<I>Penicillium</I> Diversity in Canadian Bat

VOLUME 5 JUNE 2020 Fungal Systematics and Evolution PAGES 1–15

doi.org/10.3114/fuse.2020.05.01

Penicillium diversity in Canadian bat caves, including a new species, P. speluncae

C.M. Visagie1, N. Yilmaz1, K. Vanderwolf2, J.B. Renaud3, M.W. Sumarah3, J. Houbraken4, R. Assebgui5, K.A. Seifert5, D. Malloch2

1Department of Biochemistry, Genetics and Microbiology, Forestry and Agricultural Biotechnology Institute (FABI), Faculty of Natural and Agricultural Sciences, University of Pretoria, P. Bag X20, Hatfield 0028, Pretoria, South Africa 2New Brunswick Museum, 277 Douglas Avenue, Saint John, New Brunswick, E2K 1E5, Canada 3London Research & Development Centre, Agriculture & Agri-Food Canada, London, Ontario, N5V 4T3, Canada 4Westerdijk Fungal Biodiversity Institute, Uppsalalaan 8, Utrecht, 3584 CT, Netherlands 5Biodiversity (Mycology), Eastern Cereal and Oilseed Research Centre, 960 Carling Ave., Ottawa, Ontario, K1A 0C6, Canada

*Corresponding author: [email protected]

Key words: Abstract: Penicillium species were commonly isolated during a fungal survey of bat hibernacula in New Brunswick Thysanophora and Quebec, Canada. Strains were isolated from arthropods, bats, rodents (i.e. the deer mouse Peromyscus sect. Fasciculata maniculatus), their dung, and cave walls. Hundreds of fungal strains were recovered, of which Penicillium Genealogical Concordance represented a major component of the community. Penicillium strains were grouped by colony characters on Phylogenetic Species Blakeslee’s malt extract agar. DNA sequencing of the secondary identification marker, beta-tubulin, was done Recognition (GCPSR) for representative strains from each group. In some cases, ITS and calmodulin were sequenced toconfirm concept identifications. In total, 13 species were identified, while eight strains consistently resolved into a unique clade with new taxon P. discolor, P. echinulatum and P. solitum as its closest relatives. Penicillium speluncae is described using macro- Pseudogymnoascus and micromorphological characters, multigene phylogenies (including ITS, beta-tubulin, calmodulin and RNA destructans (Pd) polymerase II second largest subunit) and extrolite profiles. Major extrolites produced by the new species include secondary metabolites cyclopenins, viridicatins, chaetoglobosins, and a microheterogenous series of cyclic and linear tetrapeptides. Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] Effectively published online: 9 August 2019.

INTRODUCTION The study of fungal diversity is important to determine the true impact of a potential invasive species such as Pd on fungal The study of fungi associated with bats and their habitats has community structure among bats and hibernacula (Johnson become important after the spread of White-nose Syndrome et al. 2013). Understudied environments such as caves are rich (WNS) caused by Pseudogymnoascus destructans (Pd), resulted sources of undescribed microbial species. Many new fungi have in an ongoing rapid decline of bat populations in North America. recently been described from underground environments as Much effort has focused on populations of Pd from positive more studies are conducted, although it is still unknown whether caves. White-nose Syndrome is named for characteristic white obligate troglobiotic fungi exist (Zhang et al. 2017). Previous growth caused by P. destructans, which was previously known studies commonly reported the isolation of Cladosporium, as Geomyces destructans (Gargas et al. 2009, Minnis & Lindner Fusarium, Mortierella, and Penicillium species from bat wings, 2013). Characterization of fungal populations and identification caves and mines (Johnson et al. 2013, Vanderwolf et al. 2013a, b). of other fungal species may reveal possible antagonists to Pd Penicillium is one of the most common genera isolated from caves (Micalizzi et al. 2017). on multiple substrates, particularly sediment and air, although no White-nose Syndrome was first reported in New York in new Penicillium species have been described from caves apart 2006 (Blehert et al. 2009), while the first report from Canada from P. cavernicola, which has also been found outside of caves on was from Ontario in 2010. In both cases, it led to mass mortality dairy products (Frisvad & Samson 2004, Vanderwolf et al. 2013a, of the hibernating bat populations (McAlpine et al. 2012). The b), and P. gravinicasei recently described from a cave in Italy from disease only occurs while bats hibernate. Pseudogymnoascus ripening Apulian cave cheeses (Anelli et al. 2018). Vanderwolf destructans cannot grow at temperatures above ± 20 °C (Gargas et al. (2016) studied the fungi associated with over-wintering et al. 2009), and it is thought that the cool caves and mines arthropods in Pd positive hibernacula in Canada. They isolated 87 inhabited by bats during hibernation serve as environmental fungal taxa from four arthropod genera. In the current study, we reservoirs of Pd (Lorch et al. 2013, Reynolds et al. 2015). The report Penicillium isolated from these arthropods, but also include presence of Pd in bat populations was confirmed in many strains isolated from various other substrates associated with countries in Europe and Asia but no significant mortality was caves and/or bats. The aims of this study were (1) to determine observed, despite the fact that some European bats have been the Penicillium species diversity in bat caves and hibernacula in found with clinical WNS (Wibbelt et al. 2010, Puechmaille et al. New Brunswick and Quebec, and (2) formally describe the new 2011). Why bats remain healthy in these areas is unclear. species that was isolated during the survey.

MATERIALS AND METHODS (http://tree.bio.ed.ac.uk/software/figtree) and visually edited in Affinity Designer v. 1.7.1 [Serif (Europe) Ltd, Nottingham, UK]. Strains, sampling and isolations Morphology Strains were isolated from arthropods, bats, rodents, rodent dung, and walls of bat hibernacula in New Brunswick (Berryton Morphological characters were captured using standardized Cave, Dallings Cave, Dorchester Mine, Glebe Mine, Markhamville protocols proposed by Visagie et al. (2014b). Colony characters

Mine, White Cave) and Quebec (Grotte à la Patate), Canada were captured on Czapek yeast autolysate agar (CYA), MEAbl, Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] (Vanderwolf et al. 2013b, 2016, 2017). Fungi were also isolated yeast extract sucrose agar (YES), oatmeal agar (OA) and creatine from a dead big brown bat that was found in a parking garage in sucrose agar (CREA). Strains were inoculated in a three-point Fredericton, New Brunswick. Isolation media included dextrose- pattern on these media in 90 mm Petri dishes. Plates were peptone yeast extract agar (DPYA), sabouraud agar (SD) or malt incubated for 7 d at 25 °C in darkness in perforated plastic bags. extract agar (MEA), with plates incubated at 7 °C. Representative Colour names and codes used in descriptions are from Kornerup strains for each species found were submitted to the Canadian & Wanscher (1967). Microscopic observations were made using Collection of Fungal Cultures (DAOMC) and the holotype an Olympus SZX12 dissecting microscope and Olympus BX50 specimen of the new species deposited in the Canadian National compound microscope equipped with Infinity3 and InfinityX Mycological Herbarium (DAOM). Strains isolated during this cameras driven by Infinity Analyze v. 6.5.1 software (Lumenera study are summarized in Table 1. Corp., Ottawa, Canada). Colonies were captured with a Sony NEX-5N camera. Plates were prepared in Affinity Photo v. 1.6.6 DNA extraction, sequencing and phylogenetic analysis [Serif (Europe) Ltd, Nottingham, UK]. For aesthetic purposes, micrographs were adjusted using the “inpainting brush tool” Strains were grown on Blakeslee’s (1915) malt extract agar without altering areas of scientific significance. Line drawings (MEAbl) for 7 d and DNA extracted using the UltracleanTM were prepared in Affinity Photo v. 1.7.1 [Serif (Europe) Ltd, Microbial DNA isolation Kit (MoBio Laboratories Inc., Solana Nottingham, UK] running on an iPad Pro with an Apple Pencil. Beach, USA). DNA was amplified with a PCR master mix consisting of 0.5 µL dNTPs (2 µM), 0.04 µL for each primer (20 µM), 1 µL Extrolites 10× Titanium Taq buffer (Clontech, California, USA), 0.1 µL 50× Titanium Taq enzyme (Clontech, California, USA), 0.5 µL template For extrolite analyses, all strains were grown in 9 cm polystyrene DNA and 7.82 µL sterile purified water. ITS barcodes (Schoch et Petri dishes on CYA (Pitt 1980) and YES (Frisvad 1981, Filtenborg al. 2012), partial beta-tubulin (BenA), partial calmodulin (CaM) et al. 1990) incubated at 25 °C for 14 d. Six agar plugs from each and RNA polymerase II second largest subunit (RPB2) genes fungal isolate were excised with a sterilized 7 mm cork-borer were amplified using PCR conditions and primers suggested by and transferred to a 13 mL polypropylene tube. Two mL of ethyl Visagie et al. (2014b). PCR products were verified by agarose gel acetate was then added and vortexed for 30 s, followed by electrophoresis and subsequently sequenced with the BigDye sonication at 30 °C for 30 min and vortexed again for 30 s. The Terminator Cycle Premix Kit (Applied Biosystems, Waltham, supernatants were transferred into new polypropylene tubes USA). Contigs were assembled and edited in Geneious v. 8.1.5 and dried on a centrifugal vacuum concentrator at 35 °C. Extracts (BioMatters Ltd., Auckland, New Zealand). Newly generated were then reconstituted in 1 mL of methanol:water (8:2) and sequences were submitted to GenBank and accession numbers filtered into 2 mL amber glass HPLC vials using a 0.45 µm PVDF provided in Table 1. Gene sequences of the new species were syringe filter. Extracts were immediately stored at -20 °C until compared to a reference sequence dataset built around the ex- analysis by liquid chromatography mass spectrometry (LC-MS). type sequences published in Visagie et al. (2014b), also including Extracts were analyzed in both positive and negative polarities reference sequences from (Samson et al. 2004, Houbraken et al. using a Q-Exactive Orbitrap coupled to an Agilent 1290 HPLC. 2011, 2012, 2014, 2016, Frisvad et al. 2013a, b, Visagie et al. The chemical formula of observed extrolites were determined 2014a) where needed (Suppl. Table S1). Additional unpublished with Xcalibur® software using accurate mass measurements (< sequences related to the new species were included and 3.0 ppm) and manually verified by isotopic pattern. The chemical originate from various past projects. Sequences were aligned formulae were then searched against microbial extrolite in MAFFT v. 7.407 (Katoh & Standley 2013), with the G-INS-i databases [AntiBase2013 (Wiley-VCH, Weinheim, Germany)] option and manually trimmed and adjusted in Geneious and KNApSAcK (Afendi et al. 2012) and putative matches were where needed. Datasets were subsequently analysed using scrutinized by comparing their MS/MS fragmentation with those Maximum Likelihood (ML) and Bayesian tree inference (BI). For published in the literature or predicted by CFM-ID (Allen et al. concatenated phylogenies, each gene was treated as a separate 2014). partition. ML trees were calculated in IQtree v. 1.6.8 (Nguyen et al. 2015) with the most suitable model for each gene and/ or partition calculated using Modelfinder (Kalyaanamoorthy RESULTS et al. 2017) and bootstrapping done using UFBoot (Minh et al. 2013), both integrated into IQtree. Bayesian inference trees Sampling, isolations & identifications were calculated in MrBayes v. 3.2.6 (Ronquist et al. 2012) with the most suitable model selected by ParitionFinder v. 2.1.1 During the survey, 70 Penicillium strains were isolated from six (Lanfear et al. 2017) using the corrected Akaike information different caves in New Brunswick and one in Quebec, Canada. criterion (Akaike 1974). Alignments and command blocks used Eight strains of the new Penicillium species were isolated from for analyses were uploaded to TreeBASE (https://treebase.org) walls of the Glebe Mine and White Cave in New Brunswick with accession 23575. Trees were visualized in Figtree v. 1.4.4 and Grotte à la Patate in Quebec, and three strains were

n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a RPB2 n/a n/a n/a n/a n/a n/a MG490973 MG490972 MG490971 MG490969 MG490968 MG490966 n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a CaM

Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected]

MG490901 MG490900 MG490898 MG490890 MG490947 MG490919 MG490946 MG490945 MG490941 MG490938 MG490932 MG490926 MG490949 MG490936 MG490934 MG490933 MG490929 MG490924 MG490906 MG490903 MG490897 MG490896 BenA n/a n/a n/a n/a n/a n/a MG490883 MG490882 MG490881 MG490879 MG490878 MG490876 n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a ITS ) ) ) sp) ) ) ) ) ) Meta ovalis Meta ) Scoliopteryx Scoliopteryx Cave wall Cave Cave wall Cave Cave wall Cave Cave wall Cave Eptesicus fuscus ( Eptesicus Bat Cave wall Cave Rodent dung Rodent ( Peromyscus maniculatus Rodent dung Rodent ( Peromyscus maniculatus Rodent dung Rodent ( Peromyscus maniculatus Rodent dung Rodent ( Peromyscus maniculatus Moth ( libatrix Peromyscus ( Peromyscus Rodent maniculatus Eptesicus fuscus ( Eptesicus Bat Harvestman ( Nelima Harvestman elegans ) Harvestman ( Nelima Harvestman elegans ) Harvestman ( Nelima Harvestman elegans ) Gnat ( Exechiopsis Gnat Spider ( Cave wall Cave Cave wall Cave Cave wall Cave Cave wall Cave Substrate Grotte à la Patate Patate à la Grotte Grotte à la Patate Patate à la Grotte Grotte à la Patate Patate à la Grotte Glebe Mine Fredericton parking Fredericton garage White Cave White Dorchester Mine Dorchester Dorchester Mine Dorchester Dorchester Mine Dorchester Dorchester Mine Dorchester Dallings Cave Dorchester Mine Dorchester Fredericton parking Fredericton garage Dorchester Mine Dorchester Glebe Mine Glebe Mine Glebe Mine Dorchester Mine Dorchester Berryton Cave Berryton Berryton Cave Berryton Grotte à la Patate Patate à la Grotte Dorchester Mine Dorchester Cave name Cave Anticosti Island Anticosti Anticosti Island Anticosti Anticosti Island Anticosti Sussex Fredericton Hillsborough Hillsborough Dorchester Dorchester Dorchester Dorchester Sussex Dorchester Fredericton Dorchester Sussex Sussex Sussex Dorchester Moncton Moncton Anticosti Island Anticosti Dorchester Location Quebec Quebec Quebec New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New Quebec New Brunswick New Province DPYA DPYA DPYA DPYA DPYA DPYA DPYA DPYA DPYA DPYA DPYA DPYA DPYA DPYA SD DPYA DPYA DPYA DPYA MEA DPYA MEA Isolation Isolation medium 07-Jul-2014 07-Jul-2014 07-Jul-2014 16-Apr-2015 16-Apr-2014 21-Apr-2015 21-Mar-2014 21-Mar-2014 21-Mar-2014 25-Mar-2014 16-Apr-2013 14-Mar-2014 16-Apr-2014 18-Mar-2013 11-Apr-2014 11-Apr-2014 11-Apr-2014 18-Mar-2013 30-Apr-2015 30-Apr-2015 07-Jul-2014 31-Mar-2015 Date Date collected KAS 7471, W59104 KAS 7470, W59104 DAOMC 252108, DAOMC KAS 7467, W72101 KAS 7459, W98105 DAOMC 252107, DAOMC KAS 7540, 742110 DAOMC 252106, DAOMC KAS 7505, W05100 DAOMC 252105, DAOMC KAS 7539, D1007 DAOMC 252104, DAOMC KAS 7538, D1007A DAOMC 252103, DAOMC KAS 7534, D2203 DAOMC 252102, DAOMC KAS 7531, D3303 DAOMC 252101, DAOMC KAS 7520, M26108 DAOMC 252100, DAOMC KAS 7514, P06101 KAS 7542, 742102 DAOMC 252099, DAOMC KAS 7525, H09108 KAS 7523, H26208 KAS 7522, H27101 KAS 7517, M50103 KAS 7511, S11101 KAS 7480, W24103 DAOMC 252098, DAOMC KAS 7476, W29304 DAOMC 252097, DAOMC KAS 7466, W72102 KAS 7465, W7430A Strain Robsamsonia Chrysogena

Robsamsonia

Brevicompacta Section Species isolated from Canadian bat caves. Canadian bat from Species isolated P. concentricum P. P. chrysogenum P. P. brevistipitatum P. Table 1. Table Species bialowiezense P.

n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a RPB2 n/a n/a n/a n/a n/a n/a n/a n/a n/a MG490964 MG490963 n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a CaM

Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected]

MG490931 MG490923 MG490920 MG490918 MG490917 MG490914 MG490912 MG490909 MG490907 MG490913 MG490895 MG490944 MG490943 MG490942 MG490939 MG490927 MG490925 MG490910 MG490908 MG490905 MG490904 BenA n/a n/a n/a n/a n/a n/a n/a n/a n/a MG490874 MG490873 n/a n/a n/a n/a n/a n/a n/a n/a n/a ITS n/a ) ) ) ) ) ) ) Scoliopteryx Scoliopteryx Moth ( libatrix Cave wall Cave Cave wall Cave Cave wall Cave Cave wall Cave Cave wall Cave Cave wall Cave Cave wall Cave Cave wall Cave Cave wall Cave Cave wall Cave Rodent dung Rodent ( Peromyscus maniculatus Rodent dung Rodent ( Peromyscus maniculatus Rodent dung Rodent ( Peromyscus maniculatus Rodent dung Rodent ( Peromyscus maniculatus Peromyscus ( Peromyscus Rodent maniculatus Peromyscus ( Peromyscus Rodent maniculatus Cave wall Cave Cave wall Cave Cave wall Cave Cave wall Cave Substrate Dallings Cave White Cave White White Cave White White Cave White White Cave White Berryton Cave Berryton Berryton Cave Berryton Berryton Cave Berryton Berryton Cave Berryton Berryton Cave Berryton Dorchester Mine Dorchester Dorchester Mine Dorchester Dorchester Mine Dorchester Dorchester Mine Dorchester Dorchester Mine Dorchester Dorchester Mine Dorchester Dorchester Mine Dorchester Berryton Cave Berryton Berryton Cave Berryton Berryton Cave Berryton Cave name Cave Berryton Cave Berryton Sussex Hillsborough Hillsborough Hillsborough Hillsborough Hillsborough Hillsborough Hillsborough Hillsborough Moncton Moncton Moncton Moncton Moncton Dorchester Dorchester Dorchester Dorchester Dorchester Dorchester Dorchester Moncton Moncton Moncton Moncton Location New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New Province DPYA SD DPYA DPYA DPYA SD DPYA DPYA DPYA DPYA DPYA DPYA DPYA DPYA DPYA SD DPYA DPYA DPYA DPYA SD Isolation Isolation medium 16-Apr-2013 21-Apr-2015 21-Apr-2015 21-Apr-2015 21-Apr-2015 30-Apr-2015 30-Apr-2015 30-Apr-2015 30-Apr-2015 30-Apr-2015 31-Mar-2015 21-Mar-2014 21-Mar-2014 21-Mar-2014 25-Mar-2014 14-Mar-2014 14-Mar-2014 30-Apr-2015 30-Apr-2015 30-Apr-2015 Date Date collected 30-Apr-2015 DAOMC 252116, DAOMC KAS 7519, M26109 KAS 7510, W00200 KAS 7506, W04407 KAS 7502, W05406 DAOMC 252115, DAOMC KAS 7501, W07104 KAS 7493, W16200B KAS 7489, W20103 DAOMC 252114, DAOMC KAS 7484, W22102 DAOMC 252113, DAOMC KAS 7481, W24100 DAOMC 252112, DAOMC KAS 7491, W19102 DAOMC 252111, DAOMC KAS 7464, W76401 KAS 7537, D1106 KAS 7536, D1204 KAS 7535, D2111 KAS 7532, D3301 KAS 7515, P05201 KAS 7513, P06102 KAS 7486, W20408 DAOMC 252110, DAOMC KAS 7483, W22102A DAOMC 252109, DAOMC KAS 7479, W24103A KAS 7478, W29203 Strain Penicillium Exilicaulis Exilicaulis Section (Continued). P. expansum P. P. corylophilum P. P. consobrinum P. Table 1. Table Species

MN170740 MN170739 MN170738 MN170737 MN170736 n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a RPB2 MG490958 MG490957 MG490956 MG490955 MG490954 n/a n/a n/a n/a n/a n/a MG490965 MG490962 MG490961 MG490960 n/a n/a n/a n/a n/a n/a n/a CaM

Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected]

MG490888 MG490887 MG490886 MG490885 MG490884 MG490948 MG490899 MG490950 MG490922 MG490916 MG490911 MG490921 MG490893 MG490892 MG490891 MG490935 MG490915 MG490902 MG490953 MG490952 MG490951 MG490937 BenA MG490868 MG490867 MG490866 MG490865 MG490864 n/a n/a n/a n/a n/a n/a MG490875 MG490872 MG490871 MG490870 n/a n/a n/a n/a n/a n/a ITS n/a ) ) ) ) ) Cave wall Cave Cave wall Cave Cave wall Cave Cave wall Cave Cave wall Cave Eptesicus fuscus ( Eptesicus Bat Cave wall Cave Eptesicus fuscus ( Eptesicus Bat Cave wall Cave Cave wall Cave Cave wall Cave Cave wall Cave Cave wall Cave Cave wall Cave Cave wall Cave Harvestman ( Nelima Harvestman elegans ) Cave wall Cave Cave wall Cave Perimyotis ( Perimyotis Bat subflavus Perimyotis ( Perimyotis Bat subflavus Perimyotis ( Perimyotis Bat subflavus Harvestman ( Nelima Harvestman elegans ) Substrate Glebe Mine White Cave White White Cave White Grotte à la Patate Patate à la Grotte Grotte à la Patate Patate à la Grotte Fredericton parking Fredericton garage Grotte à la Patate Patate à la Grotte Fredericton parking Fredericton garage Glebe Mine Berryton Cave Berryton Berryton Cave Berryton Glebe Mine Dorchester Mine Dorchester Dorchester Mine Dorchester Dorchester Mine Dorchester Dorchester Mine Dorchester Berryton Cave Berryton Grotte à la Patate Patate à la Grotte Markhamville Mine Markhamville Markhamville Mine Markhamville Markhamville Mine Markhamville Cave name Cave Dorchester Mine Dorchester Sussex Hillsborough Hillsborough Hillsborough Hillsborough Anticosti Island Anticosti Anticosti Island Anticosti Fredericton Anticosti Island Anticosti Fredericton Sussex Moncton Moncton Sussex Dorchester Dorchester Dorchester Dorchester Moncton Anticosti Island Anticosti Markhamville Markhamville Markhamville Dorchester Location New Brunswick New New Brunswick New New Brunswick New Quebec Quebec New Brunswick New Quebec New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New Quebec New Brunswick New New Brunswick New New Brunswick New New Brunswick New Province SD DPYA MEA DPYA DPYA DPYA DPYA DPYA DPYA SD DPYA SD DPYA DPYA DPYA DPYA SD DPYA DPYA DPYA DPYA DPYA Isolation Isolation medium 16-Apr-2015 21-Apr-2015 21-Apr-2015 07-Jul-2014 07-Jul-2014 16-Apr-2014 07-Jul-2014 16-Apr-2014 16-Apr-2015 30-Apr-2015 30-Apr-2015 16-Apr-2015 31-Mar-2015 31-Mar-2015 31-Mar-2015 18-Mar-2013 30-Apr-2015 07-Jul-2014 04-Apr-2013 04-Apr-2013 04-Apr-2013 Date Date collected 18-Mar-2013 DAOMC 251700, DAOMC KAS 7504, W05202 DAOMC 251699, DAOMC KAS 7503, W05404 DAOMC 251698, DAOMC KAS 7500, W07302 DAOMC 251697, DAOMC KAS 7474, W54102 DAOMC 251696, DAOMC KAS 7473, W54119 DAOMC 252125, DAOMC KAS 7541, 742105 KAS 7468, W71101 DAOMC 252124, DAOMC KAS 7543, 741102 KAS 7509, W02400 KAS 7495, W16200 DAOMC 252123, DAOMC KAS 7488, W20104 DAOMC 252122, DAOMC KAS 7508, W04210 KAS 7462, W88405 DAOMC 252120, DAOMC KAS 7461, W88405 DAOMC 252119, DAOMC KAS 7460, W88411 KAS 7524, H11101 DAOMC 252118, DAOMC KAS 7494, W16200A DAOMC 252117, DAOMC KAS 7475, W54101 KAS 7547, 701106 KAS 7546, 701115 KAS 7545, 702115 KAS 7529, H06108 Strain Fasciculata Brevicompacta Chrysogena Thysanophora Aspergilloides Section (Continued). P. speluncae P. P. spathulatum P. P. rubens P. P. glaucoalbidum P. P. glabrum P. Species Table 1. Table

isolated from a deer mouse (Peromyscus maniculatus) and its dung from the Dorchester Mine in New Brunswick (Table 1). Based on the n/a n/a MN170743 MN170742 MN170741 RPB2 BenA phylogeny (Fig. 1), and in some cases additional ITS and CaM BLAST searches, the remaining strains were identified as Penicillium bialowiezense (n = 10), P. brevistipitatum (n = 6), P. chrysogenum (n = n/a n/a MG490970 MG490967 MG490959 CaM 2), P. concentricum (n = 14), P. consobrinum (n = 2), P. corylophilum (n = 4), P. expansum (n = 9), P. glabrum (n = 3), P. glaucoalbidum (n = 4), P.

rubens (n = 4), P. spathulatum (n = 2), and P. westlingii (n = 2). Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected]

MG490930 MG490894 MG490940 MG490928 MG490889 BenA Phylogeny

A multigene phylogeny was used to show identities of strains isolated during this study (Fig. 1). The alignment contained 175 taxa and was n/a n/a MG490880 MG490877 MG490869 ITS 2 375 bp long (BenA 1–453; CaM 454–1019; RPB2 1020–1823; ITS 1824–2375). The most appropriate substitution model for each partition was: BenA TIM2e+I+G4; CaM TNe+I+G4; RPB2 TNe+R3; ITS TIM2+F+I+G4. Generally, BenA sequences from strains isolated during ) ) ) this study matched well with reference sequences in terms of resolving ) Scoliopteryx Scoliopteryx in a particular clade. However, many of the newly generated sequences represented minor deviations from previously known sequences. In Moth ( libatrix Cave wall Cave Rodent dung Rodent ( Peromyscus maniculatus Peromyscus ( Peromyscus Rodent maniculatus Peromyscus ( Peromyscus Rodent maniculatus Substrate some cases, variation was such that calmodulin was sequenced to make a final identification of a species (e.g.P. consobrinum, P. brevistipitatum). One clade was found to represent a new species in section Fasciculata. To demonstrate the genealogical concordance of the new species in relation to its close relatives, phylogenies of all known species from sectionFasciculata were calculated based on BenA, CaM and RPB2 (Fig. Dorchester Mine Dorchester Dorchester Mine Dorchester Dorchester Mine Dorchester Dorchester Mine Dorchester Dorchester Mine Dorchester Cave name Cave 2). To demonstrate the overall phylogenetic relationship, a concatenated dataset, based on ITS, BenA, CaM and RPB2 was calculated. Alignment metadata is summarised in Suppl. Table S2. As expected, ITS (not shown) lacked sufficient variation to distinguish among species. For example, P. speluncae shared similar Dorchester Dorchester Dorchester Dorchester Dorchester Location ITS sequences with P. cavernicola, P. echinulatum, P. discolor and P. solitum, noting that strains DAOMC 251696 and DAOMC 251697 formed a distinct clade because of an A-T transversion.BenA, CaM and RPB2 distinguished among close relatives much better. The exception was the clade associated with cheese; P. biforme, P. camemberti (1 bp New Brunswick New New Brunswick New New Brunswick New New Brunswick New New Brunswick New Province difference), P. caseifulvum, P. commune and P. palitans had identical CaM sequences, while P. camemberti had 1 bp difference from these species. RPB2 sequences for P. caseifulvum and P. commune were identical (albeit with limited sampling). BenA was not helpful to DPYA SD DPYA SD SD Isolation Isolation medium distinguish between P. camemberti and P. commune, supporting the hypothesis that the former is a domesticated form of the latter (Pitt et al. 1986, Polonelli et al. 1987). Much sequence variation was observed within the clade containing P. speluncae, P. discolor, P. echinulatum 14-Mar-2014 31-Mar-2015 25-Mar-2014 12-Mar-2014 14-Mar-2014 Date Date collected and P. solitum. This resulted in all phylogenies having poor backbone support in both ML and BI, mainly because of the strains identified , T as P. speluncae. Nonetheless, all phylogenies resulted in three distinct clades corresponding with P. discolor, P. solitum and P. echinulatum. CBS 271.97 and CBS 278.97 previously considered typical of P. discolor (Frisvad & Samson 2004, Samson et al. 2004) were phylogenetically T DAOMC 252129, DAOMC KAS 7518, M34108 DAOMC 252128, DAOMC KAS 7463, W77200 DAOMC 252127, DAOMC KAS 7533, D3108 DAOMC 252126, DAOMC KAS 7516, P01202 KAS 7512, P06201 DAOMC 251701 DAOMC Strain resolved distinct from the ex-type CBS 474.84 within the broad concept applied to P. speluncae. Citrina Section Fig. 1. ML tree based on ITS, BenA, CaM & RPB2 showing identities and diversity of Penicillium associated with bats or bat caves. Bootstrap values ≥ 80% are shown above branches while thickened branches indicate 100 % support. Sequences (Continued). obtained from ex-type cultures are indicated by T. Sequences obtained from strains during this study are indicated by blue text, while the new species, P. DAOMC: Culture collection of the National Mycological Collections, Agriculture & Agri-Food Canada, Ottawa, Canada; KAS: Internal working culture collection at DAOMC; Remaining acronyms represent internal isolate numbers from numbers internalisolate represent acronyms Remaining at DAOMC; collection collection working Internal of Collections, Canada;Culture Ottawa, Mycological culture the KAS: Agriculture Canada, & Agri-Food DAOMC: National Malloch. and Dave Vanderwolf Karen of collection the personal P. westlingii P. Table 1. Table Species speluncae, is in bold blue text. The tree was rooted to Talaromyces pinophilus.

P. expansum KAS 7506 P. expansum KAS 7546 P. expansum KAS 7545 P. expansum KAS 7529 P. expansum CBS 325.48T P. expansum CV 2861 P. expansum CV 2860 P. expansum KAS 7510 P. expansum KAS 7547 P. expansum CBS 481.84 P. expansum KAS 7502 P. expansum DAOMC 252116 P. expansum DAOMC 252115 P. expansum CBS 281.97 sect. Penicilliu m Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] P. speluncae DAOMC 251696 P. speluncae DAOMC 251697 P. speluncae DAOMC 251700 98 P. speluncae DAOMC 251698 P. speluncae DAOMC 251699 P. speluncae DAOMC 251701T 95 P. speluncae DAOMC 252127 P. speluncae DAOMC 252126 97 P. discolor CBS 474.84T P. solitum CBS 424.89T P. echinulatum CBS 317.48T P. crustosum CBS 115503T sect. Fasciculata P. osmophilum CBS 462.72T sect. Osmophila P. rubens CBS 319.59 P. rubens CBS 129667T P. rubens CBS 401.92 P. rubens CBS 111216 P. rubens CBS 478.84 P. rubens CBS 132210 P. rubens CBS 349.48 P. rubens CBS 339.52 96 P. rubens KAS 7509 P. rubens DAOMC 252123 P. rubens DAOMC 252124 P. rubens KAS 7495 P. chrysogenum CBS 776.95 P. chrysogenum CBS 132217 P. chrysogenum CBS 282.97 91 P. chrysogenum DAOMC 252107 P. chrysogenum DAOMC 252106 P. chrysogenum CBS 111215 92 P. chrysogenum CBS 906.70 P. chrysogenum CBS 306.48T P. chrysogenum CBS 259.29 P. chrysogenum CBS 109613 P. tardochrysogenum CBS 132200T P. allii-sativi CBS 132074T 87 P. allii-sativi DTO 149A9 sect. Chrysogena P. roqueforti CBS 221.30T sect. Roquefortorum 99 P. concentricum DAOMC 252108 P. concentricum KAS 7486 P. concentricum KAS 7532 P. concentricum KAS 7513 P. concentricum DAOMC 252110 P. concentricum KAS 7535 P. concentricum KAS 7515 P. concentricum DAOMC 252109 P. concentricum KAS 7537 P. concentricum KAS 7536 P. concentricum KAS 7478 P. concentricum CBS 477.75T 98 P. concentricum KAS 7471 P. concentricum CBS 191.88 P. concentricum KAS 7459 98 P. concentricum KAS 7470 P. robsamsonii CBS 140573T P. brevistipitatum DAOMC 252100 P. brevistipitatum DAOMC 252103 92 P. brevistipitatum DAOMC 252101 P. brevistipitatum DAOMC 252104 P. brevistipitatum DAOMC 252105 P. brevistipitatum AS 3.6887T 93 P. brevistipitatum DAOMC 252102 sect. Robsamsonia P. paradoxum CBS 527.65T sect. Paradoxa P. turbatum CBS 383.48T sect. Turbata P. bialowiezense KAS 7465 P. bialowiezense DAOMC 252099 P. bialowiezense DAOMC 252098 P. bialowiezense KAS 7511 P. bialowiezense DAOMC 252097 P. bialowiezense KAS 7523 P. bialowiezense KAS 7517 P. bialowiezense KAS 7480 P. bialowiezense CBS 227.28T P. bialowiezense KAS 7542 P. bialowiezense NRRL 32207 P. bialowiezense KAS 7522 P. bialowiezense NRRL 32205 x2 P. brevicompactum CBS 257.29T 95 P. spathulatum CBS 116975 P. spathulatum DAOMC 252125 P. spathulatum CBS 117192T P. spathulatum CBS 116972 x4 P. spathulatum CBS 116976 P. spathulatum CBS 116977 P. spathulatum KAS 7468 sect. Brevicompacta P. soppii CBS 226.28T sect. Ramosa P. canescens CBS 300.48T sect. Canescentia P. sacculum CBS 231.61T sect. Eladia

P. corylophilum DAOMC 252113 P. corylophilum KAS 7493 P. corylophilum KAS 7489 P. corylophilum DAOMC 252114 99 P. corylophilum CBS 127808 P. corylophilum CBS 312.48T 96 P. consobrinum CBS 139144T 99 P. consobrinum CV 1457 P. consobrinum DAOMC 252111 P. consobrinum DAOMC 252112 sect. Exilicaulis P. glabrum CBS 328.48 P. glabrum CBS 126333 P. glabrum CBS 127704

Editor-in-Chief T Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. P. glabrum CBS 125543 E-mail: [email protected] P. glabrum CBS 129606 P. glabrum CBS 127703 P. glabrum CBS 129784 P. glabrum CBS 126336 P. glabrum CBS 127700 P. glabrum CBS 138165 P. glabrum CBS 138166 P. glabrum CBS 129602 P. glabrum CBS 171.81 P. glabrum CBS 138164 P. glabrum CBS 131040 P. glabrum CBS 115810 P. glabrum DAOMC 252118 P. glabrum KAS 7524 P. glabrum DAOMC 252117 93 T 99 P. bussumense CBS 138160 P. frequentans CBS 105.11T T 92 P. pulvis CBS 138432 P. purpurascens CBS 366.48T P. rudallense CBS 130049T P. armarii CBS 138171T T 97 P. spinulosum CBS 374.48 T sect. Aspergilloides 91 P. thomii CBS 225.81 P. sclerotiorum CBS 287.36T sect. Sclerotiora P. charlesii CBS 304.48T sect. Charlesia P. cinnamopurpureum CBS 429.65T sect. Cinnamopurpurea P. glaucoalbidum DAOMC 252119 P. glaucoalbidum DAOMC 252121 P. glaucoalbidum WCN 1043 P. glaucoalbidum WCN 1016 x4 P. glaucoalbidum WCN 1077 99 P. glaucoalbidum WCN 1246 P. glaucoalbidum WCN 1129 P. glaucoalbidum WCN 1128 P. glaucoalbidum DAOMC 252122 P. glaucoalbidum NBRC 9011 99 P. glaucoalbidum CBS 314.56 P. glaucoalbidum CBS 348.64 P. glaucoalbidum DAOMC 252120 P. glaucoalbidum WCN 1152 x2 P. hennebertii CBS 334.68T 90 P. taxi CBS 206.57T sect. Thysanophora 90 P. cyaneum CBS 315.48T sect. Ramigena P. ochrosalmoneum CBS 489.66T sect. Ochrosalmonea P. lagena CBS 185.65T sect. Torulomyces P. westlingii CBS 127037 P. westlingii DAOMC 252129 P. westlingii DAOMC 252128 T x4 P. westlingii CBS 231.28 P. westlingii CBS 127003 99 P. cosmopolitanum CBS 122406 98 P. cosmopolitanum CBS 126995T T x16 P. citrinum CBS 139.45 sect. Citrina P. gracilentum CBS 599.73T sect. Gracilenta 97 P. javanicum CBS 341.48T sect. Lanata-divaricata P. stolkiae CBS 315.67T sect. Stolkia P. fractum CBS 124.68T sect. Fracta Talaromyces pinophilus CBS 631.66T 0.1

Fig. 1. (Continued).

Extrolites species in Penicillium subgenus Penicillium (Frisvad et al. 2004). Chaetoglobosins are a large class of metabolites biosynthesised As analysed by LC-MS, there were five classes of compounds by a polyketide derived macrocycle fused to a modified produced by P. speluncae under the reported growth conditions: tryptophan amino acid and are produced by Chaetomium cyclopenins, viridicatins, chaetoglobosins, cyclic dipeptides, globosum as well as P. discolor (Frisvad et al. 1997), P. expansum and tetrapeptides. Cyclopenins and viridicatins are derived (Frisvad & Filtenborg 1989) and P. marinum (Frisvad et al. 2004). from a shared biosynthetic pathway (Simonetti et al. 2016) One of the major chaetoglobosins produced by these isolates and are among the most widely distributed extrolites across is a newly described natural product, tetrahydrochaetoglobosin

Fig. 2. ML trees of Penicillium sectionFasciculata , based on concatenated, BenA, CaM and RPB2 alignments, showing the relationship ofP. speluncae within the section. PP and BS values ≥ 0.95/80 are shown above thickened branches (* = 1.00/100; - = <0.95/80). Sequences obtained from ex-type cultures are indicated by T. Strains of the new species characterised based on morphology and extrolites is indicated by bold blue text. Trees were rooted to Penicillium robsamsonia.

P. speluncae CBS 271.97 P. speluncae DAOMC 251696 */* P. speluncae CBS 278.97 P. speluncae DAOMC 251701T */.96 P. speluncae DTO 037C9 P. speluncae DAOMC 252126 */* P. speluncae DTO 046G4 P. speluncae DTO 046I9 P. speluncae DTO 046G5 P. speluncae DAOMC 252127 P. speluncae DAOMC 251698 P. speluncae DAOMC 251700 P. speluncae DAOMC 251699 P. speluncae DAOMC 251697 P. speluncae DAOMC 251696 90/* P. speluncae DAOMC 251698 94/- P. speluncae DAOMC 251697 P. speluncae DAOMC 251699 P. speluncae DTO 046I9 P. speluncae CBS 271.97 98/.98 91/.99 P. speluncae DAOMC 251700 */* P. speluncae DTO 037C9 P. speluncae DAOMC 251701T 85/* P. speluncae CBS 278.97 */* P. speluncae DAOMC 252127 P. speluncae DTO 046G4 P. speluncae DAOMC 252126 P. speluncae DTO 046G5 98/* P. speluncae DTO 037D2 */.97 P. speluncae DTO 037D2 P. speluncae DTO 332H8 P. speluncae DTO 332H8 P. solitum DTO 161H9 P. discolor DTO 046I4

P. solitum DTO 321F7 P. discolor DTO 047A3 Editor-in-Chief 98/.98 87/* Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] P. solitum DTO 247B8 P. discolor DTO 047A2 P. solitum DTO 376D5 P. discolor CBS 474.84T 91/- P. solitum DTO 235G1 91/.96 P. solitum CBS 146.86 P. solitum CBS 147.86 P. solitum DTO 234I5 97/.99 98/- P. solitum CBS 146.86 P. solitum CBS 147.86 */* P. solitum DTO 234I5 P. solitum DTO 235G1 P. solitum DTO 046I6 P. solitum DTO 046I6 P. solitum CBS 424.89T P. solitum CBS 424.89T T */* 98/.95 P. discolor CBS 474.84 P. solitum DTO 161H9 96/* */* P. discolor DTO 047A3 P. solitum DTO 321F7 P. discolor DTO 046I4 P. solitum DTO 376D5 P. discolor DTO 047A2 P. solitum DTO 247B8 T T P. echinulatum CBS 317.48 85/- P. echinulatum CBS 317.48 P. echinulatum DTO 228I4 P. echinulatum DTO 228I4 98/* P. echinulatum CBS 337.59 98/* P. echinulatum CBS 337.59 P. echinulatum CBS 101027 P. echinulatum CBS 101027 P. crustosum CV 0241 P. crustosum CV 0241 */* P. crustosum CV 0251 */* P. crustosum CV 0251 T T P. crustosum CBS 115503 94/* P. crustosum CBS 115503 */* T T */* P. palitans CBS 107.11 96/* P. palitans CBS 107.11 99/- P. palitans DTO 046I5 P. palitans DTO 046I5 P. biforme CBS 297.48T P. biforme CBS 297.48T 99/- T T P. camemberti CBS 299.48 94/* P. camemberti CBS 299.48 */* P. commune CBS 311.48T P. commune CBS 311.48T T T 96/- P. caseifulvum CBS 101134 P. caseifulvum CBS 101134 P. cavernicola DTO 046I3 P. cavernicola DTO 046I3 */* P. cavernicola DTO 046I8 */* P. cavernicola DTO 046I8 P. cavernicola CBS 100540T P. cavernicola CBS 100540T T T 96/* P. aurantiogriseum CBS 249.89 */* P. radicicola CBS 112430 T T 93/- 80/.98 P. cellarum NRRL 66633 P. tulipae CBS 109555 T T 95/* P. freii CBS 476.84 P. albocoremium CBS 472.84 -/* P. neoechinulatum CBS 169.87T P. allii CBS 131.89T T T 99/* -/.99 P. tricolor CBS 635.93 P. hirsutum CBS 135.41 P. viridicatum CBS 390.48T P. hordei CBS 701.68T T T */* 99/* P. cyclopium CBS 144.45 95/* P. freii CBS 476.84 x2 P. polonicum CBS 222.28T 87/.98 P. neoechinulatum CBS 169.87T T 87/* T 99/* P. melanoconidium CBS 115506 P. viridicatum CBS 390.48 T T */* P. nordicum ATCC 44219 P. aurantiogriseum CBS 249.89 Concat */* T T x2 P. verrucosum CBS 603.74 P. cellarum NRRL 66633 P. thymicola CBS 111225T P. tricolor CBS 635.93T P. venetum IBT 10661T */* P. melanoconidium CBS 115506T T T */* P. albocoremium CBS 472.84 BenA 96/* P. cyclopium CBS 144.45 88/.98 P. allii CBS 131.89T x2 P. polonicum CBS 222.28T 98/.97 T T P. hirsutum CBS 135.41 */* P. nordicum ATCC 44219 P. hordei CBS 701.68T */* P. verrucosum CBS 603.74T T x2 T 99/* P. radicicola CBS 112430 P. thymicola CBS 111225 P. tulipae CBS 109555T P. venetum IBT 10661T 86/- P. gladioli CBS 332.48T P. gladioli CBS 332.48T P. robsamsonii CBS 140573T P. robsamsonii CBS 140573T 0.008 0.02 P. speluncae DAOMC 251696 P. solitum DTO 161H9 P. speluncae DTO 046I9 P. solitum DTO 247B8 96/.97 P. speluncae DAOMC 251700 */.97 P. solitum DTO 321F7 P. speluncae DAOMC 251697 P. solitum DTO 235G1 P. speluncae DAOMC 251699 */* P. solitum CBS 424.89T P. speluncae DAOMC 251698 P. solitum DTO 234I5 96/* P. speluncae CBS 271.97 P. speluncae DTO 046G4 P. solitum DTO 046I6 95/.96 P. speluncae DTO 037C9 P. discolor DTO 046I4 91/* P. speluncae CBS 278.97 P. discolor DTO 047A2 80/- P. speluncae DTO 046G5 */* P. discolor CBS 474.84T P. speluncae DAOMC 251701T P. discolor DTO 047A3 */* P. speluncae DAOMC 252127 P. speluncae DAOMC 251698 89/* P. speluncae DAOMC 252126 85/- P. speluncae DTO 037D2 P. speluncae DTO 037D2 P. speluncae DTO 332H8 P. speluncae DTO 332H8 T P. speluncae DTO 037C9 */* P. echinulatum CBS 317.48 98/* P. echinulatum DTO 228I4 */.99 P. speluncae DTO 046G5 P. solitum CBS 424.89T P. speluncae DTO 046G4 95/* P. solitum DTO 234I5 P. speluncae DAOMC 251701T P. solitum DTO 235G1 */* P. speluncae DAOMC 252127 81/- P. solitum DTO 046I6 P. speluncae DAOMC 252126 P. solitum DTO 161H9 P. speluncae DAOMC 251696 */.97 P. solitum DTO 321F7 P. speluncae DTO 046I9 P. solitum DTO 376D5 P. speluncae DAOMC 251700 P. solitum DTO 247B8 T P. speluncae DAOMC 251699 */.98 P. discolor CBS 474.84 */* P. discolor DTO 047A3 P. speluncae DAOMC 251697 T 88/* P. discolor DTO 046I4 */* P. echinulatum CBS 317.48 P. discolor DTO 047A2 P. echinulatum DTO 228I4 P. biforme CBS 297.48T P. caseifulvum CBS 101134T T P. commune CBS 311.48 P. commune CBS 311.48T T P. caseifulvum CBS 101134 T 98/* P. biforme CBS 297.48 88/- P. palitans CBS 107.11T P. palitans CBS 107.11T 98/* P. camemberti CBS 299.48T */* 95/.98 P. palitans DTO 046I5 P. palitans DTO 046I5 T P. cavernicola CBS 100540T P. cavernicola CBS 100540 */* P. cavernicola DTO 046I8 */* P. cavernicola DTO 046I8 P. cavernicola DTO 046I3 P. cavernicola DTO 046I3 P. crustosum CV 0241 P. aurantiogriseum CBS 249.89T */* P. crustosum CV 0251 P. cyclopium CBS 144.45T T P. crustosum CBS 115503 P. polonicum CBS 222.28T T P. aurantiogriseum CBS 249.89 T 92/* P. freii CBS 476.84 90/.96 P. neoechinulatum CBS 169.87T 91/.98 P. neoechinulatum CBS 169.87T 99/* P. freii CBS 476.84T T P. viridicatum CBS 390.48T P. cellarum NRRL 66633 T T 99/* P. cyclopium CBS 144.45 95/* */* P. tricolor CBS 635.93 P. polonicum CBS 222.28T P. viridicatum CBS 390.48T */* T T x2 P. tricolor CBS 635.93 P. melanoconidium CBS 115506 T T P. melanoconidium CBS 115506 99/* P. nordicum ATCC 44219 T 90/- */* P. nordicum ATCC 44219 */* P. verrucosum CBS 603.74T 93/.99 */* T x2 P. verrucosum CBS 603.74 P. thymicola CBS 111225T CaM P. thymicola CBS 111225T 87/- x2 P. venetum IBT 10661T P. venetum IBT 10661T P. allii CBS 131.89T P. gladioli CBS 332.48T RPB2 x2 T T 97/* P. hirsutum CBS 135.41 */* P. albocoremium CBS 472.84 -/.98 T P. allii CBS 131.89T P. albocoremium CBS 472.84 P. hordei CBS 701.68T P. hordei CBS 701.68T P. hirsutum CBS 135.41T P. gladioli CBS 332.48T P. robsamsonii CBS 140573T P. robsamsonii CBS 140573T 0.02 0.006

Table 2. Extrolites produced by Penicillium speluncae. Extrolite name Formula m/z [M+H]+ RT % strains producing

cyclopenin C17H14N2O3 295.1076 3.03 86 %

cyclopenol C17H14N2O4 311.1025 2.69 86 %

cyclopeptine C17H16N2O2 281.1285 3.11 100 %

dehydrocyclopeptine C17H14N2O2 279.1130 3.17 86 %

Editorviridicatin-in-Chief C H NO 238.0865 3.36 100 % Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. 15 11 2 E-mail: [email protected]

viridicatol C15H11NO3 254.0812 2.95 100 %

chaetoglobosin F C32H38N2O5 531.2852 3.54 100 %

tetrahydrochaetoglobosin A C32H40N2O5 533.3009 3.27 100 %

chaetoglobosin A C32H38N2O5 531.2852 3.63 100 %

chaetoglobosin C C32H36N2O5 529.2698 3.78 57 %

prochaetoglobosin I C32H38N2O2 483.3005 4.41 100 %

cyclo(VP) C10H16N2O2 197.1286 2.32 100 %

cyclo(LP) C11H18N2O2 211.1441 2.50 100 %

cyclo(IP) C11H18N2O2 211.1443 2.55 100 %

cyclo(FP) C14H16N2O2 245.1285 2.62 100 %

fungisporin C28H36N4O4 493.2809 3.77 100 %

cyclo(Phe-Val-Phe-Val) C28H36N4O4 493.2809 3.55 100 %

Val-Phe-Val-Phe C28H38N4O5 511.2919 2.87 100 %

cyclo(Phe-Phe-Val-Ile) C29H38N4O4 507.2360 4.00 100 %

Phe-Val-Ile-Phe C29H4N4O5 525.3074 2.96 100 %

cyclo(Phe-Tyr-Val-Val) C28H36N4O5 509.2761 3.42 100 %

Phe-Val-Val-Tyr C28H38N4O6 527.2866 2.68 100 %

Phe-Ile-Val-Tyr C29H40N4O6 541.3022 2.74 100 %

cyclo(Phe-Trp-Val-Val) C30H37N5O4 532.2914 3.68 100 %

Phe-Val-Val-Trp C30H39N5O5 550.3024 2.89 100 %

cyclo(Tyr-Trp-Val-Val) C30H37N5O5 548.2866 3.39 100 %

Tyr-Val-Val-Trp C30H39N5O6 566.2973 2.68 100 %

cyclo(Trp-Trp-Val-Val) C32H38N6O4 571.3025 3.62 100 %

(Walsh et al. 2018). In addition to these extrolites, a series of Colony characters: CYA 25 °C, 7 d: Colonies moderately deep, cyclic and linear tetrapeptides, composed of combinations sulcate; margins low, narrow to wide, entire; mycelia white; of valine, phenylalanine, leucine/isoleucine, tyrosine and texture velutinous to fasciculate; sporulation moderately dense, tryptophan were consistently detected across all tested strains conidia en masse greyish green (25E7), dull green (26D3–4); (Table 2). These peptides could be putatively characterized by soluble pigments absent; exudates absent; reverse greyish yellow de novo sequencing and are likely similar to the series of linear (4B6), greyish orange (5B4), yellowish white (4A2). MEA 25 °C, and cyclic tetra peptides previously identified in cultures of P. 7 d: Colonies low, plain; margins low, narrow, entire; mycelia chrysogenum, including fungisporin; cyclo(D-Phe-L-Phe-D-Val-L- white; texture velutinous to fasciculate; sporulation moderately Val) (Ali et al. 2014). dense, conidia en masse greyish green (25E5–26E5); soluble pigments forming a yellow halo surrounding colony; exudates Taxonomy absent; reverse greyish yellow (4B6), light yellow (3A5), greyish green (29C6). YES 25 °C, 7 d: Colonies low, sulcate; margins low, Penicillium speluncae Visagie & Yilmaz, sp. nov. MycoBank wide, entire; mycelia white; texture velutinous to fasciculate; MB828614. Figs 3, 4. sporulation moderately dense, conidia en masse greyish green (25C5–D5), dull green (26D3); soluble pigments absent; Etymology: Latin, speluncae, meaning from a cave. exudates absent; reverse orange yellow to orange (4A7–6A7). OA 25°C, 7 days: Colonies moderately deep, plain; margins low, ITS barcode: MG490869. Alternative identification markers: narrow, entire; mycelia white; texture fasciculate; sporulation BenA = MG490889, CaM = MG490959, RPB2 = MN170741. dense, conidia en masse greyish to dark green (25E7–F7); soluble pigments forming a yellowish halo surrounding colony; Colony diam, 7 d (at 25 °C; in mm): CYA 30–35; CYA 15 °C (12–) exudates absent. CREA 25 °C, 7 d: Growth strong, acid produced, 17–22(–25); CYA 30 °C 12–21(–26); CYA 37°C no growth; CYAS colony reverse orange. 29–32(–37); MEAbl 25–30; YES 45–47; OA 25–27; CREA 25–26.

30 °C. Penicillium solitum has several synonyms examined before (Frisvad & Samson 2004), and showed no growth on CYA at 30 °C. Of the extrolites produced in this clade, chaetoglobosins are produced by only P. speluncae and P. discolor, territrems only by P. echinulatum, compactin only by P. solitum, while penitrem and roquefortine are produced by P. crustosum and other distantly related Penicillia. Penicillium speluncae produces cytoglobosin and prochaetoglobosin, which are absent in P. discolor, while

Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] palitantin was not detected for the new species (comparisons summarised in Table 3; data from Frisvad et al. 2004).

Additional materials examined: Canada, New Brunswick, Dorchester, Dorchester copper mine, from rodent fur (Peromyscus maniculatus), 12 Mar. 2014, K. Vanderwolf (culture DAOMC 252126 = KAS 7516 = P01202); Dorchester mine, from rodent dung (Peromyscus maniculatus), 25 Mar. 2014, K. Vanderwolf (culture DAOMC 252127 = KAS 7533 = D3108); Hillsborough, White Cave (gypsum), from cave wall, 21 Apr. 2015, K. Vanderwolf (cultures DAOMC 251698 = KAS 7500 = W07302, DAOMC 251699 = KAS 7503 = W05404); Sussex, Glebe mine (limestone), from cave wall, 16 Apr. 2015, K. Vanderwolf (culture DAOMC 251700 = KAS 7504 = W05202); Quebec, Anticosti Island, Grotte à la Patate (limestone), from cave wall, K. Vanderwolf (cultures DAOMC 251696 = KAS 7473 = W54119, DAOMC 251697 = KAS 7474 = W54102).

DISCUSSION

This study focused on Penicillium species isolated from six Pd- positive bat hibernacula in New Brunswick, Canada and one Pd- negative bat hibernaculum in Quebec, Canada. The isolates were collected from arthropods, bats, rodents and their dung (i.e. the Fig. 3. Line drawing of Penicillium speluncae. Scale bar = 10 µm. deer mouse Peromyscus maniculatus), cave walls, and one dead bat found in a parking garage. During the survey, hundreds of fungal strains were obtained and Penicillium represented one of Micromorphology: Conidiophores terverticillate, minor the most frequently isolated genera, probably because of the proportion bi- and quarterverticillate; stipes rough, 180–600 ability of these species to grow at low temperatures (Frisvad × 3.5–4.5 μm; branches 15–29 μm; metulae (2–)3–4, 10–16 × & Samson 2004, Vanderwolf et al. 2016). Previous studies had 3–4.5 μm; phialides ampulliform, 4–6 per metula, 8.5–11 × 3–4 similar results, but the diversity of Penicillium in caves is even μm (9.9±0.7 × 3.3±0.2); average length metula/phialide 1.3; greater than previously reported and several of these species conidia smooth, broadly ellipsoidal, 3–4 × 2.5–3.5 μm (3.6±0.2 × have never been reported from caves or mines, including P. 3±0.2), average width/length = 0.82, n = 72. bialowiezense, P. brevistipitatum, P. consobrinum, P. rubens, P. spathulatum and P. westlingii (Nováková 2009, 2018, Vanderwolf Extrolites: cyclopenins, viridicatins, chaetoglobosins, fungisporin, et al. 2013b, 2016, Anelli et al. 2018). Other species identified cyclic and linear tetrapeptides (See Table 2). were P. chrysogenum, P. concentricum, P. corylophilum, P. expansum, P. glabrum and P. glaucoalbidum. One species could Typus: Canada, New Brunswick, Dorchester, Dorchester mine, not be identified based on DNA reference sequences and further from a swab of deer mouse fur (live Peromyscus maniculatus), study showed it to represent a new species, described above as 14 Mar. 2014, K. Vanderwolf (holotype, DAOM 745788 (dried P. speluncae, classified in section Fasciculata. culture); ex-type strain DAOMC 251701 = KAS 7512 = P06201). Phylogenetic analyses of sect. Fasciculata revealed a large degree of genetic variation within P. speluncae. Single gene Notes: Penicillium speluncae is resolved in a clade with P. discolor, trees based on BenA, CaM and RPB2 resulted in inconsistent P. echinulatum and P. solitum (Fig. 2). Of these, P. speluncae groupings and poor backbone support for this clade meaning showed relatively good growth on CYA at 30 °C, compared to that genealogical concordance could not be applied to delimit poor growth observed for the others. Both P. discolor and P. segregate species. The basal branch encompassing this clade echinulatum produce roughened globose to subglobose conidia, was relatively well supported in the concatenated tree. DAOMC in contrast to the new species’ smooth, broadly ellipsoidal strains were characterized based on morphology and extrolite conidia. Penicillium solitum is morphologically most similar to data, with very few differences noted. For example, colony the new species. Both species have smooth conidia and produce growth rates varied on CYA at 30 °C and CYAS, but a similar a striking yellow orange reverse on YES. However, P. speluncae variation was previously observed inP. solitum (Frisvad & Samson produces broadly ellipsoidal conidia (globose to subglobose 2004). Extrolite data also distinguish among this group of species. in P. solitum), grows faster on YES compared to P. solitum (45– Chaetoglobosins are produced only by P. speluncae and P. discolor, 47 mm vs 25–39 mm) and has the ability to grow on CYA at while the former produces cytoglobosin and prochaetoglobosin,

Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected]

which are absent in P. discolor. Chaetomium globosum is the - - + + Palitantin + best-known producer of chaetoglobosins, including the major + - - - chaetoglobosins A, C, and F, also shown here to be produced by Prochaetoglobosin - P. speluncae. A distinguishing feature between chaetoglobosin + - - - production by C. globosum and P. speluncae is a newly described Cytoglobosin - natural product, tetrahydrochaetoglobosin A (Walsh et al. 2019), - + - - Roquefortine - which was not observed in C. globosum. Our data hint that P. - + - - speluncae may be a species complex with so far cryptic species Penitrem -

Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] that may be resolved with additional data. Considering the - - - - available data, we conservatively propose the name P. speluncae Compactin + for this clade. In principle, an analogous situation occurred Chaetoglobosins + - + - - with P. glabrum (sect Aspergilloides). This complex was studied several times morphologically but a satisfactory conclusion was never found (Pitt et al. 1990). Houbraken et al. (2014) provided an extensive phylogenetic analysis and distinguished between P. glabrum and P. frequentans using a concatenated phylogeny of BenA, CaM and RPB2, even though these species had poor backbone support in the single gene trees for BenA and CaM and could not be distinguished. Even though we adopt a consilient species concept for Penicillium, there is often bias towards DNA sequences for making a species identification or deciding whether strains are new or not. This situation is a direct consequence of the accepted Yes reverse Yes orange to yellow Orange Strongly yellow Strongly species list and associated ex-type reference sequences published with age deep red turning into Orange yellow yellow to orange to yellow by Visagie et al. (2014b) and resulted in a generally aggressive approach to describing new species or reinstating old names. Many of the resulting taxa are often based on a single strain. This in turn complicates sequence-based identifications because the reference data do not encapsulate infraspecies variation. We thus encourage a more holistic approach to introducing new species, noting that singleton species will always be a part of our science. An example is P. brevistipitatum, which before this study was known only from ex-type sequences. BenA sequences obtained from our strains differed at several nucleotide positions YES soluble pigment YES None Pale brown or none brown Pale Brilliant red diffusible colour diffusible red Brilliant on YES None None and only after CaM was sequenced could we identify strains as this species. The additional reference sequences generated here will thus aid future identifications of P. brevistipitatum. Several new genotypes were also discovered for P. bialowiezense, P. consobrinum, P. glabrum, P. glaucoalbidum and P. spathulatum (Fig. 1). Several strains were identified as P. glaucoalbidum (≡ Thysanophora glaucoalbida). Although this species is often encountered as an endophyte of conifer needles, the name is not currently accepted because no type material is available (Visagie et al. 2014b). Lectotypification is complicated by the large degree of variation observed in available sequences CYA texture CYA fasciculate to Velutinous Velutinous to weakly fasciculate, fasciculate, weakly to Velutinous crustose becoming Velutinous to fasciculate to Velutinous Velutinous to weakly fasciculate weakly to Velutinous (Iwamoto et al. 2005); this will be the focus of a future study. Velutinous

ACKNOWLEDGEMENTS

We acknowledge DNA sequencing support from Lisa James of the Molecular Technologies Laboratory of ORDC, AAFC, Ottawa, and research support from AAFC project J-1848. We also acknowledge Martin Meijer for sequencing support provided at the Westerdijk Fungal Biodiversity Institute. This manuscript was written while the first author worked at ORDC, and was subsequently first submitted while he Conidia ellipsoidal Smooth, broadly Smooth, globose to subglobose Smooth, globose to Rough, globose to subglobose globose to Rough, Rough, globose to subglobose globose to Rough, Smooth to finely rough, globose finely Smooth to subglobose to worked at the Agricultural Research Council, South Africa. . speluncae to Penicillium related of species closely features Distinguishing Fig. 4. Penicillium speluncae. A. Colonies, from left to right, top row: CYA, MEA, YES, OA; bottom row: reverse on CYA, MEA, YES, CREA. B–F. Table 3. Table speluncae P. P. crustosum P. P. discolor P. P. echinulatum P. Conidiophores. G. Conidia. Scale bars = 10 µm. solitum P.

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&lt;I&gt;Penicillium&lt;/I&gt; Diversity in Canadian Bat

<I>Penicillium</I> Diversity in Canadian Bat