UvA-DARE (Digital Academic Repository) DNA barcoding in Mucorales: an inventory of biodiversity Walther, G.; Pawłowska, J.; Alastruey-Izquierdo, A.; Wrzosek, W.; Rodriguez-Tudela, J.L.; Dolatabadi, S.; Chakrabarti, A.; de Hoog, G.S. DOI 10.3767/003158513X665070 Publication date 2013 Document Version Final published version Published in Persoonia - Molecular Phylogeny and Evolution of Fungi Link to publication Citation for published version (APA): Walther, G., Pawłowska, J., Alastruey-Izquierdo, A., Wrzosek, W., Rodriguez-Tudela, J. L., Dolatabadi, S., Chakrabarti, A., & de Hoog, G. S. (2013). DNA barcoding in Mucorales: an inventory of biodiversity. Persoonia - Molecular Phylogeny and Evolution of Fungi, 30, 11-47. https://doi.org/10.3767/003158513X665070 General rights It is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulations If you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: https://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. UvA-DARE is a service provided by the library of the University of Amsterdam (https://dare.uva.nl) Download date:29 Sep 2021 Persoonia 30, 2013: 11–47 www.ingentaconnect.com/content/nhn/pimj RESEARCH ARTICLE http://dx.doi.org/10.3767/003158513X665070 DNA barcoding in Mucorales: an inventory of biodiversity G. Walther1,2,3, J. Pawłowska4, A. Alastruey-Izquierdo5, M. Wrzosek4, J.L. Rodriguez-Tudela5, S. Dolatabadi3,6, A. Chakrabarti7, G.S. de Hoog3,6,8,9 Key words Abstract The order Mucorales comprises predominantly fast-growing saprotrophic fungi, some of which are used for the fermentation of foodstuffs but it also includes species known to cause infections in patients with severe Backusella immune or metabolic impairments. To inventory biodiversity in Mucorales ITS barcodes of 668 strains in 203 taxa biodiversity were generated covering more than two thirds of the recognised species. Using the ITS sequences, Molecular clinical relevance Operational Taxonomic Units were defined by a similarity threshold of 99 %. An LSU sequence was generated DNA barcoding for each unit as well. Analysis of the LSU sequences revealed that conventional phenotypic classifications of the intraspecific variability Mucoraceae are highly artificial. The LSU- and ITS-based trees suggest that characters, such as rhizoids and ITS sporangiola, traditionally used in mucoralean taxonomy are plesiomorphic traits. The ITS region turned out to be an LSU appropriate barcoding marker in Mucorales. It could be sequenced directly in 82 % of the strains and its variability Mucor was sufficient to resolve most of the morphospecies. Molecular identification turned out to be problematic only for Mucorales the species complexes of Mucor circinelloides, M. flavus, M. piriformis and Zygorhynchus moelleri. As many as 12 nomenclature possibly undescribed species were detected. Intraspecific variability differed widely among mucorealean species Rhizomucor ranging from 0 % in Backusella circina to 13.3 % in Cunninghamella echinulata. A high proportion of clinical strains taxonomy was included for molecular identification. Clinical isolates of Cunninghamella elegans were identified molecularly Zygorhynchus for the first time. As a result of the phylogenetic analyses several taxonomic and nomenclatural changes became necessary. The genus Backusella was emended to include all species with transitorily recurved sporangiophores. Since this matched molecular data all Mucor species possessing this character were transferred to Backusella. The genus Zygorhynchus was shown to be polyphyletic based on ITS and LSU data. Consequently, Zygorhynchus was abandoned and all species were reclassified in Mucor. Our phylogenetic analyses showed, furthermore, that all non-thermophilic Rhizomucor species belong to Mucor. Accordingly, Rhizomucor endophyticus was transferred to Mucor and Rhizomucor chlamydosporus was synonymised with Mucor indicus. Lecto-, epi- or neotypes were designated for several taxa. Article info Received: 7 September 2012; Accepted: 1 January 2013; Published: 4 March 2013. INTRODUCTION is increasing in hosts with severe immune or metabolic impair- ment, e.g. due to hemomalignancy, hematopoietic stem cell The order Mucorales represents a phylogenetically ancient transplantation or uncontrolled ketoacidotic diabetes mellitus group of fungi comprising predominantly saprotrophs inhabiting (Skiada et al. 2011). Infections often take a dramatic course and soil, dung and dead plant material, as well as several parasites have a high mortality rate. In risk group patients such as those on plants and on other fungi. Mucoralean strains have been with leukemia or allogenic bone marrow transplant an increase used for centuries in the fermentation of traditional Asian and of 8 % and 2 %, respectively, has been noted (Greenberg et al. African food such as tempeh or furu (fermented tofu) (Nout 2004). In part the clinical strains belong to the same species as & Aidoo 2010), and they also play a role in the production of the ones used in food fermentation. For example, Mucor cir- several kinds of cheese (Hermet et al. 2012). On the other cinelloides is used for starter cultures in Asian food (Hesseltine hand, some members of the Mucorales are responsible for the 1983, Nout & Aidoo 2010), but is also able to infect patients with spoilage of fresh and manufactured food (Pitt & Hocking 2009). an impaired immune system (e.g. Khan et al. 2009). Mucoralean fungi are also known to be involved in human Mucorales are among the best represented groups in fungal cul- infection. Mucormycoses are still very rare, but their incidence ture collections. They easily grow in axenic culture and they 1 Institute of Microbiology, Department of Microbiology and Molecular Biology, have been used as model organisms since the late 19th cen- University of Jena, Jena, Germany; tury. A large share of all species described in the order are corresponding author e-mail: [email protected]. represented today by living cultures publicly available in fungal 2 Leibniz-Institute for Natural Product Research and Infection Biology – Hans- reference collections. For example, the Centraalbureau voor Knöll-Institute, Jena Microbial Resource Collection, Jena, Germany. 3 CBS-KNAW Fungal Biodiversity Centre, Utrecht, The Netherlands. Schimmelcultures (www.cbs.knaw.nl) possesses 135 ex-type 4 Department of Systematics and Plant Geography, University of Warsaw, or authentic strains out of 227 currently accepted species. This Warsaw, Poland. is a unique situation, compared e.g. with dermatophytes which 5 Instituto de Salud Carlos III Mycology Department, Spanish National Center were described around the same period (Sabouraud 1910). for Microbiology, Madrid, Spain. 6 Institute for Biodiversity and Ecosystem Dynamics, University of Amster- Problematic for the nomenclatural stability of the Mucorales is dam, Amsterdam, The Netherlands. the practice of many early authors to designate a living strain 7 Departments of Medical Microbiology, Postgraduate Institute of Medical as ‘type’ although this was permitted by the International Code Education and Research, Chandigarh, India. of Botanical Nomenclature. Since 2000 Art. 8.4 of ICBN has 8 Peking University Health Science Center, Research Center for Medical Mycology, Beijing, China. allowed deposition of metabolically inactive cultures as types 9 Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou, China. (Greuter et al. 2000). In order to link these original strains to the © 2013 Naturalis Biodiversity Center & Centraalbureau voor Schimmelcultures You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. 12 Persoonia – Volume 30, 2013 respective names we designated the vial with the lyophilised between members and is not alignable over the entire order. strain that was prepared at time of its accession as lectotypes. ITS sequences of some species differ to such an extent that If the original strain was not lyophilized in the year of its ac- they could not be aligned confidently with their putative sibling cession we lectotypified the name by the original illustration species. To establish the phylogenetic position of species and designated the original strain as epitype. In the case of and to acquire an overview of the entire order that includes Zygorhynchus