Giancarlo Perrone. CONFERENZA DiSBA-2017 I FRUTTI DELLA RICERCA BIO-AGROALIMENTARI"

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1 Giancarlo Perrone CONFERENZA DiSBA-2017 I FRUTTI DELLA RICERCA BIO-AGROALIMENTARI"

2 Key topics Aspergillus genus Background: ICN and phylogenetic approach for single nomenclature Two proposals for Aspergillus New multilocus sequences analysis Conclusions Six genes analysis Nine genes analysis

3 Importance and distribution of Aspergillus genus Pharmaceutics Fermentation Industry A. fumigatus, A. terreus A. oryzae, A. sojae, A. niger Citric acid and organic enzymes A. fumigatus, A. flavus Human infection Plant pathogens MYCOTOXIN PRODUCTION Food spoilage

4 asexual morphology Aspergillus sexual morphology Conidia Phialides (secondary sterigmata) Petromyces uniseriate biseriate Neopetromyces Vesicle Conidiophore Metulae (primary sterigmata) Eurotium Foot cell Sclerotia Conidial head radiate or columnar Chaetosartorya Neosartorya Fennellia Emericella

5 Aspergillus Currently, 4 subgenera and 19 sections are accepted in Aspergillus. This subdivision was revised and supported by morphological features and phylogenetic data.

6 Key topics Background: ICN and phylogenetic approach for single nomenclature Two proposals for Aspergillus

7 ICN and Phylogeny approach International Code of Botanical Nomenclature adopted by the Melbourne Congress, July 2011 resulting in a new code named: 100 A. niger CBS T (AY583536) A. lacticoffeatus CBS T (AY819998) A. brasiliensis CBS T (AY820006) 100 A. foetidus CBS (AY585534) A. costaricaensis CBS T (AY820014) 97 A. tubingensis CBS T (AY820007) 100 A. vadensis CBS T (AY585531) 100 A. carbonarius CBS T (AY585532) 98 A. sclerotioniger CBS T (AY819996) 100 A. ibericus IMI T (AM419748) A. ellipticus CBS NT (AY585530) A. heteromorphus CBS (AY585529) A. uvarum IMI (AM745753) 100 A. uvarum IMI (AM745752) 93 A. uvarum IMI T (AM745751) 95 A. uvarum IMI (AM745750) 100 A. aculeatus CBS (AY585539) A. aculeatus CBS T (AY585540) 99 A. aculeatus CBS (AY585538) 100 A. japonicus CBS T (AY585542) A. homomorphus CBS T (AY820015) A. flavus CBS T (AY819992) International Code of Nomenclature for Algae, Fungi and Plants Concept of dual nomenclature is deleted and anamorph have the same priority of teleomorph names (Norvell, 2011). One Fungus One Name: all morphs belonging to one fungus. If anamorph genus represents the oldest valid and legitimate name, and it is the most widely used and preferred, then this name can be applied for all morphs (Braun, 2012; Norvell, 2011). Then Aspergillus and Penicillium would have priority over their competing teleomorphs. ICPA in 2012 was unanimously in the use of the name Penicillium. Although the majority of ICPA voted for the use of Aspergillus this decision was objected by Pitt & Taylor Phylogeny has been widely used to choose the right name for large groups of fungi through the identification of monophyletic clades Metarhizium gene - Kepler et al Glomeromycota taxa in mycorrhizal fungi - Redecker et al Curvularia and Bipolaris Magnaporthaceae taxa Zhang et al. 2013

8 Two proposal for Aspergillus 1 Aspergillus is a monophyletic group proposal n 5 of ICPA members April 14, 2012 Houbraken and Samson Phylogeny of Penicillium and the segregation of Trichocomaceae into three families. Studies in Mycology. Approach used: Sequences of four genes (Cct8, Tsr1, RPB1, RPB2), MSA aligned with MUSCLE (translated protein) and analysed with MrBayes and RaxML, Coccidioides immitis as outgroup. Samson et al Phylogeny, identification and nomenclature of the genus Aspergillus. Studies in Mycology. Approach used: Sequences of tree genes (RPB2, Tsr1, Cct8), MSA analysed with MrBayes and RaxML, Talaromyces flavus as outgroup.

9 Bayesian and RAxML phylogenetic analyses made with four genes: Cct8, Tsr1, RPB1, RPB2

10 Bayesian and RAxML phylogenetics analyses of Aspergillus and Penicillium taxa with tree genes: RPB2, Cct8, Tsr1 0.99/- */ /- */99 */* */* 0.99/- */* CBS Aspergillus aculeatus CBS Aspergillus niger CBS Aspergillus flavipes NIH 2624 Aspergillus terreus CBS Aspergillus ochraceus CBS Aspergillus candidus CBS Aspergillus robustus CBS Aspergillus togoensis NRRL 3357 Aspergillus flavus CBS Aspergillus avenaceus */86 CBS Aspergillus versicolor */* FGSC A4 Aspergillus nidulans CBS Aspergillus calidoustus CBS Aspergillus funiculosus CBS Aspergillus sparsus */94 CBS Aspergillus cejpii ( Dichotomomyces cejpii) */* NRRL 1 Aspergillus clavatus */98 */* Af293 Aspergillus fumigatus NRRL 181 Aspergillus fischeri */- * 83 */81 */82 */- */ /- */* */* */99 */* Aspergillus s. str. CBS Aspergillus cervinus */* CBS Aspergillus glaucus CBS Aspergillus montevidensis CBS Aspergillus restrictus CBS Aspergillus penicillioides CBS Aspergillus caninus ( Phialosimplex caninus) CBS Aspergillus baarnensis ( Basipetospora halophila) CBS Aspergillus sclerotialis ( Phialosimplex sclerotialis) CBS Aspergillus insolitus ( Polypaecilum insolitum) CBS Aspergillus arxii ( Cristaspora arxii) CBS Aspergillus pulvinus CBS Aspergillus wentii CBS Aspergillus brunneo-uniseriatus CBS Penicillium inflatum ( Aspergillus inflatus) CBS Monascus purpureus CBS Xeromyces bisporus CBS Penicillium megasporum CBS Hamigera avellanea CBS Aspergillus clavatoflavus 0.99/- */97 CBS Aspergillus zonatus CBS Penicilliopsis clavariiformis */* CBS Sclerocleista thaxteri CBS Sclerocleista ornata */* CBS Penicillium arenicola CBS Phialomyces macrosporus CBS Penicillium fractum CBS Penicillium ramusculum CBS Penicillium taxi CBS Penicillium ochrosalmoneum */* CBS Penicillium stolkiae CBS Penicillium janthinellum CBS Penicillium restrictum CBS Penicillium gracilentum CBS Penicillium cinnamopurpureum */* CBS Penicillium herquei CBS Penicillium glabrum CBS Penicillium charlesii */98 CBS Penicillium digitatum CBS Penicillium expansum */* */* CBS Penicillium viridicatum CBS Penicillium chrysogenum */* CBS Penicillium paradoxum */* */* */* CBS Penicillium lanosum CBS Penicillium olsonii CBS Penicillium sacculum */92 CBS Penicillium canescens CBS Penicillium citrinum CBS Penicillium lagena CBS Talaromyces flavus 0.1 Miscellaneous Aspergillaceae Penicillium

11 Both phylogenetic analysis give evidence of monophyly of Aspergillus but with Low ML Support */51

12 Two proposal for Aspergillus 2 Aspergillus is a paraphyletic group proposal n 4 of ICPA members April 14, 2012 Pitt and Taylor Aspergillus, its sexual states and the new International Code of Nomenclature. Mycologia. Approach used: Sequences of four genes (RPB1, RPB2, Tsr1, Cct8) aligned with MUSCLE, MSA analysed with MrBayes and Mega5, Thermoascus aurantiacus as closest outgroup (used the same sequences of Houbraken and Samson 2011) Taylor et al Choosing one name for pleomorphic fungi: the example of Aspergillus v. Eurotium, Neosartorya and Emericella. Taxon Approach used: Sequences of four genes (RPB1, RPB2, Tsr1, Cct8), MSA (removed regions in Tsr1) analysed with RaxML, Talaromyces flavus as outgroup.

13 Bayesian and ML phylogenetic analyses of Aspergillus and related taxa made with four genes: RPB1, RPB2, Tsr1, Cct8

14 97 Eupenicillium_lassenii_ Penicillium_cryptum_ Penicillium_lagena_ Eupenicillium_catenatum_ Penicillium_dimorphosporum_ Eupenicillium_katangense_ Penicillium_citreonigrum_ Eupenicillium_lapidosum_ Penicillium_namyslowskii_ Eupenicillium_gracilentum_ Penicillium_macrosclerotiorum_ Eupenicillium_stolkiae_ Penicillium_simplicissimum_ Eupenicillium_javanicum_ Penicillium_janthinellum_ Eupenicillium_cinnamopurpureum_ Eupenicillium_idahoense_ Eupenicillium_euglaucum_ Eupenicillium_shearii_ Penicillium_citrinum_ Eupenicillium_fractum_ Penicillium subgen Aspergilloides 100 Eupenicillium_inusitatum_ Eupenicillium_ornatum_ Penicillium_ramusculum_ Penicillium_isariiforme_ Penicillium_ochrosalmoneum_ Thysanophora_canadensis_ Thysanophora_taxi_ Penicillium_griseolum_ Penicillium_charlesii_ Penicillium_coffeae_ Penicillium_glabrum_ Penicillium_thiersii_ Penicillium_adametzii_ RAxML phylogenetic analyses of Aspergillus and related taxa made with four genes: RPB1, RPB2, Cct8, Tsr Chromocleista_malachitea_ Eupenicillium_hirayamae_ Eladia_saccula_ Penicillium_canescens_ Penicillium_lanosum_ Eupenicillium_tularense_ Penicillium_olsonii_ Hemicarpenteles_paradoxus_ Penicillium_expansum_ Penicillium_chrysogenum_ Penicillium_griseofulvum_ Aspergillus_conjunctus_ Aspergillus_biplanus_ Aspergillus_sparsus_ Aspergillus_funiculosus_ Aspergillus_ochraceoroseus_ Aspergillus_bisporus_ Aspergillus_calidoustus_ Aspergillus_amylovorus_ Aspergillus_egyptiacus_ Emericella_nidulans_ Aspergillus_sydowii_ Aspergillus_versicolor_ Aspergillus_aculeatus_ Aspergillus_niger_ Aspergillus_arenarius_ Aspergillus_candidus_ Aspergillus_janus_ Aspergillus_terreus_NIH2624 Aspergillus_aureofulgens_ Aspergillus_flavipes_ Aspergillus_robustus_ Aspergillus_ochraceus_ Aspergillus_steynii_ Aspergillus_avenaceus_ Aspergillus_leporis_ Aspergillus_coremiiformis_ Aspergillus_togoensis_ Aspergillus_flavus_ Penicillium subgen Penicillium Emericella Aspergillus section Circumdati Aspergillus_oryzae_RIB Aspergillus_cervinus_ Aspergillus_kanagawaensis_ Aspergillus_fumigatus_Af293 Neosartorya_fischeri_NRRL18 Dichotomomyces_cejpii_ Aspergillus_clavatus_ Neocarpenteles_acanthosporum_ Aspergillus_brunneouniseriatus_ Aspergillus_wentii_ Aspergillus_pulvinus_ Cristaspora_arxii_ Aspergillus_penicillioides_11C3 Aspergillus_restrictus_ Eurotium_amstelodami_ Eurotium_herbariorum_ Basipetospora_halophilica_ Phialosimplex_caninus_ Phialosimplex_chlamydosporus_ Phialosimplex_sclerotialis_ Polypaecilum_insolitum_ Polypaecilum_pisci_ Leiothecium_ellipsoideum_ Neosartoria Cristaspora Eurotium Phyalosimplex, Polypaecilum Chrysosporium_inops_ Xeromyces_bisporus_ Monascus_purpureus_ Monascus_argentinensis_ Monascus_lunisporas_ Aspergillus_clavatoflavus_ TreeBASE Tr94200 Aspergillus_zonatus_ Penicilliopsis_clavariiformis_ Penicillium_megasporum_ Warcupiella_spinulosa_ Hamigera_striata_ Aspergillus_penicilliformis_ Hamigera_avellanea_ Hemicarpenteles_ornatus_ Hemicarpenteles_thaxteri_ Outgroup Penicillium_arenicola_ Talaromyces_flavus_ Phialomyces_macrosporus_ Thermoascus_aurantiacus_ Thermoascus_aurantiacus_ Byssochlamys_verrucosa_ Thermoascus_crustaceus_ Thermoascus_thermophilus_528.71

15 Both phylogenetic analysis give evidence of paraphyly of Aspergillus but with Low ML Support 1/60 0,99/- 1/- 1/- 0,98/-

16 Results of the two proposal for Aspergillus Proposal n 5 of ICPA members Proposal n 4 of ICPA members Aspergillus

17 Key topics New multilocus sequences analysis Six genes analysis Nine genes analysis

18 Two different multilocus phylogenetic sequences analyses With the aim to untangle the monophyly vs non-monophyly discussion, we adopted two independent approaches to try to resolve definitely the phylogenetic incongruence of the previous studies: 1) Preliminary analysis with six loci phylogeny study on the coding regions of RPB1, RPB2, Cct8, Tsr1, BenA, Cam on 96 species (the same used by previous authors) 2) Extended analysis performed a nine loci phylogeny study on the coding regions of 18S rrna, 5.8S rrna, 28S rrna (D1-D2), RPB1, RPB2, CaM, BenA, Tsr1 and Cct8 on 204 species

19 Methods used in the Six genes analysis Same set of taxa of the previous studies (96 isolates), but only the CDS sequences were retrieved from database. Algorithm MUSCLE and ClustalW: multi-align sequences Transitive Consistence Score (Chang et al. 2015) Models RogueNaRock: an Efficient and Exact Algorithm rogue taxa search and goodness of MSA (Aberer et al. 2013) JModelTest: best-fit model of nucleotide substitution (Darriba et al. 2012) PartitionFinder: partitioning schemes and substitution models (Lanfear et al. 2012) Phylogenetic Inference model used MrBayes: Bayesian Inference (Cipres, simba) (Ronquist et al. 2012) RAxML: Maximum Likelyhood (Cipres, raxmlgui) (Stamakis et al. 2014) IQ-Tree: Ultra Fast Maximum Likelyhood (partitioning schemes, substitution models, confidence of trees)- (Nguyen et al. 2015)

20 Penicillium UFML, MrBayes and RAxML support of Aspergillus, Penicillium and related taxa with coding regions of six genes: RPB1, RPB2, Cct8, Tsr1, BenA (CDS), Cam (CDS) Aspergillus

21 UFML 100 MB 100 RAxML 100

22 UFML 94 MB 100 RAxML 77 UFML 93 MB 99 RAxML 63

23 Phylogeny inferred from nine loci : 18S rrna, 5,8S rrna, 28S rrna (D1-D2), RPB1, RPB2, CaM, BenA, Tsr1 and Cct8 The dataset containing sequences from 204 strains was partitioned according to loci. Algorithm Methods used in the Nine genes analysis Multiple Sequence Alignments by PRANK v (Loytynoja 2014) Models Gaps of the highly variable region of the TSr1 gene were coded by 2matrix and added to the analysis by using SequenceMatrix 1.8 (Vaidya et al., 2010) JModelTest: best-fit model of nucleotide substitution (Darriba et al. 2012) Phylogenetic Inference model used MrBayes: Bayesian Inference (Cipres, simba) (Ronquist et al. 2012) RAxML: Maximum Likelyhood (Cipres, raxmlgui) (Stamakis et al. 2014)) IQ-Tree: Ultra Fast Maximum Likelyhood (partitioning schemes, substitution models, confidence of trees)- (Nguyen et al. 2015)

24 Phylogeny inffered by RAxML I Aspergillus 87 Aspergillus 76 Penicillium 79 Talaromyces Thermoascus

25 Phylogeny inffered by RAxML II 72 Aspergillus Penicillium Penicillium 70 Talaromyces Thermoascus

26 Single branch tests by IQ-TREE 99/98/*/*/* Values above braches are SH-aLRT support (%)/ local bootstrap support (%)/ parametric alrt support/ abayes support/ ultrafast bootstrap support (%) 96/94/*/*/99 99/99/*/*/* 95/94/*/*/98 Aspergillus Penicillium 82/80/*/*/90 * 85/81/0.99/0.99/* *

27 Branch support analysis The branch supports of the principal nodes of six and nine-genes phylogeny for the monophyletic topology were evaluated. Three categories of branch support were considered: parametric (Bpp, alrt-chi2, 200 abayes) nonparametric (Rbs, SH-aLRT) hybrid (Ibs) (Anisimova et al. 2011, Minh et al. 2013) Confidence of trees: Tree-Puzzle - Consel The best trees obtained with the respective shafts consent of each analysis, and a forced tree with the paraphyletic topology were compared. Tree-Puzzle calculated respective Log / Likelihood and statistical analysis of KH, SH and ELW, Consel was used to statistically calculate KH, SH, WKH, WSH and AU parameter (Schmidt et al. 2009)

28 Collapsed phylograms showing the support values of the principal nodes involved in the monophyly of Aspergillus based on six (A) and nine (B) genes. The tables are summarizing the values of these nodes obtained by different methods.

29 Testing Trees with Tree-Puzzle and Consel Trees Obs. log/l difference KH WKH SH WSH c-elw AU IQ-Tree (partitioned dataset) RAxML (partitioned dataset) Monophyly RAxML MrBayes Non- Monoph yly IQ-Tree RAxML (partitioned dataset) constraint

30 Extrolites profile of Aspergillus genus At least xanthocillins, terphenyllins and emodin are in common within all the subgenera of the genus

31 Key topics Conclusions

32 Conclusions The results of these efforts lead to the inference of good supported trees and clearly showing the monophyly of the genus Aspergillus (sub.: Circumdati, Nidulantes, Fumigati, Aspergillus, and two new subgenera were described: subgen. nov Cremei subgen nov Polypaecilum Both conducted statistical analyses reject the hypothesis that Aspergilli are nonmonophyletic There is no phylogenetic evidence supporting the splitting of Aspergillus genus Recently the hypothesis of paraphyly by Pitt and Taylor was rejected General Committee of Nomenclature (TAXON December 2017)

33 Acknowledgments Donato Magistà Institute of Sciences of Food Production CNR Sándor Kocsubé and János Varga Dept. of Microbiology, Faculty of Science and Informatics, University of Szeged Robert A. Samson and Jos Houbraken CBS - Fungal Biodiversity Centre Jens C. Frisvad Department of System Biology DTU

34 Acknowledgments Donato Magistà Institute of Sciences of Food Production CNR Sándor Kocsubé and János Varga Dept. of Microbiology, Faculty of Science and Informatics, University of Szeged Robert A. Samson and Jos Houbraken CBS - Fungal Biodiversity Centre Jens C. Frisvad Department of System Biology DTU

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