Porites rus from a hologenomic sample and compared it to traditional approaches.Many cnidarians, including stony corals, engage in complex symbiotic associations, comprising the eukaryotic host, photosynthetic algae, and highly diverse microbial communities—together referred to as holobiont. This taxonomic complexity makes sequencing and assembling coral host genomes extremely challenging. Therefore, previous cnidarian genomic projects were based on symbiont-free tissue samples. However, this approach may not be applicable to the majority of cnidarian species for ecological reasons. We therefore evaluated the performance of an alternative method based on sequence binning for reconstructing the genome of the stony coral P. rus. An assembly evaluation based on operational criteria showed results that were comparable to symbiont-free approaches in terms of completeness and usefulness, despite a high degree of fragmentation in our assembly. In addition, we found that binning provides sufficient data for exploratory k-mer estimation of genomic features, such as genome size and heterozygosity.Our results demonstrate that binning performs well for hologenomic data, producing sufficient reads for assembling the draft genome of Binning constitutes a powerful approach for disentangling taxonomically complex coral hologenomes. Considering the recent decline of coral reefs on the one hand and previous limitations to coral genome sequencing on the other hand, binning may facilitate rapid and reliable genome assembly. This study also provides an important milestone in advancing binning from the metagenomic to the hologenomic and from the prokaryotic to the eukaryotic level. Symbiotic associations are common across the tree of life, being fundamental to nearly all aspects of host function and fitness . The ecoSymbiodinium spp.). The latter provide up to 90% of the energy requirements of the coral [Well-known examples of symbiotic relationships can be found in reef-building stony corals (order Scleractinia) . The suche coral . Furtherhe coral , 9, endohe coral , fungi [he coral , archaeahe coral , and virhe coral  has beenin hospite symbiont cells [The complexity of the symbiotic assemblage makes sequencing and assembly of coral genomes extremely challenging. Reasons include the high taxonomic diversity of the hologenome , the firnt cells , and thent cells . As a cont cells , 19. Thent cells .in situ reduction of taxonomic complexity. Respective approaches included the use of gametes , larvalectensis ), tissue pallida ), and/oroma spp. ). Whereaoma spp. , making oma spp. ) and/or oma spp. , symbionoma spp. . This pric units  based onic units , differeic units , and/or ic units . It thusic units . Howeveric units . In coranas spp. ). HowevePorites rus; Scleractinia) as an alternative to prevalent symbiont-free approaches. In the first step, we performed a 16S ribosomal RNA (16S rRNA) gene amplicon analysis in order to assess the P. rus hologenome complexity. In the second step, we conducted high-throughput DNA and RNA sequencing (RNA-seq) of a hologenomic sample. The third step included hologenome assembly, binning, and reconstruction of the P. rus host genome. In the final step, we compared the quality of our binning-based assembly with assemblies based on traditional, symbiont-free DNA approaches -European Bioinformatics Institute (EBI) Annotare project ID for the holotranscriptome data is E-MTAB-6535.Hologenomic RNA was extracted from an adult P. rus adult colony for whole-hologenome sequencing, binning, and coral host genome assembly. The hologenome raw data are available under the Bioproject ID PRJEB23570.Hologenomic DNA was extracted from an adult in silico approach for reducing the taxonomic complexity in cnidarians compared to in situ approaches. The EMBL-EBI accession numbers are OKRP01000001-OKRP01014982 for the genome. The transcriptome data are available via the Annotare project ID E-MTAB-6535  and the chloroplast 16S rRNA gene of various algae such as Synedra spp. and Ostreobium spp. (∼21%). The bacterial superkingdom was represented by ∼38% of the reads  were obtained and taxonomically classified from kingdom to genus level based on 16S rRNA gene amplicons. The majority of reads (∼60%) were assigned to eukaryotic replicons. This included the 16S rRNA gene of the A. digitifera, E. pallida, and N. vectensis, all bins were classified as P. rus, thus representing its draft genome. Using these genome bins as a reference, ∼148 million reads were assigned to the coral bins through read mapping. These reads were used for a single genome assembly adapted to diploid organisms, resulting in a 470-Mb draft genome (∼72x coverage), comprising 14,982 scaffolds (N50 = 137 kb) and  81,422 contigs (N50 = 5.3 kb) and featuring a guanine-cytosine (GC) content of 38.86%  were generated. After quality trimming and filtering, the metagenome assembly resulted in  1,829,146 contigs (>100 bp) (N50 = 1.3 kb) with a size of 947 Mb. About 45% of the ∼212 million metagenome reads were mapped back onto these contigs. The resulting bam-file was used for binning with MetaBat. In total, 13 host genome bins were predicted, ranging from 250 kb to 370 Mb. Based on a manual inspection using Basic Local Alignment Search Tool (BLASTx), r2cat, and the reference genomes of 6% Table .de novo gene prediction tool Genemark-ES. RNA-sequencing validation confirmed 9,662 (∼25%) of the predicted gene models, whereas for the remaining predicted genes,  21,865 homologous reference genes were detected in the National Center for Biotechnology Information database . They mainly refer to other coral genomes  and hence represent potential orthologs. InterProScan identified 31, 611 genes with known functional domains. Additionally,  17,754 of these genes could be assigned to Gene Ontology term numbers. InterPro numbers could be assigned to  25,966 genes and enzyme commission number to 1,191 enzyme-encoding genes.In total , 39,453 protein-coding genes were predicted using the k-mer size has been suggested as adequate in previous comparative experiments [k-mer frequency displayed a double-peak profile, with the first peak  taller than the second peak , indicating high heterozygosity [k-mer occurrences corresponded to repetitive sequences , particularly between the Poisson-based approaches (Waterman and gce) and the negative binomial-based GenomeScope  performed best, followed by the two stony coral assemblies (A. digitifera and P. rus)  , Fig. 3aa level” , Fig. 3bP. rus assembly did not overperform in any of these parameters and underperformed in the following four of seven parameters: NG50 scaffold length, NG50 contig length, N content, and contigs >10 kb. According to the cumulative z-score analysis for the contiguity metrics, the A. digitifera assembly was ranked best, followed by the E. pallida, N. vectensis, and P. rus assemblies   was inclmed best , Fig. 4.semblies .P. rus genome had superior performance for the criterion usefulness (together with the A. digitifera assembly), average performance for the criterion completeness, and inferior performance for the criterion contiguity , which was similar to those reported for the stony coral A. digitifera (13%) [E. pallida (26%) [N. vectensis (26%) [k-mer-based methods of genome size estimation. Although the Waterman and gce methods are based on the assumption that the k-mer frequency profile should approximate a Poisson distribution [P. rus genome, Poisson-based k-mer approaches may produce reliable genome size estimations.Of interest for hologenomic projects is the fact that binned reads enable the estimation of genomic features , based on assembly-independent methods such as the ra (13%)  and the da (26%)  and N. vribution , which wribution , 41. Thek-mer approaches, may produce comprehensive estimations of genomic features such as repeat content, degree of heterozygosity, and genome size. This information, in turn, may help define library types, insert sizes, and sequencing coverage required for sequencing and assembling high-quality genomes [In conclusion, coral-binned reads, together with  genomes .Comparing our binning-based host genome assembly with assemblies based on symbiont-free DNA, all approaches showed a similar performance in terms of completeness and usefulness Fig. . In partFinally, as we compared assemblies from different cnidarian species with distinct genome features , our study did not aim at comparing assembler performances. Rather, we were interested in whether binning can provide sufficient data for assembling host genomes of comparable quality to assemblies based on symbiont-free DNA. Our results demonstrate that binning performs well in reducing hologenomic data complexity aimed at assembling host genomes. However, it is recommended that several assembly strategies be tested in order to improve final assembly contiguity based on the features of the binned data.In conclusion, our study demonstrate that both symbiont-free and hologenomic binning-based strategies enable host genome reconstruction. The choice of either approach for future coral genome assemblies thus depends on the study taxon , the scientific question of interest, and whether the study has a genomic, metagenomic, or hologenomic focus.Despite the high taxonomic complexity of coral holobionts, binning performed well in our hologenomic dataset. However, it is important to note that our hologenomic sample turned out to be rich in coral DNA, which may have contributed to the good performance of our binning approach. At this point, it is difficult to tell whether hologenomic samples with a higher fraction of symbiont-DNA may be equally suited for host genome reconstruction or whether they require a prior enrichment step.Symbiodinium symbionts, as recently implemented [It is also important to note that binning may be sensitive to problems associated with genomic interactions within a holobiont. In particular, horizontal gene transfer between the coral host and the microbial community  may redulemented . Alternalemented . In addilemented .P. rus. A comparative assessment of the operational criteria contiguity, completeness, and usefulness across cnidarian genome projects revealed that the binning approach displayed comparable results to symbiont-free approaches in terms of completeness and usefulness, despite a high degree of fragmentation. In addition, we demonstrate that binning might supply data for exploratory k-mer-based estimations of genomic features such as genome size, heterozygosity, and repetitive content. Thus, binning constitutes a powerful tool for reducing the taxonomic complexity of holobiontic samples. Moreover, even though binning was originally designed for metagenomic analyses, we showed that it is capable of handling eukaryotic data.In conclusion, we demonstrate that binning might handle cnidarian hologenomic sequencing samples well, providing enough binned reads for assembling the draft genome of the stony coral per se be the first choice for future coral genome projects. Whether to use in silico approaches for reducing the taxonomic complexity of holobiontic samples or alternative in situ approaches largely depends on the study taxon and the scientific question of interest. Nonetheless, given the methodological challenges in hologenomics on the one hand and the low number of cnidarian host-genome assemblies on the other hand, binning may open the door to rapid and reliable host genome reconstructions in cnidarian holobionts. This in turn might help generate the genomic data necessary for assessing the complex genetic interactions within the coral holobiont and thus the genetic base of coral disease susceptibility and resistance.However, binning may not in silico approach may also be applicable for disentangling other holobiont assemblages, particularly when in situ taxonomic complexity reduction is not desired or feasible. Taking into account that eukaryotes such as plants and animals are no longer regarded as isolated entities [Our study shows that sequence binning is a valid tool for reducing the high taxonomical complexity of a coral holobiont. This entities , 5, 44, P. rus colony was imported from Indonesia in 2007 (CITES permit number 14 846/IV/SATS-LN/2007) and kept in the marine facilities at Justus Liebig University Giessen. The colony was maintained in a seawater system at approximately 26°C on a 10:14 h light–dark cycle. A fragment of ∼9 cm2 was taken from the colony for hologenomic DNA/RNA isolation. The tissue was removed by scraping the fragment's surface with a sterilized razor blade as previously recommended [P. rus colony is in long-term culture at Justus Liebig University Giessen and thus available for further investigation.An adult ommended  and immeHigh-molecular-weight genomic DNA was isolated from ∼30 mg of tissue using the DNeasy Blood and Tissue Kit  according to the manufacturer's instructions. DNA quality and integrity were assessed with a Nanodrop 2000 photometer  and through visual inspection on a 2% agarose gel. Exact quantity was determined using the Quant-iT PicoGreen dsDNA Assay Kit . Total RNA was isolated by incubating 30 mg of tissue with 1 mL TRIzol (Invitrogen) in a 2 -mL tube for 1 hour. Further tissue disruption and homogenization was performed in three 1-minute cycles with grinding beads using a TissueLyser II (Qiagen). RNA was purified from the homogenized sample with Direct-Zol RNA minipreps  following the manufacturer's protocol. Quantity and quality were assessed using a RNA Pico chip on a Bioanalyzer 2100 .In order to determine the taxonomic complexity of the hologenome, high-throughput 16S rRNA gene amplicon sequencing was performed as described by Maus et al. . Primersde novo metagenome assembly was performed using the Ray Meta assembler v.2.3.0 [k-mer size of 41 and default settings. All processed raw reads were aligned to the assembled metagenome contigs using Bowtie v.2.2.4 [P. rus draft genome was done with GeneMark v.4.3.2. [To obtain hologenome sequence data, a whole-genome-shotgun PCR-free  and 10-kb mate-pair libraries  were generated based on the manufacturer's protocols. Both libraries were sequenced in two HiSeq 1500 rapid runs in paired-end mode (2 × 250 bp). After sequencing and processing of the raw data with Trimmomatic v.0.32  and an i v.2.3.0  with a k v.2.2.4 . The res v.2.2.4 . To sort v.2.2.4  was usedv.4.3.2. . The funv.4.3.2. . To assev.4.3.2. ) to the v.4.3.2. .P. rus genome assembly using tophat v.2.1.1 [P. rus genome. The sequence read analysis platform ReadXplorer v.2.2.3. [single best match option for each library. In addition, gene prediction results were confirmed with ReadXplorer. The RNA-seq and the draft genome data are available on the EBI Annotare server  and in European Nucleotide Archive , respectively.In total, 2.25 μg of RNA were used for library preparation with the TruSeq Stranded mRNA Sample Preparation Kit (Illumina). Sequencing of the complementary DNA library was carried out on the Illumina HiSeq 1500 platform following a modified protocol of Verwaaijen et al. . The sam v.2.1.1 . Two misv.2.2.3.  was usedP. rus genome was estimated through short-sequence substring (k-mers) frequencies of the reads used for the assembly. Different k-mer sizes were implemented, ranging from 17 to 25 bp. Their k-mer count distributions were calculated with Jellyfish v.2.0 [P. rus [k-mers obtained. The second approach implemented the heterozygous mode of the gce program v.1.0 [k-mer distribution into account. The third approach utilized the GenomeScope web interface tool v.1.0. [k-mer frequency profiles. In addition, the latter approach measures the relative abundances of heterozygous, homozygous, unique, and two-copy sequences. The performance of these three approaches was evaluated based on paired-end reads from the two genomic datasets for which flow cytometry estimations are available , Ciona intestinalis (KH), Ciona savignyi (CSAV 2.0), Drosophila melanogaster (BDGP6), and Saccharomyces cerevisiae (R64–1-1).Standard statistics for assessing the assembly's contiguity were calculated using QUAST v. 4.4  and the l script . Completl script  in the gl script ) using Bl script ). Finalll script  and usinl script  for CaenN. vectensis v.1.0 [A. digitifera v.1.1 [E. pallida v.1.1 [sensu Bradnam et al. [In order to assess the quality of our hologenome binning-based assembly, we compared it to literature data from the symbiont-free-based genome assemblies of is v.1.0 , A. digira v.1.1 , 75, andda v.1.1   were caGIGA-D-18-00072.pdfClick here for additional data file.Response_to_Reviewer_Comments_Original_Submission.pdfClick here for additional data file.Reviewer_1_Report_ -- Beate Slaby04/19/2018 ReviewedClick here for additional data file.Reviewer_2_Report_ -- Thomas Hackl04/20/2018 ReviewedClick here for additional data file.Additional FilesClick here for additional data file.


Dactylis glomerata L., Festuca rubra L. and Lolium perenne L.) over two consecutive years. Our results showed that the management practices influenced fungal communities in the plant holobiont, but observed effects differed between grass species and sampling year. Phylogenetic diversity of fungal endophytes in D. glomerata was significantly affected by mowing frequency in 2010, whereas fertilizer application and the interaction of fertilization with mowing frequency had a significant impact on community composition of L. perenne in 2010 and 2011, respectively. Taken together, our research provides a basis for future studies on responses of fungal endophytes towards management practices. To the best of our knowledge, this is the first study simultaneously assessing fungal endophyte communities in aerial parts of three agriculturally important grass species over two consecutive years.Despite the importance of endophytic fungi for plant health, it remains unclear how these fungi are influenced by grassland management practices. Here, we investigated the effect of fertilizer application and mowing frequency on fungal endophyte communities and their life strategies in aerial tissues of three agriculturally important grass species ( Endophytic fungi have been found in every plant species investigated so far ,2,3. In Several endophytic fungi play important roles in plant health by promoting plant growth and providing resistance to multiple stresses, such as drought or nutrient deficiency ,8,9. SomLolium perenne L. were not affected by land-use intensity , though these assemblages did differ among study regions and season [Recent research has shown how management can influence foliar fungal assemblages. For instance, Pancher et al.  showed td season .Zea mays L. using denaturing gel gradient electrophoresis (DGGE). On the contrary, nitrogen addition had an impact on the overall community composition of fungi associated with shoots of three boreal moss species [Seghers et al.  found no species . As most species ,15 or fu species ,18,19, iDactylis glomerata L., Festuca rubra L. and L. perenne) over two consecutive years were investigated by DGGE and pyrotag sequencing [D. glomerata and L. perenne have a higher tolerance against mowing compared to F. rubra, while L. perenne has a higher tolerance against higher nitrogen supply than the other two species [Neotyphodium species was investigated [In two previous studies, the effect of grassland management practices on bacterial endophyte communities in aerial parts of three grass species  of the endophytic fungi found were determined using the FUNGuild database [In the present study, we used Illumina-based sequencing of the internal transcribed spacer region 2 (ITS2) region to assess the effect of management practices  on fungal endophyte communities in aerial parts of database . This isWe focused on three main hypotheses: (i) fungal endophyte diversity and community composition differ among the investigated grass species and between the sampling years, (ii) fungal endophyte communities in the grass species respond differently towards management practices and (iii) the putative life strategies of fungal endophytes are associated with grass species and management practices. To our knowledge, this is the first study simultaneously assessing fungal endophyte communities in aerial parts of three agriculturally important grass species over two consecutive years.The Grassland Management Experiment (GrassMan) was a long-term field experiment to study the effect of grassland management practices and intensity on biodiversity. It was established in spring 2008 at a semi-natural, moderately species-rich, temperate grassland site at the Relliehausen Experimental Farm in the Solling Mountains in Lower Saxony, Germany . The full-factorial design of GrassMan included three levels of plant species richness  as well as two management practices. The plant species richness gradient was achieved by selective herbicide application which either reduced dicot or monocot species diversity . One thManagement practices for the three plant species richness levels included two mowing frequencies  and two fertilization treatments (fertilizer application with nitrogen/phosphorous/potassium (NPK) vs no fertilizer application). The combination of these management practices resulted in four different management levels: (1) no fertilizer application and mown once (NPK- 1x), (2) no fertilizer application and mown three times (NPK- 3x), (3) fertilizer application and mown once (NPK+ 1x), and (4) fertilizer application and mown three times (NPK+ 3x). Each treatment was replicated six times on 15 × 15 m plots, resulting in 72 plots. The plots were arranged in a Latin Square design with six rows and six blocks. Please see Petersen et al.  for a deL. perenne, F. rubra and D. glomerata were randomly collected from each management level and grass species from dicot-reduced plots on 19 September 2010 and the 12 September 2011. Only plants from dicot-reduced plots were sampled due to the higher abundances of the target grass species on these plots. The plants were collected with at least 1 m distance from each other. Ten plants per grass species and plot were sampled, with one exception: L. perenne samples were collected only from two non-fertilized and three fertilized plots mown once in 2010 due to the absence of this grass species in the other plots. Only plants without obvious disease symptoms, such as chlorosis, leaf spots or pathogen-induced lesions, were collected. Plant samples were immediately cooled down to 4 °C and transferred to the laboratory. During the study period, precipitation and mean temperature were 93.6 mm and 11.42 °C in September 2010 and 54.75 mm and 14.75 °C in September 2011, respectively.Plant samples were taken from the GrassMan experimental field as described previously . In brieμl aliquots of the water used in the final washing step were plated on common laboratory media plates. The plates were incubated in the dark at 25 °C for at least 2 weeks. No growth of microorganisms was observed. As a second control, water from the final washing step was subjected to polymerase chain reaction (PCR) targeting the ITS region. No amplification was detected. The surface-sterilized plant material was ground to a fine powder in liquid nitrogen using an autoclaved mortar and pestle. Aliquots of the ground tissue were subsequently stored at −20 °C until DNA extraction. DNA was extracted employing the peqGOLD Plant DNA Mini kit  according to the manufacturer’s instructions with two modifications as described previously [Surface sterilization of collected aerial plant material was performed as described in Wemheuer et al. . To confTCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGATGAAGAACGYAGYRAA-3′) and MiSeq-ITS4 (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTCCTCCGCTTATTGATATGC-3′). Negative controls in both PCRs were performed using the PCR reaction mixture without template.Fungal communities in aerial plant parts were assessed by nested PCR targeting the ITS region using the same template DNA as in two previous studies investigating bacterial endophytes ,21. The L. perenne samples from non-fertilized plots mown once in 2010. In total, 71  samples were analyzed in this study  and pooled in equal amounts. Quantification of the purified PCR products was performed using the Quant-iT dsDNA HS assay kit and a Qubit fluorometer (Thermo Scientific) as recommended by the manufacturer. The best three of the six replicates with the strongest specific PCR signal and highest DNA concentrations were chosen for sequencing with one exception: as described above, we had only two is study . Quantifdenovo using the UCHIME algorithm during clustering [Sequencing data were initially quality filtered with the Trimmomatic tool version 0.36 . Low-quaustering . Subsequustering  in referustering . To assign taxonomy, unique and chimera-free sequences were classified by BLAST alignment  against P ≤ 0.05 and P ≤ 0.1, respectively. Differences in sequencing depth among management practices, sampling year and grass species were tested using a Kruskal Wallis test. There were no significant differences in library size; and thus, data were not normalized prior to further analysis . OTU tables were rarefied to 5741 fungal sequences per sample prior to alpha diversity analysis using the rrarefy function in vegan 2.5-2 [vegan [picante version 1.7 [drc version 3.0-1 [rarecurve function in vegan [MM2 model within the drc package [All analyses were conducted in R version 3.5.0 . Differean 2.5-2 . Alpha d2 [vegan , picantesion 1.7  and drc on 3.0-1 . To calcin vegan . The Mic package . All alpshapiro function) and for homogeneity of variance using a Levene test (leveneTest function) via the R package car version 3.0-0 [The impact of grass species and sampling year on fungal richness and diversity was tested using a repeated measures analysis of variance (ANOVA) with plot as error term . The infon 3.0-0 . Becausevegdist and adonis function within the vegan package [metaMDS function in the vegan package [p-values in PERMANOVA, we tested for differences in group homogeneity using permutational analysis of multivariate dispersions (PERMDISP) with 999 permutations using the function betadisper in the R package vegan [P value adjustment for multiple comparison. Differences in community composition between the management intensities and between the grass species were tested using pairwise PERMANOVA .Potential differences in community structure were investigated by permutational multivariate analysis of variance (PERMANOVA) with 1000 random permutations using the  package . Four di package . In casege vegan , followemultipatt function from the indicspecies package [multipatt function analyses the association between species patterns and combinations of groups of sites, which provides an extra flexibility to qualitatively model the habitat preferences of the species of interest [R) with a particular management practice or grass species were corrected for unequal sample size using the function r.g [To identify zOTUs that are significantly associated with each grass species and the four management levels, multipattern analyses were applied with  package . The mulinterest . The bistion r.g . The poition r.g . Accordition r.g , the poiCytoscape version 3.6.1 [t-test with P value adjustment (Benjamini-Hochberg) for multiple comparisons. To enhance reliability of the indicator analysis, only fungal zOTUs found in at least three samples were considered. All zOTUs present in all samples of a particular grass species were defined as core microbiome. In addition, we identified zOTUs found in all 71 samples. The core community and the fungal zOTUs associated with each grass species as well as with management practices and grass species were visualized using on 3.6.1 . The rest-test with Benjamini-Hochberg correction for multiple testing.Functional information was assigned to fungal zOTUs using FUNGuild . SamplesSequence data are deposited in the Sequence Read Archive (SRA) of the National Center for Biotechnology Information (NCBI) under the accession number SRA750152.D. glomerata, F. rubra and L perenne from the GrassMan Experimental Field over two consecutive years. Diversity and composition of fungal endophyte communities in 71 grass samples were assessed by Illumina-based sequencing targeting the fungal ITS2 region  and Cladosporium (11.3%) were predominant. Other abundant fungal genera were, for example, Candida (6.6%), Epicoccum (5.0%), Pyrenochaetopsis (2.5%), Vishniacozyma (2.2%), Penicillium (2.2%), and Chrysosporium (1.8%). However, we detected differences in the relative abundances between the two sampling years. In 2010, Cladosporium was the predominant fungal genus in L. perenne (16.73%) and D. glomerata (26.78%), while Talaromyces dominated the fungal community in F. rubra in 2010 (12.67%) and 2011 (13.25%). Talaromyces (14.03%) and Pyrenochaetopsis (7.44%) were the two predominant fungal genera in L. perenne in 2011, while Talaromyces (19.03%) and Candida (8.46%) dominated the endophyte community of D. glomerata.Fungi were mainly represented by two phyla: Ascomycota (65.5%) and Basidiomycota (27.7%) . The preF. rubra and D. glomerata had the highest and the lowest alpha diversity values, respectively (P = 0.0638). Sampling year significantly affected fungal diversity , richness , and marginally the phylogenetic diversity . The management practices and their interaction had no effect on the alpha diversity measures.To address our hypothesis (i) that fungal endophyte diversity and community composition would differ between the grass species and the two sampling years, we compared fungal diversity (represented by the Shannon diversity index H’), richness (number of observed zOTUs) and phylogenetic diversity (represented by Faith’s PD). In general, ectively . HoweverP = 0.04). Sampling year also significantly affected endophytic community composition (P = 0.03). However, both findings were only true when employing Bray-Curtis dissimilarities. Grass species and sampling year explained 4.48% and 2.82% (Bray-Curtis dissimilarities) and 3.82% and 2.05% (weighted UniFrac dissimilarities) of the variance in the dataset, respectively. We did not observe any direct influence of mowing frequency on fungal community composition in both sampling years  and the interaction of mowing frequency with fertilization (P = 0.07) had a marginally significant impact on fungal community composition in 2010, but only when using Bray-Curtis dissimilarities. In 2011, we observed a significant effect of fertilizer application and/or the interaction of the two management practices when employing Bray-Curtis and weighted UniFrac dissimilarities. The interaction of mowing frequency with fertilization explained 12.16% or 14.29% (Bray-Curtis dissimilarities) and 10.49% or 18.26% (weighted UniFrac dissimilarities) of the variance in the dataset in 2010 and 2011, respectively.Nonetheless, fertilizer application  higher abundances of Myrmecridium were recorded in F. rubra (1.29%) compared to D. glomerata (0.27%) and L. perenne (0.18%) in 2010. In addition, Pyrenochaetopsis had higher abundances in 2010 samples of F. rubra (2.51%) than in D. glomerata (0.64%) and L. perenne (0.98%). However, this was only marginally significant (P ≤ 0.1). In contrast, significantly (P = 0.04) higher abundances of this genus were detected in L. perenne (7.44%) compared to D. glomerata (2.10%) and F. rubra (1.28%) in 2011.Most of the predominant genera including ng years . MarginaD. glomerata and F. rubra, respectively. Specifically, two zOTUs affiliated with Rhynchosporium orthosporum were significantly associated with D. glomerata (average relative abundance in this grass species: 0.52%), whereas zOTUs belonging to Rodwayella citrinula (0.85%) and Pyrenochaetopsis sp. (1.35%) showed a unique association with L. perenne. In addition, several zOTUs affiliated to Blumeria graminis (0.63%), Phragmocephala garethjonesii (0.18%), Phomatospora dinemasporium, (0.18%) and Ascochyta sorghi (2.57%) were significantly associated with F. rubra.Additionally, we performed an indicator species analysis to identify fungal taxa responsible for the observed differences among the grass species . The lowC. cladosporioides, Candida subhashii, Cuniculitrema polymorpha and Talaromyces sp.  was detected in L. perenne plants collected in 2010 and 2011. Four and two zOTUs were found in F. rubra and D. glomerata, respectively.We were also interested in the core communities of each grass species and if there are fungal zOTUs found in all 71 samples. In total, 16 zOTUs  were identified as core community observed in one, two or three grass species in both sampling years. Although the zOTU number was low, more than 30% of all sequences were assigned to these 16 zOTUs. These zOTUs belonged to yces sp. . Only a yces sp. . The twoD. glomerata grown on plots mown three times compared to those mown once (P = 0.04) in 2011. In addition, there was a marginally significant effect of management (P = 0.09) on the phylogenetic diversity of endophytes in D. glomerata in 2011, but not in 2010. Fertilization had a marginally significant impact on fungal richness of D. glomerata (P = 0.08) and F. rubra (P = 0.08) in 2011, while fungal richness of L. perenne was not affected (P = 0.52).We further expected that fungal endophyte communities in the grass species respond differently towards management practices (hypothesis ii). In general, management practices had only limited effects on alpha diversity measures. Nonetheless, a significantly higher phylogenetic diversity of endophytes was observed in P = 0.11; weighted UniFrac: P = 0.22) and 34%  of the variance in the dataset in 2010 and 2011, respectively. To identify the impact of management practices on fungal community composition in each grass species for each sampling year, we performed non-metric multidimensional scaling (nMDS) analysis for the six subsets. We observed no consistent pattern among the management levels  and the interaction of fertilizer application with mowing frequency (P = 0.04) had a significant effect on the fungal community composition of L. perenne in 2011 and 2010, respectively, but only when using Bray-Curtis dissimilarities. In addition, the fungal community composition of L. perenne was marginally affected by mowing frequency in 2010  and by fertilizer application in both sampling years . Although mowing frequency alone did not result in any significant impact on community composition in L. perenne in both sampling years, the interaction of this management practice with fertilizer application explained 41.51% or 36.72% (Bray-Curtis dissimilarities) and 41.07% or 38.61% (weighted UniFrac dissimilarities) of the variance in 2010 and 2011, respectively.Conversely, fertilizer application  and 2011 (P < 0.05). Moreover, significantly (P < 0.002) higher abundances of Penicillium were observed in L. perenne samples grown on fertilized plots mown once in 2010. The genera Alternaria and Cladosporium had significantly (P < 0.05) higher abundances in non-fertilized plots mown three times compared to plants collected from fertilized plots 2011. As we observed contrasting effects of management practices on the grass species, we next compared the relative abundance of the predominant fungal genera between the four management levels for each grass species in 2010 and 2011 . NumerouP < 0.004) higher abundances of Vishniacozyma were observed in L. perenne samples grown on plots mown three times compared to plants from plots mown once. In addition, we detected higher abundances of Phragmocephala in F. rubra grown on fertilized plots mown three times compared to all other plots in 2010. However, this was only marginally significant (P = 0.06). In 2011, significantly (P < 0.03) higher abundances of Talaromyces were observed in F. rubra grown on fertilized plots mown once compared to non-fertilized plots. In contrast, the relative abundances of the predominant fungal genera in D. glomerata plants were not affected by management level in both sampling years.In the same year, significantly  and L. perenne (n = 45), respectively  was significantly affiliated with D. glomerata grown on fertilised plots mown once and three times. We further identified one zOTU belonging to A. sorghi and one zOTU affiliated with R orthosporum, which showed unique associations with F. rubra grown on non-fertilized plots mown once and with D. glomerata grown on fertilized plots mown once a year, respectively. In contrast, two zOTUs affiliated with B. graminis and two zOTUs belonging to P garethjonesii were associated with F. rubra grown on fertilized plots mown three times.Only four fungal zOTUs were significantly associated with We hypothesized that the putative life strategies of fungal endophytes are associated with grass species and management practices (hypothesis iii). To test this hypothesis, life strategies  of endophytic fungi under different management levels were determined using FUNGuild (20). In total, highly-probable and probable life strategies for 1009 of the 3230 zOTUs (= 31.2%) were predicted . The curP = 0.08) between D. glomerata and in F. rubra in 2010, but not in 2011  in plants from fertilized plots mown three times compared to those from non-fertilized plots mown once in 2010. The life strategies of the fungal endophytes did not differ in their sequence abundance between the grass species in 2010 and 2011 . Neverth in 2011 B. The efP = 0.052) in F. rubra grown on fertilized plots mown once compared to plants from non-fertilized plots mown once and from fertilized plots mown three times in 2011. In the same year, a marginally significant (P = 0.052) increase in the relative abundance (sequence richness) of saprotroph-symbiotrophs was observed in F. rubra plants from fertilized plots mown once. Moreover, we detected a marginally significant (P = 0.051) higher abundance of saprotrophs in L. perenne from fertilized plots mown once compared to plants collected from non-fertilized plots mown three times in 2011. We further detected a marginally significant higher fungal zOTU richness of saprotrophs (F. rubra and L. perenne plants between the management levels in 2010 and in D. glomerata for both sampling years (P = 0.03) lower richness of zOTUs assigned as pathotrophs were detected in F. rubra grown on fertilized plots mown once than in fertilized plots mown three times and in those from non-fertilized plots mown once in 2011. In the same year, we observed a significantly (P = 0.02) higher sequence richness of pathotrops in L. perenne grown on non-fertilized plots mown three times than from fertilized plots mown once. However, no differences were detected when investigating the sequence richness.Interestingly, we detected no differences in the relative abundance of pathotrophs in ng years . NonetheOverall, we found different responses of fungal endophytes towards fertilizer application and mowing frequency . In addiL. perenne, D. glomerata and F. rubra over consecutive years. To our knowledge, this is the first study using culture-independent approaches to assess the impact of two management regimes on three agricultural-important grass species.Fungal endophytes can play an important role for plant growth and health ,8,10. HoTalaromyces and Cladosporium. Members of the genus Talaromyces are well-known for its secondary metabolites, with some having antimicrobial activities [Cladosporium was mainly represented by zOTUs of the species C. cladosporioides and C. herbarum. Both species are common, widely distributed fungi, which can be found on dead leaves of numerous plant species or as secondary invader of necrotic plant tissues [C. cladosporioides have antifungal activity against important plant pathogens of the genera Colletotrichum and Phomopsis. In line with previous work on fungal endophytes in grass and other plant species ,48,49, ftivities ,51. The  tissues ,53. Wang tissues  showed tCryptococcus, Rhizosphaera, and Mycopappus dominated fungal endophyte communities associated with four vascular plant species [Goidanichiella and Candida dominated the aerial plant parts [The majority of fungal species in this study belonged to Class 3 endophytes, rather than to well-studied clavicipitaceous endophytes . In con species . In anotnt parts . We postnt parts ,48,56. Agave species [Acer species [Triticum dicoccoides and Aegilops sharonensis [Fungal alpha diversity measures were not affected by grass species, which is not consistent with previous observations on foliar fungal endophytes of crop plants  or tree  species , in leav species  and in sronensis , respectronensis  found noR. orthosporum was significantly associated with D. glomerata, which is known as host grass species of this fungus [B. graminis and A. sorghi were significantly associated with F. rubra. The fungus B. graminis includes important, host-specific fungal pathogens of various grasses and cereals of the family Poaceae [A. sorghi causes leaf spots on numerous grasses and cereals [T. aestivum L. and observed that the most common endophytes in these leaves were wheat pathogens. The pathogen s fungus , while B Poaceae , whereas cereals . As we c cereals ,63,64. FL. perenne grown under different land-use intensities  by Illumina-based sequencing and found no effect of land-use intensity on the composition of fungal assemblages. This is partly in line with our study, as the interaction of fertilizer application with mowing frequency had a significant effect on fungal community composition in L. perenne in 2010, but not in 2011. A possible explanation for the contrasting finding is that König and colleagues [Our study further demonstrated grass species-specific responses of fungal endophytes towards management practices. Previous studies investigating the effect of agricultural practices on foliar fungal communities had contrasting findings ,18,19,47lleagues  analysedD. glomerata plants grown on plots mown three times in 2011. We suggest that frequent mowing promote the growth of distantly related, horizontal transmitted endophytic fungi  in aerilleagues , niche dC. subhashii or several Talaromyces species, which were significantly associated with one grass species and one management level. For example, Hilber-Bodmer et al. [C. subhashii strongly antagonized a wide range of filamentous fungi. In addition, we identified several pathogenic fungi such as R. orthosporum, B graminis and A. sorghi [A possible explanation for the contrasting findings in our and the above-mentioned studies is that fungal endophytes from different grass species and/or genotypes from different regions were investigated, suggesting that the responses of these communities towards management practices might differ. Another explanation is that the grass species select for beneficial microorganisms as they provide a selective advantage for their host plant to better adapt to environmental constraints as suggested by Vandenkoornhuyse and colleagues . These ar et al.  showed r. sorghi ,61,62. H. sorghi ,68. In aC. cladosporioides were present in all plant samples. These findings suggest that these fungi are seed-borne. This is supported by a previous study on seed fungal endophytes of the coastal plant Suaeda salsa assessed by pyrotag-based sequencing [C. cladosporioides significantly promoted host seed germination and plant growth, which might play a role in the present study. The authors stated that the transmission mode of C. cladosporioides in S. salsa remains elusive. In a previous study on endophytes from six forb species, the two endophyte species Alternaria alternata and C. sphaerospermum were vertically transmitted [Neotyphodium [Interestingly, we found only a few zOTUs as core community in one or two of the grass species investigated. Moreover, two abundant zOTUs belonging to quencing . Here, tnsmitted . Verticansmitted ,72 and byphodium ,73 is weIn the present study, the effects of grassland management practices on endophyte communities differed not only among the grass species, but also between the sampling years. This is in line with previous studies showing that sampling time influenced fungal endophyte communities ,59. We sWe observed differences between the two dissimilarities used, as significant effects were mainly detected when using Bray-Curtis dissimilarities. Bray-Curtis dissimilarities are taxonomy-independent, whereas UniFrac dissimilarities incorporate the phylogenetic relationships between zOTUs. Our results indicate that fungal endophyte communities were taxonomically related to each other, i.e., different but closely related fungal species colonized the grass species in the two sampling years, and that closely related taxa are potentially ecologically equivalent. Moreover, these results highlight that the use of two different dissimilarities for beta diversity analysis can give an additional insight into fungal community composition. We applied FUNGuild  to betteContrary to changes in community composition, the ecological life strategies did not differ among the grass species. However, we observed differences when analysing the sequence richness  and zOTU richness (the proportion of zOTUs assigned to a specific trophic mode), supporting the suggestion of Nguyen et al.  that comIn the current study, we showed that sampling year and grass species had a significant impact on fungal endophyte community composition. In addition, sampling year significantly affected fungal diversity and richness but not the phylogenetic diversity. These results are only partly in line with our first hypothesis that fungal endophyte diversity and community composition differ among the investigated grass species and between the sampling years. We further observed grass species-specific responses of fungal endophytes towards management regimes, which supports our second hypothesis. However, the interaction of grass species and management practices explained only 36% and 34% of the variance in the dataset in 2010 and 2011, respectively, indicating that other factors than those investigated are important drivers of fungal endophyte communities in the grass species analysed in this study. Contrary to our third hypothesis that putative life strategies of fungal endophytes are determined by grass species and management practices, grass species had no effect on the fungal life strategies. However, we observed grass species-specific responses of the fungal life strategies towards management practices, which were not consistent in both sampling years. We found a few zOTUs, which were present in all plant samples, suggesting that these fungi were vertically transmitted. We suggest that future studies should include endophytes from seeds and grass seedlings to identify vertically transmitted fungi. In addition, the number of grass species should be extended to get a more comprehensive picture on how fungal endophytes respond to different management practices. Nonetheless, our results provide a basis for further studies on responses of fungal endophytes and their life strategies in agriculturally important grass species towards management practices.


Kerstersia gyiorum, a potential etiological agent of clinical infections, was isolated from several human patients presenting clinical symptoms. Its significance as a possible pathogen has been previously overlooked as no disease has thus far been definitively associated with this bacterium. To better understand how the organism contributes to the infectious disease, we determined the complete genomic sequence of K. gyiorum SWMUKG01, the first clinical isolate from southwest China.The Gram-negative bacterium K. gyiorum. Phylogenomic comparisons with Alcaligenaceae species indicated that K. gyiorum SWMUKG01 had a close evolutionary relationships with Alcaligenes aquatilis and Alcaligenes faecalis.The genomic data obtained displayed a single circular chromosome of 3, 945, 801 base pairs in length, which contains 3, 441 protein-coding genes, 55 tRNA genes and 9 rRNA genes. Analysis on the full spectrum of protein coding genes for cellular structures, two-component regulatory systems and iron uptake pathways that may be important for the success of the bacterial survival, colonization and establishment in the host conferred new insights into the virulence characteristics of K. gyiorum, which is expected to provide useful information for subsequent studies on pathogenesis of this species.The comprehensive analysis presented in this work determinates for the first time a complete genome sequence of  Kerstersia gyiorum is a Gram-negative coccobacillus that is occasionally isolated from clinical samples of human infections. On nutrient agar, colonies of K. gyiorum are usually characterized by spreading edge morphology, exhibiting flat or slightly convex with smooth margins [et al., since the organism was primarily isolated from lower-extremity wounds. The novel genus Kerstersia, initially described by Coenye et al., is grouped in the family Alcaligenaceae along with Alcaligenes, Achromobacter, Bordetella, and Pigmentiphaga spp. [Kerstersia members resemble Alcaligenes faecalis phenotypically, except that isolates of Kerstersia are oxidase-negative and do not produce a fruity odor. margins –3. The waga spp. . KersterK. gyiorum by Coenye et al. in 2003 reported six isolates recovered from leg wounds, sputum, and feces. After 2012, case reports documenting Kerstersia spp. infection began to emerge again. Since then, there have been one case of infection with a second species of Kerstersia, Kerstersia similis [K. gyiorum [K. gyiorum was also reported to be isolated from patients with chronic tracheostomy [K. gyiorum from other microorganisms using conventional methods, such as traditional biochemical tests and automated identification systems, which may lead to K. gyiorum being identified incorrectly or unsuccessfully in the past in most clinical laboratories [K. gyiorum may therefore be overlooked. For the current, with the development of (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) MALDI–TOF MS and 16S rRNA gene sequencing, K. gyiorum is expected to be identified in laboratories more accurately.The initial publication describing  similis  and 12 p gyiorum –13. Amon gyiorum , 11, 13, gyiorum , 10. Addheostomy . In factK. gyiorum from specimens in most cases [K. gyiorum has an affinity towards causing chronic mixed infections in patients [K. gyiorum because of a lack of pathogenesis-related information on this organism in the literature. Reported cases of human Kerstersia infections showed that the patients gained remission when treated with an antimicrobial agent to which this organism was susceptible [K. gyiorum could contribute significantly as a possible aetiology. To better understand how the organism contributes to infections, virulence factors and pathogenic mechanisms of K. gyiorum need to be investigated. Whole genome sequencing represents a valuable approach in an in-depth exploration of potential virulence factors and may answer important questions concerning the evolution of this bacterium [K. gyiorum was announced by Greninger et al [K. gyiorum SWMUKG01, which was recovered from the sputum of a patient with respiratory infection. We reported and analyzed the complete genome sequence of K. gyiorum SWMUKG01, accompanied by a detailed annotation of its genome organization and expression strategy, with an emphasis on the investigation of genes and operons related to potential virulence factors.As previous reports indicated, other bacterial species were co-isolated with st cases , 3, 6, 9ceptible , 8, 9. Tacterium . Though er et al , a comprK. gyiorum strain SWMUKG01 was recovered from the sputum of a 70-year-old female patient of respiratory infection with a history of tracheotomy and epilepsy in May, 2018 from the affiliated hospital of Southwest Medical University. The isolate SWMUKG01 was identified as K. gyiorum by MALDI-TOF MS and 16S rRNA gene sequencing. For genomic DNA extraction, one colony of K. gyiorum SWMUKG01 was transferred into 5 ml Tryptic Soy Broth  and cultured at 37°C. After an overnight incubation, the culture was diluted 1:100 into fresh TSB for sub-cultivation until mid-exponential growth phase was reached. The bacterial cells were collected by centrifugation at 5000 g for 10 min.The K. gyiorum SWMUKG01 was extracted using Rapid Bacterial Genomic DNA Isolation Kit  according to the manufacturer’s protocol. The genomic DNA was sent to Sangon Biotech  for de novo whole genome sequencing. A combination of HiSeq 2500 Sequencer  and the PacBio RSII platforms  was employed for whole genome sequencing. De novo genome assembly of filtered reads was conducted using the Hierarchical Genome Assembly Process workflow  [Total genomic DNA of ciences) .K. gyiorum SWMUKG01 complete genome with Prokka [E < 1e-5) [http://www.pathogenomics.sfu.ca/islandviewer/) [Protein coding sequences (CDSs), tRNAs and rRNAs were predicted in h Prokka . BLAST [h Prokka  was emplh Prokka , 20, Kyoh Prokka , SwissPrh Prokka , NR, andh Prokka . The genh Prokka . BLAST wh Prokka , and the < 1e-5) . Genomicviewer/) .K. gyiorum SWMUKG01 were obtained from the NCBI including K. gyiorum CG1 (NZ_LBNE00000000.1), A. faecalis ZD02 (NZ_CP013119.1), Alcaligenes aquatilis BU33N (NZ_CP022390.1), Achromobacter xylosoxidans NCTC10807 (NZ_LN831029.1), Achromobacter denitrificans USDA-ARS-USMARC-56712 (NZ_CP013923.1), Achromobacter insolitus FDAARGOS_88 (NZ_CP026973.1), Bordetella bronchiseptica 253 (NC_019382.1), Bordetella pertussis Tohama I (NC_002929.2), Bordetella holmesii ATCC 51541 (NZ_CP007494.1), Bordetella hinzii ASM107827v1 (NZ_CP012076.1), Bordetella petrii ASM6720v1 (NC_010170.1), Bordetella parapertussis Bpp5 (NC_018828.1), Bordetella avium 197N (NC_010645.1), Bordetella trematum H044680328 (NZ_LT546645.1), and Bordetella pseudohinzii HI4681 (NZ_CP016440.1). A phylogenetic tree based on the 16S rRNA gene sequences was generated using the tool FastTree [K. gyiorum SWMUKG01 and selected strains were compared using Pan-Genomes Analysis Pipeline (PGAP) [http://jspecies.ribohost.com/jspeciesws/#analyse).For phylogenetic and comparative analysis, the genome sequences of the closest genetic relatives of 5 NC_0188.1, BordeNZ_LN8310.1, AchroNZ_LN8310.1, AchroK. gyiorum SWMUKG01 were deposited in GenBank with the accession number CP033936. Raw sequencing data were deposited under a BioProject with accession number PRJNA497911.All relevant data are within the paper and its Supporting Information files. The genome sequence data of The current study was approved by the Ethics Committee of Southwest Medical University . Written informed consent was exempted, since this retrospective study mainly focused on bacteria and patient intervention was not required.Kerstersia gyiorum SWMUKG01 is composed of 3, 945, 801 base pairs (bps) with a single circular chromosome  of SWMUKG01 chromosome was identified to be located from 3, 914, 889 to 3, 915, 821 bp, with the web-based system Ori-Finder [K. gyiorum), ZD02 , BU33N (A. aquatilis), NCTC10807 (A. xylosoxidans), FDAARGOS_88 (A. insolitus), 253 (B. bronchiseptica), Tohama I (B. pertussis), and Bpp5 (B. parapertussis)  .K. gyiorum SWMUKG01. Of the 3441 protein-coding genes in SWMUKG01, 2801 were categorized into 23 COG functional codes   , both of species , based os tested . The str species .K. gyiorum. The genomic characteristics of the specific pathogenesis/virulence factors are described in detail below:326 potential virulence factors were annotated in strain SWMUKG01 genome and these proteins fell into 134 VF terms. Among all these potential virulence factors, proteins involved in flagella and pili production, biosynthesis of lipopolysaccharide and capsule, iron acquisition, secretion and efflux pump systems as well as two-component systems were included . FurtherAlcaligenaceae, flagella have been widely identified in the genera of Alcaligenes, Achromobacter and Pigmentiphaga [Bordetella, the flagellar operons of both B. pertussis and B. parapertussis are inactivated, leading to inability to make flagella [Kerstersia, K. similis was reported to have no motility due to lacking flagella [K. gyiorum SWMUKG01 in this study  in SWMUKG01, approximately 52 kb, encodes proteins involved in flagella biosynthesis, export, motor and bacterial chemotaxis  genes encode the machinery that is essential for the assembly of adhesive Flp  pili, which are required for autoaggregation, colonization, biofilm formation and pathogenesis in the genera Actinobacillus, Haemophilus, Pseudomonas, Yersinia and perhaps others [tad gene cluster tadZABCD (PROKKA_01524–01528) in the strain SWMUKG01  that is predicted to be involved in the biosynthesis and secretion of Flp pili [flp and tadV of the cluster were missing in CG1. By a further search in the SWMUKG01 genome, we found another incomplete subset of tad operon, tadE-orf-rcpCA-tadABC (PROKKA_01139–01145) , pilF (PROKKA_00008), pilD (PROKKA_00160) and pilP (PROKKA_02422). However, these CDs seem to be incomplete for the type IVa pili production, as the gene coding for the main pilus subunit pilin (PilA), a central component, was not found [Furthermore, we found that the Flp pili  (Table 29–01145) . Due to IVb pili . To explot found . BesidesSurface polysaccharides are extremely diverse and occur in multiple forms in Gram-negative bacteria, such as lipopolysaccharides (LPSs), the essential components of the outer membrane structurally and functionally, and capsular polysaccharides, surface layers or capsules that are associated with the cell .K. gyiorum showing no less than 94% identities with those in CG1. These CDSs are scattered throughout the SWMUKG01 chromosome, just like most Gram-negative bacteria, such as E. coli, Neisseria meningitidis, Y. pestis and Pseudomonas aeruginosa. The majority of genes essential for the synthesis of lipid A  and KDO core oligosaccharide  are highly conserved among different species within the family Alcaligenaceae. However, kdsB in K. gyiorum SWMUKG01, encoding CMP-2-keto-3-deoxyoctulosonic acid synthetase involved in lipid A synthesis, showed little similarity with that in other Alcaligenaceae members, but shared 71% sequence identity in Pseudomonas monteilii and Pseudomonas citronellolis. lpsE, encoding glycosyltransferase in core OS biosynthesis, showed 67% identity (98% sequence coverage) with that in Stenotrophomonas rhizophila but only 23% coverage in B. pertussis. The gene msbA (PROKKA_01899) is also identified encoding a putative lipid A export ATP-binding/permease protein (583aa) that is required for the fiipping of lipid A / core moiety of LPS from the cytoplasmic side of the IM to the periplasmic face.All genes encoding enzymes for biosynthesis of the lipid A and core OS in the SWMUKG01 genome were identified , which arfb operon , glycosyltransferase genes wcaG, wecC, and wecA (PROKKA_02964–02966) and oligosaccharide repeat unit processing genes wzy (PROKKA_03380) and wzx (PROKKA_02023). Of these CDSs, only genes encoding the dTDP-glucose 4, 6-dehydratase RfbB and UDP-N-acetyl-D-glucosamine 6-dehydrogenase WecC are conserved across a wide range of species in Alcaligenaceae family, such as B. bronchiseptica and B. pertussis. It is known that in the Wzy-dependent pathway implicated in the assembly or export of O-antigen, O-unit polymerase Wzy, O-unit flippase Wzx and the O-chain length determinant Wzz are included [wzz homolog in K. gyiorum SWMUKG01. Further experimental works are required to verify the O-antigen-processing process.9 CDSs, showing more than 96% identities with those in CG1, that are likely associated with O-antigen biogenesis were predicted in the SWMUKG01 genome. These CDSs could be classified into three groups: nucleotide sugar synthetases by a potential included . HoweverK. gyiorum SWMUKG01 seem to be allelic to many LPS biosynthetic loci, and multiple enzymes are the same between these pathways, such as flippase Wzx, glycosyltransferase RfaG and UDP-N-acetyl-D-mannosaminuronate dehydrogenase WecC. It is known that Wza, Wzb, and Wzc are translocation proteins that specifically export group I or IV CPS to the outer surface. The identification of wza, wzb, and wzc genes suggested a type of group 1 or IV CPS in K. gyiorum SWMUKG01. It is reported that the initiating glycosyltransferase is the distinguishing factor between group I and IV CPS biosynthesis pathways, with WbaP catalyzing group I and WecA for group IV CPS biosynthesis. By searching in the SWMUKG01 genome, we could not identify a wbaP homologue, but two wecA (PROKKA_02966 and PROKKA_00500) were identified though it locates outside the CPS gene cluster. We proposed that the strain SWMUKG01 appears to have a group IV CPS. As group IV CPS biosynthesis is Wzy-dependent, the O-antigen polymerase Wzy appears to participate in both CPS and LPS biosynthesis in K. gyiorum SWMUKG01, asin Vibrio vulnificus [A cluster of genes coding for proteins involved in CPS biosynthesis and export were identified in the strain SWMUKG01 ranging from PROKKA_01910 to PROKKA_01935 . Howeverlnificus .Analysis on the SWMUKG01 genome sequence identified three genomic islands (GIs) , which wprsD1-prsE1-tolC1 (PROKKA_00929–00931), tolC2-prsD2-prsE2 (PROKKA_01512–01514) and prsE3-prsD3-tolC3 (PROKKA_02054–02056), were identified on this genome  and two-arginine (Tat) pathways , which as genome . The ideK. gyiorum harbors a putative gsp operon encoding proteins putatively involved in the biosynthesis of T2SS  inserted into the operon. It seems that GspC and GspO proteins were lacking in the strain SWMUKG01, for that a typical T2SS apparatus bears 12 core components (T2SS CDEFGHIJKLMO) [ of T2SS . This gsHIJKLMO) . These gHIJKLMO) . More wovir1 (PROKKA_00412–00423) and vir2 (PROKKA_02490–02502) locus  with those of A. aquatilis, and other homologous T4SS were also found in Bordetella petrii, Bordetella trematum, B. bronchiseptica and P. aeruginosa etc  is placed between virB5 and virB6. Similar gene arrangements also occur in other homologous T4SSs. This suggested that the hypothetical protein may play similar roles as the VirB7 does. If this is true and both vir1 and vir2 are functional, it would be interesting to learn these two T4SS systems coexist in SWMUKG01. Furthermore, the presence of secretion systems in CG1 was investigated; the results showed that all the secretion systems discussed above could be detected except the vir1 locus of T4SS.An interesting finding on the SWMUKG01 genome was the presence of two sets of genes encoding proteins homologous to the conjugation paradigm VirB/D T4SS, which we designated the 2) locus . The nuce latter . Both vinosa etc . It was y number . And, weefaciens , an arch3+), ferrous iron (Fe2+) and heme-bound iron. By searching the genome, we found that the SWMUKG01 harbors genes encoding proteins for all three categories of iron-uptake systems.Three broad categories of iron-uptake systems have been currently identified: systems for the utilization of ferric iron  for the transport of ferric catechols, FecBCDE (PROKKA_02160–02163) for ferric citrate and FhuBCD (PROKKA_02518–02521) for ferric hydroxamates. In addition, a putative fbp operon (PROKKA_01345–01347) encoding the FbpABC system was found on SWMUKG01 genome. It was previously shown that FbpABC is likely to be a ferric iron transporter that is involved in the translocation of iron delivered by transferrin and lactoferrin, across the cytosolic membrane [Approximately 88 genes (2.5%) of the SWMUKG01 genome are involved in iron uptake , all of membrane .K. gyiorum SWMUKG01, five genes encoding CDSs homologous to heme-binding receptors were identified  in this bacterium encodes a ferrous iron permease EfeU and two periplasmic proteins EfeB and EfeO in sequential order, with a slight difference in gene order comparing with previous studies [tonB-exbB-exbD (PROKKA_01872–01870), were identified in SWMUKG01 genome. In some bacteria, such as V. cholerae [P. aeruginosa [It is demonstrated that Fenditions . Only onSWMUKG01 , which i studies . In Gram studies . A set ocholerae  and P. aruginosa , there iK. gyiorum SWMUKG01, a total of 23 open reading frames were identified as putative RRs, 19 of which are adjacent to genes encoding probable HKs, forming 21 HK/RR pairs, all of which could be identified in CG1 but the gene PROKKA_00858. These histidine kinase and response regulator proteins could be categorized into five groups  and cefuroxime (>16 μg/ml). Our genomic analysis showed that 48 (1.39%) out of 3441 potential CDSs were identified in CARD database , sulfonamide, rifampicin, and fosfomycin were included, as well as some other multidrug resistance proteins. However, no plasmid was detected in the strain SWMUKG01, thus plasmid-carried drug resistance genes are not part of drug resistance for this pathogen.Clinical reports showed that some of floxacin , 8, colifloxacin , cefepimfloxacin , which sdatabase . Among tBy searching in the SWMUKG01 genome, we found a total of 10 sets of genes encoding multidrug efflux pump systems , among wK. gyiorum strain SWMUKG01, the first clinical isolate from southwest China, was sequenced in our present study. The length of the genome is about 3.9 million bps with genomic GC content of 62%. Genomic and phylogenetic comparisons indicated that K. gyiorum, A. aquatilis and A. faecalis may derive from a recent common ancestor. A total of 3441 CDSs were annotated, of which 326 potential virulence factors were predicted by VFDB database. Genes and operons related to bacterial surface polysaccharides, flagella, pili, iron acquisition systems, secretion systems, and TCSs as well as efflux pumps were analyzed at the genomic level and compared with those from other pathogens, which underlined the genetic basis of the pathogenesis and virulence of K. gyiorum. This work allows the identification of a new bacterial species at the genetic level and provides a foundation for future research into the mechanisms of pathogenesis of K. gyiorum.In conclusion, the complete genome of S1 Fig(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.


Ipomoea L. is the largest genus within the Convolvulaceae and contains 600–700 species. Ipomoea species (morning glories) are economically valuable as horticultural species and scientifically valuable as ecological model plants to investigate mating systems, molecular evolution, and both plant–herbivore and plant–parasite interactions. Furthermore, the dried seeds of I. nil or I. purpurea are used in Korean traditional herbal medicines. In this study, chloroplast (cp) genomes were sequenced from six Ipomoea species, namely, I. nil and I. purpurea and, for the first time, I. triloba, I. lacunosa, I. hederacea, and I. hederacea var. integriuscula. The cp genomes were 161,354–161,750 bp in length and exhibited conserved quadripartite structures. In total, 112 genes were identified, including 78 protein-coding regions, 30 transfer RNA genes, and 4 ribosomal RNA genes. The gene order, content, and orientation of the six Ipomoea cp genomes were highly conserved and were consistent with the general structure of angiosperm cp genomes. Comparison of the six Ipomoea cp genomes revealed locally divergent regions, mainly within intergenic spacer regions . In addition, the protein-coding genes accD, cemA, and ycf2 exhibited high sequence variability and were under positive selection (Ka/Ks > 1), indicating adaptive evolution to the environment within the Ipomoea genus. Phylogenetic analysis of the six Ipomoea species revealed that these species clustered according to the APG IV system. In particular, I. nil and I. hederacea had monophyletic positions, with I. purpurea as a sister. I. triloba and I. lacunosa in the section Batatas and I. hederacea and I. hederacea var. integriuscula in the section Quamoclit were supported in this study with strong bootstrap values and posterior probabilities. We uncovered high-resolution phylogenetic relationships between Ipomoeeae. Finally, indel markers (IPOTY and IPOYCF) were developed for the discrimination of the important herbal medicine species I. nil and I. purpurea. The cp genomes and analyses in this study provide useful information for taxonomic, phylogenetic, and evolutionary analysis of the Ipomoea genome, and the indel markers will be useful for authentication of herbal medicines. Chloroplasts (cp) are among the most important organelles in plants, having important roles in photosynthesis and carbon fixation as well as in the biosynthesis of starch, fatty acids, amino acids, and pigments . Cp genoIpomoea is the largest genus in the Convolvulaceae family, with 600–700 species  or regulatory modifications , 2016]. Pharbitidis Semen is designated as a medicine in Korea, and is regulated by the Ministry of Food and Drug Safety due to its pharmaceutical activity and potential toxicity   . Analysi5.25 MYA . ExaminaI. nil and I. purpurea and four closely related Ipomoea species with similar seed structures were collected and their cp genomes were compared. This study aimed to (1) characterize six Ipomoea cp genomes and identify genetically variable regions by comparison of their global structures, (2) develop novel molecular markers for use in authentication of herbal medicine species, and (3) understand evolutionary relationships within tribe Ipomoeeae through enhanced phylogenic studies in conjunction with previously reported cp genomes.Here, samples of herbal medicine species Ipomoea species were collected from native habitats in Korea and used for cp genome sequencing. I. nil, I. purpurea, I. hederacea, I. hederacea var. integriuscula, I. lacunosa, and I. triloba were assigned identification numbers, and specimens were registered in the Korean Herbarium of Standard Herbal Resources (Index Herbariorum code KIOM) at the KIOM. The plant samples used for cp genome analysis and indel validation in this study are listed in Supplementary Table Fresh leaves of six De novo assembly was used to construct cp genomes from low-coverage whole-genome sequences. Trimmed paired-end reads (Phred scores ≥20) were assembled using CLC genome assembler  with default parameters. SOAP de novo gap closer was used to fill gaps based on alignment of paired-end reads (I. nil (AP017304) as a reference  according to the manufacturer’s instructions. Illumina short-insert paired-end sequencing libraries were constructed and generated using the NextSeq platform . nd reads . Principnd reads , and alieference .Ipomoea cp genomes was performed using GeSeq  and Capsicum annuum var. glabriusculum (GenBank acc. KJ619462.1) as outgroups. Of these, 32 cp genome sequences were downloaded from the NCBI GenBank  with the following amplification parameters: initial denaturation at 95°C for 2 min; 35 cycles at 95°C for 50 s, 62°C for 50 s, and 72°C for 50 s; and final extension at 72°C for 5 min. PCR products were separated on a 2% agarose gel for 40 min at 150V. DNA fragments were extracted from agarose using a Gel Extraction Kit, subcloned into the pGEM-T Easy vector , and sequenced on a DNA sequence analyzer . The six Ipomoea species germplasms used are listed in Supplementary Table Indel regions were detected by the alignment of six Ipomoea species  separated by LSC  and SSC  regions . The six Ipomoea cp genomes had similar GC contents, with higher GC contents observed in the IR regions (41%) than in the single-copy regions , consistent with previously reported cp genomes . Seventeen of the genes contained introns, fourteen with a single intron and two (ycf3 and clpP) with two introns  and non-coding regions . Mononucleotide motifs were the most abundant repeat type, with dinucleotide motifs, the second most abundant in the six Ipomoea cp genomes . SSRs were compared between the genomes to identify common and species-specific SSRs. I. purpurea contained seven specific SSRs, whereas no I. hederacea-specific SSRs were identified. The Ipomoea cp SSRs encompassed abundant variation and will be useful genomic resources for marker development and population genetic studies of Ipomoea species. Tandem repeat sequences influence genome structure with respect to genome size, genome rearrangement, and gene duplication . Of these, most were located in non-coding LSC and SSC regions . On average, long tandem repeats (>70 bp) constituted 27% of all tandem repeats in the six Ipomoea cp genomes. I. triloba contained the longest repeat (484 bp). While most of the tandem repeats were conserved, a small number of specific tandem repeats were detected in the six Ipomoea cp genomes . In particular, indel regions with different repeat copy numbers were identified for I. nil in trnN-ycf1 (68 bp × one copy in I. nil / 68 bp × two copies in other Ipomoea) and I. purpurea in ycf1 (18 bp × two copies in I. purpurea / 18 bp × three copies in other Ipomoea). These characteristics allowed indel markers for distinguishing I. nil and I. purpurea from other Ipomoea species to be developed in this study.SSRs (1–6 nucleotide repeats) were distributed abundantly across the cp genome. SSRs from cp genomes can be used for analysis of phylogenetic relationships and population genetics due to their high polymorphism rates and stable reproducibility . Most SSlication . Here, 2Ipomoea cp genomes were highly conserved, with 98.5–99.8% similarity, conserved genomic structure, and conserved gene order and orientation . In general, non-coding regions were more diverged than coding regions. Five non-coding regions, petN-psbM, trnI-CAU-ycf2, ndhH-ndhF, psbC-trnS, and ccsA-ndhD, exhibited high divergence among the six Ipomoea. Coding regions were generally more conserved, with the exception of ycf1, matK, and rbcL, which are commonly used as representative plant DNA barcoding regions . As expected, IR regions were more conserved than the LSC and SSC regions, with average Pi values of 0.003 for IR and 0.006 in SC . The average Pi value for coding regions was 0.00315 . The Pi value for intron-containing IGS averaged 0.00752 . In the SSC, ccsA-ndhD exhibited a Pi value of 0.03, higher by an order of magnitude. Although the six Ipomoea cp genomes were generally highly conserved, the intergenic regions were particularly divergent. This is consistent with previous research with angiosperm cp genome  ratios from collinear genes as a marker of evolution , and the highest Ka/Ks ratio was 2.654 (for accD between I. purpurea and I. lacunosa). Thus, although the six Ipomoea cp genomes exhibited highly conserved organization, positive selection pressure (Ka/Ks > 1) was observed for accD, cemA, and ycf2. Positive selection of these three genes suggests that they are undergoing essential adaptations to their environment despite the weak selection pressures experienced by Ipomoea. Previous studies show that these genes are generally lost from, or are highly divergent in, angiosperms  and chalcone synthase (CHS) in Ipomoea are under positive selection pressure  experienced selective pressure to promote divergence via increasing gene duplication in Ipomoea. Ipomoea has extremely diverse flower colors, a rapid generation time, and various growth forms. We suggest that these ecological characteristics of Ipomoea reflect their remarkable adaptability to various environments due to diverse positive selection pressure on genes in the nucleus or plastid.IR regions were more highly conserved than SC regions due to copy correction by gene conversion in IR regions . However genomes . Althougiosperms . McNeal pressure . DFR is Ipomoea species within the Convolvulaceae, we aligned 48 protein-coding sequences shared by 38 cp genomes  share closer relationships with I. hederacea and I. hederacea var. integriuscula than with I. triloba and I. lacunosa. Thus, there is a strong possibility for confusion between I. hederacea and I. hederacea var. integriuscula. We suspect that the most frequent adulterations of Pharbitidis Semen are seeds of I. hederacea and I. hederacea var. integriuscula.Cp genomes are valuable genomic resources for reconstruction of accurate and high-resolution phylogenies, and have been used as such in several studies , for exasections . Most Ippomoeeae . Quamoclpomoeeae , whereasV system . The resI. nil and I. purpurea are used in traditional herbal medicine in Korea . The primer pairs, respectively named IPOTY and IPOYCF, successfully amplified sequences from I. nil and I. purpurea . The markers were tested with other Ipomoea germplasms (23 samples for IPOTY and 22 samples for IPOYCF), and the five I. nil samples and six I. purpurea samples were clearly distinguishable. Amplified fragments from all of the tested Ipomoea samples were sequenced to identify exact amplicon size. IPOTY primers yielded a 525 bp amplicon with I. nil, and IPOYCF primers yielded a 467 bp fragment with I. purpurea. Predicted deletion or insertion sizes from cp genomes were consistent with those resulting from the Ipomoea germplasms used in this study. Indel markers for variable copy numbers at tandem repeats were also used previously to distinguish closely related Fagopyrum and Chenopodium species, indicating the utility of these markers in species identification (Ipomoea species identification and authentication of herbal medicines.Dried seeds of in Korea . Howeverfication . Copy nuIpomoea cp genomes were sequenced in this study. Overall, the cp genomes were highly conserved with respect to gene content, gene orientation, and GC content, but local variations in sequence and structure were observed. Tandem repeats and SSRs were identified with the aim of developing molecular markers for species identification and authentication of herbal medicines. The most divergent regions among the six genomes were found in non-coding regions petN-psbM, trnI-CAU-ycf2, ndhH-ndhF, psbC-trnS, and ccsA-ndhD, and coding regions accD, cemA, and ycf2. The accD, cemA, and ycf2 genes exhibited positive selection. Phylogenetic analysis of cp genome sequences yielded more accurate phylogenetic relationships within the Ipomoea genus than previous studies. Novel indel markers based on copy number variation at tandem repeats were developed for identification of I. nil and I. purpurea. These markers, named IPOTY and IPOYCF, were able to discriminate between authentic I. nil and I. purpurea and other inauthentic Ipomoea species, respectively, and will be useful for authentication of herbal medicines containing these two species. The cp genomes and analyses in this study are valuable for species identification, clarification of taxonomy, and understanding evolutionary history in the Ipomoea genus.Six IP designed the experimental framework and drafted and revised the manuscript. SY and BCM collected and identified plant materials. WJK and PN performed the experiments. HOL carried out sequence analysis. BCM revised the manuscript. All authors contributed to the experiments and approved the final manuscript.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. The reviewer AZ and the handling Editor declared their shared affiliation.


Erwinia tracheiphila is a bacterial plant pathogen emerging in eastern North America. To aid in understanding genetic variation within E. tracheiphila, here we sequence the first reference genome of an infected muskmelon (Cucumis melo). Erwinia tracheiphila is a bacterial plant pathogen emerging in eastern North America. To aid in understanding genetic variation within E. tracheiphila, here we sequence the first reference genome of an infected muskmelon (Cucumis melo). The genome assembles into a single chromosomal contig, three plasmid contigs, and one bacteriophage contig. Erwinia tracheiphila Smith (Enterobacteriaceae) is a bacterial plant pathogen that causes losses from species from only two Cucurbitaceae crop plant genera, Cucumis spp. (muskmelon and cucumber) and Cucurbita spp.   and cucumber (Cucumis sativus). gourds) , 2. E. tt appear . E. tracE. tracheiphila isolate from a muskmelon (Cucumis melo) host plant and the first draft genome sequence of an isolate from lineage Et-melo. MDCuke was isolated in 2009 from an infected muskmelon in Maryland.Here, we report the high-quality draft genome of MDCuke, the first sequenced E. tracheiphila isolate BuffGH, which was originally isolated from an infected wild gourd (Cucurbita pepo subsp. texana) and falls within the phylogenetic lineage Et-C1  medium to stationary phase. Genomic DNA was extracted following the same methods for generating the reference sequence of ge Et-C1 , 5, 6. Bge Et-C1 . This inCP013970. The version described in this paper is the first version, CP013970.1. The raw reads are available under BioSample accession no. SAMN10144255 and run no. SRR7942800 in the Sequence Read Archive.This whole-genome shotgun project has been deposited in GenBank under the accession no.


Leishmania species without using expensive equipment. This method was applied to 92 clinical samples collected at 33 sites in 14 provinces of Ecuador, which have been identified at the species level by the kinetoplast cytochrome b (cyt b) gene sequence analysis, and the results obtained by the two analyses were compared. Although most results corresponded between the two analyses, PCR-RFLP analyses revealed distribution of hybrid strains between Leishmania (Viannia) guyanensis and L. (V.) braziliensis and between L. (V.) guyanensis and L. (V.) panamensis, of which the latter was firstly identified in Ecuador. Moreover, unexpected parasite strains having the kinetoplast cyt b gene of L. (V.) braziliensis and nuclear genes of L. (V.) guyanensis, L. (V.) panamensis, or a hybrid between L. (V.) guyanensis and L. (V.) panamensis were identified. This is the first report of the distribution of a protozoan parasite having mismatches between kinetoplast and nuclear genes, known as mito-nuclear discordance. The result demonstrated that genetically complex Leishmania strains are present in Ecuador. Since genetic exchanges such as hybrid formation were suggested to cause higher pathogenicity in Leishmania and may be transmitted by more species of sand flies, further country-wide epidemiological studies on clinical symptoms, as well as transmissible vectors, will be necessary.PCR-Restriction Fragment Length Polymorphism (RFLP) analyses targeting multiple nuclear genes were established for the simple and practical identification of  Leishmania is a neglected tropical disease widely distributing worldwide, especially in tropical and subtropical areas. Approximately 20 species are known to be pathogenic to humans, of which eight species have been recorded as causative agents of cutaneous and mucocutaneous leishmaniases in Ecuador. Since infecting species are the major determinant of clinical outcomes, identification at the species level is important for the treatment and prognosis. The parasite species have been identified conventionally by multilocus enzyme electrophoresis (MLEE) and recently by genetic analysis such as sequencing and genotyping. In the present study, PCR-Restriction Fragment Length Polymorphism (RFLP) targeting multiple nuclear genes was employed, and the results were compared with those obtained by kinetoplast cytochrome b (cyt b) gene sequence analysis, which is widely applied to species identification. Although most results corresponded between the two analyses, PCR-RFLP revealed presence of unexpected genetically complex Leishmania strains having characteristics of hybrid and mito-nuclear discordance. Since hybrid strains of Leishmania were suggested to increase disease severity and may be transmitted by a wider range of sand fly species, careful epidemiological research, including clinical courses and vector research, will be needed.Leishmaniasis caused by intracellular protozoa of the genus  Leishmania, is a neglected tropical disease widely distributed worldwide, especially in tropical and subtropical areas, affecting at least 12 million people in 96 countries  that were isolated from CL patients in Ecuador and identified at the species level by MLEE ..Leishman6pgd gene with Bsp1286I resulted in distinct gene fragment patterns of L. (V.) guyanensis/L. (V.) panamensis, L. (V.) braziliensis, L. (V.) naiffi, L. (L.) major-like and L. (L.) mexicana; however, the patterns between L. (V.) guyanensis/L. (V.) panamensis and L. (V.) naiffi were similar and difficult to discriminate because of only about a 50 bp difference in a fragment of approximately 1 kbp (HinfI (Digestion of the ly 1 kbp . The twop (HinfI .L. (V.) guyanensis and L. (V.) panamensis were not discriminated by PCR-RFLP of mpi and 6pgd genes, PCR-RFLP of the hsp70 gene with a restriction enzyme, BccI, successfully differentiated the two species as reported previously (Although eviously  27]..L. (V.) mpi gene with restriction enzymes, HaeIII and HpaI, and 6pgd gene with Bsp1286I and HinfI were applied to 92 clinical samples collected at 33 sites in 14 provinces of Ecuador. PCR-RFLP analysis of the hsp70 gene with a restriction enzyme, BccI, was used for differentiation between L. (V.) guyanensis and L. (V.) panamensis. The results obtained by PCR-RFLP analyses were compared with those obtained by the cyt b gene sequence analysis. The results of the species identification obtained by the two nuclear genes always agreed with each other. The identification by PCR-RFLP analyses completely matched with that obtained by the cyt b gene sequence analysis in all of L. (V.) naiffi (2 samples) and L. (L.) mexicana (3 samples) (L. (V.) guyanensis by cyt b gene analysis, 72 samples were identified as L. (V.) guyanensis by PCR-RFLP analyses, whereas one sample from a Pacific coast area showed a hybrid pattern of L. (V.) guyanensis and L. (V.) panamensis based on the PCR-RFLP of the hsp70 gene  guyanensis but “T” in L. (V.) panamensis, whereas a sample having a hybrid RFLP pattern had both “C” and “T” peaks at the corresponding position (L. (V.) guyanensis and L. (V.) panamensis in Ecuador. On the other hand, of the 14 samples identified as L. (V.) braziliensis by cyt b gene analysis, only 6 samples were identified as L. (V.) braziliensis by RFLP analyses (L. (V.) braziliensis by the cyt b gene analysis, three samples showed hybrid patterns in PCR-RFLP analyses of both the mpi and 6pgd genes  guyanensis but “T” in L. (V.) braziliensis of the mpi gene, and “T” in L. (V.) guyanensis but “C” in L. (V.) braziliensis of the 6pgd gene. On the other hand, the mpi and 6pgd genes from the three samples with hybrid RFLP patterns had both “C” and “T” peaks at the corresponding position (L. (V.) braziliensis and L. (V.) guyanensis (L. (V.) braziliensis by sequence analysis of the cyt b gene, PCR-RFLP analyses showed that one sample from a Pacific coast area was L. (V.) guyanensis, three samples from the northern Pacific coast and Amazonian areas were L. (V.) panamensis, and one sample from a northern Pacific coast area had a hybrid pattern of L. (V.) guyanensis and L. (V.) panamensis  braziliensis and L. (V.) guyanensis in this study since this region has not been well-analyzed in subgenus Viannia species. The sequences from parasites with mito-nuclear discordance showed 98.9–99.1% and 98.5–98.9% identities with those of L. (V.) braziliensis and L. (V.) guyanensis, respectively (accession numbers: LC475135-LC475142). When partial COII gene sequences in the obtained COII-ND1 region sequences were analyzed on the GenBank database, the sequences from parasites with mito-nuclear discordance showed 99.5% and 98.9% identities with those of L. (V.) braziliensis and L. (V.) guyanensis, respectively. This result strongly suggested that the kinetoplast genes of these parasites originated from L. (V.) braziliensis, corresponding to the result of cyt b gene analysis.PCR-RFLP analyses of samples) . Of the position , indicatanalyses . In the gd genes . The seqposition . From thyanensis , Fig 4. namensis , Fig 4.  Ecuador , Fig 4. Leishmania species distributing in Ecuador in order to develop a simple and practical way for species identification independent of expensive equipment such as a genetic analyzer. As a result, mpi and 6pgd genes, for which encoding enzymes have been widely used as the gold standard of species identification, were identified as suitable targets for this purpose in the tested samples. The results obtained by the PCR-RFLP analyses of multiple nuclear targets were compared to those of cyt b gene sequence analysis  and a hybrid of L. (V.) braziliensis and L. (V.) peruviana circulating in the Department of Huanuco, Peru [mpi and 6pgd gene fragments (>1000bp) were successfully established and applied to 92 clinical samples in Ecuador. Although a polymorphic RFLP pattern, which does not affect the identification, was detected in the mpi of one L. (L.) mexicana strain, the variant RFLP pattern was not detected in the present clinical samples identified as L. (L.) mexicana. Further sample analyses from different areas and different countries will be important to confirm the utility of this analysis, although polymorphic RFLP profiles may be detectable in these genes. Since polymorphism was also reported in the hsp70 gene of several Leishmania species [PCR-RFLP analysis has been employed for species identification of accepted . The rRNld World , 37–41.  species . In thiseruviana , 44. In co, Peru . In the  species , PCR-RFLLeishmania species. It is due to the following reasons: 1) It is little or no chance to be infected by more than one parasite in a cutaneous lesion because the lesion is typically developed at the site bitten by a sand fly transmitting specific Leishmania species, 2) Even if mixed infection occurs, either parasite becomes dominant in the lesion, resulting in the presence of dominant allele by the genetic analysis. However, both alleles were comparably amplified as observed in the PCR-RFLP analysis, which is indicative of a putative hybrid strain. In addition, similar results were obtained on electrograms of the direct sequencing, showing comparable fluorescence intensities of polymorphic nucleotides derived from both species. 3) The presence of hybrid strain has been reported in the same area as described below [Several samples showing hybrid RFLP patterns were identified as hybrid strains rather than mixed infection of different ed below . Isolaticyt b sequence analyses showed corresponding results in most clinical samples, the present study revealed the distribution of several unexpected strains in Ecuador, including hybrid and mito-nuclear discordance strains. Since hybrid strains cannot be identified by the cyt b gene analysis after molecular cloning, this is another advantage of identifying parasite species by PCR-RFLP. Distribution of a hybrid strain of L. (V.) guyanensis/panamensis complex and L. (V.) braziliensis was reported in Zumba, a province of Zamora-Chinchipe in a southern part of Ecuador by using MLEE and random amplified polymorphic DNA (RAPD) [L. (V.) guyanensis and L. (V.) panamensis was detected in northern Pacific areas of Ecuador. This is the first report of the presence of a hybrid strain of L. (V.) guyanensis and L. (V.) panamensis in Ecuador. L. (V.) guyanensis and its closely related L. (V.) panamensis have been reported to be endemic in northern Pacific areas of Ecuador by MLEE; however, only L. (V.) guyanensis was identified in the same areas by cyt b gene analysis in recent studies [L. (V.) guyanensis is dominantly present in these areas, suggesting that endemic species may change, or that there may be discordance between MLEE and genetic analysis. However, the identification of a hybrid of L. (V.) guyanensis and L. (V.) panamensis as a minor population suggests that parental L. (V.) panamensis may still be present in some of these areas. Another unexpected finding was identification of mito-nuclear discordant strains of Leishmania species in northern Pacific and Amazonian areas. Interestingly, mito-nuclear discordant strains were identified only in the species identified as L. (V.) braziliensis by cyt b gene analysis. This finding supports a recent study using cyt b gene analysis reporting increasing cases of L. (V.) braziliensis infection in Pacific coast areas when compared to previous studies using enzymatic MLEE analysis [L. (V.) braziliensis and L. (V.) peruviana was suggested to increase disease severity when compared to parental species in an animal model [Leishmania species would be another interesting subject [Wolbachia infection in insects, and 6) human actions [Schistosoma turkestanicum between populations [Taenia solium between lineages [T. saginata and T. asiatica [Leishmania species in Ecuador. Although the present PCR-RFLP analyses was shown to be practical for identification of Leishmania species in Ecuador, further study focusing on other Leishmania species and clinical samples from different countries will be needed to enhance the utility of this approach. PCR-RFLP analyses of clinical samples and subsequent comparison with kinetoplast cyt b sequence analysis revealed the distribution of genetically complex Leishmania strains having genetic characteristics of hybrid and mito-nuclear discordance. Although intraspecies genetic variation observed in the cyt b gene resulted in this gene as an unsuitable target for RFLP analysis, there is no doubt about the utility of cyt b gene sequence analysis for species identification and phylogenetic analysis since distinct interspecies genetic diversity of this gene overcomes the disadvantage of the intraspecies variation. However, the present study points to the importance of applying multiple target approaches as the combination of cyt b and the PCR-RFLP assays presented here, enhancing the reliability of species identification and characterization of genetic properties including hybrid and mito-nuclear discordance. Further studies are needed to reveal the parasitological characteristics of hybrid and mito-nuclear discordance, clinical outcomes caused by these parasites, and the range of vector species of these parasites. In addition, studies on mito-nuclear discordance in Leishmania and other protozoa may provide further insights into the mechanism of genetic exchanges of these parasites.Although multiple PCR-RFLP and A (RAPD) . The pre studies , 21, 46.analysis . The hybal model . Therefo subject –51. Mito actions . It provulations , and ceslineages , and betasiatica –57. ThisS1 Fighttp://english.freemap.jp/)The dark gray areas show the Andean plateau , and the light gray areas show highland jungle or Andean slopes . 1. San Lorenzo, 2. Esmeraldas, and 3. Atacames, Province of Esmeraldas; 4. Pedernales, 5. Montalvo, and 6. Pedro Pablo Gomez, Province of Manabi; 7. Cielo Verde, Province of Imbabura; 8. Puerto Quito, 9. Pedro Vicente Maldonado, 10. Los Bancos, 11. Nanegalito, 12. Pachijal, and 13. Quinche, Province of Pichincha; 14. Valle Hermoso, Province of Santo Domingo; 15. Balsapamba, Province of Bolivar; 16. Chanchan, Province of Chimborazo; 17. La Troncal, Province of Cañar; 18. El Triunfo, 19. Naranjal, and 20. Balao, Province of Guayas; 21. Santa Rosa, Province of El Oro; 22. Cascales, 23. Lago Agrio, and 24. Palma Roja, Province of Scumbios; 25. Coca, 26. Shangrila, 27. La Joya de los Sachas, 28. Pompeya, 29. Union Milagrena, and 30. Loreto, Province of Orellana; 31. Puyo, Province of Pastaza; 32. Palanda, and 33. Zumba, Province of Zamora-Chinchipe. (Adapted from a map available at (TIF)Click here for additional data file.S2 FigL. (V.) guyanensis, 2. L. (V.) panamensis, 3. L. (V.) lainsoni.1. (TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 FigLeishmania mpi gene (A) or 6pgd gene (B) fragments.Direct sequence analysis showing a species-specific polymorphic site of (TIF)Click here for additional data file.