In this paper, a new method in forecasting the horizontal displacement of diaphragm wall (D.W.) for high-rise buildings is introduced. A new stochastic optimizer, called Planet Optimization Algorithm (P.O.A.), is employed to assess how proper finite element (F.E.) simulation is against field data. The process is adopted for a real phased excavation measured at the field. To automatically run the iterative optimization tasks, a source code is constructed directly in the Geotechnical Engineering Software (PLAXIS) by using Python to ensure that the operation between optimization algorithm and F.E. simulations are smooth to guarantee the accuracy of the complex calculation for the soil problem. The proposed process consists of two steps. (1) The parameters will be optimized at the early phases of the excavation. (2) The responses of D.W. displacements are forecasted at the subsequent phases. The aim of the process is to predict the displacements of D.W. of the building from the result of the nearby excavation or to provide early warning about the risks of excavation that may happen under vital phases. The proposed procedure also provides an effective method for optimization-based soil parameters updating in real engineering practice. With growing challenges that require A.I. algorithms to be constantly improved4, many swarm-inspired algorithms such as Particle Swarm Optimization algorithm (P.S.O.)5, Genetic Algorithm (G.A.)6, or physics-inspired algorithm , even behavior-inspired of human (e.g. Human Behavior-Based Optimization8) have been proposed. Optimization algorithms are employed in most fields in daily life. In difficult and obscure fields such as soil and foundation mechanics, it is even more necessary to apply the strengths of both to maximize efficiency.With the drastic development in the field of Computer Science in the past several decades, dealing with complex problems becomes easier by using a combination of Artificial Intelligence (A.I.) and finite element modelsSkyscrapers have emerged as a solution to meet workplace or residence needs. While the used space expands upwards, the space inside the ground is also exploited for entertainment, storage, museums, or simply parking at the same time. Excavated construction for projects with two to four basements has become quite popular, in which the traditional method of digging with the anti-system has existed for a long time. This solution is simple and easy to run; it requires, nevertheless, a large area for construction.9. A semi-Top-Down construction solution is more suitable for excavation in urban space. This method has two advantages; short construction time and smaller displacement of the diaphragm wall than the braced excavation method.Additionally, displacement of the soil and the diaphragm wall is usually large10 studied the effects of subway station construction on surrounding buildings. Huang et al.11 presented a method combining S.T.D. excavation technique with steel bracing for the Shanghai's subway station. Peck12 proposed the inverse analysis or observational method. This technique is typically adopted to evaluate the dominant or representative characteristics or parameters of soil via observed data at the site.Many previous studies show the influence of displacement of the diaphragm wall on adjacent buildings with the Semi-Top-Down (S.T.D.) constructed technique. Tan et al.Applying the nature-inspired optimization algorithm for the inverse analysis of the problem brings several benefits. This gives us a practical view of the suitable parameters for soil behavior.13, Qi and Zhou14, Zhang and Mahadevan15, Hsiao et al.16 or Juang et al.17 employing Bayesian updating of soil parameters for prediction settlement or displacement of D.W., and Špačková and Straub18 using Bayesian for the tunnel excavation problem, etc.). Nevertheless, in the application of a model to update Bayesian, more than 10,000 calculations for the Markov chain of Monte Carlo sampling may be required, which makes the method impractical to use. In the last two decades, a new, more suitable method that has been widely used is the optimization algorithm. Specifically, it is particularly an intelligent algorithm to solving optimization problems. Yin and Jin19 demonstrated that an efficient multi-objective optimization-based updating framework could be constructed. Tang and Kung20 presented a study called a Nonlinear Optimization Method (NOM) to inversely analyze geotechnical engineering problems.One of the inverse analyses is a Bayesian updating procedure  simulation F.E. using PLAXIS, (2) novel optimization algorithm (P.O.A.), and (3) the link between (1) and (2) based on the environment of the Python language.In deep excavation problems, settlement and displacement of the D.W. are the two important information on the construction of the structure of a high-rise building. In practice, the deformation ratio, which is defined as the maximum surface settlement over the maximum lateral displacement of D.W. introduced by Kung et al.The geotechnical engineering software PLAXIS version 2020 is employed to calculate the behavior of the soil under the excavation. In more detail, the Hardening Soil Model (H.S.M.) is the soil model employed to characterize the soil in the PLAXIS simulation of the excavation. The stiffness of soil commonly is indicated by a set three modulus, namely the secant modulus in standard drained triaxial test , see Fig. 7 kN/m, 2/m and Poisson’s ratio is 0.15. The excavation of Lancaster was performed employing the Semi -Top-Down technique. The excavation process underwent 5 main steps, in which the D.W. is supported by concrete slabs of 250 mm thick with material properties: 6 kN/m and Poisson’s ratio is 0.15. The steps of excavation are summarized in Table 2. Figure 2.The excavation width at the L.L.T. is 70 m, and the length of the 0.8 m thick diaphragm wall is 32 m with material properties: mentclass2pt{minimmentclass2pt{minimmentclass2pt{minimWhile the three first excavation stages are employed to search soil parameters by using PLAXIS and Python, the lateral displacement of D.W. and settlement under two final-stages excavation are predicted based on these optimized parameters at early-phases.28 tried to determine the stiffness and strength parameters for H.S.M. Some scholars made a concentrated effort to back calculate the ratio of the stiffness modulus of the undrained shear strength . The P.O.A. theory will be fully developed in the other paper, and consequently is not reproduced here. In the paper, a brief description of the algorithm is, however, also presented including flowchart.The basic concept of gravitation laws (or Newton's law) will be presented. Next, inspired by the motion of planet in the universe by this law, a mathematical model for an optimal algorithm will be constructed.In this study, the parameter The optimized model is illustrated simply by a system consists of the Sun, the Earth and the Moon as shown in the Fig. Stages 1: The best start. The algorithm's first step is to search for an effective solution to operate as the best solution. The aim of this step is to improve the convergence and accuracy in the first iterations.Stages 2: Calculation  factor.ith or jth planet, the worst planet and the sun, respectively. This means that the objective function value of a planet is smaller, the mass of this planet is larger. The distance i and j at Stages 3: Global search. Mi/Mmax is a coefficient, which depends on Mi is the sun's gravity on a planet ith at t iteration, and entclass1pt{minimaStages 4: Local search. When the distance between the Sun and a planet is small, the local search process is operated. The planet with the biggest mass will operate like the sun. It means that the planet moves a small distance between it and the hypothetical Sun at iteration t instead of going straight towards the hypothetical Sun. This is illustrated and Eq.  summarized in the Fig. ith.ith.ith of the adjacent building.ith of the adjacent building.The meta-heuristic optimization algorithm (P.O.A.), which is integrated into geotechnical engineering software, is adopted to solve this problem. Each set of parameter values is sent to the simulation model using PLAXIS, which has been calibrated for the particular soil system. Then, the simulation model is solved to obtain the resulting response in the form of displacements of diaphragm wall at phases. This displacements values and observed data at field are utilized by P.O.A. to solve the objective functions n in Eq. . With thUp to sum, this methodology that combine of F.E. method using PLAXIS and the optimization algorithm (P.O.A.), in which Python language is operated as a strong connection both together.In this subsection, the comparison between P.O.A. and two well-known algorithms (P.S.O. and G.A.) for forecasting real excavation problems is presented. A thorough investigation is conducted for comparison between computed displacements and field data via observations from inclinometers of the excavation. The results of the P.O.A. are presented for comparison with the results of the G.A. and P.S.O. in this problem. It means that the proposed methodology replaces P.O.A. with G.A. or P.S.O. Furthermore, by doing that, we can evaluate the effect of optimization algorithm on the proposed method, check the performance of P.O.A. at the same time. Figure Figure The inverse analysis D.W. displacements of each excavation phase in the L.L.T. are indicated in Fig. Up to the sum, the P.O.A. provides the prediction of horizontal displacements of retaining wall is more suitable than G.A. and P.S.O. Although the difference of value of displacement D.W. is not large between algorithms, it also verifies that P.O.A. explores effectively and strongly in optimization.33. The back-figured values of modulus for both layer 3 and 4 in  are essentially suitable for recommendation of researchers33. The identification iteration paths illustrated in Fig. In practice, the inverse analysis combination with excavation is to make a forecast the final soil behavior of excavation on what happened in early-phase observations. First, the target parameters have to be optimized at the early phases of the excavation. Next, the responses of D.W. displacement at the subsequent phases, especially the final phase of excavation, are predicted under target parameters optimized at early phases. Consequently, the results of inverse analysis of the L.L.T. is adopted to further experiment the capacity of the developed method. The In this case, the inverse analysis is employed for only one target parameter for each soil layer, however, this back computing provides the essential accuracy and satisfaction for inverse analysis D.W. displacements. It is clear that for back-figured parameters for D.W. displacement in early-stage excavation are approximately identical with field observation despite the differences. This is acceptable in real geotechnical engineering problems because these errors are quite small. Additionally, the shape of the displacement line of D.W. is very proper with field monitoring data. Furthermore, these errors at the maximum displacement points do not exceed 3 mm, which are highly desirable results in the design field of real geotechnical engineering problems. Generally, the soil parameters inverse analysis at early phases can be employed to predict the maximum D.W. displacements at stage 5 satisfactorily. It means that the similar soil parameters inverse analysis at different phases may be representative and is approaching the real soil parameters.Based on the analyses and comparisons presented herein, the following conclusions can be drawn:(1) The inverse analysis procedure, which is a fully automated process integrated the optimization algorithm (P.O.A.) with the Python code into geotechnical software (PLAXIS) platform, is effectively employed for the model calibration of lateral movements at the excavation. Simultaneously, it is also employed in conjunction with field observations to increase practicality. (2) From early observations about lateral movements of D.W. and surface settlement in the first excavation, stages are employed to recalibrate the model, which can "adequately" estimate the responses of the soil for the subsequent excavation stages. This method is significant in that a successfully updated model at the first excavation stages, which affects all processes, is the automatic forecast of the soil behaviour whole excavation. (3) The growing accuracy of the computed results of the objective function and relative fit enhancement are statistics that proof this technique's efficiency.With the inverse analysis method based on monitored data in the field at the early phases of excavation or from nearby excavation buildings, model parameters may be obtained easily and reliably instead of the traditional approaches, which depend mainly on the pure experience of engineers. Forecasting accidents due to extraordinary displacement D.W. and the surface settlement in time provides a good risk forecasting guarantee for the proper prevention. This research provides a comprehensive methodology for predicting risk, enhancing safety, saving time and budget, and effectively designing and constructing high-rise buildings. Furthermore, with the drastic development of the Computer Science field and Artificial Intelligence (A.I.) in recent decades, we firmly believe that the technique will be trending in the future inevitably.


We sincerely apologize for the inconvenience of updating the authorship. The previous authorship was Jianjia Wang, Xichen Wu, Mingrui Li, and the updated authorship is Jianjia Wang, Xichen Wu, Mingrui Li, Hui Wu, Edwin R. Hancock. Ms. Hui Wu and Professor Edwin R. Hancock should be added to the list of contributing authors. Ms. Hui Wu contributed to the formal analysis, methodology, and investigation in the original manuscript. Professor Edwin R. Hancock contributed to the supervision, methodology, and the review and editing of the manuscript. We have added them to the authorship of our paper  and upda


Lycium barbarum L. (goji berry) is used as traditional Chinese medicine, and has the functions of immune regulation, anti-tumor, neuroprotection, anti-diabetes, and anti-fatigue. One of the main bioactive components is L. barbarum polysaccharide (LBP). Nowadays, LBP is widely used in the health market, and it is extracted from the fruit of L. barbarum. The planting of L. barbarum needs large amounts of fields, and it takes one year to harvest the goji berry. The efficiency of natural LBP production is low, and the LBP quality is not the same at different places. Goji berry-derived LBP cannot satisfy the growing market demands. Engineered Saccharomyces cerevisiae has been used for the biosynthesis of some plant natural products. Recovery of LBP biosynthetic pathway in L. barbarum and expression of them in engineered S. cerevisiae might lead to the yeast LBP production. However, information on LBP biosynthetic pathways and the related key enzymes of L. barbarum is still limited. In this review, we summarized current studies about LBP biosynthetic pathway and proposed the strategies to recover key enzymes for LBP biosynthesis. Moreover, the potential application of synthetic biology strategies to produce LBP using engineered S. cerevisiae was discussed.The fruit of  Lycium species in the world, and they are widely distributed in Asia, Europe, North America, and other regions .,67.S. ceynthesis . Though S. cerevisiae strain can synthesize LBP, selecting suitable substrates, enhancing the supply of cofactors and precursors, and the heterologous expression of key genes will direct the metabolic flux to LBP synthesis and improve the production of LBP. For industrial-scale production, optimization of yeast cultivation medium and conditions might further increase LBP production in S. cerevisiae. Some plant natural products produced with engineered yeasts, such as artemisinin, have been approved for medicinal use [S. cerevisiae is GRAS, the S. cerevisiae biomass might be directly used as prebiotics for poultry or other animals without further bioprocess. In the future, the synthetic biology will help introduce the redesigned LBP biosynthetic pathway in yeast and lead to the high-level production of LBP in the future [When the engineered inal use ,44, but e future .S. cerevisiae provides a possible green strategy for LBP supply. The completed synthetic pathways and key enzymes are unclear now, which limits yeast production of LBP. Finding the key enzymes and designing the intact pathways using omics data are possible now. In the near future, construction of an optimized redesigned LBP synthetic pathway in S. cerevisiae will lead to large-scale production of LBP, which would provide a sustainable way for LBP supply.As the main bioactive component, LBP has a variety of pharmacological effects, but its supply depends on the extraction from goji berry. Production of LBP by engineered


Rheumatology Advances in Practice, Samnaliev et al. [Although new therapies have been licensed for the treatment of SLE, we do not yet know to what extent these will improve mortality, quality of life and health-care costs. To date, these therapies have mostly been trialled and prescribed to patients with an SLE diagnosis of several years, and after other immunosuppressants. But is this the best time to use our most effective therapies? In this issue of v et al.  conductePrevious work has already suggested this. The standardized mortality ratio is highest in the first year of SLE diagnosis, at 5.4 . This iset al. [The CPRD used in the study by Samnaliev et al.  captureset al. .et al. [vs 3.8 per year respectively) in the 5 years preceding diagnosis, which was attributable, in part, to clinical features of SLE [et al. [S.d.) unadjusted direct health-care cost peaked in the first year after diagnosis at £7532 (£9634) per patient and remained high for the next 9 years. In the first year after diagnosis alone, the adjusted total mean annual increase in costs per patient was £4476  greater compared with the year before the diagnosis. Second, the authors used models of individual trajectories of mean all-cause health-care costs and reported that the increase in costs per year was 4.7- and 1.6-fold higher among patients with severe SLE compared with those who had mild or moderate disease [Rheumatology Advances in Practice, Langham et al. [S.d.) time to initiation of any medication being 177 (385.3) days. Consequently, almost all patients  experienced at least one flare episode during the follow-up. In year 1 of diagnosis, the mean (S.d.) overall flare rate was 3.5 (2.5), and the median time to first flare was 63  days. Patients with moderate or severe disease had the shortest median time to first flare compared with those who had mild SLE [Samnaliev et al.  reportedet al. . This cos of SLE . In the  [et al. , the risCollectively, these findings present a concerning picture. Not only do highly severe manifestations present early in the disease and impact on quality of life and mortality, but also these diagnoses are made late. This suggests that a radically different approach to referral, diagnosis and intensity of early treatment is warranted to improve outcomes, including the prevention of irreversible damage; the availability of a new therapy might not be sufficient unless the entire early treatment pathway is revised. In other diseases, such as RA, the 2016 EULAR guidelines advocate for patients with suspected inflammatory arthritis to be referred to, and seen by, a rheumatologist, within 6 weeks of the onset of symptoms . SpecifiHowever, we must consider some limitations in both studies. The use of electronic health records depended on whether a diagnosis was coded accurately in both the primary and secondary care. Hence, a misclassification of SLE is still possible and is more likely for the mild patients, which might exaggerate differences between severe and mild patient groups. Furthermore, the severity of SLE could have been underestimated for the severe category and overestimated for the mild to moderate categories. For example, a criterion for severe disease was oral prednisolone ≥60 mg/day, whereas the intention to treat of >20 mg/day in other established indices, such as the BILAG-2004, would be considered as severe. The study also lacks age- and gender-matched healthy controls for comparison. Lastly, the study also did not examine the impact of ancestry and ethnicity, which are factors that are known to impact on lupus severity and health-care utilization patterns.Future treatment innovations in SLE should focus on early diagnosis and treatment. A broad range of research questions must be answered. Better diagnostics during the pre-clinical and early phase  are needM.Y.M.Y. is a Senior Research Fellow and is funded/supported by the the Wellcome Trust Institutional Strategic Support Fund Fellowship (204825/Z/16/Z) and the National Institute for Health Research (NIHR) Doctoral Research Fellowship (DRF-2014-07-155). E.M.V. is an NIHR Clinician Scientist (CS-2013-13-032). This article/paper/report also presents independent research funded/supported by the NIHR Leeds Biomedical Research Centre. The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care.Funding: No specific funding was received from any bodies in the public, commercial or not-for-profit sectors to carry out the work described in this article.Disclosure statement: M.Y.M.Y. has received consultancy fees from Aurinia Pharmaceuticals. E.M.V. has received honoraria, consultancy fees and research grant support from Aurinia, Roche, GSK, Lilly, Novartis and AstraZeneca.All data underlying this article are available in the manuscript.


Trifolium is one of the largest genera of the legume family Fabaceae with ca. 255 species. The genus is divided into eight sections; the section Trifolium is a major section of the genus, comprising 73 species mainly distributed in the Mediterranean region. We used nuclear ribosomal DNA internal transcribed spacer (ITS) and morphological variation to reconsider the delimitation and phylogenetic relationships of species in the section Trifolium with reference to chromosomal variations. Bayesian analysis of ITS data delimited the species as three clades based on the analysis of ITS sequence and informative indels in combination with morphological variation. The phylogeny of the species by different analyses methods does not support their current delimitation in 17 subsections. The basic chromosome number x = 8 is the number for the genus Trifolium, from which x = 7, 6 and 5 were derived through successive aneuploidy events. With reference to the distribution of these numbers in the species of the section Trifolium, species in clade III and clade II are more evolved than species in clade I.The genus  Trifolium L. is one of the most important genera of the family Fabaceae. It comprises approximately 255 herbaceous, perennial and annual species distributed in different geographic regions, particularly in the Mediterranean region, East Europe, Eurasia, the highlands of eastern Africa and western North America [Rhizobium leguminosarum biovar. Trifolii [Trifolium [T. pallescens) mated with a diploid coastal species  to produce tetraploid T. repens. This has spurred interest in the evaluation of the agronomic potential of locally utilized and currently uncultivated species as a potential genetic resource for crops [The clover genus  America ,2,3. TheTrifolii . Fertilerifolium ,7,8,9. Wrifolium  supporteor crops .Trifolium sensu lato as one large genus of eight sections [Chronosemium, Mistyllus, Paramesus, Trichocephalum, Trifolium, Vesicaria), one to the New World (Involucrarium) and one occurs in both hemispheres (Lotoidea). The later section comprises over 95 species and has traditionally been considered ancestral to all other sections due to its worldwide distribution and morphological heterogeneity [Most contemporary classifications treat sections ,13,14, oTrifolium section has a native distribution throughout continental Europe, extending to Eurasia and south to North Africa. Species of this section show diverse specializations in seed dispersal mechanisms [The chanisms . The secchanisms ,16, highchanisms  and hetechanisms .Trifolium is contradictory. Zohary and Heller [The available literature about the subsectional delimation of the section d Heller  proposedd Heller ,7,8.Trifolium species showed the Trifolium section to be polyphyletic, and the placement of the Mediterranean section Chronosemium (21 species) remained unresolved with conflicting hypotheses such as being derived from within the section Trifolium in a nrDNA phylogeny vs. sister to the entire genus in a cpDNA phylogeny [Trifolium using parsimony and Bayesian phylogenetic analyses based on nuclear ribosomal DNA internal transcribed spacer and chloroplast trnL intron sequences. The authors proposed dividing the genus into two subgenera, subg. Chronosemium and subg. Trifolium; the latter was divided into eight sections which are not in agreement with the sub-sectional delimitations of Zohary and Heller [Chronosemium and indefinite in subg. Trifolium. However, transitions between the annual and perennial habit are common [Molecular phylogeny of Old World hylogeny . Ellisonosemium 2 species d Heller . The ance common .Trifolium species [Trifolium sections, which contradict the sub-sectional delimitation based on molecular phylogeny. Moreover, the sub-sectional delimitation of species in the section Trifolium is not congruent with the number of clades resolved by Watson et al. [Trifolium and an inferred 19 instances of aneuploidy and 22 of polyploidy in the genus [x = 8, 7, 6 and 5 [Trifolium.Comparative seed characteristics of  species  and comp species  indicaten et al.  and by En et al. . The usen et al. . Characthe genus ,22. All  6 and 5 ,24,25,26Trifolium at the sectional level [The internal transcribed spacer sequence (ITS) of the ribosomal DNA has been demonstrated to be an accurate source of information for examining molecular phylogeny in many plant families including the Fabaceae ,28. Indeal level ,16 and aal level . Jreisatal level  recommenTrifolium sect. Trifolium aims to reconsider the sub-sectional delimitation of species in this section, based on a comprehensive sampling of species, ITS analysis and variation in selected morphological traits in the light of variations in chromosome numbers of the examined species.Therefore, the objective of the present study is to construct a phylogenetic relationship of species in the Trifolium based on the ITS sequence and informative indels, using MrBayes 3.2 [Trifolium are clearly isolated from the two outgroups of Trigonella, i.e., Trigonella gladiata and Trigonella spinosum. The Trifolium species are divided into two major clades: clade I comprises 26 species and clade II 28 species, in addition to two minor clades, clade III of 4 species and clade IV of 2 species. Clade I with a strongly supported bootstrap value of 100 is differentiated into two subclades, one of 7 species and the other of 19 species. The seven species of the first subclade are T. alexandrinum, T. echinatum, T. scutatum, T. vavilovii, T. clypeatum, T. pallidum and T.pannonicum. In the second subclade, T. berytheum is grouped with the two samples of T. carmeli 1, 2, and T. meironense, while T. squarrosum is clustered with T. miegeanum and T. obscurum. The other 12 species in this subclade are differentiated as single branches , one of three species  and two small subclades of two species each, i.e., T. molinerii grouped with T. phleoides and T. striatum with T. wettsteinii . On the other hand, chromosome numbers based on x = 5, x = 6, and x = 7 have been reported in fourteen species in clade II and all the six species in clades III and IV.ayes 3.2  applyingayes 3.2 . This Fibranches . In cladtsteinii . On the Trifolium constructed using TreeGraph 2 [T. dichroanthum, T. vavilovii and T. scutatum, while T. pallidum and T. pannonicum form a small subclade. Of the remaining species, T. alexandrinum, T. echinatum and T. clypeatum are clustered from the other eighteen species, which are differentiated as one large subclade of seven species  and a subclade of the four species T. berytheum, T. carmeli 1 and 2, and T. meironense). The remaining seven species are differentiated as T. caucasicum, one subclade of T. plebeium, T. dasyrum and T. constantinopolitanum and another of T. miegeanum, T. obscurum and T. squarrosum. The clustering of these species is congruent with their clustering in eGraph 2  and geneT. latinum and T. ligusticum are delimited as a small subclade from the other species in both T. affine, T. arvense, T. bocconei, and T. dalmaticum form a separate subclade in T. incarnatum, T. leucanthum, T. stellatum, T. striatum, T. wettsteinii, in addition to T. molinerii and T. phleoides. The same species are recognized as three small clades in T.ochroleucum and T. pratense is recognized in both T. lappaceum 1 and 2 and a second of the five species T. alpestre, T. medium, T. caudatum, T. diffusum and T. rubins, and the third of six species . The clustering of these species, except T. pallidum and T. pannonicum, is congruent with views by a number of authors that represent the genetic resources of the cultivated species T. alexandrinum, known as Egyptian clover (Berseem). Aaronsohn [T. echinatum M. B. from Palestine. Bobrov [T. apertum is the progenitor of T. alexandrinum based on morphological similarities. Putiyevsky et al. [T. alexandrinum such as T. vavilovii, T. apertum, T. salmoneum, T. meironense and T. berytheum. On the other hand, Trabut [T. berytheum from the coastal plains of Lebanon as the ancestor of T. alexandrinum. More recently, AFLP data analysis supported a close relationship of T. alexandrinum accessions from Syria and Egypt to T. apertum, T. berytheum and T. salmoneum as well as T. clypeatum, T. plebeium, T. echinatum, T. constantinopolitanum and T. meironense [In the current study, the analysis of ITS data alone and in combination with variation in morphological traits produced different delimitation of species in the section d Heller  who divisections . The anaaronsohn  suggeste. Bobrov  claimed y et al.  consider, Trabut  and Eig , Trabut  proposedironense .T. alexandrinum, T. apertum and T. berytheum as well as T. scutatum, T. plebeium, T. vavilovii, T. echinatum and T. salmonium in addition to T. constantinopolitanum. The grouping of T. plebeium, T. berytheum and T. apertum with T. alexandrinum is not well supported in the current results of the section Trifolium phylogeny. In the work of Zohary and Heller [T. alexandrinum, is placed in subsection Alexandrina with other four species, i.e., T. apertum, T. berytheum, T. meironense and T. vavilovii, whereas T. plebeium is placed in subsect. Clypeata with T. clypeatum and T. scutatum. In the meantime, T. pallidum is placed in the subsection Trifolium with T. diffusum and T. pratense, whereas T. pannonicum is placed with other six species in subsection Ochroleuca; these species are T. canescens, T. caucasicum, T. caudatum, T. longidentatum, T.ochroleucum and T. trichocephalum. However, in the current study, only T. canescens, T. caucasicum are in clade I while the others are in clade II. The delimitation of these species is generally congruent with their classification in Ellison et al. [Ellison et al.  also repd Heller , T. alexn et al. .T. berytheum, T. carmeli 1 and 2 and T. meironense, and the other of T. miegeanum, T. obscurum and T. squarrosum. The 12 species in this subclade are differentiated as single branches . The same subclade is also differentiated in the other trees based on the analysis of informative indels of ITS sequence alone or in combination with the morphological data. The delimitation of T. affine, T. arvense, T. bocconei agrees with the results of Ellison et al. [T. affine, T. arvense are placed in subsect, Arvensia by Zohary and Heller [T. bocconei was placed in subsection Trichoptera with T. trichopterum and T. dalmaticum was placed in subsection Scabroidea with T. lucanicum and T. scabrum. In the study of Ellison et al. [T. dalmaticum was clustered with T. scabrum and T. lucanicum.The clade II of 28 species is differentiated in small subclades based on the ITS data analysis including one clade of four species  generally corresponds to clade I of the present study, whereas clade B generally corresponds to clade II and clade III of this study. Ellison et al. [T. haussknechtii, T. sylvaticum and T. trichopterum, associated with T.cherleri and T. hirtum, representing five of the six species differentiated as clade III in the tree constructed using ITS sequence and informative indels in combination with morphological data  are grouped together in clade II. The remaining species are distributed in the three clades. In the genus Trifolium, the basic number of x = 8 is the number from which x = 7, 6 and 5 were derived through successive aneuploidy events. With reference to the distribution of these numbers in the section Trifolium, species in clade III and clade II appear to be more evolved than species in clade I.In conclusion, the grouping of species as reported here and by Ellison et al.  does notd Heller . The speTrifolium was obtained from a variety of sources . The sequence boundaries of the spacers were determined by comparison to published Trifolium sequences [The ITS region was amplified using primers of White et al. . The spaequences ,16 and cequences , with maequences  using thequences .Two approaches were used to express the phylogenetic relationships of the examined species based on the molecular ITS sequencing alone and in combination with the morphological characters coded as multistate matrix . The Bay


Taxodium distichum and Taxodium ascendens) in the hydro-fluctuation zone of the Three Gorges Reservoir (TGR) region, China. The stoichiometrical contents of C, N, and P in fine roots, leaves, and branches, and the growth conditions of T. distichum and T. ascendens were measured in July 2019. The results showed that periodic submergence affected the stoichiometric characteristics and growth conditions of these two woody species, and the impact was restrained, but both grew well. The effects of inundation on the C, N, and P ecological stoichiometric characteristics differed in different parts of trees. In general, the C contents showed the following pattern: leaves > branches > fine roots. The N and P content showed the following pattern: leaves > fine roots > branches, while the C/N and C/P ratios showed an opposite trend to that of N and P. The N and P content in all parts of T. distichum  and T. ascendens  were higher than the national average level . Except for N and P contents in the leaves of T. distichum, there was a significant correlation between N and P elements in other parts (p < 0.05). Nevertheless, the N/P ratio  was lower than the critical ratio of 14. The growth conditions of T. distichum and T. ascendens were significantly negatively correlated with their leaf C contents and significantly positively correlated with their fine root N and P contents. This study showed that T. distichum and T. ascendens could maintain their normal growth needs by properly allocating nutrients between different organs to adapt to the long periodic submergence in the hydro-fluctuation zone of the TGR region.Ecological stoichiometric studies can be useful for managing the deteriorated riparian zones of mega-reservoirs in which nutrients significantly impact the balanced vegetation cover. The present study aims to explore the effects of periodic submergence on the stoichiometric ecological characteristics of carbon (C), nitrogen (N), and phosphorus (P), as well as the growth conditions of two leading conifer species ( As a levation . Due to levation . Prolonglevation , land-uslevation . As a relevation , vegetattability ,10,11.T. distichum and T. ascendens under flooding stress have been evaluated using in situ and controlled garden experiments [T. distichum and T. ascendens. However, under flooding stress conditions, many physiological processes related to plant C, N, and P can be constrained, such as C-sequestration, N-fixation, and nutrient mineralization. The balance of nutrient elements in interactions and processes is known as ecological stoichiometry. Ecological stoichiometry is an important tool for studying ecological processes and functions, which is used to explore the dynamic balance of various elements and their interactions, as well as the limitations on plant growth [T. distichum and T. ascendens deserve more attention, as they are the leading tree species in the area. C, N, and P are essential elements of plant bodies, and C, N, and P ecological stoichiometry represents an organism’s demand for natural resources and connects different levels of biogeochemical cycling; furthermore, their changes affect the growth and development of plants [The growth, photosynthetic response, physiological metabolism, and nutrient levels of eriments ,10,12, wt growth ,14. Thusf plants  and the f plants . Studiesf plants . This wiPlant ecological stoichiometry has been widely used in the study of plant adaptation mechanisms and nutrition strategies under stressed environments, mainly including the characteristics of biological nutrient allocation, cycle, supply, and demand in the forest , desert T. distichum and T. ascendens. The current study also determines their responses to periodic submergence in the hydro-fluctuation zone of the TGR region. Thus, we hypothesized that (1) both species have strong flooding tolerance, but high-intensity flooding would inhibit plant growth to some degree, (2) the growth was significantly correlated with the C, N, and P content of the two species, and (3) the two species may have different adaptation strategies to the periodic submergence environment in the hydro-fluctuation zone of the TGR region. This study could improve the theory of ecological stoichiometry and provide the basis for vegetation protection and restoration in the hydro-fluctuation zone of the TGR.The present study focuses on the patterns of C, N, and P stoichiometry among plant organs and the growth conditions of T. distichum were significantly restricted .After experiencing seven annual cycles of periodic submergence, the growth of plants in the three submergence treatment groups showed certain differences , and theT. distichum were not affected by different submergence treatments (p > 0.05). The C, N, and P contents and stoichiometric ratio of T. distichum were significantly affected by different submergence treatments, different organs, and their interaction (p < 0.01) , and the C contents, which were unaffected by the interaction of different submergence treatments and different organs (p > 0.05), the C, N, and P contents, as well as the stoichiometric ratio of T. ascendens, were significantly affected by different submergence treatments, different organs, and their interaction (p < 0.01)  . Except  < 0.01) .T. distichum , whereas it had no significant effects on the N contents in the fine roots of T. distichum. The P content in the fine roots of T. ascendens in the DS group was significantly lower than that in the MS and SS groups. In all submergence treatment groups, the N and P contents in the fine roots of T. ascendens were significantly higher than those of T. distichum (p < 0.05). In the MS group, the C content in the fine roots of T. distichum was significantly higher than that of T. ascendens (p < 0.05), but the C content in the fine roots of both was not affected by flooding. Therefore, the effects of flooding on the C/N ratio and C/P ratio in fine roots were opposite to the N and P content trend. There was a significant difference in the N/P ratio in the fine roots of T. distichum among different submergence treatment groups (p < 0.05). With the increase in submergence depth, it showed a trend of first increasing and then decreasing. There was no significant difference in the N/P ratio in the fine roots of T. ascendens between the MS and SS groups. Nevertheless, they were significantly lower than that of the DS group (p < 0.05) and substantially lower than that of T. distichum (p < 0.05) (Flooding significantly reduced the P contents in the fine roots of  < 0.05) .T. distichum and T. ascendens. In the DS group, the C content in the leaves of T. ascendens was significantly higher than that of T. distichum (p < 0.05). With the increase in submergence depth, the N content in the leaves of T. distichum significantly decreased. There was no significant difference in the N content in the leaves of T. ascendens between the DS and SS groups, but they were substantially higher than those in the MS group (p < 0.05). The N content in the leaves of T. distichum in all treatment groups was significantly (p < 0.05) or extremely significantly (p < 0.01) higher than that of T. ascendens. The P content in leaves of T. distichum in the DS and SS groups was significantly higher than that of the MS group (p < 0.05). The P content in T. ascendens in the DS group was considerably higher than that of the MS and SS groups. In the DS group, the P content in the leaves of T. ascendens was significantly higher than that of T. distichum (p < 0.001); however, in the SS group, it was substantially lower than that of T. distichum (p < 0.01). The C/N and C/P ratios in the leaves of T. distichum and T. ascendens showed opposite trends to the N and P contents, respectively. The N/P ratio in the leaves of T. distichum in the MS group was significantly higher than that in the DS group (p < 0.05), and the N/P ratio in the leaves of T. ascendens in the SS group was considerably higher than that in the DS and MS groups (p < 0.05). In the DS and MS groups, the N/P ratio in the leaves of T. distichum was significantly higher than that of T. ascendens (p < 0.001).As shown in T. distichum and T. ascendens (T. distichum among different treatment groups. The C content in the branches of T. ascendens in the MS group was significantly lower than in the DS group (p < 0.05). In the DS and SS groups, the C content in the branches of T. ascendens was significantly higher than that of T. distichum (p < 0.01). The N and P content in the branches of T. distichum and T. ascendens showed significant differences in different submergence treatment groups. With the increase in submergence depth, the N and P contents in the branches of T. distichum and the P content in the branches of T. ascendens showed a trend of first increasing and then decreasing. The N content in the branches of T. ascendens showed a trend of first decreasing and then increasing. The C/N and C/P ratios in the branches of T. distichum and T. ascendens showed opposite trends to the N and P contents, respectively. With the increase in submergence depth, the N/P ratio in the branches of T. distichum and T. ascendens showed a trend of first decreasing and then increasing, and they were significantly different. In the DS group, the N/P ratio in the branches of T. distichum was significantly lower than that of T. ascendens. At the same time, it was significantly higher than that of T. ascendens in the MS and SS groups.Different submergence treatments have different effects on the C, N, and P stoichiometric characteristics in the branches of scendens . There wT. distichum are significantly positively correlated with fine root N and P contents and leaf N content, and significantly negatively correlated with leaf C content  with fine root N content with coefficient values of 0.588 *, 0.516 *, and 0.524 *, respectively. The correlation between plant height, crown area, and DBH and leaf N content was more significant (p < 0.01) with peak coefficient values of 0.712 **, 0.685 **, and 0.669 **, respectively. Plant height, crown area, and basal diameter were extremely significantly positively correlated (p < 0.01) with fine root P with peak coefficient values of 0.904 **, 0.642 **, and 0.649 **, respectively. Leaf C content was extremely significantly negatively correlated (p < 0.01) with plant height (−0.785 **) and crown area (−0.678 **), and significantly negatively correlated (p < 0.05) with basal diameter (−0.578 *) and DBH (−0.598 *). DBH was significantly positively correlated (p < 0.05) with fine root C content (0.537 *) and branch N content (0.533 *). However, there was some difference in T. ascendens (p < 0.01) with plant height (−0.662 **), crown area (−0.687 **), and basal diameter (−0.699 **). The correlation between growth conditions and fine root N content was more significant (p < 0.01) in T. ascendens, and the highest coefficient values were those of plant height (0.785 **) and crown area (0.697 **).Pearson’s correlation coefficients determined the correlations between growth conditions and C, N, and P content. Results showed there were significant correlations between growth conditions and C, N, and P contents. All growth indices of  content . Specifiscendens . Leaf P Flooding is an environmental stress in many natural and manufactured ecosystems worldwide , especiaT. distichum was better than that of T. ascendens  are closely related to organ functions . Differet groups , reflecto leaves , enablino leaves , and allT. distichum and T. ascendens in each submergence treatment group showed the following pattern: branches > fine roots > leaves, indicating that T. distichum and T. ascendens have higher N and P utilization efficiency in the hydro-fluctuation zone of the TGR region. N and P are the main limiting elements for plant growth, and the N/P ratio can characterize the nutrient limitation of plants. When plant N/P < 14, it means that the N element mainly restricts plant growth; when N/P > 16, it means that the P element mainly determines plant growth; when 14 < N/P < 16, it means that plant growth is subjected to the common limitation of N and P elements [T. distichum and T. ascendens were all less than 14 in all treatments, indicating that the N element may restrict the growth of the two species. However, the concentrations of N and P were higher, except for the N content in the leaves of T. ascendens, in the MS group. The N and P contents in branches, leaves, and fine roots in all treatment groups were higher than the national average level [T. distichum and T. ascendens have good adaptability to environmental changes [The ratio of C, N, and P in plants can reflect the adaptation of plants to the environment in which they live . In geneelements . In the ge level , which ige level . From th changes . The cor changes .T. distichum and T. ascendens showed a negative correlation with leaf C content  than in non-photosynthetic organs (branches and fine roots). Within an appropriate range, higher N and P content in the leaves leads to a greater net photosynthetic rate, faster growth rate, and stronger resource competition [T. distichum. Therefore, N (the main element that constitutes protein) and P (the main element that constitutes nucleic acid) acquisition is essential for growth [T. ascendens. The growth indices were negatively correlated with the leaf N and P content. The possible reason is that the concentration of N and P in T. ascendens leaves was diluted [T. ascendens can carry out sufficient photosynthesis and rapid metabolism under sufficient light and rainfall conditions, promoting the plant’s vegetative organ construction. The carbohydrates produced by photosynthesis in the leaves of T. ascendens were not transported to other parts in time, thus diluting the N and P concentration, but T. ascendens still proliferated. This also confirmed part of our third hypothesis. Compared with thick roots, fine roots are more complex in structure, have higher physiological activity, and are in a state of constant renewal. The enzyme system in the cell and respiration and metabolism were active [T. distichum and T. ascendens was positively correlated with the N and P content in fine roots, and more N and P elements in fine roots can promote rapid growth of fine roots, absorb more nutrients from the soil, and provide raw materials for the synthesis of proteins, nucleic acids, and other substances needed for the rapid growth of plants [Our results showed that petition . Higher petition  and ensur growth . However diluted . Since Je active . The grof plants .2. The annual precipitation is 1200 mm, and the relative humidity is 80% [T. distichum and T. ascendens saplings were planted at this site at an altitude of 165–175 m in March 2012, and both species were planted in an alternating pattern with a spacing of 1 m × 1 m.The study site  was located in the Ruxi River basin in Gonghe village of Shibao Township, Zhong County, Chongqing municipality of China . The RuxT. distichum and T. ascendens were randomly selected at each elevation to sample the leaves, branches, and fine roots. We used high branch shears to collect mature leaves and branches of T. distichum and T. ascendens in four directions in the upper–middle layers of the canopy. The leaves and branches were mixed evenly and sealed in a ziplock bag. Fine roots (d ≤ 2 mm) were collected using a flat shovel, excavating the fine roots of T. distichum and T. ascendens in a radius of 0.25 m in the sample square, carefully cleaning the soil and impurities on the surface of the root system, and finally mixing the collected root system and placing the quartered part into a ziplock bag. At the same time as sampling, a height measuring rod was used to measure the plant’s height, a tape measure was used to measure the crown width of the plant, and a vernier caliper was used to measure the plant’s basal diameter and diameter at breast height (DBH).According to the difference in winter flooding intensity, the study site was divided into three transects; 165 m (DS), 170 m (MS), and 175 m (SS) . In situAll samples were refrigerated and transported to the laboratory, cleaned with tap water and deionized water, and placed in an oven. After 30 min of drying at 105 °C, the samples were dried to a constant weight at 65 °C. Plant samples were crushed by a Lech MM400 Ball Mill  to measure the C, N, and P contents. All samples’ C and N contents were measured using an element analyzer . The P contents were digested by a microwave digestion apparatus  and then measured using an inductively coupled plasma emission spectrometer .p < 0.05 level was used to analyze the effects of submergence treatment on plant growth and C, N, and P contents and stoichiometry in various parts of plants. Two-way ANOVA was used to analyze the effects of submergence treatment, different organs, and their interactions on C, N, and P contents, as well as the stoichiometry of T. distichum and T. ascendens. The independent sample t-test was used to analyze the differences in C, N, and P content and stoichiometry between different species under the same submergence treatment. The Pearson correlation was used to analyze the connection between the growth conditions and the C, N, and P contents of T. distichum and T. ascendens, as well as the correlation between the content of N and P in various plant organs. All images were drawn with Origin 9.0 software. Plant C, N, and P element concentrations were calculated by dry weight (g/kg), and all C/N/P ratios were calculated by mass ratio.All statistical analyses were performed using SPSS 25.0 and Microsoft 2019 software. One-way ANOVA followed by Tukey’s test to determine significant differences at the Taxodium distichum and Taxodium ascendens differed under the three submergence treatments after years of periodic flooding. Flooding significantly affected their growth but did not endanger their health. Our findings also showed that the nutrient allocation strategies of the two species were similar. The submergence treatments, different organs, and their interactions significantly affected both species in terms of improving their stress resistance by increasing their leaves’ carbon content and coordinating the nitrogen and phosphorus contents in their organs to maintain normal functions under submergence conditions. When the submergence depth increased, the T. ascendens transferred more N and P elements from the fine roots to the leaves for adaptation to the periodic flooding. However, the N/P ratio in all organs of T. distichum and T. ascendens was lower than the critical ratio of 14 in all treatments, indicating that the N element may restrict the growth of the two species. In contrast, the N and P content in all organs of the two species were higher than the national average level, making up for the limitation of the N element. In summary, Taxodium distichum and Taxodium ascendens can respond positively to submergence and coordinate the allocation of nutrient elements among various organs to better adapt to the complex hydrological conditions in the hydro-fluctuation zone of the Three Gorges Reservoir.The results showed that the C, N, and P ecological stoichiometric characteristics and growth of


Lactobacillus and an increase in anaerobic bacteria. While Gardnerella spp. are considered a major cause of BV, they are also detected in the vaginal microbiome of healthy women. G. vaginalis was the only recognized species of Gardnerella until a recent study characterized three new species, G. leopoldii, G. piotii, and G. swidsinskii. This review describes the different types and genetic diversity of Gardnerella, as well as new findings on the correlation between different Gardnerella spp. and BV.Bacterial vaginosis (BV) is the most common infectious disease of the reproductive tract in women of childbearing age. It often manifests as an imbalance in the vaginal microbiome, including a decrease in  Gardnerella vaginalis , Megasphaera spp., Fannyhessea vaginae , Dialisella spp . Althougella spp . In addiella spp . Seriousella spp , adverseella spp , pelvic ella spp  and sexuella spp , gonorrhella spp , human pella spp  and humaella spp .Haemophilus vaginalis , and the name was changed to H. vaginalis vaginitis  in recognition of the absence of recognized agents of vaginitis . In 1955aginitis . Howevervaginale . This navaginale . In 1980vaginale . This re with BV . It wasn with BV .Gardnerella spp. is highly detected in BV, it appears to have a special role in vaginal microbiota dysbiosis  or in a candleflame extinction jar , NanH2, and NanH3. NanH2 and NanH3 appear to be the main sources of sialidase activity in Gardnerella spp  specific for Gardnerella spp. and were the first to show that these species form biofilms on vaginal epithelium in women with BV. This finding revealed the nature of clue cells, epithelial cells covered by a Gardnerella spp. biofilm, and their etiological role in Gardnerella spp. infection  using different restriction enzymes  typing can be used on uncultured clinical specimens, allowing for quantitative assessment of bacterial load and qualitative identification of subtypes . Since t species . Balashopecimens .Gardnerella spp. comprises four subgroups A, B, C, and D, based on cpn60 barcode sequences and whole-genome sequences , and MALDI-MS protein signatures  among groups based on clinical Nugent scores  and self-reported symptoms in the 2 weeks prior to the swab collection  were compared  and the “intermediate state” microbiome was observed by cpn60 sequencing and qPCR , sialidase and biofilm formation   .Gardnerella  and the National Natural Science Foundation of China (81971342).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.


Surveillance of human leishmaniasis in Europe is mostly limited to country-specific information from autochthonous infections in the southern part. As at the end of 2021, no integrated analysis has been performed for cases seen across centres in different European countries.To provide a broad perspective on autochthonous and imported leishmaniasis cases in endemic and non-endemic countries in Europe.We retrospectively collected records from cutaneous, mucosal and visceral leishmaniasis cases diagnosed in 15 centres between 2014 and 2019. Centres were located in 11 countries: Belgium, France, Germany, Italy, the Netherlands, Norway, Portugal, Spain, Sweden, Switzerland and the United Kingdom. Data on country of infection, reason for travelling, infecting species, age and sex were analysed.We obtained diagnostic files from 1,142 cases, of which 76%, 21% and 3% had cutaneous, visceral, and mucosal disease, respectively. Of these, 68% were men, and 32% women, with the median age of 37 years (range: 0–90) at diagnosis. Visceral leishmaniasis was mainly acquired in Europe , while cutaneous leishmaniasis was primarily imported from outside Europe . Sixty-two percent of cutaneous leishmaniasis cases from outside Europe were from the Old World, and 38% from the New World. Geographic species distribution largely confirmed known epidemiology, with notable exceptions.Our study confirms previous reports regarding geographic origin, species, and traveller subgroups importing leishmaniasis into Europe. We demonstrate the importance of pooling species typing data from many centres, even from areas where the aetiology is presumably known, to monitor changing epidemiology. Leishmania cause a variety of diseases in humans, collectively known as the leishmaniases [Kinetoplastid parasites of the genus maniases . VisceraL. infantum and L. tropica are the only species known to be transmitted in Mediterranean Europe, but globally between 15 and 20 Leishmania species are pathogenic to humans [Transmission of the parasite occurs through blood-feeding female sandflies. In Europe, the vector mainly occurs in the southern countries, primarily those of the Mediterranean basin. Several papers have described the epidemiology of endemic leishmaniasis in Europe ,3. Howevo humans .The European LeishMan network was established in 2010, with the aim of sharing leishmaniasis case management data, and harmonising diagnosis and treatment . To reveFifteen centres within the European LeishMan network from 11 countries in west and north Europe  shared tBecause the exact infection date is unknown, the date of sampling for disease confirmation was used. Age and sex were recorded; the age of the patient on this date was recorded as the age at time of diagnosis, which was analysed in periods of 10 years. As the study uses retrospective data, the travel and disease history was not uniformly recorded across centres, but we aimed to collect the first disease episode for every case. We took utmost care to avoid inclusion of relapses from infections dating before 2014, even though this cannot be guaranteed if the patient visited other clinics before consulting one of the centres participating to the study. For ML, however, some cases are likely a relapse from an earlier CL manifestation, especially those imported from the New World.Leishmania infection were discriminated: VL, CL, and ML. A case was interpreted as ML if mucosal tissue was affected, irrespective of concomitant or earlier cutaneous lesions elsewhere on the body. The same case definitions are used throughout the LeishMan network. The definitions of these main leishmaniasis manifestations are straight-forward, and this study did not stratify according particular sub-categories of CL and ML where classification would be more ambiguous.The three main clinical manifestations of a An autochthonous case was considered as an infection that took place in the country where it was diagnosed. All other cases were considered as imported or travel related, e.g. an infection diagnosed in France but acquired in Spain was considered an imported infection, even though it was acquired in Europe. For imported cases, we further subdivided this category into migrants, tourists, military personnel, and people visiting friends or relatives (VFR). If there was another reason for travelling, or the reason for travelling was not known, the case was categorised as ‘Traveller unspecified’, to make the distinction with an autochthonous case. The classification ‘Unknown’ was reserved for those cases without information distinguishing an imported or autochthonous infection.The ‘country of infection’ was defined as the country where the case was most likely infected. Some cases, especially migrants and tourists, often travelled through various endemic regions, so the exact country of infection was impossible to establish. For migrants, we assumed they were infected in their home country unless other countries were mentioned in the diagnostic records. Whenever the exact country of infection was unclear from the medical record, we pragmatically grouped them into the following regions from the Old World: (i) Mediterranean, (ii) Sub-Saharan Africa and (iii) the Middle East including Pakistan, and from the New World: (iv) South America and (v) Middle America . If no information on the source country was available, or patients visited several of the aforementioned regions, the case was categorised as (vi) ‘Unknown’.Leishmania genus as used in this paper is listed in L. (Leishmania) and L. (Viannia).The taxonomy of the Several methods and genome loci were used for genomic typing purposes. These include sequencing, restriction fragment length polymorphism (RFLP) analysis, and/or subgenus-specific PCR of the following: heat-shock protein 70 gene (HSP70) -17, the Supplementary Material: Ethics statements). All data were shared and analysed anonymously in accordance with respective national guidelines. If needed, specific ethical clearance was obtained from the ethical committees or institutional review boards from the respective centres , France (n = 166), Sweden (n = 133), and Belgium (n = 124) . BecauseL. donovani complex , followed by VL ; only 3% (n = 33) showed mucosal involvement . Two cas complex . In 997  complex , and 75%https://microreact.org/project/leishman-2014-2019) [The full dataset including GenBank accession can be downloaded and explored interactively in the MicroReact platform (14-2019) .The probable country of infection was determined for 946 of 1,142 (83%) cases. These countries were pragmatically grouped into the endemic regions . Of the L. donovani complex were found both in the New and Old World. Most of these were L. infantum , while L. donovani was identified only from the Horn of Africa and Afghanistan. L. tropica was imported from northern Africa and the Middle East, with one case from Eritrea, while L. aethiopica was identified only from Ethiopia. L. major had a broad distribution in the Middle East and in Africa north of the equator. The L. braziliensis species complex was imported to Europe from Central and South America, with L. peruviana identified in one case from Peru. The L. guyanensis complex showed the same distribution, with L. panamensis originating only from the pacific coastline. L. naiffi was found in Belize and French Guiana. The L. lainsoni cases originated from Brazil and Peru. The L. mexicana complex was identified from several Latin American countries, with a marked dichotomy between L. mexicana in Middle America and L. amazonensis in South America. The L. (enriettii) species complex was identified in one case that visited several leishmaniasis endemic zones and could have been infected in either Guyana, Ghana, or Grenada in the Caribbean area.Species from the We examined relevant trends for different sub-categories of the variables . VL was L. donovani complex  and elderly people (> 60 years) were most often affected, which could be explained by the fact that VL is primarily an autochthonous disease caused by immunity .We observed that twice as many cases were male as opposed to female, both in VL and CL, which can be explained by two factors. First, in many parts of the world, males are more often engaged in outdoor activities and behaviour that could render them more prone to vector contact and thus infection and, second, females are less susceptible to severe disease .L. infantum as the only causative species, confirming other reports [We confirmed the findings of a literature review by Mansueto et al.  showing  reports . The vas reports . This re reports . Boggild reports  analysed reports . This isMost CL cases were imported from outside the European territory. Boggild et al. found that Old World CL is imported primarily by migrants and VFR . They idIn the Boggild et al. study , the thrL. donovani or L. braziliensis complex. Where the species were identified, the latter were all L. braziliensis except for a single case of L. peruviana. The L. donovani complex species can cause both ML and VL, while L. braziliensis can lead to ML [L. guyanensis complex, occasionally causing ML as well [L. tropica, again indicating the limited risk. In contrast, of the 122 tourists infected with species from the L. donovani complex, 49 (40%) developed VL, underscoring a high risk.Our analysis shows that among travellers, tourists may be at greatest risk for developing ML or VL, as 65% (166/254) were infected with species from the L. infantum were reported from Cameroon, Guinea, Kenya, India, the Dominican Republic, and the United States (US). In Cameroon and Guinea, L. major is the known aetiological agent of CL [L. donovani complex was previously isolated from a sandfly vector in Cameroon [L. donovani and L. major [L. infantum in West Africa, as was previously evidenced from human VL and CL in various countries, the presence of and parasite isolation from the vector, and identification of leishmaniasis in dogs, typically considered the species’ reservoir [L. major, L. tropica, and L. aethiopica, while VL is caused by L. donovani [L. infantum, which is unique for East Africa. Even though L. infantum had been described based on multilocus microsatellite data, further analysis revealed they were all L. donovani [L. infantum infection acquired in India, while L. donovani is the causative agent in the country [L. donovani species complex, consisting of strains from Kenya and a subset of India and Ethiopia [L. infantum. Finally, autochthonous CL from L. infantum has never been documented in the Dominican Republic or the US, where two tourists were presumably infected. In both countries, autochthonous CL is sporadically caused by the L. mexicana complex [L. infantum is common [Some of our typing records presented unexpected results in the context of the known epidemiology of species and disease distribution ,30. Casent of CL . The L. Cameroon , but thiL. major . The feweservoir . In Kenydonovani . We detedonovani ,35. Equa country . EvidentEthiopia ,36. At p complex ,37,38. Is common , but no s common , the onlL. major and L. tropica infections is common [L. donovani complex have been documented in travellers and soldiers [L. donovani complex were identified, one of which was typed to the species level as L. donovani. Further, we counted three noteworthy CL cases caused by L. major, i.e. from Spain, Kazakhstan, and Ethiopia. Foci of L. major CL have been described in Kazakhstan, but the caseload is probably under-reported [L. major infection has been described, and L. infantum is known as the only aetiological agent of leishmaniasis. The case in our database concerns a tourist who returned with many sandfly bites from the Balearic island of Ibiza, and did not travel to any other known place endemic for leishmaniasis. The species typing was confirmed twice using HSP70 sequencing. In addition, a migrant from Eritrea was diagnosed with the L. tropica complex, probably the L. tropica species rather than L. aethiopica. Little information on the species in this country is known, even though both VL and CL are present [L. aethiopica infection [L. siamensis / L. martiniquensis VL case in our dataset was described as originating from Guyana [In Afghanistan, CL from s common , but CL soldiers ,41. In oreported . In Ethireported . In Spaireported , not a s present  and one nfection . Finallym Guyana , but coum Guyana . Even thThe main limitation of our study is that the 15 participating centres are from 11 countries in western Europe and Scandinavia, which generally do not capture all medically attended leishmaniasis cases in their respective countries. We estimate that in each of the 11 countries we overall reported between 10 and 100% of all imported infections, which is a representative sample in terms of species distribution, geographic origin, and type of travel. As is the case for all travel-related diseases, when and where a patient was infected often cannot be determined with absolute certainty, which highlights the need to pool data from many institutes to confirm individual extraordinary observations. As many CL manifestations are self-healing or can be efficiently treated in a peripheral dermatology practice, our data collection probably missed relatively more CL compared with VL cases, as happens even in countries where the diseases are notifiable [When comparing our data to those of the World Health Organization (WHO) Global Observatory  (consultThis study highlights the value of permanent monitoring of autochthonous and travel-related leishmaniasis cases in Europe. On one hand, such monitoring can confirm previous observations, on the other, surveillance can also draw attention to changes in the epidemiology of the diseases over time, or to endemic regions that have been under-studied and where awareness campaigns are needed. Cases imported into high-income countries, where effective species typing tools are available, are a rich source of information, provided that data from many medical centres are pooled to increase the number of observations. Resources like the WHO Global Health Observatory are excellent tools to implement such continuous surveillance. Since 2017, the Department of Control of Neglected Tropical Diseases of the WHO headquarters and the WHO Regional Office for Europe have provided financial and technical support to the European LeishMan network. With that support, databases have been aligned, allowing LeishMan centres to share their data of diagnosed leishmaniasis cases with WHO. This will improve surveillance of the epidemiology of the leishmaniases not only in Europe, but also in other parts of the world frequented by European citizens.


Clematis taxa from South Korea. The petiole surface features were observed with the help of stereomicroscopy and scanning electron microscopy, and the anatomical features are studied via microtomy and light microscopy. The results of this study showed that the presence/absence and abundance of trichomes, petiole cross-section outlines, upper surface wings and grooves, and the number of vascular bundles were useful for species discrimination in Clematis. Among the studied taxa, C. hexapetala was the only species with a glabrous petiole surface. Two types of trichomes were observed in the other 18 taxa: long, non-glandular and flagelliform trichomes and short, glandular capitate trichomes. We found four to six major vascular bundles and a maximum of eight interfascicular vascular bundles (C. heracleifolia and C. urticifolia) in the 19 taxa. A cluster analysis based on UPGMA identified six clusters with 18 nodes. Although the number of taxa investigated was limited, taxa from the sections Tubulosae, Viorna, and Astragene clustered with each other in the UPGMA phenogram due to the overall similarity of petiole features. Based on this observation, we can conclude that most of the petiole features are limited to the species level, and thus, the data obtained could be used as descriptive and/or diagnostic features for particular taxa, which may be useful for the investigation of problematic taxa in the genus.We assessed the micro-morphological and anatomical structures of the petioles of 19  Ranunculaceae is one of the larger families of eudicots, comprising nearly 2,500 species within 50–60 genera . The famClematis L. is classified under the tribe Anemoneae DC. in the subfamily Ranunculoideae Hutch. The petiole morphology, including the indumentum, trichome type and abundance, and upper surface groove was observed under a stereomicroscope and a scanning electron microscope (SEM). A Leica MZ16 FA microscope  was used for the observations and digital images of the best-represented part of the petiole were taken with a Leica DFC420 C multifocal camera attached to the microscope. Before SEM imaging, petiole pieces were immersed in 100% ethanol and sputter-coated with gold in a KIC-IA COXEM Ion-Coater . SEM imaging was carried out with a COXEM EM-30 PLUS+ table scanning electron microscope (COXEM) at 20 kV at the seed testing laboratory of the Korea National Arboretum.At least three petioles of each taxon were subjected to microtome sectioning according to the following procedure used in Thirteen quantitative characters were categorized and coded binary and/or multistate. The character states and their codes are provided in Supplementary File S1. Principal component analysis (PCA) and cluster analysis using the unweighted pair group (UPGMA) clustering method using the Gower general similarity coefficient were carried out with MultiVariate Statistical Package 3.1 software (MVSP Version 3.1) .Clematis petioles observed in this study include the petiole indumentum, trichome type and abundance, petiole outline in cross-section (CS), upper surface wings and groove of the petiole, sclerenchyma region, and vascular bundles. All the characters are summarized in The morphological and anatomical characters of Clematis hexapetala, which has an almost glabrous surface (a few trichomes occur in the region from which leaflets arise) , or ‘pilose’, which are covered with soft, weak, thin, and separated hairs, as found in the rest of the species . The non-glandular trichomes are either unicate  or flabelliform . Based on the trichome density per unit area, the trichome abundance was categorized as high, medium, or low.The petiole surface of the studied species is pubescent except in s arise) –1S. Clemrichomes –2J. GlanC. terniflora) , sub-flattened (11 species), U-shaped (three species), or V-shaped (three species). The formation of the upper surface groove is due primarily to the dorsal surface wings, although some species with inconspicuous wings have a slight groove in the petiole .The studied species show considerable variation in their CS petiole outline . The speshown in –6. ClemaThe epidermis is single-layered and cutinized in all species –6. The cClematis species. The 19 taxa have four to six major vascular bundles and a maximum of eight interfascicular vascular bundles (C. heracleifolia and C. urticifolia). Twelve species have five major vascular bundles, six species have six, and only C. patens has four  among has four . The majThe petiole of each species has a large region of the ground tissue, i.e., the pith. The cells in the pith are thin-walled, rounded, ovoid, or angular and parenchymatous and are comparatively larger than those in the cortex –6.Clematis fusca var. fusca and C. fusca var. violacea, representing the first node in the sixth cluster of the phenogram, showed 93.6% similarity in petiole features, whereas C. hexapetala and C. serratifolia, representing the 18th node in the first cluster in the phenogram, shared only 52.7% similarity in petiole features with the rest of the species.The similarities among the species based on the 13 petiole features were revealed using PCA and cluster analysis. The first three components of the PCA explained 74.01% of the total variation in the analysed data. The first axis of the first complete set explained 42.66% of the total variation and showed strong positive loadings for the trichome abundance and the number of vascular bundles  . The secClematis is morphologically diverse in terms of leaf phyllotaxy, types of compound leaves, and leaflet number. The anatomy of the petiole, therefore, is expected to be equally diverse. Previously, Clematis species. Thus, reasonable diversity in petiole anatomical features, including variation in the number of vascular bundles, is certainly useful for the taxonomic treatment of the genus. The overall anatomical organization of the petioles in the investigated species was comparable to that described in Clematis species and a discussion of their taxonomic relevance within the genus, which have not been included in previous studies.The genus Clematis species can be differentiated based on the petiole indumentum. The presence/absence and/or type of trichomes in the petiole have also been used for species differentiation in other taxa , whereas C. taeguensis and C. serratifolia have subglabrous or sparsely pubescent indumentum. The remaining species have sparsely or thickly pubescent petioles. In sparsely pubescent taxa such as C. terniflora, C. terniflora var. mandshurica, C. fusca var. fusca, and C. fusca var. violacea, the trichomes are restricted to mainly the upper groove region. Additionally, the stem of these taxa is either subglabrous or puberulous only at nodes . Glandular pubescence on the stem, petiole and leaf of C. gattingeri Small has been reported in a few older studies . Petiole features that are considered to be useful for taxonomic discrimination in various taxa  confirmed the presence of glandular pubescence on the stem, petiole, and leaf, which had never been observed in other species of subsection Viornae. The recognition of C. gattingeri as a species has been based primarily on the glandular pubescence and small flower size of Gattinger’s specimens. The results from this study also revealed that the petiole indumentum and types of trichomes appear to have taxonomic value for species delimitation in Clematis.Most of the species have both types of trichomes, although glandular trichomes are very scarce and restricted to the upper surface groove of the petiole. We observed both glandular capitate and simple non-glandular trichomes on the leaf surface of most of the investigated ous taxa  are geneis genus . In a taClematis include the petiole outline in CS, the upper surface groove and wings, and the phloem fibre cap. Of these features, the petiole outline and upper surface groove and wings have already been proven to be useful in the taxonomic discrimination of species in some eudicot genera , Atragene , and Pterocarpa (C. brachyura). In a recent phylogenetic classification of the genus, C. fusca var. fusca, C. fusca var. violacea, and C. fusca var. flabellata were classified under clade L; C. ochotensis, C. calcicola, and C. koreana were classified under clade H: and C. brachyura was classified under clade K . However, in this study, it remained connected to the Viorna and Atragene sections. In fact, this uncertainty is a common and typical interpretation for this large genus, as previous morphological and molecular studies also found a similar tendency  and C. brachurya (section Pterocarpa), whereas C. fusca var. fusca and C. fusca var. violacea remained together in a separate sub-cluster. The PCA biplot also inferred similar relationships among these three subspecific taxa, with C. fusca var. flabellata was positioned on the positive side of axis 1, while C. fusca var. fusca and C. fusca var. violacea remained closer on the negative side of axis 1. The petioles of C. fusca var. flabellata differ from those of C. fusca var. fusca and C. fusca var. violacea in terms of the trichome abundance, phloem fibre cap height, and number of vascular bundles. In terms of morphology, C. fusca var. flabellata is an erect herb with ternate leaves, whereas C. fusca and C. fusca var. violacea are woody vines with pinnately foliate leaves. In addition, of the three species in section Clematis, C. trichotoma and C. brevicaudata formed a sub-cluster with C. patens (section Viticella), while C. apiifolia allied with C. terniflora (section Flammula) in another sub-cluster. The PCA showed a slightly different pattern, as C. apiifolia remained closer to C. serraatifolia and C. patens on the negative side of both axes, whereas C. brevicaudata allied with C. taeguensis and C. terniflora var. mandshurica on the positive side of axis 1 and the negative side of axis 2. Morphologically, the petiole of C. apiifolia differs from those of the other two species in its trichome abundance, outline in CS, phloem fibre cap, intrafascicular sclerenchyma, and number of vascular bundles. Notably,petiole features alone can be more useful for species delimitation than for distinguishing infrageneric relationships and thus are not indicative of infrageneric classifications. Similar explanations were provided by Clematis, respectively.According to PCA, the close affinity of the taxa from section n axis 1 . PreviouClematis. The results of this study also indicated that taxa from the sections Tubulosae, Viorna, and Atragene could be grouped based on the overall similarity of their petiole features. Moreover, we found that many of the petiole characters such as the abundance of trichomes, upper surface wings and grooves, and number of vascular bundles, that were neglected in taxonomic and systematic considerations within and among Clematis taxa can be useful for species delimitation. We do not consider these petiole morphological data to be strong enough to provide useful information on infrageneric relationships with the Clematis genus; however, our results provide new and interesting insights into these data, which can be used as a source of descriptive and/or diagnostic features for particular taxa in the genus.In conclusion, the presence/absence and abundance of trichomes, petiole outline in CS, upper surface wings and groove, and number of vascular bundles were determined to be useful for species discrimination in Korean 10.7717/peerj.11669/supp-1Supplemental Information 1Click here for additional data file.


Borrelia burgdorferi sensu lato (s.l.) from Ixodes spp. ticks to a diverse group of vertebrate hosts. Small mammal reservoir hosts are primarily responsible for maintenance of B. burgdorferi s.l. across the United States. Nevertheless, birds can also be parasitized by ticks and are capable of infection with B. burgdorferi s.l. but their role in B. burgdorferi s.l. transmission dynamics is understudied. Birds could be important in both the maintenance and spread of B. burgdorferi s.l. and ticks because of their high mobility and shared habitat with important mammalian reservoir hosts. This study aims to better understand the role of avian hosts in tick-borne zoonotic disease transmission cycles in the western United States. We surveyed birds, mammals, and ticks at nine sites in northern California for B. burgdorferi s.l. infection and collected data on other metrics of host community composition such as abundance and diversity of birds, small mammals, lizards, predators, and ticks. We found 22.8% of birds infected with B. burgdorferi s.l. and that the likelihood of avian B. burgdorferi s.l. infection was significantly associated with local host community composition and pathogen prevalence in California. Additionally, we found an average tick burden of 0.22 ticks per bird across all species. Predator and lizard abundances were significant predictors of avian tick infestation. These results indicate that birds are relevant hosts in the local B. burgdorferi s.l. transmission cycle in the western United States and quantifying their role in the spread and maintenance of Lyme disease requires further research.Globally, zoonotic vector-borne diseases are on the rise and understanding their complex transmission cycles is pertinent to mitigating disease risk. In North America, Lyme disease is the most commonly reported vector-borne disease and is caused by transmission of  Borrelia burgdorferi sensu stricto (s.s.) between Ixodes spp. ticks and their vertebrate hosts . AdB. burgdorferi s.l. during their journey and underscore the importance of studying both resident and migratory birds [B. burgdorferi s.l. infection prevalence of 15.8% in attached nymphs. While it is unclear if ticks are able to establish after relocation via avian hosts, there is clear potential for birds to increase existing tick populations and introduce novel genetic variants of B. burgdorferi s.l. [B. burgdorferi s.l. detected in bird species in our study could lead to an increased likelihood of pathogen spread to migratory birds through shared habitat and spread of pathogen infected attached ticks to new regions [While our study focused mostly on resident songbird communities that are not likely to move more than 15–95 km, one species included in our analyses, the pacific slope flycatchers, are part of the over 65 million land birds that migrate through California each year and could transport ticks and ry birds ,88,89,90ry birds  found theri s.l. . The hig regions ,31.B. burgdorferi s.l. infection prevalence and tick parasitism among resident bird populations in the western United States. Many of the bird species that were examined in this study are resident species with limited dispersal behavior and our analyses indicate that these birds are likely acquiring infection locally. Our findings that rodent richness is driving avian B. burgdorferi s.l. infection status and that predator and lizard abundances are driving avian tick burden highlight the importance of considering full community dynamics when evaluating birds, or any other host, as a potential B. burgdorferi s.l. reservoir. Future studies should focus on the reservoir competency and ability of key bird species to infect ticks in order to further resolve the role of birds in local B. burgdorferi s.l. transmission dynamics and their potential for maintaining and spreading B. burgdorferi s.l. to new environments.Our study examines how complex community interactions drive avian