Skip to main content
PMC home page
Fungal Systematics and Evolution logoLink to Fungal Systematics and Evolution
. 2019 Aug 9;5:1–15. doi: 10.3114/fuse.2020.05.01

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

CM Visagie 1,*, N Yilmaz 1, K Vanderwolf 2, JB Renaud 3, MW Sumarah 3, J Houbraken 4, R Assebgui 5, KA Seifert 5, D Malloch 2
PMCID: PMC7250010  PMID: 32467912

Abstract

Penicillium species were commonly isolated during a fungal survey of bat hibernacula in New Brunswick and Quebec, Canada. Strains were isolated from arthropods, bats, rodents (i.e. the deer mouse Peromyscus maniculatus), their dung, and cave walls. Hundreds of fungal strains were recovered, of which Penicillium represented a major component of the community. Penicillium strains were grouped by colony characters on Blakeslee’s malt extract agar. DNA sequencing of the secondary identification marker, beta-tubulin, was done for representative strains from each group. In some cases, ITS and calmodulin were sequenced to confirm identifications. In total, 13 species were identified, while eight strains consistently resolved into a unique clade with P. discolor, P. echinulatum and P. solitum as its closest relatives. Penicillium speluncae is described using macroand micromorphological characters, multigene phylogenies (including ITS, beta-tubulin, calmodulin and RNA polymerase II second largest subunit) and extrolite profiles. Major extrolites produced by the new species include cyclopenins, viridicatins, chaetoglobosins, and a microheterogenous series of cyclic and linear tetrapeptides.

Keywords: Thysanophora, sect. Fasciculata, Genealogical Concordance Phylogenetic Species Recognition (GCPSR) concept, new taxon, Pseudogymnoascus destructans (Pd), secondary metabolites

INTRODUCTION

The study of fungi associated with bats and their habitats has become important after the spread of White-nose Syndrome (WNS) caused by Pseudogymnoascus destructans (Pd), resulted in an ongoing rapid decline of bat populations in North America. Much effort has focused on populations of Pd from positive caves. White-nose Syndrome is named for characteristic white growth caused by P. destructans, which was previously known as Geomyces destructans (Gargas et al. 2009, Minnis & Lindner 2013). Characterization of fungal populations and identification of other fungal species may reveal possible antagonists to Pd (Micalizzi et al. 2017).

White-nose Syndrome was first reported in New York in 2006 (Blehert et al. 2009), while the first report from Canada was from Ontario in 2010. In both cases, it led to mass mortality of the hibernating bat populations (McAlpine et al. 2012). The disease only occurs while bats hibernate. Pseudogymnoascus destructans cannot grow at temperatures above ± 20 °C (Gargas et al. 2009), and it is thought that the cool caves and mines inhabited by bats during hibernation serve as environmental reservoirs of Pd (Lorch et al. 2013, Reynolds et al. 2015). The presence of Pd in bat populations was confirmed in many countries in Europe and Asia but no significant mortality was observed, despite the fact that some European bats have been found with clinical WNS (Wibbelt et al. 2010, Puechmaille et al. 2011). Why bats remain healthy in these areas is unclear.

The study of fungal diversity is important to determine the true impact of a potential invasive species such as Pd on fungal community structure among bats and hibernacula (Johnson et al. 2013). Understudied environments such as caves are rich sources of undescribed microbial species. Many new fungi have recently been described from underground environments as more studies are conducted, although it is still unknown whether obligate troglobiotic fungi exist (Zhang et al. 2017). Previous studies commonly reported the isolation of Cladosporium, Fusarium, Mortierella, and Penicillium species from bat wings, caves and mines (Johnson et al. 2013, Vanderwolf et al. 2013a, b). Penicillium is one of the most common genera isolated from caves on multiple substrates, particularly sediment and air, although no new Penicillium species have been described from caves apart from P. cavernicola, which has also been found outside of caves on dairy products (Frisvad & Samson 2004, Vanderwolf et al. 2013a, b), and P. gravinicasei recently described from a cave in Italy from ripening Apulian cave cheeses (Anelli et al. 2018). Vanderwolf et al. (2016) studied the fungi associated with over-wintering arthropods in Pd positive hibernacula in Canada. They isolated 87 fungal taxa from four arthropod genera. In the current study, we report Penicillium isolated from these arthropods, but also include strains isolated from various other substrates associated with caves and/or bats. The aims of this study were (1) to determine the Penicillium species diversity in bat caves and hibernacula in New Brunswick and Quebec, and (2) formally describe the new species that was isolated during the survey.

MATERIALS AND METHODS

Strains, sampling and isolations

Strains were isolated from arthropods, bats, rodents, rodent dung, and walls of bat hibernacula in New Brunswick (Berryton Cave, Dallings Cave, Dorchester Mine, Glebe Mine, Markhamville Mine, White Cave) and Quebec (Grotte à la Patate), Canada (Vanderwolf et al. 2013b, 2016, 2017). Fungi were also isolated from a dead big brown bat that was found in a parking garage in Fredericton, New Brunswick. Isolation media included dextrose-peptone yeast extract agar (DPYA), sabouraud agar (SD) or malt extract agar (MEA), with plates incubated at 7 °C. Representative strains for each species found were submitted to the Canadian Collection of Fungal Cultures (DAOMC) and the holotype specimen of the new species deposited in the Canadian National Mycological Herbarium (DAOM). Strains isolated during this study are summarized in Table 1.

Table 1.

Species isolated from Canadian bat caves.

Species Section Strain Date collected Isolation medium Province Location Cave name Substrate ITS BenA CaM RPB2
P. biolowiezense Brevicompacta KAS 7465, W7430A 31-Mar~2015 MEA New Brunswick Dorchester Dorchester Mine Cave wall n/a MG490896 n/a n/a
DAOMC 252097, KAS 7466, W72102 07-Jul-2014 DPYA Quebec Anticosti Island Grotte à la Patate Cave wall n/a MG490897 n/a n/a
DAOMC 252098, KAS 7476, W29304 30-Apr-2015 MEA New Brunswick Moncton Berryton Cave Cave wall n/a MG490903 n/a n/a
KAS 7480, W24103 30-Apr-2015 DPYA New Brunswick Moncton Berryton Cave Cave wall n/a MG490906 n/a n/a
KAS 7511, S11101 18-Mar-2013 DPYA New Brunswick Dorchester Dorchester Mine Spider (Meta ovalis) n/a MG490924 n/a n/a
KAS 7517, M50103 ll-Apr-2014 DPYA New Brunswick Sussex Glebe Mine Gnat (Exechiopsis sp) n/a MG490929 n/a n/a
KAS 7522, H27101 ll-Apr-2014 DPYA New Brunswick Sussex Glebe Mine Harvestman (Nelima elegans) n/a MG490933 n/a n/a
KAS 7523, H26208 ll-Apr-2014 SD New Brunswick Sussex Glebe Mine Harvestman (Nelima elegans) n/a MG490934 n/a n/a
DAOMC 252099, KAS 7525, H09108 18-Mar-2013 DPYA New Brunswick Dorchester Dorchester Mine Harvestman (Nelima elegans) n/a MG490936 n/a n/a
KAS 7542, 742102 16-Apr-2014 DPYA New Brunswick Fredericton Fredericton parking garage Bat (Eptesicus fuscus) n/a MG490949 n/a n/a
P. brevistipitotum Robsamsonia DAOMC 252100, KAS 7514, P06101 14-Mar-2014 DPYA New Brunswick Dorchester Dorchester Mine Rodent (Peromyscus maniculotus) MG490876 MG490926 MG490966 n/a
DAOMC 252101, KAS 7520, M26108 16-Apr-2013 DPYA New Brunswick Sussex Dal lings Cave Moth (Scoliopteryx libatrix) MG490878 MG490932 MG490968 n/a
DAOMC 252102, KAS 7531, D3303 25-Mar-2014 DPYA New Brunswick Dorchester Dorchester Mine Rodent dung (Peromyscus maniculotus) MG490879 MG490938 MG490969 n/a
DAOMC 252103, KAS 7534, D2203 21-Mar-2014 DPYA New Brunswick Dorchester Dorchester Mine Rodent dung (Peromyscus maniculotus) MG490881 MG490941 MG490971 n/a
DAOMC 252104, KAS 7538, D1007A 21-Mar-2014 DPYA New Brunswick Dorchester Dorchester Mine Rodent dung (Peromyscus maniculotus) MG490882 MG490945 MG490972 n/a
DAOMC 252105, KAS 7539, D1007 21-Mar-2014 DPYA New Brunswick Dorchester Dorchester Mine Rodent dung (Peromyscus maniculotus) MG490883 MG490946 MG490973 n/a
P. chrysogenum Chrysogena DAOMC 252106, KAS 7505, W05100 21-Apr-2015 DPYA New Brunswick Hillsborough White Cave Cave wall n/a MG490919 n/a n/a
DAOMC 252107, KAS 7540, 742110 16-Apr-2014 DPYA New Brunswick Fredericton Fredericton parking garage Bat (Eptesicus fuscus) n/a MG490947 n/a n/a
P. concentricum Robsamsonia KAS 7459, W98105 16-Apr-2015 DPYA New Brunswick Sussex Glebe Mine Cave wall n/a MG490890 n/a n/a
DAOMC 252108, KAS 7467, W72101 07-Jul-2014 DPYA Quebec Anticosti Island Grotte à la Patate Cave wall n/a MG490898 n/a n/a
KAS 7470, W59104 07-Jul-2014 DPYA Quebec Anticosti Island Grotte à la Patate Cave wall n/a MG490900 n/a n/a
KAS 7471, W59104 07-Jul-2014 DPYA Quebec Anticosti Island Grotte à la Patate Cave wall n/a MG490901 n/a n/a
KAS 7478, W29203 30-Apr-2015 SD New Brunswick Moncton Berryton Cave Cave wall n/a MG490904 n/a n/a
DAOMC 252109, KAS 7479, W24103A 30-Apr-2015 DPYA New Brunswick Moncton Berryton Cave Cave wall n/a MG490905 n/a n/a
DAOMC 252110, KAS 7483, W22102A 30-Apr-2015 DPYA New Brunswick Moncton Berryton Cave Cave wall n/a MG490908 n/a n/a
KAS 7486, W20408 30-Apr-2015 DPYA New Brunswick Moncton Berryton Cave Cave wall n/a MG490910 n/a n/a
KAS 7513, P06102 14-Mar-2014 DPYA New Brunswick Dorchester Dorchester Mine Rodent (Peromyscus maniculotus) n/a MG490925 n/a n/a
KAS 7515, P05201 14-Mar-2014 SD New Brunswick Dorchester Dorchester Mine Rodent (Peromyscus maniculotus) n/a MG490927 n/a n/a
KAS 7532, D3301 25-Mar-2014 DPYA New Brunswick Dorchester Dorchester Mine Rodent dung (Peromyscus maniculotus) n/a MG490939 n/a n/a
KAS 7535, D2111 21-Mar-2014 DPYA New Brunswick Dorchester Dorchester Mine Rodent dung (Peromyscus maniculotus) n/a MG490942 n/a n/a
KAS 7536, D1204 21-Mar-2014 DPYA New Brunswick Dorchester Dorchester Mine Rodent dung (Peromyscus maniculotus) n/a MG490943 n/a n/a
KAS 7537, D1106 21-Mar-2014 DPYA New Brunswick Dorchester Dorchester Mine Rodent dung (Peromyscus maniculotus) n/a MG490944 n/a n/a
P. consobrinum Exilicoulis DAOMC 252111, KAS 7464, W76401 31-Mar-2015 DPYA New Brunswick Dorchester Dorchester Mine Cave wall MG490873 MG490895 MG490963 n/a
DAOMC 252112, KAS 7491, W19102 30-Apr-2015 DPYA New Brunswick Moncton Berryton Cave Cave wall MG490874 MG490913 MG490964 n/a
P. corylophilum Exilicoulis DAOMC 252113, KAS 7481, W24100 30-Apr-2015 DPYA New Brunswick Moncton Berryton Cave Cave wall n/a MG490907 n/a n/a
DAOMC 252114, KAS 7484, W22102 30-Apr-2015 DPYA New Brunswick Moncton Berryton Cave Cave wall n/a MG490909 n/a n/a
KAS 7489, W20103 30-Apr-2015 DPYA New Brunswick Moncton Berryton Cave Cave wall n/a MG490912 n/a n/a
KAS 7493, W16200B 30-Apr-2015 SD New Brunswick Moncton Berryton Cave Cave wall n/a MG490914 n/a n/a
P. exponsum Penicillium DAOMC 252115, KAS 7501, W07104 21-Apr-2015 DPYA New Brunswick Hillsborough White Cave Cave wall n/a MG490917 n/a n/a
KAS 7502, W05406 21-Apr-2015 DPYA New Brunswick Hillsborough White Cave Cave wall n/a MG490918 n/a n/a
KAS 7506, W04407 21-Apr-2015 DPYA New Brunswick Hillsborough White Cave Cave wall n/a MG490920 n/a n/a
KAS 7510, W00200 21-Apr-2015 SD New Brunswick Hillsborough White Cave Cave wall n/a MG490923 n/a n/a
DAOMC 252116, KAS 7519, M26109 16-Apr-2013 DPYA New Brunswick Sussex Dallings Cave Moth (Scoliopteryx libatrix) n/a MG490931 n/a n/a
KAS 7529, H06108 18-Mar-2013 DPYA New Brunswick Dorchester Dorchester Mine Harvestman (Nelima elegans) n/a MG490937 n/a n/a
KAS 7545, 702115 04-Apr-2013 DPYA New Brunswick Markhamville Markhamville Mine Bat (Perimyotis subflavus) n/a MG490951 n/a n/a
KAS 7546, 701115 04-Apr-2013 DPYA New Brunswick Markhamville Markhamville Mine Bat (Perimyotis subflavus) n/a MG490952 n/a n/a
KAS 7547, 701106 04-Apr-2013 DPYA New Brunswick Markhamville Markhamville Mine Bat (Perimyotis subflavus) n/a MG490953 n/a n/a
P. globrum Aspergilloides DAOMC 252117, KAS 7475, W54101 07-Jul-2014 DPYA Quebec Anticosti Island Grotte à la Patate Cave wall n/a MG490902 n/a n/a
DAOMC 252118, KAS 7494, W16200A 30-Apr-2015 SD New Brunswick Moncton Berryton Cave Cave wall n/a MG490915 n/a n/a
KAS 7524, H11101 18-Mar-2013 DPYA New Brunswick Dorchester Dorchester Mine Harvestman (Nelima elegans) n/a MG490935 n/a n/a
P. gloucoolbidum Thysonophoro DAOMC 252119, KAS 7460, W88411 31-Mar-2015 DPYA New Brunswick Dorchester Dorchester Mine Cave wall MG490870 MG490891 MG490960 n/a
DAOMC 252120, KAS 7461, W88405 31-Mar-2015 DPYA New Brunswick Dorchester Dorchester Mine Cave wall MG490871 MG490892 MG490961 n/a
KAS 7462, W88405 31-Mar-2015 DPYA New Brunswick Dorchester Dorchester Mine Cave wall MG490872 MG490893 MG490962 n/a
DAOMC 252122, KAS 7508, W04210 16-Apr-2015 SD New Brunswick Sussex Glebe Mine Cave wall MG490875 MG490921 MG490965 n/a
P. rubens Chrysogeno DAOMC 252123, KAS 7488, W20104 30-Apr-2015 DPYA New Brunswick Moncton Berryton Cave Cave wall n/a MG490911 n/a n/a
KAS 7495, W16200 30-Apr-2015 SD New Brunswick Moncton Berryton Cave Cave wall n/a MG490916 n/a n/a
KAS 7509, W02400 16-Apr-2015 DPYA New Brunswick Sussex Glebe Mine Cave wall n/a MG490922 n/a n/a
DAOMC 252124, KAS 7543, 741102 16-Apr-2014 DPYA New Brunswick Fredericton Fredericton parking garage Bat (Eptesicus fuscus) n/a MG490950 n/a n/a
P. spothulotum Brevicompacta KAS 7468, W71101 07-Jul-2014 DPYA Quebec Anticosti Island Grotte à la Patate Cave wall n/a MG490899 n/a n/a
DAOMC 252125, KAS 7541, 742105 16-Apr-2014 DPYA New Brunswick Fredericton Fredericton parking garage Bat (Eptesicus fuscus) n/a MG490948 n/a n/a
P. speluncoe Fasciculata DAOMC 251696, KAS 7473, W54119 07-Jul-2014 DPYA Quebec Anticosti Island Grotte à la Patate Cave wall MG490864 MG490884 MG490954 MN170736
DAOMC 251697, KAS 7474, W54102 07-Jul-2014 DPYA Quebec Anticosti Island Grotte à la Patate Cave wall MG490865 MG490885 MG490955 MN170737
DAOMC 251698, KAS 7500, W07302 21-Apr-2015 MEA New Brunswick Hillsborough White Cave Cave wall MG490866 MG490886 MG490956 MN170738
DAOMC 251699, KAS 7503, W05404 21-Apr-2015 DPYA New Brunswick Hillsborough White Cave Cave wall MG490867 MG490887 MG490957 MN170739
DAOMC 251700, KAS 7504, W05202 16-Apr-2015 SD New Brunswick Sussex Glebe Mine Cave wall MG490868 MG490888 MG490958 MN170740
DAOMC 251701T, KAS 7512, P06201 14-Mar-2014 SD New Brunswick Dorchester Dorchester Mine Rodent (Peromyscus maniculatus) MG490869 MG490889 MG490959 MN170741
DAOMC 252126, KAS 7516, P01202 12-Mar-2014 SD New Brunswick Dorchester Dorchester Mine Rodent (Peromyscus maniculatus) MG490877 MG490928 MG490967 MN170742
DAOMC 252127, KAS 7533, D3108 25-Mar-2014 DPYA New Brunswick Dorchester Dorchester Mine Rodent dung (Peromyscus maniculatus) MG490880 MG490940 MG490970 MN170743
P. westlingii Citrina DAOMC 252128, KAS 7463, W77200 31-Mar-2015 SD New Brunswick Dorchester Dorchester Mine Cave wall n/a MG490894 n/a n/a
DAOMC 252129, KAS 7518, M34108 14-Mar-2014 DPYA New Brunswick Dorchester Dorchester Mine Moth (Scoliopteryx libatrix) n/a MG490930 n/a n/a
Open in a new tab

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 the personal collection of Karen Vanderwolf and Dave Malloch.

DNA extraction, sequencing and phylogenetic analysis

Strains were grown on Blakeslee’s (1915) malt extract agar (MEAbl) for 7 d and DNA extracted using the UltracleanTM Microbial DNA isolation Kit (MoBio Laboratories Inc., Solana 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 10× Titanium Taq buffer (Clontech, California, USA), 0.1 µL 50× Titanium Taq enzyme (Clontech, California, USA), 0.5 µL template DNA and 7.82 µL sterile purified water. ITS barcodes (Schoch et al. 2012), partial beta-tubulin (BenA), partial calmodulin (CaM) and RNA polymerase II second largest subunit (RPB2) genes were amplified using PCR conditions and primers suggested by Visagie et al. (2014b). PCR products were verified by agarose gel electrophoresis and subsequently sequenced with the BigDye Terminator Cycle Premix Kit (Applied Biosystems, Waltham, USA). Contigs were assembled and edited in Geneious v. 8.1.5 (BioMatters Ltd., Auckland, New Zealand). Newly generated sequences were submitted to GenBank and accession numbers provided in Table 1. Gene sequences of the new species were compared to a reference sequence dataset built around the ex-type sequences published in Visagie et al. (2014b), also including reference sequences from (Samson et al. 2004, Houbraken et al. 2011, 2012, 2014, 2016, Frisvad et al. 2013a, b, Visagie et al. 2014a) where needed (Suppl. Table S1). Additional unpublished sequences related to the new species were included and originate from various past projects. Sequences were aligned in MAFFT v. 7.407 (Katoh & Standley 2013), with the G-INS-i option and manually trimmed and adjusted in Geneious where needed. Datasets were subsequently analysed using Maximum Likelihood (ML) and Bayesian tree inference (BI). For concatenated phylogenies, each gene was treated as a separate 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 et al. 2017) and bootstrapping done using UFBoot (Minh et al. 2013), both integrated into IQtree. Bayesian inference trees were calculated in MrBayes v. 3.2.6 (Ronquist et al. 2012) with the most suitable model selected by ParitionFinder v. 2.1.1 (Lanfear et al. 2017) using the corrected Akaike information criterion (Akaike 1974). Alignments and command blocks used for analyses were uploaded to TreeBASE (https://treebase.org) with accession 23575. Trees were visualized in Figtree v. 1.4.4 (http://tree.bio.ed.ac.uk/software/figtree) and visually edited in Affinity Designer v. 1.7.1 [Serif (Europe) Ltd, Nottingham, UK].

Table. S1.

Supplementary Table S1. Strains used for phylogenetic analyses.

Species Strain Section ITS_GB BenA_GB CaM_GB RBP2_GB
P. armarii CBS 138171 (ex-type) Aspergilloides KM189758 KM089007 KM089394 KM089781
P. bussumense CBS 138160 (ex-type) Aspergilloides KM189458 KM088685 KM089070 KM089457
P. frequentans CBS 105.11 (ex-type) Aspergilloides KM189525 KM088762 KM089147 KM089534
P. glabrum CBS 125543 (ex-type) Aspergilloides KM189530 KM088767 KM089152 KM089539
P. glabrum CBS 129784 Aspergilloides KM189736 KM088985 KM089372 KM089759
P. glabrum CBS 129606 Aspergilloides KM189733 KM088982 KM089369 KM089756
P. glabrum CBS 126336 Aspergilloides KM189537 KM088775 KM089160 KM089547
P. glabrum CBS 127700 Aspergilloides KM189540 KM088778 KM089163 KM089550
P. glabrum CBS 126333 Aspergilloides KM189536 KM088774 KM089159 KM089546
P. glabrum CBS 127704 Aspergilloides KM189533 KM088771 KM089156 KM089543
P. glabrum CBS 328.48 Aspergilloides KM189790 KM089040 KM089427 KM089814
P. glabrum CBS 138165 Aspergilloides KM189546 KM088784 KM089169 KM089556
P. glabrum CBS 138166 Aspergilloides KM189611 KM088855 KM089242 KM089629
P. glabrum CBS 129602 Aspergilloides KM189732 KM088981 KM089368 KM089755
P. glabrum CBS 131040 Aspergilloides KM189785 KM089035 KM089422 KM089809
P. glabrum CBS 115810 Aspergilloides KM189477 KM088712 KM089097 KM089484
P. glabrum CBS 138164 Aspergilloides KM189544 KM088782 KM089167 KM089554
P. glabrum CBS 171.81 Aspergilloides KM189468 KM088700 KM089085 KM089472
P. glabrum CBS 127703 Aspergilloides KM189534 KM088772 KM089157 KM089544
P. pulvis CBS 138432 (ex-type) Aspergilloides KM189632 KM088876 KM089263 KM089650
P. purpurascens CBS 366.48 (ex-type) Aspergilloides KM189561 KM088801 KM089186 KM089573
P. rudallense CBS 130049 (ex-type) Aspergilloides KM189744 KM088993 KM089380 KM089767
P. spinulosum CBS 374.48 (ex-type) Aspergilloides KM189448 KM088672 KM089057 KM089444
P. thomii CBS 225.81 (ex-type) Aspergilloides KM189560 KM088799 KM089184 KM089571
P. bialowiezense CBS 227.28 (ex-type) Brevicompacta EU587315 AY674439 AY484828 JN406604
P. bialowiezense NRRL 32205 Brevicompacta AY484904 DQ645791 AY484836
P. bialowiezense NRRL 32207 Brevicompacta AY484905 DQ645792 AY484837
P. brevicompactum CBS 257.29 (ex-type) Brevicompacta AY484912 AY674437 AY484813 JN406594
P. spathulatum CBS 117192 (ex-type) Brevicompacta JX313165 JX313183 JX313149 JN406636
P. spathulatum CBS 116976 Brevicompacta JX313161 JX313179 JX313145
P. spathulatum CBS 116975 Brevicompacta JX313160 JX313178 JX313144
P. spathulatum CBS 116977 Brevicompacta JX313162 JX313180 JX313146
P. spathulatum CBS 116972 Brevicompacta JX313157 JX313175 JX313141
P. canescens CBS 300.48 (ex-type) Canescentia AF033493 JX140946 KJ867009 JN121485
P. charlesii CBS 304.48 (ex-type) Charlesia AF033400 JX091508 AY741727 JN121486
P. allii-sativi CBS 132074 (ex-type) Chrysogena JX997021 JX996891 JX996232 JX996627
P. allii-sativi DTO 149A9 Chrysogena JX997022 JX996892 JX996233 JX996628
P. chrysogenum CBS 306.48 (ex-type) Chrysogena AF033465 AY495981 JX996273 JN121487
P. chrysogenum CBS 776.95 Chrysogena JX997114 JX996933 JX996283 JX996678
P. chrysogenum CBS 132217 Chrysogena JX996997 JX996871 JX996211 JX996606
P. chrysogenum CBS 111215 Chrysogena JX997070 JX996922 JX996266 JX996661
P. chrysogenum CBS 259.29 Chrysogena JX997089 JX996924 JX996270 JX996665
P. chrysogenum CBS 906.70 Chrysogena JX997117 JX996934 JX996284 JX996679
P. chrysogenum CBS 282.97 Chrysogena JX996925 JX996271 JX996666
P. chrysogenum CBS 109613 Chrysogena KJ866978 KJ866990
P. rubens CBS 129667 (ex-type) Chrysogena JX997057 JF909949 JX996263 JX996658
P. rubens CBS 339.52 Chrysogena JX997098 JX996929 JX996277 JX996672
P. rubens CBS 111216 Chrysogena JX997071 JX996923 JX996267 JX996662
P. rubens CBS 132210 Chrysogena JX996984 JX996859 JX996198 JX996593
P. rubens CBS 319.59 Chrysogena JX997097 JX996928 JX996276 JX996671
P. rubens CBS 349.48 Chrysogena JX997100 JX996930 JX996278 JX996673
P. rubens CBS 401.92 Chrysogena JX997103 JX996931 JX996280 JX996675
P. rubens CBS 478.84 Chrysogena JX997109 JX996932 JX996282 JX996677
P. tardochrysogenum CBS 132200 (ex-type) Chrysogena JX997027 JX996898 JX996239 JX996634
P. cinnamopurpureum CBS 429.65 (ex-type) Cinnamopurpurea EF626950 EF626948 EF626949 JN406533
P. citrinum CBS 139.45 (ex-type) Citrina AF033422 GU944545 GU944638 JF417416
P. cosmopolitanum CBS 126995 (ex-type) Citrina JN617691 JN606733 JN606472
P. cosmopolitanum CBS 122406 Citrina JN606754 JN606481
P. westlingii CBS 231.28 (ex-type) Citrina GU944601 JN606718 JN606500 JN606625
P. westlingii CBS 127037 Citrina JN606720 JN606496
P. westlingii CBS 127003 Citrina JN606711 JN606490
P. sacculum CBS 231.61 (ex-type) Eladia KC411707 KJ834488 KU896849 JN121462
P. consobrinum CBS 139144 (ex-type) Exilicaulis JX140888 JX141135 JX157453 KP064619
P. consobrinum CV 1457 Exilicaulis JX141146 JX157486 KP064630
P. corylophilum CBS 312.48 (ex-type) Exilicaulis AF033450 JX141042 KP016780 KP064631
P. corylophilum CBS 127808 Exilicaulis KP016813 KP016752 KP016776 KP064613
P. albocoremium CBS 472.84 (ex-type) Fasciculata AJ004819 AY674326 KU896819 KU904344
P. allii CBS 131.89 (ex-type) Fasciculata AJ005484 AY674331 KU896820 KU904345
P. aurantiogriseum CBS 249.89 (ex-type) Fasciculata AF033476 AY674296 KU896822 JN406573
P. biforme CBS 297.48 (ex-type) Fasciculata KC411731 FJ930944 KU896823 KU904346
P camemberti CBS 299.48 (ex-type) Fasciculata AB479314 FJ930956 KU896825
P. caseifulvum CBS 101134 (ex-type) Fasciculata KJ834504 AY674372 KU896826 KU904347
P. cavernicola CBS 100540 (ex-type) Fasciculata KJ834505 KJ834439 KU896827 KU904348
P. cavernicola DTO 046I3 = IBT 25514 Fasciculata MN149916 MN149935 MN149955
P. cavernicola DTO 046I8 = IBT 25513 Fasciculata MN149920 MN149939 MN149959
P. cellarum NRRL 66633 (ex-type) Fasciculata KM249068 KM249108 KM249117
P. commune CBS 311.48 (ex-type) Fasciculata AY213672 AY674366 KU896829 KU904350
P. crustosum CBS 115503 (ex-type) Fasciculata AF033472 AY674353 DQ911132
P. crustosum CV 0241 Fasciculata JX091403 JX091536 JX141576
P. crustosum CV 0251 Fasciculata JX091404 JX091530 JX141577
P. cyclopium CBS 144.45 (ex-type) Fasciculata JN942742 AY674310 KU896832 JN985388
P. discolor CBS 474.84 (ex-type) Fasciculata AJ004816 AY674348 KU896834 KU904351
P. discolor DTO 046I4 = IBT 22523 Fasciculata MN149917 MN149936 MN149956
P. discolor DTO 047A2 = IBT 5736 Fasciculata MN149922 MN149941 MN149961
P. discolor DTO 047A3 = IBT 5744 Fasciculata MN149923 MN149942 MN149962
P. echinulatum CBS 101027 Fasciculata AY674342
P. echinulatum CBS 317.48 (ex-type) Fasciculata AF033473 AY674341 DQ911133 KU904352
P. echinulatum CBS 337.59 Fasciculata KC411742 AY674340
P. echinulatum DTO 228I4 Fasciculata MN149925 MN149944 MN149964
P. freii CBS 476.84 (ex-type) Fasciculata JN942696 AY674290 KU896836 JN985430
P. gladioli CBS 332.48 (ex-type) Fasciculata AF033480 AY674287 KU896837 JN406567
P. hirsutum CBS 135.41 (ex-type) Fasciculata AY373918 AF003243 KU896840 JN406629
P. hordei CBS 701.68 (ex-type) Fasciculata AJ004817 AY674347 KU896841 KU904355
P. melanoconidium CBS 115506 (ex-type) Fasciculata AJ005483 AY674304 KU896843 KU904358
P. neoechinulatum CBS 169.87 (ex-type) Fasciculata JN942722 AF003237 KU896844 JN985406
P. nordicum ATCC 44219 (ex-type) Fasciculata KJ834513 KJ834476 KU896845 KU904359
P. palitans CBS 107.11 (ex-type) Fasciculata KJ834514 KJ834480 KU896847 KU904360
P. palitans DTO 046I5 Fasciculata MN149918 MN149937 MN149957
P. polonicum CBS 222.28 (ex-type) Fasciculata AF033475 AY674305 KU896848 JN406609
P. radicicola CBS 112430 (ex-type) Fasciculata KJ834516 AY674357
P. robsamsonii CBS 140573 (ex-type) Fasciculata KU904339 KT698885 KT698894 KT698904
P. solitum CBS 146.86 Fasciculata AY674356
P. solitum CBS 147.86 Fasciculata HQ225713 AY674355
P. solitum CBS 424.89 (ex-type) Fasciculata AY373932 AY674354 KU896851 KU904363
P. solitum DTO 046I6 = IBT 22216 Fasciculata MN149919 MN149938 MN149958
P. solitum DTO 161H9 Fasciculata MN149924 MN149943 MN149963
P. solitum DTO 234I5 Fasciculata MN149926 MN149945 MN149965
P. solitum DTO 235G1 Fasciculata KJ775670 KJ775163 MN149946 MN149966
P. solitum DTO 247B8 Fasciculata MN149927 MN149947 MN149967
P. solitum DTO 321F7 Fasciculata MN149928 MN149948 MN149968
P. solitum DTO 376D5 Fasciculata MN149930 MN149950
P. speluncae CBS 271.97 Fasciculata AY674350 MN170734
P. speluncae CBS 278.97 Fasciculata AY674349 MN170735
P. speluncae DAOMC 251696 Fasciculata MG490864 MG490884 MG490954 MN170736
P. speluncae DAOMC 251697 Fasciculata MG490865 MG490885 MG490955 MN170737
P. speluncae DAOMC 251698 Fasciculata MG490866 MG490886 MG490956 MN170738
P. speluncae DAOMC 251699 Fasciculata MG490867 MG490887 MG490957 MN170739
P. speluncae DAOMC 251700 Fasciculata MG490868 MG490888 MG490958 MN170740
P. speluncae DAOMC 251701 (ex-type) Fasciculata MG490869 MG490889 MG490959 MN170741
P. speluncae DAOMC 252126 Fasciculata MG490877 MG490928 MG490967 MN170742
P. speluncae DAOMC 252127 Fasciculata MG490880 MG490940 MG490970 MN170743
P. speluncae CBS 551.95 = DTO 037C9 Fasciculata MN149912 MN149931 MN149951
P. speluncae CBS 112559 = DTO 037D2 Fasciculata MN149913 MN149932 MN149952
P. speluncae CBS 112569 = DTO 046G4 Fasciculata MN149914 MN149933 MN149953
P. speluncae CBS 112568 = DTO 046G5 Fasciculata MN149915 MN149934 MN149954
P. speluncae DTO 046I9 = IBT 22369 Fasciculata MN149921 MN149940 MN149960
P. speluncae DTO 332H8 Fasciculata MN149929 MN149949 MN149973
P. thymicola CBS 111225 (ex-type) Fasciculata KJ834518 AY674321 FJ530990 KU904364
P. tricolor CBS 635.93 (ex-type) Fasciculata JN942704 AY674313 KU896852 JN985422
P. tulipae CBS 109555 (ex-type) Fasciculata KJ834519 AY674344
P. venetum IBT 10661 (ex-type) Fasciculata AJ005485 AY674335 KU896855 KU904366
P. verrucosum CBS 603.74 (ex-type) Fasciculata AY373938 AY674323 DQ911138 JN121539
P. viridicatum CBS 390.48 (ex-type) Fasciculata AY373939 AY674295 KU896856 KY989209
P. fractum CBS 124.68 (ex-type) Fracta KC411674 KJ834452 JN121441
P. gracilentum CBS 599.73 (ex-type) Gracilenta KC411768 KJ834453 JN121537
P. javanicum CBS 341.48 (ex-type) Lanata-Divaricata GU981613 GU981657 KF296387 JN121498
P. ochrosalmoneum CBS 489.66 (ex-type) Ochrosalmonea EF626961 EF506212 EF506237 JN121524
P. osmophilum CBS 462.72 (ex-type) Osmophila EU427295 AY674376 KU896846 JN121518
P. paradoxum CBS 527.65 (ex-type) Paradoxa EF669707 EF669683 EF669692 EF669670
P. expansum CBS 325.48 (ex-type) Penicillium AY373912 AY674400 DQ911134 JF417427
P. expansum CV 2860 Penicillium FJ230989 JX091539 JX141580
P. expansum CV 2861 Penicillium FJ230990 JX091540 JX141581
P. expansum CBS 481.84 Penicillium AY674399
P. expansum CBS 281.97 Penicillium AY674401
P. cyaneum CBS 315.48 (ex-type) Ramigena AF033427 JX091552 JN406575
P. soppii CBS 226.28 (ex-type) Ramosa AF033488 DQ285616 KJ867002 JN406606
P. brevistipitatum AS 3.6887 (ex-type) Robsamsonia DQ221696 DQ221695 KU896824 JN406528
P. concentricum CBS 477.75 (ex-type) Robsamsonia KC411763 AY674413 DQ911131 KT900575
P. concentricum CBS 191.88 Robsamsonia AY674412
P. robsamsonii CBS 140573 (ex-type) Robsamsonia KU904339 KT698885 KT698894 KT698904
P. roqueforti CBS 221.30 (ex-type) Roquefortorum EU427296 AF000303 HQ442332 JN406611
P. sclerotiorum CBS 287.36 (ex-type) Sclerotiora JN626132 JN626001 JN626044 JN406585
P. stolkiae CBS 315.67 (ex-type) Stolkia AF033444 JN617717 AF481135 JN121488
P. glaucoalbidum WCN 1129 Thysanophora AB175275
P. glaucoalbidum WCN 1128 Thysanophora AB175273
P. glaucoalbidum WCN 1043 Thysanophora AB175259
P. glaucoalbidum WCN 1246 Thysanophora AB175268
P. glaucoalbidum WCN 1016 Thysanophora AB175254
P. glaucoalbidum CBS 314.56 Thysanophora AB213277
P. glaucoalbidum CBS 348.64 Thysanophora AB213275
P. glaucoalbidum WCN 1152 Thysanophora AB175272
P. glaucoalbidum WCN 1077 Thysanophora AB175262
P. glaucoalbidum NBRC 9011 Thysanophora AB213279
P. hennebertii CBS 334.68 (ex-type) Thysanophora KJ834507 KJ834454 JN121493
P. taxi CBS 206.57 (ex-type) Thysanophora KJ834517 KJ834495 JN121454
P. lagena CBS 185.65 (ex-type) Torulomyces KF303665 KF303619 KF303634 JN121450
P. turbatum CBS 383.48 (ex-type) Turbata AF034454 KJ834499 KU896853 JN406556
Talaromyces pinophilus (outgroup) CBS 631.66 (ex-type) JN899382 JX091381 KF741964 KM023291
Open in a new tab

AS: Internal culture collection at CGMCC, China General Microbiological Culture Collection Centre, Beijing, China; ATCC: American Type Culture Collection, Manassas, VA, USA; CBS: Culture collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, Netherlands; CV: Working collection of Prof Karin Jacobs, from the Dept of Microbiology, Stellenbosch University, Stellenbosch, South Africa; DAOMC: Culture collection of the National Mycological Collections, Agriculture & Agri-Food Canada, Ottawa, Canada; DTO: Internal culture collection of Westerdijk Fungal Biodiversity Institute; IBT: Culture collection of Center for Microbial Biotechnology (CMB) at Department of Systems Biology, Technical University of Denmark; NRBC: Culture collection of the National Institute of Technology and Evaluation, Tokyo, Japan; NRRL: ARS Culture Collection, U.S. Department of Agriculture, Peoria, Illinois, USA; WCN: Working collection of Susumu Iwamoto, Tokyo, Japan.

Morphology

Morphological characters were captured using standardized protocols proposed by Visagie et al. (2014b). Colony characters were captured on Czapek yeast autolysate agar (CYA), MEAbl, yeast extract sucrose agar (YES), oatmeal agar (OA) and creatine sucrose agar (CREA). Strains were inoculated in a three-point pattern on these media in 90 mm Petri dishes. Plates were incubated for 7 d at 25 °C in darkness in perforated plastic bags. Colour names and codes used in descriptions are from Kornerup & Wanscher (1967). Microscopic observations were made using an Olympus SZX12 dissecting microscope and Olympus BX50 compound microscope equipped with Infinity3 and InfinityX cameras driven by Infinity Analyze v. 6.5.1 software (Lumenera Corp., Ottawa, Canada). Colonies were captured with a Sony NEX-5N camera. Plates were prepared in Affinity Photo v. 1.6.6 [Serif (Europe) Ltd, Nottingham, UK]. For aesthetic purposes, micrographs were adjusted using the “inpainting brush tool” without altering areas of scientific significance. Line drawings were prepared in Affinity Photo v. 1.7.1 [Serif (Europe) Ltd, Nottingham, UK] running on an iPad Pro with an Apple Pencil.

Extrolites

For extrolite analyses, all strains were grown in 9 cm polystyrene Petri dishes on CYA (Pitt 1980) and YES (Frisvad 1981, Filtenborg et al. 1990) incubated at 25 °C for 14 d. Six agar plugs from each fungal isolate were excised with a sterilized 7 mm cork-borer and transferred to a 13 mL polypropylene tube. Two mL of ethyl acetate was then added and vortexed for 30 s, followed by sonication at 30 °C for 30 min and vortexed again for 30 s. The supernatants were transferred into new polypropylene tubes and dried on a centrifugal vacuum concentrator at 35 °C. Extracts were then reconstituted in 1 mL of methanol:water (8:2) and filtered into 2 mL amber glass HPLC vials using a 0.45 µm PVDF syringe filter. Extracts were immediately stored at -20 °C until analysis by liquid chromatography mass spectrometry (LC-MS). Extracts were analyzed in both positive and negative polarities using a Q-Exactive Orbitrap coupled to an Agilent 1290 HPLC. The chemical formula of observed extrolites were determined with Xcalibur® software using accurate mass measurements (< 3.0 ppm) and manually verified by isotopic pattern. The chemical formulae were then searched against microbial extrolite databases [AntiBase2013 (Wiley-VCH, Weinheim, Germany)] and KNApSAcK (Afendi et al. 2012) and putative matches were scrutinized by comparing their MS/MS fragmentation with those published in the literature or predicted by CFM-ID (Allen et al. 2014).

RESULTS

Sampling, isolations & identifications

During the survey, 70 Penicillium strains were isolated from six different caves in New Brunswick and one in Quebec, Canada. Eight strains of the new Penicillium species were isolated from walls of the Glebe Mine and White Cave in New Brunswick and Grotte à la Patate in Quebec, and three strains were isolated from a deer mouse (Peromyscus maniculatus) and its dung from the Dorchester Mine in New Brunswick (Table 1). Based on the 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 = 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).

Fig. 1.

Fig. 1.

Fig. 1.

Open in a new tab

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 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. speluncae, is in bold blue text. The tree was rooted to Talaromyces pinophilus.

Phylogeny

A multigene phylogeny was used to show identities of strains isolated during this study (Fig. 1). The alignment contained 175 taxa and was 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 in a particular clade. However, many of the newly generated sequences represented minor deviations from previously known sequences. In 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 section Fasciculata were calculated based on BenA, CaM and RPB2 (Fig. 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.

Fig. 2.

Fig. 2.

Open in a new tab

ML trees of Penicillium section Fasciculata, based on concatenated, BenA, CaM and RPB2 alignments, showing the relationship of P. 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.

Table. S2.

Supplementary Table S2. Metadata related to the phylogenetic analysis of sect. Fasciculata.

Dataset Nr of taxa Bp length Partition scheme
BenA 67 406 1st, 2nd & 3rd codon positions (HKY+G)
CaM 60 500 1st, 2nd & 3rd codon positions (SYM+G)
ITS 44 505 1st, 2nd & 3rd codon positions (GTR+I)
RPB2 54 937 1st codon position (F81), 2nd codon position (HKY+G), 3rd codon position (GTR+I)
Concat 67 2348 1st, 2nd & 3rd codon positions of ITS (GTR+I), 1st codon position of BenA (SYM+I); 2nd & 3rd codon positions of CaM & BenA (SYM+G); 1st codon position of CaM (K80+I); 1st codon position of RPB2 (F81); 2nd codon position of RPB2 (HKY+G); 3rd codon position of RPB2 (GTR+I)
Open in a new tab

As expected, ITS (not shown) lacked sufficient variation to distinguish among species. For example, P. speluncae shared similar 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 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 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 and P. solitum. This resulted in all phylogenies having poor backbone support in both ML and BI, mainly because of the strains identified 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 resolved distinct from the ex-type CBS 474.84T within the broad concept applied to P. speluncae.

Extrolites

As analysed by LC-MS, there were five classes of compounds produced by P. speluncae under the reported growth conditions: cyclopenins, viridicatins, chaetoglobosins, cyclic dipeptides, and tetrapeptides. Cyclopenins and viridicatins are derived from a shared biosynthetic pathway (Simonetti et al. 2016) and are among the most widely distributed extrolites across species in Penicillium subgenus Penicillium (Frisvad et al. 2004). Chaetoglobosins are a large class of metabolites biosynthesised by a polyketide derived macrocycle fused to a modified tryptophan amino acid and are produced by Chaetomium globosum as well as P. discolor (Frisvad et al. 1997), P. expansum (Frisvad & Filtenborg 1989) and P. marinum (Frisvad et al. 2004). One of the major chaetoglobosins produced by these isolates is a newly described natural product, tetrahydrochaetoglobosin (Walsh et al. 2018). In addition to these extrolites, a series of cyclic and linear tetrapeptides, composed of combinations of valine, phenylalanine, leucine/isoleucine, tyrosine and tryptophan were consistently detected across all tested strains (Table 2). These peptides could be putatively characterized by de novo sequencing and are likely similar to the series of linear and cyclic tetra peptides previously identified in cultures of P. chrysogenum, including fungisporin; cyclo(D-Phe-L-Phe-D-Val-L-Val) (Ali et al. 2014).

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 %
viridicatin C15H11NO2 238.0865 3.36 100 %
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 %
Open in a new tab

Taxonomy

Penicillium speluncae Visagie & Yilmaz, sp. nov. MycoBank MB828614. Figs 3, 4.

Fig. 3.

Fig. 3.

Open in a new tab

Line drawing ofPenicillium speluncae. Scale bar = 10 µm.

Fig. 4.

Fig. 4.

Open in a new tab

Penicillium speluncae. A. Colonies, from left to right, top row: CYA, MEA, YES, OA; bottom row: reverse on CYA, MEA, YES, CREA. B–F. Conidiophores. G. Conidia. Scale bars = 10 μm.

Etymology: Latin, speluncae, meaning from a cave.

ITS barcode: MG490869. Alternative identification markers: BenA = MG490889, CaM = MG490959, RPB2 = MN170741.

Colony diam, 7 d (at 25 °C; in mm): CYA 30–35; CYA 15 °C (12–) 17–22(–25); CYA 30 °C 12–21(–26); CYA 37°C no growth; CYAS 29–32(–37); MEAbl 25–30; YES 45–47; OA 25–27; CREA 25–26.

Colony characters: CYA 25 °C, 7 d: Colonies moderately deep, sulcate; margins low, narrow to wide, entire; mycelia white; texture velutinous to fasciculate; sporulation moderately dense, conidia en masse greyish green (25E7), dull green (26D3–4); soluble pigments absent; exudates absent; reverse greyish yellow (4B6), greyish orange (5B4), yellowish white (4A2). MEA 25 °C, 7 d: Colonies low, plain; margins low, narrow, entire; mycelia white; texture velutinous to fasciculate; sporulation moderately dense, conidia en masse greyish green (25E5–26E5); soluble pigments forming a yellow halo surrounding colony; exudates absent; reverse greyish yellow (4B6), light yellow (3A5), greyish green (29C6). YES 25 °C, 7 d: Colonies low, sulcate; margins low, wide, entire; mycelia white; texture velutinous to fasciculate; sporulation moderately dense, conidia en masse greyish green (25C5–D5), dull green (26D3); soluble pigments absent; exudates absent; reverse orange yellow to orange (4A7–6A7). OA 25°C, 7 days: Colonies moderately deep, plain; margins low, narrow, entire; mycelia white; texture fasciculate; sporulation dense, conidia en masse greyish to dark green (25E7–F7); soluble pigments forming a yellowish halo surrounding colony; exudates absent. CREA 25 °C, 7 d: Growth strong, acid produced, colony reverse orange.

Micromorphology: Conidiophores terverticillate, minor proportion bi- and quarterverticillate; stipes rough, 180–600 × 3.5–4.5 μm; branches 15–29 μm; metulae (2–)3–4, 10–16 × 3–4.5 μm; phialides ampulliform, 4–6 per metula, 8.5–11 × 3–4 μm (9.9±0.7 × 3.3±0.2); average length metula/phialide 1.3; conidia smooth, broadly ellipsoidal, 3–4 × 2.5–3.5 μm (3.6±0.2 × 3±0.2), average width/length = 0.82, n = 72.

Extrolites: cyclopenins, viridicatins, chaetoglobosins, fungisporin, cyclic and linear tetrapeptides (See Table 2).

Typus: Canada, New Brunswick, Dorchester, Dorchester mine, from a swab of deer mouse fur (live Peromyscus maniculatus), 14 Mar. 2014, K. Vanderwolf (holotype, DAOM 745788 (dried culture); ex-type strain DAOMC 251701 = KAS 7512 = P06201).

Notes: Penicillium speluncae is resolved in a clade with P. discolor, P. echinulatum and P. solitum (Fig. 2). Of these, P. speluncae showed relatively good growth on CYA at 30 °C, compared to poor growth observed for the others. Both P. discolor and P. echinulatum produce roughened globose to subglobose conidia, in contrast to the new species’ smooth, broadly ellipsoidal conidia. Penicillium solitum is morphologically most similar to the new species. Both species have smooth conidia and produce a striking yellow orange reverse on YES. However, P. speluncae produces broadly ellipsoidal conidia (globose to subglobose in P. solitum), grows faster on YES compared to P. solitum (45–47 mm vs 25–39 mm) and has the ability to grow on CYA at 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 palitantin was not detected for the new species (comparisons summarised in Table 3; data from Frisvad et al. 2004).

Table 3.

Distinguishing features of species closely related to Penicillium speluncae.

Conidia CYA texture YES soluble pigment Yes reverse Chaetoglobosins Compactin Penitrem Roquefortine Cytoglobosin Prochaetoglobosin Palitantin
P. speluncae Smooth, broadly ellipsoidal Velutinous to fasciculate None Orange yellow to orange + - - - + + -
P. crustosum Smooth, globose to subglobose Velutinous to weakly fasciculate, becoming crustose Pale brown or none Strongly yellow - - + + - - -
P. discolor Rough, globose to subglobose Velutinous to fasciculate Brilliant red diffusible colour on YES Orange turning into deep red with age + - - - - - +
P. echinulatum Rough, globose to subglobose Velutinous to weakly fasciculate None yellow - - - - - - +
P. solitum Smooth to finely rough, globose to subglobose Velutinous None yellow to orange - + - - - - +
Open in a new tab

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 Pdpositive bat hibernacula in New Brunswick, Canada and one Pdnegative bat hibernaculum in Quebec, Canada. The isolates were collected from arthropods, bats, rodents and their dung (i.e. the 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 the most frequently isolated genera, probably because of the ability of these species to grow at low temperatures (Frisvad & Samson 2004, Vanderwolf et al. 2016). Previous studies had similar results, but the diversity of Penicillium in caves is even greater than previously reported and several of these species have never been reported from caves or mines, including P. bialowiezense, P. brevistipitatum, P. consobrinum, P. rubens, P. spathulatum and P. westlingii (Nováková 2009, 2018, Vanderwolf et al. 2013b, 2016, Anelli et al. 2018). Other species identified were P. chrysogenum, P. concentricum, P. corylophilum, P. expansum, P. glabrum and P. glaucoalbidum. One species could not be identified based on DNA reference sequences and further study showed it to represent a new species, described above as P. speluncae, classified in section Fasciculata.

Phylogenetic analyses of sect. Fasciculata revealed a large degree of genetic variation within P. speluncae. Single gene trees based on BenA, CaM and RPB2 resulted in inconsistent groupings and poor backbone support for this clade meaning that genealogical concordance could not be applied to delimit segregate species. The basal branch encompassing this clade was relatively well supported in the concatenated tree. DAOMC strains were characterized based on morphology and extrolite data, with very few differences noted. For example, colony growth rates varied on CYA at 30 °C and CYAS, but a similar variation was previously observed in P. solitum (Frisvad & Samson 2004). Extrolite data also distinguish among this group of species. Chaetoglobosins are produced only by P. speluncae and P. discolor, while the former produces cytoglobosin and prochaetoglobosin, which are absent in P. discolor. Chaetomium globosum is the best-known producer of chaetoglobosins, including the major chaetoglobosins A, C, and F, also shown here to be produced by P. speluncae. A distinguishing feature between chaetoglobosin production by C. globosum and P. speluncae is a newly described natural product, tetrahydrochaetoglobosin A (Walsh et al. 2019), which was not observed in C. globosum. Our data hint that P. speluncae may be a species complex with so far cryptic species that may be resolved with additional data. Considering the available data, we conservatively propose the name P. speluncae for this clade. In principle, an analogous situation occurred 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 species list and associated ex-type reference sequences published 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 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 (Iwamoto et al. 2005); this will be the focus of a future study.

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 worked at the Agricultural Research Council, South Africa.

Supplementary Material: http://fuse-journal.org/

REFERENCES

  1. Afendi FM, Okada T, Yamazaki M, et al. (2012). KNApSAcK Family Databases: Integrated Metabolite–Plant Species Databases for Multifaceted Plant Research. Plant and Cell Physiology 53: e1. [DOI] [PubMed] [Google Scholar]
  2. Akaike H. (1974). A new look at the statistical model identification. IEEE Transations on Automatic Control 19: 716–723. [Google Scholar]
  3. Ali H, Ries MI, Lankhorst PP, et al. (2014). A non-canonical NRPS is involved in the synthesis of fungisporin and related hydrophobic cyclic tetrapeptides in Penicillium chrysogenum. PLoS ONE 9: e98212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Allen F, Pon A, Wilson M, et al. (2014). CFM-ID: A web server for annotation, spectrum prediction and metabolite identification from tandem mass spectra. Nucleic Acids Research 42: W94–W99. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Anelli P, Peterson SW, Haidukowski M, et al. (2018). Penicillium gravinicasei, a new species isolated from cave cheese in Apulia, Italy. International Journal of Food Microbiology 282: 66–70. [DOI] [PubMed] [Google Scholar]
  6. Blakeslee AF. (1915). Lindner’s roll tube method of separation cultures. Phytopathology 5: 68–69. [Google Scholar]
  7. Blehert DS, Hicks AC, Behr M, et al. (2009). Bat White-Nose Syndrome: An Emerging Fungal Pathogen? Science 323: 227. [DOI] [PubMed] [Google Scholar]
  8. Frisvad JC, Filtenborg O. (1989). Terverticillate Penicillia: Chemotaxonomy and mycotoxin production. Mycologia 81: 837–861. [Google Scholar]
  9. Frisvad JC, Samson RA, Rassing BR, et al. (1997). Penicillium discolor, a new species from cheese, nuts and vegetables. Antonie van Leeuwenhoek 72: 119–126. [DOI] [PubMed] [Google Scholar]
  10. Frisvad JC, Samson RA. (2004). Polyphasic taxonomy of Penicillium subgenus Penicillium. A guide to identification of food and air-borne terverticillate Penicillia and their mycotoxins. Studies in Mycology 49: 1–174. [Google Scholar]
  11. Frisvad JC, Smedsgaard J, Larsen TO, et al. (2004). Mycotoxins, drugs and other extrolites produced by species in Penicillium subgenus Penicillium. Studies in Mycology 49: 201–241. [Google Scholar]
  12. Frisvad JC, Houbraken J, Popma S, et al. (2013a). Two new Penicillium species Penicillium buchwaldii and Penicillium spathulatum, producing the anticancer compound asperphenamate. FEMS Microbiology Letters 339: 77–92. [DOI] [PubMed] [Google Scholar]
  13. Frisvad JC, Yilmaz N, Thrane U, et al. (2013b). Talaromyces atroroseus, a new species efficiently producing industrially relevant red pigments. PLoS ONE 8: e84102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gargas A, Trest MT, Christensen M, et al. (2009). Geomyces destructans sp. nov. associated with bat white-nose syndrome. Mycotaxon 108: 147–154. [Google Scholar]
  15. Houbraken J, Frisvad JC, Samson RA. (2011). Taxonomy of Penicillium section Citrina. Studies in Mycology 70: 53–138. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Houbraken J, Frisvad JC, Seifert KA, et al. (2012). New penicillinproducing Penicillium species and an overview of section Chrysogena. Persoonia 29: 78–100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Houbraken J, Visagie CM, Meijer M, et al. (2014). A taxonomic and phylogenetic revision of Penicillium section Aspergilloides. Studies in Mycology 78: 373–451. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Houbraken J, Wang L, Lee HB, et al. (2016). New sections in Penicillium containing novel species producing patulin, pyripyropens or other bioactive compounds. Persoonia 36: 299–314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Iwamoto S, Tokumasu S, Suyama Y, et al. (2005). Thysanophora penicillioides includes multiple genetically diverged groups that coexist respectively in Abies mariesii forests in Japan. Mycologia 97: 1238–1250. [DOI] [PubMed] [Google Scholar]
  20. Johnson LJAN, Miller AN, McCleery RA, et al. (2013). Psychrophilic and psychrotolerant fungi on bats and the presence of Geomyces spp. on bat wings prior to the arrival of White Nose Syndrome. Applied and Environmental Microbiology 79: 5465–5471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kalyaanamoorthy S, Minh BQ, Wong TKF, et al. (2017). ModelFinder: Fast model selection for accurate phylogenetic estimates. Nature Methods 14: 587–589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Katoh K, Standley DM. (2013). MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Molecular Biology and Evolution 30: 772–780. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kornerup A, Wanscher JH. (1967). Methuen Handbook of Colour. 2nd edn Methuen & Co Ltd, London, England. [Google Scholar]
  24. Lanfear R, Frandsen PB, Wright AM, et al. (2017). PartitionFinder 2: New methods for selecting partitioned models of evolution for molecular and morphological phylogenetic analyses. Molecular Biology and Evolution 34: 772–773. [DOI] [PubMed] [Google Scholar]
  25. Lorch JM, Muller LK, Russell RE, et al. (2013). Distribution and environmental persistence of the causative agent of white-nose syndrome, Geomyces destructans, in bat hibernacula of the eastern United States. Applied and Environmental Microbiology 79: 1293–1301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. McAlpine DF, Vanderwolf KJ, Forbes GJ, et al. (2012). Consumption of bats (Myotis spp.) by raccoons (Procyon lotor) during an outbreak of white-nose syndrome in New Brunswick, Canada: Implications for estimates of bat mortality. Canadian Field-Naturalist 125: 257–260. [Google Scholar]
  27. Micalizzi EW, Mack JN, White GP, et al. (2017). Microbial inhibitors of the fungus Pseudogymnoascus destructans, the causal agent of white-nose syndrome in bats. Plos One 12: e0179770. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Minh BQ, Nguyen MAT, Von Haeseler A. (2013). Ultrafast approximation for phylogenetic bootstrap. Molecular Biology and Evolution 30: 1188–1195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Minnis AM, Lindner DL. (2013). Phylogenetic evaluation of Geomyces and allies reveals no close relatives of Pseudogymnoascus destructans, comb. nov., in bat hibernacula of eastern North America. Fungal Biology 117: 638–649. [DOI] [PubMed] [Google Scholar]
  30. Nguyen LT, Schmidt HA, Von Haeseler A, et al. (2015). IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Molecular Biology and Evolution 32: 268–274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nováková A. (2009). Microscopic fungi isolated from the Domica Cave system (Slovak Karst National Park, Slovakia). A review. International Journal of Speleology 38: 71–82. [Google Scholar]
  32. Nováková A, Hubka V, Valinová Š, et al. (2018). Cultivable microscopic fungi from an underground chemosynthesis-based ecosystem: a preliminary study. Folia Microbiologica 63: 43–55. [DOI] [PubMed] [Google Scholar]
  33. Pitt JI, Cruickshank RH, Leistner L. (1986). Penicillium commune, P. camembertii, the origin of white cheese moulds, and the production of cyclopiazonic acid. Food Microbiology 3: 363–371. [Google Scholar]
  34. Pitt JI, Klich MA, Shafer GP, et al. (1990). Differentiation of Penicillium glabrum from Penicillium spinulosum and other closely related species: an integrated taxonomic approach. Systematic and Applied Microbiology 13: 304–309. [Google Scholar]
  35. Polonelli L, Morace G, Rosa R, et al. (1987). Antigenic characterization of Penicillium camemberti and related common cheese contaminants. Applied and Environmental Microbiology 53: 872–878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Puechmaille SJ, Wibbelt G, Korn V, et al. (2011). Pan-European distribution of white-nose syndrome fungus (Geomyces destructans) not associated with mass mortality. PLoS ONE 6: e19167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Reynolds HT, Ingersoll T, Barton HA. (2015). Modeling the environmental growth of Pseudogymnoascus destructans and its impact on the white-nose syndrome epidemic. Journal of Wildlife Diseases 51: 318–331. [DOI] [PubMed] [Google Scholar]
  38. Ronquist F, Teslenko M, van der Mark P, et al. (2012). MrBayes 3.2: Efficient bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61: 539–542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Samson RA, Seifert KA, kuijpers AFA, et al. (2004). Phylogenetic analysis of Penicillium subgenus Penicillium using partial B-tubulin sequences. Studies in Mycology 49: 175–200. [Google Scholar]
  40. Schoch CL, Seifert KA, Huhndorf S, et al. (2012). Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proceedings of the National Academy of Sciences of the USA 109: 6241–6246. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Simonetti SO, Larghi EL, Kaufman TS. (2016). The 3,4-dioxygenated 5-hydroxy-4-aryl-quinolin-2(1H)-one alkaloids. Results of 20 years of research, uncovering a new family of natural products. Natural Product Reports 33: 1425–1446. [DOI] [PubMed] [Google Scholar]
  42. Vanderwolf KJ, Malloch D, Mcalpine DF, et al. (2013a). A world review of fungi, yeasts, and slime molds in caves. International Journal of Speleology 42: 77–96. [Google Scholar]
  43. Vanderwolf KJ, McAlpine DF, Malloch D, et al. (2013b). Ectomycota associated with hibernating bats in Eastern Canadian caves prior to the emergence of White-Nose Syndrome. Northeastern Naturalist 20: 115–130. [Google Scholar]
  44. Vanderwolf KJ, Malloch D, McAlpine DF. (2016). Ectomycota associated with arthropods from bat hibernacula in Eastern Canada, with particular reference to Pseudogymnoasucs destructans. Insects 7: 16. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Vanderwolf KJ, Malloch D, McAlpine DF. (2017). Psychrotolerant microfungi associated with deer mice (Peromyscus maniculatus) in a white-nose syndrome positive bat hibernaculum in eastern Canada. Canadian Field-Naturalist 131: 238–245. [Google Scholar]
  46. Visagie CM, Hirooka Y, Tanney JB, et al. (2014a). Aspergillus, Penicillium and Talaromyces isolated from house dust samples collected around the world. Studies in Mycology 78: 63–139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Visagie CM, Houbraken J, Frisvad JC, et al. (2014b). Identification and nomenclature of the genus Penicillium. Studies in Mycology 78: 343–371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Walsh JP, Renaud JB, Hoogstra S, et al. (2019). Diagnostic fragmentation filtering for the discovery of new chaetoglobosins and cytochalasins. Rapid Communications in Mass Spectrometry 33: 133–139. [DOI] [PubMed] [Google Scholar]
  49. Wibbelt G, Kurth A, Hellmann D, et al. (2010). White-nose syndrome fungus (Geomyces destructans) in bats, Europe. Emerging Infectious Diseases 16: 1237–1242. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Zhang ZF, Liu F, Zhou X, et al. (2017). Culturable mycobiota from Karst caves in China, with descriptions of 20 new species. Persoonia 39: 1–31. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Articles from Fungal Systematics and Evolution are provided here courtesy of Westerdijk Fungal Biodiversity Institute

RESOURCES