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Table of Contents
Abstract
Background
Objectives
Methods
Results
Conclusion
Introduction
Materials And Methods
Fungal Isolates
Species Identification
Antifungal Susceptibility Testing
Molecular Strain Typing
Whole Genome Sequencing
Phylogenomic Reconstruction
Analysis Of Genetic Polymorphism
Quantitative RT-PCR
Results
Discussion
Author Contributions
Declarations Of Interest
Data Accessibility
Supplementary Data
Competing Interest Statement
Competing Interest Statement
Abstract
Background
The Trichophyton rubrum species group consists of prevalent causative agents of human skin, nail and hair infections, including T. rubrum sensu stricto and T. violaceum , as well as other less well established or debatable taxa like T. soudanense , T. kuryangei and T. megninii . Our previous study provided limited evidence in favour of the existence of two genetic lineages in the Russian T. rubrum sensu stricto population.
Objectives
We aimed to study the genetic structure of the Russian population of T. rubrum , and to identify factors shaping this structure.
Methods
We analysed the polymorphism of 12 simple sequence repeat (SSR, or microsatellite) markers and single-nucleotide polymorphism in the TERG_02941 protein-coding gene in 70 T. rubrum isolates and performed a phylogenomic reconstruction.
Results
All three types of data provided conclusive evidence that the population consists of two genetic lineages. Clustering, performed by means of microsatellite length polymorphism analysis, was strongly dependent on the number of nucleotide repeats in the 5’-area of the fructose-1,6-bisphosphate aldolase gene. Analysis of molecular variance (AMOVA) on the basis of SSR typing data indicated that 22–48% of the variability was among groups within T. rubrum . There was no clear connection of population structure with types of infection, places of geographic origin, aldolase gene expression or urease activity.
Conclusion
Our results suggest that the Russian population of T. rubrum consists of two cosmopolitan genetic lineages.
Introduction
Trichophyton rubrum is an ascomycete from the order Onygenales. It is prevalent fungus of medical importance in most countries due to its major contribution to the etiology of toenail onychomycosis and foot skin mycosis. 1 Trichophyton rubrum sensu stricto is a part of species group, which also includes T. violaceum and T. soudanense along with several forms, potentially deserving species status. All of these can be differentiated by ribosomal DNA internal transcribed spacer (ITS) region sequences. 2 – 6 Statistical analysis of genetic polymorphism in simple sequence repeats (SSR, or microsatellites) revealed clonal mode of reproduction in the species. 7 – 8 It is expectedly accompanied by low genetic polymorphism. 9 – 11 Several studies of mating type locus idiomorphs distribution in T. rubrum population found a single mating type locus variant, out of two, which are necessary for mating. 12 – 14 A certain proportion of T. rubrum isolates, belonging to the so-called “raubitschekii” morphotype, possess urease activity. This trait is believed to be ancestral, 15 but has unclear value for taxonomy. To the best of our knowledge, by the moment there are two papers, reporting bipartite population structure in T. rubrum sensu stricto . In a previous study, we reported a concordance between ribosomal non-transcribed spacer region (NTS) genotypes and nonsynonymous single nucleotide polymorphisms (SNPs) in two protein-coding loci, TERG_02941 and TERG_03298 in T. rubrum isolates from a city in North-Western Russia. 16 The two genetic lines were named ST1 and ST2. Also, Zheng et al. 17 studied a sample of T. rubrum isolates, covering major part of China, by whole genome sequencing (WGS). A number of other studies obtained T. rubrum WGS data from wide variety of geographic locations, covering Europe, Asia and North America. 4 , 14 , 18 , 19 Clear understanding of the potential of low throughput methods to uncover genetic population structure can be achieved when their results are cross-validated and compared with whole genome sequence-based trees. However, there have been no studies, bringing together data on SNP, SSR, WGS and NTS polymorphism in T. rubrum . Our study aimed to verify the presence of previously shown genetic structure in Russian population of T. rubrum sensu stricto , to find whether this structure is linked to particular characteristics and, finally, to identify factors, which shape the structure.
Materials and Methods
Fungal isolates
Clinical isolates of T. rubrum have been obtained in the clinic of Kashkin Research Institute of Medical Mycology, Saint Petersburg (n = 44) and in Sverdlovsk Regional Dermatovenereologic Dispensary, Yekaterinburg (n = 23) from October 2015 to January 2018 (Supplement S1) and subcultured on Sabouraud agar medium, with the addition of dextrose to a final concentration of 2%. We also used two reference strains RCPF 1280 and RCPF 1408 and the type strain CBS 392.58 up to a total of 70 strains in the sample. This sample shared 19 isolates with our earlier study. 16 Major part of strains originated from toenail onychomycosis and tinea pedis cases (87%), 6% were from tinea corporis, 4% from tinea cruris and 3% from fingernail onychomycosis. The following clinical T. rubrum isolates were deposited in the Russian Collection of Pathogenic Fungi (RCPF): D15P33, RCPF 1864; D15P44, RCPF 1865; D15P50, RCPF 1866; D15P62, RCPF 1867; D15P71, RCPF 1868; Y1013, RCPF 1869; D15P139, RCPF 1870. The urease activity was tested twice in Christensen’s urea broth, supplemented with chloramphenicol to a final concentration of 40 μg/ml.
Species identification
was performed by phenotypic methods and confirmed by DNA sequencing of ITS region. To extract genomic DNA, we used GeneJET Plant Genomic DNA Purification Mini Kit (Thermo Fisher Scientific, Lithuania). Aerial mycelium and fungal spores were scraped by pipette tips, placed into microtubes containing Lysis Buffer A, beaten with glass beads in Minilys tissue homogenizer (Bertin Technologies, France), supplemented with Lysis Buffer B and RNase A and further treated according to the kit manufacturer’s protocol. Amplification of ITS region with ITS5 and ITS4 primers 20 ( Table 1 ) was performed in 50-μl volumes, using standard kit for PCR diagnostics (Syntol, Russia). Reaction mixture consisted of 100 mM Tris/HCl (pH 9.0), 250 mM KCl, 10 mM Tween-20, 2.5 mM MgCl 2 , 250 μM each dNTP and contained 5 pmol of primers, 1 U Taq polymerase, and 2.5 μl of template DNA. The thermal cycler C1000 Touch (Bio-Rad, USA) was programmed as follows: initial denaturation step for 5 min at 95 °C, 34 cycles of amplification: denaturation for 30 s at 95 °C, annealing 30 s at 56 °C, extension for 1 min at 72 °C, and a final extension step for 10 min at 72 °C. The products of PCRs were purified by isopropanol precipitation in the presence of glycogen. PCR products were sequenced on both strands using BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, USA) on a 3500 Genetic Analyzer (Thermo Fisher Scientific, USA).
Antifungal susceptibility testing
was performed according to EUCAST E.Def 9.3.1 document, 22 in RPMI-1640 medium with the addition of glucose to a final concentration of 2%. We tested the activity of terbinafine, fluconazole and itraconazole against six T. rubrum isolates with newly obtained WGS sequences. Terbinafine was tested at concentration range 0.007-4 μ g/ml, fluconazole, itraconazole and voriconazole were tested at 0.015-8 μ g/ml, posaconazole was tested at 0.008-4 μ g/ml.
Molecular strain typing
We employed two strain typing approaches. Firstly, we probed the G793A nucleotide substitution in protein-coding locus TERG_02941 (A7C99_6411) by Sanger DNA sequencing. The amplification was carried out under the following conditions: 5 min at 95 °C; 39 cycles of 30 s at 95 °C, 1 min 5 s at 64 °C; and final elongation for 5 min at 72 °C. All other steps were performed as described in the section 2.2 for ITS region sequencing. To optimize the genotyping process, we developed TaqMan SNP genotyping assay with two allele-specific probes ( Table 1 ). Samples were run using a C1000 Touch thermal cycler equipped with a CFX96 real-time PCR detection system (Bio-Rad, USA) according to the following program: initial denaturation step for 3 min at 95 °C; and 39 cycles of 10 s at 95 °C and 30 s at 59 °C. The assay generated fluorescent signal from both probes in every sample, specific signal reached the threshold value with 4-cycle outrunning. Secondly, we performed microsatellite typing. We used eight microsatellite loci from the papers by Gräser et al. 7 and Gong et al. 8 and developed four additional loci ( Table 2 ). To find new polymorphic loci, we searched genome contigs for repeat sequences and prepared nucleotide alignments. In total, 170 loci were screened. Primers for amplification of nine candidate loci were designed with the use of Primer-BLAST tool. 23 Five loci had uniform lengths in 21-56 isolates and were excluded from further experiments. Amplification of microsatellite loci was performed in 25-μl volumes. The primers used are listed in the Table 2 . The amplification program for microsatellites included initial denaturation at 95 °C for 10 min, followed by 30 cycles of 50 s at 95 °C, 1 min at 58 °C, and 1 min at 72 °C, and a terminal extension step of 72 °C for 10 min. Fluorescent PCR products were subjected to fragment analysis on a 3500 Genetic Analyzer (Thermo Fisher Scientific, USA) in the presence of Red DNA Size Standard (Molecular Cloning Laboratories, USA). The set of fluorescent dyes used in this work appeared to be suboptimal because of the need to check data for issues of spectral overlapping between TAMRA and ROX channels.
Whole genome sequencing
Genomic DNA of six T. rubrum isolates was extracted using GeneJET Plant Genomic DNA Purification Mini Kit (Thermo Fisher Scientific, Lithuania). Preparation of DNA sample library was done with the use of Kapa HTP Library Preparation Kit (Roche, USA), according to the manufacturer’s instructions. Paired end sequencing (250 × 250 cycles) was performed using the MiSeq sequencer (Illumina, USA). The reads obtained were prepared for the analyses as described earlier. 24 Quality control of the reads was performed using FastQC 0.11.8. 25 It was followed by trimming low-quality reads and TruSeq2 adapters with Trimmomatic 0.39. 26 De novo assembly was performed with SPAdes 3.9.0 with the “--careful option”. 27 Raw reads were submitted to the Sequence Read Archive of National Center for Biotechnology Information (BioProject ID PRJNA552357).
Phylogenomic reconstruction
We analyzed six original whole genome sequences, as well as the genome of isolate CBS 118892, NCBI BioProject accession ACPH00000000; 18 isolates CBS 202.88, AOKX00000000; CBS 288.86, AOKU00000000; CBS 289.86, AOKV00000000; CBS 100081, AOKT00000000; D6, AOLB00000000; MR850, AOKR00000000; MR1448, AOKZ00000000; MR1459, AOLC00000000; 14 isolate CMCC(F)T1i, LHPM00000000; 4 IGIB-SBL-CI1, JPGR00000000. 19 The raw read data for eight strains from the project of Zheng et al. 17 have been downloaded from NCBI SRA database and assembled de novo for the analysis. This number included the strains LZ164 (NCBI SRA accession SRX5814844), GZ0580 (SRX5814815), NJ785 (SRX5814822) from one cluster, and the strains LZ155 (SRX5814845), NJ786 (SRX5814823), NJ823 (SRX5814824), DL7506 (SRX5814836) from another cluster, revealed in the original publication. The eighth strain GZ0663 (SRX5814819) from the study of Zheng et al., analyzed here, had unclear cluster affiliation. The sequences of T. rubrum morphotype “megninii” CBS 735.88 JHQM00000000, T. soudanense CBS 452.61 AOKW00000000 and T. violaceum CMCC(F)T3l LHPN00000000 were used as an outgroup. Single-nucleotide polymorphism analysis in core genome was done in Harvest Parsnp 1.2 software. 28 Parsnp run with T. rubrum CBS 118892 assembly GCA_000151425.1 as a reference and with default parameters. Whole-genome phylogenetic tree was reconstructed by Maximum Likelihood method in FastTree2, 29 as implemented in Harvest suite. Branch support values were calculated by performing Shimodaira-Hasegawa test on three alternate topologies around particular splits, with a total of 1000 resamples.
Analysis of genetic polymorphism
A matrix of mutation probability-based genetic distances 30 was calculated in polysat 1.7-3 package for R 31 using microsatellite polymorphism data. Distances in terms of weighted divergence of microsatellite loci were calculated in Microsoft Excel 2010 according to the formula, modified for haploids from. 32 The dissimilarity d i between haplotypes A and B at particular locus i was calculated as absolute value of arithmetic difference between the lengths of PCR products A i and B i The genetic distance D between haplotypes A and B was based on the sum of dissimilarities d between haplotypes at all loci, weighted by dividing this sum by the number of loci taken into analysis, k Both distance matrices were further employed in the construction of phylogenetic network in SplitsTree 4.14.2 program. 33 Neighbor-Joining tree on the basis of Bruvo distances was calculated in Mega 5.2. 34 The allele association index was calculated from the lengths of microsatellite loci in poppr 2.8.1 package for R, 35 using the Multilocus Style Permutation method, after deleting one non-informative locus and performing clonal correction. The null hypothesis was the absence of association between the lengths of microsatellite loci. Simpson’s index of genotype diversity within upper and lower bounds for a 95% confidence interval was calculated in the package polysat on the basis of Bruvo genetic distances, in equal groups of 21 isolates for ST1 and ST2 lineages. The analysis of molecular variation (AMOVA) was done on the basis of Bruvo and Rozenfeld distance matrices in GenAlEx 6.5 package 36 , 37 with 999 standard permutations. TERG_03298 genotypes, MAT idiomorphs and TERG_05790 urease gene variants were determined through Blast searches in whole genome sequences. 38 For mating type determination, TERG_02406 gene coding for α-domain protein was probed by AB759084 sequence. 13 The absence of HMG protein-coding gene was verified by the searches with T. simii HMG gene, GenBank accession number AB605766 . 39
Quantitative RT-PCR
We hypothesized the influence of number of STR Tr 9 repeats on FBA gene expression under normal conditions. The sample of six T. rubrum isolates was grown on Sabouraud agar medium for two weeks at 28 °C. RNA was isolated with the use of GeneJet Plant RNA Purification Mini Kit (Thermo Fisher Scientific, Lithuania). The quantitative RT-PCR experiments were performed in triplicates on two independently grown colonies for each isolate in the sample with the use of OneTube RT-PCR SYBR Kit (Evrogen, Russia). The reactions were carried out on CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA) with the following cycling conditions: reverse transcription step at 55 °C for 15 min, polymerase activation and reverse transcriptase inactivation step at 95 °C for 1 min, and main amplification stage for 40 cycles, including denaturation at 95 °C for 15 s, annealing at 60 °C for 20 s and elongation at 72 °C for 15 s. Melting curve analysis was performed to ensure that a single PCR product was obtained. Relative gene expression levels were assessed using beta-tubulin and DNA-dependent RNA polymerase II for normalization. The sequences of primers used are given in the Table 1 .
Results
The majority of 69 T. rubrum isolates from Russia and the type strain CBS 392.58 had the same ribosomal ITS region sequence, identical to KT285224. There were two exceptions, the isolate D15P139 had 247insC mutation, and another isolate had additional TA element in the microsatellite near the 3’-end of the sequence. Our preliminary study showed discrepancies between the results of strain typing by TERG_02941 locus polymorphism and the microsatellite typing assay of Gong et al., 8 based on eight loci. 40 We hypothesised that this discrepancy was due to insufficient informativeness of the assay and used four additional polymorphic loci, up to a total of 12. The stability of microsatellite loci was assessed in 20 isolates for the time span of 1.5 years. Observed differences of less than one repeat length were considered insignificant, since those differences could be associated with the rounding of measurements, fluctuating near the half-integers. At the endpoint, mutations were observed in five loci. The most extensive changes were noticed in Tr(CT) 20 locus, with 30% of isolates having mutations. The most pronounced indels were in Tr006 and Tr008 loci, covering up to 3 repeat lengths. Our first approach for calculating microsatellite polymorphism-based genetic distances employed the probability of mutation events, described with the use of stepwise mutation model. 30 The split network, inferred using mutation probability-based distances, was divided into three major partitions. One partition contained TERG_02941 793A isolates and another two partitions contained TERG_02941 793G isolates ( Fig. 1A ). The second approach was based on the comparison of the lengths of microsatellite loci. 32 The resulting phylogenetic network also had pronounced division into TERG_02941 793A and 793G parts ( Fig. 1B ). In Rozenfeld distances-based network, further subdivision of TERG_02941 793G partition was less evident, but still discernable. From 22 TERG_02941 793G isolates, 18-21 were located in the same subgroups in both networks. For the two genetic lines, the average lengths of PCR products of Tr001, Tr002, Tr003, Tr004, Tr005, Tr007, Tr008, Tr009, Tr(CT) 20 and Tr(GA) 25 loci differed by the length of less than one repeat unit. Average Tr006 lengths differed by a single unit length. For STR Tr 9 locus, this difference was 7.5 unit lengths ( Fig. 2A ). This difference should be obviously explained by the pressure of natural selection. Since the analysis of intraspecific divergence in most cases is carried out using neutral loci, 41 we performed calculation of genetic distances without STR Tr 9 length data. This broke clear grouping of isolates in both phylogenetic networks. However, a certain tendency to clustering of TERG_02941 793G isolates persisted ( Figs. 1C, 1D ). The analysis of molecular variance with 12 microsatellite loci revealed neglectable contribution of TERG_02941 793G subdivision to intergroup and overall variability ( Table 3 ). With the removal of STR Tr 9 from the analysis, the percent of variability, explained by the differences between genetic lineages, dropped drastically. But, this was accompanied by significant increase in explanatory power of TERG_02941 793G group subdivision. A half of the between-group variability in the dataset with 11 loci depended on it. In the sample of 70 isolates, the association index value laid beyond the right boundary of the distribution of expected frequencies. The probability was estimated at 0.002, which made it possible to reject the null hypothesis of the absence of connections between the markers. Hence, the studied T. rubrum population was considered to be clonal. Also, six Russian isolates, subjected here to whole genome sequencing, harbored MAT1-1 idiomorph of mating type locus. Simpson index was calculated on the basis of data from 11 neutral SSR loci. Its value within 95% confidence interval was estimated at 0.59-1.03 for ST1 lineage and 0.38-0.91 for ST2 lineage. The Fisher exact test showed no significant association between TERG_02941 genotypes and the localization of the lesion on patient’s body. Also, Yates-corrected χ 2 test showed no connection between TERG_02941 genotype and a city of origin. The CAAG repeats of STR Tr 9 ended at the position −278 in the 5’-region of fructose 1,6-bisphosphate aldolase gene TERG_06744, wherein the adenine of the ATG translation initiation codon is considered +1. Therefore, we hypothesized that STR Tr 9 locus promotes microevolution in T. rubrum through regulation of aldolase gene expression, which in its turn is one of the key enzymes of glycolysis. 42 We determined relative TERG_06744 gene expression in six T. rubrum isolates. The isolate D15P44 with maximal length of STR Tr 9 locus demonstrated the lowest level of TERG_06744 transcripts ( Fig. 2B ). The highest levels of TERG_06744 transcripts were observed in D15P62 isolate with the microsatellite length very close to minimal one. In the same time, three isolates with the same STR Tr 9 length demonstrated significantly lower levels of TERG_06744 gene expression. Also, there were no clear differences between TERG_06744 transcript levels in isolates of different sequence types. Russian sample of T. rubrum isolates was placed in global context by whole genome sequencing in six T. rubrum isolates. To get a representative sample, we selected isolates D15P33, D15P44, D15P50, D15P62, D15P71 and Y1013, covering the whole span of microsatellite distances-based Neighbour-Joining tree (not shown). These isolates were tested for susceptibility to antifungals ( Table 4 ). The phylogenomic tree included T. rubrum sensu stricto genomes from Europe, Asia and North America ( Fig. 3 ) and contained well-supported monophyletic branch of ST1 isolates and basal paraphyletic ST2 branch. Both branches contained European and Asian isolates. In a representative selection of seven isolates from the study of Zheng et al., 17 cluster affiliation, determined in the original study, matched their TERG_02941/03298 genotypes. Depending on the presence of T. violaceum CMCC(F)T3l and T. rubrum “megninii” CBS 735.88 in the phylogenomic analysis, the depth of branches within T. rubrum sensu stricto clade related to T. rubrum sensu stricto — T. soudanense CBS 452.61 branch as 1:63 or 1:337 (with the two strains excluded). Since urease activity has been regarded phylogenetically important trait in the dermatophytes, 43 we assessed urease activity in our sample and found three urease-positive strains. For the isolate D15P50, whole genome sequence was available. There is an observation of the 68-bp intron and the 9-bp insertion in a urease-positive T. rubrum strain. 44 However, in line with the results of Adamski et al., 45 we found identical urease gene sequences in all T. rubrum sensu stricto isolates, shown on Fig. 3 , including urease-positive strains D15P50 and CBS 202.88. Trichophyton rubrum “megninii” CBS 735.88 harbored two point mutations in the urease gene, A685G in the intron and synonymous substitution G803A in the second exon. All our three isolates, positive for urease activity, came from the cases of toenail and foot skin infections and belonged to both major genetic lineages of T. rubrum .
Discussion
Until recently, low genetic diversity of T. rubrum precluded persuasive description of its population structure. For this species, our earlier work seemingly represented the first successful attempt to obtain congruent results by two independent molecular strain typing techniques. 16 However, we have not performed DNA sequencing of ribosomal ITS region to clarify whether the sample contained only T. rubrum sensu stricto isolates or not. Here we filled this gap by demonstrating that Russian population of the fungus is represented by only «classic» T. rubrum genotype. The two genetic lines of Russian T. rubrum population, found here, demonstrated significant divergence. The AMOVA analysis showed relatively high proportion of variability, explained by the division of T. rubrum sample into ST1 and ST2 groups, 22-48% depending on the distance metrics. It was not less, than in other studies on clonal filamentous ascomycetes with 19-21% of variability between groups 46 , 47 or filamentous ascomycetes with mixed reproduction mode, with 2-13% of total variability explained by differences between intraspecific groups. 48 , 49 Intraspecific genetic lines in fungi sometimes demonstrate restricted geographic ranges. 50 – 53 Here, we compiled the map of global distribution of T. rubrum genetic lineages ( Fig. 4 ). We largely built upon existing literature on NTS typing in T. rubrum and departed from the observation that single 450 bp PCR product of TRS-1 locus is associated with ST1 genetic lineage. 16 We didn’t observe a specific distribution pattern of the two genetic lineages, but in most parts of the World, the derived ST1 lineage of T. rubrum presumably had higher prevalence and therefore demonstrated evolutionary success. The affiliation of T. rubrum isolates to genetic lineages can be probed not only by NTS, SNP and WGS-based typing techniques, but also by our modified scheme of microsatellite analysis. But, this result may be of limited interest for healthcare practitioners, since there is seemingly no association of lineage affiliation and common isolate descriptors, like geography of origin and clinical picture of respective infection. Also, in the study of Zheng et al., 17 there was no association between cluster affiliation and antifungal susceptibility levels. It is widely accepted that truly independent species of clonal microbes occupy distinct ecological niches. 62 – 64 In present study, there seemingly have been no clinical differences between the subpopulations, contrary to what we have claimed earlier. 16 In this previous study, the association of STs and clinical forms of dermatophytosis might have occurred due to some random factors. From this perspective, the two genetic lineages of T. rubrum sensu stricto belong to the same fungal species. The STR Tr 9 locus was paramount for the analysis of the structure of the T. rubrum population by microsatellite analysis. Its length significantly differed between the isolates of the two genetic lines, implying the pressure of natural selection. There were significant differences in relative aldolase gene expression levels between different isolates. At the scale of our experiments, these differences could not be associated with population structure. Also, in our sample there were three urease-positive isolates, a proportion of 4% from the total. We did not find peculiarities in urease gene sequences of urease-positive isolates D15P50 and CBS 202.88. Positive strains belonged to both ST1 and ST2 groups. Therefore, our results suggest that urease-positive strains present in certain proportion in the population of the fungus, and represent natural variation of T. rubrum sensu stricto , not an independent entity. Lackner et al. 65 obtained comparable results, studying virulence potential of closely related Aspergillus species from the section Terrei . They concluded that virulence is a strain-specific rather than species-specific feature. Lineage-specific differences in STR Tr 9 locus length may still hallmark different adaptation strategies, given that glycolysis pathway regulation is connected with mechanisms of keratin degradation. 66 Urease activity is an ancestral trait, commonly observable in geophilic dermatophytes 43 and we expected to find it only in basal ST2 lineage. However, in our sample one of three urease-positive isolates belonged to ST1 lineage. Also, genetic diversity in the two lineages proved to be the same, which is contrary to what might be expected if ST2 is an ancestral lineage. It can be probably due to a sort of bottleneck, occurred in the population on the way from South-East Asia to Russia. In future studies, it is necessary to sample isolates from South and South-East Asia, in order to find the territory with highest genetic diversity and thereby conclusively determine the geographic region of the origin of the species. Thus, we provided the description of the two intraspecific global lineages of T. rubrum sensu stricto . They can be revealed by SNP, SSR and WGS-based typing techniques (and NTS, with a certain amount of ambiguity). The species is polymorphic in terms of ecology and physiology, but this polymorphism is not associated with genetic population structure.
Author contributions
I.P. designed the study. I.P., Y.M., I.V., S.R. and T.B. performed the experiments. G.C. performed morphological species identification and maintained fungal cultures. I.P., D.A., V.Z. and Y.M. analyzed the data with the participation of A.T. N.V., A.T. and S.A. performed supervision and funding acquisition. I.P. wrote the manuscript with the help of V.Z.
Declarations of interest
none
Data accessibility
Raw WGS reads were submitted to NCBI SRA database, BioProject PRJNA552357: the isolate D15P33, accession SRX6389058; D15P44, SRX6389060; D15P50, SRX6389057; D15P62, SRX6389059; D15P71, SRX6389062; Y1013, SRX6389061.
Supplementary data
Supplement S1. Molecular strain typing in 70 T. rubrum isolates.
Competing Interest Statement
Competing Interest Statement
The authors have declared no competing interest.
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