Preventive medicine is important in an aging society. Presarcopenia is the preliminary stage of sarcopenia. Recent advances in bioelectrical impedance analysis (BIA) devices have enabled automatic estimation of neck circumference (NC). However, the agreement between and interchangeability of NC measured manually and that calculated with BIA have not been evaluated. We performed these analyses in the context of health checkups and investigated their associations with presarcopenia. 2).  We enrolled 318 participants who underwent anthropometric measurements, including NC measured manually and by BIA; assessment of physical function; and blood testing. We used Bland-Altman analysis to calculate the agreement between and interchangeability of NC measurements by BIA and by the manual method. We then statistically compared normal participants and those with presarcopenia. Using multivariable analysis, we subsequently investigated significant risk factors for presarcopenia. We defined presarcopenia according to the appendicular skeletal muscle index  was negative overall (−1.07), for male participants (−1.23), and for female participants (−0.96). This finding suggests that BIA measurement is an underestimate in comparison with manual measurement. NC measurement by BIA was found to be interchangeable with that by manual methods, inasmuch as the percentage error was less than 5% overall (4.38%), for male participants (3.81%), and for female participants (4.58%). Univariable analysis revealed that NC was significantly smaller in the participants with presarcopenia than in those without. Multivariable analysis, adjusted for confounding factors, revealed that a decrease in NC was significantly correlated with presarcopenia.  BIA measurements of NC are interchangeable within about 95% with manual measurements. The decrease in NC measured by BIA was significantly associated with presarcopenia in both genders. NC measurement can be used for early detection of presarcopenia. Sarcopenia, a syndrome characterized by progressive and generalized loss of skeletal muscle mass and strength, is associated with adverse outcomes such as physical disability, poor quality-of-life (QOL), and death , 2. To mPresarcopenia is the preliminary stage of sarcopenia. To establish better public health policy and devise prevention strategies, the prevalence and temporal trends of presarcopenia and related body composition measurements must be understood in relation to sex, age, and race. Evidence-based prevention strategies for sarcopenia and musculoskeletal diseases must be based on the incidence of disability, risk factors, and other epidemiological data . Few sucNeck circumference (NC), an anthropometric marker for detecting risk for metabolic disorders , 9, can Bioelectrical impedance analysis (BIA), a method of easily measuring water content and body fat mass, is commonly performed during general medical examinations , 13. RecTherefore, the purpose of this study was to validate the measurement of NC by both BIA and manual methods and to evaluate whether these two measurements are interchangeable. We also investigated whether NC measurement by BIA is correlated with presarcopenia according to gender during general health checkups in a large, prospective population.The study participants were volunteers who underwent health checkups supported by the local government of Yakumo, Japan, in 2019. Of Yakumo's population of approximately 17,000 people, 28% are older than 65 years. A sizable proportion of its residents are engaged in agriculture and fishery. Since 1982, health checkups have been conducted annually in Yakumo; they consist of voluntary orthopedic and physical function examinations, internal medical examinations, and psychological tests, and a health-related QOL survey is administered as well –19. In oWe excluded from this study subjects with neck masses , thyroid diseases, a history of spine and limb joint surgery, severe knee injury, severe osteoarthritis, a history of hip or spine fracture, neurological disorders, severe mental illness, diabetes, kidney or heart disease, and severe disability in walking or standing or any dysfunction of the central or peripheral nervous system. We also excluded data from participants who did not fast before testing. In addition, we excluded patients with sarcopenia (defined as reduced muscle mass and either reduced muscle strength or performance) and severe sarcopenia  in order to focus on presarcopenia. Of the 537 individuals who underwent the health checkups, 318 participants (125 men and 193 women) met the selection criteria.The study protocol was approved by the ethics committee of human research and the institutional review board of our university . All participants provided written informed consent to participate before the study. The study procedures were carried out in accordance with the principles of the Declaration of Helsinki.Through BIA, we collected anthropometric data: weight, body mass index (BMI), percentage of body fat (PBF), appendicular skeletal muscle index (aSMI) to represent muscle mass, and NC. We used the InBody 770 body composition and body water analyzer , a BIA unit, to differentiate tissues  according to their electrical impedance , 13. Mea2 for men and 5.7 kg/m2 for women, calculated with BIA) were based on the diagnostic criteria described by the AWGS [Presarcopenia is characterized by a reduction in muscle mass that does not affect muscle strength or physical performance, according to the EWGSOP . The cutthe AWGS , 24.While the participants stood with the head positioned in the Frankfort horizontal plane and the shoulders relaxed, we used a nonstretchable plastic tape to manually measure NC from the level just below the laryngeal prominence perpendicular to the long axis of the neck; measurements were performed twice by two independent investigators, and mean data were recorded in centimeters and rounded to the nearest millimeter –27. IntrTo measure grip strength, we used the Toei Light Handgrip Dynamometer  , 28. ParWe analyzed venous blood samples from each participant for levels of albumin , total cholesterol and triglycerides (which can indicate the presence of metabolic syndrome), and C-reactive protein (a marker of inflammation). Biochemical analyses of the blood samples were performed with the use of an autoanalyzer  , 13.U test and the chi-square test to evaluate between-group differences, as appropriate for the data distribution. We examined correlations between manual and BIA measurements of NC by using the Spearman r and ICC . To interpret Spearman correlations, cutoff values of less than 0.20 were considered very weak; 0.20 to 0.39, as weak; 0.40 to 0.59, as moderate; 0.60 to 0.79, as strong; and 0.80 to 1.0, as very strong [x-axis and the difference  between measured values on the y-axis. To determine whether BIA measurements were interchangeable with manual measurements, we used a percentage error  as 8% or less [p < 0.01) in the univariable analyses, logistic regression analysis using a stepwise method was performed using the aforementioned variables as covariables. A p value of < 0.05 was considered significant in all analyses.All data were analyzed with SPSS statistical software . We calculated continuous variables as means and standard deviations (SDs) and categorical variables as percentages. We used the Mann–Whitney y strong , 30. To y strong . To examy strong . The mea or less , 32. To 2, and the average PBF was 29.4%. The average age of the 318 participants was 63.4 years , the average BMI was 23.7 kg/mr, ICC, and Bland-Altman analysis results for the NCs as measured by the two methods. BIA measurements were found to be interchangeable with manual measurements, inasmuch as the percentage error was less than 5% . The Bland-Altman plots showed that BIA measurements of NC were in almost perfect agreement with manual measurements, according to ICC , for men , and for women , body weight (p < 0.001), grip strength (p < 0.001), back muscle strength (p < 0.001), PBF (p = 0.003), and female gender (p = 0.025) were significantly associated with presarcopenia. The results of the logistic regression model according to sex are listed in The results of the logistic regression model for presarcopenia in all participants are listed in r = 0.89, p < 0.0001), for men , and for women (r = 0.80, p < 0.0001; BIA measurement of NC was significantly and very strongly positively correlated with aSMI overall (α; and social and mental causes, such as decline in cognitive function or decreased social activity [Sarcopenia is an important barometer of disability and frailty in elderly people, inasmuch as it exacerbates poor general health or frailty , 5. Presactivity –37. Lossactivity . InterveInterest in NC as a clinical measurement has increased substantially . Unlike In general, NC measurement is performed manually. In large-scale health checkups, manual measurements of many patients within a limited time may lead to interrater error because of the increasing numbers of clinicians who perform the measurements. To solve such problems, a device that can be used to perform automatic measurements in a short time must yield measurements that correspond to manual measurements. Portable BIA devices have been used to measure body composition and anthropometric markers . CliniciMoreover, BIA devices have been used to measure PBF and aSMI in various studies, and so we hypothesized that additional evidence could be accumulated , 22. aSMWith regard to accuracy, BIA and manual measurements of NC must be in agreement because manual measurement can introduce errors. If the agreement is high, it is possible that the measurement modalities are interchangeable. In general, Spearman correlations and ICC are used to validate the values obtained with the two measurement methods. However, correlation analysis concerns the relationship between two different events, and the value of the correlation coefficient cannot reveal differences between or variations in measurements . TherefoFurthermore, because we confirmed that BIA measurement of NC is interchangeable with manual measurement, we examined the results of a previous report in which NC was not measured with BIA and presarcopenia as the preliminary stage of sarcopenia was not considered . The uniThis study had some limitations. First, data from only one race of people in a single center were analyzed, and their demographic characteristics did not reflect those of people dwelling in the general community. The subjects were healthy middle-aged and elderly people who lived in a relatively rural area, and many had jobs in agriculture or fishing; therefore, they did not represent people living in an urban environment , 29. SecThe progress of BIA technology has been remarkable; depending on the device, it is possible to measure NC, and a large amount of data can be obtained in a short time from one measurement. The results of this study indicate that the relationship between NC and various diseases and conditions might be investigated on a large scale. We hope that the simple measurement of NC will play an important role in screening for presarcopenia and that early intervention in the form of muscle-strengthening exercises may prevent the conversion to sarcopenia.BIA measurements of NC are interchangeable within about 95% with manual measurements. The decline in NC as measured by BIA was significantly associated with presarcopenia in both genders. NC measurement may play a role in the early detection of presarcopenia.


To develop an artificial intelligence (AI) system that can predict optical coherence tomography (OCT)-derived high myopia grades based on fundus photographs.In this retrospective study, 1,853 qualified fundus photographs obtained from the Zhongshan Ophthalmic Center (ZOC) were selected to develop an AI system. Three retinal specialists assessed corresponding OCT images to label the fundus photographs. We developed a novel deep learning model to detect and predict myopic maculopathy according to the atrophy (A), traction (T), and neovascularisation (N) classification and grading system. Furthermore, we compared the performance of our model with that of ophthalmologists.When evaluated on the test set, the deep learning model showed an area under the receiver operating characteristic curve (AUC) of 0.969 for category A, 0.895 for category T, and 0.936 for category N. The average accuracy of each category was 92.38% (A), 85.34% (T), and 94.21% (N). Moreover, the performance of our AI system was superior to that of attending ophthalmologists and comparable to that of retinal specialists.Our AI system achieved performance comparable to that of retinal specialists in predicting vision-threatening conditions in high myopia via simple fundus photographs instead of fundus and OCT images. The application of this system can save the cost of patients' follow-up, and is more suitable for applications in less developed areas that only have fundus photography. Myopia has been recognized as an important public health problem worldwide . The gloThe atrophy, traction, and neovascularisation (ATN) classification and grading system proposed by Jorge Ruiz-Medrano is currently a common clinical diagnostic criterion, allowing a simple and systematic grading of myopic maculopathy that is both understandable and widely applicable . It contA potential solution is development of artificial intelligence (AI) technology, which has been applied to identify various ophthalmic diseases –16. PrevThis study aimed to develop an AI system that can predict OCT-derived ATN classification using monoscopic fundus photographs.This study was approved by the Ethics Committee of Zhongshan Ophthalmic Center  (ID: 2021KYPJ175) and the requirement of informed consent was waived in this retrospective study. And this study was performed in accordance with the principles of the Declaration of Helsinki; all private information that could identify individuals was excluded.Patients with high myopia presenting to the retinal clinic at ZOC between January 2014 and February 2021 were reviewed and analyzed. High myopia was defined as refractive error (RE) ≤ −6.0 D or axial length (AL) ≥ 26.0 mm. None of the patients had previously undergone surgery. Patients with coexisting or previous ocular disorders, such as diabetic retinopathy, retinal vascular abnormalities, uveitis, and age-related macular degeneration, were excluded.Fundus photographs were collected using a Topcon fundus camera , and one single macula-centered 50°color fundus photograph was obtained for each eye. All fundus photographs were downloaded using the standard tiff or jpeg compression formats. The corresponding spectral domain OCT images  were collected. The OCT images with horizontal and vertical slices through the fovea were downloaded in a standard image format according to the manufacturer's software instructions, and the scan length was 6 mm. These slices can detect most vision-threatening conditions associated to high myopia. For some patients, the same eye was examined multiple times to monitor disease progression. If the interval between the two examinations exceeded 1 month and the multiple examinations showed that the disease is progressing, the images of the multiple examinations will be included as separate data in this study. There were no exclusion criteria based on age, sex, or race.Three Chinese board-certified retinal specialists with over 10 years of experience were invited to label each fundus photograph according to the corresponding OCT images of each eye . InitialWe used binary classification to simplify the ATN system, which can screen out patients who need close follow-up or treatment. Specifically, retinal atrophy (A1), macular hole or retinal detachment (T1), and pathological myopic choroidal neovascularisation (PMCNV) (N1) were detected or predicted . In addiThe devised framework used the residual network ResNet-34 model as the backbone to build a multi-branch ResNet-34 structure, which is utilized to extract rich high-level feature information , 23. We We used accuracy, precision, sensitivity, specificity, and F1 score to evaluate the performance and stability of the classification model. Accuracy was measured by dividing the number of correct predictions by the total number of samples. Precision was measured by dividing the number of true positives by the total number of true positives and false positives. Sensitivity was determined by dividing the number of true positives by the total number of true positives and false negatives. Specificity, was determined by dividing the number of true negatives by the total number of true negatives and false positives. The F1 score was calculated as twice the product of precision and sensitivity divided by their sum and measures the balance of the positive and negative samples at the same time. For further evaluation, two retinal specialists and two attending ophthalmologists were invited to classify the images to compare with the AI system. The area under the receiver operating characteristic (ROC) curve (AUC) was calculated to assess the total performance of AI models and clinicians.A total of 2,014 fundus photographs were collected, including 1,814 high myopia fundus photographs (704 subjects) and 200 normal fundus photographs (116 subjects). After quality control, 161 poor quality images were excluded. Finally, 1,853 fundus photographs were selected to develop and evaluate our AI system. We split the data assigning 1,483 images to the training dataset and 370 to the test dataset. The demographic characteristics of included subjects are shown in The AUCs of the proposed method were 0.969, 0.895, and 0.936 for categories A, T, and N, respectively, and the accuracies were 92.38, 85.34, and 94.21%, respectively ; Table 3In addition, we trained the model to perform a multi-classification task for category A. Compared with the binary classification model, this multiclass model can distinguish tessellated fundus independently, which is more conducive to the identification of early lesions. The accuracies of the four methods  were 89.05, 90.71, 90.95, and 91.67%, respectively. We compared the classification performance of the AI system with that of ophthalmologists, including two retinal specialists and two attending ophthalmologists. To illustrate the superiority of the proposed method, we plotted the ROC curves and marked the operating points of the four ophthalmologists as well . The avePrevious studies have shown that AI technology can use fundus photographs to predict cardiovascular risk, refractive errors, and ci-DME, significantly outperforming specialists –19. VaraIn previous researches, Li et al.  developeAutomatic diagnosis of retinal detachment and/or PMCNV by fundus photographs has been reported in some studies , 26. HemSome researchers have performed automatic identification and segmentation of myopic lesions based on fundus photographs, and achieved promising performance –28. Tan Due to the large population of patients with myopia, it is challenging for ophthalmologists to conduct large-scale screenings , 29. If This study had some limitations. First, the ATN classification in this study has been simplified. However, the system can accurately identify patients who need referral and can save the cost of patient follow-up. Second, all images in our study were collected from the same hospital. Images from multiple centers could be obtained as external data to further improve and assess our AI system. In addition, this AI system can detect PMCNV, but it is difficult to estimate the activity of the lesion. This problem could be solved by collecting more images from the long-term follow-up of patients with PMCNV to compare the different features over time.In conclusion, the AI system we developed can predict ATN classification of high myopia using the cheaper and more widely available fundus photographs, with performance comparable to that of retinal specialists. Its clinical applications would provide a comprehensive diagnosis to triage and referral and reduce the individual and social costs. Such promising performance recommends its extensive application as a large-scale screening tool in primary medical institutions.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Ethics Committee of Zhongshan Ophthalmic Center  (ID: 2021KYPJ175). Written informed consent was not required for this study, in accordance with the local legislation and institutional requirements.ZW, WC, and SC: conception and design. LL: funding obtainment and provision of data. ZW, SC, and YW: collection and assembly of data. HX and BL: data analysis and interpretation. YZ, LL, and SC: manuscript revision. All authors wrote the manuscript and approved the final version of the manuscript.This work was supported by National Natural Science Foundation of China (No.62106153) and Guangdong Province Science and Technology Projects (No.3030901010066).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Annual species show traits, such as shortleaf lifetimes, higher specific leaf area, and leaf nutrient concentrations, that provided a more rapid resource acquisition compared to perennials. However, the comparison of root traits between the annuals and perennials is extremely limited, as well as the trade-offs of leaf and fine root traits, and resource allocation between leaf and root, which may provide insight into the mechanism of species changes in arid and semi-arid areas. With lab analysis and field observation, 12 traits of leaf and fine root of 54 dominant species from Horqin Sandy Land, Northeastern China were measured. The organization of leaf and fine root traits, and coordination between leaf and fine root traits of annual and perennial plants were examined. Results showed that there were differences between annuals and perennials in several leaves and fine root traits important in resource acquisition and conservation. Annuals had higher leaf area (LA), specific LA (SLA), and specific root length (SRL) but lower leaf dry-matter content (LDMC), leaf tissue density (LTD), leaf carbon concentration (LC), and fine root dry-matter content (FRDMC) than perennials. Leaf nitrogen (LN) concentration and fine root nitrogen concentration (FRN) were negatively related to LTD and FRDMC in annuals, while FRN was positively related to FRTD and fine root carbon concentration (FRC), and LA was positively related to LN in perennials. These implied that annuals exhibited tough tissue and low palatability, but perennials tend to have smaller leaves to reduce metabolism when N is insufficient. Annuals showed significant positive correlations between FRC/FRDMC and LDMC/LTD/LC, suggesting a proportional allocation of photosynthate between leaf and fine root. In perennials, significant negative correlations were detected between LN, LC, and SRL, fine root tissue density (FRTD), as well as between LA and FRTD/FRC. These indicated that perennials tend to allocate more photosynthate to construct a deeper and rigid roots system to improve resource absorption capacity in resource-limited habitats. Our findings suggested that annuals and perennials differed considerably in terms of adaptation, resource acquisition, and allocation strategies, which might be partly responsible for species changes in desertified grasslands. More broadly, this work might be conducive to understand the mechanism of species changes and could also provide support to the management and restoration of desertified grassland in arid and semi-arid areas. Species changes are universal in degraded ecosystems, especially in desertified grassland in arid and semi-arid areas . Dominan2 generally capture resources and complete their life cycles more rapidly , differences in leaf-root traits coordination between annuals and perennials are expected, with leaf-root traits exhibiting extravagant resource allocation strategy between above- and belowground components in annuals, and resource conservation allocation strategy in perennials. Growing evidence of consistent trait syndromes between leaf and root indicates coordination between above- and belowground organs in terms of the acquisition and allocation of limited resources . HoweverThis study investigated 12 traits of leaf and fine root of 54 dominant species in Horqin Sandy Land, Northeastern China, to analyze leaf and fine root traits organization and leaf-root traits coordination in annuals and perennials, respectively. Horqin Sandy Land is one of the four well-known Sandy Lands in northern China, has been subjected to serious desertification in the last 40 years . Studies4 km2 in the northeastern part of Inner Mongolia, China. The area has a continental semi-arid to a semi-humid monsoon climate. The mean annual temperature ranges from 5.8°C to 6.4°C, with mean monthly temperatures from −12.6°C in January to 23.5°C in August, and mean annual precipitation from 343 mm to 451 mm, of which approximately 70% falls from June to August. The mean annual potential evaporation ranges from 1,500 to 2,500 mm. The mean annual wind velocity ranges from 3.5 to 4.5 m s–1, and the mean wind velocity in spring  ranges from 4.2 to 5.9 m s–1. Windy days with a velocity greater than 17 m s–1 is from 25 to 40 days per year, which results in 10 to 15 days of sandstorms and dust storms, mainly  in the spring.The study was conducted in the Horqin Sandy Land , which covers an area of 12.90 × 10Agriophyllum squarrosum , Setaria viridis , Artemisia halodendron (sub-shrub), Caragana microphylla (shrub), Bassia dasyphylla , Artemisia annua , Chenopodium acuminatum , Artemisia frigid , Periploca sepium (shrub), and Cynanchum thesioides .The Horqin Sandy Land is a sandy dune landscape due to desertification, with a mosaic distribution of flat or undulating sandy land, mobile dunes, semi-fixed dunes, fixed dunes, and interdune lowlands. The soil in the study area is classified as a Cambic Arenosol , in whicA. halodendron and C. microphylla, it was necessary to excavate to a depth of 1 m to obtain complete fine root tissue. Plants were transported to the laboratory and cleaned with distilled water to remove adherent soil, debris, and root of surrounding plants. For leaves, intact and mature leaves were collected for each sample. Part of the collection was oven-dried at 65°C for chemical analysis, while the other  was placed in a plastic bag for traits analysis. For fine root, we extracted undamaged fine root (<2 mm) of each sample. Part of the collection was oven-dried at 65°C for chemical analysis, while the other was placed in a plastic bag for traits analysis. Then, the collection of leaves and fine roots of each sample were put in the water in the dark for 24 h at 4°C to allow water saturation. For the leafless species Ephedra sinica, the plant part functionally similar to the leaf was measured.In August 2018, we investigated the traits of leaf and fine root of 54 dominant plants on a sand streak in Horqin Sandy Land. We measured leaf and fine root traits on five samples for each species, and at least five healthy and mature individuals were collected as one sample. To obtain intact fine roots, we excavated each plant by digging soil range 25 cm diameter and 25 cm deep, and wider and deeper when necessary to obtain fine roots for deeper-rooted species or taprooted species. For some sand-fixing shrubs, such as Before measurement, fresh and water-saturated leaf and fine root were dried with clean filter paper gently and weighted for water-saturated leaf and fine root. The leaf and fine root of each sample were then scanned with a scanner  for the digital image. Image analysis was conducted with WinRHIZO software  to measure the LA, length, and volume of the fine root. After scanning, these materials were oven-dried at 65°C to a constant weight and weighted. Leaf thickness (LT) was measured with a digital caliper as the mean of 3 measurements for each leaf, and 10 leaves were measured for each sample. SLA and SRL were calculated as the ratio between leaf area and leaf dry mass, and between root length and root dry mass, respectively. LDMC and FRDMC were calculated as the ratio between leaf and fine root dry mass and their water-saturated mass, respectively. LTD and FRTD were calculated as the ratio between leaf dry mass and leaf volume, and between fine root dry mass and root volume, respectively. The leaf and fine root of each sample were ground into a fine powder using a ball mill . The C and N concentrations of leaf and fine root  were measured by an elemental analyzer .K) in all leaf and fine root traits was tested using the K statistic, where K is calculated as the ratio of the observed phylogenetically correct mean-square error divided by the mean-square error of the data, standardized by the expectation under Brownian motion  in leaf and fine root traits were calculated using Equation 1:P = 0.05) and the homogeneity of error variance . An independent-sample t-test was used to test for the differences in leaf and fine root traits between annuals and perennials. The Pearson correlation tests were used to analyze the correlations among leaf and fine root traits. We repeated this correlation analysis using PIC of traits to test the effect of species evolutional history in the correlation of leaf and fine root traits. To test the correlations both within and among traits defining leaf and fine root, a principal component analysis (PCA) was performed on 12 traits (with ‘vegan’ package in R). To account for the influence of species evolutionary histories, we conducted PCA both with and without the PIC. Both PIC correlations and PCA with PIC were analyzed based on standardized contrasts, calculating relationships through the origin and adjusting degrees of freedom.The 54 species in Horqin Sandy Land were classified into two categories: annuals (27) and perennials (27) . For allP < 0.05), suggesting that these traits were strongly influenced by their evolutionary history. However, the K values of these traits were relatively small (K < 1).The LT and LA showed large variation (CV) among species, while FRDMC, LC, FRC, LN, and FRN had low variation in 54 dominant species in Horqin Sand Land . SignifiP < 0.05). But the perennial species had higher LDMC, LTD, LC, FRN, and FRC than annual species significantly (P < 0.05). Unexpectedly, there was no significant LN difference between annuals and perennials, as well as LT, LA, FRDMC, and FRTD (P > 0.05).Annuals showed significantly higher SLA and SRL than perennials (P < 0.05). LDMC was positively related to LTD and LC but negatively related to SLA and LN (P < 0.05). The correlation of fine root traits had a similar pattern as the leaf. FRDMC was positively related to FRTD but negatively related to SRL (P < 0.05). When PIC was included in the analysis of pairwise correlation of leaf and fine root traits, the different patterns of correlation were observed (P < 0.05).Pairwise correlation among leaf and fine root traits of overall species were analyzed, significant correlations were found (observed . The corP < 0.05). But in perennials, LN was positively correlated with LA (P < 0.05). And FRN is positively correlated with FRTD and FRC.Both for annuals and perennials, species with high LT tend to low LDMC and LTD, and species with high SRL means low FRTD , 4. HoweP < 0.05). However, SLA and LTD did not correlate to SRL and FRTD, respectively (P > 0.05). When PIC was included, the SLA and SRL were still not correlated, while the relationship between LN and FRN exhibited concordant patterns as ahistorical correlations, although the magnitude was increased. The LTD was positively correlated with FRTD, but the LDMC was decoupled from FRDMC. Somewhat unexpected was the positive relationship between LC and FRC in ahistorical correlations, while the negative relationship was observed in evolutionary correlations . And the LDMC and LTD were related to FRDMC and FRC positively (P < 0.05). The LN and LC were related to FRN and FRC, respectively, suggesting the proportional distribution of photosynthate and nutrients between the aboveground and belowground components in annuals. But for perennials, the LN was positively related to FRN, but negatively related to SRL and FRTD (P < 0.05). A positive relationship between LA and SRL and a negative relationship between LA and FRTD and FRC were also detected in perennials (P < 0.05).The coordination between leaf and fine root traits of annuals differed from those of perennials. For annuals, LT was negatively related to FRDMC and FRC  in carbon-use strategies previously reported . As expevia higher tissue density and dry matter content, which is the advantage of survival in harsh conditions of desertified grassland , which capture additional N through biological N fixation. These legumes would have a growth advantage in N limited desertified grassland, which is theoretically one of the reasons why these species could colonize in desertified grassland. Furthermore, a positive correlation between LA and LN was observed in perennials. Leaf nitrogen is integral to protein factors in photosynthetic machinery, especially Rubisco  and/or Agriophyllum squarrosum dominated the final state, virtually excluding other species (In the last 40 years, Horqin Sandy Land has been subjected to serious desertification, accompanied by a sharp decline in soil nutrients and marked changes in the vegetation. The dominant species olonized . This pr species .The differences in adaptation, resource acquisition, and allocation strategies between annuals and perennials can also provide guidance for the restoration and management of desertified grassland. In severely desertified grassland, it is necessary to establish artificial sand-fixing vegetation with perennial plants, which have a conservative strategy, such as smaller leaves and a deeper and rigid roots system, and thus can gradually colonize in desertified grassland. The colonization of perennials can create better conditions for the recolonization of annuals, which adopt an opportunistic strategy. Annuals can utilize the short-term availability of water in upper soil layers and rapidly grow to complete their life cycle once the rainfall events occur. Annuals and perennials might show a dynamically balanced state and coexist stably, due to diversification of their both vertical and horizontal spatial resource utilization. Thus, a new artificial-natural ecosystem might be established in the former moving dune-dominated landscape, which is important to the mitigation of desertification and restoration of desertified grassland in arid and semi-arid areas.via the presence of large leaves, high SLA and SRL, low-density leaf and fine root, and high N concentration, while perennials exhibited a conservative strategy relatively. Second, annuals tend to establish tough tissue and low palatability, but perennials tend to have smaller leaves to reduce metabolism and water loss in resource-limited conditions. Third, there was a proportional allocation of photosynthate between leaf and fine root in annuals. But perennials showed a conservative resource allocation strategy that species tend to allocate more photosynthate to roots to improve resource absorption capacity in resource-limited habitats, resulting in a deeper and rigid roots system and smaller leaves. Perennials gradually colonized and coexisted with annuals in desertified grassland in arid and semi-arid areas, due to their different conservative adaptation and resource acquisition strategies, as well as their diversified use of spatial resources. These different strategies of annuals and perennials might be useful for the prediction of the succession of vegetation community in desertified grassland in arid and semi-arid areas, and thus provide support for the management and restoration of desertified grassland. We suggest the establishment of artificial sand-fixing vegetation with perennials in desertified grassland, which would facilitate the recolonization of annuals, and is expected to form a stable artificial-natural ecosystem.Our study shows important implications for an in-depth understanding of the mechanism of species changes in desertified grassland in arid and semi-arid areas. Annuals and perennials differed considerably in terms of adaptation, resource acquisition, and allocation strategies, which might be partly responsible for species changes in desertified grassland. First, annuals differed from perennials in terms of several leaves and fine root traits important in resource uptake and conservation. Annuals displayed an opportunistic strategy associated with enhanced resource acquisition The original contributions presented in the study are included in the article/XZ conceived and designed the study based on discussions involving ZN and YL. ZN, YL, DH, and JZ performed the experiments. ZN and YL analyzed the results. ZN drafted the manuscript. All co-authors had a chance to review the manuscript before submission and contributed to discussion and interpretation of the data. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


To examine the associations of serum albumin, a nutrition indicator, with disability in activities of daily living (ADL), mobility, and objective physical functioning among Chinese older adults.Cross-sectional data of 2233 older adults (≥65 years) who participated in the 2011/2012 main survey of the Chinese Longitudinal Healthy Longevity Survey (CLHLS) and the 2012 biomarker sub-study was used. Serum albumin was measured by immunoturbidimetric assay. Physical functioning included subjectively (ADL and mobility) and objectively measured disability . Multivariable logistic regression models were performed.After adjusting for age and sex, compared with participants in the lowest quartile group of serum albumin, those in the highest quartile group had 45% lower odds of disability in ADL ; 48% lower odds of disability in mobility ; 46% lower odds of disability in standing up from a chair ; and 37% lower odds of disability in picking up a book from the floor . We did not observe a statistically significant interaction effect between serum albumin and vitamin D on disability in physical functioning.Serum albumin level was associated with physical functioning among Chinese older adults, regardless of vitamin D level. The findings indicate that appropriate management of poor nutritional status, in particular low serum albumin levels, may contribute to maintaining physical functioning in older adults. By 2050, China will have 400 million adults aged 65+ years . Among tAlbumin, the most abundant protein in the blood, is a significant indicator of malnutrition. Recently, lower serum albumin level has been linked to a decline in physical functioning among older adults in various studies –13. HoweIn the context of rapid population aging in China, this study aimed to examine the associations of serum albumin with disability in ADL, mobility, and objective physical functioning among Chinese older adults. Furthermore, we aimed to evaluate the interaction between serum albumin and vitamin D on physical functioning in the same population. We used data from the 2011/2012 main survey and the 2012 biomarker sub-study of the Chinese Longitudinal Healthy Longevity Survey (CLHLS), a national survey in China.CLHLS was established in 1998 as a national survey and was designed to investigate determinants of healthy longevity in Chinese older adults. Details of the methodology and the study design have been previously reported . In shorN = 2439). We excluded participants aged below 65 years (N = 85) or with missing data on serum albumin levels (N = 121), and had a sample of 2233 participants, with no missing data on covariates. Further, due to the varying numbers of missing data on each outcome, different analytic samples for each outcome were assembled , respectively. Further details are provided elsewhere , 26.Serum albumin levels were divided into quartiles and the cutoff values were 36.8, 40.1, and 43.3 g/L. In addition, we divided serum albumin and vitamin D into a dichotomous variable. The cutoff value of serum albumin was 35 g/L, recommended to define hypoalbuminemia in literature . SimilarIn the CLHLS survey, six items of daily self-care ability were collected from each participant based on the Katz index: dressing, bathing, transferring, toileting, continence, and eating . Each itThe status of mobility function was assessed by asking participants the question: “Can you walk continuously for 1 kilometer at a time by yourself?” As done in previous studies, mobility disability was defined as present if participants reflected any difficulty in this task .Objective physical functioning was measured through three tasks (protected by trained technicians) in every participant's home. These tasks included picking up a book from the floor, standing up from a chair, and turning around 360°. Thus, three dichotomous indicators were used to represent the presence or absence of disability in objective physical functioning. If participants were not able to perform one task independently, they were defined as having a disability in this task .2). The height (in centimeter) and body weight (in kilogram) of participants were measured by interviewers during the survey  –39 and le survey .2 test was used to compare basic characteristics among four quartile groups of serum albumin levels.Basic characteristics of study participants were summarized in the total sample and by serum albumin quantile groups. Continuous variables were summarized by mean ± standard deviation (SD) and categorical variables were summarized by numbers and percentage. The analysis of variance (ANOVA) or χWe used multivariable logistic regression models to examine the associations of serum albumin levels with ADL disability, mobility disability, and disability in objective physical functioning. The odds ratios (OR) and corresponding 95% confidence intervals (CI) were documented. The lowest quartile group of serum albumin levels was considered as the reference group and two models were considered. In model 1, we adjusted for age and sex. In model 2, we additionally adjusted for ethnicity, current marital status, residence, education, BMI, lifetime primary occupation, economic independence, being in receipt of adequate medication, co-residence with adult children, currently consuming alcohol, currently smoking, and regular exercise. In model 3, to determine the effect of serum vitamin D levels on associations of serum albumin with physical disability, we further adjusted for serum vitamin D levels based on model 2.Next, we examined the association of serum albumin and disability in physical functioning by serum vitamin D status. The significance of the multiplication interaction between serum albumin and serum vitamin D levels was estimated by adding cross-product terms in models. We evaluated three indices and their 95% CI to determine if there was an additive interaction between serum albumin and vitamin D on physical functioning. The three indices included the interaction contrast ratio (ICR), attributable proportion due to interaction (AP), and the synergy index (S). If there was no statistically significant additive interaction, the 95% CI of ICR and AP included 0 and the 95% CI of S included 1 . In addiTo examine whether the associations of serum albumin with outcomes differed by age, sex, and the status of comorbidity, we repeated the main analyses by age (aged 65–79 years or ≥80 years), sex, and comorbidity with the same covariates adjusted in model 2. In addition, we conducted several additional analyses to assess the robustness of our results. First, in model 4, we repeated all analyses additionally adjusted for five chronic diseases  based on model 2. Second, in model 5, we repeated the main analysis further adjusting for MCI based on model 2. All analyses were performed using SAS version 9.4 . A P <0.05 was considered to be statistically significant.N = 1231) were females. Compared with participants with lower serum albumin levels (quartile 1), participants with higher serum albumin levels tended to be older, and more likely to be female, Han, residents of rural areas, to have a formal education, and to have more social contact and support . Meanwhile, they were more likely to have a lower proportion of regular exercise, to consume alcohol, to have adequate medication, economic independence, and chronic diseases . Participants with higher serum albumin levels tended to have a higher BMI and higher serum vitamin D levels, compared with participants with lower serum albumin levels. Details about the characteristics of study participants are shown in The mean age (± SD) of study participants was 86.3 ± 12.2 years, and 55.1%  when treating serum albumin levels as continuous variables. The results of stratified analyses by age, sex, and comorbidity were shown in In this study, we observed statistically significant associations between serum albumin level and various indicators of disability in physical functioning among Chinese older adults. These associations were regardless of vitamin D level since no interaction between serum albumin and vitamin D level on disability in physical functioning was observed in our study. These findings suggest that serum albumin level could be a potentially useful biomarker of disability in physical functioning among older adults.Our findings of the associations between serum albumin level and disability in physical functioning are consistent with previous studies , 48–53. per se reflects an unhealthy status. Previous studies demonstrated that serum albumin was associated with many diseases such as diabetes  for the publication of any potentially identifiable images or data included in this article.XL: methodology, data curation, software, visualization, writing—original draft preparation, and writing—review and editing. XC: methodology, data curation, software, writing—original draft preparation, and writing—review and editing. ZY: investigation, methodology, and writing—review and editing. JZ, XS, and EH: methodology and writing—review and editing. ZL: conceptualization, methodology, data curation, software, visualization, and writing—review and editing. All authors contributed to the article and approved the submitted version.This study was supported by a grant from the National Natural Science Foundation of China (Grant No. 82171584), and the 2020 Milstein Medical Asian American Partnership Foundation Irma and Paul Milstein Program for Senior Health project award (ZL), the Fundamental Research Funds for the Central Universities (ZL), a project from the Natural Science Foundation of Zhejiang Province (Grant No. LQ21H260003), Alibaba Cloud, Key Laboratory of Intelligent Preventive Medicine of Zhejiang Province (Grant No. 2020E10004), and Zhejiang University Global Partnership Fund (Grant No. 188170–11103). The data used in this study were from the Chinese Longitudinal Healthy Longevity Survey (CLHLS), which is managed by the Center for Healthy Aging and Development Studies, Peking University. The CLHLS was supported by funds from the U.S. National Institutes on Aging (Grant No. R01AG023627), China National Natural Science Foundation (Grant Nos. 71233001 and 71110107025), China Social Science Foundation, and UNFPA. The funders had no role in the study design; data collection, analysis, or interpretation; in the writing of the report; or in the decision to submit the article for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Objectives: A systematic review and meta-analysis was performed to evaluate the potential prognostic role of serum uric acid (SUA) in patients with chronic heart failure (CHF).Methods: The Embase, PubMed, Web of Science and Cochrane Library databases were searched up to 5 April 2021 for relevant publications. Random effects model was used to pool data. STATA15.0 software was used to perform meta-analysis. Heterogeneity was assessed using the Cochran Q statistic (significance level of P < 0.10) and I2 statistics (significance level of 50%).Results: Ultimately, 18 publications reporting adverse events in CHF patients were included. The results indicate reveal associations between a high level of SUA and the risk of all-cause mortality , cardiovascular mortality , and the composite of death or cardiac events  in CHF patients. A 1 mg/dL increase in serum uric acid led to 4%  and 9%  increases in the risk of all-cause mortality and the composite endpoint of death or cardiac events in CHF patients, respectively.Conclusion: Serum uric acid is positively associated with the risk of adverse events in CHF patients. This study protocol has been registered at PROSPERO as CRD42021247084 (https://www.crd.york.ac.uk/PROSPERO).Systematic Review Registration:https://www.crd.york.ac.uk/PROSPERO. Chronic heart failure (CHF) is one of the most significant public health problems, and its prevalence is increasing as the population ages . CHF leaTo predict the prognosis and progression of HF, several biomarkers have been used. Biomarkers (BNP and troponin), ejection fraction (EF), NYHA, etc., provide good predictive value for HF mortality. However, in the early stages of heart failure, their ability to discriminate is still uncertain. Serum uric acid (SUA), a byproduct of purine catabolism, which is well-known as a diagnostic indicator of gout. In recent years, many epidemiological studies have reported the relationship between SUA levels and a variety of cardiovascular and cerebrovascular diseases, including heart failure , hyperteBased on the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines, a literature review and meta-analysis of the scientific peer-reviewed literature were carried out. We searched the PubMed, Web of Science, Embase, and Cochrane Library databases from the establishment of the database to 5 April 2021. The language was limited to English. The key search words used were (“left-sided heart failure” or “heart failure” or “congestive heart failure” or “cardiac failure” or “chronic heart failure”) and “urate” or “uric acid” or “hyperuricemia”). Supplementary document  describepost-hoc analyses of randomized controlled trials; 2. studies reporting the prognostic value of adverse outcomes between SUA levels and CHF; 3. multivariate adjusted hazard ratios (HRs) with corresponding 95% confidence intervals (CIs) were published; 4. follow-up time more than 3 months. The exclusion criteria were as follows: 1. enrolment of AHF patients; 2. trial subjects were the same and did not report longer follow-up times; 3. letters, editorials, and review articles. 4. Studies of drug trials.The inclusion criteria were as follows: 1. observational studies or The following data were extracted by two reviewers independently: first author's name, sample size, design, location of study, year of publication, baseline patient profiles, SUA cutoff value, event number, follow-up duration, and unadjusted or multivariate adjusted effect sizes [HRs] for outcomes.Quality assessments were independently conducted by two authors. Dispute was settled by further discussion. Should the dispute continue, a third party would be invited to the discussion for settlement. The Newcastle-Ottawa scale was used to assess quality of observational studies and cohort studies . The scoP < 0.10) and I2 statistics (significance level of 50%). All combined effect sizes were analyzed by using the random effects model. Meta-regression analysis and subgroup analyses of the primary outcome were performed according to publication year, patient age, sample size, whether to adjust the confounding, quality score, and follow-up time. The subgroup differences were tested by the Z test (significance level at p < 0.05). When the number of analyzed studies exceeded 10, Begg's test , non-English literature (n = 2), unavailable full text (n = 5), prognosis study of non-CHF (n = 16), lack of data (n = 14), conference abstract and review (n = 10). Thus, 18 studies  (I2 = 94.6%)  . Twelve = 94.6%) . PublicaP = 0.006; P = 0.001) and funnel plot , but the other result is stable . The results of sensitivity analysis showed that there was no significant change in the effect size when we deleted any of the studies, which indicated that our results were stable and reliable   . The sen= 67.8%) . Publica= 67.8%) ., but substantial statistical heterogeneity was found (I2 = 77.5%) (I2 = 93.4%)  . A 1 mg/= 77.5%) , 28–30. = 93.4%) . The res= 93.4%) , 13. Pub= 93.4%) .To explore the heterogeneity of the study, we conducted a subgroup analysis of all-cause mortality based on publication year, patient age, sample size, quality score, and follow-up time. The results of subgroup analysis show that none of these factors are the main sources of heterogeneity. The test of differences between subgroups shows that the effect size of the results may be affected by the sample size, follow-up time, publication year, and whether to adjust factors , 3.p > 0.05), so they were not the main sources of heterogeneity  Functional upregulation of a key enzyme in purine metabolism, xanthine oxidase (XO), which derives reactive oxygen species that may be responsible for a series of harmful processes in the pathophysiology of CHF, such as cardiac hypertrophy, myocardial fibrosis, left ventricular remodeling and impaired contractility. (ii) Uric acid impairs vascular endothelial cells and causes poor prognosis . A previIt is worth noting that the heterogeneity of the included studies was high. The potential sources of differences are age, sample size, variable adjustment, follow-up time, literature quality score and publication year. Based on the above factors, we conducted a subgroup analysis to explain the heterogeneity, but this did not find the source of heterogeneity. However, the results of our subgroup analysis did not change the conclusion that elevated uric acid would increase the risk of all-cause death. In addition, according to the results of the subgroup analysis, we also found that the size of the effect may be affected by factors such as sample size, follow-up time, publication year, and age of the research subjects. The reason may be as follows: there is a significant difference between the research results before and after 2010. Combined with the subgroup analysis based on sample size, it can be inferred that the main reason may be related to the sample size. The sample size studied before 2010 was small, while the sample size studied after 2010 was larger. Subgroup analysis of studies with adjusted or unadjusted variables showed that unadjusted studies found that the risk of all-cause death in patients with heart failure was higher than that in adjusted studies. Although multivariate adjustments have been made in most observational studies, residual confounding factors still inevitably exist. Among them, the use of diuretics and the degree of renal damage may play a key role in variable factors. Uric acid is the final metabolite of purine and is catalyzed by xanthine oxidase. Under normal circumstances, the production and excretion of uric acid are in dynamic balance, and all factors that can affect the production or excretion of uric acid, such as the activation of xanthine oxidase, the use of diuretics, and the injury to renal function, can affect the concentration of uric acid in the body. Diuretics are the basic drugs for the treatment of HF. The extensive use of diuretics can not only increase uric acid, but also produce many adverse reactions, such as electrolyte disturbance, hemodynamic instability, and neuroendocrine activation. These adverse reactions can increase the risk of death and lead to poorer prognosis in patients with heart failure. Other factors that influence uric acid excretion, such as reduced glomerular filtration rate, hyperinsulinemia, renal vasoconstriction, and factors that increase uric acid production, such as tissue ischemia or oxidative stress, may influence the prognosis of uric acid and chronic heart failure.Although there have been many studies on the relationship between uric acid and cardiovascular disease, a crucial question about how to determine the cut-off of cardiovascular uric acid needs to be answered. The actual cut-off of hyperuricemia (>6 mg/dL in women and 7 mg/dL in men) is mainly based on the saturation point of uric acid, but previous evidence suggests that low levels of uric acid may also have a negative impact on the cardiovascular system. The recently published URRAH study reported the uric acid cut-off for total and cardiovascular mortality, which indicated that the optimal cut point for total mortality and cardiovascular mortality was 4.7 and 5.6 mg/dL, respectively . This reTo the best of our knowledge, this is the first meta-analysis focusing on the relationship between uric acid level and the prognosis of CHF. Our meta-analysis showed that uric acid level is an independent predictor of all-cause death, cardiovascular death and the composite end point of death or cardiovascular events in CHF patients. Moreover, included studies from different countries were included, and the sample size was large, so the results were relatively stable and reliable. All the quality of the included studies was relatively high. Although there was heterogeneity in the results, the results of our subgroup analysis and sensitivity analysis were stable.However, our study also has limitations. First, the heterogeneity in our study was significant. Even though the sensitivity analysis and subgroup analysis were adopted, we still failed to explore the origin of heterogeneity. Second, the lack of repeated measurements of serum uric acid levels was a common limitation in the included studies. One time determination of serum uric acid levels may not accurately reflect the changes with time. Third, individual studies have different threshold for high and low uric acid, which would have an impact on our research results and may be a reason for the heterogeneity of outcome indicators. Four, considering the quality of the included research, we did not include some conference studies and gray literatures with small sample sizes, which may cause the publication bias. Finally, due to the insufficient of data, the relationship between serum uric acid levels and different stages of chronic heart failure cannot be evaluated in our meta-analysis.High SUA levels independently predicted the risk of all-cause mortality, cardiovascular death and combined death or cardiac events in CHF patients.The original contributions presented in the study are included in the article/JD and DS: conceptualization and supervision. LM and MG: data curation, formal analysis, investigation, and methodology. ZC: project administration. LM, YY, and PC: software and writing . MG and DS: writing (review and editing). LM, DP, and JG: modified the final version. All authors contributed to the article and approved the submitted version.This work was supported by the project of Major New Drug Creation (No. 2018ZX09301-011-001), National Natural Science Foundation of China , and Fundamental Research Funds for the Central Public Welfare Research Institutes Grant (Grant No. ZZ13-YQ-016 and ZZ13-YQ-016-C1).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Carex pulicaris is considered an endangered species, and further losses are forecast under the influence of even moderate climate change. Local studies indicate that temporal declines in C. pulicaris abundance are positively correlated to decreases in precipitation and increases in air temperature. Determining ecological properties on larger scales than local ones can help develop effective protection programs for the species. We hypothesize that the local relationships observed between C. pulicaris abundance and precipitation, air temperature and soil properties will be confirmed in a spatially-oriented large-scale study performed in situ. Therefore, the present study takes a novel, large-scale integrated approach to (1) precisely characterize the ecological requirements of C. pulicaris within its eastern distribution range, and (2) determine the influence of its community type, soil properties and climatic conditions on its abundance. It was found that C. pulicaris is not a dominant or codominant species in the studied phytocoenoses in the eastern distribution range. Five natural vegetation groups including C. pulicaris, with significantly diverse species compositions, were resolved: well supported Estonian, Polish, Slovak and Radecz groups, and a weakly-supported Ambiguous group. The abundance of C. pulicaris was found to be positively correlated with the composition of the geographically-diversified plant communities and atmospheric precipitation, and to be also negatively associated with latitude and soil pH. Although the species is adapted to a relatively wide range of soil types, such adaptation requires appropriate substrate moisture level and light conditions. The species prefers moist organic and mineral soils and grows on both acid and neutral medium, characterized by a narrow C:N ratio, with various amounts of digestible total P, Mg and N, and low levels of digestible K. Climate change, manifested by reduced rainfall, may be one of the most important predictors negatively affecting the occurrence of C. pulicaris. Such changes will be reflected in species distribution and phenology, as well as in interspecies interactions, among others, and they are expected to become the main threat to biodiversity. Of these species, the most vulnerable will be the rare and endangered ones, as they are usually characterized by a narrower niche width5. Therefore, one of the core goals of conservation biology is to understand the distribution, abundance and ecological requirements of rare species7. Such studies are urgently needed for species whose natural habitat is fragmented and vulnerable under anthropogenic pressure. Some regional differences related to climate, geology and the history of land use may occur within the species range9. It is also important to note that conservation efforts should focus not only on populations from the centers of their distribution, but even more on those located at the edges, where the risk of extinction is highest10.All conservation initiatives are subject to the influence of climate change, which will undoubtedly have a dramatic impact on many ecosystems11. As a result, many fen species are believed to be at risk of extinction, and species conservation has become a priority in wetland ecosystems.Global climate change has increased the risk of summer droughts, which makes fen habitats one of the most sensitive habitats in Europe. In many cases, this damage is exacerbated by anthropogenic land transformations of varying intensity, such as extensive landscape degradation and drainage. Unfortunately, habitat restoration programmes may fail to recover the biodiversity of degraded peatlands12.Wetland ecosystems, and peatlands in particular, are a refuge for many rare plant species associated with specific habitat conditions and which significantly enrich the biodiversity of a given region. Many of these are stenotopic organisms, with a very narrow ecological niche. As a result of the transformation and disappearance of peatland habitats, the population of these plants decreases, which, in turn, may lead to the disappearance of their localities and their withdrawal from previously-occupied areas. Such losses may cause disjunctions within their species range and, in drastic cases, even a shrinkage of the range itselfCarex pulicaris (flea sedge) is a typical fen species, occurring in western and northern Europe, ranging from Spain and Ireland in the west to Scandinavia in the north, with its eastern distribution limits in Baltic countries , Belarus and central Europe (Slovakia); the species does not reach the Mediterranean region13. The occurrence of C. pulicaris has a fragmented, island-like character at the eastern edge of its distribution range, with a disjunction in Poland east of the Bay of Puck between Atlantic and Eastern Baltic sites. Several studies have already documented a loss of C. pulicaris localities in recent decades, e.g. in southern Germany, in Slovakia, northwest Poland and Estonia17. Indeed, the species is classified in various threat categories in many European countries, being considered endangered in Central Europe20 or vulnerable in Fennoscandia22. In Estonia, according to the last Red Data Book assessment, the species is near threatened23.C. pulicaris are mainly associated with wet and periodically wet habitats, and occasionally strongly transformed ones. They occur in low and transitional bogs, laggs of raised bogs, peaty meadows and wet forest habitats, and less often on impermeable loam and clay slopes24. The species is found on both acidic and neutral soils, preferring peat soils. It is less common on soil-glial soils16.Populations of Carex pulicaris does not usually contribute much to the construction of plant communities. It is considered a species typical for the Caricetalia davallianae order and the Caricion davallianae alliance25. It is a frequent element of plant communities classified into the new, recently-distinguished Sphagno warnstorfiani—Tomenthypnion alliance27, belonging to the Caricetalia davallianae order. It is also a part of the Caricion nigrae and Caricion lasiocarpae alliance of the Scheuchzerio-Caricetea nigrae class, and encroaches into the Calthion and Molinion alliance of the Molinio-Arrhenatheretea class29.Carex pulicaris is a highly vulnerable species. Distribution models suggest that it will be lost from many regions along its eastern distributional limits in response to moderate climate change30. Further studies also suggest that assisted migration may be a beneficial management strategy for the species, with reintroduction being another potential management strategy31. As the species is a weak competitor, low nutrient availability and low competition for light may favour its long-term survival. A case study performed at the regional scale found that negative changes in hydrological regimes occurring since 1950, influenced mainly by lower summer precipitation and higher mean annual temperature, both driven by climate change, are significantly associated with predicted extinctions of C. pulicaris populations occurring at lower altitude limits14. However, all these results were obtained from modelling based on climate-only data or regional studies. There is a lack of detailed studies of habitat conditions across a larger area of occurrence; such research is extremely important, especially at the edges of the distribution range, where the species is less likely to find favorable conditions for development, and its sites are more vulnerable to disappearance.C. pulicaris with more comprehensive studies of the habitat parameters and floristic diversity of plant communities over a larger area, including the climate conditions. Such data can be used to identify appropriate protective measures to prevent the emergence of disjunctions and the projected shrinkage of the eastern range as a result of projected climate change. We expect that in the eastern range of C. pulicaris, its abundance is significantly influenced by temperature and rainfall14, vegetation type and substrate properties . We also hypothesise that both the abundance of C. pulicaris and the diversity of plant communities in which it occurs may be related to the region of its occurrence.Hence, there is a pressing need to identify the factors determining the abundance of The present study addresses the following questions:1. Do the plant communities in which C. pulicaris occurs vary regionaly, and is species abundance related to community diversity?C. pulicaris and its abundance at the eastern end of its range, with regard to different latitudes and climatic conditions?2. How can climatic factors affect the communities of C. pulicaris, and do they affect its abundance?3. What are the properties of the soil in the eastern range of C. pulicaris were performed at 17 different locations: three in Denmark (Bornholm), six in Poland and six in Estonia, and two in Slovakia , other phytosociological relevés of Poland (13 relevés), which are recorded in the Polish Vegetation Database32, as well as previously published data from Slovakia (13 relevés)29. In total, our analyses were based on 43 phytosociological relevés recorded from 27 localities . The syntaxonomic approach to communities was adopted after Matuszkiewicz (2006)25.Phytosociological relevés were performed in patches of 33: ecological , sociological  frequency and risk . In the case of 17 of the examined locations, the soil data were also analysed.The habitat conditions were assessed based on Ellenberg indicator values (EIVs)https://worldgrids.org library, developed for the needs of the “Soilgrids” project, organized by the International Soil Reference and Information Centre . The data are currently available in the archive at https://web.archive.org/web/20170619054443/http://www.worldgrids.org/doku.php/start.Climatic conditions were evaluated for all the analysed locations based on the rasters collected in the The analyses included latitude, longitude, daily minimal temperatures, daily mean temperatures, daily mean temperatures in December/January, and annual mean monthly precipitation Table . The ras34  for each of the natural sites34 Table . In the −3 HCl 36 and soluble in concentrated HNO3 and HClO4, at the ratio 1:1  was determined using an ICE series 3000 spectrometer with flame atomization (FAAS)37. The content of Na, Ca and K was determined by flame atomic emission spectrometry, while that of the other elements was determined using flame atomic absorption spectrometry. The limits of detection were (mg kg−1): Ca—0.004; Mg—0.002; K—0.001; Na—0.004; Fe—0.004; Cd—0.003; Co—0.010; Cu—0.005; Ni—0.008; Pb—0.013; Zn—0.003 and Mn—0.002. The content of available and total P was determined with spectrophotometric molybdenum blue method (690 nm wave length)37 using a Marcel MEDIA™ spectrophotometer.In addition, the content of Mg, K, Ca, Na, Cd, Co, Cu, Ni, Pb, Mn, Fe and Zn soluble in 0.5 mol dm−3.The accuracy and precision of the analytical methods and procedures used were confirmed using certified reference material: CRM036-050 Loamy Sand 4 . The effectiveness of the process was validated with 90–95% efficiency. The results shown are the mean values of three measurements, with working standards made from Merck standards at a concentration of 1000 mg dm40.The soil type classification follows the international and regional standardsCarex pulicaris was compared with the phytosociological composition of sites and three sets of environmental variables: mean Ellenberg indicator values (EIVs), climate variables (CLIM) and soil variables (SOIL).The abundance of vegan package in R environment: non-metric multidimensional scaling (NMDS) of the species composition data matrix (43 sites × 310 species), and principal component analysis (PCA) of the soil data matrix (17 sites × 34 soil variables)42. The raw species composition data matrix and Bray dissimilarity index were subjected to NMDS, and raw data were centred and scaled prior to PCA. Cluster analysis  was performed to delineate natural phytosociological groups; the grouping pattern was used throughout results. The relationship of C. pulicaris to site structure and its relationship with possible explanatory variables were assessed by fitting C. pulicaris abundance data over NMDS or PCA ordinations using the envfit function of the vegan package42.The species composition and soil types of the sites were compared by multivariate analyses using the In addition, species composition on the sites was compared with environmental data. The collected data were utilised to construct six data matrices; three types of environmental data matrices: (1) mean Ellenberg indicator values (EIVs), (2) climate variables, (3) soil variables, and three corresponding species-composition data matrices. The species composition data in each locality were averaged per locality to obtain a reduced dataset corresponding to database-mined climate data ; this dataset was independent of the species composition in each location.vegan42 to formally test for the ecological interpretation (EIVs and CLIM) of the sites and the species similarity assemblages. ANOVA was applied for statistical testing, and the global CCA model was statistically significant. The parsimonious CCA model was selected by a permutation test using forward model selection.Canonical correspondence analysis (CCA) was performed in vegan42) to superimpose SOIL variables over NMDS ordination of species composition data .SOIL data were not subjected to CCA, since the number of sites  was much lower than the number of soil variables (n = 34). Instead, it was tested whether soil data may explain species composition data; briefly, the envfit function was used  and Tukey's HSD pairwise multiple comparison were used to test for differences between means of particular parameters of different vegetation groups. Prior to analysis, assumptions of ANOVA, data normality and homogeneity of variance, were tested applying the Shapiro–Wilk test and Levene's test, respectively. Slight deviations from data normality were tolerated. The level of significance was set at p < 0.05 in all analyses. Statistical analysis of the obtained results was performed using R or in Past 3.1043.To characterise each of the localities, plant diversity indices, i.e. the number of species recorded per relevé, Shannon’s H diversity index, and Pielou`s Evenness were also calculated in the , the statistical significance of the differences between means was determined by testing the normality of distribution in each group and homogeneity of variance in all groups, followed by ANOVA with Tukey's post hoc test. The significance was set at p < 0.05. These analyses were performed using Statistica 12.5 PL software .Regarding the tested chemical properties of the soilCarex pulicaris locations were found to demonstrate a range of species compositions. Our cluster analysis of the similarity in species composition between sites . C. pulicaris. These are Potentilla erecta , Carex panicea (83.72%), Briza media (79.07%), Succisa pratensis (65.12%), Molinia caerulea (62.79%), Galium uliginosum (55.81%), Cirsium palustre (53.49%), Calliergonella cuspidata (51.16%), and Holcus lanatus (51.16%).Most of the recorded species are very rare in the dataset, with 69.03% being present in fewer than five plots, i.e. approximately 10% of all of investigated plots. In contrast, only ten species are present in more than half of all plots with C. pulicaris in particular vegetation types. Carex davalliana dominates in the Estonian and Slovak groups , Anthoxanthum odoratum is common in the Polish and Ambiguous groups , and Lotus uliginosus is also common in the Polish group (76.92%). The Slovak group is floristically very rich (results presented below), with Campylium stellatum (92.31%), Fissidens adianthoides (92.31%), Bryum pseudotriquetrum (76.92%), Eriophorum angustifolium (76.92%), Carex echinata (69.23%), Carex flava (69.23%), Parnassia palustris (69.23%) and Plagiomnium elatum (69.23%) commonly found together with C. pulicaris. Many species are commonly observed with C. pulicaris in the Radecz group, but these data were obtained from only three related relevés.Other species were found to accompany C. pulicaris are moderately rich, with the number of species per relevé varying between 13 and 59. Comparatively high species richness was also observed in the Polish (35.38 ± 8.62), Ambiguous (35.00 ± 5.55), Slovak (42.92 ± 9.31) and Radecz groups (41.67 ± 1.53), but lower in the Estonian group (18.00 ± 3.95)  and Slovak groups ; however, they are rare or even absent in the rest, where number of moss species per site ranged from 0 to 8.. the Estonian, Slovak, Polish and Radecz groups, are compact and well resolved on the NMDS ordination biplot  out of a total of 16  and humus mineral soils (Umbric Gleysols).The studies on soils associated with C. pulicaris also grew in fen, but strongly decayed bogs with high ash content (> 40%) and slightly acidic or neutral pH. The rhizospheres of the bogs differed significantly with regard to total nitrogen content  and magnesium  and a very low content of available potassium. The Estonian, Polish and Slovak bogs did not differ significantly in their available K or Na content. The peatlands from Poland contained significantly more available P than those from Slovakia, which were richer in available Ca and Mg , they were characterized by low ash content < 20%) and acidic or slightly acidic pH. In Slovakia, 0% and acCarex pulicaris was also found on moist mineral soils (Umbric Gleysols) with a mean organic matter content of 10 to 16% and a C/N ratio ranging from 13 to 14. Despite their similar systematic affiliation, the surface soil layers (rhizospheres) differed in their chemical composition between Estonia, Denmark and Poland   found that the site groupings reflect generally geographic locations and soil types; in addition, this patterning appears to be irrespective of plant community type, and of the abundance of ris Fig. A,B. In cris Fig. C. The on01) Fig. C.Figure C. pulicaris, were characterized by a homogeneous type of vegetation. The Estonian group was represented by communities of the Molinion caeruleae alliance, sometimes with a significant share of Carex davallianae, and the other two by communities of the Caricetalia davallianae order. In Slovakia, C. pulicaris is associated with non-forest communities of minerotrophic fens, particularly with the Sphagno warnstorfiani-Tomenthypnion alliance29. In turn, the Polish and Ambiguous groups showed greater diversity: their composition included communities from both of the above-mentioned syntaxonomic units, with the Polish group including some from the Caricetalia nigrae order, with some tendency towards the Nardo-Callunetea class.The Estonian, Slovak and Radecz groups, which were most closely related to a particular region within the range of C. pulicaris is commonly derived from the communities of the Molinion alliance, and this can be seen in Slovenia, the Netherlands and Sweden45. In France, it was recorded in acidic fen grasslands 46, and in Great Britain, in communities such as calcicolous grassland community Sesleria albicans—Galium sterneri in the Carex pulicaris—Carex panicea subcommunity, mire community Carex dioica—Pinguicula vulgaris and Molinia caerulea—Cirsium dissectum fen-meadow (according to the UK NVC47). The last of these communities is the British equivalent of Cirsio-Molinietum Sissingh & de Vries 1942, described from the Netherlands, Belgium, Germany and Ireland48.Throughout its entire range, Molinia caerulea in the studied phytocoenoses. This is a highly competitive grass, and its presence in large clumps not only limited the number of safe places for the germination of other plant species diaspores, but also hindered their development. This was especially true for annual plants, and to a lesser extent, the perennials, which show vegetative reproduction. Moreover, most of the plant patches were partially shaded by the crowns of trees growing in the immediate vicinity. Under these conditions, C. pulicaris often grew in the Molinia clumps. We observed this phenomenon not only in the locations in Estonia, but also in the Rozwarowskie Marshes in Poland (PL_BagR).The groups of plant communities separated as a result of the analyses were generally characterized by considerable plant species diversity, with the only exception being the Estonian group Fig. ; the lowC. pulicaris was often accompanied by species associated with grassland communities, particularly meso- and eutrophic hay meadows and riverside herbaceous plant communities; these are permanently, or at least periodically, moist . The significant share of these species in some of the examined patches of C. pulicaris communities from the Caricetalia davallianae order (e.g. in Slovakia) may indicate the presence of fluctuations in the water regime and/or a slightly increased availability of nutrients for plants. Such coexistence of species may take place over a longer period of time, and in the case of a permanent change in habitat conditions, it may lead to another stage of succession towards meadow communities from the Molinion caeruleae alliance29. Hence, it is sometimes difficult to unequivocally define the syntaxonomic affiliation of plant communities from C. pulicaris.Regardless of the research area, C. pulicaris in the analyzed groups was diversified, and it was neither a dominant nor a codominant species in any of the phytocoenoses. In contrast to the eastern end of its range, C. pulicaris can be found abundantly in the remainder of its distribution area; it has been reported as the dominant or codominant species in some phytocoenoses, for instance in Great Britain and Ireland50. As C. pulicaris is a poorly-germinating51, light-requiring species that is weak in competition, its abundance is favored by extensive land use, which limits the development of tall, expansive perennials. Our findings indicate that the mowing of meadows in areas where C. pulicaris occurs (Domysłów and Rozwarowskie Marshes) favors its survival, even under conditions of high competition from Molinia caerulea, and in the vicinity of Phragmites australis plantations. It is classified as a species dependent on mowing in the area of the fenoscandian limestone forest meadows52, and was only found in mowed areas in the costal grasslands in the Stockholm archipelago45. Also in the Netherlands, this type of land use is necessary to maintain the structure and species composition of meadows belonging to the Molinion caeruleae alliance, where C. pulicaris is present7. This prevents the succession towards scrub and forest, especially when the habitat is fragmented.The share of C. pulicaris is correlated with greater precipitation, albeit to a small extent  calculated for sea level55 with the species distribution data53, a clear relationship can be seen between the limit of its range and the -4 °C isotherm. The results of local studies from mountain areas in southern Germany14 highlight the significant role played by both temperature and precipitation on the occurrence of C. pulicaris. In contrast, while our present findings do not show any clear relationship between species abundance and air temperature, the CCA analysis  of the soils in the tested areas demonstrated great variation in their physical and chemical compositions. Murshic Histosols were acidic to slightly acidic, while Umbric Gleysols were acidic to neutral38.Certain chemical and physical properties of soils have a strong influence on the occurrence of plantsC. pulicaris occurs demonstrate similar variations in soil pH61. However, C. pulicaris generally preferred slightly acidic soils, and did not favour strongly acidic and alkaline soils. Our study showed that soil pH was significantly correlated with the grouping of plant communities with C. pulicaris participation. Soil acidity affects such features as the trophic nature of the habitat, the quantity and quality of humus, and the abundance of assimilable macro- and microelements. The wide range of soil pH values tolerated by C. pulicaris may indicate a well-developed adaptability to various environmental conditions; however, this contrasts with previous observations, i.e. that the species occurs in bogs of a narrow ecological amplitude29.The areas across Europe where 64. The results of our analyses showed that both organic and mineral soils were generally low in available P and K, which may support the growth of C. pulicaris. These habitats have previously been found to have low nitrogen content65, and data from areas within the C. pulicaris range suggest that its communities require low N, P and K concentrations69; however, our present results do not indicate any significant relationship between the abundance of C. pulicaris itself and the P, N and K content of the soil. The plant communities of C. pulicaris are sensitive to excess N-NH4 levels and N-NO3 deficiency; this relationship is related to long-term stagnation of water on the soil surface, resulting in the development of anaerobic conditions70.Nitrogen, K and P levels are particularly important for plant community stability. In wet habitats, an increase of P, K and N levels in soil can lead to a decline in the diversity and abundance of plant speciesC. pulicaris abundance is not significantly affected by soil Ca, Mg and salt content. The species was recorded in soils with various Mg, Ca and salinity levels65, and its associated plant communities have been observed in habitats relatively rich in nutrients67. It has also been found to grow in soils containing low levels of heavy metals40.Our own studies indicate that Carex pulicaris appears quite resistant to anthropogenic influences known to increase habitat trophy, as indicated by our finding that macro- and micronutrient content appears to have no clear effect on its abundance in plant communities. On the other hand, distinct changes in soil moisture and the cessation of pratotechnical practices may limit its abundance70.Carex pulicaris responds well to surface floods in the autumn-spring period as well as to strong falls in groundwater level in summer71. Short periodic floods cause a reduction in the mineralization of organic matter; they also drive the release of nutrients, which are beneficial for the habitat, and stimulate the formation of Fe, Ca and P complexes, which are normally inaccessible to plants72. However, if maintained for too long, excessive soil moisture levels result in changes in the oxidoreductive and chemical properties of the soil that are unfavorable for the growth of C. pulicaris70. In contrast, a rise in the level of groundwater may favor paludification processes, which in turn will lead to the disappearance of C. pulicaris as a result of the reconstruction of plant communities with more competitive members. In addition, excessive dehydration and oxygenation of the habitat causes rapid mineralization of organic matter and the release of large quantities of mineral nutrients; this is also unfavorable for C. pulicaris, and favors the entry of expansive perennials, trees and shrubs.C. pulicaris. The specific humidity requirements described above explain the sensitivity of this species to changes in humidity in the habitat.Finally, most of the Estonian sites were characterized by large shares of trees in the vegetation patches; this can increase shading and thus may be the reason for the low abundance of C. pulicaris is present in five natural phytocoenological groups: four closely correspond to geographic regions, viz. Estonia, Poland, Slovakia and Radecz (Poland), and a fifth Ambiguous group scattered throughout the study region. In general, the groups show a significant diversity of species composition. The Estonian group was represented by communities from the Molinion caeruleae alliance, the Slovak and Radecz groups by communities from the Caricetalia davallianae order, while the other two showed greater diversity with regard to their syntaxonomic units. However, C. pulicaris was not dominant or codominant in any of the groups with regard to the studied phytocoenoses.Within the eastern end of its range, C. pulicaris is positively correlated with the composition of its geographically-diversified plant communities and atmospheric precipitation. In the eastern part of its range, C. pulicaris grows on organic (Murshic Histosols) and mineral (Umbric Gleysols) soils associated with high groundwater conditions. These soils are characterized by an acidic to neutral reaction, narrow C:N ratio, low salinity, mostly low levels of available P and K and micronutrients, and varying levels of available Mg, Ca and Na. Carex pulicaris appeared to grow most abundantly in plant communities which prefer slightly acid soils.Our findings indicate that the abundance of