In recent years, immune checkpoint inhibitors (ICIs) have become the standard treatment option for tumors. With the widespread application of ICIs, immune-related adverse events (irAEs) have gradually attracted the attention of researchers. Owing to the characteristics of ICIs, irAEs can affect each organ of the human body. Thromboembolism is uncommon in cancer patients receiving ICIs, but it may affect their survival. Most thromboembolic events do not cause serious effects after early prediction and treatment, but life-threatening toxic reactions are also observed. This condition should not be ignored because of vague and atypical symptoms, which make early diagnosis more challenging. This article focuses on the high-risk factors, underlying mechanisms, incidence, and prognosis of thromboembolism in patients using ICIs and briefly describes the intervention and treatment measures. This information would allow patients to effectively manage the side effects of thromboembolism during Immune checkpoint inhibitors treatment, ensuring the efficacy of ICIs and reducing mortality. Immune checkpoint inhibitors (ICIs) are widely used in tumor treatment. They inhibit the negative costimulation of T cells, help reactivate T cells, enhance their anti-tumor effects, and improve the survival of patients . There aWith the widespread use of ICIs, adverse reactions in organs have attracted the attention of doctors and researchers. One of the most important complications in cancer is thromboembolism (TE) . A serieTE includes VTE and arterial TE (ATE). VTE includes acute symptomatic or occasional deep vein thrombosis, pulmonary embolism (PE), visceral vein thrombosis, and fatal PE . ATE is The coagulation-fibrinolysis process maintains a balanced state of normal physiological functions, but its function changes in malignant tumors . The mecThe biomarkers observed in another study may provide a possible mechanism for the occurrence of VTE in patients receiving ICIs . MyeloidThe above studies revealed a possible mechanism of VTE in tumor patients treated with ICIs. The relationship between ICI and atherosclerosis is well-established ; therefoTE in cancer patients receiving ICI therapy may be caused by different high-risk factors, but this has not been fully studied. The following sections summarize some common high-risk factors based on published research.Previous studies have shown that a history of VTE or ATE is a risk factor in cancer patients receiving ICI therapy . AnotherThe presence of a stage IV tumor is also one of the reasons for the increased risk of TE in cancer patients receiving ICI treatment. In a retrospective cohort study, patients with stage IV tumors had an increased risk of TE . SimilarKS, a risk stratification tool, has been used for VTE risk assessment . It inclp = 0.048) (p = 0.760)  . In cont= 0.760) . Patient= 0.760) .A previous study showed that combined ICI therapy is significantly associated with VTE . Two patAssociations between the type of cancers and the risk of thrombosis have been compared. The prediction score includes the location of the primary tumor as a variable for predicting TE . Lung cap < 0.05)  . So far,ICI therapy has demonstrated profound clinical efficacy for multiple malignancies, which may also introduce additional risks. An increasing number of issues are of great interest in the incidence of TE and their impact on the prognosis of cancer patients treated with ICIs. Data from the retrospective studies summarized in this review suggest that the incidence of VTE is between 2.6 and 30.3% . It has We summarized the incidence of TE events in cancer patients receiving ICI therapy from published articles . Evidencp = 0.44) (p = 0.725) (p < 0.008)  . The inf = 0.44) . Ibrahim= 0.725) . Another= 0.725) . However= 0.725) . A retro= 0.725) . The med= 0.725) . Moik et= 0.725) . The inc= 0.725) . The inc= 0.725) . After c= 0.725) . In anot< 0.008) .The data available for ATE are limited. In one study, only six (1.1%) and one (0.18%) patient had ischemic stroke and arterial thrombosis, respectively . Similarp = 0.675)  . Therefo= 0.675) . In addip = 0.00004)  who received anticoagulation therapy, two (4%) developed intracranial hemorrhage. There was no significant difference between these patients and those who did not receive anticoagulation therapy. In addition, VTE patients who received anticoagulant therapy achieved longer OS . However0.00004) . The ris0.00004) . In view0.00004) . TherefoAccording to the data summarized in this study, TE incidence is relatively high incidence in cancer patients treated with ICI, even exceeding that in patients receiving chemotherapy . This reThe included studies have some limitations. They included patients with tumor heterogeneity, such as inconsistent tumor types. In addition, the retrospective nature of these studies and the short follow-up time may have led to the loss of key data. Moreover, the ATE events are not sufficient to perform additional data analysis on susceptibility factors and survival analysis of specific tumor types. Therefore, future studies with larger sample sizes and prospective designs are essential to better describe TE in patients treated with ICIs. Preclinical studies should focus on the identification of risk factors and biomarkers for developing TE risk assessment models and screening patient groups at risk. The specific mechanism of bleeding during ICI treatment needs further elucidation. A unified thrombosis risk assessment standard for patients treated with ICIs should be established, for grading the risk level and to help obtain reliable and comparable data in the clinic. These would ensure that the risk of thromboembolism is avoided as much as possible, the therapeutic effect of ICIs is maximized, and the OS is prolonged.


Taxus wallichiana var. mairei response to AR stress. Our results showed that low Ca level leads to photosynthesis, and antioxidant defense system decreases in T. wallichiana var. mairei leaves; however, these negative effects could be reversed at high Ca level. In addition, proteomic analyses identified 44 differentially expressed proteins in different Ca level treatments of T. wallichiana var. mairei under AR stress. These proteins were classified into seven groups, which include metabolic process, photosynthesis and energy pathway, cell rescue and defense, transcription and translation, protein modification and degradation, signal transduction, etc. Furthermore, the study found that low Ca level leads to an obvious increase of Ca-related gene expression under AR stress in T. wallichiana var. mairei using qRT-PCR analyses and however can be reversed at high Ca level. These findings would enrich and extend the Ca signaling pathways of AR stress in AR-resistant woody plants and are expected to have important theoretical and practical significance in revealing the mechanism of woody plants tolerating AR stress and protecting forestry ecosystem in soil environment under different Ca levels.As one of the serious environmental problems worldwide, acid rain (AR) has always caused continuous damage to the forestry ecosystem. Studies have shown that AR can leach calcium ions from plants and soil. Calcium (Ca) is also a crucial regulator of the plant stress response, whereas there are few reports on how Ca regulates the response of AR-resistant woody plants to AR stress. In this study, by setting different exogenous Ca levels, we study the physiological and molecular mechanism of Ca in regulating the  As a serious environmental problem, acid rain (AR) affects seriously normal growth and development of forest tree species, which includes balance of leaf nutrient, growth indices, chlorosis, and necrosis in leaves, chlorophyll content, restrict photosynthesis, and the antioxidant enzyme activity . FurtherTaxus wallichiana var. mairei is a well-known gymnosperm with great ornamental and medicinal value , respectively (–1), medium Ca level (19.65 mmol kg–1), and low Ca level (1.85 mmol kg–1).In our experiment, the substrate soil was lateritic soil, and the soil samples were collected from in southern forest areas of China where AR is much too harmful. Ca content in the soil was leached for 6 months of simulated AR, and the specific soil leaching process is referred to ectively . The finT. wallichiana var. mairei seedlings  were transplanted into plastic pots. The seedlings were grown in a greenhouse with a light–dark regime of 16/8 h, temperature of 27/21°C (day/night), relative humidity of 60–70%, and photosynthetically active radiation of 210 μmol m–2 s–1. After 2 weeks of recovery, the T. wallichiana var. mairei seedlings were sprayed once each day with simulated AR solution (pH 3.0) with medium Ca level as control group and low or high Ca level for treatment group as described by the previous study  was performed with a portable photosynthesis system  as described by Pn measurements. For Ca element analysis, T. wallichiana var. mairei leaf tissue was dried at 80°C for 72 h. Ca concentration in the leaves was measured using ICP-MS , following the study by Chlorophyll in 2O2, and O2•– content were measured as described by Soluble protein, proline, HT. wallichiana var. mairei leaves was extracted using the method of phenol extraction according to the study of Protein in t-test (p < 0.05), which were considered for mass spectrometry analysis.Two-dimensional electrophoresis (2-DE) was conducted according to the study of T. wallichiana var. mairei was performed according to the study of T. wallichiana var. mairei were searched against the National Center for Biotechnology Information non-redundant (NCBInr) database, and the taxonomy of green plants was selected using the MASCOT interface . The following parameters were used for database search: no molecular weight restriction, permitting one missed cleavage, fixed modification of cysteine by carbamidomethylation, oxidation (Met) as a variable modification, the peptide tolerance of 100 ppm, and fragment ion mass tolerance of ±0.3 Da. At least three peptides were matched for protein identification, and the protein scores of MOWSE threshold were set greater than 73 with the NCBInr database (p < 0.05). As described by 1 UniProt,2 and published literature.Protein digestion and protein identification in Hierarchical clustering was performed on density value of differentially expressed proteins according to the study of T. wallichiana var. mairei leaves (0.1 g) was extracted using RNA purification reagent  in liquid nitrogen according to the study of T. wallichiana var. mairei (GAPDH) in T. wallichiana var. mairei was used as the internal control for each sample. Three independent biological replicates were performed for each sample.Total RNA from . mairei . The Ca-. mairei , and extp < 0.05.All data were presented as the mean ± SE of three replicated samples. The statistical significance was analyzed using a univariate analysis of variance . Statistical significance was considered at T. wallichiana var. mairei was treated with simulated AR (pH 3.0) for 2 months. Physiological changes are shown in Pn) were also tested and analyzed. After 2-month AR treatment, Ca content and Pn were significantly decreased in T. wallichiana var. mairei leaves at low Ca level; however, high Ca treatment can reverse the decline of the physiological indicators.To study the responses of woody plant to AR stress at different Ca levels, T. wallichiana var. mairei at high Ca level. We found that the levels of MDA, H2O2, and O2•– were significantly stimulated by low Ca treatment  showed a downward trend with the increase of Ca concentration under AR stress. Signal transduction-related proteins  showed a significant upregulation trend under low Ca-AR treatment. The expression trend of these proteins was reversed to downregulated with the increase in Ca concentration. More interestingly, we found that a protein  related to photosynthesis process was significantly downregulated in low Ca condition, but turned to upregulated in high Ca condition, which indicates that high Ca treatment helps to improve T. wallichiana var. mairei leaves photosynthesis, which is one of the ways to enhance plant resistance to AR stress. The specific information of the differential proteins is listed in In addition, we used a hierarchical cluster analysis to find the regular pattern of different protein expression changes during AR stress in T. wallichiana var. mairei , and ATP synthase  was increased in high Ca-AR-treated . mairei . As showT. wallichiana var. mairei, we further analyze calmodulin gene (CaM1), touch 3 gene (TCH3), calreticulin 3 gene (CRT3), CDPK-related kinase gene (CDPK1), glutamate dehydrogenase 2 gene (GDH2), calcineurin B-like calcium sensor protein 1 gene (CBL1), calnexin 1 gene (CNX1), and respiratory burst oxidase homolog A gene (RbohA). As shown in To evaluate the expression abundances of the Ca-related genes at different Ca levels in AR-treated T. wallichiana var. mairei  and AR-sensitive tree species (L. formosana and P. massoniana) in response to AR stress. Further study is needed to clarify the different metabolic processes at various Ca levels in AR-tolerance woody plants.The environmental stresses severely affect the material metabolism of plants . UDP-glu. mairei . Under Aormosana . It has ormosana . In thisormosana . We specormosana . In thisormosana , which iormosana . These fT. wallichiana var. mairei response to AR stress; however, this expression trend can be reversed at high Ca level , ribulose-1,5-bisphosphate carboxylase–oxygenase large subunit (spot 16), light-harvesting complex II protein Lhcb1 (spot 17), and ferredoxin-NADP reductase (spot 18) were decreased in protein expression abundance with low Ca-AR treatment and increased with high Ca-AR treatment in T. wallichiana var. mairei  have the increased abundance of energy production-related proteins with high Ca-AR treatment  was upregulated by high Ca level with AR treatment. Based on these results, high Ca condition may stimulate the resistance of T. wallichiana var. mairei to AR stress by modulating special energy-related pathway. These findings indicate that with high Ca-AR treatment, the mechanisms of energy pathway in AR-resistant tree species  are different from AR-sensitive tree species (L. formosana and P. massoniana). Further investigations are needed to clarify the different energy pathways in AR-tolerance woody plants.On the other hand, sufficient ATP is necessary for nversion . Previounversion . Our resreatment ,2016. Inreatment , and wesn plants . Expressn plants . Our stu2O2 and O2•–], which suggests the ROS enrichment in T. wallichiana var. mairei; however, high Ca supplementation can reverse these effects , e.g., H effects . In addiT. wallichiana var. mairei.To further reveal the different strategies to cope with AR stress at various Ca levels, many cell rescue and defense-related proteins were also identified by proteomic analysis in this study. As stress response proteins and heat shock proteins can be induced by many abiotic stresses , 2013, oL. formosana seedlings in response to AR stress  and glutathione S-transferase (spot 24) were also observed at high Ca levels. It was reported that thioredoxin can reduce intramolecular disulfide bridges of target proteins, maintain suppression of apoptosis, and supply reducing equivalents to the antioxidant systems . Under bR stress . In addiR stress . Previoueedlings ,2016. Aseedlings . In thisT. wallichiana var. mairei, which suggests that expression changes in maturase K may play a part in linking change of Ca level under AR stress. Moreover, T. wallichiana var. mairei. In addition, T. wallichiana var. mairei under AR stress. Transcription and translation control the expression of stress responsive genes, which play crucial roles in plants in response to various abiotic stresses  signaling pathway  and AR-resistant  tree species under high Ca condition; however, further studies are needed to elucidate the specific function of the signal transduction-related proteins in AR-stressed tree species.Under environmental stresses, woody plants initiate multiple signaling pathways through sensing and transducting several stress signals, further regulate the expression level of many functional proteins, and finally exhibit corresponding physiological response to against the abiotic stresses . Membran pathway . The TOR pathway . We founreatment , which s2+ level is a universal second messenger of signal transduction in plants in response to environmental stresses (2+ levels (T. wallichiana var. mairei with high Ca-AR treatment. It has been observed that Ca-binding proteins are regarded as crosstalk key nodes for environmental stress signaling pathways (T. wallichiana var. mairei, which provides informative hints on interactions between Ca-associated signal transduction and AR tolerance. In T. wallichiana var. mairei.Moreover, Ca plays an important role in plants coping with a series of environmental stresses . In the stresses . Ca-bind+ levels ,2016. Inpathways . These fT. wallichiana var. mairei response to simulated AR treatment. Our data reveal that low Ca treatment activated the cell rescue- and defense-related proteins and Ca-related gene level to respond to AR stress in T. wallichiana var. mairei. These proteins might operate in a dynamic network in the response of AR-resistant tree species under AR stress, and these identified proteins might be useful in revealing insights into the different defense mechanisms of AR-resistant tree species to AR stress. In addition, exogenous Ca supply against AR stress by increasing the abundance of the proteins involved in photosynthesis and energy pathway, translational factors, which suggest that AR-tolerant woody plants can equip themselves better to respond to AR stress by provoking related proteins and gene expression. As shown in T. wallichiana var. mairei responding to AR damage at different Ca levels. These results would deepen insights into the adaptation strategies of AR-resistant tree species under AR stress at different Ca levels and furthermore provide theoretical support for the scientific prevention of AR stress.In this study, physiological and proteomic evidence is carried out to clarify that Ca plays an important role in AR-resistant tree species The original contributions presented for this study are included in the article/supplementary material. For details, please refer to H-LZ, W-JH, and G-XS: conceptualization and funding acquisition. W-JH and QW: methodology. T-WL: software and visualization. C-KJ: validation. JW: formal analysis. QW and H-LL: investigation. LC: resources. C-QZ: data curation. W-JH and C-QZ: writing – original draft preparation. H-LZ and G-XS: writing, reviewing, and editing, and project administration. H-LZ: supervision. All authors have read and agreed to the published version of the 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Colletotrichum gloeosporioides, can conventionally form a germ tube (GT) and develop into a fungal colony. Under certain conditions, they tend to get connected through a conidial anastomosis tube (CAT) to share the nutrients. CAT fusion is believed to be responsible for the generation of genetic variations in few asexual fungi, which appears problematic for effective fungal disease management. The physiological and molecular requirements underlying the GT formation versus CAT fusion remained underexplored. In the present study, we have deciphered the physiological prerequisites for GT formation versus CAT fusion in C. gloeosporioides. GT formation occurred at a high frequency in the presence of nutrients, while CAT fusion was found to be higher in the absence of nutrients. Younger conidia were found to form GT efficiently, while older conidia preferentially formed CAT. Whole transcriptome analysis of GT and CAT revealed highly differential gene expression profiles, wherein 11,050 and 9786 genes were differentially expressed during GT formation and CAT fusion, respectively. A total of 1567 effector candidates were identified; out of them, 102 and 100 were uniquely expressed during GT formation and CAT fusion, respectively. Genes coding for cell wall degrading enzymes, germination, hyphal growth, host-fungus interaction, and virulence were highly upregulated during GT formation. Meanwhile, genes involved in stress response, cell wall remodeling, membrane transport, cytoskeleton, cell cycle, and cell rescue were highly upregulated during CAT fusion. To conclude, the GT formation and CAT fusion were found to be mutually exclusive processes, requiring differential physiological conditions and sets of DEGs in C. gloeosporioides. This study will help in understanding the basic CAT biology in emerging fungal model species of the genus Colletotrichum. The conidia of a hemibiotrophic fungus,  The asexual spore or conidium is essential in the life cycle of many fungi because it is the key for dispersal and serves as a safehouse for the fungi in unfavorable environmental conditions. Under favorable conditions, the conidia germinate to form hyphae and finally a fully grown fungal colony. The mechanics of conidial germination are diverse and vary between different fungal species . In geneColletotrichum lindemuthianum, Neurospora crassa, and Fusarium oxysporum [Colletotrichum fructicola, Colletotrichum nymphaeae, and Colletotrichum theobromicola [N. crassa and F. oxysporum [C. lindemuthianum, C. gloeosporioides, and C. siamense [C. lindemuthianum and C. graminicola, which only occurs in water/water agar media [N. crassa and F. oxysporum, CAT induction requires some nutrients to occur [The CAT has shown to be morphologically and physiologically distinct from GT in xysporum ,5,6. In xysporum . It has romicola . The CATxysporum ,5,9. Intsiamense ,11. The siamense . Howeverar media . In N. cto occur ,5. ThereF. oxysporum and C. lindemuthianum [C. lindemuthianum and C. gossypii resulted in the formation of some hybrids, which exhibited phenotypic variations; one hybrid was highly pathogenic [C. gloeosporioides and C. siamense generated genetic and phenotypic diversity in these fungal pathogens [CAT fusion was shown to play a role in the generation of phenotypic and genetic variations in few fungi. The heterokaryotic progenies have been generated through CAT fusion with distinct phenotypes compared to parental strains in uthianum ,13. Intethogenic  Furthermathogens . PathogeC. gloeosporioides starts its usual life cycle from conidial germination to the formation of melanized infection structures, appressoria [C. gloeosporioides were reported in both in vitro and in vivo conditions [C. gloeosporioides can be considered as an alternative model organism and as a proxy to understand the underlying physiological and molecular requirements for GT formation and CAT fusion. In the present study, we systematically investigated the in vitro dynamics and the nutritional and physiological requirements of GT formation and CAT fusion in C. gloeosporioides. Furthermore, a comparative transcriptome analysis of CAT fusion and GT formation was done to decipher the underlying differential molecular prerequisites for these two processes in C. gloeosporioides. ressoria . The fornditions ,16. DespC. gloeosporioides, the GT and CAT induction assays were carried out by following a protocol developed in our lab [C. gloeosporioides (CBS 953.97) culture was inoculated on bean pod agar medium (autoclaved French bean pods submerged in 2% water agar) and was incubated in the dark at 25 °C to induce conidiation. Post-inoculation, the conidia were harvested at different time points, viz., 6, 10, 13, and 17 days. Conidial cells were counted using Neubauer’s chamber and conidial suspension of 4 × 105 per ml was prepared in distilled water and potato dextrose broth (PDB) for CAT and GT induction, respectively. One ml of such conidial suspensions was placed in a 24-well tissue culture plate  and incubated in the dark at 25 °C for different time points, viz., 3, 6, 12, 18, and 24 h for GT formation and 24, 48, 72, and 96 h for CAT fusion. They were then examined under a microscope  using differential interference contrast (DIC) optics. The GT formation and CAT fusion were quantified as the percentage of conidia (n = 300) involved in GT formation or CAT fusion [To study the effect of conidial age on GT formation and CAT fusion and their mutual exclusiveness in  our lab . The C. C. gloeosporioides. Different concentrations of 6-day-old conidia were incubated in PDB for 18 h in the dark for GT induction. In contrast, for CAT induction, different concentrations of 17-day-old conidia were incubated in dH2O for 72 h in the dark. The concentrations of conidia tested were 4 × 102, 4 × 103, 4 × 104, 4 × 105, and 4 × 106 per mL [Different conidial numbers were tested to determine the threshold of conidial concentrations required for CAT fusion and GT formation in 6 per mL .C. gloeosporioides are dependent on the availability of nutrients and starvation stress conditions, CAT fusion (in 17-day-old conidia) and GT formation (in 6-day-old conidia) were induced in nutrient-limiting conditions (water), and high nutrient conditions such as PDB, 2% glucose. Furthermore, to study whether the CAT induction or GT formation in C. gloeosporioides is mediated through MAP kinase (MEK) pathway, CAT fusion and GT formation percentage was also determined in the presence of an MEK inhibitor InSolution™ PD98059 (5 µM).To study whether CAT fusion and GT formation in 5 conidia/mL density in PDB incubated for 18 h in the dark, and the CAT induction was carried out in 17-day-old conidia at 4 × 105 conidia/mL density in water incubated for 72 h in the dark. After that, total RNA was isolated from resting conidia (6-day-old conidia grown on bean pod agar media), germinating conidia (GTs), and fused conidia via CAT (CATs) (3 replicates of each sample) of C. gloeosporioides using NucleoSpin RNA Plant kit  according to manufacturer’s guidelines. DNAse treatment was carried out using the Amplification grade DNase I kit . Isolated total RNA was quantified and qualified using a Nanodrop ND-1000 spectrophotometer and Qubit fluorometer. RNA quality was also assessed using formaldehyde agarose gel electrophoresis. RNA integrity was checked using a Bioanalyzer chip and Agilent RNA TapeStation .GT formation was induced in 6-day-old conidia at 4 × 10C. gloeosporioides, were pooled, and independent library preparations were carried out for these three different life stages. Pooling of the RNA samples for library preparation was done due to economic constraints. RNA-seq library preparation was performed as per Illumina-compatible NEBNext UltraTM Directional RNA Library Prep. Sequencing for 150 bp length paired-end (PE) reads was performed in an Illumina HiSeq sequencer.Three µg of total RNA from the three technical replicates of each life stage, namely, resting conidia, GTs, and CATs of Reads were processed for quality assessment and low-quality filtering before the assembly. The raw data generated was checked for the quality using FastQC, and preprocessing of the data, which includes removing the adapter sequences and low-quality bases (<q30), was done with Cutadapt .Processed reads were assembled using a graph-based approach by the rnaSPAdes program . rnaSPAdC. gloeosporioides Nara gc5 isolate (ftp://ftp.ncbi.nlm.nih.gov/genomes/genbank/fungi/Colletotrichum_gloeosporioides/ (accessed on 8 April 2019) was used as a reference genome. For each sample, the count of mapped reads was derived and normalized to RPKM (reads per kilobase of exon model per million mapped reads). The count data was further used to identify differentially expressed genes at each time point using the DESeq package [P values were adjusted for multiple testing with the procedure described by [To assess the quality of the assembly, an evaluation of the read content approach was used. Processed reads from all three libraries were aligned back to the final assembly using Bowtie2  with end package . Sequencribed by  to contrp < 0.05), selecting the NCBI blast Fungi as a taxonomy filter and the default parameters [Assembled transcripts were functionally enriched and categorized based on blast sequence homologies and gene ontology (GO) annotations describing biological processes and molecular functions using Blast2GO software  for C. gloeosporioides to find out more accurate molecular and biological functions.Multiple databases  were used to annotate the transcripts and determine the possible roles of the annotated genes/proteins. Clustered transcripts were annotated using the homology approach to assign functional annotation using BLAST tool  against http://ftfd.snu.ac.kr (accessed on 4 October 2019). Transcription factor analysis was performed based on homology approach using NCBI-blast program. Assembled transcripts were translated into protein using TransDecoder. The putative transcription factors (TFs) were predicted using the Fungal Transcription Factors Database  with Tukey’s post hoc test using GraphPad Prism 5 Statistics Software. Differences with a C. gloeosporioides. Initially, conidia become primed for CAT fusion as a result of CAT induction   A,B, GT ion (hpi) B,C, GT eon (hpi) D,E, and on (hpi) F. When CAT fusion and GT formation was studied in differently aged conidia, it was observed that young conidia (6 days) in PDB were efficient in forming GT, while older conidia (17 days) in sterile distilled water were good at forming CAT A. The yo4 and 4 × 105 conidia per ml of water was found to be optimal for CAT fusion in C. gloeosporioides. The CAT fusion percentages were 9.8 ± 3% and 12.3 ± 3.4% in 4 × 104 and 4 × 105 conidia per ml of water, respectively  greater than 9.0. An average of 41.62 million raw sequencing PE reads were produced in total for three samples, out of which an average of 39 million reads were used for the downstream analysis after preprocessing. Thus, for every sample on an average of 95% of high-quality data was retained and used for analysis . A totalThe assembly of high-quality reads was performed separately for resting conidia, GT, and CAT. Sample wise transcripts were further clustered into 121,260 transcripts with an average length of 603 bp and N50 of 1849 bp . The lenThe assembled transcripts for GT and CAT were individually grouped into ten molecular functions (MF) and ten biological processes (BP) . The traThe DEGs were further analyzed between GT and CAT. The transcripts which show a log2fold change less than −1 are represented as downregulated, the values greater than 1 are defined as upregulated, and between −1 to 1 are termed as neutrally regulated.C. gloeosporioides  families and 1044 TF proteins were predicted using the Fungal Transcription Factors Database; out of theses, 38 TF families  and 220 C. gloeosporioides involve the deployment of secreted effector proteins. Effector candidates are often <200 amino acids in length and cysteine-rich [GT formation and CAT fusion in ine-rich ; therefoDifferentially expressed secretory proteins were manually annotated and categorized in four and three major groups for GT formation and CAT fusion, respectively. Few selected uniquely expressed effector candidates during GT formation include hydrolytic enzymes, adhesion, germination, hyphal development associated proteins, transport, signaling-related proteins, and proteins responsible for virulence. Some examples of these groups of effector proteins uniquely secreted during GT formation and their possible functions are given in 2 = 0.736; p < 0.01) with transcriptome Log2fold data . We observed a strong correlation between the qRT-PCR and RNA-Seq analysis of 17 DEGs in GT versus CAT formation A. Similaold data B. It shoC. gloeosporioides. We observed significant CAT fusion in 17-day-old conidia and GT formation in 6-day-old conidia  for CAT fusion and younger conidia (6 d old) for GT formation were preferred in C. gloeosporioides  compared to nutrient-rich conditions such as PDB. The presence of glucose in water also reduces CAT fusion percentage in this fungus  and even in nutrient-poor Vogel’s medium, while it occurs only in water media [N. crassa and F. oxysporum, CAT fusion occurs in the presence of nutrients, e.g., Vogel’s medium for N. crassa and YNB+KNO3/1% PDB +NaNO3 for F. oxysporum [C. gloeosporioides is inhibited by a MAP kinase inhibitor InSolution™ PD98059 , availability of external nutrients, and/or starvation stress conditions. We have shown that the older conidia were superlative for CAT fusion while younger conidia were ideal for GT formation. We believe that increased CAT fusion reduces the internal resources for GT formation; however, the conidia’s decision to either form CAT or GT does not only rely solely upon the internal resources. We had shown that when the older conidia (17 days) were incubated in water (no nutrients), they could undergo extensive CAT fusion and no GT formation. However, when the same aged older conidia were incubated in PDB, they could also form GT but at a reduced rate compared to younger conidia (6 and 10 days old) A. In C. t stress . TherefoC. gloeosporioides by undertaking RNA-sequencing and analysis. As a result, we observed significant differences in transcriptomes of GT formation and CAT fusion. Furthermore, we identified many differentially expressed genes, transcription factors, and secretory effector proteins during GT formation and CAT fusion in C. gloeosporioides. After understanding the differential physiological requirements of CAT and GT formation, we attempted to decipher the differential molecular prerequisites of these two processes in C. fructicola [C. gloeosporioides, which are probably involved in conidial adhesion. Fungal hydrophobins are small, secreted hydrophobic proteins involved in the adhesion of conidia to the host surface prior to GT formation [A. oryzae and C. fructicola [Genes involved in cell wall degradation, host-fungus interaction, germination, transport, signaling, and virulence were found to be upregulated during GT formation. Previously, it has been shown that genes responsible for plant cell wall degradation, secondary metabolism, and detoxification were upregulated during GT formation in ucticola . We alsoormation . Fungal ucticola ,39. OtheC. gloeosporioides [Fungal transporter proteins play a broad range of biological functions and an essential role in the pathogenicity of phytopathogens ,41,42. Iorioides ,43.C. gloeosporioides [C. gloeosporioides  are known to play an important role in redox signaling pathways, including cell differentiation, development, and cytoskeleton remodeling . NADPH-o dahliae ,49,50,51T fusion . In our revisiae . Furtherrevisiae . Our stuC. gloeosporioides. The CAT fusion involves sensing of quorum through signaling and transport followed by cell wall and cell membrane remodeling. Many membrane transporters and cell wall modulation genes, including MFS transporter, transmembrane amino acid transporter, peptidoglycan-N-acetylglucosamine deacetylase, and Glycosyltransferase family 2 protein, were found to be upregulated during CAT fusion. In addition, other important transcripts, viz., alcohol dehydrogenase GroES-like domain, flippase-like domain-containing protein, and acetyl-CoA carboxylase, were upregulated during CAT fusion, which probably play roles in protein folding, pheromone response, cytoskeletal dynamics, cell division, lipid metabolism, and fatty acid synthesis in N. crassa, the actin cables and patches are significantly increased at CAT tips during chemotropic interactions between germlings or conidia [N. crassa [C. gloeosporioides, suggesting that actin is critically essential for polarized growth and CAT fusion. We have previously shown that an inter-specific CAT fusion between C. gloeosporioides and C. siamense involved nuclear transfer [C. gloeosporioides also involves nuclear exchange. Corroborating to this belief, we have observed that several cell cycle and cell rescue genes were highly upregulated during CAT fusion in C. gloeosporioides, which include the F-box domain gene, which is thought to be involved in mitosis or cell division cycle; DNA repair; and the MutS domain V gene which is probably involved in the protection of cells from DNA damage-induced genome instability by repairing the DNA mismatch. The cytoskeleton actin is important for several functions, including cell polarity, exocytosis, endocytosis, septation, movement of organelle, and chemotropic growth of fungi . In N. c conidia . However. crassa . We obsetransfer . TherefoC. gloeosporioides (N. crassa [C. gloeosporioides.Transcription factors play important roles in various biological processes. Out of the total 220 annotated TF genes, 82 were uniquely expressed during CAT fusion, and these genes are probably involved in the regulation of CAT fusion in orioides . Two suc. crassa ,57. SimiC. gloeosporioides. The WSC domain-containing protein-coding gene has previously been shown to be important in germling and hyphal fusion in N. crassa [C. gloeosporioides. Calcium and some calcium-dependent genes have been shown to play a critical role during the membrane fusion in germling communication in various fungi [N. crassa [C. gloeosporioides. We also detected some transcripts of cell division cycle genes, which uniquely secreted during CAT fusion, viz., coronin, which is known to play a role in the organization and dynamics of actin and F-actin remodeling, and Wlm domain-containing genes, which is associated with sister chromatid separation and segregation during mitosis.We have also found various unique effector genes specifically secreted during CAT fusion, and interestingly the major transcripts could be grouped in similar categories such as general DEGs, which included stress-associated proteins, cell wall, membrane transport, signaling related proteins, cytoskeleton, cell cycle, and fungal development-associated proteins. We observed a few stress-associated effector genes, including Acetyl-CoA acetyl transferase, Calcineurin-like phosphoesterase, LipA, NB-ARC domain-containing protein, and WSC domain-containing protein-coding gene, which might be involved in the physiological stress response and nutrient availability signals during CAT fusion in . crassa . We obseus fungi . Calcium. crassa ,60. We dC. gloeosporioides. It would be interesting to know whether other fungal species capable of forming CAT also exhibit similar transcriptome profiles. We propose a model to explain the mutual exclusiveness of GT formation and CAT fusion and their dependency on conidial age, availability of external nutrients, and differential RNA profiles . The you


Vibrio cholerae isolated from different regions in Tanzania.Cholera continues to cause morbidity and mortality in developing countries, including Tanzania. Since August 2015, Tanzania Mainland has experienced cholera outbreaks affecting 26 regions and a 1.6% case fatality rate. The current study determined the virulence factors, genetic relatedness and antimicrobial susceptibility patterns of the V. cholerae isolates from eleven regions in Tanzania was carried out. There were 99 V. cholerae isolates collected between January 2016 and December 2017. The study perfomed a Multi-locus Variable-number tandem-repeat analysis for genetic relatedness and Mismatch Amplification Mutation Analysis polymerase chain reaction for analyzing toxin genes. All the isolates were tested for antimicrobial susceptibility using the Kirby Bauer disk diffusion method. Data were generally analyzed using Microsoft excel, where genetic relatedness was analyzed using eBurst software v3.A cross-sectional study that involved the genetic characterization of V. cholerae O1. Ogawa was the most predominant 97(98%) serotype. Isolates were genetically related with a small genetic diversity and were positive for ctxA, tcpA El Tor virulence genes. All isolates (100%) were sensitive to doxycycline, trimethoprim-sulphamethoxazole, tetracycline, ceftriaxone, and chloramphenicol, while 87.8% were sensitive to ciprofloxacin. A high resistance rate (100%) was detected towards erythromycin, nalidixic acid, amoxicillin, and ampicillin.All isolates were V.cholerae O1 serotypes Ogawa, El Tor variant predominantly caused cholera outbreaks in Tanzania with strains clonally related regardless of the place and time of the outbreak. Most of the isolates were susceptible to the antibiotic regimen currently used in Tanzania. The high resistance rate detected for the other common antibiotics calls for continuous antimicrobial susceptibility testing during outbreaks.The  V. cholerae, continues to cause morbidity and mortality in low-income countries. Out of 200 serogroups of V. cholerae reported, only V.cholerae O1 and O139 are associated with the epidemic [V. cholerae strains contain a cholera toxin prophage that carries the genes encoding for cholera toxin, a vital virulence factor responsible for the typical clinical symptoms of the disease [Cholera, a disease caused by V. cholerae O1 serotype commonly exists as Classical and El Tor biotypes [V. cholerae O1 are closely related in their O-antigen biosynthetic genes suggesting the genetic hybrids [V. cholerae O1 strain that appear to have classical and El Tor traits, generically termed as atypical El Tor biotypes, have been reported elsewhere [biotypes . Classic hybrids . Variantlsewhere –8.V. cholerae strains that caused the outbreak that started from August 2015 onwards.Since 1974 when the first case of cholera was reported in Tanzania [V. cholerae isolates collected from 11 out of 26 regions of Tanzania Mainland. Ethical clearance to conduct this study was obtained from the Senate Research and Publications Committee of Muhimbili University of Health and Allied Sciences (MUHAS) in Dar es Salaam, Tanzania. Specimens were collected with permission from the National Health Laboratory Quality Assurance Training Center. The patient’s consent was obtained as part of routine surveillance by MoHCDGEC, in Tanzania. A laboratory identification number was assigned to each specimen to maintain patient confidentiality.A cross-sectional study involving eleven regions that experienced cholera outbreaks from January 2016 to December 2017 was carried out. The regions included were Dar-es-Salaam, Mwanza, Kigoma, Arusha, Pwani, Mara, Dodoma, Katavi, Mbeya, Songwe, and Ruvuma. The study included 99 V.cholerae isolates and fresh stool samples were used in this study. Forty-nine preserved V.cholerae isolates were revived by inoculating into alkaline peptone water at 35.5°C for 6 hours and then sub-cultured on Thiosulfate-Citrate-Bile salt-Sucrose (TCBS) Agar , and incubated aerobically at 35.5°C overnight. Stool samples collected from 50 patients with cholera during the outbreak were also cultured similarly. Isolates were identified by biochemical tests (oxidase and string tests) using pure culture from Tryptone Soya Agar . Isolates were serogrouped using V. cholerae polyvalent O1 and O139 sera (Denka Seiken Ltd-Japan) and serotyped by monovalent antisera Ogawa and Inaba (Denka Seiken Ltd-Japan). The procedure included saline controls to detect auto agglutination [Preserved tination .V.cholerae growth on Luria Bertani (LB) broth culture medium was used as per the Qiagen-DNeasy DNA extraction kit protocol [V. cholerae DNA was subjected to Multi-locus variable-number tandem-repeat Analysis (MLVA) for genetic relatedness and Mismatch Amplification Mutation Analysis (MAMA) PCR for toxin genes.DNA extraction was performed using Qiagen-QIAamp DNA Mini-Kit . Briefly, 5μl of overnight protocol . ExtractV. cholerae isolates based on the Variable Numbers of Tandem Repeats (VNTR). Each VNTR locus was amplified using specific primers, as described previously [http://eburst.mlst.net) [MLVA analysis was performed for five loci  of eviously , 13. PCRlst.net) , 13.V. cholerae O1. MAMA-PCR designed to detect the nucleotide sequence difference at position 203 of the ctxB gene to identify cholera toxin of classical and El Tor biotype was used. A conserved forward primer  and two allele-specific primers, Rv-elt 5′-CCTGGTACTTCTACTTGAAACA-3′) and Rv-cla (5′-CCTGGTACTTCTACTTGAAACG-3′) were used (14). Seventy-nine V. cholerae strains were subjected to MAMA-PCR to detect the V. cholerae O1 that harbors classical ctxB or El Tor ctxB separately. N16961 El Tor biotype and 569B classical biotype were used as reference strains. DNA template for PCR, specific primers, Taq polymerase, and nitrogenous bases were prepared. The automated thermal cycler was programmed as follows: Initial denaturation at 94°C for 2 minutes, followed by 30 cycles of denaturation at 94°C for 30 seconds, annealing at 60°C for 20 seconds, and externsion at 72°C for 25 seconds and a final extension at 72°C for 4 minutes [MAMA-PCR was perfomed to distinguish the cholera toxin B subunit of El Tor and Classical biotypes of ere used . Seventy minutes .ctxA responsible for cholera toxin and tcpA responsible for toxin-co-regulated pilus specific for El Tor and classical strains [The isolates were examined for the presence of genes encoding virulence using multiplex PCR. The virulence included  strains . BrieflyS. aureus (ATCC 25923) for erythromycin and E.coli (ATCC 25922) for the rest of the antibiotics were used as reference strains for quality control.Antimicrobial susceptibility testing (AST) was performed using Kirby Bauer disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines . The antData was generally analyzed using the Microsoft Excel program, where eBurst software was used to analyze genetic relatedness.V. cholerae isolates were investigated; Thirty-two (32.3%) isolates were from Mbeya region with Dar es Salaam contributing 24 (24.2%) of the isolates. Katavi region had only one isolate. All isolates were V. cholerae serogroup O1, with 97 (98.0%) being Ogawa serotype, and only 2 (2%) were Inaba serotype. Mbeya was the only region that reported both Ogawa and Inaba serotypes  had 36 (85.7%) genotypes , while CctxA gene that codes for cholera toxin subunit A and tcpA (El Tor) that codes for toxin co-regulated pili for intestinal colonization , and 569B  generated PCR products which indicated that the primers were working optimally.nization . All 79 Tor type . The ctxisolates . The useAll 99 isolates (100%) were sensitive to trimethoprim-sulphamethoxazole, tetracycline, doxycycline, ceftriaxone, and chloramphenicol while 88% were susceptible to ciprofloxacin. Resistance to nalidixic acid, ampicillin, erythromycin, and amoxicillin was 100% .V.cholerae isolated from various regions in Tanzania during the cholera outbreak that occurred between 2016 and 2017. V. cholerae O1 Ogawa was the predominant serotype. Generally, isolates were genetically related, irrespective of the time of the outbreak. All eleven regions had serotype Ogawa, while serotype Inaba was only detected in Mbeya region. Wide distribution of V. cholerae O1 Ogawa was reported in 2011 and 2015 outbreaks that occurred in Tanzania Mainland [The current study determined the genetic relatedness, virulence genes, and antimicrobial susceptibility patterns of the Mainland . On the Mainland . The difMainland .V.cholerae demonstrated an extensive genetic diversity [V.cholerae O1, though, other atypical variants of El Tor biotypes exist [ctxA, tcpA, and ctxB) of V. cholerae O1 strain detected in this study are consistent with findings in Malaysia [ctxB [The current study demonstrated the close genetic relatedness among isolates, causing outbreaks between 2016 and 2017 with a slight genetic diversity in the affected regions. Of the 42 genotypes detected, the isolate that determined the group occurred in Dar es Salaam, spread to Mbeya and Ruvuma regions, and then to other regions. The findings signify that the Dar es Salaam region was the source of the cholera outbreaks that occurred from 2016 to 2017. The current findings differ from those reported in the earlier outbreaks that occurred between 2011 and 2015, where iversity –26. Clases exist . El Tor es exist . Our finMalaysia . Moreoveia [ctxB , 25.V. cholerae isolates in this study were susceptible to the antibiotics currently used in the treatment of cholera in Tanzania, including doxycycline and ciprofloxacin. However, resistance to fluoroquinolones, including ciprofloxacin, has been reported [V.cholerae isolates being phenotypically susceptible to trimethoprim, ciprofloxacin, ceftazidime, tetracycline and chloramphenicol but genotyically resistant to trimethoprim [Most reported . Notablyreported . The isoethoprim . On the ethoprim . A situaethoprim . The emeV.cholerae O1, serotype Ogawa, El tor variant. The outbreaks have a single primary source where the interventions should be focused to stop further spread of the disease. Most isolates were susceptible to the antibiotics currently used to manage cholera cases in Tanzania. However, high resistance rate detected for previously used antibiotics regime calls for continuous monitoring of the trend of antimicrobial susceptibility testing during the outbreaks.The cholera outbreaks in Tanzania between 2016 and 2017 were mainly caused by clonally related S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 Raw images(TIF)Click here for additional data file.


Mycobacterium tuberculosis (Mtb), with 10.4 million new cases per year reported in the human population. Recent studies on the Mtb transcriptome have revealed the abundance of noncoding RNAs expressed at various phases of mycobacteria growth, in culture, in infected mammalian cells, and in patients. Among these noncoding RNAs are both small RNAs (sRNAs) between 50 and 350 nts in length and smaller RNAs (sncRNA) < 50 nts. In this review, we provide an up-to-date synopsis of the identification, designation, and function of these Mtb-encoded sRNAs and sncRNAs. The methodological advances including RNA sequencing strategies, small RNA antagonists, and locked nucleic acid sequence-specific RNA probes advancing the studies on these small RNA are described. Initial insights into the regulation of the small RNA expression and putative processing enzymes required for their synthesis and function are discussed. There are many open questions remaining about the biological and pathogenic roles of these small non-coding RNAs, and potential research directions needed to define the role of these mycobacterial noncoding RNAs are summarized.Tuberculosis (TB) is an infectious disease caused by  Mycobacterium tuberculosis (Mtb) remains one of the leading infectious causes of human mortality, supplanted only in 2020 by the COVID-19 pandemic triggered by the SARS-CoV-2 virus. Mtb evolved from an ancestral smooth tubercule bacillus , acquiring virulence elements to attain its preferred pathogenicity towards humans . La. LaM. smegulated  10]..M. smegmrv3282, fadA3, and lipB translation by binding a 7–11 nucleotide region upstream of the start codon. Supplementing fatty acids in the mycobacterial cultures overrides this regulatory process, revealing a feedback loop to control metabolic functions in Mtb. The regulatory role for sRNAs in Mtb metabolism is also revealed with ncRv11846 (106 nts). An ortholog of the E. coli sRNA RhyB, ncRv11846 is termed mycobacterial regulatory sRNA in iron (MrsI) , later named Mcr11, upregulates several genes required for Mtb fatty acid production  30]. Th. Th30]. n (MrsI)  12]. nc. ncrv328esxA and espI. Over-expressing sncRNA-1 alters the Mtb transcriptome, with multiple genes required for fatty acid biogenesis increased in expression. Screening putative targets of sncRNA-1 by seed-sequence complementarity searches reveals two targets of this sRNA, rv0242c, and rv1094. These encode two proteins involved in the oleic acid biogenesis pathway. Both genes have putative sncRNA-1 binding sites within their 5′ UTRs. Substituting selected nucleotides involved in Watson‐Crick base pairing, either within the 5′ UTR or in the sncRNA seed sequence, eliminated the positive regulation. One novel approach for studying microRNA functions is the use of locked nucleic acid power inhibitors (LNA-PIs). These have modified RNA sequences that prevent their cleavage by RNA processing enzymes. They also have chemical modifications to enable uptake into cells without any transfection or liposome-based carrier needs . Th. Th13]. er needs . They hyer needs . IncubatM. smegmatis and has homology to the 6S sRNA . Al. Al58]. E. coli and B. subtilis, used as model bacteria for RNA processing, Mtb encodes for RNase P, which is involved in the initial processing of the 5′ end of tRNA molecules [rv2681), and a divergent functional and structural ortholog of RNase T (rv2179c) [E. coli, RNase PH is implicated in degradation of structured RNAs, which accumulate in the mutant lacking this RNase [Transfer RNAs (tRNAs) share some common features with small RNAs, being relatively short and highly structured non-coding RNA molecules. tRNA maturation involves several steps, with both 3′ and 5′ ends being extensively processed in an orchestrated, sequential order. Besides the core RNA degradosome components, tRNA processing enzymes likely play roles in maturation and turnover of certain sRNA species. In eukaryotes, many RNP complexes involved in tRNA biology participate in the generation and subcellular trafficking of other small structured RNAs such as snRNA, snoRNA, 5S RNA, and others . In manyolecules . The suiolecules , RNase Drv2179c) . As a lais RNase . In the Streptomyces coelicolor, another actinomycetes related to Mycobacteria [Gln32-CUG and tRNALeu3-CAG [tRNA cleavage is seen in all kingdoms of life as a regulatory mechanism, adding another layer to the complexity of gene regulation mechanisms. This has been observed in bacteria . tRNA clbacteria . TubercuLeu3-CAG . Mtb encLeu3-CAG . Direct Leu3-CAG , but theM. smegmatis growth when overexpressed, a screen for RNA chaperons that mediate the interaction between 6C, and its targets was developed [M. smegmatis clones overexpressing 6C were exposed to saturation mutagenesis, there was some growth. However, the few colonies that recovered were those that had mutations in the overexpression cassette, indicating the lethality of 6C. No other genetic mutations were observed in the colonies from the saturation mutagenesis library. Presuming a chaperon was targeted, the growth inhibition by B11/6C was not overcome, suggesting a chaperone protein was not involved [In the majority of bacteria species, trans-encoded sRNAs require RNA chaperones, either Hfq or ProQ, to ensure appropriate sRNA/mRNA base pairing . Taking eveloped . In expeinvolved . It remaBacillus subtilis [CsrA, a conserved small RNA binding protein, was recently shown to assist in a complex between the sRNA and its mRNA targets in subtilis . The RNAsubtilis . Howeversubtilis . While tE. coli and S. aureus have revealed that cold shock domain containing proteins (CSPs), involved in binding and melting RNA species [E. coli has reported the requirement of the CsdA DEAD-box helicase for low temperature riboregulation of rpoS mRNA via sRNA-mediated mechanism, where the activity of Hfq was not sufficient for translational activation of rpoS expression [Recent studies from  species ,77, also species . Hence,  species . Future pression .M. smegmatis, M. dioxanotrophicus, and M. goodie encode a eukaryotic-like protein with a full length TROVE domain (KEGG database search www.genome.jp (accessed on 27 May 2021)), sharing over 35% identity with the human 60 kDa SS-A/Ro ribonucleoprotein ortholog ). It remains unknown whether the TROVE protein has acquired some functions related to sRNA metabolism in fast growing mycobacterial species or whether it is simply a useless remnant of a previous bacteriophage infection.Intriguingly,  results ). The 60 results . It is t results . In mycoMtb-encoded small RNAs are emerging as new regulators of mycobacterial growth, survival, and pathogenesis. To date, the functions for only a handful of these sRNAs have been described. Given their distinct sizes, their functional contributions likely differ when comparing those <50 nt and those between 50 and 350 nt. The recently developed CRISPR interference-based assays hold great potential for studying the function of sRNAs and sncRNAs ,26,61. I


T. albomarginatus s.l. is mainly distributed in the lowlands of South China, as currently identified. A total of 40 localities , which covers most of its known range, were sampled. Molecular phylogenetic analyses based on concatenated mtDNA and nuclear genes recover nine highly supported lineages clustering into eight geographic populations. The integration of molecular evidence, morphological data, and geographic distribution demonstrates the delineation of T. albomarginatus s.l. as eight putative species. Four species, namely, T. albomarginatus, T. lani, T. analis, and T. zhangfei sp. nov. and the T. similis complex are taxonomically recognized herein. Moreover, T. zhangfei sp. nov. comprises two genetically distinct lineages with no morphological and geographical difference. This study also reveals aspects of estimation of divergence time, distribution, and ecological adaption within the T. albomarginatus group. The unraveling of the hidden species diversity of this lowland bagrid fish highlights the need for not only the molecular scrutiny of widely distributed species of South China but also the adjustment of current biodiversity conservation strategies to protect the largely overlooked diversity of fishes from low-elevation rapids.There is increasing evidence that species diversity is underestimated in the current taxonomy of widespread freshwater fishes. The bagrid species  Freshwater ecosystems not only harbor incredible amounts of biodiversity but also provide significant ecosystem services for mankind . HoweverThe delineation of hidden diversity within widespread species has the great potential to further our understanding of macroecology, evolutionary processes, and conservation biology . The practice of integrative taxonomy can yield many more putative species than traditional taxonomy, which would result in improved biodiversity assessments allowing better modeling of macroecological processes and increased resolution of spatial heterogeneity . The detHemicculter leuciclus   . The preognition . A thoroTachysurus albomarginatus s.l. utilizing integrative approaches. Molecular phylogenetic relationships among intraspecific populations were inferred based on the concatenated mitochondrial and nuclear genes of specimens extensively sampled throughout the majority of its known range. Morphometric measurements and meristic counts were analyzed to test for morphological differentiations among genetic clustering groups. Species delineation within T. albomarginatus s.l. was predicated on the congruence between morphological and genetic datasets. The findings of all the above analyses highlight a need for taxonomic reevaluation of currently identified widely distributed species and reconsideration of current protection strategies of freshwater fishes from South China, particularly those found in relatively lower elevations.The objective of this study is to explore the possible hidden species diversity within Tachysurus albomarginatus s.l. were selected, spanning 14 river basins ] and one nuclear gene [recombination activating 2 (RAG2)] were amplified using polymerase chain reaction (PCR). The information for the primers used in this study is shown in Genomic DNA was extracted from alcohol-preserved fin clips using the DNeasy Tissue Kit . Two mitochondrial genes [cytochrome b (Cyt. b) and cytochrome b, and RAG2 genes of each analyzed specimen were concatenated to form a dataset for phylogenetic tree reconstructions with Bayesian inference (BI) and Maximum likelihood (ML). The general time-reversible model with invariant sites and a gamma distribution variation across sites (GTR + G) was selected as the best-fitting model for COI, Cyt. b, and RAG2 in Jmodeltest2 . Bootstrap values (BS) of nodes were estimated by 1,000 bootstrap replicates. All trees were rooted by Hemibagrus macropterus and visualized in FigTree v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).COI, Cyt. deltest2  based ondeltest2 . MrBayesdeltest2  was applHaplotype networks were constructed for the concatenated mtDNA sequence datasets to better visualize relationships in multifurcations and reticulations . A maximT. albomarginatus s.l.: the automatic barcode gap detection (ABGD) and the Poisson tree process (PTP) model. The automatic barcode gap discovery (ABGD) is a barcode species delineation method aiming to establish a minimum gap that probably corresponds to the threshold between interspecific and intraspecific processes  in which a 0.1 value assumes a maximum intraspecific variability, suggesting that all sequences belong to only one species, whereas a 0.001 value assumes a very small intraspecific variability suggesting that each distinct haplotype represents a different species. After performing the ABGD analysis, additional molecular, morphological, or ecological characters are needed to infer the correct number of species. The ABGD analysis was run on the ABGD server website (https://bioinfo.mnhn.fr/abi/public/abgd/abgdweb.html).Two independent methods relying on different operational criteria were applied to infer molecular species delineation for rocesses . The majhttp://species.h-its.org/ptp/).The PTP is a tree-based species delineation method under the assumption that the intraspecific substitution number is significantly less than the interspecific substitution number . The BayT. albomarginatus s.l. using the birth/death speciation process model in Beast v1.10.4 with concatenated data of Cyt. b and COI sequences  to 0.18–0.30% per million years for substitution rate. The mean rate and standard deviation of COI were estimated in the MCMC run. A single MCMC run was performed with 200,000,000 generations, sampling every 1,000 generations. Sufficient convergence and burn-in of runs were checked in Tracer v1.7 (http://tree.bio.ed.ac.uk/software/tracer) to ascertain that all effective sampling size (ESS) values are above 200. The first 10% of samples were discarded as burn-in in TreeAnnotator v1.10.4 in the Beast package (http://tree.bio.ed.ac.uk/software/figtree/).We estimated the divergence time of equences . Best-fi package . A conseTwenty-six morphometric measurements utilized in this study are presented in PCA was run with SPSS 16 . Prior to the analysis, the method of The counts of vertebrae were taken from photographs of Micro-CT (X-ray-based micro-computed tomography) scanning to explore internal morphology. Micro-CT scanning was made in a Siemens SOMATOM Definition X-ray machine . Micro-CT data were imported into the Mimics medical imaging software , and a 3D digital reconstruction of each block was created. The individual reconstructions were tidied, smoothed, and stitched together utilizing Adobe Photoshop .The sequence information for the collection used in the molecular phylogenetic analysis is shown in T. albomarginatus s.l. are inferred from the combined mtDNA (COI and Cyt. b) and nuclear (RAG2) genes using two different inference approaches (ML and BI). Analyses based on these two approaches results in the same general topology  and posterior probability (hereafter pp) and cluster into five clades  well supported by 100% bv and 100% pp. While Clades B, C, and E include a single lineage , Clades A and D comprise four and two lineages, respectively. Clade E is the earliest diverging lineage including samples from the Liu-Jiang of the Zhu-Jiang basin. It is supported by 96% bv and 72% pp to be sister to the group formed by the remaining four clades where Clade D is the basal lineage and the weakly supported (less than 50% bv and 81% pp). Paired clades (B and C) receive a strong support of 100% bv and 91% pp to be sister to Clade A. Clade D is constituted by samples from the Yuan-Jiang and Xiang-Jiang, two affluents of Lake Dongting linked to the middle Chang-Jiang, and comprises two sister lineages (D1 and D2), sympatrically occurring or having a entirely overlapping distribution. Clade C is made up of samples from the Xin-Jiang, Fu-He, and Gan-Jiang, three affluents of Lake Poyang connected with the mainstem of the lower Chang-Jiang. Clade B is composed of samples from the lower Huai-He basin, the main stream of the lower Chang-Jiang, and coastal river basins of Zhejiang Province such as the Qiantang-Jiang and Ou-Jiang. There are four lineages (A1-A4) clustered within Clade A where Lineage A4 is sister to the remaining three ones constituting a polytomy. These lineages are each weakly supported but exhibited a significant regional clustering as they correspond to four coastal river basins of Fujian Province. Lineages A1-A4 are each found only in the Min-Jiang, Jin-Jiang, Han-Jiang, and Jiulong-Jiang basins, respectively.Phylogenetic trees for topology . All samT. albormarginatus s.l. species group  , is shown in 79–6.01) . FollowiThe ABGD analysis supports six molecular operational taxonomic units (MOTUs) at a prior maximal distance of 0.0100, and these six MOTUs correspond to four clades  and two lineages (D1 and D2), recovered in the molecular phylogenetic analysis based on the combined mtDNA and nuclear genes . ConcurrThe nine distinct lineages detected in the molecular phylogenetic analysis correspond to eight geographical populations . Paired There are two morphometric measurements  with main loadings on the first axis (PC1), three  on the second axis (PC2), and two (inner and outer mandibular-barbel lengths) on the third axis (PC3) . Stable Clade C and Lineage A1 differ from all other lineages in having more vertebrae numbers  and nuclear (RAG2) genes, and the use of different molecular species delineation methods results in the detection of six or nine MOTUs , despite their small genetic divergences.The hidden species diversity of the morphology-based species ne MOTUs . The misne MOTUs , fails tne MOTUs . In contne MOTUs . In the T. albomarginatus s.l. correspond to eight geographic populations. Sympatric lineages D1 and D2, both indistinguishable morphologically, represent one geographical population. Each of the remaining seven lineages forms its own geographical one. These eight allopatric populations are distinguished by the combination of two meristic counts and three morphometric measurements  are truly cryptic species, as both exhibit a distinct sequence divergence of 2.2% for the Cyt. b gene, a complete overlap in their ranges, and no external morphological differences. External morphology and allopatric distribution patterns distinguish the species represented by Lineages A2-A4 from the rest of this species group, and no previous studies have ever described them after a review of literature, thus making them pseudocryptic species.There is growing evidence for the existence of underestimated diversity when morphology-based species are scrutinized under a molecular phylogenetic context . Two or T. albomarginatus species group forms a monophyletic assemblage under rapid diversification , inhabiting in the lower Chang-Jiang basin and coastal rivers of Southeast China, are phylogenetically allied to each other but exhibit low sequence divergence and low resolution of phylogenetic relationship, thus indicating an explosive speciation event. The emergence of the most recent common ancestor of these three clades was estimated to be about 3.91 Ma, and the divergence time between Clades B and C was roughly 3.45 Ma. This period is broadly consistent with the intense uplifting of the Qinghai–Tibetan Plateau and the accompanying occurrence of Asian monsoons during the mid-Pliocene . It exhibits a similarity of genetic structure , 3, therears ago . The canT. albomarginatus s.l. as including eight putative species. A conservative stance is adopted to recognize five taxa including four species, namely, T. albomarginatus (Clade B), T. analis (Clade C), T. lani (Clade E), and T. zhangfei, sp. nov. (Clade D), and a species complex (Clade A): the T. similis complex. Taxonomic confusions surrounding T. albomarginatus have been clarified by T. albomarginatus.In this study, integrative taxonomy supports the detection of b gene among Clades A-E range from 1.9 to 2.4%, which fulfill the barcode threshold of 2% , Fu-He, and Gan-Jiang of the Poyang Lake system clustered into Clade C which was distantly allied with Clade B, representing a species distinct from T. albomarginatus. These samples also have a narrow, slightly tapering, rounded caudal fin  as a valid species and also marked differences in the counts of vertebrae and anal-fin rays . In reality, the new species represents a cryptic species complex, within which two cryptic species having overlapping distribution and indistinguishable morphology are included (Lineages D1 and D2).Recently, fin rays  with allTachysurus similis, initially described by Tachysurus also show that T. analis is the only species with 47–49 vertebrae. Samples from Min-Jiang, with 19–22 anal-fin rays  in the phylogenetic trees  depicts a species with a weakly forked instead of a slightly emarginate caudal fin , clustered within Clade A, represent three pseudocryptic species. However, it is premature to assign a specific status to each of these lineages, given that their sample sizes and number of sampling localities in this study are relatively small, especially for Lineages A2 and A3 . The namTachysurus albomarginatus s.l. in this analysis has many implications for biodiversity conservation. The species, as conventionally defined, has so far been listed as LC (least concern) in the recent assessment of Chinese freshwater fish red list  strong serrations along posterior margin. Pectoral-fin margin straight anteriorly, convex posteriorly. Cleithral process triangular with a sharp pointed tip. Pelvic fin with five soft branched rays, inserted equidistant from vertical through caudal-fin base and tip of snout. Tip of depressed pelvic fin slightly extending beyond anal-fin origin. Pelvic-fin distal margin convex. Anus and urogenital opening halfway between anal-fin origin and pelvic-fin insertion.Anal fin shorter than adipose-fin base in length, with 15 (4), 16 (12), 17 (9), or 18 (5) branched rays; inserted anterior to adipose-fin origin. Anal-fin origin nearer to caudal-fin base than to tip of snout. Distal margin of anal fin convex; anterior rays shortest.Caudal fin broadly rounded or slightly emarginate, with 8 + 9 principal rays; upper lobe roughly equidistant from lower one; procurrent rays extending slightly anterior to fin base.In formalin-preserved specimens , body daTachysurus zhangfei is currently known from the Xiang-Jiang and Yuan-Jiang, and possibly Dongting Lake, in the middle Chang-Jiang basin.The specific epithet, used as a noun, is named after Zhang Fei, a character of the Romance of the Three Kingdoms—one of four famous literature works in China, in allusion to the presence of a blackish or brown body. He is endowed with an imagination of a black face in China opera.Pseudobagrus (=Tachysurus) predicated on mitochondrial sequence data. Clade I  recovered by them was formed by species with a smooth anterior margin of the pectoral spine and short maxillary barbels not extending to the base of the pectoral spine. Twenty-one constituent species can be subdivided into two groups based on the caudal-fin shape (Tachysurus albomarginatus are referred, includes the following nine species: T. adiposalis, T. brachyrhabdion, T. gracilis, T. omeihensis, T. tenuis, T. trilineatus, T. truncatus, and T. ussuriensis. All these species except T. adiposalis and T. omeihensis were included in the ingroup of our molecular phylogenetic analysis. The topology of the phylogenetic tree (T. albomarginatus group. It is apparent that the caudal-fin coloration can be used as a character uniquely diagnostic for this species group. A narrow white mark along the distal margin of the caudal fin is present in T. tenius and T. ussuriensis (T. brachyrhabdion and T. gracilis (The taxonomic usefulness of the presence or absence of serrations along the anterior margin of the pectoral spine and length of maxillary barbels was suggested in in shape . The spetic tree  shows thuriensis . A shortgracilis .


Ancylonema nordenskioeldii or unicellular Mesotaenium berggrenii var. alaskanum, were sampled. Molecular analysis showed that both species are closely related, sharing identical chloroplast morphologies , thus the unicellular species was renamed Ancylonema alaskana. Moreover, an ecophysiological comparison of the two species was performed: pulse–amplitude modulated (PAM) fluorometry confirmed that they have a high tolerance to elevated solar irradiation, the physiological light preferences reflected the conditions in the original habitat; nonetheless, A. nordenskioeldii was adapted to higher irradiances while the photosystems of A. alaskana were able to use efficiently low irradiances. Additionally, the main vacuolar polyphenol, which effectively shields the photosystems, was identical in both species. Also, about half of the cellular fatty acids were polyunsaturated, and the lipidome profiles dominated by triacylglycerols were very similar. The results indicate that A. alaskana is physiologically very similar and closely related but genetically distinct to A. nordenskioeldii.Melting polar and alpine ice surfaces frequently exhibit blooms of dark pigmented algae. These microbial extremophiles significantly reduce the surface albedo of glaciers, thus accelerating melt rates. However, the ecology, physiology and taxonomy of cryoflora are not yet fully understood. Here, a Swiss and an Austrian glacier dominated either by filamentous  Mesotaenium berggrenii var. alaskanum and Ancylonema nordenskioeldii . Both species are closely related but morphologically distinguishable, the first being more roundish after cell cleavage and practically unicellular − palmitic acid in the negative ESI. The mass accuracy was better than 0.9 ppm. The chemical structure of the compounds was confirmed with the help of the spectral database LIPID MAPS® Lipidomics Gateway (http://www.lipidmaps.org/ (accessed on 15 December 2020)).An LTQ-Orbitrap Velos mass spectrometer , a high-resolution hybrid mass spectrometer equipped with a heated electrospray interface (H-ESI), was operated in positive and negative ionization mode. Flow injection analysis (FIA) was used for sample introduction into the H-ESI ion source. Acetonitrile/water  were confirmed by comparison with gas chromatography/mass spectrometry retention times and fragmentation patterns with those of standard FAMEs  . ProceduA. nordenskioeldii and some scattered unicells of M. berggrenii var. alaskanum. In contrast, the blooms at Gurgler Ferner in Austria, which were situated approximately 90 km northeast from the Swiss site, consisted microscopically exclusively of M. berggrenii var. alaskanum. Based on the acquired morphological and genetic data from both field sites, the latter was moved to the genus Ancylonema and the rank of a variety is raised to species status (see below).Microalgal ice surface blooms at Morteratsch Glacier in Switzerland were mainly caused by filaments of Ancylonema alaskana (Kol) Procházková and Remias comb. et stat. nov. BASIONYM: EPITYPE (here designated): The field vegetative cells (specimen WP167) deposited in a lyophilized (non-viable) state at the Culture Collection of Algae of Charles University in Prague, Czech Republic. HABITAT: polar and alpine glacial ice surfaces, occasionally in melting snow and slush.DISTRIBUTION : Alaska ,29,30, ErbcL (MW922840).SPECIES DESCRIPTION: Cells single or in temporary connected pairs after cell division, cell walls smooth, cells 7–11.6 µm wide and 8.1–20.6 µm long, cylindrical cell shape with apexes broadly rounded, one (or two) parietal, discoid, cup-shaped or elongate-hemispherical, weakly lobed chloroplast with one pyrenoid, sometimes causing a bulge. Cytosol with central unpigmented nucleus and peripheral vacuoles, the latter occupied by characteristic dark phenols. Chloroplast division precedes cytokinesis. Sexual reproduction by conjugation between two cells results in a regular or irregular, spherical or angular to ellipsoidal zygote. DNA sequences available: nuclear 18S rDNA (JF430424) and plastid FIELD SAMPLES EXAMINED: WP167, WP251.A. nordenskioeldii occurs in the same habitat as A. alaskana and has the same brownish vacuolar pigmentation, but differs by significantly larger cell widths and cell lengths , trichal habitus  ..5 cells A. alaskana from A. nordenskioeldii. Interestingly, generally smaller A. nordenskioeldii were reported from Arctic Svalbard , these rbcL phylogenies showed a very close relationship between A. nordenskioeldii and A. alaskana, pointing to a common ancestor, and they formed the Ancylonema clade. In contrast, the third prominent species of glacial algae, Cylindrocystis brebissonii, is situated in its own zygnematophycean clade which was named after this organism [Molecular mechanisms during terrestrialisation of the Zygnematophyceae were highlighted by , namely organism .A. alaskana from Austria was very closely related to four unaffiliated environmental sequences isolated from an Alaskan glacier. Appropriately, this species (respectively its basionym) was initially described from Alaska [rbcL sequences obtained in this study confirmed the placement of the genus Ancylonema in the larger “Mesotaenium 1”-clade sensu [rbcL sequences belonged to semi-terrestrial Mesotaenium AG-2009-1 isolated from wet rocks in a forest at Eifel, Germany . It was placed into the Ancylonema clade, indicating that this clade is not exclusively glacial algae. The further closest relatives are the high alpine semi-terrestrial alga Zygogonium ericetorum from an intermittent streamlet in the Austrian Alps [Fottea pyrenoidosa SAG 1.88, isolated from soil at subantarctic Signy Island [Zygogonium exhibited striking purple vacuoles [m Alaska . The rbcde sensu . The cloian Alps  and Fotty Island . While tvacuoles  caused bMesotaenium. Traditionally, the chloroplast morphology was crucial to distinguish between genera within the Zygnematales. Mesotaenium was typified by a single, central  plate-like chloroplast (and prior cell division there are two of them) [Ancylonema species is clearly parietal, from discoid through slightly cup-shaped to flattened-lobed. A. nordenskioeldii was initially described as possessing a single chloroplast per cell [Cylindrocystis spp. [A. alaskana nor A. nordenskioeldii yet, but its presence cannot be excluded.This study confirms, in accordance with earlier work , the poof them) . In contper cell . Howeverper cell  may eithtis spp. . Such a A. nordenskioeldii from Switzerland. Using different protocols, no drop of the light-dependent oxygen evolution took place in Arctic populations of filamentous Ancylonema, even at high VIS irradiation of 2000 µmol photons m−2 s−1 and more [A. alaskana from Austria performed well up to approximately 1400 µmol PAR m−2 s−1 , compared to A. alaskana. The low alpha and high Ik of A. nordenskioeldii from Switzerland is in agreement with values from a community at the Greenland Ice Sheet [A. alaskana and the Morteratsch Glacier site for A. nordenskioeldii), and further measurements should reveal the variability within and between the species.Fluorometric photophysiology showed no decline of activity at higher irradiation in and more ,12. Simi sensor, ). In thice Sheet . Here, ak ∼46 μmol photons m−2 s−1; [Glacial algae appear dark due to the massive accumulation of brownish aqueous phenols , and them−2 s−1; . The phom−2 s−1; .A. nordenskioeldii and A. alaskana were identical by means of retention time, spectral absorption, molecular weight and MS fragmentation pattern [Cylindrocystis brebissonii [Temnogametum iztacalense [The main chromatographic peaks of  pattern . It remabissonii  or Temnoacalense .A. nordenskioeldii and A. alaskana, reflecting that these species live in the same type of habitat. Here, a snapshot of lipid profiles of natural populations is presented which may be utilized as a reference for future laboratorial strain studies. One of the major thylakoid membrane lipids in algal chloroplasts are glycolipids, namely MGDG, DGDG and SQDG, and phospholipid PG [A. alaskana and A.nordenskioeldii was 5.81 and 5.95, respectively. In general, a ratio of more than 2 is typical for microalgae grown under balanced growth [Ancylonema species, and the same was reported also for leaves of land plants [Regarding the proportion of the lipid classes and relative proportions of lipid species, a very similar lipidome was found for lipid PG . Microallipid PG . The MGDd growth , while td growth ,55. Membd growth . In the d growth . The mosd growth . Additiod growth . These cd growth . Namely,d plants .Zygnema [A. nordenskioeldii and A. alaskana, taken close to the end of the melting season at alpine glaciers , showed that the predominant fatty acids of both glacial blooms were palmitic acid, oleic acid, linoleic, α-linolenic and steriadonic acid. Similarly, these five FAs were the most abundant and accounted altogether for 93.8% of all fatty acids in the subantarctic soil alga Fottea pyrenoidosa SAG 1.88 [Generally, the knowledge of FA composition of conjugating algae is limited . In a stZygnema , young aZygnema . The snaSAG 1.88 . In thisSAG 1.88 . HoweverAncylonema can no longer be regarded as a monotypic genus for a filamentous glacial alga. Except from molecular traits, the main diagnostic feature of this genus are parietal hemispheric, cup-shaped or elongate-lobed chloroplasts while “true” Mesotaenium are characterized by an axial, plate-like chloroplast. Unicellular glacial alga A. alaskana  is widespread in European glaciers, but it has to be tested how much species are actually behind the phenomenon in a global point of view. Physiologically, A. nordenskioeldii and A. alaskana are closely related organisms with similar or identical cellular lipid profiles and identical kind of phenolic secondary pigmentation. The photophysiologies of both species have to be compared with caution since the sampling sites were not the same. Nonetheless, the photosystems of A. alaskana were able to use efficiently low irradiances but showed higher ETR rates throughout the rapid light curve as compared to A. nordenskioeldii. Finally, the ecological reason why A. alaskana dominates communities at central European glaciers and, vice versa, A. nordenskioeldii dominates Arctic ones, remains open.This study demonstrated that


Bineurus Simpson 1900 and Thaiconcha Bolotov et al., 2020 primarily inhabit small and medium-sized tributaries of these rivers. Here, we present an integrative taxonomic review of these genus-level clades. We show that Bineurus contains four species: B. mouhotii , B. exilis  stat. rev., B. anodontinum  stat. rev., and B. loeiensis sp. nov. In its turn, Thaiconcha comprises three species: T. callifera , T. munelliptica sp. nov., and T. thaiensis sp. nov. Two species, Pseudodon ovalis Morlet, 1889 and P. thomsoni Morlet, 1884, are considered here as questionable taxa. These findings further highlight that Southeast Asia represents a significant evolutionary hotspot of freshwater mussels, which requires further international collaborative research and conservation efforts.The Mekong and Chao Phraya rivers harbor a species-rich freshwater mussel assemblage containing a large radiation of the Pseudodontini species. Members of the genera  Several recent phylogenetic studies have substantially revised the systematics of various freshwater mussel taxa from the Mekong basin, especially among representatives of the tribes Indochinellini , Rectidentini , and Contradentini . Less taxonomic focus has been paid to the most species-rich tribe in Southeast Asia, the Pseudodontini. It was shown that the genus- and species-level diversity of this clade needs greater systematic attention, as it was largely underestimated12.The Mekong is the largest freshwater basin in Southeast Asia harboring a species-rich fauna of the UnionidaeBineurus Simpson 1900 represents a divergent clade within the Pseudodontini, with at least two valid species: B. mouhotii  and B. exilis 12. Originally, it was described as a subgenus within Pseudodon Gould, 1844 with three species: Pseudodon mouhotii, P. exilis, and P. avae 14. Later, Prashad15 transferred Pseudodon avae to the genus Indopseudodon Prashad, 1922. Afterwards Haas16 synonymized Pseudodon exilis with P. mouhotii and placed only two species in the section Bineurus, i.e. P. mouhotii and P. hageni . Bineurus has remained to be a valid subgenus/section until a comprehensive revision of Brandt1 who recognized the single genus Pseudodon (without subgenera and sections) containing four species, i.e. P. mouhoti, P. inoscularis , P. cambodjensis , and P. vondembuschianus  . Recently, Bolotov et al.17 resurrected Bineurus as a valid genus based on the results of an integrative taxonomic analysis. However, the species-level taxonomy of Bineurus taxa remains poorly understood, and yet to be revised using DNA sequence data. Furthermore, the genus Thaiconcha Bolotov et al., 2020 was introduced to separate the Pseudodon callifera/P. ellipticum group from the Bineurus clade12. Pseudodon callifera  was assigned as the type species for the genus, whereas P. ellipticum Conrad, 1865 and P. thomsoni Morlet, 1884 were synonymized with the first species12. Finally, the nominal taxon Pseudodon ovalis Morlet, 1889 was also placed in Thaiconcha but that solution was based solely on conchological features12.The genus Bineurus and Thaiconcha using integrative approach combining DNA-based, morphological, and biogeographic evidence, and (2) describe three freshwater mussel species new to science discovered in the Mekong and Chao Phraya basins.The present study aims to (1) revise the genera COI + 16S rRNA + 28S rRNA) included 143 individuals from the Pseudodontini . We identified four species-level Bineurus subclades corresponding to B. mouhotii, B. exilis, B. anodontinum, and B. loeiensis sp. nov. The Thaiconcha clade contained three species-level subclades, including T. callifera and two undescribed lineages, i.e. T. munelliptica sp. nov. and T. thaiensis sp. nov. All the novel subclades represented well-supported species-level groups with diagnostic nucleotide substitutions (Table COI p-distance) among these species varies from 2.4 to 5.8% , and it is characterized by having a smooth periostracum without wrinkles, and higher posterior margin.Past studies on the genus eth Fig. . ProspecThaiconcha species share elliptical shells, small umbo, more or less developed pseudocardinal teeth and muscle scars, and elongated posterior end   Figs. E,F, 4 diBineurus includes the Mekong basin in Laos, Thailand, Cambodia, and southern Vietnam. Bineurus exilis is the most widespread species, and is distributed throughout the Middle and Lower Mekong  around Nong Khai in Thailand , although it represents a rather divergent intraspecific lineage  was discovered from the Loei River, a tributary of the Upper Mekong  also occurs in at least one northern tributary of the Chao Phraya  from the Chao Phraya population were genetically identical, or very similar (2 nucleotide difference), to specimens from the Kong Si River of the Upper Mekong  and P. ovalis Morlet, 1889 (syntype Cat. no. MNHN-IM-2000-35801) have demonstrated that these species more likely do not belong to the genus Thaiconcha. Both species share a rather specific shell shape that seems to be higher posteriorly than that of Thaiconcha taxa. The type specimen of Pseudodon ovalis differs from Thaiconcha by having a compressed circular shell with weakly developed wing. A syntype of Pseudodon thomsoni is obovate, moderately inflated, truncated posteriorly, and has a distinct ridge on the posterior slope. These nominal taxa seem to be representatives of Monodontina Conrad, 1853. However, due to the lack of molecular data we transfer these taxa to the questionable group (see Taxonomic account below).Additional morphological research using the type specimens of Finally, a taxonomic key was designed for more accurate identification of the genera, belonging to the tribe Pseudodontini.Family Unionidae Rafinesque, 1820.Subfamily Gonideinae Ortmann, 1916.Tribe Pseudodontini Frierson, 1927.Pseudodon Gould, 1844 .Type genus: Monocondyloea mouhotii Lea, 1863 .Type species: Bineurus can be distinguished from its sister genera by an elongated rhomboid/kidney-shaped, inequilateral, compressed, and rather thin shell, usually with straight or concave ventral margin. Hinge plate narrow, with a small, tubercle-like pseudocardinal tooth in each valve. Muscle scars are usually more or less developed.Diagnosis: Distribution: Mekong River basin.Comments: This genus contains four species Table , one of Monocondyloea mouhotii Lea (1863): 19023. = Monocondylaea mouhotiana Lea (1866): 6524. = Pseudodon (Bineurus) mouhoti : 19025. = Pseudodon mouhoti : 265–2661. = Figure Type and type locality: Holotype USNM 86339  and northern Thailand.Monocondyloea mouhotii and Unio laosensis Lea, 1863 23. An expanded re-description of these taxa was published three years later23. Henri Mouhot, an adventurous French naturalist, collected this sample somewhere during his travel throughout Siam, Cambodia and Laos in the 1858–1861. Lea24 noted that Mouhot’s collection contained only five or six freshwater mussel species with the two new taxa. The collector died near Luang Prabang (Laos) in 1861, two years before the description of the taxa. Mouhot’s field diary and correspondence were published in a two-volume work entitled “Travels in the central parts of Indo-China (Siam), Cambodia, and Laos, during the years 1858, 1859, and 1860”27. It is likely that the vague type locality of the two taxa was taken by Lea (1863)17 from the general route of Mouhot’s travel. In the field notes, Mouhot27 did not mention any samples of freshwater shells, although several samples of land snails and seashells were noted. However, Samuel Stevens, a well-known British naturalist, in a letter to Mouhot’s brother (p. 294)27 wrote that Mouhot collected freshwater shells only from the Laos Mountains.Comments: Isaac Lea briefly described two new freshwater mussel species using a shell sample labelled as “Laos Mountains, Cambodia, Siam. Monsieur Mouhot, per H. Cuming, Esq.”, i.e. Monocondylaea exilis Morelet (1866): 6328  , Pfeiffer and Page leg : 4129. = Pseudodon (Bineurus) mouhoti : 265–2661. = Pseudodon mouhoti : 265–2661. = Figure Type and type locality: Syntype MNHN-IM-2000-1638  sp.2.Comments: Bolotov et al.Anodonta callifera Martens, 1860 .Type species: Diagnosis: Shell large, elliptical, moderately thick, and inflated. Pseudocardinal teeth rather well developed, muscle attachment scars deep.Distribution: Mekong and Chao Phraya basins in Thailand and Cambodia.Comments: This genus contains three species Table , two of Anodonta callifera Martens (1860): 1530. = Pseudodon ellipticum Conrad (1865): 352 : 10, 1411. = Type and type locality: Syntype MNHN-IM-2000-1800 : 166, 19734. = Pseudodon (Monodontina) ellipticus Haas (1969) 13416. = Pseudodon vondembuschianus ellipticus Brandt (1974): 2701. = Thaiconcha ovalis Bolotov et al. (2020): 1012. = Type and type locality: Syntype MNHN-IM-2000-35801  and B. anodontium in the southern part of distribution, . These species are morphologically indistinguishable but are divergent phylogenetically. Despite the morphological similarity, their sympatry and high level of genetic divergence suggest that these lineages represent biological species and are intrinsically reproductively isolated. The presence of morphologically similar but phylogenetically distant species among unionids has been demonstrated for a number of genera, e.g. Pleurobema35, Hyriopsis36, Sinanodonta40, Fusconaia41, and Etheria42.Study of the morphology, phylogeny and distribution of the Pseudodontini from Mekong and Chao Phraya revealed four distant species-level taxa in the genus Thaiconcha are rather variable morphologically and mainly differed by shell shape, especially within T. callifera. Such shape plasticity could be driven by environmental conditions and hydrology which may influence the conchological characteristics45. Bineurus and Thaiconcha species were mainly collected from small and medium sized rivers, as well as at the confluence of small tributaries of the Mekong proper, usually with soft  substrate. Less specimens were found in the mainstreams of the Mekong and Chao Phraya.The representatives of the genus Bineurus loeiensis sp. nov., Thaiconcha munelliptica sp. nov. and T. thaiensis sp. nov. were recognized and described under the framework of this study. The minimum level of genetic divergence was 2.4%  is rather divergent phylogenetically (based on the COI gene p-distance = 2.2%), and it may belong to a separate taxon  were observed in the Mekong (Chiang Rai) and Chao Phraya 47. The cyprinid fish Garra theunensis Kottelat, 1998, known from the Mekong basin, was found in the Upper Nan River belonging to the Chao Phraya drainage48. Among freshwater mussels, Lens contradens  represents an opposite example to Thaiconcha thaiensis as it primarily ranged throughout the Chao Phraya, with a few geographically disjunctive populations in the Mekong 7.The genera 53. The Wang River represents a part of the Northern Chao Phraya drainage, raising from the Phi Pan Nam Mountain Range and flowing through Chiang Rai and Lumpang to the Tak Province, where it joins the Ping River54. The tributaries starting from the northern slopes of this range belong to the Mekong basin, while tributaries of the western slopes fell to the Salween River55. The Phi Pan Nam Mountains serve as a geographic barrier separating these large river basins. Hence, a disjunctive range of Thaiconcha thainesis sp. nov. may reflect past rearrangements of the northern paleo-Chao Phraya and the paleo-Mekong. A small genetic divergence between the COI haplotypes of Thaiconcha thaiensis sp. nov. from the Wang River and a tributary of the Upper Mekong indicated that these drainage rearrangements could have been triggered by relatively recent geological events (e.g. Pliocene to Pleistocene)56. It was found that Lens contradens also shares shallow molecular divergence between its populations from the Mekong and Chao Phraya7.The rearrangements of Southeast Asian river systems such as separations, connections, and stream captures repeatedly occurred in the past mainly caused by tectonic processes and sea level fluctuationsIn any case, a more extensive sampling is required for these remote water bodies to reconstruct the history of past drainage rearrangements and to exclude a possibility of recent human-mediated introduction events for given taxa.Bineurus and Thaiconcha species were collected from different water bodies in Thailand, Laos, and Cambodia (Supplementary Table Bineurus exilis from a tributary of the Tonle Sap River in Cambodia and of B. anodontinum from the Lower Mekong basin (upstream of Sambour) were collected. We studied the type specimens of nominal taxa under discussion and shell lots of other species in the following museum collections: Natural History Museum, London, Great Britain (BMNH); National Museum of Natural History, Smithsonian Institution, Washington D.C., USA (NMNH); Muséum national d’histoire naturelle, Paris, France (MNHN); Florida Museum of Natural History, Gainesville, USA (UF); and Russian Museum of Biodiversity Hotspots, Federal Center for Integrated Arctic Research of the Ural Branch of the Russian Academy of Sciences, Arkhangelsk, Russia (RMBH). Images of the type specimens were obtained from Academy of Natural Sciences of Drexel University, Philadelphia, USA (ANSP), BMNH, and MNHN.The specimens of COI, 16S rRNA, and 28S rRNA gene fragments. It was carried out using PCR primers and laboratory protocols as described in our earlier papers57. The newly generated sequences were checked using a sequence alignment editor (BioEdit v. 7.2.558) and aligned through MUSCLE algorithm in MEGA759. The alignments of three gene fragments were joined to the combined dataset using an online fasta sequence toolbox FaBox v. 1.4160.Molecular analysis was based on three molecular markers, i.e. the COI + 16S rRNA + 28S rRNA) of 147 sequences. Four representatives of the subfamily Gonideinae were used as outgroup, i.e. Gonidea angulata , Potomida littoralis , Leguminaia wheatleyi , and Lamprotula leaii  (Supplementary Table B. exilis (ICH-00734) was deleted from the dataset due to having some doubtful regions. The maximum likelihood (ML) analysis was performed through IQ-TREE (W-IQ-TREE) server61 using an automatic identification of most appropriate evolutionary models62 and an ultrafast bootstrap (UFBoot) algorithm with 5000 replicates63. Models of sequence evolution for each partition calculated through Model Finder64 based on Bayesian Information Criterion (BIC) were following: 1st codon COI − F81 + I; 2nd codon COI − TPM2u + G + I; 3rd codon COI − TN + G + I; 28S − HKY + G + I; TIM2e + G. Bayesian inference analysis (BI) was carried out in MrBayes v. 3.2.765 with four runs, each with three heated (temperature = 0.1) and one cold Markov chain, during 25 million generations and sampling every 1000th generation. The first 15% of trees were discarded as burn-in. The calculation was performed at San Diego Supercomputer Center through the CIPRES Science Gateway66. A trace analysis tool (Tracer v. 1.7) was used to check a convergence of the MCMC chains to a stationary distribution67. The effective sample size (ESS) for all the parameters was recorded as > 3000. To study the phylogeography of Bineurus and Thaiconcha species, a median joining network was constructed through Network v. 4.6.1.3 software21 based on 102 COI haplotypes , the online registration system for the ICZN. The LSID for this publication is: http://zoobank.org/urn:lsid:zoobank.org:pub:EE6080B0-3444-46D8-B24B-4B5D6B97EF4C. The electronic edition of this paper was published in a journal with an ISSN, and has been archived and is available from PubMed Central.The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature (ICZN), and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank (Supplementary Information.