements were reliable as the individual ANOVA variation was significantly higher in the shape of the centroid (MS = 0.000095) compared to the digitizing error (MS = 0.000017)  and 41 males 31%), with a mean age of 21.88 ± 5.58 years old. The principal component analysis displayed a multivariate analysis and major features in shape variation. In this study, PCA yielded 60 dimensions of hard and soft tissue shape variations, represented by PC1 to PC60. PC1 and PC2 represent the two highest variations within the whole data  shows the highest variations and describes them in the vertical dimension; the facial profile varies from hypodivergent to hyperdivergent. The gonion angle and the maxilla and mandibular plane angles vary from acute to obtuse. The upper lip lengths vary from long to short. PC2 (16.5%), on the other hand, describes variations in the anteroposterior dimension, where the severity of the Class II skeletal pattern varies from mild to severe. The facial profile varies from straight to convex, while lip protuberance varies from everted to inverted . After orthodontic treatment, observable significant changes included an increase in the interincisal angle . There was also a reduction in the SNA (0.66o), SNB (0.03o), and ANB (0.64o), but an increase in MMPA (0.45o). However, these changes were not statistically significant (p > 0.05) (Table p < 0.0005) in the maxilla, with a Procrustes distance of 0.105. After treatment, significant retroclination of the upper incisors  and remodelling of the alveolar bone following the incisors were observed  with a Procrustes distance of 0.021. Lower incisors were uprighted by 3.73o (p < 0.0005), and there was intrusion of the lower incisors with downward remodelling of the B point  in the soft tissue, with a Procrustes distance of 0.045. The NLA significantly increased by 8o (p < 0.0005), and the upper lip thickness increased by 1.55 mm (p < 0.0005). However, the lower lip thickness reduced significantly by 0.77 mm (p < 0.0005). A reduction of interlabial distance was also observed  Table ; Fig. 4aThe highest variation (PC1) was observed in the vertical dimension, which showed hypodivergent and hyperdivergent facial profiles. This finding aligns with other studies showing that Class II division 1 maloclusion is associated with a range of vertical skeletal patterns . The secWhile Class II division 1 malocclusion are usually presented with protruded and incompetent lips, the present study indicates that the soft tissue lips can also be everted, long or short. The relationship between the hard and soft tissue profiles varies because some soft tissue structures are closely related to those of hard tissues, while others are influenced by their length, thickness, and function .Morphometric analysis has been shown to be reliable in detecting skeletal, dental, and soft tissue shapes. Skeletally, DA showed a slight reduction of the SNA and SNB, while dentally, there was uprighting of both upper and lower incisors. Proclined incisors, such as those in Class II division 1 malocclusion, are often treated by extraction of premolars and retraction of anterior teeth with anchorage. It is often believed that alveolar bone remodelling occurs spontaneously with orthodontic tooth movements. When the incisors are retracted more posteriorly, the alveolar bone will be retracted as well, causing a reduction in SNA and SNB value . A studyThe present study observed a significant reduction in the inclination of the upper and lower incisors, together with an increase in the interincisal angle. The upper incisor uprighting in Class II division 1 malocclusion was in line with other studies, where the reduction of the upper incisor inclination varies from 7.9° to 25° , 34, 35.When the lower arch is well aligned with a flat curve of Spee and an increased overjet, extractions may be limited only to the upper premolars. However, when extraction is warranted in crowding cases, second premolars are preferred in the lower arch to preserve the position of the lower incisors, which helps overjet reduction. It is also believed that extractions of the first mandibular premolars produce more incisor retractions, whereas extractions of the second mandibular premolars enable more mesial movement of the first mandibular molars . In thisWhile orthodontic treatment resulted in an increase in the MMPA, the observed effects were not significant. Differential outcomes have resulted from theories regarding the impact of orthodontic treatment on MMPA, or vertical height. This can be explained by the extrusion of the molars or a “wedging effect,” especially when Class II elastics were extensively used . The useMany studies have also attempted to quantify the effect of upper incisor retraction on the lips. In the present study, a flattening of the lips at T2 was observed with the retraction of the upper incisors, accompanied by the upper lip retraction at a mean ratio of 2:1. Kasai  stated tIn orthodontics, GMA has been found to be more beneficial for investigating and describing complex hard and soft tissue shapes compared to traditional analysis . RecentlSkeletally, the facial profile varies from hypodivergent to hyperdivergent, as well as mild to severe skeletal II.The incisor inclination varies from upright to procline, and the alveolar bone height ranges from short to long.The nasolabial angle varies from acute to obtuse. The upper lip length varies from long to short, while the thickness of both upper and lower lips varies from thick to thin, as well as everted or inverted.After orthodontic treatment, the upper and lower incisors become significantly upright. The interincisal angle, NLA, and the upper lip thickness increase, whereas the lower lip thickness reduces significantly.GMA improves the diagnostic accuracy of lateral cephalometric radiographs in orthodontic treatment by enabling the identification and description of complex shapes and changes.Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


Bronchial Dieulafoy's disease (BDD) is a rare disease that causes massive hemoptysis. This paper reports a case of BDD treated surgically. At the same time, we summarize the data of BDD patients reported in domestic and foreign literature to improve the understanding, diagnosis and treatment of this disease.A case of BDD with hemoptysis during bronchoscopy was reported. In addition, we searched for "bronchial Dieulafoy disease" through Pubmed, Web of Science, CNKI and Wanfang databases, covering the literature related to BDD that was definitely diagnosed or highly suspected from January 1995 to December 2021, and summarized the clinical characteristics, chest imaging, bronchoscopic manifestations, angiographic characteristics, pathological characteristics, treatment and outcome of patients.The patient was a 68 year old male. Tracheoscopy revealed nodular and mass like changes in the basal segment of the left lower lobe, which appeared massive hemorrhage when touching the surface. The computed tomography angiophy of the bronchial artery confirmed that the branches of the left bronchial artery were tortuous and dilated, and then the left lower lobe of the lung was resected. During the operation, 3 thick tortuous nutrient artery vessels were sent out from the descending aorta, and 1 thick tortuous nutrient artery was sent out from the autonomic arch. All of them were ligated and cut. The pathology after the operation was in accordance with BDD; The patient did not have hemoptysis after discharge and is still under follow-up. The database identified 65 articles from January 1995 to December 2021. After removing repeated reports, meetings, incomplete information and nursing literature, 60 articles were included to report 88 cases of BDD. BDD can occur at all ages, with a male to female ratio of about 1.6:1. It mainly starts with hemoptysis, and can also be seen due to cough, infection, and respiratory failure; Inflammatory changes such as pulmonary patch shadow, exudation shadow and ground glass shadow of pulmonary hemorrhage were more common in chest imaging; The diagnosis of BDD is mainly based on the bronchoscopy, bronchial angiography and pathological findings of surgical or autopsy specimens. Bronchoscopic findings were mostly non pulsating, smooth nodular or mucosal processes. Bronchial angiography mainly showed tortuous dilatation of bronchial artery, and the lesions were mainly located in the right bronchus, more from the bronchial artery; Diagnosis depends on pathology, showing submucosal expansion of bronchus or abnormal artery rupture and bleeding; 54 cases underwent selective bronchial artery embolization, 39 cases underwent pulmonary lobectomy, 66 cases improved, and 10 cases died .BDD is rare, but may cause fatal massive hemoptysis. Bronchial angiography is considered to be an effective method to diagnose BDD. Since pathological biopsy may lead to fatal bleeding, the necessity of pathological diagnosis remains controversial. Interventional and surgical treatment plays an important role in patients with cough accompanied by massive hemoptysis. Dieulafoy's disease was first reported by French doctor Georges Dieulafoy  in 1898.A 68 year old male smoking for 30 pack-year was admitted to the hospital on December 5, 2018 due to "left lung shadow was found for 4 months, and cough with left subcostal pain for 2 months". After admission, chest enhanced CT showed that there was a leaf like solid density increase shadow in the lower left lung with obvious enhancement, and the left lower lobe bronchus was partially occluded Fig. , 8 and 9Massive hemoptysis is a common critical disease in respiratory medicine. The common causes include bronchiectasis, pulmonary tuberculosis or lung tumor. BDD is one of the rare causes of massive hemoptysis. Since Sweerts  and otheThere are 54 males and 34 females in 88 BDD patients, with a male to female ratio of 1.6:1. The minimum age of the patients is 9 months + 3 days, the maximum age is 85 years old, and the average age is 46.1 years old. All age groups have diseases, and about 1/3 of the patients are aged 60–75 years.The etiology and pathogenesis of BDD remain to be clarified. At present, the possible mechanisms include: (1) congenital bronchial artery dysplasia ; (2) ChrThe most common symptom of BDD is repeated hemoptysis. It is reported that the maximum hemoptysis volume is 2000 ml . HoweverAuxiliary examinations of BDD include chest X-ray and computed tomography (CT), bronchoscopy and bronchial angiography. 36 patients received chest X-ray , 56, 60.Summarizing the lesions of BDD, we found that there were 62 cases of right bronchus ; 25 cases of left bronchus ; There were 5 cases of bilateral bronchi and 1 case near the carina , lobectomy and argon plasma coagulation via bronchoscope. At present, SBAE is the preferred surgical method. For SABE or recurrent hemoptysis after embolization, lobectomy of the diseased lung is used. Eighty eight patients with BDD were summarized, including: (1) conservative drug therapy (9 cases); (2) Endobronchial intervention (7 cases); (3) Bronchoscopic arterial embolization only (30 cases); (4) Pulmonary lobectomy only 15 cases); (5) Bronchial artery embolism + pulmonary lobectomy (24 cases) (Table  cases; (In conclusion, the case we reported and the 88 cases we summarized support that BDD should be considered when recurrent and unexplained hemoptysis occurs and the lesion is limited to the right bronchus. Bronchial biopsy should be avoided and bronchial arteriography should be performed as early as possible. SBAE can be used for stable or intolerant patients to reduce the risk of life-threatening hemoptysis. Although it can retain some functions of the diseased lung, it is easy to recur after treatment. For uncontrollable cases or cases with poor prognosis, lobectomy can be the best choice to save lives. Although it will affect the quality of life of patients, it can eliminate the possibility of recurrence and obtain histopathological confirmation.


Optical genome mapping (OGM) has developed into a highly promising method for detecting structural variants (SVs) in human genomes. Complex chromosomal rearrangements (CCRs) and cryptic translocations are rare events that are considered difficult to detect by routine cytogenetic methods. In this study, OGM was applied to delineate the precise chromosomal rearrangements in three cases with uncertain or unconfirmed CCRs detected by conventional karyotyping and one case with a cryptic translocation suggested by fetal chromosomal microarray analysis (CMA).In the three cases with CCRs, OGM not only confirmed or revised the original karyotyping results but also refined the precise chromosomal structures. In the case with a suspected translocation not detected by karyotyping, OGM efficiently identified the cryptic translocation and defined the genomic breakpoints with relatively high accuracy.Our study confirmed OGM as a robust alternative approach to karyotyping for the detection of chromosomal structural rearrangements, including CCRs and cryptic translocations. Genomic structural variations (SVs) usually refer to structural and quantitative chromosomal rearrangements that are longer than 100 bp . UnbalanCCRs describe SVs involving at least three breakpoints on two or more chromosomes, especially multiple translocations. Cryptic chromosomal rearrangements are characterized by chromosomal aberrations that are very subtle or located in the terminal zone and cannot be identified by conventional karyotyping, including cryptic translocations, inversions, insertions and other forms. The published data indicate that the unique and complex events involved in these rearrangements could be more complex than initially assumed , 9, but Individuals with apparently balanced SVs are expected to be asymptomatic, but in fact, they are commonly at a high risk of infertility, recurrent spontaneous abortion, stillbirth, and delivering newborns with congenital malformations caused by chromosome unbalanced recombination events during the gametogenesis period . Recent A total of four patients who suffered from a history of adverse pregnancy or fertility, including two males and two females, were recruited to this study at the First Affiliated Hospital of Jinan University. These individuals had previously received peripheral blood chromosome karyotyping, which revealed that 3 of the patients carried unique CCRs and defined chromosomal break points at the microscopic level. The remaining patient was identified to have a normal karyotype, but a cryptic chromosomal rearrangement was highly suspected based on fetal CMA results. The patient demographic details are shown in Table Routine G-banded karyotyping of peripheral blood lymphocytes from the 4 patients was performed using standard procedures. The metaphase chromosomes were analyzed according to the International System for Human Cytogenomic Nomenclature (ISCN) 2020 at 450-band resolution.In case 4, CMA was performed on the fetus (amniotic fluid) and the parents  using a CytoScan 750 K chip  following the routine experimental procedure previously described . The rawOGM was performed on the 4 cases. The brief experimental procedures were as follows, briefly: Ultra-high molecular weight (UHMW) gDNA was isolated from peripheral blood following the manufacturer's guidelines . Thereafter, the direct label and stain (DLS) technique was used according to the manufacturer’s instructions  to label the DNA. The direct labeling enzyme 1 (DLE-1) reaction was carried out using 750 ng of high-molecular-weight gDNA to tag a specific 6-bp sequence (CTTAAG). Subsequently, the fluorescently labeled gDNA molecules were loaded on Saphyr chips for linearization and sequential imaging in massively parallel nanochannel arrays. De novo assembly and structural variant (SV) calling were performed via a de novo assembly pipeline through Bionano Access v1.2.1  and compared with Genome Reference Consortium GRCh38 (hg 38). Variant types such as insertions, deletions, inversion breakpoints, translocation breakpoints, and CNVs were detected with this pipeline , 23.Our study used the OGM technique to reveal the refined genomic structures of three individuals with CCRs originally identified by conventional karyotyping.A 30-year-old man was diagnosed with infertility and cryptozoospermia, and his wife had a history of miscarriage at 8 gestational weeks. Karyotyping of his peripheral blood indicated a karyotype of 46,X,Yqh+ ,?del21)(q21),add(22)(p13), suggestive of a large deletion ranging from chromosome 21q21 to the terminus of the long arm and attachment of a large unknown chromosome segment to chromosome 22p13 ,addUnexpectedly, further delineation of the chromosomes using OGM identified a breakpoint on chromosome 3q27.1, two breakpoints on chromosome 21 at q21.1 and q21.2 , and an assumed breakpoint on chromosome 22p11.2  on the basis of initial karyotyping . Cytogenetic investigation revealed an apparently balanced karyotype of 46,XX,tp31;q21),ins,der(5)inv(5)(q12q13.1)ins. The complex rearrangement was found to have an apparent translocation between chromosomes 1p31 and 14q21, an insertion of chromosome 5q15q13.1 material within the 16q21 region, while the derivative chromosome 5 generated by the insertion exhibited more than one rearrangement, involving a paracentric inversion with the breakpoints at 5q12 and 5q13.1. This complex rearrangement was predicted to involve 4 chromosomes and 7 breakpoints  results, suggesting a high risk of deletion at chromosome 9p. Karyotyping and CMA analysis of amniotic fluid cells were routinely performed. CMA detected a 4.37-Mb terminal duplication of chromosome 2q37.3  × 3) and a 4.39-Mb terminal deletion of chromosome 9p24.3p24.1  × 1) in the fetus  are rare events that require at least three breaks (two on the donor chromosome and one at the insertion site on the acceptor chromosome) and thus can be defined as CCRs. A recent study utilizing mate-pair genome sequencing (GS) revised previously designated karyotypes in 75.0% of the cases and identified additional cryptic rearrangements in 68.8% of the cases . SimilarIn case 1, a CCR involving three chromosomes  without genomic gain and deletion was discovered. The refined karyotype was 46,X,Yqh+ ,t,t. Although the translocated 3q27.1-qter fragment (~ 15 Mb) is expected to be within the resolution of karyotyping, it might have been masked as multiple segments were involved in the complex rearrangements and thus were not recognized in the initial karyotyping. Due to the limitations of the OGM technique , the shoIn case 2, the abnormal morphology of chromosomes X and 8 on karyotyping was initially speculated to be due to an insertion event between the two chromosomes. This may be attributed to the difficulty of interpreting various chromosome morphologies and the subjective error on the part of the analyst. Encouragingly, OGM showed a powerful ability to correct these errors and detected multiple rearrangement events containing 7 chromosome breakpoints. The corrected virtual chromosome acceptor breakpoint for the inserted chromosome Xq23q23 was revealed at chromosome 10q26.12; two disconnected deletion segments were identified at chromosomes Xq22.2q23 and Xq23q24, and an inversion event was identified at chromosome 8q21.2q24.3.In case 3, the OGM assay confirmed the rough karyotyping result, with refinement revealing a more complex rearrangement originating from 9 breakpoints, and provided a molecular characterization of karyotypically apparent simple insertions to demonstrate previously underappreciated complexities.Additionally, we described a cryptic balanced chromosomal translocation in the father in case 4 that caused unbalanced genomic dosage changes in the fetus. In this case, a cryptic translocation t was clearly identified by OGM but not by conventional karyotyping. The translocated fragments on chromosome 2 and chromosome 9 are less than 5 Mb in length; therefore, they were undetectable by karyotyping, the routine resolution of which is estimated to be 5 ~ 10 Mb on average. The fetus inherited der(9)t from the father. The unbalanced genomic changes were expected to contribute to adverse pregnancy outcomes for the fetus and pregnancy. Carriers of cryptic balanced chromosomal translocations are prone to adverse reproductive events, such as infertility, recurrent abortion, suspended embryo development, stillbirth, teratogenesis and birth defects –32. ThusThe main techniques for detecting structural variations include G-banded karyotyping, CMA, FISH, and NGS, each of which has some well-known advantages and disadvantages. As the most basic analysis of chromosome morphology, karyotyping is capable of detecting most types of balanced and unbalanced SVs as well as numerical chromosomal aberrations, but its resolution is limited to approximately 5–10 Mbp. CMA, which is based on DNA hybridization, offers a resolution of several hundreds to thousands of base pairs in the form of deletions and duplications, but it is not able to detect balanced SVs; FISH can only detect anticipated rearrangements when targeted probes are available; and SKY analysis has limited resolution in delineating cryptic chromosomal rearrangements. Recently, WGS paired with second-generation  or third-generation  technology has been applied to detect SVs. Nevertheless, the relatively short DNA strands used for second-generation sequencing present significant limitations, as long regions of high similarity tend to be difficult or impossible to analyze, whereas high costs and computational challenges currently impede the widespread application of third-generation sequencing. In contrast to these techniques, OGM has extremely high resolution and is capable of comprehensively detecting balanced and unbalanced SVs as small as 30 kb , which aDespite its high accuracy in resolving all types of chromosomal SVs, OGM has inherent limitations relative to karyotyping in detecting breakpoints lying within large, unmappable repetitive regions, such as centromeres, the p arm of acrocentric chromosomes, or stretches of constitutive heterochromatin. Hence, Robertsonian translocations (RTs) and other whole-arm translocations are considered beyond the current technical capability of OGM and might be prone to false positive and false negative results from this technique . The ideOverall, due to the hidden nature and uncertainty of SVs, the comprehensive analysis of all SVs requires a combination of OGM with other current techniques. The OGM demonstrated highly effective performance in clinical practice in identifying CCRs and cryptic translocations in clinical practice. Our study confirmed OGM as a solid alternative approach to karyotyping, FISH, and CMA for the detection of chromosomal structural rearrangements, including CCRs and cryptic translocations.


The analysis of left ventricular function is predominantly based on left ventricular volume assessment. Especially in valvular heart diseases, the quantitative assessment of total and effective stroke volumes as well as regurgitant volumes is necessary for a quantitative approach to determine regurgitant volumes and regurgitant fraction. In the literature, there is an ongoing discussion about differences between cardiac volumes estimated by echocardiography and cardiac magnetic resonance tomography. This viewpoint focuses on the feasibility to assess comparable cardiac volumes with both modalities. The former underestimation of cardiac volumes determined by 2D and 3D echocardiography is presumably explained by methodological and technical limitations. Thus, this viewpoint aims to stimulate an urgent and critical rethinking of the echocardiographic assessment of patients with valvular heart diseases, especially valvular regurgitations, because the actual integrative approach might be too error prone to be continued in this form. It should be replaced or supplemented by a definitive quantitative approach. Valid quantitative assessment by echocardiography is feasible once echocardiography and data analysis are performed with methodological and technical considerations in mind. Unfortunately, implementation of this approach cannot generally be considered for real-world conditions. GuideThe volume assessment of right ventricular (RV) volumes by 2D echocardiography is not reliable, due to the triangular shell shape of the RV . TherefoEspecially in patients with valvular heart diseases (VHD), cardiac volume assessment is important to properly assess the hemodynamic conditions of the individual patient, which is the prerequisite to guide further therapeutic decisions ,5,13. A ○Do these methodological discrepancies between both imaging modalities really exist?○If LV volumes determined by echocardiography and CMR under comparable circulatory conditions are not similar, how can the problem of underestimation of cardiac volumes by echocardiography or overestimation by CMR be explained?○In addition, if echocardiography is always reputed to measure smaller LV volumes than CMR, how can the data of some previous trials , in whic○In contrast, if LV volumes determined by echocardiography and CMR under circulatory comparable conditions are similar, are physiological aspects during image acquisition the reason for potential irrelevant differences?○If the assessment of comparable cardiac volumes is possible, does the underestimation of cardiac volumes by echocardiography then constitute a methodological failure?○Assuming that cardiac volumes are underestimated by echocardiography in the past, what are the clinical implications?(1)both modalities measure the same target values if both methods are used properly;(2)echocardiography generally underestimates cardiac volumes implying that cardiac volumes generally cannot be properly assessed by echocardiography.The answers to these questions basically offer two main options:While the first option implies that echocardiography offers conclusive diagnostic results by proper cardiac volume analyses, especially for VHD, the second option implies that current echocardiographic recommendations may be based on incorrect cardiac volumes. In consequence, all echocardiographic recommendations—at least for VHD—need to be completely rewritten by defining new normal ranges and cut-off values—particularly for regurgitant volumes and fractions, because both parameters are based on proper LVEDV, LVESV, and RV end-diastolic and end-systolic volume  measurements. Furthermore, the second option implies that echocardiography does not generally enable a verifiable quantitative assessment of cardiac volumes.It is obvious that image acquisition with both modalities—echocardiography and CMR—is often limited in real life. Therefore, this viewpoint focuses on proper and standardized image acquisition using optimized settings which are especially necessary in clinical science. The practical limitations in clinical practice are obvious to introduce a verifiable quantitative approach of cardiac volume assessment. However, this challenge must be accepted to improve echocardiography diagnostics—especially in valvular heart disease.Former phantom studies have shown that quantitative assessment of cardiac anatomy by 2D echocardiography is possible . RecentlAll these studies were performed with older ultrasound technology, so that an even better agreement with modern techniques can be assumed. Overall, either no significant or only small volume differences of maximum 1–2% were found compared with the true volume. However, phantom volumes were overestimated by up to 5% using, e.g., MSCT . In concThe frequent reports of underestimated cardiac volume by 2D echocardiography ,35,36,37Several old as well as recent studies have shown that the determination of cardiac volumes by 3D echocardiography is comparable to CMR, with LV volumes determined by 3D echocardiography having no or only an irrelevant underestimation of less than 10% compared with CMR ,44,45,46How to interpret these different findings? Minor volume differences between 3D echocardiography and CMR can be explained by both methodological and physiological causes. Smoothing of regional endocardial irregularities and blurring of the endocardial contour due to low spatial resolution may result in lower cardiac volumes ,50. LimiThe preliminary summary of these aspects suggests that echocardiography—especially 3D echocardiography due to improvements in modern imaging technology—allows accurate assessment of cardiac volumes. Future reviews about this topic should therefore distinguish between old and new data. Strong discrepancies between cardiac volumes determined by echocardiography and CMR can only be explained by methodological limitations. In general, cardiac volume assessment by 2D echocardiography can only be valid and reliable if measurements were performed in standardized views. Then, even comparability between 2D and 3D echocardiography can be assumed if spatial and temporal resolution of 3D data sets are sufficient, echocardiographic views are standardized, endocardial surface rendering is performed properly , and alg2 for Doppler echocardiography, and 5.0 L/min and 2.6 L/min m2 for 2D planimetry, respectively [Despite the knowledge that cardiac volumes can, in principle, be measured just as well by echocardiography as by CMR, the described scenario had an impact on echocardiographic recommendations. Validation studies of cardiac output (CO)/cardiac index (CI) determination by Doppler echocardiography using the velocity time integral (VTI) of the left ventricular outflow tract (LVOT) and the LVOT cross-sectional area compared with CO/CI assessment by thermodilution and the Fick method yield comparable values ,62,63. Iectively .2 ) are significantly lower than the comparable ranges determined by Doppler echocardiography) [2) [In most reported normal ranges of LVSV, CO/CI determined by 2D/3D echocardiography in recent recommendations  ,67,68. Iphy) [2) , indirec2 [The same observations were reported in terms of RV volumes obtained by 3D echocardiography ,71. AssuThe summary of these reports suggests that current echocardiographic reference may be too low. Therefore, echocardiographic reference values for cardiac volumes need to be critically verified for plausibility, in comparison with other modalities such as CMR. Further, especially for modern 3D echocardiography, the calibrations of the analysis software must be checked with respect to proper delineation of endocardial border. Two-dimensional echocardiography can also serve as a reference in controls, as the mathematical calculations have been validated in normal cardiac geometry .Understanding hemodynamics is essentially related to the proper and quantitative assessment of cardiac volumes, CO/CI ,72,73,74Valvular regurgitations complicate the interpretation of hemodynamic measurements. For example, flow conditions in AS patients with combined aortic regurgitation (AR) can be analyzed by the forward LVSV or by the forward RVSV. In this scenario, the effective LVSV and the regurgitant volume of the AR is characterized by the forward LVSV, whereas the forward RVSV represents both the effective RVSV and the effective LVSV, if no pulmonary regurgitation is present . In pureIn general, in VHD patients, total LVSV is always characterized by 2D or 3D LV planimetry, and the effective LVSV is always characterized by Doppler echocardiography using the VTI of the right ventricular outflow tract (RVOT) or the pulmonary trunk cross-sectional area, respectively, if there is no pulmonary stenosis as well as no or only trace pulmonary regurgitation. The proper LV planimetry in standardized views can objectified by postprocessing in 3D data sets. Thus, foreshortening and consecutive errors of 2D planimetry in non-standardized views can be detected .MR ≥ 60 mL and a RF ≥ 50%. Additional information about RegVolMR is “may be lower in low flow conditions” [Recent recommendations propose an integrative approach to assess the severity of valvular regurgitations ,5. In adThis approach is understandable because of the reported discordance between echocardiography and CMR in terms of grading MR severity ,78,79. TMR determination in echocardiography is predominantly based on 2D PISA method, the effect of underestimating total LVSV is substantial.The question remains if the quantitative assessment of MR severity by echocardiography is based on incorrect small cardiac volumes, where does the general assumption come from that LVEF can be properly assessed if LV volumes cannot be correctly determined? If RFMR of 50% , if severe MR is defined by a RegVolMR of 60 mL [2, corresponding to cardiogenic shock and situations incompatible with daily life [A total LVSV of 120 mL is necessary to ensure almost normal CO/CI at normal heart rates of approximately 65/min at a RFily life ,81,82,832 at the lower limit to the shock index, MR of any severity might be hemodynamically relevant and would result in cardiac decompensation. Thus, even grading by RFMR would be questionable. Consider again the scenario of a true LVEDV of 200 mL with a LVEF of 30% in a patient with optimal medical conservative treatment and a heart rate of 65/min. With a BSA of 1.8 m2, CO/CI would be (60 mL × 65/min = 3.9 L/min) 3.9 L/min/2.15 L/min m2, respectively. If even mild MR with a RegVolMR of 20 mL develops according to recommendations, the hemodynamic situation corresponds to cardiogenic shock . Clearly, myocardial dysfunction is the predominant pathology in this case. However, the cause of acute decompensation is the development of clinically relevant MR, which would have to be classified as formally mild even according to recent recommendations.Another thought-provoking aspect of current echocardiographic recommendations is the severity grading of regurgitation based on fixed RegVol especially when the estimation of RegVol is based on results of the 2D PISA method and the total LVSV is ultimately based on questionably low LVEDV and LVESV. If a patient with severely reduced LVEF and normal valve function had a CI of 1.8 L/min mFirst, the patient’s baseline data, symptoms, and history must be known and considered.2, blood pressure: 130/80 mmHg) with sinus rhythm presented to hospital because his daughter, herself a physician, was advised by a cardiologist to undergo interventional mitral valve therapy due to severe MR. The severity in terms of high-grade MR was based on eyeballing of the regurgitant jet area  facilitates the interpretation. Thus, the maximum possible RegVolMR to be above shock limit is (90 mL − 63 mL = 27 mL), which corresponds to a RF of 30%.At this point in the examination, the findings should be checked for hemodynamic plausibility. Calculating the minimum effective LVSV to enable a required effective CO and CI at a heart rate of 63/min The actual, integrative approach is too error prone to be continued in this form. It should be replaced or supplemented by a definitive quantitative approach comparable to CMR.(2)Valid quantitative assessment by echocardiography is feasible once echocardiography and data analysis are performed with methodological and technical considerations in mind.(3)Unfortunately, implementation of this approach cannot generally be considered for real-world conditions.(4)Cardiac volumes, particularly total and effective LVSV and RegVol, should be checked for plausibility and valid hemodynamics.(5)Apparently, incorrect former echocardiographic data of cardiac volume measurements should not be considered in future meta-analyses.(6)New studies about normal ranges of cardiac volumes measured by echocardiography and validated by CMR are necessary.(7)The echocardiographic algorithm for analysis of VHD, particularly valvular regurgitations, should be revised to include hemodynamically valid CO and CI.This viewpoint aims to stimulate an urgent and critical rethinking of the echocardiographic assessment of patients with VHD, especially valvular regurgitations:


Melipona rufiventris, Scaptotrigona postica and Tetragonisca angustula. Simultaneously, we evaluate the local floristic components through seventeen rapid botanical surveys conducted at different distances from the nests. We discovered a broad set of explored floral sources, with 46.3 plant species per bee species in honey samples and 53.67 in pollen samples. Plant families Myrtaceae, Asteraceae, Euphorbiaceae, Melastomataceae and Malpighiaceae dominated the records, indicating stingless bee preferences for abundant resources that flowers of these families provide in the region. Results also reinforce the preference of stingless bees for forest trees, even if only available at long distances. Our high-resolution results encourage future bee-plant studies using pollen and honey metabarcoding in hyper-diverse tropical environments.Stingless bees are major flower visitors in the tropics, but their foraging preferences and behavior are still poorly understood. Studying stingless bee interactions with angiosperms is methodologically challenging due to the high tropical plant diversity and inaccessibility of upper canopy flowers in forested habitats. Pollen DNA metabarcoding offers an opportunity of assessing floral visitation efficiently and was applied here to understand stingless bee floral resources spectra and foraging behavior. We analyzed pollen and honey from nests of three distantly related stingless bee species, with different body size and social behavior:  The current decline in pollinator abundance and diversity worldwide threatens pollination services, with direct consequences to nature conservation and food security2. Therefore, understanding interactions between pollinators and flowering plants became crucial, since it can provide an information framework to subsidize conservation policies and decisions in a changing world. The most important group of animal pollinators—the bees—are completely dependent on floral resources to complete their life cycles3, while 87.5% of the animal-pollinated flowering plants depend on bees to reproduce4. Interaction between bees and angiosperms has been a major research focus in the last two decades, due to the potential threats of massive insect and bee declines5 on ecosystem services and food security7. In this context, pollen, and honey DNA metabarcoding emerged as an efficient technique to identify plant taxa visited by pollinators based on samples extracted from bees' bodies or nests11. DNA metabarcoding of pollen and honey has been largely applied to temperate systems, and recently to (sub) tropical species of stingless bees13.Plant-pollinator interactions mediate most flowering plant reproduction, maintaining terrestrial ecosystems and crops14. The role of stingless bees as pollinators of the neotropical flora could become even more relevant under climate change scenarios, if the predicted expansion of warmer temperatures pushes the distribution of predominantly temperate bees, such as Apis and Bombus, into cooler regions15. Although domestication is still restricted to a few species, stingless bees are also explored commercially for honey production16, which can reduce the use of introduced honeybees and their impact on native species in these regions12.The stingless bees  comprise c. 500 species of eusocial bees, most of which occur throughout tropical America (more than 400 species), but also in Africa, Asia and Australia17. Particularities exist though, since stingless bees exhibits a huge diversity of body size (1.8 to 13.5 mm)3, flight distance (0.3 to 3 km)18, and foraging behavior19. However, the breadth of stingless bees diet—that is, how many different species of flowering plant they can forage on—and the extent of their role as pollinators of tropical plants are still largely open questions. Most studies aiming to answer these questions face methodological difficulties due to inaccessibility of visited flowers, which mostly occupy upper canopy strata especially in rainforests20, and hyper diversity of tropical plants, thus hampering easy identification through bee-pollen morphology22.As all eusocial bees, stingless bees show a predominantly generalist pattern of floral exploitation, i.e. they visit a large number of species in several plant families, supposedly disregarding specific floral traits22In this study, we explored the diet breadth of three distantly related species of stingless bees of different body sizes and flight ranges in a hyper-diverse tropical ecosystem, the Cerrado savannas of central South America. The Cerrado is the most species-rich savanna in the world and a hotspot of biodiversity23. The flora encompasses > 13,000 native plant species24 in a highly patchy vegetation with several different physiognomies, ranging from grasslands, marshlands, and typical savanna to closed canopy riverine forests along waterways25. The proportion of pollinator-dependent species in the Cerrado flora is still unknown, although this number is likely to be similar to that of tropical forests4, with some authors estimating c. 60% of angiosperm species being bee dependent27.Melipona rufiventris Lepeletier, 1836, Scaptotrigona postica  and Tetragonisca angustula ) native to the Cerrado to investigate: (i) How broad is the diet breadth of stingless bees in a hyper-diverse flora? (ii) Which plant species and families are the most important sources of pollen and/or nectar for stingless bees in the area? (iii) What can pollen and honey metabarcoding reveal, when combined with floristic surveys of the area, about stingless bees foraging behavior, particularly foraging distances, and floral preferences? We also discuss how efficiently pollen and honey metabarcoding identified plants visited by bees in the area, considering the low DNA sequence coverage of neotropical plant species in public databases28, and the potential role of this technique in improving ecological understanding of bee-plant interactions in the tropics.We analyzed pollen and honey from the pots of the nests (henceforward pot-pollen and pot-honey respectively) from three commonly managed stingless bee species (cerrado sensu stricto), palm swamps (veredas), grasslands (campo limpo and campo sujo) and riverine forests , surrounded by natural and agricultural areas  (15°56′41″ S and 47°53′07″ W) that, together with the contiguous Brasilia Botanic Garden and the University of Brasília Experimental Field Station, preserves an area of c. 10,000 ha of native Cerrado in the Distrito Federal, Brazil. The IBGE reserve occupies a central position within the Cerrado domain and was chosen as our study site for being one of the most well-studied areas of Cerrado, with good prospects of building a relatively robust plant DNA reference library, a requirement for our analyses (see below). The climate in the area is typical tropical savanna climate (Aw Köppen classification system) with dry winters and rainy summers with an average annual precipitation of 1453 mm and altitude ranging from 1048 to 1160 m. The IBGE reserve contains the main vegetation types typical of the Cerrado domain: savannas 14.Three native stingless bee species were chosen for our study based on differences in body size, differences in foraging behavior, and phylogenetic relationships. M. rufiventris, eight of S. postica and seven of T. angustula. The nests were installed at a distance of about 5 m from each other and c. 150 cm above ground level, in typical savanna or cerrado sensu stricto29 where most plant species are subshrubs, shrubs or small trees.The three species are commonly managed by local beekeepers and were chosen also for their relatively easy management in artificial colonies Figure . The decM. rufiventris, 81 of S. postica and 74 of T. angustula.Nests were moved to the study area eight weeks prior to the first sampling to allow bees time to accumulate pollen and honey from local species in the artificial nests. Pot-pollen and pot-honey samples were collected from the nests Figure  once eveDNA extractions of pollen samples from pot-pollen and pot-honey followed different methodologies, due to the different natures of the samples. For pot-honey, we extracted DNA using the Machery-Nagel  NucleoSpin Food Kit; for pot-pollen we used the Machery-Nagel  NucleoSpin Plant II.To extract pollen genomic DNA, we added to the pooled samples (weight ranging from 0.1 g to 2 g) 4 mL of deionized and autoclaved water and homogenized it using a vortex. We then placed 200 µL of this emulsion in a 1.5 mL microcentrifuge tube and centrifuged it for 15 min at 8000 rpm. We discarded the supernatant material, froze the pellet obtained in liquid nitrogen, and then used mortar and pestle to break the pollen exine, and the NucleoSpin Plant II Kit to promote cell lysis and to isolate the DNA according to the manufacturer’s instructions.To extract pollen genomic DNA from honey, we added deionized and autoclaved water to the samples until the volume of each sample tube reached 1.5 mL. We incubated the tubes at 65 °C for 30 min and, over that period, inverted the tubes slowly to homogenize the material. We then pooled the honey samples collected from the same nest and day by pouring them into falcon tubes, to which deionized and autoclaved water was added until completing 10 mL. Afterwards, we centrifuged these pooled samples for 15 min at 5000 rpm and discarded the supernatant material. Each precipitated pooled honey sample was resuspended in 200 µL deionized and autoclaved water and placed in a 1.5-mL microcentrifuge tube. This procedure was done twice. Finally, we centrifuged the samples for 15 min at 5000 rpm, discarded the supernatant material, dried the pellet in a drying cabinet at 35 °C, and then ground the samples inside the microcentrifuge tube using micro-pestles and liquid nitrogen. We then used the NucleoSpin Food Kit to promote cell lysis and to isolate the DNA according to the manufacturer’s instructions.9 to amplify the ITS2 region, using the primers ITS-S2F and ITS4R. Primer sequences, references and other amplification methodological details can be found in Sickel (2015)9 and Campos et al. (2021)31. The triplicate PCR reactions were combined per samples, well mixed and checked on 1% agarose gel using 5 uL of the combined products for quality. PCR products of each sample were then normalized to ensure library concentrations were approximately equivalent across samples using the SequalPrep Normalisation kit  according to the manufacturer's protocol. After pooling the multiplex-index samples, we quantitated the pools with a dsDNA High Sensitivity Assay on a Qubit Fluorometer and assessed fragment lengths with a Bioanalyzer High Sensitivity DNA Chip . For library dilution, we followed the Illumina Sample Preparation Guide for a 2 nM library where 5% consisted of PhiX control library. In addition, the reagent cassette of the sequencing kit was spiked with the Read1, Read 2 and index primers according to Sickel et al. (2015). Sequencing was then performed on the Illumina MiSeq system at the University of Würzburg. Sequence data are available at NCBI (Bioproject 976708).The protocol of amplification utilizes a dual-indexing strategy32 to join paired ends of forward and reverse reads and to remove reads shorter than 150 bp, quality filtering (EE < 1)33, de-novo chimera filtering (following UCHIME3)34, and determination of amplicon sequence variants (ASVs)34, as previously done for pollen metabarcoding networks12. Reads were first directly mapped iteratively with global alignments using VSEARCH against five flowering plant ITS2 reference databases (see below for construction details) for the study region and an identity cut-off threshold of at least 97% (higher percentages are prioritized). These references databases were created with BCdatabaser, then automatically curated35 from GenBank entries with default parameters , from the following species lists: 1) all plant species recorded from IBGE. This database was then manually curated to remove voucher-less entries for greater trustworthiness. Remaining unclassified sequences were then tracked by iterative searches against geographically broadening public sequence reference data, i.e., 2) species lists of the flora of the Distrito Federal, then 3) the large, neighboring state of Goiás, and 4) the entire Cerrado domain flora to increase completeness of reads. These reference databases were created with the BCdatabaser36 from GenBank entries given above mentioned species lists and default parameters . For still unclassified reads, we used SINTAX37 to assign taxonomic levels as deep as possible. Finally, we used 5) global reference database36. After classification, we performed plausibility checks according to geolocation and phenology with the results to verify validity. Thirteen species were automatically matched to genus level only, but were manually attributed to species based on these being the only species of the genus to occur in the Distrito Federal.We used VSEARCH v2.14.2in loco as homogeneous to vegetation type. These plots were exhaustively surveyed for all flowering plant species of all life forms, fertile or not, by a team of 3–5 researchers, where one was the booker, i.e. the most experienced person in the group, who identified the plants in the field and discarded duplicated species; other team member collected and pressed the vouchers .To improve our knowledge of the flora surrounding the nests, we conducted Rapid Botanical Surveys (RBS) in small plots that were demarcated 18. These eleven RBS plots mostly fell in areas of well-preserved savanna within the IBGE Reserve, ranging from the more open, grass and herb-rich areas with few shrubs and trees (campo sujo), to dense savanna woodland (cerradão); one outer pentagon plot fell in disturbed cerrado and another in heavily degraded secondary vegetation out of the IBGE. Because none of the plots fell in riverine gallery forest, we included six additional RBS plots in this vegetation type: three in the riverine gallery forest nearest to the nests , and three in a more distant gallery forest , measured as the crow flies, totaling 17 RBS plots. Lastly, we also surveyed the plants and weeds growing in the ornamental gardens associated with the Main Building and Seat of the Reserva Ecológica do IBGE, which is located c. 650 m from the nests. All specimens collected in RBS inventories were identified by the author CEBP and JEQF and were deposited in the UB Herbarium (University of Brasilia) and the records are available online in the Species Link Network (https://specieslink.net/search/) by searching on the collector name "Projeto Barcode Cerrado". From all species collected, three are categorized as endangered at some level: Virola urbaniana Warb. and Cedrela fissilis Vell. (vulnerable) and Anemopaegma arvense (Vell.) Stellfeld ex de Souza (endangered). The vast majority of herbarium collections  did not require uprooting.Eleven RBS plots had been initially chosen to correspond to one plot near the nests (henceforward nest plot) and ten other plots established at the vertices of two pentagons; the inner pentagon was established with its vertices at 700 m from the nests and the outer pentagon with vertices at 1500 m from the nests. These distances were chosen based on the literature of the flight capabilities of other stingless beesThe 30 most abundant plant species in the pollen and honey samples were classified by ubiquity . We then crossed this information with data from the RBS floristic surveys: distance from the nests, i.e., if they were sampled at nest plot, inner pentagon plots, outer pentagon plots, nearest or furthest gallery forest plots, or the gardens. These 30 species were also characterized from the literature in terms of type of resources , their habitat  and habit  Table .Table 1T39 and the packages phyloseq40 vegan41, bipartite42, circlize43 and viridis44 . In R, non-plant sequences were removed from the dataset, as well as the data transformed to relative read abundances (RRAs) per sample. ASVs that were classified as the same plant species were accumulated at the species level. Low abundance taxa that contributed less than 1% to a sample were removed from those samples. The Shannon diversity index was calculated for each sample (pollen and honey) for each bee species. The diversity was tested for significant differences between stingless bee species using the Kruskal-Wallace test, separately for pollen and honey samples. We also performed an NMDS ordination to visualize clustering of samples of pollen and nectar using Bray–Curtis beta-diversity dissimilarities. The ordination represented by proximity of points shows how similar two samples are in terms of composition and abundance of taxa. We tested for differences between species by using a PERMANOVA, separately for honey and pollen samples. We further calculated network indices of the three stingless bee species to account for their overlap and complementarity in the visited plant resources, i.e. the d’ for each bee species and H2’ for the entire network.Data was processed for analyses using R 4.2.229; 12 of the 15 absent taxa were exotic cultivated or weedy species. A detailed list of all significant plant species present in pot-pollen and pot-honey samples is available in Table Pollen and honey metabarcoding yielded a total of 5,079,123 quality filtered reads, with mean throughput per sample of 27,307.11 reads ±1756.635 (SE). Significant reads (more than 1% of reads in any sampling) accounted for 110 ASVs, in 86 genera and 40 plant families; c. 36% of these reads were only matched to generic level or above. In total, 95 out of the 110 ASVs recovered from the samples had been previously recorded in the IBGE Reserve floraM. rufiventris d’ = 0.22, S. postica d’ = 0.04, and T. angustula d’ = 0.22) , and consequently also that of the three species within the network  Skeels, Myrcia linearifolia Cambess. and Myrcia pinifolia Cambess.; Loranthaceae: Struthanthus/Psittachanthus, Anacardiaceae: Tapirira guianensis Aubl., Phyllanthaceae: Richeria grandis Vahl, Sapindaceae: Matayba guianensis Aubl., and Melastomataceae: Miconia stenostachya DC. Most of them offer pollen and nectar, except the pollen-only Miconia and the two Myrcia species. Thirteen of these ubiquitous species were nectar or oil flowers . Five highly abundant reads were incompletely matched, i.e. could not be identified to species level  but Croton, Eucalyptus, Psittacanthus and Struthanthus are known to produce floral nectar. Pollen-only flowers were found in honey samples of all three species: Myrsine sp, Blepharocalyx salicifolius (Kunth) O.Berg, Piper aduncum L., Miconia leucocarpa DC. and several Myrcia species, thus indicating some kind of mixing nectar and pollen trips, manipulation or spill-over inside the nests. Pollen records include similar diversity numbers of pollen-only flowers and flowers offering nectar and pollen. Only four out of the 110 ASVs were not recorded in our RBSs: Baccharis dracunculifolia DC. and M. pinifolia Cambess., both native Cerrado species that occur in IBGE, and exotic Eucalyptus sp. and Toxicodendron succedaneum (L.) Kuntze.The 30 ubiquitously found plant species in pot-honey and pot-pollen samples belong to the following families: Myrtaceae, Loranthaceae, Anacardiaceae, Phyllanthaceae, Sapindaceae, Melastomataceae, Euphorbiaceae, Primulaceae, Nyctaginaceae, Rosaceae, Asteraceae, Malpighiaceae, Cloranthaceae, Piperaceae, Fabaceae, and Clusiaceae Fig. . Out of Group 1 is composed of riverine forest species that offer pollen and nectar, recorded as very common in the Forest RBS surveys: Syzigium cumini, Tapirira guianensis, Richeria grandis, Matayba guianensis, Rubus urticifolius Poir.  Group 2 includes Cerrado shrubs or trees offering only pollen and recorded as common around the nests, in the Cerrado RBS surveys: Myrcia linearifolia, Blepharocalyx salicifolius and Maprounea guianensis Aubl. . They could be grouped into two dominant groups according to a combination of the habitat and floral resources. Melipona, Scaptotrigona and Tetragonisca revealed a broad generalized set of used floral sources regarding number of species and plant families explored. We recovered 110 plant species in pot-honey and pot-pollen retrieved from nests of the three stingless bee species. This reveals a broader spectrum of food sources than found by previous surveys on neotropical stingless bees that relied on non-DNA based methods such as field observations, field collections, and palynological studies. For instance, non-DNA based studies in another hyper diverse area in the Neotropics, the Amazon, revealed from 80 to 122 pollen types in nests and pollen loads of 10–15 species of stingless bees45. Other similar studies in species-rich areas of the Neotropics show comparatively lower numbers22. While these studies recorded a maximum of five to eight plant species per bee species, we found a mean of 46.3 plant species per bee species in honey samples and 53.67 in pollen samples. The interaction network and high number of species found in honey and pollen of the three analyzed stingless bee species point to a generalist foraging behavior, known to be common in eusocial bees and in stingless bees in particular20. It also points to probable scouting investigative trips, followed by heavy recruitment and opportunistic behavior when a high-quality resource is located, with most plant species with low abundance and a few highly abundant. Note that our results may still be an underestimation, since samples were collected during only 6 months, i.e., did not include all seasons.Pollen and honey metabarcoding of three stingless bee species in the genus Tetragonula laeviceps  using pollen metabarcoding coupled with light microscopy revealed 99 plant species46. Similarly, a study with Tetragonula carbonaria  in Queensland retrieved 302 plant species in pollen samples across seven sites at different seasons of the year over a two-year period13. These are promising results, especially when considering expanding this technique to tropical and subtropical forests in the Neotropics. Studies of pollination and floral biology in these habitats is often very difficult because flowers are often in the upper canopy and difficult to reach. Therefore, direct observations of bees on flowers in tropical and subtropical forests are rare20, and records of stingless bee—flower interaction in these environments became almost restricted to palynological analyses of pollen loads or pot pollen analyses22. Although their utility is undeniable47, morphological identification of pollen may become obsolete for pollination biology studies when compared with the efficiency of DNA metabarcoding to identify different plant species in extremely rich floras.The power of pollen DNA metabarcoding in revealing broad food sources for stingless bees had only been demonstrated before in Southeast Asia and Australia. In Sumatra, a study of Acalypha, Amaranthaceae: Amaranthus, Urticaceae: Cecropia, Cannabaceae: Trema), and a conifer genus, the introduced Pinus , widely cited in the literature as wind-pollinated51. This species was not only recorded in the pollen samples of all three species of bees, but was amongst the 10 most abundant records for T. angustula in our results. These results reinforce the theory that anemophilous plants, which account for 10% of angiosperms and most gymnosperms, produce enough pollen52 to be attractive to social bees, under certain conditions of colony size and food demands. However, the role of bees and other insects as true pollinators of anemophilous plants remains unresolved, in spite of the importance of wind-pollinated crops53 and of the several records showing that anemophilous plant pollen is important for several bee species (see references above).Pollen analyses via DNA metabarcoding also have the advantage of revealing unexpected food sources used by bees that would perhaps be unnoticed in studies using other methodologies. For instance, our analyses revealed that DNA from 13 wind-pollinated plant species were found among the 50 most abundant species in the sample of the three species, including monocots , eudicots (Euphorbiaceae: us Table . The preDumortiera hirsuta (Sw.) Nees found in pot pollen from the three studied stingless bee species 3, but because they show clear preferences towards some groups of plants62. Melipona are also the only stingless bees capable of buzzing to harvest pollen63, but pollen-flowers that require buzz-pollination for pollen harvesting were not abundant in the samples, even though species with poricidal anthers were observed flowering around the nests during the months of collection  Triana, Solanum falciforme Farruggia).19. In the case of T. angustula, which is considered a solitary forager, the range of pollen sources is wider and seems less biased towards mass blooming plants. In the Atlantic rainforest, another hyper diverse neotropical ecosystem, stingless bees have a preference for upper canopy stratum with small hermaphroditic or monoecious whitish flowers and abundant resources (pollen and/or nectar)29. Importantly, most of their preferred trees flower in mass, i.e. produce a large number of flowers over a short period of time20. In the Cerrado savannas, where the nests were, we observed the typical high frequency of shrubs and herbaceous species in stingless bees pollen (ca. 38% of samples), which reflects the savanna physiognomy where herbs and shrubs are predominant64. However, despite the high availability of flowers in the savanna surrounding their nests, they still flew up to riverine forests at least 630 m far from the nests to collect resources where mass-flowering species were more common.Our botanical surveys also reinforced the patterns of floral exploitation among the three species, such as the apparent preference for trees with mass flowering by stingless bees, even though their exploitation demands a long flight range. Some stingless bees' sophisticated communication abilities allow a massive recruitment of foragers when mass blooming plants are available65 and social behavior 66. Given that the closest riverine forest is located at a distance of 630 m to the nests, and that species from this habitat were among the most abundant in the samples, this suggests that all three stingless bee species will forage and probably recruit at least 630 m from their nests, supporting the hypothesis of long-distance foraging when attractive rewards are available20. This distance is well within the known flight range of Melipona whose typical flight distance is about 2 km, but can be extended up to 10 km18, but it is more surprising for Scaptotrigona and Tetragonisca whose reported maximum flight distances are 1.7 to 0.6 km, respectively18.Flight distance in bees is usually related to body size (larger bees tend to have wider flight ranges)Syzygium cumini, an introduced species that only occurs in a small portion of the nearest riverine forest to the nests. Other highly abundant species in our samples are common in the Distrito Federal riverine forests 69 and were only found in our surveys of the riverine forests  in the flora29, but it is surprising that other diverse plant families in the IBGE area, i.e. Fabaceae, Lamiaceae and Orchidaceae, which also represent close to 300 species combined29, are less conspicuous or totally absent from our most frequent 30 taxa. This means that, although the important floral sources for stingless bees partially overlap with the most common plants in the area, indicating that abundant sources are preferred, this is not always the case. This could simply mean that species within these families were not flowering at the time of sampling, but it is worth noting that Lamiaceae, papilionoid legumes and orchids share complex floral morphologies that are different from those of the families recorded as most abundant in our samples These three families tend to present flowers with bilateral symmetry, specialized petals and androecia, and deep, hidden resources that often forces floral visitors to approach and handle the flowers in a specific way70. Our results confirm the hypothesis that stingless bees may be specialized in exploiting small, open resource “bowl-type” flowers52, with exposed stamens and nectar, that are produced in large numbers20. They may also favor plant species with a “big bang” flowering phenology i.e., that that undergo mass blooming for short periods. Floral morphology, floral chemistry and phenology of plants exploited by stingless bees deserve further investigation. Investigations of plant resources exploited by stingless bees using metabarcoding over a longer time periods, in other types of vegetation, and of other bee species, would also be desirable to consolidate our knowledge of stingless bee ecology in the Neotropics.Some plant families stand out as the most important floral sources for the three stingless bee species, i.e. have one or more species amongst the 30 most frequent ASVs. Amongst them, Myrtaceae, Anacardiaceae, Sapindaceae, Melastomataceae, Euphorbiaceae, and Asteraceae are well-known as common resources for stingless bees globally28. The method has been prooved useful not only for pot-pollen, but also for pollen loads carried by bees in their scopae or corbiculae12. Given the importance of social and solitary bees for pollination of tropical crops and natural ecosystems, this methodology has the power to answer various important ecological questions, regarding the use of floral resources by bees, the dependance of bees on specific floral sources, their role as pollinators in crops or how well they compete with different species.In conclusion, our high-resolution results should encourage future studies on bee-plant interactions in hyper diverse tropical environments using pollen or honey DNA metabarcoding, even considering the currently low DNA sequence coverage of tropical plant species in public databasesSupplementary Information 1.Supplementary Figure S1.Supplementary Figure S2.