Mycopathologia

https://doi.org/10.1007/s11046-019-00338-7 (0123456789().,-volV)( 0123456789().,-volV)

ORIGINAL ARTICLE

Updating the of of the BCCM/ IHEM Collection According to the New Standard: A Phylogenetic Approach

F. Baert . D. Stubbe . E. D’hooge . A. Packeu . M. Hendrickx

Received: 23 January 2019 / Accepted: 30 April 2019 Ó Springer Nature B.V. 2019

Abstract Recent taxonomical revisions based on floccosum as the only representative, fell within the multilocus gene sequencing have provided some Nannizzia clade, whereas the phylogenetic analysis, clarifications to () based on the ITS region alone, differentiates Epider- family tree. These changes promoted us to investigate mophyton from Nannizzia as a separate genus. Re- the impact of the changed nomenclature of the identification and reclassification of many strains in dermatophyte strains in the BCCM/IHEM fungal the collection have had a profound impact on the collection, which contains strains of all dermatophyte composition of the BCCM/IHEM dermatophyte col- genera except for Ctenomyces. For 688 strains from lection. The biggest change is the decline of preva- this collection, both internal transcribed spacer region lence of Arthroderma strains; starting with 103 strains, (ITS) and partial b-tubulin (BT) sequences were only 22 strains remain in the genus after reassessment. aligned and a multilocus phylogenetic tree was Most Arthroderma strains were reclassified into Tri- constructed. The ITS ? BT phylogentic tree was able chophyton, with A. benhamiae and A. van- to distinguish the genera Arthroderma, Lophophyton, breuseghemii leaving the genus. The amount of Microsporum, Paraphyton, Nannizzia and Trichophy- Microsporum strains also dropped significantly with ton with high certainty. Epidermophyton, which is most of these strains being reclassified into the genera widely considered as a well-defined genus with E. Paraphyton and Nannizzia.

Keywords Arthrodermataceae Á Dermatophytes Á Handling Editor: Sybren de Hoog. Taxonomy Á Phylogeny Á Beta-tubulin Á Internal transcribed spacer Á BCCM Á IHEM Á Trichophyton Á Electronic supplementary material The online version of Nannizzia Á Arthroderma Á Paraphyton Á this article (https://doi.org/10.1007/s11046-019-00338-7) con- tains supplementary material, which is available to authorized Epidermophyton Á Microsporum Á Lophophyton users.

F. Baert (&) Á D. Stubbe Á E. D’hooge Á A. Packeu Á M. Hendrickx Sciensano, Service of Mycology and Aerobiology, Introduction Brussels, Belgium e-mail: [email protected] The dermatophytes (, Arthrodermataceae) are a group of closely related filamentous fungi mainly F. Baert Á D. Stubbe Á E. D’hooge Á M. Hendrickx BCCM/IHEM Fungal Collection, Sciensano, Service of characterized by their capacity to invade and infect Mycology and Aerobiology, Brussels, Belgium keratinized tissues [1, 2]. These infections of the skin, 123 Mycopathologia hair or nail tissues are called dermatophytoses, or the topology of the family, and some species that can when nails are involved and are also be found in ancestral clades are morphologically close commonly known as tinea or ringworm. Diseases to anthropohilic species found in a derived position in caused by this group of fungi have a worldwide the phylogeny. Because geophilic, anthropophilic and distribution in both humans and animal hosts [3]. zoophilic dermatophytes also differ clinically, it is Since the mid-nineteenth century, the taxonomy of important to accurately distinguish these species. An the dermatophytes has extensively been investigated infection by a geophilic species is most often highly and was based on both their clinical symptoms and inflammatory but quickly resolved, an infection by a in vitro morphological characteristics. Since this zoophile is moderately inflammatory and self-limit- classification was phenotype-based, mutants or mor- ing, while anthropophilic dermatophytes cause mild, photypes of single species were often mistaken for non-inflammatory and chronic infections [5, 11]. separate species. Moreover, due to the lack of De Hoog et al. have formalized the dermatophyte reference strains, many species were described sepa- topology in their recent paper, where seven main rately by several independent researchers introducing clades of dermatophytes were identified and judged to many synonymic taxa to this specific fungal family represent genera using a multilocus phylogenetic [4]. Historically, more than 300 names were at some approach [5]. The distinguished genera are Arthro- point defined for the 54 species of dermatophytes that derma, Microsporum, Lophophyton, Nannizzia, Epi- exist according to the latest taxonomic reports [5]. dermophyton, Paraphyton and Trichophyton. In this Since the 1980’s, molecular techniques have been classification, most geophilic species can be found in used for the identification of fungal strains, providing the ancestral Arthroderma genus, while the anthro- valuable insights into the taxonomy of the family. pophiles are almost exclusively located within the Phylogenetic analysis using different molecular mark- more derived Epidermophyton and Trichophyton ers has clarified much of the dermatophyte taxonomy, genera. although mainly the rDNA internal transcribed spacer The BCCM/IHEM collection of biomedical and (ITS) region is informative [6–9]. The dermatophytes veterinary fungi holds more than 2000 different strains are a closely related and highly conserved family of of dermatophytes and is thus a valuable resource in fungi, so while the molecular approach has been very dermatophyte research. The major taxonomical successful at determining the main clades of the changes and developments in this family demanded dermatophyte family, problematic species complexes a thorough revision of the taxonomy and species re- such as Trichophyton rubrum and Trichophyton men- identification of the BCCM/IHEM strains. Firstly, the tagrophytes still exist [7, 10]. Therefore, a combina- analysis of this large dataset allows to assess whether tion of different approaches including ecological, taxonomical changes proposed by de Hoog et al. are morphological, clinical and molecular data is recom- confirmed, and secondly, the impact of these taxo- mended for the delineation of species boundaries. nomical changes can be evaluated [5]. We aim to re- The use of biological sequence data for phyloge- evaluate the nomenclature of the collection strains netic inference has shown that the dermatophytes can based on their phylogenetic characteristics. No mor- be grouped more accurately based on clinical and phological, physiological, mating or data of the origin ecological traits than based on morphological charac- of the strains were taken into account. teristics [11]. Ecologically speaking, the dermato- phytes can be divided into three broad groups, namely the anthropophilic, zoophilic and geophilic species. M&M This is reflected in the main topology of the Arthro- dermataceae family [5, 6]. Geophilic species are In this study, a total of 688 strains of the BCCM/IHEM separated from the other taxa and located in the collection were analyzed. All sequences used in this ancestral position of the phylogenetic tree, zoophilic study were uploaded to genbank (see Online Resource species are found scattered throughout the middle of 1). Since for some species type material was not the tree, while the anthropophilic species can be found available in the collection, type sequences from the in the derived clades. Important to note is that the reference collection of the Westerdijk Fungal Biodi- morphology of these species does not correspond to versity Institute were included when available. In 123 Mycopathologia total, 37 sequences of CBS type strains were incorpo- Results rated (see Online Resource 2). Strains were cultured on Sabouraud dextrose broth The ML analysis of the concatenated dataset (Fig. 1) for at least 5 days. Genomic DNA of the strains was shows strong support for Arthroderma, Paraphyton, extracted using the Invisorb Spin Plant Mini Kit Lophophyton, Trichophyton and Microsporum. The (Invitek, Berlin, Germany). The extraction kit was relationship between Epidermophyton and Nannizzia used according to the manufacturer’s instructions, remains unresolved. In the obtained topology, Epi- with some adaptations: (1) Before lysis, a lyophiliza- dermophyton is nested within the Nannizzia genus, in a tion step and subsequent bead beating was added to clade which receives high bootstrap support. facilitate the disruption of the fungal cell wall and (2) Epidermophyton is considered a well-defined genus the lysis time was raised to more than 2 h. with E. floccosum as its only representative. However, Two gene regions of the genomic DNA were in the ITS-BT analysis, the species fell within the amplified and sequenced: (1) The primers Bt2b and Nannizzia clade. Phylogenetic analysis based solely Bt2a described by Glass and Donaldson [12] were on the ITS gene region does distinguish Epidermo- used for amplification and sequencing of the partial b- phyton from Nannizzia as a separate clade, but adding tubulin (BT) gene and (2) the ITS region was amplified BT information to the analysis casted doubt on this using primers ITS5 and ITS4 [13]. BT was chosen as a classification. secondary region since it has been shown to provide Although the genus Nannizzia is not distinguishable the highest resolution when determining clades in the from Epidermophyton, the individual species clades dermatophyte family when choosing among the most are well supported throughout the entire genus. Most commonly used markers, with the exception of species of this genus were formerly classified in barcoding region ITS [5, 14]. Microsporum. In the IHEM collection, the strains of 6 PCR amplicons were purified using ExoSAP-IT species are now transferred from Microsporum to PCR Product Cleanup (Affymetrix, Santa Clara, CA, Nannizzia as a result of the new taxonomy: N. praecox USA). Sanger sequencing was performed with an ABI (= M. praecox), N. gypsea (= M. gypseum), N. nana 3130xl Genetic Analyser (Applied Biosystems, Wal- (= M. nanum), N. persicolor (= M. persicolor),N. tham, MA, USA). fulva (= M. fulvum), N. duboisii (= M. duboisii) and N. Using BT and ITS sequences, a multilocus phylo- incurvata (= M. incurvatum). The genus is also home genetic analysis was performed involving all 688 to N. aenygmatica [5], but no sequences for this strains (see Online Resource 3 for full phylogeny). The species were included in the dataset. Arthroderma multiple sequence alignment was constructed with corniculatum should also be transferred to Nannizzia, MAFFT version 7.394 using the FFT-NS-i iterative N. corniculata. Some strains of A. racemosum, refinement method. The scoring matrix for nucleotide considered a synonym of P. cookei by De Hoog sequences was set to 1PAM/j = 2. Afterward, the et al., from the BCCM/IHEM collection formed a fully alignment was manually assessed and checked for supported clade together with the type strain (CBS inconsistencies. On the basis of this dataset, a max- 450.65) within the Nannizzia clade [5]. imum likelihood (ML) phylogeny was constructed Within the genus Arthroderma, all examined using RAxML-HPC Blackbox version 8.2.1 hosted on species are well resolved. It is the genus with the the CIPRES Science Gateway [15]. Branch support highest interspecific variability in the Arthrodermat- was calculated using 1000 bootstrap replicates. The aceae. The dataset did not contain any strains for the dataset was subdivided into 5 gene partitions: ITS1 ? species A. redelli and did not contain the type ITS2, 5.8S ? 28S, BT introns codon 1 and 2, BT sequence for A. quadrifudum. introns codon 3 and BT exons. For each partition, the Paraphyton is a recently defined genus consisting GTRCAT approximation of rate heterogeneity was of three species. The genus is fully supported in this estimated. study, as are the species clades P. mirabile, P. cookiellum and P. cookei. The sequences of the type strain of P. cookei (CBS 228.58) were included in our analyses. Arthroderma cajetani is confirmed as a synonym of P. cookei. 123 Mycopathologia

Fig. 1 Maximum likelihood analysis of the concatenated dataset of ITS and BT sequences of 688 BCCM/IHEM strains and reference strains. Bootstrap support is mentioned below each branch

The genus Microsporum is fully supported. The M. BCCM/IHEM collection has changed significantly canis clade received weak support in contrast to M. (see Fig. 2). The majority of name changes is seen in audouinii. Two strains, currently designated as A. otae the genera Arthroderma and Microsporum. In total, 81 within the BCCM/IHEM collection, did not cluster out of 103 Arthroderma strains are reclassified to five with either of these two clades nor cluster with each different genera, leaving only 22 strains in Arthro- other. Since no BT sequences were available for M. derma (see Fig. 3). The number of strains in Tri- ferrugineum, the species is not included in the chophyton is largely unchanged, and the amount of multilocus analysis, but the species is clearly distin- Epidermophyton strains remains the same. guished and highly supported in the single locus ITS Figure 4 shows the distribution of the origin of the analysis. analyzed strains, classified into the categories human, Trichophyton is the genus in the most derived animal and environmental. Arthroderma has the position of the phylogenetic tree and contains the highest proportion of strains from environmental majority of analyzed strains in this study. The genus origin. Paraphyton also shows a high proportion of consists of eighteen closely related species that are all environmental strains as two out of three representa- represented in this study. Their relationship is not tives are geophylic, namely P. cookei and P. cookeil- always resolved in this multilocus analysis. Tri- lum. The proportion of strains from human infection is chophyton tonsurans and T. equineum are phyloge- visibly higher in the more derived clades. Strains from netically indistinguishable; similar situation is veterinary origin are found in each genus except observed between T. quinckeanum and the T. schoen- Epidermophyton. Lophophyton contains only one leinii. The T. benhamiae species clade is also not species, L. gallinae, which is zoophilic on poultry, resolved, with a group of 23 strains clustering with T. and thus, almost all strains were of animal origin. concentricum, while a minority of eight strains cluster with T. bullosum. The species clades T. simii, T. interdigitale, T. eriotrephon, T. verrucosum and T. Discussion erinacei were well resolved. The T. rubrum complex contains T. violaceum, T. yaoundei, T. kuryangei, T. Combined analysis of ITS and BT genes provides soudanense and T. rubrum. Trichophyton rubrum strong support for the major clades of the Arthroder- strains cluster together with high support. Strains matacae family. Combining these genes into one belonging to the other species in this complex, group multilocus analysis provided a phylogenetic tree with in paraphyletic clades with inconclusive support. more significant support than using any one gene By applying the recently proposed changes in separately. The backbone of the dermatophyte phy- dermatophyte taxonomy by de Hoog et al. [5], the logeny is firmly supported with bootstrap values above composition of the dermatophyte strains in the 85 for all internal nodes. The genera Arthroderma,

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Fig. 2 Number of strains 1000 per genus before and after revision of the taxonomy based on BT and ITS molecular phylogeny. 100 Logarithmic scale

10 Prior revision A er revision

1

100.00% 90.00% 80.00% 70.00% 60.00% 50.00% 40.00% 30.00% 20.00% 10.00% 0.00% Arthroderma Epidermophyton Microsporum Trichophyton Trichophyton 59.55% 0.00% 0.00% 97.98% Paraphyton 4.49% 0.00% 8.82% 0.00% Nannizzia 17.98% 0.00% 29.41% 0.00% Microsporum 2.25% 0.00% 56.86% 0.00% Lophophyton 1.12% 0.00% 4.90% 0.00% Epidermophyton 0.00% 100.00% 0.00% 0.00% Arthroderma 14.61% 0.00% 0.00% 2.02% Name in new classificaon Name in old classificaon

Fig. 3 Depiction of the proportional flow of strains in the BCCM/IHEM collection for each genus from the old classification to the new classification of genera as proposed by de Hoog et al. [5].

Paraphyton, Microsporum and Lophophyton and level, except for the position of the genus Epidermo- Trichophyton are all well supported, which is in line phyton relative to Nannizzia. ITS analysis alone with the findings of recent publications [5, 14]. The indicates that Epidermophyton is a sister genus of tree topologies of the single and multiple locus Nannizzia, while based on addition of the BT gene, E. analyses did not show any conflicts on the genus floccosum (the only representative of the genus)

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Fig. 4 Percentage of strains 100% in each genus having an 90% environmental, animal- infectious or human- 80% infectious origin 70% 60% 50% 40% Environmental 30% Animal 20% Human 10% 0%

should be considered a species belonging to Nan- Within the genus Microsporum, phylogenetic res- nizzia. This result is consistent with findings from olution on the species level was low. Although the Rezaei-Matehkolaei et al., who analyzed 54 BT examined M. audouinii and M. canis strains formed sequences and found E. floccosum to be closely separate clades, the 2 strains previously designated to related to species now comprised in Nannizzia [8]. A M. otae (IHEM 26531, IHEM 04242) did not fall similar result was also obtained using the genes for within one of these clades. Intraspecies variation calmodulin [16] and translation elongation factor between the strains previously named M. otae, 1 - a [17]. Seemingly, the protein-encoding genes amounting to a difference of 6 basepairs, is close to paint a different picture regarding the classification of the number of base differences per sequence from E. floccosum than the non-encoding barcoding region, averaging over all sequence pairs between strains ITS. Since protein-encoding genes are under more previously named M. otae and M. canis/M. audouinii selective pressure and thus less variable, ITS is most (amounting to an average of 6 and 9 base differences, suited to delineate these closely related genera. Use of respectively). This problem hindered species delin- other non-encoding and variable regions could provide eation within Microsporum. more definitive insights into the exact phylogeny. Trichophyton contains several phylogenetically An interesting case is that of M. racemosum. Based hard to distinguish species. Even though they clearly on syntype strains CBS 423.74 and CBS 424.74, de differ on an ecological basis, with only one or two Hoog et al. concluded this name was a synonym for P. different nucleotides, T. tonsurans and T. equinum cookei [5]. There exists, however, a type strain IHEM were phylogenetically indistinguishable in this study. 21235 (= CBS 450.65 = ATCC 16135 = IMI There exists, however, a C/T polymorphism at posi- 128984) isolated by Borelli and which has different tion 18 in the ITS gene that can be used to distinguish ITS and BT sequences. Rush-Munro et al. [18] these species [19]. This difference was present in the originally introduced IMI 135823 (= CBS strains analyzed here, but proved insufficient to 424.74 = ATCC 18911) and IMI 135822 (= CBS distinguish them in the phylogenetic tree. The BT 423.74 = ATCC 18910) as single spore isolates of IMI sequences showed no interspecific variation, in con- 128984 representing both mating types in 1970, but trast to the observations of Rezaei-Matehkolaei [8], did not mention them being syntypes. In our study, it is detecting a difference of one nucleotide. The same clear that the type strain (IHEM 21235) belongs to situation was observed for T. quinckeanum and T. Nannizzia, and that N. racemosa cannot be considered schoenleinii; as previously observed by Beguin et al. a synonym of P. cookei, but should be accepted as a [20], their ITS sequences only differ by 3 basepairs, separate species. namely a GC insertion at position 533 and a C/T

123 Mycopathologia polymorphism at position 568. The BT sequences are The impact of the updated taxonomy of the completely identical. These differences are conse- dermatophytes remains limited for frequently encoun- quent though, so they are sufficient to differentiate tered clinical species. The nomenclature for T. between the 2 species, in addition to their morpho- rubrum, T. tonsurans, M. canis and M. audouinii stays logical and clinical differences [20]. The exact topol- unchanged, while the position of some closely related ogy of the clade remained unclear as the more basal species is more clear. As mentioned by de Hoog and nodes were only weakly supported. confirmed by the present study, anthropophilic strains Multigene phylogenetic analysis by Suh et al. causing low-inflammatory infections correspond with subdivided T. benhamiae into 3 phylogroups, one of T. interdigitale while more inflammatory human which turned out to be very distinct from the other two infections are caused by isolates of the zoophilic T. and consisted of 2 African strains of T. benhamiae mentagrophytes s. st.[5]. Another important change [21]. This phylogroup was judged to represent a novel for the routine identification of dermatophytes is the species separate from the typical T. benhamiae strains. reclassification of A. benhamiae into T. benhamiae and Our analysis corroborates this claim: while most T. of A. vanbreuseghemii into T. mentagrophytes. benhamiae strains clustered together with T. concen- In conclusion, analyzing the ITS and BT genes of tricum as a subclade in the ITS ? BT phylogeny, all the BCCM/IHEM strains provides great support for African strains plus some strains of European origin the taxonomic changes proposed by De Hoog et al. [5]. clustered with T. bullosum forming a highly supported Regarding the genera Epidermophyton and Nannizzia, clade. phylogeny based on BT was contradictory to the ITS Trichophyton rubrum and T. violaceum formed phylogeny, casting doubt on the status of these genera. highly supported clades even though the average To obtain conclusive results concerning this topic, a interspecies variation amounted to only 7 SNP’s. more extensive set of genes will be needed. This is also While the species T. soudanense,T.kuryangei and T. the case on the species level, where some clearly yaoundei are closely related to both T. rubrum and T. distinct species were not distinguishable genetically. violaceum, there are few though distinct SNP’s The species complexes of interest that need a more in differentiating these taxa. However, these differences depth, polyphasic approach than solely based on were insufficient to produce monophyletic clades in molecular data for species delineation are the T. the phylogenetic analysis, rather T. soudanense, T. mentagrophytes complex (e.g., T. tonsurans, T. kuryangei and T. yaoundei appear to reside in equinum, T. benhamiae) and the T. rubrum complex. paraphyly with T. violaceum. Due to the reclassification and re-identification of Funding Funding was provided by the Belgian Science Policy many strains, the composition of the BCCM-IHEM (Belspo). dermatophyte collection has changed significantly Compliance with Ethical Standards (see Figs. 2, 3). The decline of the amount of Arthroderma strains is remarkable, but mainly caused Conflict of interest The authors declare that they have no by reclassification of some important species such as conflict of interest. A. vanbreuseghemii and A. benhamiae into Trichophy- Ethical Approval This article does not contain any studies ton, A. fulvum and A. corniculatum into Nannizzia, etc. with human participants or animals performed by any of the In total 79% of Arthroderma strains were reclassified authors. to a different genus, leaving only 22 of 103 analyzed strains in the Arthroderma genus. The number of strains in Microsporum also declined by 45%, mainly due to reclassification to the genera Nannizzia and References Paraphyton (Fig. 3). The number of strains in Tri- 1. Weitzman I, Summerbell RC. The dermatophytes. Clin chophyton remained largely unchanged, while the Microbiol Rev. 1995;8(2):240–59. amount in Epidermophyton remained exactly the 2. De Vroey Ch. Epidemiology of ringworm (dermatophyto- same. The collection does not contain any sequences sis). Semin Dermatol. 1985;4:185–200. for strains of the genera Guarromyces and Ctenomyces. 123 Mycopathologia

3. Seebacher C, Bouchara J-P, Mignon B. Updates on the protocols: a guide to methods and application, vol. 18, no. 1. epidemiology of dermatophyte infections. Mycopathologia. 1990. p. 315–322. 2008;166(5-6):335–52. 14. Zhan P, et al. Phylogeny of dermatophytes with genomic 4. de Hoog GS, et al. Atlas of clinical fungi. Electronic version character evaluation of clinically distinct Trichophyton 4.1. 4. Utrecht: CBS-KNAW Fungal Biodiversity Centre; rubrum and T. a´violaceum. Stud Mycol. 2018;89:153–75. 2017. 15. Miller, MA, Pfeiffer W, Schwartz, T. Creating the CIPRES 5. de Hoog GS, et al. Toward a novel multilocus phylogenetic science gateway for inference of large phylogenetic trees. taxonomy for the dermatophytes. Mycopathologia. In: Proceedings of the gateway computing environments 2017;182(1–2):5–31. workshop (GCE), 14 Nov 2010, New Orleans, LA. 2010, 6. Gra¨ser Y, et al. Phylogeny and taxonomy of the family p. 1–8. Arthrodermataceae (dermatophytes) using sequence analy- 16. Ahmadi B, et al. Phylogenetic analysis of dermatophyte sis of the ribosomal ITS region. Med Mycol. species using DNA sequence polymorphism in calmodulin 1999;37(2):105–14. gene. Sabouraudia. 2016;54(5):500–14. 7. Gra¨ser Y, et al. Molecular taxonomy of the Trichophyton 17. Mirhendi H, et al. Translation elongation factor 1-a gene as rubrum complex. J Clin Microbiol. 2000;38(9):3329–36. a potential taxonomic and identification marker in der- 8. Rezaei-Matehkolaei A, et al. Nucleotide sequence analysis matophytes. Med Mycol. 2014;53(3):215–24. of beta tubulin gene in a wide range of dermatophytes. 18. Rush-Munro FM, Smith JMB, Borelli D. The perfect state of Medical mycology. 2014;52(7):674–88. Microsporum racemosum. Mycologia. 1970;62:856–9. 9. Makimura K, et al. Phylogenetic classification and species 19. Summerbell RC, et al. ITS barcodes for Trichophyton ton- identification of dermatophyte strains based on DNA surans and T. equinum. Sabouraudia. 2007;45(3):193–200. sequences of nuclear ribosomal internal transcribed spacer 1 20. Beguin H, et al. The taxonomic status of Trichophyton regions. J Clin Microbiol. 1999;37(4):920–4. quinckeanum and T. interdigitale revisited: a multigene 10. Symoens F, et al. Molecular analysis and mating behaviour phylogenetic approach. Med Mycol. 2012;50(8):871–82. of the Trichophyton mentagrophytes species complex. Int J 21. Suh Sung-Oui, Grosso Kendra M, Carrion Miguel E. Mul- Med Microbiol. 2011;301(3):260–6. tilocus phylogeny of the Trichophyton mentagrophytes 11. Summerbell RC. Form and function in the evolution of species complex and the application of matrix-assisted laser dermatophytes. Biology of dermatophytes and other ker- desorption/ionization–time-of-flight (MALDI-TOF) mass atinophilic fungi. Rev Iberoam Micol. 2000;17:30–43. spectrometry for the rapid identification of dermatophytes. 12. Glass NL, Donaldson GC. Development of primer sets Mycologia. 2018;110(1):118–30. designed for use with the PCR to amplify conserved genes from filamentous ascomycetes. Appl Environ Microbiol. Publisher’s Note Springer Nature remains neutral with 1995;61(4):1323–30. regard to jurisdictional claims in published maps and 13. White, TJ, et al. Amplification and direct sequencing of institutional affiliations. fungal ribosomal RNA genes for phylogenetics. In: PCR

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