Mycopathologia https://doi.org/10.1007/s11046-019-00338-7 (0123456789().,-volV)( 0123456789().,-volV) ORIGINAL ARTICLE Updating the Taxonomy of Dermatophytes 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 dermatophyte (Arthrodermataceae) 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 (Onygenales, 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 onychomycosis 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