Treponema phylogroups implicated in bovine digital dermatitis (BDD). However, a polymicrobial aetiology involving other gram-stain-negative anaerobes is suspected.Non-healing bovine foot lesions, including non-healing white line disease, non-healing sole ulcer and toe necrosis, are an increasingly important cause of chronic lameness that are poorly responsive to treatment. Recent studies have demonstrated a high-level association between these non-healing lesions and the Fusobacterium necrophorum, Porphyromonas endodontalis, Dichelobacter nodosus and Treponema pallidum/T. paraluiscuniculi was performed.A PCR-based bacteriological survey of uncomplicated BDD lesions (n=10) and non-healing bovine foot lesions (n=10) targeting P. endodontalis DNA was detected in 80.0% of the non-healing lesion biopsies (p=<0.001) but was entirely absent from uncomplicated BDD lesion biopsies. When compared to the BDD lesions, F. necrophorum was detected at a higher frequency in the non-healing lesions , whereas D. nodosus was detected at a lower frequency . Conversely, T. pallidum/T. paraluiscuniculi DNA was not detected in either lesion type.P. endodontalis and F. necrophorum should be further investigated as potential aetiological agents of non-healing bovine foot lesions. A failure to detect syphilis treponemes in either lesion type is reassuring given the potential public health implications such an infection would present.The data from this pilot study suggest that  Treponema medium phylogroup, Treponema phagedenis phylogroup and Treponema pedis) in the aetiology of BDD and the non-healing lesions.Bovine digital dermatitis (BDD), an inflammatory disease of the interdigital skin with an infectious aetiology, is one of the most frequently encountered causes of lameness.Dichelobacter nodosus and Fusobacterium necrophorum, the aetiological agents of ovine footrot,Porphyromonas genera is highly associated with BDD lesionsPorphyromonas endodontalis has previously been detected in 80% of surveyed CODD lesions,Treponema pallidum subs. pallidum and T. pallidum subs. pertenue DNA. This PCR was also designed to detect Treponema paraluiscuniculi, the aetiological agent of venereal syphilis in rabbits, which has previously been identified in BDD lesion microbiome datasets.To investigate this possibility further, non-healing bovine foot lesion biopsies were surveyed for the two other key bacterial species known to be associated with ruminant foot disease, namely The non-healing lesion samples used here were collected between February and July 2009 from nine Holstein Friesian cows living on nine dairy farms in the UK (seven in Gloucestershire and two in Cambridgeshire) and comprised of nhWLD (n=3), nhSU (n=3) and TN (n=4) lesion punch biopsies. Samples were processed as described previously.F. necrophorum,D. nodosusP. endodontalis.T. pallidum 16S rRNA gene was developed as part of this study. The T. pallidum PCR thermal profile was as follows: an initial denaturation at 95°C for 7 min followed by 35 cycles of 95°C for 1 min, 71°C for 2 min and 72°C for 3 min and a final elongation step at 72°C for 10 min. DNA extracted from T. pallidum subsp. pallidum  was used as a positive control. To validate this assay, purified water and genomic DNA from the three BDD-associated Treponema phylogroups were used as negative controls. Each 25 µL PCR reaction included 1 µL of DNA template and was performed using Taq polymerase  in accordance with the manufacturer’s instructions. All PCRs were performed in duplicate with relevant genomic DNA controls. Amplicons were visualised by gel electrophoresis and ethidium bromide staining. Statistical analysis was performed using the Fisher’s exact test in Minitab V.18 .All lesion biopsy samples were subjected to the following PCR assays, performed as described previously: The dataset  shows thP. endodontalis DNA was found to be highly associated  with the non-healing lesions but entirely absent from typical, uncomplicated BDD lesions. F. necrophorum DNA was also detected at a higher frequency in non-healing lesions relative to BDD lesions , whereas D. nodosus was detected at a lower frequency , although these differences were not statistically significant. Neither T. pallidum nor T. paraluiscuniculi DNA was detected in either lesion type.These bacteriological profiles, while confirming the ubiquity of BDD treponemes in both digital dermatitis lesions and non-healing lesions, also reveal certain distinctions. Most strikingly, F. necrophorum and P. endodontalis may also contribute to pathogenesis of TN, nhWLD and nhSU. F. necrophorum is considered to be an opportunistic pathogen and has been implicated in several animal diseases, including interdigital phlegmon,The aetiologies of non-healing bovine foot lesions are poorly understood. A high-level association with the BDD-associated treponemes supports their involvement in the pathogenesis of these lesions, but the roles played by other pathogenic bacteria remains unknown. Based on the data presented here, it is hypothesised that in addition to the BDD treponemes, infection with P. endodontalis is strongly associated with chronic oral infections where it participates in tissue destructive processes.P. endodontalis-derived virulence factors, including lipopolysaccharide, are potent stimulators of inflammatory cytokine release, and are thought to have a role in the initiation and development of periapical periodontitis, odontogenic abscesses and alveolar bone abnormalities.P. endodontalis in the non-healing bovine foot lesions (and its complete absence from BDD lesions) suggests a potential pathogenic role here, too. To the best of our knowledge, P. endodontalis colonisation distal to the oral cavity has hitherto only been observed in CODD,P. endodontalis colonisation may be a prominent feature of aggressive foot lesions in ruminants. We hypothesise that synergy between P. endodontalis, F. necrophorum and the BDD-associated treponemes may lead to enhanced lesion pathology. Conversely, no association between the two T. pallidum subspecies, T. paraluiscuniculi and either lesion type was identified. This is in contrast to the findings of a recent microbiome study that reported the presence of T. paraluiscuniculi in the BDD lesion biopsies of North American cattle.T. paraluiscuniculi in BDD is considered improbable.In humans, P. endodontalis and F. necrophorum were both detected at a greater frequency in the non-healing bovine foot lesions relative to uncomplicated BDD lesions. Further studies are required to elucidate the precise relationship between these fastidious gram-stain-negative anaerobes and other relevant species of bacteria in the aetiopathogenesis of these atypical lesions. A failure to detect syphilis treponemes in either lesion type is reassuring given the potential public health implications such an infection would present.In summary,


Melaleuca have been widely used as traditional medicine mainly because of their broad spectrum antimicrobial activity. In this study, we reported the complete chloroplast genome of Melaleuca cajuputi subsp. cumingiana. The chloroplast genome of this species is 158,855 bp in length, including a pair of inverted repeat regions (IRs)  that is divided by a large single-copy (LSC) area  and a small single-copy (SSC) area . The circular chloroplast genome of M. cajuputi subsp. cumingiana contains 135 unique genes, composing of 87 protein-coding genes, 40 tRNA genes, and eight rRNA genes. Phylogenetic analysis indicates that M. cajuputi subsp. cumingiana was clustered with species in the tribe Melaleuceae. This complete chloroplast genome of M. cajuputi subsp. cumingiana will provide a powerful tool to accelerate breeding, biotechnological and phylogenetic study.Plants in the genus  Melaleuca L. genus (Myrtaceae family) have been used as traditional medicine for many years  has been reported , and deposited at the herbarium of South China Botanical Garden (accession number: SCBG-CF-2071). Total genomic DNA was extracted from fresh leaves using the CTAB-chloroform protocol (Doyle and Doyle rbcL) gene sequence from M. alternifolia (GenBank accession no. MN310606) was used as seed sequence, and the whole cp genome sequence of M. alternifolia and Eucalyptus grandis (GenBank accession no. NC_014570) was used as a reference to resolve the inverted repeat (IR) in the chloroplast genome of M. cajuputi subsp. cumingiana. The assembled chloroplast genome was annotated using PGA  of 26,727 bp that is insulated by a large single-copy  and a small single-copy . The total GC content of complete chloroplast genome, LSC, SSC, IR regions is 36.8%, 55.0%, 11.4%, and 33.6%, respectively. The complete chloroplast genome of M. cajuputi subsp. cumingiana contains 135 unique genes, including 87 protein-coding genes, 40 tRNA genes, and eight rRNA genes. Introns are present in 19 of the annotated genes. Five of the intron containing genes contain three exons. Most of these genes are single-copy genes. However, 19 genes were duplicated in IR regions.The complete chloroplast genome of M. cajuputi subsp. cumingiana, the complete chloroplast genomes of 16 published species within Myrtaceae and one outgroup  were downloaded from the NCBI GenBank database. Ninety-four chloroplast genes shared by all species in this analysis were extracted, and were aligned by using MUSCLE (Edgar M. cajuputi subsp. cumingiana was closely related to species in tribe Melaleuceae (Melaleuca rigidus, initially described as Callistemon rigidus, also supported the sinking of Callistemon into Melaleuca (Craven M. cajuputi and it will provide a solid foundation for phylogenetic and evolutionary studies in Melaleuca and is expected to contribute to improving M. cajuputi breeding.To confirm the phylogenetic position of LE Edgar . We concLE Edgar  using IQLE Edgar . Phylogealeuceae , which ialeuceae . In addia Craven . In conc


Beauveria bassiana (B. bassiana) is a famous entomopathogenic fungus that could parasitize on hundreds of insect species, which are being used as an environmentally friendly mycoinsecticide. Nevertheless, the possible effect of genetic diversity of these B. bassiana isolates from different hosts on virulence has not been explored before. In order to explore that issue, we compared the genome sequences among seventeen B. bassiana isolates from 17 different insects using whole genome re-sequencing, with B. bassiana strain ARSEF 2860 as the reference genome.B. bassiana. Among these, two genes with high frequency mutations encode the toxin-producing non-ribosomal peptide synthase (NRPS) protein. Seven genes undergoing positive selection were enriched in the two-component signaling pathway that is known to regulate the fungal toxicity. In addition, the domain changes of three positively selected genes are also directly related to the virulence plasticity. Besides, the functional categorization of mutated genes showed that most of them involved in the biological functions of toxic proteins involved in.There were a total of 10,098 missense mutated genes, 720 positively selected genes were identified in 17 strains of B. bassiana towards hosts during infection process, which provide an insight into the potential effects of natural variation on the virulence of B. bassiana, which will be useful in screening out potential virulence factors in B. bassiana.Based on our data, our results indicate that several mutated genes and positively selected genes may underpin virulence of  Beauveria bassiana (B. bassiana), a well-known entomopathogenic fungus, is belonged to the family Clavicipitaceae, order Hypocreales [B. bassiana exhibits high adaptability to various environmental conditions and thus could parasitize a wide range of insect populations [B. bassiana is actively developed as a biological control tool for various insects in all regions of world [B. bassiana to reduce the occurrence of forest insects in China every year [B. bassiana is thus a generalist with no strict host preference and can be used as an effective and sustainable biological control agent against a myriad of insect pests [ocreales . Owing tulations . Currentof world , 4. Accoery year . Generalct pests .B. bassiana strains from a specific insect host varies frequently [B. bassiana isolated from a given insect showed low virulence against other insects, and showed stronger virulence against these insects after domestication [B. bassiana to hosts has a great plasticity. Genome structure and DNA sequence variation in these isolates could be related to many factors in the biology of this fungus. Recently, the re-sequencing of numerous individuals within pathogen species has been as a forceful tool to explore the genetic mechanism in fungal and fungal-host interaction studies [B. bassiana isolates with different levels of virulence and repellence to the termite, and then some differences were found in the nucleotide sequences of the two isolates of B. bassiana, suggesting a genetic basis for the observed intra-specific differences in their virulence against the termite [B. bassiana with low/high virulence, and finally identified several genes and molecular processes that effect the virulence towards Mosquitoes [B. bassiana isolates isolated from different hosts.Virulence is variable and not universal for a prescribed host and pathogen . Increasequently . It has tication , indicat studies . For exa termite . Valero-squitoes . These sB. bassiana strains by a comparative genomics analysis. Thus, we sequenced the 17 B. bassiana isolates from different hosts compared to the reference genome B. bassiana strain ARSEF 2860. Based on the analysis, understanding the potential factors of genetic variation on the virulence of B. bassiana will improve our methods to use this fungus as an effective and sustainable biological control agent against.In the present study, we aim to gain our knowledge in understanding genomic diversity involved in virulence and host-pathogen interaction for 17 B. bassiana strains were summarized in Table SB. bassiana strain ARSEF 2860 reference genome, with varying 67.30 to 75.30% among different strains  are synonymous and 820,233(29.51%) are nonsynonymous. In addition, a total of 884,811 Indels were obtained, with 423,409 (47.85%) InDel-Ins and 461,402 (52.15%) InDel-Del  in seventeen samples  have been widely applied to characterize potentially adaptive genetic variation [B. bassiana. Among the Indels, the number of InDel-Ins variants  is almost equal to the InDel-Del variants , and most of the variants were located in the upstream region of the genes (79.15%). Furthermore, structural variation is known to be a key component of phenotypic diversity in organisms [Firstly, 17 strains of SEF 2860 , which iariation , revealirganisms . In thisrganisms .B. bassiana isolates. However, significant nucleotide variations were identified in four genes  in all samples, and two of them encoding the common protein non-ribosomal peptide synthase . NRPS is a key mechanism for the biosynthesis of bioactive metabolites in fungi and plays an important role in the production of fungaltoxin. For example, NRP synthetase Pes3 has been found to contribute to Aspergillus. fumigatus virulence, and disruption of pes3 resulted in an augmented virulence phenotype in invertebrate for invasive aspergillosis. NRP synthetase mgoA has a role in the virulence of Pseudomonas. syringae pv. Syringae, and A Tn5 mutant disrupted in mgoA was defective in mangotoxin production and virulence. NRP synthetase gliP was predicted to be involved in gliotoxin production, and mutants with a disrupted gliP locus failed to produce gliotoxin [B. bassiana trains, which may be closely related to the maintenance of the pathogenesis during the evolution process, but the specific virulence effects of each mutation need to be further explored. Furthermore, GO annotations showed that the functions of mutated genes were mainly related to binding, catalytic activity, extracellular region, metabolic process, and nucleic acid binding transcription transporter activity. Mutated genes involved in binding, transport and other biological processes may be associated with increased interaction between virulence associated proteins and hosts when B. bassiana exerts a toxic infection. Additionally, among these groups, the variation involved in the metabolic process may be related to the production of antibacterial compounds, it is very important for B. bassiana to resist microbial competition in the process of killing insects [Secondly, a hierarchical clustering analysis was carried out on the top 50 genes with missense SNPs in 17 samples. The results showed that there was no significant difference in the frequency of missense SNP mutations among different hosts, which revealing the population identity of the seventeen sequenced liotoxin –23. Base insects .B. bassiana were determined using the whole missense SNPs. Three distinct B. bassiana clusters were formed, namely the isolates of S2\S3\S4\S5\S6\S8\S14\S19, the isolates of S2\S9\S10\S13\S15\S18\S19\S20\S21, and the isolates of S7/S11. Previously, the study of Ghikas et al. revealed that seven phylogenetic clusters in B. bassiana that consisted of isolates which had common climate features but did not exhibit any geographic distribution [B. bassiana is not monophyletic, but rather is composed of several clusters that are morphologically identical [B. bassiana. Additionally, the results showed that similar mutations occurred in B. bassiana strains in each cluster during evolution, which indicated a close relationship among B. bassiana isolates isolated different hosts in each cluster, but the relationship between the virulence of B. bassiana isolates in each cluster is not clear and further studies are needed. Furthermore, in the PCA plot, an obvious separation of three clusters was found, which also revealed a clear genetic differentiation among these 17 samples.Thirdly, the phylogenetic relationships and principal component analysis (PCA) between isolates of ribution . Our findentical –27, whicB. bassiana. Finally, a total of 7050 genes with Ka/Ks > 1 and 720 genes with Ka/Ks < 1 are confirmed, respectively. Among genes predicted to be under strong purification selection, there is enrichment for GO categories involving NAD + ADP-ribosyltransferase activity, chromatin, chromatin assembly or disassembly, chitin catabolic process, and actin cytoskeleton. In Salmonella, spv genes can establish successful systemic infection in experimental animals, and SpvB virulence-associated protein has recently been shown to contain the ADP-ribosyltransferase domain, suggesting that NAD + ADP-ribosyltransferase activity is associated with the regulation of virulence [Candida albicans [B. bassiana could secrete several chitin-degrading enzymes, such as Endo chitosanase and Chitinase D [B. bassiana virulence [B. bassiana strains with positive selection were abundantly enriched in this pathway. Increasing studies have revealed the involvement of this pathway in regulating resistance processes and diverse virulence in a wide range of pathogenic bacteria, such as Mycobacterium tuberculosis [Salmonella typhimurium [Staphylococcus aureus [Pseudomonas aeruginosa [B. bassiana strains.In previous study, genes with a signature of positive selection is possible to result in the adaptation to a dynamic environment and may be associated with fungi virulence . Herein,irulence . Chromatalbicans . Earlieralbicans . Chitin tinase D , which airulence . Additioirulence . In the rculosis , Salmonehimurium , Staphyls aureus , Pseudomruginosa . Taken tSalmonella was the direct result of the inactivation of both of the mutS and recD gene products, however, S. typhimurium carrying mutations in either one of these genes behaved similarly to the wild-type strain and without attenuating virulence [Mycobacterium tuberculosis, which could regulate many intracellular metabolic processes and interfere with signaling pathways of the infected host cell [B. bassiana strains may be closely related to its plasticity of virulence. Besides, other genes deletion or genes acquisition may also be critical in virulence evolution. Of course, further study is needed in the future.Finally, a comprehensive protein domain analysis was performed on 720 positively selected genes with missense mutations. Significant differences in 13 domains encoded by 13 protein-coding genes were identified. Among these, three domain proteins involved in the host-pathogen interaction, such as ankyrin repeat domain protein, Metallophos domain protein and MutS domain V that were encoded by gene-7731, gene-5369, and gene-10,314 may affect host-pathogen interactions. For example, Zahrt et al. found that the alteration in virulence on irulence . Ankyrinirulence , 39. Besirulence . The Metirulence . Earlierost cell . In summB. bassiana isolates obtained from different insect hosts. Subsequently, the relationship between stable genetic variation and virulence function shared by 17 strains of B. bassiana in different hosts, and the correlation between mutated genes and virulence effect of B. bassiana were analyzed. A number of genes with stable mutations in these B. bassiana isolates have been established. Among them, some key factors including NRPS mutations, domain changes of ankyrin repeat domain protein, Metallophos domain protein, and MutS domain occurred in B. bassiana isolates may be crucial for the virulence of B. bassiana. Besides, the 720 positive selection genes and the top 50 genes with missense mutations were significantly enriched in some important biological processes, and pathways may also be related to the virulence of B. bassiana against hosts. The study provided the relevant theoretical basis for targeted changes in the virulence effect of B. bassiana. In this case, we think the virulence of B. bassiana might be influenced by natural variation in the process of evolution, which may benefit future molecular investigations of insect-fungus interactions and promote the development of B. bassiana as cost-effective and sustainable mycoinsecticides. However, do these genetic mutations really play a role in virulence or how do it regulate virulence in biological processes, which is interested and will need to further study.According to the plasticity and variability of fungal virulence, we sequenced and compared the genome of seventeen B. bassiana that were obtained from 17 different insects (This study employed seventeen isolates of  insects . All isoB. bassiana was extracted using the CTAB method as the previous report [http://hannonlab.cshl.edu/fastx_toolkit) to filter the low-quality reads (< 10), reads shorter than 50 nt and duplicated reads [B. bassiana strain ARSEF 2860 [Genomic DNA of 17 isolates of s report . DNA coned reads . After tSEF 2860 . The aliSEF 2860 .http://picard.sourceforge.net) was used to discard duplicates, and the obtained BAM files were further processed using SAMtools 1.4.1 (samtools.sourceforge.net) [broadinstitute.org/gatk/) UnifiedGenotyper [http://snpeff.sourceforge.net/) [Picard tools version 1.92 (rge.net) . SNPs anenotyper . After tge.net/) .B. bassiana originates from different hosts. The optimal nucleic acid replacement mode was found by modelfinder. Then a phylogenetic tree was conducted using iqtree with the bootstrapping of 100 replicates [To determine the evolutionary relationship among plicates , and furplicates  on the mB. bassiana isolates were searched using the bidirectional best hit (BBH) method [The positively selected genes among the genomes of 17 ) method . The rathttp://www.genome.jp/kegg/) database to explore main biological functions [http://pfam.xfam.org/) annotation [A hierarchical clustering analysis of the top 50 genes with missense SNPs was conducted by the clustergram function in the Matlab Bioinformatics toolbox with minor changes. GO analysis was performed with Fisher’s Exact Test and classified under three categories: biological process (BP), cellular component (CC), and molecular function (MF) . Meanwhiunctions , and thenotation .Structural variants was determined using BreakDancer (v1.1.2) software , containB. bassiana database using hmmsearch in HMMER (v3.1b) with e-value < 0.00001 [To explore the effect of nonsynonymous SNPs in the positively selected genes on protein domains, we first identify the mutant amino acid sequences using Bioperl. Then, the hidden Markov model (HMM) profile for the NBS domain was applied to search the protein sequences in the  0.00001 .Additional file 1: Table S1. Quality control results of sequencing data of 17 B. bassiana strains. Table S2. Mutation information of the seven positively selected genes that are enriched in the two component systems. Table S3. The formation of the structure variants shared by seventeen B. bassiana isolates genomic sequences. Table S4. The information of the hosts of seventeen B. bassiana isolates. Table S5. The number of genes shared between the 17 isolates presented in each category in Fig.


Aegilops tauschii Cosson , which is naturally distributed in Central Eurasia, ranging from northern Syria and Turkey to western China, is considered a potential genetic resource for improving bread wheat. In this study, the chloroplast (cp) genomes of 17 Ae. tauschii accessions were reconstructed. The cp genome sizes ranged from 135,551 bp to 136,009 bp and contained a typical quadripartite structure of angiosperms. Within these genomes, we identified a total of 124 functional genes, including 82 protein-coding genes, 34 transfer RNA genes and eight ribosomal RNA genes, with 17 duplicated genes in the IRs. Although the comparative analysis revealed that the genomic structure  is conserved, a few variant loci were detected, predominantly in the non-coding regions (intergenic spacer regions). The phylogenetic relationships determined based on the complete genome sequences were consistent with the hypothesis that Ae. tauschii populations in the Yellow River region of China originated in South Asia not Xinjiang province or Iran, which could contribute to more effective utilization of wild germplasm resources. Furthermore, we confirmed that Ae. tauschii was derived from monophyletic speciation rather than hybrid speciation at the cp genome level. We also identified four variable genomic regions, rpl32-trnL-UAG, ccsA-ndhD, rbcL-psaI and rps18-rpl20, showing high levels of nucleotide polymorphisms, which may accordingly prove useful as cpDNA markers in studying the intraspecific genetic structure and diversity of Ae. tauschii.The D genome progenitor of bread wheat,  Aegilops tauschii Cosson , which is the D genome progenitor of common bread wheat, is characterized by abundant genetic variation and is noted for its strong tillering ability and high plant tolerance  . In addition, as a farmland weed that co-occurs with common wheat, Ae. tauschii is also found in the middle reaches of the Yellow River   showes more genetic differentiation than does the D genome of the common wheat  . Ae. tau, China) . On the ectively . Previouectively , as wellectively . Given ton wheat . Thus, ion wheat . MoreoveAe. tauschii , moving eastwards into northern Xinjiang  using the HiSeq X ten sequencing platform  with 150 bp read length. Low quality reads with a phred score <30 and 0.001 probability error were removed using Trimmomatic v0.36 (http://www.usadellab.org/cms/index.php?page=trimmomatic), and the remaining high quality fragment s were assembled into contigs using the SOAPdenovo v2.21 (http://soap.genomics.org.cn/soapdenovo.html). The assembled contigs were further aligned to the AL8/78 reference genome (GenBank No. KJ 614412) using BLASTN (http://www.ncbi.nlm.nih.gov). Finally, gaps in the genome sequence were filled using GapCloser v1.12 (http://soap.genomics.org.cn/soapdenovo.html).For each of the 17 Ae. tauschii was annotated using the Dual Organellar GenoMe Annotator program . Using AL8/78 as the reference genome, the cp genomes of T093 and AY81 were performed to visualize the sequence variations by mVISTA.Ae. tauschii accessions were examined using the complete cp genome sequences of the 17 accessions. In addition, in order to establish the origin of Ae. tauschii within the family Triticeae, we performed phylogenetic analysis using the genome sequences of the following 99 accessions: the 17 accessions newly sequenced in the present study, 56 accessions in the genus Aegilops, 24 accessions in the genus Triticum, and two accessions of Hordeum vulgare as the outgroups.The phylogenetic relationships among The sequences of these cp genomes were aligned using Geneious 9.0.5 software . Gaps were adjusted manually or removed. The alignment lengths with all gap positions removed were determined to be 135,984 bp (17 accessions) and 131,116 bp (99 accessions). We constructed two corresponding phylogenetic trees based on the maximum-likelihood (ML) method utilizing MEGA7.0 software . The booAe. tauschii accessions was performed by employing MAFFT alignment based on homologous genomic regions  and a LSC region   . The int,140 bp) .matK), an envelope membrane protein (cemA), a C-type cytochrome synthase (ccsA) as well as a protease (clpP) , of which 17 were duplicated genes . Among te (clpP) . As showe (clpP)  which wee (clpP) .representative genomes of three Ae. tauschii accessions were relatively conserved, and any translocations or inversions among these genomes were not identified. The IR regions showed lower sequence variation than either LSC or SSC regions , three accessions from Xinxiang , and a further five accessions from India, Iran, Turkmenistan, Turkistan and Pakistan, respectively.The two cp genome datasets (the 17 complete cp genome sequences assembled in this study and the 99 selected cp genome sequences) were used to reconstruct the corresponding phylogenetic trees. As shown in Triticum and Aegilops clusters  and the remaining 18 loci were found in coding regions. A relatively high value of nucleotide variability (Pi) was thus determined for non-coding regions, ranging from 0.00008 to 0.00635 with an average of 0.00133, which was approximately three times greater than that in the coding regions (average: 0.000432) (rpl32-trnL-UAG (0.00478), ccsA-ndhD (0.00483), rbcL-psaI (0.00492), and rps18-rpl20 (0.00635), which are located in intergenic regions (the former two in the SSC region and the latter two in the LSC region), displayed the highest nucleotide polymorphisms (Ae. tauschii.In order to elucidate variant characteristics among the 17 .000432) . Among torphisms . Moreoveorphisms , which wAe. tauschii accessions, all of which display the typical angiosperms structure and harbor an identical component of 107 unigenes arrayed in the same order .In this study, we sequenced the cp genomes of 17 geographically dispersed me order . These gme order . Differeme order , with acme order . Comparame order , we deteAe. tauschii in China, the Yili Area of Xinjiang is undisputedly considered to represent the easternmost boundary of the natural distribution of wild Ae. tauschii population  clustered closely in a small branch with AY22 from Pakistan (South Asia), wheras Xinjiang appeared to clustered together with Central Asian (Ae. tauschii accessions collected from the Yellow River region (Henan and Shaanxi) could have originated from South Asian populations rather than from those in Xinjiang Province in China  and Ae. speltoides (B genome), we found that Ae. tauschii firstly clustered together with the other Aegilops specie. If the D genome is derived from an ancient hybridization between the A and B genomes, Ae. tauschii should be closely clustered with T. urartu  or Ae. speltoides . Thus, we proposed that Ae. tauschii is derived from monophyletic speciation rather than ancient hybridization. In brief, these phylogeny results will serve as a reference framework for future studies on Triticeae or Ae. tauschii.In order to gain further insights into the origin of the D genome origin of common wheat, we we performed phylogenetic analysis based on the cp genomes of 99 accession in the e family , and foueciation . Howevereciation . On the rpl32-trnL-UAG, ccsA-ndhD, rbcL-psaI and rps18-rpl20) located in the non-coding regions showed notably high levels of nucleotide polymorphisms, two of which  are located in the SSC region and the other two loci  are found in the LSC region  with relatively high levels of intraspecific variation, which can be used for population genetic analyses or serve as specific DNA barcodes of Ae. tauschii  of coding genes, intron regions and intergenic regions using DnaSP. The results revealed that the sequence divergence of the IR regions appeared to be lower than that of the LSC and SSC regions, which has also been noted in other angiosperms and may possibly be attributed to copy correction of the IR regions via gene conversion . The IR C region . PreviouhF-rpl32 . Here, wtauschii .Ae. tauschii accessions we assembled using Illumina next-generation DNA sequencing technology. Intraspecific differences among the cp genomes were detected primarily in non-coding regions (intergenic spacer regions) which are the main features contributing to the observed differences in genome size. An analysis of the phylogenetic relationships among the accessions based on the complete genome sequences indicated that Ae. tauschii accessions from the Yellow River region in China might have originated directly from South Asia. We also confirmed that Ae. tauschii is derived from monophyletic speciation rather than hybrid speciation. Furthermore, we identified four cpDNA marker sequences  that can be used to study inter- and intraspecific genetic structure and diversity of Ae. tauschii.In this study, with respect to gene order, gene number, and IR/SC boundary regions, we demonstrate that genomic structure is well conserved among the cp genomes of the 17 10.7717/peerj.8678/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.8678/supp-2Table S1Click here for additional data file.10.7717/peerj.8678/supp-3Table S2Click here for additional data file.10.7717/peerj.8678/supp-4Table S3Click here for additional data file.10.7717/peerj.8678/supp-5Table S4Click here for additional data file.10.7717/peerj.8678/supp-6Figure S1Click here for additional data file.10.7717/peerj.8678/supp-7Figure S2The vertical scale of mVISTA visualization image denotes the percentage of identity, ranging from 50% to 100%. The horizontal axis marked color code as protein-coding genes, rRNA, tRNA or conserved non-coding regions.Click here for additional data file.10.7717/peerj.8678/supp-8Fgirue S3Click here for additional data file.


Trypanosoma cruzi, the causative agent of Chagas disease, and T. rangeli are kinetoplastid parasites endemic to Latin America. Although closely related to T. cruzi and capable of infecting humans, T. rangeli is non-pathogenic. Both parasite species are transmitted by triatomine bugs, and the presence of T. rangeli constitutes a confounding factor in the study of Chagas disease prevalence and transmission dynamics. Trypanosoma cruzi possesses high molecular heterogeneity: seven discrete typing units (DTUs) are currently recognized. In Ecuador, T. cruzi TcI and T. rangeli KP1(-) predominate, while other genetic lineages are seldom reported.T. cruzi and/or T. rangeli in different developmental stages of triatomine bugs from two communities of southern Ecuador was evaluated via polymerase chain reaction product size polymorphism of kinetoplast minicircle sequences and the non-transcribed spacer region of the mini-exon gene (n = 48). Forty-three mini-exon amplicons were also deep sequenced to analyze single-nucleotide polymorphisms within single and mixed infections. Mini-exon products from ten monoclonal reference strains were included as controls.Infection by Trypanosoma cruzi genetic richness and diversity was not significantly greater in adult vectors than in nymphal stages III and V. In contrast, instar V individuals showed significantly higher T. rangeli richness when compared with other developmental stages. Among infected triatomines, deep sequencing revealed one T. rangeli infection (3%), 8 T. cruzi infections (23.5%) and 25 T. cruzi + T. rangeli co-infections (73.5%), suggesting that T. rangeli prevalence has been largely underestimated in the region. Furthermore, deep sequencing detected TcIV sequences in nine samples; this DTU had not previously been reported in Loja Province.T. cruzi and T. rangeli, or when multiple T. cruzi DTUs are present. Additionally, our analysis showed extensive overlap among the parasite populations present in the two studied localities (c.28 km apart), suggesting active parasite dispersal over the study area. Our results highlight the value of amplicon sequencing methodologies to clarify the population dynamics of kinetoplastid parasites in endemic regions and inform control campaigns in southern Ecuador.Our data indicate that deep sequencing allows for better parasite identification/typing than amplicon size analysis alone for mixed infections containing both  Trypanosoma cruzi and affects 6–7 million people worldwide [Chagas disease (CD) is caused by the kinetoplastid hemoflagellate parasite orldwide . This neorldwide . Transmiorldwide .Trypanosoma cruzi has high molecular and phenotypic heterogeneity, and the existence of genetic lineages has been long recognized. Initially, three “zymodemes”  were identified via multilocus enzyme electrophoresis [phoresis , 5. Subsphoresis –12. In Ephoresis , 14 and phoresis . Only twphoresis , 17.Triatoma dimidiata, T. carrioni, Panstrongylus chinai, P. rufotuberculatus and Rhodnius ecuadoriensis are epidemiologically relevant vector species [Rhodnius ecuadoriensis is widely distributed in the western lowlands and the southern provinces of the country, as well as in northern Peru [In Ecuador,  species , 19. Rhoern Peru , both inern Peru .Trypanosoma rangeli, a related kinetoplastid parasite whose geographical distribution overlaps with that of T. cruzi, may also be found in the same triatomine vectors and mammalian hosts. Mixed infections with both species are often reported [T. rangeli is epidemiologically relevant since it may cause false-positive results in microscopical and serological tests used for diagnosis of T. cruzi infection [T. cruzi, transmission of T. rangeli to vertebrate hosts occurs via the salivary route, although the possibility of transmission through infected feces has also been suggested [T. rangeli trypomastigotes accumulate in the digestive tract of triatomines, replicate, transform into epimastigotes, cross the intestinal epithelium into the hemocoel and migrate to the salivary glands. Infective metacyclic trypomastigotes are released with the saliva during a blood meal [T. rangeli in Ecuador. PCR minicircle amplification of > 3600 samples revealed that 10% of triatomines and mammals from Manabí and Loja provinces were infected with T. rangeli, while 1.25% presented T. cruzi + T. rangeli co-infections [reported , 22, 23.nfection , 25. In uggested , 27. Upoood meal . Little fections .T. cruzi, there are four copies of the conserved sequence [Trypanosoma rangeli may present one, two or four copies of the mini-repeat. These three different minicircle classes in T. rangeli are known respectively as KP1, KP2 and KP3 [SSU rRNA), the internal transcribed spacer of rDNA (ITS rDNA) and the intergenic region of the splice leader, has unveiled a more complex population structure for T. rangeli, with five groups defined as A, B, C, D and E, each of which shows a strong association with vector species and geographical distribution [T. rangeli: (i) the Amazonian group, associated to Rhodnius brethesi and vertebrate hosts; (ii) the KP1(-) group, linked to the R. pallescens complex; and (iii) the KP1(+) group, related to the R. robustus complex.The kinetoplast DNA of trypanosomatid parasites is composed of maxicircles and minicircles, whose number varies between species. Minicircles possess a conserved 100–200 bp repetitive region, whose number of repeats also differs among species. In sequence . Trypano and KP3 , 32. Furribution , 33, 34.ribution  suggesteT. cruzi in mammalian reservoirs and invertebrate vectors is frequently multiclonal, involving several intra-specific genotypes with genetically dissimilar profiles [Infection by profiles –39. Multprofiles , diseaseprofiles . They caprofiles .Trypanosoma, mini-exon genes are present in several tandemly arranged copies and encode the splice leader, a 35-nucleotide sequence translocated to the 5’-end of every newly synthesized mRNA [Trypanosoma cruzi mini-exon genes include conserved, semi-conserved and highly variable regions, which have been used for phylogenetic analysis [T. cruzi DTU detection and identification [In the genus zed mRNA , 44 in azed mRNA . Trypanoanalysis , discrimanalysis , populatanalysis , and moranalysis . Nonethefication . Given ification –55.T. cruzi infections in humans [T. cruzi and T. rangeli in two localities of southern Ecuador via analysis of the mini-exon gene. We analyzed 48 intestinal DNA extracts from triatomines collected in Loja Province as well as DNA from ten cloned isolates of reference strain DTUs I, III, IV, V and VI in order to identify relationships between mini-exon diversity and DTU identity. Mini-exon amplicon sequence reads generated by NGS were sorted into haplotype clusters to measure genetic richness, diversity, variability and substructure in the landscape. Moreover, the dataset included samples isolated from different developmental stages of the vector to evaluate a potential correlation with mini-exon diversity. We also compared genotyping results from NGS and PCR product size polymorphism analysis in terms of sensitivity for discrimination between T. cruzi and T. rangeli parasites.Next-generation sequencing (NGS) has helped provide insights into multiclonal n humans  and to an humans . In the T. cruzi (330 bp fragment size expected) and T. rangeli (380 bp). All samples appeared to be infected exclusively with T. cruzi based on visualization of 121/122 amplicons (data not shown).Archived DNA samples collected between years 2009–2013 by the Center for Research on Health in Latin America (CISeAL) were employed in the study. Details of entomological searches, intestinal content DNA extractions and corresponding approved protocols and collection permits have been reported elsewhere , 23. SamForty-eight samples from two rural communities from southern Ecuador (Loja Province) were included in the sample panel: 31 from Bramaderos ; and 17 from Bellamaría   following a custom protocol . Paired-RCH v8.1 . ChimeriT. cruzi and T. rangeli) independently and combined. Data corresponding to samples TBM2823, TBR1487 and TBR1489 were excluded from this analysis because they are the only representatives of instar IV. TBR1446 and TBR1503 were also excluded to avoid bias because the sequence reads from these samples has been merged across four sequencing reactions.Richness was defined as the total number of haplotypes found in each sample. Shannonʼs index was defined as -Ʃ(Piln(Pi)) . As mentpost-hoc test. A P-value of ≤ 0.05 was considered significant.An analysis of variance (ANOVA) was performed when data showed normality and homoscedasticity. When data violated normality, a Kruskal-Wallis test was employed, followed by a Bonferroni-adjusted Dunn’s The monoclonal reference strains included in the study were Chilec22 cl.6 (TcI), M5521 cl.3 and cl.5 (TcIII), Arma18 cl.1 and cl.5 (TcIII), Saimiri3 cl.8 (TcIV), Para6 cl.1 (TcV), Para7 cl.3 (TcVI), Chaco9 cl.15 (TcVI) and LHVA cl.1 (TcVI) , 50, 64.n = 18) of the samples were co-infected with T. cruzi and T. rangeli, while 52.1% (n = 25) presented infection exclusively with T. cruzi and 6.25% (n = 3) were infected only with T. rangeli. Samples TBM2873 and TBM2983 showed faint bands in agarose gels and no clear pattern was obtained. Meanwhile, NGS was performed only on 43 samples from the Ecuadorian dataset; 5 were not sequenced due to technical issues. An additional 9 samples were excluded from further analysis because they did not reach the 20,000 reads threshold, indicating insufficient sequencing depth. Among the 34 samples for which NGS was successfully performed, taxonomic assignment in GenBank identified sequence types associated with both T. cruzi and T. rangeli in 25, while 8 were deemed to contain exclusively T. cruzi-like sequences, and only one had exclusively T. rangeli-like sequences (TBR1455). Remarkably, sequence types matching DTU TcIV were found in samples TBR1410, TBR1422, TBR1445, TBR1475, TBR1492, TBR1510, TBM2873, TBM2903 and TBM2983  tree alongside selected sequence types derived from Ecuadorian strains and ces Fig. .Fig. 4NJT. rangeli haplotypes yielded a rather uninformative clustering with several polytomies. Therefore, only selected T. rangeli haplotypes were included along with available sequence data retrieved from NCBI . The Bonferroni-adjusted Dunn’s post-hoc test showed significant differences between instar V nymphs and adult females (P = 0.0124) and among instar V nymphs and the merged group of adult females and males (P = 0.0138). In both cases, instar V showed significantly higher richness than the remaining developmental stages.Chao richness and Shannonʼs diversity indices were calculated for each parasite species within each sample. No significant differences in richness or diversity were found between samples from the two studied localities (Bramaderos and Bellamaria) for either parasite species. Analyses were performed merging both male and female adults and with adults from each sex taken independently. In both cases, we found significant differences for T. cruzi based on PCR product size polymorphism of kinetoplast minicircle sequences, with no evidence of co-infection with the sympatric sister species T. rangeli. The sample set was subsequently analyzed by two additional molecular techniques; first by PCR product size polymorphism analysis of the non-transcribed spacer region of the mini-exon gene, then by deep sequencing of these mini-exon amplicons. Incongruences were detected among the results obtained by the three methods, and our results suggest that simple amplicon visualization of kinetoplast minicircle sequences may not suffice for detection of T. cruzi + T. rangeli co-infections.We compared the results from three different types of molecular analysis for 48 intestinal content DNA samples from triatomine bugs collected in two rural communities of Loja Province, southern Ecuador. All samples selected for the study had been previously characterized as infected with T. cruzi + T. rangeli co-infections in our sample panel, which the kinetoplast minicircle PCR [T. cruzi and T. rangeli in vector and mammalian hosts are frequent [T. rangeli infection in sampled triatomines and mammals and just ~1.25% in co-infection with T. cruzi [T. rangeli-like sequences in 26 out of 34 (76.47%) infected samples characterized in our study.The PCR-based method for mini-exon gene analysis without sequencing  uncoverercle PCR  did not frequent , 65, 66 frequent . PreviouT. cruzi . In starR. ecuadoriensis (45 out of 48 DNA samples were isolated from this vector species), one of the two most important vectors of CD in the study region and in Ecuador at large [T. rangeli and Rhodnius spp. has been established in the past, where 12 out of 15 species of the genus have shown vectorial capacity [T. rangeli infection in R. ecuadoriensis may underestimate the presence of this parasite in southern Ecuador, where a wider distribution of T. rangeli in Loja, and perhaps the rest of the country, may have been overlooked. Furthermore, although only a ~100-bp fragment was analyzed, a wide T. rangeli genetic diversity was encountered (79 haplotypes in 26 triatomines). Consistent with previous reports, where 99% of Ecuadorian T. rangeli were classified as KP1(−), and 80% as lineage C [T. rangeli amplicon and the limited number of informative SNPs found within the sequence (some haplotypes differ by only one SNP). Notably, we did not identify any KP1(+) haplotypes, although previous reports indicate KP1(+) and (−) lineages are sympatric and may be found in the intestinal contents of Rhodnius spp. [Our sample panel was heavily biased toward at large . A closecapacity . Our resineage C , NJ analius spp. .T. cruzi and T. rangeli occurred in 73.5% of analyzed triatomines. Despite earlier reports suggesting T. rangeli infection is pathogenic to triatomines, more recent studies point to the need of more solid evidence before generalizing this claim [R. prolixus survival, reproduction and fitness, which in turn would favor transmission of both parasite species [R. ecuadoriensis and its possible impact over the epidemiology of Chagas disease in the south of Ecuador warrants further investigation.Co-infection with is claim . Indeed,T. cruzi intra-strain variability [T. cruzi diversity in the intestinal content of T. dimidiata [T. cruzi naturally infecting a group of captive non-human primates in the southern USA. In our study, 14.5 T. cruzi and 14.4 T. rangeli haplotypes were detected on average per triatomine. However, we do note from our analysis of monoclonal reference samples that mini-exon sequences may not accurately resolve T. cruzi phylogenetic diversity, thus reflecting to an extent the lack of reliability of this locus in identifying DTUs. From monoclonal strain haplotypes, our data showed that some low abundance sequence types are shared by almost all DTUs, with important implications for those using the mini-exon splice leader locus to define intra-host DTU diversity /C, in congruence with previous reports. Additionally, we provide the first report for the presence of DTU TcIV in Loja Province. In conclusion, we demonstrate the power of NGS to scrutinize parasite populations with high resolution. However, we do note that the mini-exon marker must be approached with caution, and further deep sequencing analyses targeted at additional markers, potentially conserved house-keeping genes [T. cruzi transmission in southern Ecuador and must be considered in disease control efforts in the region.Our results indicate that techniques based solely on PCR amplification of minicircle or mini-exon sequences are not optimal for detection and characterization of mixed infections with kinetoplastid parasites in triatomines. Employing deep sequencing of the non-transcribed spacer region of the mini-exon gene, we have encountered 73.5% (considering the 34 samples with NGS results) of studied triatomines to be co-infected with ng genes , are reqAdditional file 1: Table S1. Raw data for monoclones and Ecuadorian samples. Haplotype ID, identity and number of reads are included.Additional file 2: Table S2. Correspondence between FLDs* (Illumina sequencer output) and samples belonging to intestinal contents.Additional file 3: Alignment S1. Sequences employed for the phylogenetic tree displayed in Fig. Additional file 4: Alignment S2. Sequences employed for the phylogenetic tree displayed in Fig. Additional file 5: Table S3. Accession numbers of downloaded sequences from NCBI database used in the generation of the NJ tree for T. rangeli. The retrieved sequences were trimmed to match with the amplicons generated with Ecuadorian samples. The discarded portions of the sequences are shown. Lineage  and KP1(+/−) information was included when available. Geographical origin of the samples is also in the file.


Neisseria gonorrhoeae.It is unclear how important bystander selection is in the genesis of antimicrobial resistance (AMR) in We assessed bystander selection in a novel way. Mixed-effects linear regression was used to assess if country-level prevalence of gonococcal AMR in 30 European countries predicts homologous AMR in other bacteria. The data used was from the European Antimicrobial Resistance Surveillance Network.E. coli , Acinetobacter spp.  and Pseudomonas aeruginosa  but not Klebsiella pneumoniae. Azithromycin resistance in N. gonorrhoeae was positively associated with macrolide resistance in Streptococcus pneumoniae . No association was found for cephalosporins.The prevalence of gonococcal ciprofloxacin resistance was found to be positively associated with AMR prevalence in Gonococcal AMR is linked to that in other bacteria. This finding is likely explained by high antimicrobial consumption in affected populations and provides additional motivation for strengthening antimicrobial stewardship programs. Neisseria gonorrhoeae has developed antimicrobial resistance (AMR) to every major class of antimicrobials used to treat it [N. gonorrhoeae is vital to prevent the future emergence of AMR. Initially, it was thought that the major AMR determinant was exposure to antibiotics used to treat gonorrhoea [N. gonorrhoeae is asymptomatic for much or most of the time it circulates in a population means that antibiotics used for other indications needed to be considered (bystander selection) [Mycoplasma genitalium genital infections may drive AMR in N. gonorrhoeae, little direct evidence to support this notion has been published [Treponema pallidum, the bacterium responsible for syphilis, was strongly associated with population-level consumption of macrolides [treat it , 2. Thertreat it . Understnorrhoea –6. The alection) . Since glection) . While aublished . One stucrolides . Other scrolides . Understcrolides .Fig. 1CN. gonorrhoeae, with some studies finding no association [N. gonorrhoeae where travel has been shown to have played a vital role in the spread of AMR [Ecological studies have reached different conclusions regarding the association between general antimicrobial consumption and the emergence of AMR in ociation  and otheociation , 13, 14.ociation . For exaociation . Exposurociation . This prd of AMR . The prod of AMR –20. The These limitations motivated the current study where we approach the problem via a novel methodology. In addition to assessing the traditional association between antimicrobial consumption and gonococcal AMR, we analyze the association between gonococcal AMR and homologous AMR in other pathobionts. The hypothesis we test is that gonococcal AMR is associated with AMR in these pathobionts Fig. . FindingEscherichia coli, Klebsiella pneumoniae, Streptococcus pneumoniae, Acinetobacter spp., Pseudomonas aeruginosa, Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus), with the exception of Greece which did not report data on S. pneumoniae. Only data from invasive  isolates are included in EARS-Net. This is done to limit biases that may emerge if isolates from all anatomical sites were included. Further details can be found in the EARS-Net reporting protocol [https://www.ecdc.europa.eu/en/surveillance-atlas-infectious-diseasesThe AMR data was taken from the European Centre for Disease Prevention and Control (ECDC) Surveillance Atlas which reports resistance prevalence estimates from the European Antimicrobial Resistance Surveillance Network (EARS-Net) - the EU’s main surveillance system for AMR in bacteria that cause serious infections. All 28 EU Member States and two EEA countries (Iceland and Norway) participate in EARS-Net. The countries provide data for all eight species under surveillance . The following minimum inhibitory concentration (MIC) breakpoints were used to define gonococcal antimicrobial resistance: Azithromycin: > 0.5 mg/L, Cefixime: > 0.12 mg/L, Ceftriaxone: > 0.12 mg/L, Ciprofloxacin: > 0.06 mg/L [The ECDC Surveillance Atlas reports gonococcal AMR for ciprofloxacin (a fluoroquinolone), azithromycin (a macrolide), cefixime and ceftriaxone (both Extended Spectrum (ES) cephalosporins) by country and year for 30 countries from 1997 to 2018. Since our hypothesis tested homologous class bystander selection, we limited our analysis to the 5 bacterial species that reported AMR for fluoroquinolones, macrolides or ESCephalosporins  were used as a measure of national general population-level antimicrobial drug consumption , 26. ESAN. gonorrhoeae and each of the other bacterial species. The following mixed effects linear model was used:For each antibiotic class, mixed effects linear regression was used to assess the association between the prevalence of AMR in N.gonorrhoeae_resistance-to-X in year Y and country C) ~ (Species-Z_resistance-to-X in year Y and country C) + (random intercept for country C) + intercept + error, where species-Z is one of E. coli, K. pneumoniae, S. pneumoniae, Acinetobacter spp. or P. aeruginosa, and X could be fluoroquinolones, macrolides or ESCephalosporins. ~  + (random intercept for country C) + intercept + error.P-value of < 0.05 was regarded as significant.The statistical analyses were performed in Stata 16.0. A This analysis involved ecological analyses of public access data and thus no Ethics approval was necessary.There were large variations (up to 300-fold) in the consumption of cephalosporins, fluoroquinolones and macrolides between countries Table . The medE. coli and K. pneumoniae. The prevalence of fluoroquinolone resistance in N. gonorrhoeae declined over time.For each of the bacterial species, the prevalence of AMR varied considerably between countries Table . There wE. coli , Acinetobacter spp.  and P. aeruginosa  but not K. pneumoniae , Acinetobacter spp. , N. gonorrhoeae  and K. pneumoniae  but not P. aeruginosa , but not N. gonorrhoeae was associated with macrolide consumption  consumption and prevalence of antimicrobial resistance to fluoroquinolones in Neisseria gonorrhoeae (Ng) and Escherichia coli (Ec) in 30 European countries. Figure S2. Macrolide consumption and prevalence of antimicrobial resistance to azithromycin in Neisseria gonorrhoeae (Ng) and macrolides in Streptococcus pneumoniae (Sp) in 30 European countries.


Microhyla include some of the world’s smallest amphibians and represent the largest radiation of Asian microhylids, currently encompassing 50 species, distributed across the Oriental biogeographic region. The genus Microhyla remains one of the taxonomically most challenging groups of Asian frogs and was found to be paraphyletic with respect to large-sized fossorial Glyphoglossus. In this study we present a time-calibrated phylogeny for frogs in the genus Microhyla, and discuss taxonomy, historical biogeography, and morphological evolution of these frogs. Our updated phylogeny of the genus with nearly complete taxon sampling includes 48 nominal Microhyla species and several undescribed candidate species. Phylogenetic analyses of 3,207 bp of combined mtDNA and nuDNA data recovered three well-supported groups: the Glyphoglossus clade, Southeast Asian Microhyla II clade (includes M. annectens species group), and a diverse Microhyla I clade including all other species. Within the largest major clade of Microhyla are seven well-supported subclades that we identify as the M. achatina, M. fissipes, M. berdmorei, M. superciliaris, M. ornata, M. butleri, and M. palmipes species groups. The phylogenetic position of 12 poorly known Microhyla species is clarified for the first time. These phylogenetic results, along with molecular clock and ancestral area analyses, show the Microhyla—Glyphoglossus assemblage to have originated in Southeast Asia in the middle Eocene just after the first hypothesized land connections between the Indian Plate and the Asian mainland. While Glyphoglossus and Microhyla II remained within their ancestral ranges, Microhyla I expanded its distribution generally east to west, colonizing and diversifying through the Cenozoic. The Indian Subcontinent was colonized by members of five Microhyla species groups independently, starting with the end Oligocene—early Miocene that coincides with an onset of seasonally dry climates in South Asia. Body size evolution modeling suggests that four groups of Microhyla have independently achieved extreme miniaturization with adult body size below 15 mm. Three of the five smallest Microhyla species are obligate phytotelm-breeders and we argue that their peculiar reproductive biology may be a factor involved in miniaturization. Body size increases in Microhyla—Glyphoglossus seem to be associated with a burrowing adaptation to seasonally dry habitats. Species delimitation analyses suggest a vast underestimation of species richness and diversity in Microhyla and reveal 15–33 undescribed species. We revalidate M. nepenthicola, synonymize M. pulverata with M. marmorata, and provide insights on taxonomic statuses of a number of poorly known species. Further integrative studies, combining evidence from phylogeny, morphology, advertisement calls, and behavior will result in a better systematic understanding of this morphologically cryptic radiation of Asian frogs.Frogs of the genus  The tropical areas of South and Southeast Asia include biogeographic regions recognized as global centers of biodiversity . UnderstThe tectonic collision between the Indian subcontinent (ISC) and the Eurasian landmass during the Early Cenozoic is widely recognized as a key event that caused significant geologic and climatic changes, such as the rise of the Himalaya, uplift of the Tibetan plateau, and a general drying of Central Asia . This teThere is an ongoing debate on timing and topography of the ISC–Eurasian collision , 2012. SFrogs of the family Microhylidae are some of the most species rich groups of Anura, comprising 690 species in 12 subfamilies . BecauseMicrohyla is the most species rich genus in the Microhylinae, currently comprising 50 recognized species ; phylogenetic information for 12 species and a number of candidate new species is reported for the first time. We use the resulting phylogeny (based on both mtDNA and nuDNA markers) to test biogeographic hypotheses in space and time and provide a scenario for Microhyla diversification. Our study provides the first nearly complete phylogeny for the genus Microhyla and links the Indian Subcontinent with Sundaland, Indo-Burma, and East Asia, thereby allowing a better understanding of biogeographic history and diversification of the group. We also evaluate miniaturization and simulate body size evolution across different lineages of the genus, providing novel insights into morphological evolution in Microhyla.Herein, we identify unrecognized diversity and examine phylogenetic relationships among almost all described species of We used tissues from the herpetological collections of Zoological Museum of Moscow University ; Zoological Institute, Russian Academy of Sciences ; Vertebrate Zoology Department, Biological Faculty, Moscow State University ; Amphibian Research Center, Hiroshima University ; Danum Valley Conservation Area, Specimen collection ; Department of Fisheries, Bangamata Sheikh Fazilatunnesa Mujib Science & Technology University ; School of Agriculture and Natural Resources, University of Phayao ; and the Institute of Biodiversity and Environmental Conservation, Universiti Malaysia Sarawak   extraction procedures with consequent isopropanol precipitation . This marker was recently successfully applied in biodiversity and phylogenetic studies of Indian Microhyla species ;A mtDNA dataset joining 12S rRNA and 16S rRNA for all examined samples, used for assessment of species groups and estimation of cryptic diversity within BDNF gene sequences for 118 selected samples representing all major lineages within Microhyla , used for obtaining a more robust phylogenetic hypothesis, time-tree estimation, and ancestral range reconstruction for Microhyla.A concatenated mtDNA + nuDNA dataset, joining long 12S rRNA—16S rRNA mtDNA fragment and BDNF gene was constructed using median-joining method in the PopArt v1.5  using default parameters .The ABGD method is a single-gene approach to statistical detection of barcode gaps in a pairwise genetic distance distribution . BarcodeThe GMYC method is also a single-gene approach to identifying species “boundaries” associated with shifts in branching rates between intra- and interspecies branching events on a time-calibrated ultrametric tree . We usedp-distances were calculated using MEGA 6.1  Maihyla see . Analysehyla see : Langranhyla see , and thehyla see . We asseGiven the tremendous geological complexity of the region through time, we applied the following time and dispersal constraints to the analyses: we set four periods, corresponding to the main stages of gradual ISC movements northwards, formation of the first land bridges between the ISC and Southeast Asia, and final accretion between the ISC and the Asian mainland based on data from recent geologic models , five species (of nine currently recognized species) of the phylogenetically closely related genus Glyphoglossus, and 24 samples from outgroup taxa , although with generally higher node support values in our study. All analyses unambiguously supported the monophyly of the Microhyla—Glyphoglossus assemblage; however, the basal node of this radiation was not sufficiently resolved in all analyses  , which is further subdivided into two subclades: (A1) comprising species from Sundaland  and southern Vietnam (M. minuta) (0.97/68), and (A2) with species from Sundaland , Myanmar , and southern India  (1.0/87). Monophyly of clade A was not supported in the “total evidence” analysis  Clade A corresponded to ctively) . Genealo0.72/50; , while t0.91/62; . The M.  network  and was M. fissipes species group (1.0/100) and consisted of two well-supported subclades , and species from eastern India, Bangladesh and the Andaman Islands  (1.0/100); (B2) encompassing species from mainland China  and the Ryukyus  (1.0/98). In the BDNF gene haplotype network, M. okinavensis and Microhyla sp. 3 (B2) were distantly placed from members of the M. fissipes species group with a minimum of 14 mutational steps  Clade B corresponded to ubclades : (B1) inal steps , also ag0.96/55; .M. berdmorei complex and M. pulchra from Indochina and southern China, as well as M. picta from southernmost Vietnam (1.0/100). Clade C was recovered as sister clade to a clade A + B  Clade C included the al steps .M. zeylanica, M. karunaratnei) and southern India  , western Thailand (Microhyla sp. 2 from Tenasserim) and Thai-Malay Peninsula (M. superciliaris). The BDNF haplotype network suggests distant placement of Microhyla sp. 2, separated by at least seven mutational steps from other members of the M. superciliaris group ((D) Clade D encompassed species from Sri Lanka  and M. rubra complex members . In the BDNF gene haplotype networks, subclades E1 and E2 are separated from each other by at least three mutational steps ((E) Clade E included species with distribution on the Indian Subcontinent and Sri Lanka, including al steps . MtDNA dal steps , but it al steps . A similal steps , but notal steps .M. butleri species group and joined the M. butleri complex from southern China and Southeast Asia with M. aurantiventris from central Vietnam (1.0/100). Clade F was strongly supported as a sister lineage with respect to Clade D based on the “total evidence” analysis (0.96/67) (BDNF gene haplotype network ((F) Clade F corresponds to 0.96/67)  and the  network .M. palmipes species complex from Java and Sumatra; its phylogenetic position was poorly supported. In contrast to earlier data, suggesting sister relationships between M. palmipes and the group joining A + B + C + E clades  Clade G was represented by the 0.99/56; , our “to0.92/41; . The BDNal steps .Microhyla II (subclade AIII of M. annectens species group and was further subdivided in two subclades: (H1) included species from Borneo , the Thai-Malay Peninsula (M. annectens), and Annamite Mountains of Indochina  (1.0/88); and (H2) joined species from central (M. nanapollexa) and southern parts of Annamite Mountains . M. marmorata was recovered paraphyletic, with respect to M. pulverata  Clade H corresponds to 1.0/100; . In the lineated .Glyphoglossus , and a number of deep lineages within species complexes . In most cases, analyses had congruent results, however in 14 species, bGMYC proposed more groups than were recovered by ABGD. Nonetheless, species delimitation analyses strongly indicate the presence of many unrecognized species-level lineages by further partitioning M. heymonsi (into 7–8 species), M. bermodrei (3–4 species), M. malang (3 species), M. butleri (2–4 species), and M. gadjahmadai, M. okinavensis, M. palmipes, M. perparva, M. petrigena, M. achatina and M. arboricola (each with 2 species)   . For nina sp. 4) .Microhyla and Glyphoglossus originated between late Paleocene and early Eocene, ca. 50.8 Ma (44.1–57.0), and agrees with the analysis of Microhyla + Glyphoglossus radiated within a relatively narrow time period in the middle Eocene ca. 43.8 Ma (38.7–49.1), slightly younger than estimates of Microhyla I, Microhyla II, and Glyphoglossus clades started in early to mid-Oligocene (from 35 to 25 Ma), generally agreeing with The resulting BEAST chronogram see  elucidatMicrohyla using the RASP biogeographic analysis. According to both the DEC and the S-DIVA model, the MRCA of Microhyla + Glyphoglossus  and ancestral areas  for the node 12,  most liknode 30, . Microhyontinent . Resultsontinent .Microhyla and Glyphoglossus are shown in Glyphoglossus  are generally larger compared to Microhyla ; the MRCA of the Microhyla + Glyphoglossus assemblage is reconstructed as a mid-sized frog . Most species of Microhyla were found to be smaller  than their common ancestor, with five cases of miniaturization . However, both Glyphoglossus and M. berdmorei species groups of Microhyla I have subsequently increased their body size   .Microhyla, with the most complete taxon sampling including 48 of the 50 currently recognized species. The absent taxa in our study are M. fusca and M. darevskii—two enigmatic species from central and southern Vietnam. Microhyla fusca was described from a single specimen collected from southern Vietnam . Subsequent multilocus phylogenetic studies , but also from Myanmar  and southern India . This group includes tiny , Java + Bali , and Borneo . Our study also supports previously reported placement of south Indian M. kodial in one clade with Myanmar species M. irrawaddy and M. sp. 4 (M. achatina + M. gadjahmadai clade from Java and Sumatra (subgroup A2). Unfortunately, phylogenetic positions of the M. heymonsi complex and M. pineticola remain unresolved. The morphologically different semi-fossorial M. fodiens from central Myanmar, previously identified as M. rubra , Indochina, and eastern India . We also agree with M. achatina group, though monophyly of the latter is only moderately supported  and East Asian (B2) taxa  taxa  (Yuan etB2) taxa , supportnghensis , and sugnavensis .Microhyla berdmorei group , M. pulchra , and stout-bodied semi-fossorial M. picta from southern Vietnam (SVL up to 33.4 mm). The phylogenetic position of M. picta was investigated here for the first time, and was recovered as sister taxon to M. pulchra. Members of the M. berdmorei species group exhibit considerable morphological differentiation: smooth to sparsely granular skin on dorsum; finger tips rounded, toe tips rounded or bearing tiny disks with or without terminal grooves; toe webbing from rudimentary to complete reaching toe disks; inner metatarsal tubercle oval, outer metatarsal tubercle from small to large, shovel-shaped (in M. picta); lacking mid-dorsal line or skinfold; with a light streak extending from posterior corner of eye to axilla; and a characteristic bright-yellow coloration of the groin and posterior parts of the belly. Based only on morphological characteristics, the unsampled M. darevskii (absent in our phylogeny), likely belongs to this species group.The Clade C,  encompasMicrohyla superciliaris group , western Thailand (Microhyla sp. 2) and the Malay Peninsula (M. superciliaris). Morphologically members of this group are small-sized , have dorsal orientation of nostrils; smooth to granular skin on dorsum; finger disks rounded or with weak disks lacking terminal grooves; toe disks having terminal grooves; inner metatarsal tubercle oval-shaped, outer metatarsal tubercle small and rounded; toe webbing reduced or well-developed; mid-dorsal skinfold generally present; with a light streak from posterior eye corner to axilla; and often with contrasting black and white blotches on belly. M. zeylanica species group for taxa inhabiting peninsular India + Sri Lanka, but M. eos from northeastern India as a sister lineage of this clade, refraining from assigning this species to any species group. Herein, we propose recognizing the M. superciliaris species group for taxa inhabiting Southeast Asia, northeastern and southern India, and Sri Lanka.The Microhyla ornata group . Morphological data on M. palmipes are scarce; they are small-sized frogs  with lateral nostrils; shagreened skin on dorsum; weak disks on digits lacking terminal grooves; moderately developed webbing on toes; inner and outer metatarsal tubercles small; middorsal line absent; dark markings on flanks and a light eye-axilla streak present , Annamite Mountains in Vietnam , and Borneo (M. petrigena and M. perparva); and H2 including species from mountains of central and southern Vietnam . M. marmorata was found to be paraphyletic with respect to M. pulverata. Morphologically, M. annectens group members are characterized by a relatively short body; lateral position of nostrils; sparsely granular to tubercular dorsal skin; complete toe webbing with well-developed, flattened, and slightly expanded disks on digits bearing terminal grooves; inner metatarsal tubercle present, and outer metatarsal tubercle present or absent. Further morphological studies are required to examine morphological differentiation among M. annectens group members. Phylogenetic positions of M. annamensis, M. marmorata, M. pulverata, M. arboricola, and M. pulchella are for the first time reported in the present study.Finally, the Microhyla that likely represent undescribed species: Microhyla sp. 1 from Sabah, Malaysia (corresponds to Microhyla sp. 1 of Microhyla sp. 2 from western Thailand (previously not reported), Microhyla sp. 3 from Yaeyama Archipelago (previously referred to as M. okinavensis), and Microhyla sp. 4 from northern Myanmar (reported as Microhyla sp. A by N = 31). This suggests that the taxonomy of the genus Microhyla still remains largely incomplete.Our updated phylogeny reveals several lineages of The origin of Asian microhylids, including the subfamily Microhylinae, is connected with a break-up of Gondwana and the Indian Collision . Most liSeveral recent biogeographic studies suggested that collision of the ISC with the Asian mainland was a more complicated process than previously conceived, implicating early opportunities for faunal exchange between the ISC and present-day Southeast Asia . The “OuMicrohyla + Glyphoglossus into three main lineages  happened during a short time frame in the middle Eocene (ca. 43.8 Ma), slightly later than previous estimates , respectively , and two times independently colonized the Ryukyus (M. okinavensis and Microhyla sp. 3). These results also corroborate data that suggested faunal exchanges between Eurasian continent and East Asian islands  or almost complete  reduction of the first finger. Similar patterns have also been reported in other miniature microhylids . Body size increased in the Glyphoglossus clade (up to 105 mm SVL), was slightly reduced in the Microhyla I clade, and significantly reduced in the Microhyla II clade ancestors  and three species of this group reach extreme miniaturization . Microhyla I clade shows significant variation in body size: the M. berdmorei species group and, to a lesser extent, several species in other lineages  demonstrate increased body sizes, while members of two lineages within M. achatina (M. nepenthicola + M. borneensis + Microhyla sp. 1) and M. superciliaris species groups (M. superciliaris + Microhyla sp. 2) are diminutive  have stout body habitus and are excellent burrowers , compared to other Microhyla species   . Egg siz clutch) . On one  clutch) . BreedinMicrohyla as the only available systematic study addressing the genus by Microhyla species, most likely due to high frequency of homoplasies both in adult and larval morphology. Further thorough morphological and osteological studies along with a robust phylogeny are required to diagnose supraspecific-level taxa within the Microhyla + Glyphoglossus assemblage.Diminutive frogs are recognized as a source of astonishingly high undescribed cryptic diversity at different taxonomic levels due to incomplete phylogenetic information and widespread homoplasies in morphology . Until rMicrohyla species has since increased almost two-fold, many questions of Microhyla taxonomy remain unaddressed. In the present article, we used an updated and almost complete phylogeny of the genus along with species delimitation methods to resolve several long-standing areas of confusion in the genus Microhyla. Our analyses suggest that despite recent progress in Microhyla taxonomy, the current number of recognized Microhyla species is still greatly underestimated. Based on species delimitation analyses, 15 (ABGD estimate) to 33 lineages (bGMYC estimate) probably reflect new species requiring formal description , Sarawak, Borneo  is found on sandstone massifs of western Sarawak, and is diagnosable in showing a pale brown dorsum with darker subtriangular pattern on scapular region, its adjacent areas lacking dark variegation; and flanks with an elongated dark area. On the other hand, the latter population (M. borneensis), restricted to the limestone hills of the interior, shows a gray-brown dorsum, areas outside dark subtriangular pattern with dark gray variegation; and flanks with isolated dark blotches. Therefore, we propose to revalidate the species M. nepenticolaM. borneensis, M. nepenthicola, and Microhyla sp. 1 from Sabah form a clade of morphologically similar and closely related taxa. Further studies are required to fully clarify morphological differences between M. nepenthicola and Microhyla sp. 1.Below we give a brief summary of taxonomic implications of our results. The smallest member of the genus, , Borneo . Matsui accepted . We anali region , specifirneensis . The namM. achatina species group suggesting presence of cryptic diversity. Examples include M. malang ; M. orientalis ; M. mantheyi ; M. achatina (2 putative species within Java); and M. gadjahmadai (2 putative species within Sumatra)  lineages from China and northern Vietnam, Thailand and Laos, Thailand and south Vietnam, Taiwan, Myanmar, peninsular Malaysia, and Sumatra  see . Geneticheymonsi . Our new Sumatra . The taxM. fissipes species group, the bGMYC analysis indicated the presence of three cryptic species-level lineages within M. mukhlesuri, although the ABGD analysis lumped M. mukhlesuri with M. fissipes ; populations from Yaeyama Archipelago formerly treated as M. okinavensis grouped with M. mixtura and most likely represent an undescribed species, Microhyla sp. 3  and M. darevskii , are needed to estimate taxonomic statuses of these newly revealed lineages and extent of their distribution. Within the M. superciliaris group, our study confirmed occurrence of M. superciliaris in southern Thailand (Songkhla), however the deep divergence of this population from the M. superciliaris population in Malaysia (p = 1.6%) suggests that it is necessary to reevaluate the taxonomy of Thai populations  also suggests presence of two cryptic lineages.Substantial genetic divergences also uncovered cryptic species lineages within the  complex   to four (bGMYC) cryptic lineages were recovered within this complex, with the most divergent lineages being distributed in Taiwan + mainland China versus the rest of the species range in Indochina and the Malay Peninsula  and originated from Bali and Sumatra; likely they both represent distinct species.For the iventris , but alseninsula . We inclM. annectens species group, our species delimitation analyses reveal a number of cryptic and undescribed lineages. Our study confirms genetic distinctiveness of recently described M. arboricola and M. pulchella . Both species were described by M. marmorata versus dusty in M. pulverata) and skin texture. Our genetic data reveal almost no genetic differentiation between samples of M. marmorata and the topotypic M. pulverata , M. petrigena (p = 3.7% between populations from Sarawak and Sabah), and M. perparva   . It is lMicrohyla. An exhaustive taxonomic sampling for this group is challenging due to the high number of narrow-ranged or point-endemic species across South and Southeast Asia. In the present study, however, we examined mtDNA and nuDNA markers for 48 of 50 recognized Microhyla species (96%), including 12 nominal species and several undescribed candidate species that have not been examined phylogenetically before our work, thus providing the most comprehensive taxonomic sampling for Microhyla to date. Our data further highlight the importance of broad phylogenetic sampling and ground-level field research to gather an accurate picture of global biodiversity, phylogenetic relationships, and evolutionary patterns in cryptic groups such as microhylid frogs.Herein, we provide an updated phylogenetic hypothesis for the genus Microhyla—Glyphoglossus assemblage , divided into three clades of probable genus-level differentiation: Microhyla I, Microhyla II and Glyphoglossus. Further integrative research combining phylogenetic and morphological lines of evidence is required to fully diagnose these recognized groups and test our taxonomic arrangement. The basal radiation of the Microhyla—Glyphoglossus assemblage is dated to the middle Eocene and likely took place in Southeast Asia. Following drifting of the Indian Plate northwards and formation of firm land bridges between the subcontinent and Asian mainland in Oligocene, ancestors of Microhyla colonized India several times from Southeast Asia and later diversified there. Our analysis also suggests that such dispersal occurred independently in five different species groups of the Microhyla II clade. Our results further corroborate the growing set of evidence for early-Eocene land connections between the Indian Subcontinent and Southeast Asia. Progressing aridification since the late Oligocene—Miocene likely facilitated dispersal of Southeast Asian biotic elements to India including the ancestral lineage-genus Microhyla. Our study further highlights the importance of Indochina not only as a cradle of autochthonous amphibian diversity and a key evolutionary hotspot for the herpetofauna  and moderately supported  nodes, respectively; no circles indicate unsupported nodes. Color marking of species groups in Microhyla species complex corresponds to BI genealogy of Click here for additional data file.10.7717/peerj.9411/supp-3Supplemental Information 3Circle sizes are proportional to the number of haplotypes, circle numbers correspond to sample numbers summarized in Supplementary Click here for additional data file.10.7717/peerj.9411/supp-4Supplemental Information 4Node values correspond to node numbers, for estimated divergence times (in Ma) see Click here for additional data file.10.7717/peerj.9411/supp-5Supplemental Information 5Asterisk (*) denotes sequences that were included in the alignment for timetree calibration. No exact locality information is available for specimens obtained via pet trade and published in earlier works. For references see Click here for additional data file.10.7717/peerj.9411/supp-6Supplemental Information 6“F,” “L”–forward primer, “R,” “H”–reverse primer. For references see Click here for additional data file.10.7717/peerj.9411/supp-7Supplemental Information 7The optimal partitioning scheme and model fit was estimated as suggested by the Akaike information criterion (AIC).Click here for additional data file.10.7717/peerj.9411/supp-8Supplemental Information 8Node – tree node used for calibration, for node names see Click here for additional data file.10.7717/peerj.9411/supp-9Supplemental Information 9Geographic regions: (A) Mainland East Asia; (B) Eastern Indochina; (C) Western Indochina; (D) Indian Subcontinent; (E) Malayan Peninsula; (F) Sumatra - Java - Bali; (G) Borneo and Philippines; (H) Sri Lanka; (I) East Asian Islands; see Click here for additional data file.10.7717/peerj.9411/supp-10Supplemental Information 10Four time periods correspond to: (1) 100–57 MYA marks the complete isolation of the ISC from Eurasia; (2) 57–50 MYA marks the first assumed land connections between India and the modern-day Sumatra; (3) during 50–35 MYA the ISC likely continued the counter-clockwise moving northwards forming land bridges with the modern-day Indo-Burma; and (4) the period of 35–0.0 MYA corresponds to the firm collision and formation of a stable land connection between the ISC and Eurasia. Letters encode: (A) Mainland East Asia; (B) Eastern Indochina; (C) Western Indochina; (D) Indian Subcontinent; (E) Malayan Peninsula; (F) Sumatra - Java - Bali; (G) Borneo and Philippines; (H) Sri Lanka; (I) East Asian Islands; see Click here for additional data file.10.7717/peerj.9411/supp-11Supplemental Information 11Microhyla nanapollexa: ZMMU A5635; Microhyla sp. 2: ZMMU A6032–A6035, KIZ-031270–031273; Microhyla sp. 3: ZMMU NAP-6340–6341.For each species minimal, maximal and average (when available) body size data is given for both sexes. For references see File S2. Question mark denotes “no data”. Voucher IDs for specimens measured for this study are as follows: Microhyla fodiens: CAS 215851, ZMMU A5960–A5961; Microhyla irrawaddy: ZMMU A5966–A5967; ZMMU A5975–A5976; Click here for additional data file.10.7717/peerj.9411/supp-12Supplemental Information 12Total length (in b.p.), number of conservative (Cons.), variable (Var.) and parsimony-informative (Pars.-Inf.) sites are given (data presented only for the ingroup).Click here for additional data file.10.7717/peerj.9411/supp-13Supplemental Information 13Microhyla – Glyphoglossus assemblage (1–57).Uncorrected average interspecific  and intraspecific  genetic p-distances for 16S rRNA mtDNA gene fragment (in percentage) are given for species of the Click here for additional data file.10.7717/peerj.9411/supp-14Supplemental Information 14Microhyla sensu lato (1–52).Number of species-level groups recovered by bGMYC and ABGD analyses presented for each of the morphospecies within Click here for additional data file.10.7717/peerj.9411/supp-15Supplemental Information 15Node No. – estimated tree node, for node names see Click here for additional data file.10.7717/peerj.9411/supp-16Supplemental Information 16Click here for additional data file.10.7717/peerj.9411/supp-17Supplemental Information 17Click here for additional data file.10.7717/peerj.9411/supp-18Supplemental Information 18See Supplemental Table S1 for sequence details.Click here for additional data file.10.7717/peerj.9411/supp-19Supplemental Information 19See Supplemental Table S1 for sequence details.Click here for additional data file.