Accepted Manuscript Aspergillus is monophyletic: evidence from multiple gene phylogenies and extrolites profiles S. Kocsubé, G. Perrone, D. Magistà, J. Houbraken, J. Varga, G. Szigeti, V. Hubka, S.-B. Hong, J.C. Frisvad, R.A. Samson PII: S0166-0616(16)30020-3 DOI: 10.1016/j.simyco.2016.11.006 Reference: SIMYCO 40 To appear in: Studies in Mycology Please cite this article as: Kocsubé S, Perrone G, Magistà D, Houbraken J, Varga J, Szigeti G, Hubka V, Hong S-B, Frisvad JC, Samson RA, Aspergillus is monophyletic: evidence from multiple gene phylogenies and extrolites profiles, Studies in Mycology (2016), doi: 10.1016/j.simyco.2016.11.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT 1 Running title: The genus Aspergillus is monophyletic 2 3 Aspergillus is monophyletic: evidence from multiple gene phylogenies and extrolites profiles 4 5 S. Kocsubé 1#, G. Perrone 2# , D. Magistà 2, J. Houbraken 3, J. Varga 1, G. Szigeti 1, V. Hubka 4, S-B. Hong 5, J. C. 6 Frisvad 6 and R. A. Samson 3* 7 8 1Dept. of Microbiology, Faculty of Science and Informatics, University of Szeged, Szeged, Hungary; 9 2Institute of Sciences of Food Production, National Research Council, Bari, Italy; 3CBS-KNAW Fungal 10 Biodiversity Centre, Utrecht, The Netherlands; 4Department of Botany, Charles University in Prague, 11 Prague, Czech Republic; 5Korean Agricultural Culture Collection, National Institute of Agricultural Science, 12 166, Nongsaengmyeong-ro, Iseo-myeon, Wanju-gun, Jeollabuk-do, 55365, Republic of Korea; 6Department 13 of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark. 14 15 #These authors contributed equally to this work. 16 *Correspondence: R.A. Samson, [email protected] MANUSCRIPT 17 18 19 20 ACCEPTED ACCEPTED MANUSCRIPT 21 Abstract: Aspergillus is one of the economically most important fungal genera. Recently, the ICN adopted 22 the single name nomenclature which has forced mycologists to choose one name for fungi (e.g. Aspergillus , 23 Fusarium , Penicillium , etc.). Previously two proposals for the single name nomenclature in Aspergillus are 24 presented: one attributes the name “ Aspergillus ” to clades comprising seven different teleomorphic names, 25 by supporting the monophyly of this genus; the other proposes that Aspergillus is a non-monophyletic 26 genus, by preserving the Aspergillus name only to species belonging to subgenus Circumdati and 27 maintaining the sexual names in the other clades. The aim of our study was to test the monophyly of 28 Aspergilli by two independent phylogenetic analyses using a multilocus phylogenetic approach. One test 29 was run on the publicly available coding regions of six genes ( RPB1 , RPB2 , Tsr1 , Cct8 , BenA , CaM ), using 30 96 species of Penicillium , Aspergillus and related taxa. Bayesian (MrBayes) and Ultrafast Maximum 31 Likelihood (IQ-Tree) and Rapid Maximum Likelihood (RaxML) analyses gave the same conclusion highly 32 supporting the monophyly of Aspergillus . The other analyses were also performed by using publicly 33 available data by using the coding sequences of nine loci (18S rRNA, 5,8S rRNA, 28S rRNA (D1-D2), 34 RPB1 , RPB2 , CaM , BenA , Tsr1 , Cct8 ) of 204 different species. Both Bayesian (MrBayes) and Maximum 35 Likelihood (RAxML) trees obtained by this second round of independent analyses strongly supported the 36 monophyly of the genus Aspergillus . The stability test also confirmed the robustness of the results obtained. 37 In conclusion, statistical analyses have rejected the hypothesisMANUSCRIPT that the Aspergilli are non-monophyletic, and 38 provided robust arguments that the genus is monophyletic and clearly separated from the monophyletic 39 genus Penicillium . There is no phylogenetic evidence to split Aspergillus into several genera and the name 40 Aspergillus can be used for all the species belonging to Aspergillus i.e. the clade comprising the subgenera 41 Aspergillus, Circumdati, Fumigati, Nidulantes, section Cremei and certain species which were formerly part 42 of the genera Phialosimplex and Polypaecilum . Section Cremei and the clade containing Polypaecilum and 43 Phialosimplex are proposed as new subgenera of Aspergillus .The phylogenetic analysis also clearly shows 44 that Aspergillus clavatoflavus and A.zonatus do not belong to the genus Aspergillus . Aspergillus 45 clavatoflavus is therefore transferred to a new genus Aspergillago as Aspergillago clavatoflava and A. 46 zonatus was transferred to Penicilliopsis as P. zonata. The subgenera of Aspergillus share similar extrolite 47 profiles indicating thatACCEPTED the genus is one large genus from a chemotaxonomical point of view. Morphological 48 and ecophysiological characteristics of the species also strongly indicate that Aspergillus is a polythetic 49 class in phenotypic characters. 50 Taxonomic novelties: Aspergillus subgenus Cremei, subgen. nov, Aspergillus subgenus Polypaecilum , 51 subgen. nov.; Aspergillago Samson, Houbraken & Frisvad, gen . nov. New combinations : Aspergillago ACCEPTED MANUSCRIPT 52 clavatoflavus (Raper & Fennell) Samson, Houbraken & Frisvad, comb. nov.; Penicilliopsis zonatus (Kwon- 53 Chung & Fennell) Samson, Houbraken & Frisvad, comb.nov. 54 55 Introduction 56 The genus Aspergillus contains some of the most abundant and widely distributed organisms on earth, and 57 comprises approximately 350 accepted species (Samson et al . 2014). It is one of the fungal genera with the 58 highest economic importance in biotechnology (enzymes, organic acids, bioactive metabolites), but 59 members of the genus are also frequently reported as foodborne contaminants (food spoilage and 60 mycotoxin contamination), or as causal agents of human mycoses (pulmonary aspergillosis, otomycosis, 61 keratitis). Aspergillus is also one of the oldest names in fungal taxonomy since it was applied by P. Micheli 62 (1729), who gave it this name because the spore-bearing structure characteristic of the genus resembled an 63 aspergillum (a device used by the Catholic church to sprinkle holy water). However this morphological 64 characteristic resulted in a broad generic concept because it is associated to twelve quite different 65 teleomorphs demonstrating the variation in physiological and morphological features (Houbraken & Samson 66 2011, Pitt & Taylor 2014). Houbraken et al . (2014) have MANUSCRIPTreduced the number of teleomorphic names to ten 67 (Petromyces, Neopetromyces, Saitoa, Fennellia, Emericella, Hemisartorya, Neosartorya, Neocarpenteles, 68 Cristaspora , and Eurotium ) and showed that the teleomorphs Warcupiella and Sclerocleista do not belong to 69 the Aspergillus monophyletic clade. 70 The most important change in recent fungal nomenclature is the abandonment of dual 71 nomenclature for pleomorphic fungi, following the decision taken at the International Botanical Congress in 72 Melbourne (24-30 July, 2011). In the latest International Code of Nomenclature for algae, fungi and plants 73 (ICN, McNeill et al. 2012), the single name nomenclature was adopted. This has forced mycologists to 74 choose one name for each fungal genus (i.e Aspergillus, Fusarium , Penicillium, etc.). The ICN 75 recommended that either the sexual or asexual name can be chosen, in contrast to the earlier 76 recommendation thatACCEPTED the name of the sexual state should always be preferred. Several sexual names have 77 priority over the asexual ones, but the final choice among the names should also be strongly supported by 78 the (mycological) community. In general, the nomenclatural decision has been easily assigned for most 79 fungal genera, but it sometimes became complicated for economically and socially important fungi having a 80 well-established sexual and asexual name (Zhang et al . 2013). Even though taxonomy contains the rather ACCEPTED MANUSCRIPT 81 independent disciplines such as classification, nomenclature and identification, decisions concerning 82 nomenclature should take into account both the other two. In recent years cladonomy has having a more 83 and more important impact on taxonomy, to a degree where monophyly is the overruling factor in deciding 84 which taxa (clada) should be accepted and which names to give to them, rather than classificatory 85 principles. 86 Phylogenetic approaches have helped to solve taxonomical and nomenclatural problems. A clear 87 example is evident in the paper of Kepler et al . (2014) in which the robust monophyly of the genus 88 Metharrizum included the majority of species recognized in Metacordyceps as well as the green-spored 89 Nomuraea species and those in the more recently described genus Chamaeleomyces . In the same analysis 90 Pochonia was shown to be polyphyletic and the description of Metapochonia gen. nov. was done to 91 accommodate these species forming a separate clade. In this regard, a dispute on the asexual genus 92 Aspergillus and its sexual generic names, started after the International Commission of Penicillium and 93 Aspergillus (ICPA) discussed the single nomenclature and made a decision on April 12 2012 94 (www.aspergilluspenicillium.org). 95 Two proposals for the single name nomenclature