Schistosoma mansoni causes morbidity in human beings, with the highest prevalence in rural sub-Saharan Africa. Prolonged S. mansoni infection with egg deposition in intestinal blood vessels leads to liver and spleen enlargement, and thus chronic morbidity. The objective of this study was to assess whether preschool-aged children develop severe S. mansoni-related morbidity. Parasitological, clinical, and ultrasonographic examinations were carried out in 916 preschool-aged children in five schistosomiasis-endemic districts  along the Lake Victoria shoreline in east-central Uganda. Anaemia and anthropometry measurements were also taken. Using the Kato-Katz technique on one stool sample collected on three consecutive days, 74.9% (686/916) were found infected with S. mansoni; the majority were lightly infected (57.9%), while 22.7% and 19.4% were moderately and heavily infected, respectively. The overall geometric mean intensity (GMI) of infected children was 294.2 eggs per gram faeces. Mayuge and Jinja districts had the highest (51.2%) and lowest (2.2%) number of infected children, respectively. Hookworm infection was found in 7.8% (71/916) of the children. Both liver and spleen were significantly more enlarged in the infected children than in the uninfected children (p < 0.0005), as measured by ultrasonography. Physical palpation of the spleen was more often detected in the uninfected children. A significantly (p < 0.0005) higher proportion of S. mansoni-positive children were anaemic  compared to the children who had no eggs in their stool samples . Schistosoma mansoni infection did not have any severe effect on the nutrition status of preschool-aged children. Neither infected nor uninfected children were found to be underweight or stunted. Liver fibrosis with distinct Symmer’s ‘pipe stems’ was found in a few heavily-infected children (0.3%). In a linear multivariable regression analysis, age of the child, anaemia, liver fibrosis, and size of the left liver lobe were associated with S. mansoni intensity of infection . Our results demonstrate that S. mansoni-related morbidity does develop in children less than six years of age, and that older children (37–60 months) are at higher risk  compared to younger ones (12–36 months). We recommend that preschool-aged children be included in the target population for schistosomiasis mass treatment so as to prevent the childhood chronic form of schistosomiasis. Schistosoma mansoni and presents a significant health burden, particularly in sub-Saharan Africa [S. mansoni parasites, they are at risk of developing the related morbidity [Schistosoma mansoni is highly endemic in Uganda [S. mansoni eggs deposited in the liver and intestines by adult female worms living in the blood vessels surrounding these organs [Intestinal schistosomiasis is caused by infections with n Africa . Once anorbidity . Schiston Uganda ,4,5,6,7,n Uganda . Schistoe organs ,9.S. mansoni such as hepatosplenomegaly, dilation of the portal vein, anaemia, decreased physical fitness, and delayed physical and cognitive development has been characterised in school-aged children [S. mansoni was initially thought to be rare [S. mansoni infection [S. mansoni in preschool-aged children is interesting as the infection in such children is likely to be contracted recently, thereby providing an opportunity to assess the early effects of infection with S. mansoni.Morbidity associated with children ,10,11 anchildren ,8,12. Li be rare . Howevernfection ,14. A stS. mansoni living along the Lake Victoria shoreline, eastern Uganda. This description can be used as a baseline for evaluating the effect of S. mansoni control measures on these indices.In this community-based study we describe anthropometric indices, haemoglobin levels, and the sizes of liver, spleen, as well as the portal vein diameter among children aged 12–60 months infected or not infected with S. mansoni endemic communities along the north-east shoreline of Lake Victoria in eastern Uganda from April 2013 to February 2014. The study area has been described in detail elsewhere [S. mansoni infection status.The study was carried out in lsewhere , but in S. mansoni infection. Each slide from the same specimen was read by two different technicians, and eggs per slide were recorded. For quality control, 10% of the slides were randomly selected and re-examined by an independent experienced microscopist. No discrepancies were reported.Each child provided one stool sample on three consecutive days, and the samples were processed according to the modified Kato-Katz thick smear technique  using 41® photometer . Anaemia was classified according to the World Health Organization guidelines [A finger-prick blood sample was taken from all the recruited children, and their haemoglobin (Hb) concentration (g/L) was measured using a portable HemoCueidelines  as non-aidelines . Body weS. mansoni-infected children and those who did not have any traces of S. mansoni eggs in the three stool samples were recruited into the study for morbidity assessment.After Kato-Katz examinations, all S. mansoni infection. The size of a palpable liver (left lobe and right lobe) was measured at the mid-sternal line (MSL) and right mid-clavicular line (MCL), whereas a palpable spleen was measured along the mid-axillary line (MAL). A liver edge and/or splenic tip extending ≥2 cm below the costal margins were considered enlarged. The consistency of each palpable organ was recorded as soft, firm, or hard. Liver tenderness and tipped spleen were also examined. The two nurses disclosed their measurements after each examination, and in case of any variations or discrepancy, re-examinations were performed with the aim of reaching a consensus.Abdominal palpations were performed independently by two experienced nurses on each child to detect pathological changes in the liver and spleen, which may be due to ®, SonocameraSSD-500 Tokyo, Japan) with a convex 3.5 MHz transducer, according to WHO standard guidelines [S. mansoni-related ultrasonography. The children were examined lying on their backs with their legs stretched on an examination table. Measurements from the upper to the caudal margin in the left parasternal line (PSL) were done for the spleen length (SL) and the left liver lobe. The size of the right liver lobe was measured in the right mid-clavicular line (MCL), whereas the portal-vein diameter (PVD) was measured at the point where the portal vein enters the porta hepatica at the lower end of the caudate lobe. In children with texture patterns suggestive of periportal fibrosis (PPF), a picture was taken and the corresponding texture pattern score recorded. The sonographic picture of the liver texture was translated into six texture patterns (A–F), as described in WHO guidelines of the Niamey protocol [Abdominal ultrasonography was performed using a portable ultrasonographical device ’s involvement in the study. Any child that showed some dissent behaviour (like crying and resisting) was excluded from the study. Study participants who were identified as positive for intestinal schistosomiasis were treated with 40 mg/kg praziquantel by a qualified nurse after clinical and ultrasound examinations.TM version 3.1  and Microsoft Excel® spreadsheet software [Schistosoma mansoni intensities were classified according to WHO guidelines [2) test. To assess whether S. mansoni infection was associated with morbidity indices, we performed analysis at two levels. First, we compared anthropometry measurements, level of anaemia, and size of spleen, liver, and PVD among children who were infected with those who were not infected. Secondly, we determined the association between potential morbidity indicators and S. mansoni intensity using a stepwise multiple regression analysis. Log-transformed egg counts  were also used for the linear regression model. p-Values of less than 0.05 were considered statistically significant.Data were double entered using EpiDatasoftware . The dupidelines  as lightidelines . The Z-sidelines . The valS. mansoni, while 230 (25.1%) did not have any traces of S. mansoni eggs in the three stool samples. Of the 686 infected children, 397 (57.9%) were lightly infected, while 156 (22.7%) were moderately infected and 133 (19.4%) were heavily infected. The overall geometric mean intensity (GMI) among the infected children was 294.2 epg. A total of 71 children (7.8%) had hookworm infection, with 70 having light infection. Only one of the infected children had moderate hookworm intensity while none had heavy infection  were positive for nfection .S. mansoni infection (p >0.05). Older preschool-aged children (49–60 months) were more often infected with S. mansoni compared to younger ones (12–48 months), as shown in Most children—and consequently most infected children—came from Mayuge and Namayingo districts, while the other districts contributed fewer children . Mayuge S. mansoni were more likely to be anaemic (All 916 children had data on haemoglobin (Hb) concentration, anthropometry, and ultrasonography collected. Compared to the uninfected, children who were infected with  anaemic .p < 0.0005. Overall, 97.5%, 84.1%, 91.2%, and 95.4% of the participating children had normal weight-for-age, height-for-age, weight-for-height, and MUAC for age, respectively (p = 0.001) more often found in the uninfected children compared to the infected ones . More S. mansoni-infected children were observed with liver texture abnormalities compared to uninfected children  as shown in S. mansoni-infected children  compared to 35.3% of the uninfected children; ectively . There wted ones . Childrechildren .S. mansoni-infected and uninfected children.Schistosoma mansoni uninfected children had a higher rate of palpable spleen (36.1%) compared to the infected (19.2%); this difference was statically significant (p < 0.0005). Uninfected children had soft spleens significantly (p < 0.0005) more often (28.7%) compared to the infected ones (10.5%). There was a significant difference in liver palpability between S. mansoni infected and uninfected children, 17.2% vs. 10.8%; p = 0.02. Infected children had firm livers significantly (p < 0.005) more often (3.2%) compared to the uninfected ones (0.9%). S. mansoni infected and uninfected children as detected by ultrasonography.S. mansoni-infected ones . Marked splenic length (SL) exceeding mean +4SD of height-adjusted reference values was observed in 31.5% and 13.9% of infected and uninfected children, respectively (p < 0.0005). Uninfected children had a significantly (p = 0.005) higher number of moderately lengthened spleens (27.8%) compared with 16.8% found in the infected children. The left liver lobe was significantly more often (p < 0.0005) much enlarged (mean +4SD) in the infected children (26.2%) than in the uninfected children (10.4%). In contrast, the right liver lobe was significantly shrunken in a higher percentage of the uninfected children compared to the infected ones . The portal vein diameter was found significantly (p = 0.02) dilated only in the infected children. There was a strong positive relationship between liver texture patterns and S. mansoni infection intensity among the preschool-aged children .Thus, a significantly higher proportion of uninfected children had normal organs compared to children ; liver dThe children with advanced liver damage of prominent pipe stems (liver pattern C2) had an abnormally high mean intensity of infection compared to those with other liver texture patterns . LightlyUltrasound examination showed detectable liver fibrosis a,b in a S. mansoni infection intensity as the dependent variable (S. mansoni intensity (p < 0.05).In a stepwise multiple regression analysis with variable , age of S. mansoni intensity of infection by 0.33 epg. For every unit increase in anaemia , there was an increase in S. mansoni intensity of infection by 0.12 epg. For every advancement in the liver pattern (A to C) there was an increase in S. mansoni intensity of infection by 0.1 epg. For each unit increase in the size of the left liver lobe, there was an increase in S. mansoni intensity of infection by 0.12 epg. The size of the right liver lobe, spleen length, MUAC for age, weight-for-height, height-for-age, weight-for-age, district of origin, and sex were not associated with S. mansoni intensity of infection and were excluded from the model.For every one-year (12 months) increase in the age of the child there was an increase in S. mansoni than was detected by physical palpation. This is in accordance with earlier studies, which also showed that ultrasound techniques in comparison to clinical examinations provide more sensitive and precise measurements of liver and spleen enlargement associated with S. mansoni infections [S. mansoni-related liver fibrosis and splenomegaly are more easily detected by ultrasound than by abdominal palpation [S. mansoni, but studies have revealed that preschool-aged children get exposed to S. mansoni-infested waters through bathing and playing [S. mansoni-infected and -uninfected school children with respect to liver and spleen enlargement [Morbidity associated with schistosomiasis was detected in preschool-aged children aged 12–60 months using both clinical and ultrasound examination. However, sonographic examinations revealed much more disease related to infection with fections ,22,23. Aalpation ,24,25,26argement ,27.S. mansoni infection, and it is measured through liver size enlargement—especially the left liver lobe. Enlargement of the left liver lobe is a pattern related to infection with S. mansoni [S. mansoni. However, it is well known that intestinal schistosomiasis is second only to malaria in causing morbidity [The present study shows that much of the morbidity detected in children aged 12–60 months is attributed to  mansoni , and is  mansoni ,29,30, e mansoni . This maorbidity , and thaS. mansoni-related morbidity increases with increase in intensity of infection. Children with advanced liver damage of prominent pipe stems, for instance, are the same children that were found with an abnormally high mean intensity of infection. Other morbidity indicators followed suit, showing increased S. mansoni intensity associated with increased morbidity. This study further shows that older children (49–60 months) are more likely to be infected with S. mansoni than their younger counterparts (12–48 months), and are hence at a higher risk of increased intensity of infection and morbidity development. It is therefore not surprising that advanced liver damage that occurs after a long exposure to S. mansoni infection was identified in a 60-month-old child in this study. This condition is related to high parasite burden with the production of numerous eggs, as described in other studies [S. mansoni infection in preschool-aged children increases with age [This study further reveals that  studies . Other swith age ,6,34.S. mansoni in this study had detectable periportal fibrosis (PPF). This is the first study to report Symmer’s pipe stem fibrosis in children less than six years of age. This severe liver morbidity is usually observed in long-standing infection with S. mansoni [S. mansoni, the period from infection to development of PPF may be reduced. Dilation of the portal vein was found in 2.3% of children infected with S. mansoni, and none of the uninfected ones. The size of PVD has been shown to increase linearly with intensity of infection with S. mansoni and with the size of the spleen [Few children (<1%) infected with  mansoni . Our rese spleen ,36,37,38e spleen ,40, and e spleen .S. mansoni-infected children had lower haemoglobin levels than the egg-negative children. This is consistent with conventional knowledge that S. mansoni is one of the myriad causes of anaemia [S. mansoni infected and uninfected children, and yet anaemia was significantly associated with S. mansoni infection.Results in the present study indicate that  anaemia ,43. Alth anaemia ,43, high anaemia . MoreoveS. mansoni showed a significant rate of malnutrition (MUAC for age) among infected children. Other studies in school-age children found a similar positive correlation between S. mansoni infection and malnourished children [S. mansoni-negative children in this study. This is in contrast with other studies [S. mansoni when egg production starts. This indicates that there are causes of malnutrition other than S. mansoni infection in preschool-aged children; for instance, it could be caused by other illnesses or lack of adequate and proper nutrition. A similar finding was observed in school-age children [Ascaris lumbricoides infection as indicated in some studies [Ascaris eggs detected in the examined stool in this study.The relationship between nutritional status and infection with children ,45. Unex studies , which hS. mansoni may contribute to the causation of the morbidity indicators served. First, the association between S. mansoni and liver enlargement was observed more often with the left lobe compared to the right, and this is consistent with conventional knowledge that S. mansoni is more likely to affect the left lobe more than the right lobe. Secondly, the dilation of the portal vein was observed only in children who were infected with S. mansoni. Thirdly, the severity of morbidity  increased with intensity of infection with S. mansoni, thereby indicating a dose-related association that is more linked to causation than merely association [Our study had strengths that lead credence that infection with ociation . FinallyNevertheless, some limitations still remain, and caution is needed in interpreting our findings. We did not measure co-infection with malaria, which is common in the study area, and is a known predictor of some of the morbidity indicators assessed ,47,48. WS. mansoni-associated morbidity is common among preschool children aged 12 to 60 months. Further, the study shows that organ enlargement as a result of S. mansoni infection in preschool-aged children is mainly due to liver fibrosis and not splenomegaly. These children should be a target of the schistosomiasis control program and should be treated with praziquantel [In conclusion, the present study reveals that iquantel .


Pulsatilla (Ranunculaceae) comprises about 40 species, many of which have horticultural and/or medicinal importance. However, the recognition and identification of wild Pulsatilla species is difficult due to the presence of complex morphological characters. DNA barcoding is a powerful molecular tool capable of rapidly and accurately distinguishing between species. Here, we assessed the effectiveness of four commonly used DNA barcoding loci—rbcL (R), trnH-psbA ( T ), matK (M), and ITS (I)—to identify species of Pulsatilla from a comprehensive sampling group. Among the four barcoding single loci, the nuclear ITS marker showed the highest interspecific distances and the highest rate of correct identification. Among the eleven combinations, the chloroplast multi-locus R+T and R+M+T combinations were found to have the best species discrimination rate, followed by R+M. Overall, we propose that the R+M+T combination and the ITS marker on its own are, respectively, the best multi- and single-locus barcodes for discriminating among species of Pulsatilla. The phylogenetic analysis was able to distinguish species of Pulsatilla to the subgenus level, but the analysis also showed relatively low species resolution. This may be caused by incomplete lineage sorting and/or hybridization events in the evolutionary history of the genus, or by the resolution limit of the candidate barcodes. We also investigated the leaf epidermis of eight representative species using scanning electronic microscopy. The resulting micro-morphological characters were valuable for identification of related species. Using additional genome fragments, or even whole chloroplast genomes combined with micro-morphological data may permit even higher resolution of species in Pulsatilla. Pulsatilla Miller, first described in 1753, consists of about 40 species that are restricted to temperate subarctic and mountainous areas in the Northern Hemisphere   .Pulsatilla as a subgenus or section of the genus Anemone s.l.  Regel is a well-known ingredient included in the Chinese P. ambigua, P. campanella, P. cernua, P. chinensis, P. dahurica and P. turczaninovii) used in folk medicine have been found to contain pharmacologically useful chemical components, including those with anti-cancer and anti-inﬂammatory activities   pairs of lateral leaflets. Flowers nodding before anthesis is recorded as a diagnostic character of P. campanella, but this character was also found in P. ambigua, P. cernua and P. dahurica, and their flower colors show continuous transitional shades of blue (Pulsatilla difficult to identify. species . Howeveriability . The receaflets) . After c of blue . Thus, tc oxidase I (COI), can serve as the core of a global bio-identification system for animals”. Studies have continued to demonstrate that the COI gene fragment efficiently discriminates among animal species, including amphibians (rbcL), the maturase-K gene (matK), and the trnH-psbA intergenic spacer and the nuclear ribosomal internal transcribed spacers (nrITS)] — should be used as core barcode markers for the molecular identification of plants  were assessed in 19 species (representing three subgenera) of Pulsatilla. Approximately 50% of the accepted species of Pulsatilla found in Europe and the Americas were included, as were 90% of the species found in China (rbcL+matK) in Pulsatilla, evaluate the resolution of these four barcodes, and use 2- to 4-region combinations to correctly identify individuals. We also aimed to develop a protocol that could effectively discriminate among closely related species, primarily for species discrimination of medicinal plants. In addition, we added micro-morphological analyses of leaf tissue obtained using scanning electronic microscopy (SEM) to reveal the taxonomic relationships among Pulsatilla.In this study, four DNA barcode regions  were involved in this study . PCR products were run on 1% agarose gels to check whether PCR products showed a clear single band. For those PCR products that did not show a clear single band, the corresponding template DNA was amplified again with two/one pair segmented primers  protocol . For her primers S1, S2rbcL, matK, trnH-psbA, ITS, and combinations of these), three analytical methods were employed. These included the pair-wise genetic distance method (PWG-distance), the sequence similarity method (TAXONDNA), and phylogenetic-based methods . Each of these analyses is described in detail below.All sequence assemblies and adjustments were performed using Geneious v.9.0 . SequencFor the pair-wise genetic distance-based method, five parameters — i.e. average distance, average interspecific distance, average intraspecific distance, smallest interspecific distance, and largest intraspecific distance — were calculated in MEGA 7 using the Kimura two-parameter distance model (K2P), to explore intra- and inter-species variation .Furthermore, to assess the candidate barcodes for the PWG-distance method, two analytical computations were made; i.e. the barcoding gap between interspecific and intraspecific distances and the local barcoding gap for species resolution. The barcoding gap was used to test for appropriate barcode markers, which show high interspecific but low intraspecific genetic divergence . We grapWe used the proportion of correct identifications to assess the potential of all markers for accurate species identification with TAXONDNA (Species Identifier 1.8 program). The “Best Match” , and “Best Close Match” (which assigns queries to species if a threshold similarity is met) tests in TAXONDNA were run for all species that were represented by more than one individual .To evaluate the species discrimination power of the four single barcoding markers, three different tree-building analyses—i.e. the Neighbor Joining (NJ) tree, Bayesian inference (BI) tree, and Maximum likelihood (ML) analyses—were used. The NJ and ML analyses of all markers were conducted by K2P model using MEGA7. For the BI analysis, best substitution models were selected according to the Akaike information criterion (AIC) by jModeltest version 2.1.7 . BI treePulsatilla species is shown in 2, sputter-coating with gold, and imaged using a HITACHI S-3500 scanning electron microscope (SEM). The backgrounds of SEM images were edited and details were colored using Adobe Photoshop. Photographs of mature leaves were taken with a Nikon D7100 digital camera against a black background. Descriptions of leaf morphology were based on 30 mature leaves.Leaf collection information for rbcL, matK, trnH-psbA, and ITS), PCR amplification and sequencing using a universal primer pair had a high success rate — i.e. 96.15, 96.15, 100, and 100%, for rbcL, matK, trnH-psbA, and ITS, respectively. It was difficult to obtain target barcodes for herbarium samples and the success rates from these samples were low. We could not get target barcodes for the 80% of the herbarium samples even when segmented primer pairs were used. A total of 232 new sequences were obtained from 52 accessions, which included 57 sequences for each of rbcL and matK, and 59 sequences for each of trnH-psbA and ITS. All sequences were submitted to the NCBI  and trnH-psbA (299–379 bp) markers. The number of variable sites was the highest for ITS markers (16.98%), followed by trnH-psbA (4.22%), matK (3.71%), and rbcL (1.99%). Eleven indels (1–25bp) were found at the trnH-psbA locus and five were found for ITS (1–2bp), while none were found for matK or rbcL.The aligned lengths of the rbcL showed the lowest intraspecific and interspecific divergence as measured by the PWG-distance method, while ITS showed the highest interspecific divergence (0.1161), followed by trnH-psbA (0.0381) and matK (0.0207) (rbcL marker showed the highest species resolution (48.78%), followed by ITS (44.19%), with matK and trnH-psbA showing the lower species resolution . Of the eleven combinations, rbcL+matK+trnH-psbA (R+M+T) from the chloroplast genome exhibited the best species resolution (70.73%), followed by rbcL+matK (R+M) (68.29%), while trnH-psbA+ITS (T+I) had the lowest species resolution (44.19%)  for the correct identification of species, followed by matK (58.53%) and rbcL (48.78%), respectively. Among the eleven combinations, matK+ITS performed the best (83.72%). The rbcL+matK+ITS and rbcL+matK+trnH-psbA+ITS combinations had a success rate that was similar to matK+ITS.TAXONDNA analyses using the “Best Match” and “Best Close Match” methods exhibited similar discrimination successes (rbcL demonstrated the best discrimination power (NJ tree and BI tree: 48.78%), followed by ITS (BI tree: 39.02%), while matK and trnH-psbA both had lower discrimination power. When barcoding loci were combined, the rbcL+ITS combination had the highest resolution power , followed by rbcL+matK+trnH-psbA(BI tree: 58.54%) and rbcL+matK+ITS (BI tree: 56.10%). The core rbcL+matK combination recommended by COBL had relatively low resolution , and the rbcL+ITS combination was clearly better than all combinations .The discrimination power for all markers and their combinations are shown in Pulsatilla formed a monophyletic clade with high bootstrap support (PP = 0.97/1.00), and all barcodes could discriminate between subgenera of Pulsatilla.The BI phylogenetic trees based on ITS (left) and chloroplast marker data (right) are shown in Pulsatilla, some species represented by two or more individuals formed monophyletic groups, e.g. P. camoanella and P. dahurica. However, several groups were mixed with individuals of other species, e.g. P. turczaninovii continually clustered with P. tenuiloba, and they are mixed with one individual from P. ambigua (cluster I); P. latifolia_136 was clustered with samples of P. patens (cluster II); P. chinese_128 was mixed with individuals of P. cernua . Moreover, successful sequencing rates for sequences ITS and trnH-psbA were over 90% for silica-dried samples but lower for herbarium specimens. These findings are consistent with many previous studies  (Sisyrinchium (5.11%–20.41%)   and Sisy–20.41%) . The inarbcL, matK, and the rbcL+matK combination should be sufficient for a plant barcode, and that this combination should be supplemented with additional markers as required  and rbcL+matK+trnH-psbA  exhibited higher discriminatory performance than any single marker. Consequently, this highlights the need to use chloroplast multi-locus barcodes (rbcL+matK+trnH-psbA) to improve the resolution of species identification in Pulsatilla.Among the candidate barcode genes, the Consortium for the Barcode of Life (CBOL) Plant Working Group suggested that required . In addiThe nuclear ITS region provided the highest inter-and intraspecific divergences  and had a higher success rate for the correct identification of species in TAXONDNA (Best match: 79.06%). However, as for the tree-building method, the discriminatory performance of ITS is not satisfactory, as its highest resolution is 39.02% (BI).rbcL+matK+trnH-psbA+ITS) is one of the combinations that demonstrated the highest species resolution rate, e.g., Aceraceae (90.5%) (Lysimachia (95.5%) (Oberonia (62.99%) (Rhodiola (73.01%)  cannot compensate for the drawbacks of ITS because they are sourced from a different genome. Although the nuclear genome (and the ITS region) is inherited biparentally, the chloroplast genome is inherited uniparentally. Thus, the chloroplast genome experiences more complete lineage sorting than the ITS locus does. Third, hybridizations may cause conflicts between ITS and chloroplast loci, as well as problematic results in ITS phylogeny due to the possibility of homogenization to paternal copies in some lineages and maternal copies in others.As evidenced by previous studies, the multi-locus barcode ( (90.5%) , Lysimac (95.5%) , Oberoni(62.99%) , Rhodiol(73.01%)  and Schi(73.01%) . However(73.01%) Figure 4(73.01%) . Second,Pulsatilla.A combination of DNA markers from different genomes—which have different modes of inheritance and conflicting phylogenies—can hinder our understanding of species delimitation and the evolutionary processes of speciation. Because of its myriad variable sites that can reliably distinguish species, resulting from a high mutation rate and rapid concerted evolution, we recommend ITS as a good single barcode for the genus Pulsatilla . At the same time, the micro-morphological characters of the leaves are also different . Actinocytic and anomocytic stomata exist in both species, but most stomata in P. camanella are actinocytic, whereas most are anomocytic in P. ambigua. Thus, molecular data as well as micro-morphological characters can distinguish between these two species relatively well. Both types of evidence may be helpful to accurately identify specimens that are damaged or lack sufficient diagnostic characters.Within subgenus anthesis . HoweverP. turczaninovii always clustered with P. tenuiloba. They did not have distinct barcodes. The micro-morphological characters were also found to be the same, since both plants showed polygonal epidermal cells with striation, a dense distribution of stomata, and glabrous or sparsely short trichomes. In addition, the geographical distribution of these two species overlaps in Inner Mongolia. Taken together, these distinct lines of evidence collectively suggest that P. turczaninovii and P. tenuiloba are the same species.In addition to its use in identifying specimens, DNA barcoding is also useful for resolving taxonomic uncertainty . Our phyPulsatilla, we found several complex groups. The samples of P. chinensis and P. cernua in cluster III, were indistinguishable. In the Bayesian inference (BI) tree based on ITS sequences  tree based on the combination of chloroplast sequences  may improve the resolution among plant species because nuclear loci have higher overall synonymous substitution rates, thus making nuclear markers such as ITS more sensitive. In our study, P. patens samples 148, 149, 150, and 151 formed a monophyletic group with sample P. latifolia 136. However, these samples were not clustered together by chloroplast marker data  and the National Nature Science Foundation of China .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.


Angiostrongylus vasorum is a nematode living in the pulmonary arteries of canids. Infected dogs develop severe pulmonary lesions which can potentially lead to pulmonary hypertension (PH). However, reports of PH in natural infected dogs are scant. One of the possible causes of the low prevalence of PH in A. vasorum-infected dogs could be the establishment of large diameter intrapulmonary arteriovenous anastomoses (IPAVAs), which attenuate pulmonary vascular resistance, thus reducing the pulmonary arterial pressure. The present report describes the pulmonary arterial pressure (PAP) response to A. vasorum natural infection in two dogs, assessed by echocardiography and by the saline contrast echocardiographic test (SCE).A. vasorum infection resolution, the same dog showed PH and the SCE test resulted negative, which ruled out the presence of IPAVAs. Case 2 suffered from severe pulmonary arterial hypertension and right-side congestive heart failure since the day of presentation. Saline contrast echocardiography was negative both at the time of presentation and at the follow-up visit.Both dogs showed clinical signs of respiratory disease. At presentation, case 1 did not show echocardiographic signs of PH and the SCE test was positive proving the presence of IPAVAs. However, at the follow-up visit, despite A. vasorum infection, IPAVAs recruitment mechanism is able to contrast the rise of PAP until a certain level. It probably represents an initial escape mechanism of PH that, over time, exhausts its compensatory capacities allowing PAP to rise and to be detectable on echocardiography.In the two cases described above, the PH was not associated with IPAVAs. During The online version of this article (10.1186/s13071-019-3544-2) contains supplementary material, which is available to authorized users. Angiostrongylus vasorum  is a nematode living in the right chamber of the heart and pulmonary arteries of wild canids (mainly foxes), dogs and, occasionally, other animals [Angiostrongylus vasorum develops in dogs after the ingestion of infective third-stage larvae (L3) that reside in gastropod molluscs such as snails and slugs, which are needed to complete the parasite’s life-cycle. Angiostrongylus vasorum is defined as “the great pretender” [A. vasorum infection. The clinical signs can vary from an apparently healthy condition to severe clinical manifestations and the most commonly reported clinical signs are related to respiratory, coagulation [A. vasorum infection may be progressive and potentially fatal [A. vasorum develop severe pulmonary lesions which can potentially lead to pulmonary hypertension (PH) [A. vasorum infection and the severity of pulmonary lesions, reports of PH in natural infected dogs have been uncommonly described [A. vasorum infected dogs could be the recruitment of large diameter intrapulmonary arteriovenous anastomoses (IPAVAs) which attenuate pulmonary vascular resistance, reducing the pulmonary arterial pressure (PAP) [A. vasorum natural infection in two dogs, assessed by echocardiography. animals . Angiostetender”  because  larvae L that resetender” , 5 are ngulation , 7 and ngulation , 9. If nly fatal , 10. Dogion (PH)  which ision (PH) . Despiteescribed , 14. Somre (PAP) , 16. TheA. vasorum using morphometrical keys as previously described [A. vasorum . The alignment of our sequence with BLAST showed a homology of 100% with the cox1 and ITS-2 sequences of A. vasorum available in GenBank . The Knottʼs test and in-clinic Dirofilaria immitis, Anaplasma spp., Ehrlichia spp. and Borrelia burgdorferi (sensu lato) tests scored negative.Case 1: First-stage larvae were detected using the Baermann technique and subsequently identified as escribed . BiomoleHaematology and serum biochemistry profiles did not show abnormalities except for a mild hypergammaglobulinemia and mild neutrophilic leucocytosis. Urinalysis results were normal. Chest radiographies showed a multifocal mixed unstructured interstitial pulmonary pattern. At presentation (T0) echocardiography did not detect signs of PH Table . The salA. vasorum first-stage larvae at morphological and molecular characterization. The Knottʼs test and in-clinic D. immitis, Anaplasma spp., Ehrlichia spp. and B. burgdorferi (s.l.) test scored negative. A Leishmania infantum immunofluorescence antibody test (IFAT) was negative. Blood test results revealed mild normochromic and normocytic anaemia, mild hypergammaglobulinemia and mild increase of alanine aminotransferase and alkaline phosphatase. Urinalysis results were normal. The radiological examination of the thorax revealed generalized cardiomegaly with marked right heart enlargement, moderate bulging of the main pulmonary artery and slight distension of pulmonary arteries. Lateral views showed dorsal bowing of trachea cranial to carina and caudal heart border more vertical than normal. In addition, there was diffuse unstructured interstitial pattern in the lung parenchyma, most apparent in the caudo-dorsal region, and moderate pleural effusion. Abdominal ultrasonography detected moderate ascites, hepatomegaly and hepatic vein dilation. The peritoneal effusion was sampled and analysed; it was compatible with high-protein transudate. Doppler echocardiography revealed signs of severe PH such as an RV-to-RA gradient of 88.48 mmHg, PA-to-RV gradient of 46.11 mmHg and reduction of the AT/ET ratio  at 50 mg/kg every 24 h for 15 days. In addition, case 1 received amoxicillin-clavulanic acid  12.5 mg/kg every 12 h for 8 days. In case 2, the right-side congestive heart failure and PH symptoms were treated with sildenafil citrate  at 2 mg/kg every 12 h, benazepril  0.25 mg/kg every 12 h and furosemide  at 1 mg/kg every 12 h.A. vasorum larvae were detected in the Baermann examination. The dog underwent monthly clinical check-ups. Each clinical examination was normal except for the constant presence of abnormal lung sounds (inspiratory crackles), more appreciable in the left lung. Eight months after the presentation (T1) a mild systolic heart murmur, grade II/VI, was detected in the right hemithorax. Then, the dog underwent echocardiography, thoracic radiology, blood and urine analysis and parasitological examination. Parasitological examination at T1 was negative to A. vasorum infection, the radiographic bronco-interstitial pattern was less evident than at T0, blood and urine parameters were within the normal limits, echocardiography revealed signs of mild PH characterized by the presence of TR jet of 3.3 m/s (RV-RA systolic gradient: 44 mmHg), mild RV dilation and reduced AT/ET ratio , where the parasite was reported for the first time by Pipia et al. [To the best of our knowledge, these are the first descriptions of clinical cases of a et al. . Recent a et al. , 20 and a et al. –23. The a et al. . Recentla et al.  with thea et al.  where tha et al. .A. vasorum develop severe pulmonary lesions, secondary to the intense immune response to eggs and larval antigens, including granulomatous inflammation, haemorrhage, arterial thrombosis, periarteritis and interstitial fibrosis [A. vasorum infection are determinants for the development of PH [A. vasorum [A. vasorum infection, 80% of the lungs were consolidated. Thrombi associated with parasitic larvae were present in the pulmonary arteries of untreated dogs but, at the same time point, echocardiographic-derived PAP estimation was within the normal limits. Matos et al. [A. vasorum infected dogs could be the recruitment of large diameter IPAVAs. The IPAVAs are dynamic preformed vascular conduits that have the function of avoiding excessive flow and pressure increases in the pulmonary microcirculation during exercise [A. vasorum was eliminated. This may have been due to the irreversibility of the pulmonary vascular or parenchymal lesions induced by A. vasorum. Conversely, case 1, at T0, did not show symptoms or echocardiographic signs of PH and the SCE test revealed the presence of IPAVAs. The same dog eight months later (T1), despite being successfully treated against A. vasorum infection, showed echocardiographic signs of mild PH and the SCE test was negative. These results may have been due to the fibrotic evolution of the A. vasorum lesions and/or to progression of lesions caused by A. vasorum death after treatment. Whatever the cause, we hypothesized that the increase of PAP may have induced the closure of the IPAVAs with the appearance of PH. An alternative hypothesis is that IPAVAs may be localized in areas of the pulmonary circulation that are recruited under conditions of elevated pulmonary blood flow and pressure [A. vasorum infection. Our findings are consistent with those of Matos et al. in dogs naturally [A. vasorum. They observed that dogs with negative SCE results had higher PAP [A. vasorum infection. Other causes of PH, such as congenital or acquired heart diseases [A. vasorum and D. immitis was not detected, confirming the results obtained by Schnyder et al. [A. vasorum infection [A. vasorum from the infected dogs.Dogs infected with fibrosis , 27, 28.nt of PH . Chronic vasorum . A study vasorum  evidences et al. , 16 hypoexercise , followiexercise  and in rexercise  in orderexercise , 34 and exercise , 16, 31.exercise . Normallexercise . The appexercise , 16, 37.pressure . If trueaturally  infectedgher PAP , 16. Thediseases , were exr et al.  who did r et al.  as unliknfection , 41, butA. vasorum infection in naturally infected dogs. However, the precise role played by IPAVAs in the pathophysiology of PH during angiostrongylosis requires further investigation.This report confirms that echocardiography associated with the SCE test are two valid methods for assessing and monitoring the PAP response to A. vasorum infection during a parasitological faecal screening examination. Three months before, the dog started a treatment against leishmaniasis infection with oral miltefosine  at 2 mg/kg/day for 28 days and oral allopurinol  at 10 mg/kg every 12 h for six months. Then, at the time of presentation the dog was still on therapy with allopurinol. At presentation (T0), the dog was bright, alert and responsive. The physical examination evidenced an increase in respiratory rate (40 beats per minute) and the presence of moderate lung inspiratory crackles, localized in the left caudal lung. The dog did not present clinical signs relative to neurological or coagulation disorders. The kennel’s employees did not report symptoms related to respiratory disease (e.g. cough).Case 1 was 5 years-old, female, neutered, 18 kg hunting dog, guested in a public kennel in Sassari . The dog resulted positive to Case 2 was a 10 years-old, female, neutered, 19 kg dog, presented because of exercise intolerance, chronic cough, dyspnoea, weight loss and abdominal distension. The dog did not present symptoms related to neurological or coagulation disorders. On physical examination the dog showed tachypnoea, abdominal distension, pale mucous membranes, jugular vein pulse and distension, right systolic heart murmur (grade III/VI) and severe crackle sounds diffused on both lungs.Each dog underwent parasitological examination, blood analysis, chest radiography, standard echocardiography and a saline contrast echocardiography (SCE) test.c oxidase subunit 1 (cox1) and for the second ribosomal transcribed spacer region (ITS-2) of the ribosomal RNA gene as previously described [D. immitis, Anaplasma spp., Ehrlichia spp. and B. burgdorferi (s.l.) using an ELISA in-clinic antigen test . Blood samples were also analyzed using Knott’s technique as previously described [L. infantum.The Baermann technique was performed as previously described , 42. Subescribed , 44. PCRescribed . FinallyBlood analysis was performed using a clinical chemistry analyser  and an automated haematology analyser . Serum protein electrophoresis and urinalysis were performed at an external laboratory . Coagulation tests were not performed.Thoracic radiographs were obtained within 24 h of the echocardiographic examination; both dogs underwent right lateral and ventro-dorsal views. Thoracic radiographs were evaluated by an experienced veterinary radiologist.Echocardiographic exams were performed by a single experienced operator with a portable ultrasound unit  equipped with a multifrequency (1.5–3.5 MHz) phased array transducer (3S-RS) and a multi frequency (4–11 MHz) microconvex (8C-RS) probe. The unsedated dogs were positioned alternately in right and left lateral recumbence on a raised table with a central opening, designed for veterinary echocardiographic examination. A complete transthoracic echocardiographic examination, including 2D, M-mode and Doppler, was performed on each dog, according with previously published guidelines . The priAdditional file 1. Echocardiographic cine-loop of saline contrast test performed in case 1 at the time of presentation (T0). The video shows the appearance of saline contrast microbubbles in the left heart following their passage in the right heart, demonstrating the presence of intrapulmonary arteriovenous anastomosis.Additional file 2. Echocardiographic cine-loop of saline contrast test performed in case 2 at the time of presentation. The video shows the passage of saline contrast microbubbles in the right ventricle. Microbubbles did not appear in the left heart following their appearance in the right heart, ruling out the presence of IPAVAs.Additional file 3. Echocardiographic cine-loop of saline contrast test performed in case 1 eight months after presentation (T1). The video shows the passage of saline contrast microbubbles in the right ventricle. Microbubbles did not appear in the left heart following their appearance in the right heart, ruling out the presence of IPAVAs.


E. gallinarum and E. casseliflavus) gut colonization and lower post-transplant mortality. In this study, using an expanded cohort, we evaluated whether our previously observed association is species-specific. We included allo-HCT recipients with ≥1 positive rectal swab or stool culture for iVRE between days -14 and +14 of transplant. To investigate whether iVRE modulate the gut microbiota, we performed agar diffusion assays. To investigate whether iVRE differ in their ability to activate the aryl hydrocarbon receptor, we analyzed iVRE genomes for enzymes in the shikimate and tryptophan pathways. Sixty six  of the 908 allograft recipients (2011–2017) met our inclusion criteria. Overall survival was significantly higher in patients with E. casseliflavus . In multivariable analysis, E. casseliflavus gut colonization was significantly associated with reduced all-cause mortality . While agar assays were largely unremarkable, genome mining predicted that E. casseliflavus encodes a larger number of enzymes in the tryptophan metabolism pathway. In conclusion, E. casseliflavus gut colonization is associated with reduced post-HCT morality. Further research is needed to understand the mechanisms for this association.Gut dysbiosis has been associated with worse allogeneic hematopoietic cell transplantation  outcomes. We reported an association between intrinsically vancomycin-resistant enterococci (iVRE:  E. gallinarum and E. casseliflavus) and improved overall survival (OS) due to decreased non-relapse mortality (NRM) [vanC genotype [The importance of gut microbiota as a predictor of allogeneic hematopoietic cell transplantation (HCT) outcomes has become apparent in recent years . We recety (NRM) . iVRE argenotype ,4.E. gallinarum vs. E. casseliflavus). In an expanded cohort, now also including patients who were iVRE colonized early after HCT, we demonstrate that this association with improved survival is specific to E. casseliflavus. Using in vitro agar diffusion assays and bioinformatic analysis of iVRE genomes, we explored two potential mechanisms for an E. casseliflavus-mediated effect: (i) a direct effect on the host and (ii) an indirect effect on the host via modulation of the gut microbiota.The objective of the present study was to determine whether our reported association between pre-HCT iVRE gut colonization and improved outcomes is species-specific  was used for enterococcal culture. Species-level identification was done by Vitek 2 instrument and MALDI-TOF mass spectrometry  and antibiotic sensitivity were performed by broth microdilution from a subculture of the isolate using the colorimetric Vitek 2 automated system . Colonization level, classified as light, moderate, and heavy growth according to our clinical laboratory standards, was extracted from the patients’ electronic medical records. Antimicrobial prophylaxis consisted of levofloxacin, acyclovir, and an azole. Cefepime was our empiric antibiotic for neutropenic fever. OS was estimated using Kaplan-Meier curves. Relapse, grade II-IV acute graft-versus-host-disease (GVHD), chronic GVHD and NRM were estimated using cumulative incidence, treating NRM (for relapse), non-GVHD death (for GVHD), and relapse (for NRM) as competing risks. In the analysis of relapse and NRM, we excluded patients with non-malignant disorders. Fine and Gray regression was used to adjust for potential confounding [The University of Minnesota Institutional Review Board approved the study protocol. We retrospectively studied allo-HCT recipients at the University of Minnesota (2011–2017) who had ≥1 positive rectal swab or stool culture for iVRE between days -14 and +14. New admissions for HCT were screened for gut VRE colonization weekly until discharge. Spectrafounding . All anaE. casseliflavus produces bacteriocins that create colonization resistance against prototype colonizers of the gut, we screened 16 E. casseliflavus clinical isolates for activity against 6 different E. faecium and E. faecalis isolates via agar diffusion assays. These E. casseliflavus isolates were provided by the clinical laboratory at our institution and retrieved from a repository of samples collected from inpatients (not necessarily HCT patients) over the last 10 years. In addition, a subset of the E. casseliflavus isolates were tested against two E. coli isolates, two Streptococcus agalactiae (group B) isolates, and a Streptococcus gordonii isolate.Intestinal domination may increase the risk of bacteremia and worsen transplant outcomes . To inveA 300 μL aliquot of the indicated growth medium , 8 E. gallinarum (3 human disease-associated isolates), 11 E. faecium , and 7 E. faecalis genomes  were annotated with PROKKA v1.12 [Host-microbial crosstalk at the gut barrier is partially regulated by indole, indole-derived metabolites, and other shikimate/tryptophan pathway compounds, some of which are aryl hydrocarbon receptor (AhR) agonists with immunomodulatory and gut barrier protective functions ,9. To biE. casseliflavus and 43 with E. gallinarum) of the 842 allograft recipients between 2011–2017 met our inclusion criteria. As expected, since vancomycin resistance in iVRE is constitutive rather than acquired, the groups did not differ in their prior exposure to different classes of antibiotics . The abundance of iVRE in cultures were classified as light in 3 (13%), moderate in 7 (30%), heavy in 10 (43%), and not available in 3 (13%) patients with E. casseliflavus colonization. The abundance of iVRE in cultures were classified as light in 8 (19%), moderate in 9 (21%), and heavy in 26 (60%) in patients with E. gallinarum colonization. Nine (39%) patients colonized with E. casseliflavus and 34 (79%) of those colonized with E. gallinarum had at least one other sample colonized with the same species between days -14 and +14. These findings suggest that E. gallinarum colonization is more stable than E. casseliflavus colonization. Only 2 (9%) patients with E. casseliflavus and none with E. gallinarum colonization had VRE colonization between days -14 and +14. Five patients in each group (22% and 12%) had vancomycin-sensitive enterococcus (VSE) gut colonization between days -14 and +14.Sixty six . Fig 2compares transplant outcomes between the groups with E. casseliflavus gut colonization, E. gallinarum gut colonization, and the 776 control patients who did not have colonization with either species. The median follow up for the first two groups was 30 (interquartile range 22–41) months. No deaths occurred before day +14. Comparing patients with E. casseliflavus vs. E. gallinarum colonization, OS was significantly higher in patients with E. casseliflavus , due to lower NRM . Only two patients with E. casseliflavus died within 3 years post-HCT, both due to relapse. In contrast, 14 patients with E. gallinarum died within 3 years post-HCT: 7 (50%) due to relapse and 7 (50%) from NRM . Relapse rates were not significantly different between the groups . 3-year rates of OS, NRM, relapse, and grade II-IV acute GVHD among controls were 61% , 24 (21–27)%, 30 (26–34)%, and 34 (31–37)%, respectively. These rates were similar to the E. gallinarum group.There were no significant differences between the groups in patient-, disease-, or transplant-related characteristics, except a higher frequency of sirolimus-based GVHD prophylaxis in the E. casseliflavus gut colonization was significantly associated with reduced all-cause mortality . There were no significant differences between the groups in 180-day grade II-IV acute GVHD , 1-year chronic GVHD , 100-day bacteremia , or 100-day Clostridium difficile infection . The groups did not differ in time to platelet recovery  but time to neutrophil recovery was longer in the E. casseliflavus group .In multivariable analysis, Table 2shows the results of agar diffusion assays measuring susceptibility of well-characterized other enterococcal species that might be competitors in the gut to potential bacteriocins or other compounds secreted by E. casseliflavus or E. gallinarum. Other than one E. casseliflavus isolate (#10 in Table 2), which had potent activity against both E. faecium and E. faecalis, no other E. casseliflavus isolates showed consistent zones of inhibition. We found no consistent differences between the two growth media tested in these assays. To examine the possibility that E. casseliflavus might produce bacteriocins or other compounds that target potentially-competitive non-enterococcal bacteria strains in the gut, we also tested five additional clinical isolates via the agar diffusion assay—two E. coli isolates, two Streptococcus agalactiae isolates, and a Streptococcus gordonii isolate–against a subset of six of the E. casseliflavus strains noted in Table 2. None of the non-enterococcal isolates were affected by the E. casseliflavus strains tested. These results suggest that a competitive advantage over other bacteria did not contribute to E. casseliflavus dominance in the intestines of patients with improved survival.Enterococcus genomes for enzymes in shikimate and tryptophan metabolism pathways predicted that E. casseliflavus encodes a larger number of enzymes in the tryptophan metabolism pathway . This analysis predicted that four enzymes in the chorismate pathway to indole are present only in E. casseliflavus. In addition, tryptophanase, which converts tryptophan to indole, was predicted to be present in four E. casseliflavus genomes but none of the other E. casseliflavus or non-casseliflavus genomes.Our bioinformatic analysis of 41 publicly available E. casseliflavus strains, and selected isolates of E. gallinarum, E. faecium, and E. faecalis for indole production in vitro (Methods in S1 File). None of the enterococcal isolates produced detectable indole in these assays, while a control Escherichia coli strain produced abundant indole under the same conditions .Because of the known role of indole in AhR agonism ,13, poteBlautia, butyrate-producing Clostridia spp [Eubacterium limosum [E. casseliflavus. Notably, although both E. casseliflavus and E. gallinarum are motile enterococci with intrinsic vancomycin resistance and have 74% average nucleotide identity [E. casseliflavus.The presence of idia spp , and Eub limosum  in the sidentity , our resE. casseliflavus and reduced post-allo-HCT mortality. First, we hypothesized that E. casseliflavus may have a competitive advantage over pathobionts known to cause clinical infections in transplant recipients. Intestinal domination by pathogenic bacteria such as those with antibiotic resistance genes , coupled with a compromised gut barrier due to transplant conditioning and intestinal GVHD, may increase the risk of bloodstream infection [E. casseliflavus as a powerful producer of bacteriocins against E. faecium and E. faecalis (including 5 VRE and 1 vancomycin-sensitive strains). Similarly, E. casseliflavus did not have a competitive advantage against several other gut pathobionts in our agar diffusion assays.We explored two possibilities to explain the association between nfection ,18. Bactnfection . HoweverE. casseliflavus may produce metabolites that directly benefit the host. One such metabolite is the commensal microbe-derived compound butyrate, which has both local and systemic immunomodulatory effects [E. casseliflavus with other enterococci for indole production was unremarkable. However, indole is only one of the many AhR agonists produced via shikimate and tryptophan pathways. Investigating the other metabolites involved in the shikimate/tryptophan pathway may be informative.Alternatively,  effects ,21, and  effects ,22,23. A effects . We chos effects ,26. In a effects . GVHD anE. casseliflavus compared to E. gallinarum. AhR signals augment the gut barrier and modulate the immune system, potentially reducing pathogenic injury and inducing protective immune responses [E. casseliflavus patients, no specific cause of NRM was identified as the primary driver of this difference. We chose AhR ligands for our bioinformatic analysis because of their broad range of effects on the host and hence potential to explain the overall reduced NRM rates in E. casseliflavus patients. Our genomic prediction needs biochemical validation in future work.Using a genome mining approach, we predicted the potential presence of more tryptophan pathway metabolites and AhR ligands in esponses –30. AlthE. casseliflavus may not be in the causal pathway to improved transplant outcomes. Instead, it may be a surrogate for a healthier microbiome permitting colonization by a less pathogenic organism. Future metagenomics studies may identify microbiota signatures of E. casseliflavus-colonized samples and predict metabolic pathways in which these samples are enriched.Although iVRE can be isolated from stool in ~5% of hospitalized patients, clinical infections are rare –35. Gut S1 FileLack of indole in culture supernatants of enterococcal strains.(PDF)Click here for additional data file.


Bartonella henselae can cause various infections in humans, ranging from benign and self-limiting diseases to severe and life-threatening diseases as well as persistent infections that are difficult to treat. To develop more effective treatments for persistent Bartonella infections, in this study, we performed a high-throughput screen of an FDA-approved drug library against stationary phase B. henselae using the SYBR Green I/propidium iodide (PI) viability assay. We identified 110 drug candidates that had better activity against stationary phase B. henselae than ciprofloxacin, and among the top 52 drug candidates tested, 41 drugs were confirmed by microscopy to have higher activity than the current frontline antibiotic erythromycin. The identified top drug candidates include pyrvinium pamoate, daptomycin, methylene blue, azole drugs , aminoglycosides , amifostine (Ethyol), antiviral Lopinavir/ritonavir, colistin, nitroxoline, nitrofurantoin, verteporfin, pentamidine, berberine, aprepitant, olsalazine, clinafloxacin, and clofoctol. Pyrvinium pamoate, daptomycin, methylene blue, clotrimazole, and gentamicin and streptomycin at their respective maximum drug concentration in serum (Cmax) had the capacity to completely eradicate stationary phase B. henselae after 3-day drug exposure in subculture studies. While the currently used drugs for treating bartonellosis, including rifampin, erythromycin, azithromycin, doxycycline, and ciprofloxacin, had very low minimal inhibitory concentration (MIC) against growing B. henselae, they had relatively poor activity against stationary phase B. henselae, except aminoglycosides. The identified FDA-approved agents with activity against stationary phase B. henselae should facilitate development of more effective treatments for persistent Bartonella infections. B. henselae is sometimes a co-infection pathogen of Lyme disease transmitted by ticks carrying multiple pathogens causing more severe and protracted clinical manifestations [B. henselae in liquid media especially from clinical samples [B. henselae infections has been difficult with poor clinical outcomes despite antibiotic treatment for weeks and months [B. henselae infections is due to bacterial persistence, a phenomenon that is well known to underlie persistent infections like tuberculosis, brucellosis, and Q-fever [Borrelia burgdorferi [B. henselae and identified a range of drug candidates from the FDA-drug library that have much better activity against the non-replicating B. henselae than the current antibiotics used to treat bartonellosis. The implication of finding such persister drugs for more effective treatment of persistent Bartonella infection is discussed.Bartonella species are fastidious Gram-negative intracellular bacteria that are highly adapted to their mammalian reservoir hosts. Being the predominant cause of cat scratch disease in people and the second most common species causing endocarditis and bacillary angiomatosis, they have the ability to cause either acute or chronic infections and are responsible for different clinical conditions affecting humans . The bacstations . It is v samples , as it i samples . Treatmed months . The fird months ,10, and  Q-fever . Previougdorferi ,13,14, wgdorferi . In thisBartonella henselae strain JK53, NR-31834. B. henselae was cultured in Schneider’s medium supplemented with 10% fetal bovine serum (FBS) as described [2 at all times. The colony forming unit (CFU) counting was performed on Columbia sheep blood agar  after serial dilutions.The following reagent was obtained through BEI Resources (ATCC), NIAID, NIH: escribed ,17. CultB. henselae was carried out as we described previously, which we have successfully used for high throughput drug screens against stationary phase B. burgdorferi [B. henselae dead cells and live cells mixed in different proportions (107 bacteria/mL) to validate the SYBR Green I/PI assay using a B. henselae 5-day stationary phase culture. The cells were added in 96-well plate, stained with SYBR Green I/PI mixture, and incubated at room temperature in the dark for 15 min. With excitation wavelength at 485 nm, the fluorescence intensities from green emission  and red emission  were measured using HTS 7000 plus Bioassay Reader . The green/red fluorescence ratios were measured for each proportion of live/dead B. henselae as described [The SYBR Green I/PI assay for rapid viability assessment of gdorferi . BrieflyAntibiotics, including doxycycline, cefuroxime, miconazole, metronidazole, rifampin, and fluconazole, were purchased from Sigma Aldrich and were dissolved in appropriate solvents  to form B. henselae cell suspensions for assessing the viability by ratio of green/red fluorescence as described previously [The SYBR Green I/PI dye was added to drug-treated or control eviously ,13. SampB. henselae cell suspension from a 7-day old stationary phase culture was added in 96-well plates. Each compound  from the pre-diluted plate or pre-diluted stock was added to the cell suspension. Plates were sealed and placed in 37 °C incubator over of a period of 5 days. SYBR Green I/ PI viability assay was used to assess the live and dead cells after antibiotic exposure as described [For the high-throughput drug screen, 100 μL escribed . Brieflyescribed . All tesB. henselae stationary phase culture was used for drug exposure experiments. The antibiotic exposure was carried out in 1.5 mL Eppendorf tubes over the course of 5 days at 37 °C without shaking. Then the cells were collected by centrifugation and rinsed twice with fresh Schneider’s medium followed by resuspension in 1 mL fresh Schneider’s medium. The cell suspension was serially diluted and plated on Columbia blood agar plates for viable bacterial counts .A 5-day old B. henselae after a 5-day incubation period. B. henselae cells (1 × 106) were inoculated into each well of 96-well microplate containing 90 μL fresh modified Schneider’s medium per well. Each diluted drug (10 μL) was added to the culture. All experiments were run in triplicate. The 96-well plates were sealed and incubated at 37 °C with 5% CO2 for 5 days. Cell proliferation was assessed using the SYBR Green I/PI assay and a Petroff-Hausser counting chamber after the incubation.The standard microdilution method was used to determine the minimum inhibitory concentration (MIC) needed to inhibit visible growth of B. henselae grew to logarithmic growth phase in 1 day and reached a growth peak at 2 days, with the highest CFU count reaching above 109 per mL, followed by a decline to 108 per mL at 5–7 days and a sharper decline to 106 per mL at 11 days. However, when we checked the live and dead B. henselae cells after staining with SYBR Green I/ PI, all the visible cells in 1-day-old and 5-day-old B. henselae cultures fluoresced green under fluorescence microscope  to validate the SYBR Green I/PI assay using a B. henselae stationary phase culture. The results showed that the percentage of residual viability of different live and dead cell mixtures correlated well in linear relationships with the ratios of green/red fluorescence intensities of B. henselae, especially the ratio pairs of fluorescence intensities at 535 nm/650 nm and 538 nm/650 nm   . GentamiB. henselae in the primary screen, we confirmed that 41 drugs showed higher activity than erythromycin .While we found agents that had good activity against the non-growing stationary phase henselae , it is aus study . As showB. henselae was efficiently suppressed by azithromycin and pyrvinium pamoate at a concentration of 0.04–0.08 μg/mL, by methylene blue, doxycycline and erythromycin at 0.08–0.16 μg/mL, and by clinafloxacin at 0.16–0.31 μg/mL. Nitrofurantoin, nitroxoline, and pentamidine were active with MIC values of 0.31–0.63 μg/mL. B. henselae growing cells were also susceptible to clotrimazole, gentamicin, and berberine with MICs of 0.63–1.25 μg/mL, ciprofloxacin with MIC of 1.25–2.5 μg/mL, streptomycin and miconazole with MIC of 3.13–6.25 μg/mL, and amikacin with MIC of 6.25–12.5 μg/mL. Aprepitant and clofoctol could inhibit growth of B. henselae with MIC of 10–20 μg/mL and 70–140 μg/mL, respectively, which are much higher than those corresponding maximum drug concentration in serum (Cmax) in human. Interestingly, daptomycin, which is highly active against non-growing stationary phase B. henselae . While gentamicin and streptomycin could lead to no viable cells in the log phase B. henselae culture after drug treatment for 1 day and 3 days  along with control drugs  at their Cmax were added to the culture for different times of drug exposure  followed by CFU count. Daptomycin was found to be most active at killing all bacteria by Day 1, followed by methylene blue and gentamicin which killed all bacteria by Day 3 , daptomycin, methylene blue, amifostine (Ethyol), Lopinavir/ritonavir, colistin, amikacin, nitroxoline, berberine, verteporfin, pentamidine, aprepitant, clinafloxacin, and clofoctol. We then chose some of these drugs from different categories at appropriate concentrations to determine their MICs and capability of killing 5-day-old stationary phase B. henselae. The results demonstrated that B. henselae was highly susceptible to most antibiotics tested, which is consistent with previous reports [B. henselae. Rifampin, which is the most effective agent in inhibiting growing B. henselae with the lowest MIC (B. henselae cells. Furthermore, clinically used antibiotics for Bartonella infections, including azithromycin, doxycycline, and erythromycin, while exhibiting effective inhibition of growing B. henselae cells (B. henselae cells in stationary phase (In this study, we were able to culture ry phase . We then reports ,42. Howewest MIC , could nae cells , had verry phase .B. henselae, without CFU detected after drug exposure with drug concentrations close to Cmax (Fortunately, 6 agents, pyrvinium pamoate, methylene blue, daptomycin, clotrimazole, gentamicin, and streptomycin, could efficiently kill stationary phase  to Cmax . Among tB. henselae. While it is safe to take orally even with a high dose, pyrvinium pamoate could be barely detected in plasma and urine due to poor bioavailability [Pyrvinium pamoate, an antihelmintic drug, had good activity against stationary phase lability . One limB. henselae. It is interesting to note that we have previously found methylene blue has good activity against stationary phase Borrelia burgdorferi [B. henselae.Surprisingly, methylene blue showed great activity against gdorferi . Methylegdorferi . Methylegdorferi . It is igdorferi  is consiB. henselae (B. henselae, a Gram-negative bacterium. The high activity of daptomycin against both Borrelia and Bartonella indicates it may serve as a promising drug candidate in treatment of both persistent Borrelia infections and Bartonella infections, which clinically may be present as coinfections.It is worth noting that we found daptomycin had excellent activity against stationary phase henselae , althoughenselae . InteresB. henselae. Clotrimazole at a therapeutically relevant concentration had excellent activity against both growing and non-growing B. henselae and could kill stationary phase B. henselae without viable cells detected on agar plates. In addition, it is of interest that clotrimazole has been shown to be clinically useful for treating other diseases, such as sickle cell anemia, malaria, beriberi, tinea pedis, Chagas disease, and cancer [B. henselae, and the mechanisms of action of azoles, including clotrimazole, against B. henselae remains to be determined.It is interesting to note we found several antifungal azoles to have good activity against d cancer . Clotrimd cancer . As a wed cancer . There iB. henselae strain (B. henselae cells in aggregated form (B. henselae cells, had less activity against stationary phase B. henselae cells (B. henselae cells, but it had high activity against log phase B. henselae cells (B. henselae could partly be responsible for the treatment failure and relapse of bartonellosis [B. henselae cells could be more effective at treating persistent infections in vivo in animal models and in patients in the context of drug combinations.It is worth noting that discrepant efficacies of antibiotics exist between in vitro testing based on MIC data and clinical data in patients with Bartonella-related infections . One cone strain , they shted form . In addiae cells . Erythroae cells . The poonellosis . Future Another important factor that could lead to the discrepancies could be the localization of Bartonella in the host during the infection. The bacteria are able to reside and propagate inside erythrocytes and/or endothelial cells ,53. The B. henselae, they may not be used alone due to possible resistance development and need to be used in the context of drug combinations. Future studies are needed to evaluate drug combinations using the newly identified drug candidates with the current drugs used in clinic to better target diverse bacterial populations in different niches that can happen in the host as in the Yin-Yang model [B. henselae, the findings may apply to other closely related pathogenic Bartonella species, such as B. quintana and B. bacilliformis. Future studies are needed to confirm this possibility.While we found many promising drug candidates with good activity against stationary phase ng model . In addiB. henselae using the FDA-approved drug library where we identified a range of promising drug candidates that may improve the treatment of Bartonella infections. Since these drug candidates are FDA-approved they could be more readily adopted clinically for treating Bartonella infections as long as they meet appropriate safety, PK, and efficacy requirement. Further studies are needed to determine the activity of persister drug combinations with the identified drug candidates against Bartonella persisters and biofilm bacteria in vitro and in animal models of Bartonella infection.In summary, this is the first study of a high throughput drug screen against stationary phase


Aegilops umbellulata Zhuk. (2n = 14), a wild diploid wheat relative, has been the source of trait improvement in wheat breeding. Intraspecific genetic variation of Ae. umbellulata, however, has not been well studied and the genomic information in this species is limited.Ae. umbellulata and to evaluate its genetic diversity, we performed RNA sequencing of 12 representative accessions and reconstructed transcripts by de novo assembly of reads for each accession. A large number of single nucleotide polymorphisms (SNPs) and insertions/deletions (indels) were obtained and anchored to the pseudomolecules of Ae. tauschii and barley (Hordeum vulgare L.), which were regarded as virtual chromosomes of Ae. umbellulata. Interestingly, genetic diversity in Ae. umbellulata was higher than in Ae. tauschii, despite the narrow habitat of Ae. umbellulata. Comparative analyses of nucleotide polymorphisms between Ae. umbellulata and Ae. tauschii revealed no clear lineage differentiation and existence of alleles with rarer frequencies predominantly in Ae. umbellulata, with patterns clearly distinct from those in Ae. tauschii.To develop novel genetic markers distributed over all chromosomes of Ae. umbellulata accessions. The alleles with rarer frequencies might be the main source of the high genetic diversity in Ae. umbellulata.The anchored SNPs, covering all chromosomes, provide sufficient genetic markers between The online version of this article (10.1186/s12870-018-1498-8) contains supplementary material, which is available to authorized users. Aegilops umbellulata Zhuk. (2n = 14), a wild diploid wheat relative, is distributed in West Asia and is known as the U-genome donor of Ae. columnaris and Ae. triaristata [Ae. umbellulata (UU genome) has crossability with tetraploid wheat , which allows generation of synthetic hexaploids (AABBUU genome) through ABU triploids. Some combinations of interspecific crosses between Ae. umbellulata accessions and tetraploid wheat result in hybrid incompatibility, such as severe growth abortion and grass-clump dwarfness [Ae. umbellulata that potentially vary phenotypic traits.uk. 2n = 4, a wildwarfness . This obAe. umbellulata have been used for breeding of bread wheat and is a considerable resource of disease resistance genes [T. aestivum cv. Chinese Spring PhI, which carries an epistatic inhibitor of Ph1 gene, induced homologous pairing and resulted in transfer of the leaf and stripe rust resistance genes of Ae. umbellulata into the bread wheat T. aestivum. Although Ae. umbellulata provides valuable genetic resources for breeding of bread wheat, it has not been well studied and information on its genome is limited. Evaluation of intraspecific genetic diversity based on genome-wide polymorphisms in Ae. umbellulata would impart practical information for designing genetic markers, facilitating the efficient use of Ae. umbellulata for breeding.ce genes –10. Leafce genes , 8, 11 ace genes , 12 havece genes  and BansT. monococcum [Ae. tauschii [Yr15, and a wheat spot blotch resistance gene, Sb3, within 0.77 cM and 0.15 cM intervals, respectively [Since the tribe Triticeae has a large genome, most of which is occupied by repetitive sequences, development of high-quality physical maps and whole genome sequencing are challenging. RNA sequencing (RNA-seq) is one of the solutions for detection of single nucleotide polymorphisms (SNPs) and evaluation of genetic diversity by avoiding these genome complexities of the Triticeae. RNA-seq approaches for identifying novel genetic markers have been applied to several Triticeae species such as nococcum  and Ae. tauschii –16. RNA-ectively , 18.Hordeum vulgare L.) [Ae. tauschii [Ae. tauschii can be regarded as virtual chromosomes of other Triticeae species. By combining RNA-seq with positional information from this synteny, a large number of SNPs and indels can be anchored to the chromosomes, facilitating design of genome-wide genetic markers [Recently, the highest-quality genome sequences have been developed in the diploid Triticeae species barley (gare L.) , 20 and tauschii , 22. By tauschii , 24, the markers . The RNAAe. umbellulata, transcripts of 12 representative accessions of Ae. umbellulata were first reconstructed by de novo assembly of reads from RNA-seq on the Illumina MiSeq platform. Using the deduced transcript sequences, a large number of SNPs and indels between the Ae. umbellulata accessions were detected and anchored to the barley and Ae. tauschii pseudomolecules. Comparative analysis of DNA polymorphisms between Ae. umbellulata and Ae. tauschii revealed relatively high genetic diversity in Ae. umbellulata.Here, to evaluate genetic polymorphisms and capture genetic markers in Ae. umbellulata were chosen from the wheat genetic resources database of the National BioResource Project-Wheat  to represent the diversity of this species . The total RNA was treated with DNase I at 37 °C for 20 min to remove contaminating DNA. A total of 6 to 10 μg of RNA was used for constructing paired-end libraries. The libraries were constructed with TruSeq RNA Library Preparation Kit v2  according to the manufacturer’s instructions, and were sequenced with 300-bp paired-end reads on an Illumina MiSeq sequencer.Twelve accessions of ies Fig. ; Table 1Low-quality bases , adapter sequences, and reads < 100 bp were removed using the Trimmomatic version 0.33 tool . The paiHordeum vulgare L.) reference genome “ASM32608v1 masked” [Ae. tauschii genome “PRJNA341983” from the NCBI database [Ae. tauschii and barley, SNPs and indels were anchored to the chromosomes. The distribution of SNPs and indels on barley and Ae. tauschii chromosomes were visualized using CIRCOS software [The transcripts were mapped to the barley . PCR products were resolved in 17% acrylamide gels, and the products were visualized under UV light after staining by ethidium bromide.Indel markers were designed using indels longer than 3 bp that were anchored to the barley chromosomes. Primer sets were constructed with Primer3plus software . To valiAe. tauschii accessions from the Transcriptome Shotgun Assembly division of DDBJ BioProject PRJDB4683 [Ae. tauschii KU-2075, which were constructed in our previous report [Ae. umbellulata KU-4017 as reference transcripts. Quality control for the reads of Ae. tauschii and T. urartu was performed using Trimmomatic version 0.33 [Ae. umbellulata. The reads were aligned to the reference transcripts of Ae. tauschii KU-2075 and Ae. umbellulata KU-4017 using Bowtie 2 [The RNA-seq reads from the 10 RJDB4683  were uses report , and Ae.ion 0.33  in the sBowtie 2 . SNP calBowtie 2 , 30 usinBowtie 2 , and fixBowtie 2 . A neighAe. tauschii KU-2075 and Ae. umbellulata KU-4017 were estimated according to reciprocal best hits of BLAST analysis. A BLASTN search was performed using transcripts of Ae. tauschii KU-2075 as the queries against transcripts of Ae. umbellulata KU-4017, and vice versa. When the same best hit was detected and query coverage was over 80% in both BLAST analyses, the transcripts from Ae. umbellulata KU-4017 and Ae. tauschii KU-2075 were judged an orthologous pair.Orthologous pairs of the reference transcripts of The mapped reads that were concordantly aligned to the reference transcripts were chosen from the alignment file with SAMtools . FragmenAe. umbellulata, RNA-seq was performed on the 12 representative accessions, generating 3.5–6.1 million paired-end reads per one accession  non-redundant SNPs to barley chromosomes   in Ae. uAe. umbellulata accessions using the Illumina MiSeq platform. By using Ae. tauschii and barley pseudomolecules as the virtual chromosomes of Ae. umbellulata due to the conserved synteny between Triticeae species [Ae. umbellulata were successfully anchored to the chromosomes of Ae. tauschii and barley, respectively  and to develop novel genetic markers, we conducted 300-bp paired-end RNA sequencing of leaf tissues from 12 representative  species , 24, an tauschii  dramatictauschii , in whictauschii  with itstauschii . Even whequences . The elaellulata . The RNAAe. umbellulata chromosomes in an order reflecting the conserved synteny with Ae. tauschii and barley  and the Ae. tauschii KU-2075 reference transcript dataset (b). These trees were constructed by Neighbor-Joining method. Figure S4. Phylogenetic relationship between the 12 Ae. umbellulata accessions, 10 Ae. tauschii accessions and one T. urartu accession based on SNPs estimated using the Ae. tauschii KU-2075 reference transcript dataset. The tree was constructed by the maximum-likelihood method. Figure S5. Derived allele frequency distribution in Ae. umbellulata (n = 12) (a) and Ae. tauschii (n = 10) (b), respectively. Ae. tauschii KU-2075 transcripts were used as the reference. Derived alleles were estimated using the outgroup species T. urartu. (PDF 824 kb):


Fritillaria cirrhosa D. Don, whose bulb is used in a well-known traditional Chinese medicine to relieve cough and eliminate phlegm, is one of the most important medicinal plants of Fritillaria L. The species is widely distributed among the alpine regions in southwestern China and possesses complex morphological variations in different distributions. A series of newly related species were reported, based on obscure morphological differences. As a result, F. cirrhosa and its closely related species constitute a taxonomically complex group. However, it is difficult to accurately identify these species and reveal their phylogenetic relationships using traditional taxonomy. Molecular markers and gene fragments have been adopted but they are not able to afford sufficient phylogenetic resolution in the genus. Here, we report the complete chloroplast genome sequences of F. cirrhosa and its closely related species using next generation sequencing (NGS) technology. Eight plastid genomes ranged from 151,058 bp to 152,064 bp in length and consisted of 115 genes. Gene content, gene order, GC content, and IR/SC boundary structures were highly similar among these genomes. SSRs and five large repeat sequences were identified and the total number of them ranged from 73 to 79 and 63 to 75, respectively. Six highly divergent regions were successfully identified that could be used as potential genetic markers of Fritillaria. Phylogenetic analyses revealed that eight Fritillaria species were clustered into three clades with strong supports and F. cirrhosa was closely related to F. przewalskii and F. sinica. Overall, this study indicated that the complete chloroplast genome sequence was an efficient tool for identifying species in taxonomically complex groups and exploring their phylogenetic relationships. Fritillaria L. is one of the most important genera in Liliaceae, which includes approximately 140 species of perennial herbaceous plants  and nuclear ITS to explore the phylogeny with 191 taxa in the tribe Lilieae (including 57 Fritillaria species) but the boundaries between a few species of Fritillaria remained ambiguous and needed further research. Meanwhile, the combination of nrITS and cpDNA genes was also adopted to reveal inter-specific relationships and to discriminate between the species of Fritillaria. Although these gene fragments had a preliminary resolution on certain species in Fritillaria, they could not be effective in discriminating between the closely related species  classification system was constructed based on the latest progress in plant molecular phylogenetics . Althoughylogeny . Neverthhylogeny . Day et  species . Overall species . Fortuna species .Amborella, Nymphaea, Citrus, Gossypium and Oncidium  are photosynthetic organelles providing the necessary energy for growth and are fundamental in the biosynthesis of starch, fatty acids, pigments, and amino acids . TypicalOncidium . In a reFritillaria species using Illumina sequencing technology and performed comparative analyses of genomic information. Our aims were as follows: (1) to investigate the global structure patterns of eight plastid genomes in Fritillaria in this study; (2) to examine the variations of simple sequence repeats (SSRs) and other repeats  among the eight Fritillaria plastid genomes; (3) to discover highly divergent regions that could be used as specific DNA barcodes for Fritillaria; and (4) to reveal phylogenetic relationships between F. cirrhosa and its closely related species. The study might provide better understanding of phylogenetic relationships of the complex group and afford sufficient genomic information to use in further research and the application of these medicinal species.Here, we presented complete chloroplast genome sequences of several Fritillaria cirrhosa and its closed relatives were collected and used in this study  and morphologic traits  were described immediately. All voucher specimens of Fritillaria were identified carefully by Dr. Dequan Zhang and deposited at the Herbarium of Medicinal Plants and Crude Drugs of the College of Pharmacy and Chemistry, Dali University.Eight species, including is study . F. thunPlus Spectrophotometer . DNA extracts were fragmented for 300 bp short-insert library construction and sequenced –2 × 150 bp paired-end (PE) reads on an Illumina HiSeq X-Ten instrument at Beijing Genomics Institute .Total genomic DNA was extracted from about 100 mg of dried leaf material according to a modified CTAB method . DNA quaFritillaria cirrhosa (accession number: KF769143) as a reference  using MAFFT  as a reference. Default parameters were utilized to align the chloroplast genomes in Shuffle-LAGAN mode  Hamming distance of 3, (ii) 90% or greater sequence identity, and (iii) a minimum repeat size of 30 bp . MISA waectively .After using MAFFT v.7.129 to align the chloroplast genome sequences, BioEdit software was used to adjust the sequences manually . A slidiF. cirrhosa and its closely related species were used for phylogenetic analysis, to be supplemented with F. thunbergii (accession number: MH244914) and Lilium brownii F. E. Brown ex Miellez (accession number: NC_035588) as outgroups (F. unibracteata var. wabuensis (KF769142), which was a variety of F. unibracteata, was downloaded from GenBank for our phylogenetic analysis , Maximum parsimony (MP) and Maximum likelihood (ML) methods were adopted for phylogenetic inference, respectively.The eight species of utgroups . Furtheranalysis . Phylogeanalysis . LengthsMEGA v.7.0.26 was used for MP analysis with 1,000 bootstrap replicates . For BI Fritillaria chloroplast genomes ranged from 151,083 bp in F. unibracteata to 152,064 bp in F. sinica and shared the typical quadripartite structure, composed of a pair of IRs  separated by the LSC  and SSC  regions (F. cirrhosa due to the presence of eight rRAN (55%) sequences in these regions  , 12 genes for small ribosomal proteins , five genes for photosystem I , 15 genes for photosystem II , and six genes for ATP synthase   , Fig. 2.H, atpI) , Fig. 2.F. cirrhosa whole chloroplast genome, respectively, and the remaining 47.26% was non-coding regions. The 20 amino acids crucial for protein biosynthesis were encoded by 30 tRNA. Moreover, protein coding genes included 78 protein genes and the length was 68,234 bp, which comprised 22,396 codons  were detected based on the comparison of eight Fritillaria cp genomes. A total of 78 perfect SSRs were found in F. cirrhosa  and T (52.72%) motifs in the majority of dinucleotide SSRs are AT (64.29%)  .rpoC2, cemA, ndhD, ndhG, ndhH, ycf1, and ycf2 in the CDS regions of the Fritillaria cp genome  of the plastid genomes . However, almost all of the lengths of the forward and reverse repeats were less than 59 bp in eight Fritillaria chloroplast genomes  was used as a reference for visualization analysis of the pairwise chloroplast genomic alignment between F. cirrhosa and its closely related species using mVISTA  were distributed in protein coding regions.The annotation of g mVISTA . The aliFritillaria chloroplast genomes were revealed and a detailed comparison of four junctions of two IRs between F. cirrhosa and its closely related species was performed  was between 0.0005–0.0008.Evolutionary divergences and differences among the eight s 52-340 . The p-dFritillaria plastid genomes. Of these, the SSC region exhibited the highest divergence levels (0.00332) and IR regions had the least (0.00038)  and one gene region (ycf1) from the genomes, were selected as potentially suitable gene fragments for the study of species identification and phylogenetics in Fritillaria  to estimate the divergence level of different regions in the eight 0.00038) . Furthertillaria . All higtillaria . The petFritillaria thunbergii and Lilium brownie were used as outgroups). BI, MP, ML analyses were performed to construct phylogenetic trees using the datasets . Clade I contained four species with strong support, namely F. sichuanica, F. dajinensis, F. unibracteata, and F. unibracteata var. wubuensis. It was revealed that F. sichuanica had a close relationship with F. dajinensis. Fritillaria taipaiensis, and F. yuzhongensis, both of which were distributed in the northern edge of the complex group and were clustered into one clade (II). The last clade (III) was composed of F. cirrhosa, F. przewalskii and F. sinica which revealed that F. cirrhosa was a sister species to the latter two species.In this study, five datasets extracted from the eleven plastid genomes were used for phylogenetic analysis  was highest, which could be attributed to the presence of eight rRNA (55%) sequences in these regions  of the Fritillaria plastid genomes. In the previous study, matK, rpoC1, rpoC2, cemA, ndhD, ndhG, ndhH, ycf1 and ycf2). rpoC2, cemA, rpl22, ndhD, ndhE, ndhH, ycf1 and ycf2) of three cardiocrinum plastid genomes. Therefore, all the studies strongly indicated that the chloroplast genome could be used for developing lineage-specific cpSSR markers that could help for studies on population genetics of the Fritillaria species.SSRs in the chloroplast genome (cpSSRs), which are 1-6 bp repeating sequences and distributed throughout the genome, have been used for the study of population genetics because of their high variability . In thisRepeat sequences play an important role in genome rearrangement and variation due to the illegitimate recombination and slipped-strand mispairing in the chloroplast genome . In the F. cirrhosa and its closely related species was performed. The IR regions are highly conserved and structure variation was not significant in the eight Fritillaria chloroplast genomes  and the IR regions had the least (0.00038)  and one gene region (ycf1) from the chloroplast genomes, were selected as potentially suitable gene fragments to study species identification and phylogenetics in Fritillaria  . Once agtillaria  and have  based on BI, MP, and ML analysis  Bayesian analysis (BI), (B) maximum parsimony (MP), and (C) maximum likelihood (ML).Click here for additional data file.10.7717/peerj.7480/supp-2Supplemental Information 1MH244906 –MH244913).DNA sequences for the nine chloroplast whole genomes of Fritillaria spp. in this study (Click here for additional data file.10.7717/peerj.7480/supp-3Table S1Click here for additional data file.10.7717/peerj.7480/supp-4Table S2Click here for additional data file.10.7717/peerj.7480/supp-5Table S3Click here for additional data file.10.7717/peerj.7480/supp-6Table S4Click here for additional data file.10.7717/peerj.7480/supp-7Table S5Click here for additional data file.10.7717/peerj.7480/supp-8Table S6Click here for additional data file.10.7717/peerj.7480/supp-9Table S7Click here for additional data file.10.7717/peerj.7480/supp-10Table S8Click here for additional data file.