Persoonia 36, 2016: 247–280 www.ingentaconnect.com/content/nhn/pimj RESEARCH ARTICLE http://dx.doi.org/10.3767/003158516X691627
Fifteen new species of Penicillium
C.M. Visagie1,8, J.B. Renaud 2, K.M.N. Burgess2, D.W. Malloch3, D. Clark4, L. Ketch5, M. Urb6,7, G. Louis-Seize1, R. Assabgui1, M.W. Sumarah2, K.A. Seifert1,8
Key words Abstract We introduce 15 new species of Penicillium isolated from a diverse range of locations, including Canada, Costa Rica, Germany, Italy, New Zealand, Tanzania, USA and the Dry Valleys of Antarctica, from a variety of habitats, beta-tubulin including leaf surfaces in tropical rain forests, soil eaten by chimpanzees, infrabuccal pockets of carpenter ants, calmodulin intestinal contents of caterpillars and soil. The new species are classified in sections Aspergilloides (1), Canescentia internal transcribed spacer rDNA region (2), Charlesia (1), Exilicaulis (3), Lanata-Divaricata (7) and Stolkia (1). Each is characterised and described using Trichocomaceae classical morphology, LC-MS based extrolite analyses and multigene phylogenies based on ITS, BenA and CaM. Significant extrolites detected include andrastin, pulvilloric acid, penitrem A and citrinin amongst many others.
Article info Received: 28 January 2016; Accepted: 12 March 2016; Published: 9 May 2016.
Introduction species might actually function in nature. It was easy to assume that most species are generalists found wherever we care to Because of the standardised approach to delineating species of make isolations. Penicillium now available, with ex-type or representative strains In this paper, 15 species are described that originated from and barcode sequences designated for almost all described several studies in three Canadian laboratories over the past species, including those considered synonyms (Visagie et al. 20 years, which are now recognised as novel taxa because of 2014b), routine description of new species can now be ap- the more complete reference data. Three species were isolated proached with reasonable confidence. The exact set of genes from soil of termite mounds selectively eaten by chimpanzees used for multi-locus sequence typing (MLST) or Genealogical (Pan troglodytes) in the Mahale Mountains and Gombe areas of Concordance Phylogenetic Species Recognition (GCPSR) is Tanzania (Ketch 1998). Thirty-one genera were identified from not standardised among different laboratories, but most tend these termitarium soils and surrounding control soils. The most to use a similar core set of loci. Phenotypes such as irregular commonly isolated genus was Penicillium with over 42 species conidiophore branching, culture sectoring, and the relative recovered, the most common of which was Penicillium citrinum. abundance of conidia, sterile mycelium, soluble pigments or Clark (2002) isolated microfungi from the infrabuccal pockets of exudates can be interpreted as phylogenetically informative carpenter ants, especially Camponotus pennsylvaticus. These characters or artefacts of culture degeneration according to sac-like organs in the mouths of ants filter solids from their their correlation with robustly analysed DNA sequence data. liquid foods to be expelled later, and thus capture fungal mate- The historical focus of Penicillium taxonomy on strains isolated rial collected during feeding and grooming. About 60 strains of from food, soil and the indoor environment is understandable Penicillium were isolated from extracted infrabuccal masses given the importance of these fungi as spoilage agents, produc- from ants collected in New Brunswick (Canada), Germany and ers of potent mycotoxins, and sources of valuable antibiotics. the Netherlands, representing six morphospecies; three are de- Much of the expansion of the number of species in Penicillium scribed as new species here. A US National Science Foundation and its sister genus Talaromyces can be traced to the antibiotic Microbial Observatory study of caterpillar intestines from Costa revolution and the ease with which soil could be collected by Rica previously yielded two undescribed species, P. mallochii travellers and used to isolate new strains upon return to the and P. guanacastense that clearly were significant components laboratory. This practise, however, obscured how Penicillium of that niche, along with P. citrinum (Rivera et al. 2012). An ad- ditional new species, represented as a single strain among the 1 Biodiversity (Mycology), Ottawa Research and Development Centre, 88 isolated, is described here. Other new species described in Agriculture & Agri-Food Canada, 960 Carling Ave., Ottawa, Ontario, K1A this paper were isolated from leaf surfaces of tropical plants, 0C6, Canada; corresponding author e-mail: [email protected]. nuts in temperate hardwood forests, peat bogs and soil remote 2 London Research & Development Centre, Agriculture & Agri-Food Canada, London, Ontario, N5V 4T3, Canada. from agricultural regions. 3 New Brunswick Museum, 277 Douglas Avenue, Saint John, New Brunswick, Extrolite profiling brought a new dimension to Penicillium taxo- E2K 1E5, Canada. nomy about 25 years ago (Filtenborg et al. 1990), accompanied 4 268 Gledhill Ave, Toronto, Ontario, M4C 5L2, Canada. 5 Precision BioLogic Inc., 140 Eileen Stubbs Ave, Dartmouth, Nova Scotia, by a hypothesis that different isolates of a single species should B3B 0A9, Canada. produce a consistent profile of core extrolites. With the availabili 6 Department of Biology, University of Western Ontario, London, Ontario, ty of a new generation of LC-MS-MS technology, we generated N6A 5B7, Canada. extrolite profiles for all the new species described in this paper. 7 Centre de Recherche en Cancérologie de Lyon, INSERM U1052, 165 Chemin du petit Revoyet - BP 12, 69921 Oullins Cedex, France. Although reports are unavailable for the metabolites produced 8 Department of Biology, University of Ottawa, 30 Marie-Curie, Ottawa, by many species related to our new taxa, these observations Ontario, K1N 6N5, Canada. will be useful for future comparative chemotaxonomic studies.
© 2016 Naturalis Biodiversity Center & Centraalbureau voor Schimmelcultures You are free to share - to copy, distribute and transmit the work, under the following conditions: Attribution: You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). Non-commercial: You may not use this work for commercial purposes. No derivative works: You may not alter, transform, or build upon this work. For any reuse or distribution, you must make clear to others the license terms of this work, which can be found at http://creativecommons.org/licenses/by-nc-nd/3.0/legalcode. Any of the above conditions can be waived if you get permission from the copyright holder. Nothing in this license impairs or restricts the author’s moral rights. 248 Persoonia – Volume 36, 2016
Materials and Methods Newly generated sequences were submitted to GenBank under accession numbers KT887760–KT887876. Strains Gene sequences of the new species were compared with a Cultures were isolated either by direct or dilution plating from reference sequence dataset (Visagie et al. 2014b), with phylo- various substrates or niches as indicated in the species de- genies calculated for the relevant taxonomic sections in the scriptions, usually on 2 % malt extract agar with antibacterial system of Houbraken & Samson (2011). Datasets were aligned antibiotics. Strains were recovered from the working collections using MAFFT v. 7.221 (Katoh & Standley 2013), with the L-INS-i of Keith Seifert (Agriculture and Agri-Food Canada, Ottawa) algorithm selected for ITS and G-INS-i for BenA and CaM. and David Malloch (formerly University of Toronto), the latter Aligned datasets were adjusted and trimmed manually in Ge- now maintained in the Seifert laboratory. Ex-type and other neious. Datasets were subsequently analysed using Maximum representative strains were deposited into the Canadian Collec- Parsimony (MP) and Bayesian tree Inference (BI). MP heuristic tion of Fungal Cultures (DAOMC) and the CBS-KNAW Fungal searches were performed in PAUP v. 4.0b10 (Swofford 2003). Biodiversity Centre (CBS) in the Netherlands. BI analyses were run in MrBayes v. 3.2.5 (Ronquist & Huelsen- beck 2003). Model selections were made using MrModeltest v. Morphology 2.3 (Nylander et al. 2004) based on the lowest Akaike informa- tion criterion (AIC) value. Aligned datasets with the respective Colony characters were recorded from various media incu- command blocks for MP and BI were uploaded onto TreeBASE bated for 7 d, including Czapek yeast autolysate agar (CYA), (www.treebase.org) with accession no. S18638. Trees were Blakeslee’s (1915) malt extract agar (MEAbl), yeast extract prepared for publication in Adobe Illustrator CS6. sucrose agar (YES), oatmeal agar (OA) and creatine sucrose agar (CREA). BactoTM malt extract was used for MEAbl prepa- Extrolites ration. Media preparations, inoculations, incubation conditions and microscopic preparations followed the recommendations For extrolite analyses, all strains were grown in 9 cm polystyrene Petri dishes on YES (Frisvad 1981, Filtenborg et al. 1990) and by Visagie et al. (2014b). Colour names and codes used in de- CYA (Pitt 1980) incubated at 25 °C for 14 d. Strains were also scriptions are from Kornerup & Wanscher (1967). Microscopic grown in 200 mL of CYA broth incubated at 25 °C for 14 d. observations were made on an Olympus SZX12 dissecting Extrolites were extracted from the agar plates using an esta microscope and Olympus BX50 compound microscope equip blished method (Bragulat et al. 2001) with some modifications. ped with Infinity3 and InfinityX cameras driven by Infinity Ana Three agar plugs from each fungal isolate were removed with a lyze v. 6.5.1 software (Lumenera Corp., Ottawa, Canada). sterilised 7 mm cork borer and placed in a 7 mL glass scintilla- tion vial containing 1.5 mL of 75 % methanol in water. The vials DNA extraction, sequencing and phylogenetic analysis were vortexed for 30 s and sonicated at room temperature for Strains were grown on MEAbl for 7 d and DNA extracted using 30 min. The extrolites in CYA broth media were extracted using the UltracleanTM Microbial DNA isolation Kit (MoBio Laboratories 2× 150 mL ethyl acetate, combined, dried and reconstituted in Inc., Solana Beach, USA). ITS barcodes (Schoch et al. 2012), 1 mL of methanol. The solutions were filtered into 1.5 mL am- partial β-tubulin (BenA) and partial calmodulin (CaM) genes ber HPLC glass vials using 0.45 μm syringe filters. All extracts were amplified using the Illustra pureTaq Ready-To-GoTM PCR were analysed in both positive and negative ionization modes Beads (GE Healthcare Life Sciences) with conditions and pri- on a Thermo Q-Exactive Orbitrap coupled to an Agilent 1290 mers as suggested in Visagie et al. (2014b). Sequencing reac- HPLC. Extrolites were identified by comparison with authentic tions were set up with the same primer pairs and the Big Dye standards when possible. When standards were unavailable, Terminator Cycle Premix Kit with products run on the ABI compounds were putatively identified by screening the high- PRISMTM 310 DNA automated sequencer (Applied Biosys- resolution/accurate mass m/z signals with AntiBase2013 and tems, Waltham, USA). Contigs were assembled and edited in comparing product ions observed with those published in the Geneious v. 8.1.5 (BioMatters Ltd., Auckland, New Zealand). literature.
Table 1 Overview and details used for phylogenetic analyses.
Dataset
section section section section section Aspergilloides Canescentia Charlesia & Stolkiae Exilicaulis Lanata-Divaricata Length (bp) 550 510 532 513 502 Parsimony uninformative sites 34 31 22 51 41 Parsimony informative sites 43 12 100 49 59 ITS dataset Substitution model (BI) GTR+G+I HKY+I GTR+G+I GTR+G+I GTR+G+I
Length (bp) 450 403 465 489 535 Parsimony uninformative sites 52 37 17 68 80 Parsimony informative sites 168 110 197 197 215 BenA dataset Substitution model (BI) HKY+G+I HKY+G SYM+G+I HKY+G GTR+G+I
Length (bp) 521 388 557 527 571 Parsimony uninformative sites 55 41 25 47 65 Parsimony informative sites 207 130 236 241 255 CaM dataset Substitution model (BI) GTR+G+I K80+G GTR+I+G SYM+I+G GTR+G+I
Length (bp) 1521 1301 1554 1513 1608 Parsimony uninformative sites 141 109 64 164 188
Concate- nated dataset Parsimony informative sites 418 252 533 483 522 C.M. Visagie et al.: Fifteen new species of Penicillium 249
T ITS P. thiersii CBS117503 (KM189474) CaM 0.96/* P. hoeksii CBS137776T (KM089341) P. improvisum DAOMC 250546 (KT887866) T */* P. zhuangii CBS137464 (KF769422) T P. improvisum DAOMC 250547 (KT887867) -/81 P. infraaurantiacum CBS137747T (KM089317) P. improvisum DAOMC 250548 (KT887868) */* P. malmesburiense CBS137744T (KM089308) X4 T P. saturniforme CBS122276 (KM189585) P. sublectaticum CBS138217T (KM089397) T X4 */92 P. ranomafanaense CBS137953 (KM189541) T */* P. kananaskense CBS530.93 (KM089417) */* T P. verhagenii CBS137959 (KM189708) P. odoratum CBS294.62T (KM089363) X4 T P. infra-aurantiacum CBS137747 (KM189684) T */94 P. lividum CBS347.48 (KM089211) P. malmesburiense CBS137744T (KM189676) X4 */* P. improvisum DAOMC 250546 (KT887788) T P. sublectaticum CBS138217 (KM189761) P. improvisum DAOMC 250547T (KT887789) P. kananaskense CBS530.93T (KM189780) 0.99/90 P. improvisum DAOMC 250548 (KT887790) X2 T 0.96/- P. lividum CBS347.48 (KM189582) P. quercetorum CBS417.69T (KM089180) T P. odoratum CBS294.62 (KM189727) 0.96/* P. ranomafanaense CBS137953T (KM089164) T 0.99/- P. hoeksii CBS137776 (KM189707) P. verhagenii CBS137959T (KM089342) T P. zhuangii CBS137464 (KF769435) P. saturniforme CBS122276T (KM089214) T P. quercetorum CBS417.69 (KM189556) X4 P. thiersii CBS117503T (KM089094) T P. athertonense CBS138161 (KM189462) P. roseomaculatum CBS137962T (KM089391) T P. montanense CBS310.63 (KM189551) P. sterculiniicola CBS122426T (KM089078) T P. clavistipitatum CBS138650 (KM189667) */* P. grancanariae CBS687.77T (KM089151) T P. turcosoconidiatum CBS138557 (KM189645) P. palmense CBS336.79T (KM089150) T P. tsitsikammaense CBS328.71 (KM189451) P. subspinulosum CBS137946T (KM089104) T */82 P. brunneoconidiatum CBS137732 (KM189666) P. spinulosum CBS374.48T (KM089057) T P. flavisclerotiatum CBS137750 (KM189644) P. trzebinskii CBS382.48T (KM089421) T P. fuscum CBS295.62 (KM189547) */94 P. kiamaense CBS137947T (KM089128) T P. lapatayae CBS203.87 (KM189531) P. vagum CBS137728T (KM089273) T P. longicatenatum CBS137735 (KM189636) P. longicatenatum CBS137735T (KM089267) T P. austroafricanum CBS137773 (KM189610) P. armarii CBS138171T (KM089394) T P. crocicola CBS745.70 (KM189581) P. rudallense CBS130049T (KM089380) T P. subspinulosum CBS137946 (KM189483) T */- */86 P. frequentans CBS105.11 (KM089147) T P. kiamaense CBS137947 (KM189506) P. bussumense CBS138160T (KM089070) T P. vagum CBS137728 (KM189642) P. glabrum CBS125543T (KM089152) T P. armarii CBS138171 (KM189758) 0.99/83 T */95 P. pulvis CBS138432 (KM089263) T P. aurantioviolaceum CBS137777 (KM189756) P. purpurascens CBS366.48T (KM089186) T P. cartierense CBS137956 (KM189564) T */94 P. aurantioviolaceum CBS137777 (KM089392) T P. contaminatum CBS345.52 (KM189554) P. valentinum CBS172.81T (KM089173) T 0.99/91 P. purpurascens CBS366.48 (KM189561) P. fusisporum CBS137463T (KF769413) T P. roseoviride CBS267.35 (KM189549) P. roseoviride CBS267.35T (KM089172) T P. rudallense CBS130049 (KM189744) P. contaminatum CBS345.52T (KM089178) T P. thomii CBS225.81 (KM189560) P. yezoense CBS350.59T (KM089177) T P. valentinum CBS172.81 (KM189550) P. cartierense CBS137956T (KM089189) T P. yezoense CBS350.59 (KM189553) */94 P. thomii CBS225.81T (KM089184) T P. bussumense CBS138160 (KM189458) P. austroafricanum CBS137773T (KM089241) T */97 P. frequentans CBS105.11 (KM189525) P. crocicola CBS745.70T (KM089210) T P. glabrum CBS125543 (KM189530) P. grevilleicola CBS137775T (KM089261) T P. pulvis CBS138432 (KM189632) P. turcosoconidiatum CBS138557T (KM089276) T P. grancanariae CBS687.77 (KM189529) T */* P. athertonense CBS138161 (KM089075) T */- P. palmense CBS336.79 (KM189528) P. flavisclerotiatum CBS137750T (KM089275) T P. trzebinskii CBS382.48 (KM189784) P. brunneoconidiatum CBS137732T (KM089298) T P. fusisporum CBS137463 (KF769424) P. fuscum CBS295.62T (KM089170) T P. grevilleicola CBS137775 (KM189630) */88 P. lapatayae CBS203.87T (KM089153) T P. roseomaculatum CBS137962 (KM189755) P. clavistipitatum CBS138650T (KM089299) T P. spinulosum CBS374.48 (KM189448) P. montanense CBS310.63T (KM089174) T P. sterculiniicola CBS122426 (KM189464) P. tsitsikammaense CBS328.71T (KM089060) T P. ardesiacum CBS497.73 (KM189565) P. ardesiacum CBS497.73T (KM089190) T P. corylophilum CBS259.67 (KC411717) P. corylophilum CBS259.67T (KP016779) 5.0 20.0 Fig. 1 Phylogenetic trees for ITS & CaM datasets of Penicillium sect. Aspergilloides. Branch support in nodes higher than 80 % bs and/or 0.95 pp are indi- cated above thickened branches (T = ex-type; * = 100 % bs or 1.00 pp; - = support lower than 80 % bs and/or 0.95 pp). New species are indicated by coloured blocks. GenBank accession numbers are provided between brackets.
BenA T Concat P. thiersii CBS117503 (KM088709) P. thiersii CBS117503T P. saturniforme CBS122276T (KM088828) P. saturniforme CBS122276T */99 T T X4 P. ranomafanaense CBS137953 (KM088779) */* P. ranomafanaense CBS137953 X4 0.96/87 P. verhagenii CBS137959T (KM088955) T */97 P. verhagenii CBS137959 T T X4 */99 P. infra-aurantiacum CBS137747 (KM088930) P. infraaurantiacum CBS137747 X4 */* P. malmesburiense CBS137744T (KM088921) P. malmesburiense CBS137744T 0.99/- P. sublectaticum CBS138217T (KM089010) P. sublectaticum CBS138217T X4 P. improvisum DAOMC250546 (KT887827) X4 */* P. improvisum DAOMC250546 */* P. improvisum DAOMC250548 (KT887829) P. improvisum DAOMC250547T P. improvisum DAOMC250547T (KT887828) P. improvisum DAOMC250548 T */* P. hoeksii CBS137776 (KM088954) */* P. hoeksii CBS137776T P. zhuangii CBS137464T (KF769411) T X2 P. zhuangii CBS137464 P. quercetorum CBS417.69T (KM088795) T */* P. kananaskense CBS530.93 T 0.98/- T */* P. kananaskense CBS530.93 (KM089030) P. lividum CBS347.48 P. lividum CBS347.48T (KM088825) */- P. odoratum CBS294.62T P. odoratum CBS294.62T (KM088976) P. quercetorum CBS417.69T P. vagum CBS137728T (KM088886) P. longicatenatum CBS137735T P. longicatenatum CBS137735T (KM088880) P. vagum CBS137728T P. armarii CBS138171T (KM089007) P. kiamaense CBS137947T T P. rudallense CBS130049 (KM088993) 0.97/80 P. austroafricanum CBS137773T -/84 P. bussumense CBS138160T (KM088685) */98 P. crocicola CBS745.70T P. glabrum CBS125543T (KM088767) P. grevilleicola CBS137775T P. frequentans CBS105.11T (KM088762) P. aurantioviolaceum CBS137777T T -/84 T P. pulvis CBS138432 (KM088876) */* P. fusisporum CBS137463 P. purpurascens CBS366.48T (KM088801) */99 P. valentinum CBS172.81T P. kiamaense CBS137947T (KM088743) P. roseoviride CBS267.35T P. aurantioviolaceum CBS137777T (KM089005) P. cartierense CBS137956T T */98 P. roseoviride CBS267.35 (KM088787) P. thomii CBS225.81T */94 */90 P. fusisporum CBS137463T (KF769400) P. contaminatum CBS345.52T P. valentinum CBS172.81T (KM088788) P. yezoense CBS350.59T P. contaminatum CBS345.52T (KM088793) P. subspinulosum CBS137946T T T */83 P. yezoense CBS350.59 (KM088792) 0.99/- P. roseomaculatum CBS137962 T T P. thomii CBS225.81 (KM088799) 0.98/82 P. sterculiniicola CBS122426 */82 P. cartierense CBS137956T (KM088804) P. spinulosum CBS374.48T */* P. grevilleicola CBS137775T (KM088874) P. trzebinskii CBS382.48T T P. austroafricanum CBS137773 (KM088854) */* P. grancanariae CBS687.77T 0.98/- P. crocicola CBS745.70T (KM088824) P. palmense CBS336.79T T */* P. grancanariae CBS687.77 (KM088766) P. armarii CBS138171T */* P. palmense CBS336.79T (KM088765) P. rudallense CBS130049T */96 T T */- P. roseomaculatum CBS137962 (KM089004) P. purpurascens CBS366.48 T T P. spinulosum CBS374.48 (KM088672) */- P. bussumense CBS138160 P. trzebinskii CBS382.48T (KM089034) P. frequentans CBS105.11T P. subspinulosum CBS137946T (KM088719) P. glabrum CBS125543T 0.98/86 P. sterculiniicola CBS122426T (KM088693) P. pulvis CBS138432T P. montanense CBS310.63T (KM088789) P. athertonense CBS138161T P. clavistipitatum CBS138650T (KM088912) P. clavistipitatum CBS138650T */93 P. fuscum CBS295.62T (KM088785) P. turcosoconidiatum CBS138557T */* P. lapatayae CBS203.87T (KM088768) P. tsitsikammaense CBS328.71T P. flavisclerotiatum CBS137750T (KM088888) P. flavisclerotiatum CBS137750T P. tsitsikammaense CBS328.71T (KM088675) P. fuscum CBS295.62T T T P. athertonense CBS138161 (KM088690) */* */99 P. lapatayae CBS203.87 P. turcosoconidiatum CBS138557T (KM088889) P. montanense CBS310.63T P. brunneoconidiatum CBS137732T (KM088911) P. brunneoconidiatum CBS137732T P. ardesiacum CBS497.73T (KM088805) P. ardesiacum CBS497.73T P. corylophilum CBS259.67T (KP016754) P. corylophilum CBS259.67T 30.0 50.0 Fig. 2 Phylogenetic trees for BenA & Concatenated (ITS, BenA & CaM) datasets of Penicillium sect. Aspergilloides. Branch support in nodes higher than 80 % bs and/or 0.95 pp are indicated above thickened branches (T = ex-type; * = 100 % bs or 1.00 pp; - = support lower than 80 % bs and/or 0.95 pp). New species are indicated by coloured blocks. GenBank accession numbers are provided between brackets. 250 Persoonia – Volume 36, 2016
Results Phylogeny A local BLAST search placed the new species into sections As- Morphology pergilloides, Canescentia, Charlesia, Exilicaulis, Lanata-Diva- We introduce 15 new species in the Taxonomy section below. ricata and Stolkia. Phylogenies of each section were prepared These species belong to sections Aspergilloides, Canescentia, for the ITS region, BenA and CaM genes, as well as a conca Charlesia, Exilicaulis, Lanata-Divaricata and Stolkia based on tenation of the three. Phylogenies are presented in Fig. 1–8. the phylogenetic analyses. Strains conformed to the general The length, number of parsimony informative and uninforma- morphological characters previously observed for species ac- tive sites, and substitution models (for BI) for each dataset cepted in these sections. All of the new species were com- are summarised in Table 1. Tree topologies did not differ pared with respective close relatives, with notes provided on between MP and BI analyses and MP trees were thus used distinguishing characters after each species description in the for representing results, with BI posterior probability values taxonomy section.
P. canescens CBS300.48T (AF033493) */* P. nucicola DAOMC250521 (KT887768) ITS CaM T P. janczewskii CBS166.81 (KC411682) P. nucicola DAOMC250522 (KT887782) P. radiatolobatum CBS340.79T (KC411745) 0.99/84 P. canescens IMI149218 (KP027409) T P. yarmokense CBS410.69T (KC411757) P. canescens CBS300.48 (KJ867009) T P. jensenii CBS216.28 (JN617693) P. yarmokense CBS410.69 (KJ867013) T P. radiatolobatum IBT31959 (JX140833) 0.98/85 P. murcianum CBS161.81 (KP016824) P. murcianum CBS458.69 (KP016822) P. canescens IMI149218 (KP016841) 0.99/82 P. dunedinense CBS138218T (KJ775678) P. cf murcianum IBT31963 (JX157369) P. echinatum NRRL917T (KP016840) */94 P. cf murcianum CBS414.68 (KP016821) */89 T P. radiatolobatum IBT31959 (JX157354) P. janczewskii CBS221.28 (AY157487) 0.98/- P. janczewskii CBS413.68 (KP016838) P. radiatolobatum IBT31958 (JX157359) */95 P. nigricans CBS354.48T (KC411755) P. radiatolobatum CBS340.79T (KP016825) T P. griseoazureum CBS162.42T (KC411679) */95 P. echinatum NRRL917 (KJ867021) */94 T T P. murcianum CBS161.81 (KP016844) X4 P. griseoazureum CBS162.42 (KP016823) T P. radiatolobatum IBT31958 (JX140832) */98 P. nigricans CBS354.48 (KJ867012) T P. janczewskii CBS744.70 (KP016839) P. dunedinense CBS138218 (KJ775405) P. janczewskii CBS279.47 (KP016837) */98 P. janczewskii CBS166.81 (KJ866998) T P. murcianum CBS458.69 (KP016843) */96 P. janczewskii CBS221.28 (KJ867001) */96 P. jensenii CBS327.59T (AY443470) P. janczewskii CBS413.68 (KJ867014) P. cf murcianum IBT31963 (JX140834) P. janczewskii CBS279.47 (KJ867008) P. cf murcianum CBS414.68 (KP016842) P. janczewskii CBS744.70 (KJ867018) T X4 P. corvianum DAOMC250517T (KT887875) */* P. corvianum DAOMC250517 (KT887797) 0.99/- P. corvianum DAOMC250518 (KT887876) P. corvianum DAOMC250518 (KT887798) T P. nucicola DAOMC250521 (KT887846) */* P. jensenii CBS327.59 (AY443490) P. nucicola DAOMC250522T (KT887860) P. jensenii CBS216.28 (KJ867000) P. novaezeelandiae CBS137.41T (JN617688) P. novaezeelandiae CBS138942 (JX157468) X2 */* P. novaezeelandiae CBS546.77 (KP016846) P. novaezeelandiae CBS138956 (JX157402) P. novaezeelandiae CBS138942 (JX140854) P. novaezeelandiae CBS138955 (JX157401) 0.97/- P. novaezeelandiae CBS138949 (JX157353) P. novaezeelandiae CBS138936 (JX140853) */99 P. novaezeelandiae CBS138938 (JX140846) */* P. novaezeelandiae CBS138936 (JX157352) P. novaezeelandiae CBS138956 (JX140851) P. novaezeelandiae CBS138938 (JX157356) P. novaezeelandiae CBS138955 (JX140850) */99 P. novaezeelandiae CBS546.77 (KP016828) T P. novaezeelandiae CBS138949 (JX140835) P. novaezeelandiae CBS137.41 (KJ866996) T P. antarticum CBS116939 (KP016829) */* P. coralligerum CBS123.65 (KJ866994) P. antarticum CBS116938 (KP016845) P. coralligerum CBS114.69 (KJ866991) T 0.96/- P. coralligerum CBS123.65T (JN617667) */* P. atrovenetum CBS241.56 (KJ867004) P. coralligerum CBS114.69 (KP016836) P. atrovenetum CBS243.56 (JX157411) P. antarticum CBS116939 (JX157255) */83 P. atrovenetum CBS243.56 (KP016835) */* P. atrovenetum CBS241.56T (AF033492) P. antarticum CBS116938 (KP016827) T T X2 P. antarticum CBS100492 (KJ834503) P. antarticum CBS100492 (KP016826) T P. brevicompactum CBS257.29T (AY484912) P. brevicompactum CBS257.29 (AY484813) 5.0 20.0
BenA */* P. nucicola DAOMC250521 (KT887807) Concat */* P. nucicola DAOMC250522T T P. nucicola DAOMC250522 (KT887821) P. nucicola DAOMC250521 T */* P. novaezeelandiae CBS137.41 (KJ834477) */* P. canescens CBS300.48T P. novaezeelandiae CBS546.77 (KP016926) P. canescens IMI149218 */* P. novaezeelandiae CBS138942 (JX141061) T */99 P. yarmokense CBS410.69 P. novaezeelandiae CBS138956 (JX140976) */89 T */95 P. radiatolobatum CBS340.79 */96 P. novaezeelandiae CBS138955 (JX140975) 0.97/- P. radiatolobatum IBT31959 P. novaezeelandiae CBS138949 (JX140957) */98 */98 P. radiatolobatum IBT31958 P. novaezeelandiae CBS138936 (JX140956) T 0.98/- P. murcianum CBS161.81 P. novaezeelandiae CBS138938 (JX140958) P. murcianum CBS458.69 T */87 X4 */* P. canescens CBS300.48 (JX140946) */* P. cf murcianum IBT31963 P. canescens IMI149218 (JX140951) P. cf murcianum CBS414.68 */88 P. cf murcianum CBS414.68 (KP016922) */* P. jensenii CBS216.28 P. cf murcianum IBT31963 (JX140949) X4 P. jensenii CBS327.59T */- P. murcianum CBS458.69 (KP016923) P. janczewskii CBS166.81 T P. murcianum CBS161.81 (KP016924) 0.98/99 P. janczewskii CBS221.28T */90 P. radiatolobatum IBT31958 (JX140948) P. janczewskii CBS413.68 */90 P. radiatolobatum IBT31959 (JX140947) -/83 0.99/96 P. janczewskii CBS744.70 T P. radiatolobatum CBS340.79 (KP016920) P. janczewskii CBS279.47 T X4 P. yarmokense CBS410.69 (KJ834502) 0.98/99 P. dunedinense CBS138218T T */98 P. jensenii CBS327.59 (JX140954) P. echinatum NRRL917T P. jensenii CBS216.28 (KJ866963) P. griseoazureum CBS162.42T T */98 P. corvianum DAOMC250517 (KT887836) 0.98/84 */97 P. nigricans CBS354.48T P. corvianum DAOMC250518 (KT887837) */87 */* P. corvianum DAOMC250517T T P. dunedinense CBS138218 (KJ775171) X2 P. corvianum DAOMC250518 P. janczewskii CBS166.81 (KJ866967) */* P. novaezeelandiae CBS137.41T P. janczewskii CBS413.68 (KJ866969) P. novaezeelandiae CBS546.77 P. janczewskii CBS279.47 (KJ866968) */* P. novaezeelandiae CBS138942 P. janczewskii CBS744.70 (KJ866966) */* -/91 P. novaezeelandiae CBS138956 T P. janczewskii CBS221.28 (KJ834460) */95 P. novaezeelandiae CBS138955 T P. griseoazureum CBS162.42 (KP016919) P. novaezeelandiae CBS138936 T */* P. echinatum NRRL917 (KJ866964) P. novaezeelandiae CBS138938 T 0.98/85 P. nigricans CBS354.48 (KJ866965) P. novaezeelandiae CBS138949 T */* P. coralligerum CBS123.65 (KJ834444) */* P. coralligerum CBS123.65T P. coralligerum CBS114.69 (KJ866970) P. coralligerum CBS114.69 */* T 0.98/* P. atrovenetum CBS241.56 (JX140944) */* P. atrovenetum CBS243.56 P. atrovenetum CBS243.56 (JX140945) T 0.99/83 0.95/86 P. atrovenetum CBS241.56 P. antarticum CBS116939 (KP016921) P. antarticum CBS116939 */* P. antarticum CBS116938 (KP016925) */* P. antarticum CBS116938 T P. antarticum CBS100492 (KJ834432) P. antarticum CBS100492T T P. brevicompactum CBS257.29 (AY674437) P. brevicompactum CBS257.29T 20.0 30.0
Fig. 3 Phylogenetic trees for ITS, BenA, CaM & Concatenated (ITS, BenA & CaM) datasets of Penicillium sect. Canescentia. Branch support in nodes higher than 80 % bs and/or 0.95 pp are indicated above thickened branches (T = ex-type; * = 100 % bs or 1.00 pp; - = support lower than 80 % bs and/or 0.95 pp). New species are indicated by coloured blocks. GenBank accession numbers are provided between brackets. C.M. Visagie et al.: Fifteen new species of Penicillium 251 marked on relevant branches. The exception was the con- among P. radiatolobatum and P. murcianum strains. In the catenated dataset for section Aspergilloides, where P. longi- strict sense, current data assessed with the Genealogical catenatum was resolved in an unsupported clade with ML (Fig 2, Concordance Phylogenetic Species Recognition (GCPSR) indicated by red branch), whilst in the BI tree the latter two concept, suggests that these two represent synonyms and were resolved in a well-supported clade with the P. glabrum P. radiatolobatum (Publ. Soc. Nat. Rom. Pent. Stiinta Sol. 10B: and P. thomii clades, with P. longicatenatum a distant relative 435, 1971) an older name than P. murcianum (Mycopathologia to this main clade. 74: 37, 1981). Section Aspergilloides — Fig. 1, 2 Sections Charlesia and Stolkia — Fig. 4 Houbraken et al. (2014) reviewed section Aspergilloides A new species is introduced in section Charlesia, where spe- and divided it into 12 clades. All of our phylogenies show that cies are phylogenetically diverse enough for ITS to result in a P. improvisum strains comprise a well-supported 13th clade. positive identification. Phylogenies show the distinct identity of The new species can easily be identified using the ITS barcode, P. charlesii and P. fellutanum, with P. multicolor (CBS 501.73) with BenA and CaM also distinguishing the species. and P. ebenbitarianum (CBS 415.69) confirmed as synonyms Section Canescentia — Fig. 3 of the latter. The single strain of P. costaricense forms a branch Two new species belong to section Canescentia. Penicil distantly related to all other species of section Charlesia. lium corvianum is resolved in a distinct clade closely related Section Stolkia is a small section of rather rare species. As with to species of the P. canescens and P. janczewskii clades, section Charlesia, the ITS barcode can distinguish between the while P. nucicola is distinct from all other species. The ITS bar- six species and P. alogum described here. The concatenated code has poor discriminatory power in the section. BenA easily phylogeny resolves P. alogum as a close relative of P. stolkiae distinguishes the new species, but cannot reliably distinguish and P. subarcticum.
P. georgiense CBS132826T (EF422852) P. georgiense CBS132826T (EF506239) ITS */* CaM */* P. georgiense NRRL35507 (EF422850) P. georgiense NRRL35507 (EF506238) P. georgiense NRRL35508 (EF422851) P. georgiense NRRL35508 (EF506240) T */* T */* P. costaricense DAOMC250520 (KT887873) */* P. coffeae CBS119387 (AY741747) */98 P. coffeae CBS119387T (AY742702) P. phoeniceum CBS249.32T (AY741729) -/86 P. phoeniceum CBS249.32T (KC411711) P. costaricense DAOMC250520T (KT887795) -/* T T P. indicum CBS115.63 (AY742699) */* P. indicum CBS115.63 (AY741744) 0.98/98 P. indicum CBS179.81 (EU427291) P. indicum CBS179.81T (EU427283) P. fellutanum CBS229.81T (AF033399) P. fellutanum CBS229.81T (AY741753) 0.98/95 P. fellutanum NRRL35622 (EF200082) 0.98/87 P. fellutanum CBS415.69 (AY741751) */86 P. fellutanum NRRL35619 (EF200079) P. fellutanum NRRL35622 (EF198576) P. fellutanum CV1936 (JX091419) -/98 P. fellutanum NRRL35619 (EF198573) P. fellutanum CBS415.69 (KC411759) P. fellutanum CV1936 (JX141535) */* P. fellutanum CBS501.73 (KC790402) P. fellutanum CBS501.73 (JN799646) */* P. charlesii CV0646 (JX091417) P. charlesii CV2006 (JX141534) */* P. charlesii CV0744 (JX091418) P. charlesii CV1754 (JX141532) P. charlesii CV2006 (JX091416) P. charlesii CV1819 (JX141533) P. charlesii CV1754 (JX091414) -/88 P. charlesii CV0646 (JX141530) P. charlesii CV1819 (JX091415) 0.96/94 P. charlesii CV0744 (JX141531) P. charlesii CBS304.48T (AF033400) P. charlesii CBS304.48T (AY741727) T P. boreae CBS111717 (AF481122) */98 P. boreae CBS111717T (AF481138) P. pullum CBS331.48T (AF033443) P. pullum CBS331.48T (AF481134) 0.95/92 P. canariense CBS111720T (AF481121) P. stolkiae CBS315.67T (AF481135) -/83 P. stolkiae CBS315.67T (AF033444) P. subarcticum CBS111719T (AF481141) T P. subarcticum CBS111719 (AF481120) -/83 P. alogum DAOMC250543T (KT887791) */* P. alogum DAOMC250543T (KT887869) 0.99/99 P. alogum DAOMC250545 (KT887793) P. alogum DAOMC250544 (KT887870) */* P. alogum DAOMC250544 (KT887792) P. alogum DAOMC250545 (KT887871) P. canariense CBS111720T (AF481137) P. donkii CBS188.72T (AF033445) P. donkii CBS188.72T (AF481136) 10.0 40.0
BenA */* P. georgiense CBS132826T (EF506223) Concat */* P. georgiense CBS132826T */* P. georgiense NRRL35507 (EF506222) */* P. georgiense NRRL35507 P. georgiense NRRL35508 (EF506224) P. georgiense NRRL35508 */* T T P. costaricense DAOMC250520 (KT887834) */* P. costaricense DAOMC250520 */* P. indicum CBS115.63T (EU427263) */* P. coffeae CBS119387T */90 P. indicum CBS179.81 (EU427264) P. phoeniceum CBS249.32T */* P. coffeae CBS119387T (KJ834443) 0.99/87 */* P. indicum CBS115.63T -/91 P. phoeniceum CBS249.32T (KJ834483) P. indicum CBS179.81 P. fellutanum CBS229.81T (KJ834450) P. fellutanum CBS229.81T P. fellutanum CV1936 (JX091509) */94 P. fellutanum NRRL35619 */* P. fellutanum CBS501.73 (JN799645) P. fellutanum NRRL35622 X4 */* P. fellutanum CBS415.69T (JX091513) */* P. fellutanum CBS415.69 P. fellutanum NRRL35622 (EF198548) P. fellutanum CV1936 P. fellutanum NRRL35619 (EF198545) P. fellutanum CBS501.73 */* */* P. charlesii CV2006 (JX091507) P. charlesii CV2006 */* P. charlesii CV1754 (JX091505) P. charlesii CV1754 P. charlesii CV1819 (JX091506) P. charlesii CV1819 -/96 P. charlesii CV0646 (JX091503) -/98 P. charlesii CV0646 P. charlesii CV0744 (JX091504) P. charlesii CV0744 0.99/98 */* P. charlesii CBS304.48T (JX091508) P. charlesii CBS304.48T P. boreae CBS111717T (JN617715) */96 P. boreae CBS111717T 0.96/- P. canariense CBS111720T (JN617714) P. pullum CBS331.48T T */92 T -/84 P. alogum DAOMC250543 (KT887830) P. stolkiae CBS315.67 P. alogum DAOMC250545 (KT887832) */96 P. subarcticum CBS111719T -/84 P. alogum DAOMC250544 (KT887831) */98 P. alogum DAOMC250543T P. subarcticum CBS111719T (JN617716) P. alogum DAOMC250545 */* P. stolkiae CBS315.67T (JN617717) */* */* P. alogum DAOMC250544 P. pullum CBS331.48T (JN617719) P. canariense CBS111720T P. donkii CBS188.72T (JN617718) P. donkii CBS188.72T 30.0 60.0 Fig. 4 Phylogenetic trees for ITS, BenA, CaM & Concatenated (ITS, BenA & CaM) datasets of Penicillium sections Charlesia and Stolkia. Branch support in nodes higher than 80 % bs and/or 0.95 pp are indicated above thickened branches (T = ex-type; * = 100 % bs or 1.00 pp; - = support lower than 80 % bs and/or 0.95 pp). New species are indicated by coloured blocks. GenBank accession numbers are provided between brackets. 252 Persoonia – Volume 36, 2016
T ITS P. dravuni F01V25 (AY494856) CaM T T P. griseolum CBS277.58 (EF506232) P. griseolum CBS277.58 (EF422848) T T */* P. erubescens CBS318.67 (EU427281) P. catenatum CBS352.67 (KC411754) T T P. vinaceum CBS389.48 (HQ646586) P. parvum CBS359.48 (AF033460) T T */- P. rubidurum CBS609.73 (HQ646585) P. guttulosum NRRL907 (HQ646592) T P. pimiteouiense CBS102479T (AF037431) P. guttulosum NRRL907 (HQ646587) T X4 P. rubidurum CBS609.73T (AF033462) P. pimiteouiense CBS102479 (HQ646580) 0.96/- T P. menonorum NRRL50410T (HQ646591) P. menonorum NRRL50410 (HQ646584) T P. nepalense CBS203.84T (KC411692) P. parvum CBS359.48 (KF900173) */96 T P. vinaceum CBS389.48T (AF033461) P. nepalense CBS203.84 (KP016796) T P. striatisporum CBS705.68T (AF038938) P. catenatum CBS352.67 (KP016774) 0.95/95 T T */- P. striatisporum CBS705.68 (KP016807) */- P. dimorphosporum CBS456.70 (AF081804) T P. erubescens CBS318.67T (AF033464) X4 */98 P. canis NRRL62798 (KF900177) T P. canis NRRL62798T (KJ511291) P. dimorphosporum CBS456.70 (KP016783) X2 T P. ovatum CBS136664T (KF667370) */99 P. laeve CBS136665 (KF667367) T P. laeve CBS136665T (KF667369) P. ovatum CBS136664 (KF667368) T 0.98/- P. alutaceum CBS317.67T (AF033454) P. restrictum CBS367.48 (KP016803) T P. decumbens CBS230.81T (AY157490) P. meridianum CBS314.67 (KP016794) T P. cinerascens NRRL748T (AF033455) P. philippinense CBS623.72 (KP016799) T P. citreonigrum CBS258.29T (AF033456) P. kurssanovii CBS625.67 (KP016789) T X2 P. citreonigrum CBS308.48T (EF198647) P. katangense CBS247.67 (KP016788) P. arabicum CBS414.69T (KP016770) P. citreonigrum NRRL1187 (EF198646) */98 0.98/99 P. citreosulfuratum NRRL6172 (EF198650) P. albocinerascens CBS219.66T (KP016767) */94 X4 P. citreosulfuratum NRRL31271 (EF198660) P. cinereoatrum CBS222.66T (KP125335) P. citreosulfuratum IMI92228T (KP016814) P. heteromorphum CBS226.89T (KP016786) P. citreosulfuratum CBS139165 (JX140942) */* P. alutaceum CBS317.67T (KP016768) P. citreosulfuratum CBS139156 (JX140935) P. decumbens CBS230.81T (KP016782) P. citreosulfuratum CBS139158 (JX140939) -/86 P. melinii CBS139168 (JX157527) P. melinii CBS139169 (JX157528) P. citreosulfuratum CBS139162 (JX140940) 0.95/87 P. citreosulfuratum CBS139135 (JX140937) P. melinii CBS139143 (JX157452) */* P. melinii CBS280.58T (KP016801) P. citreosulfuratum CBS139140 (JX140943) */99 P. citreonigrum CBS321.59T (KP016812) P. melinii CBS139142 (JX157451) P. fundyense DAOMC250519T (KT887853) P. melinii CBS218.30T (KP016792) P. kurssanovii CBS625.67T (EF422849) */* ‘P. fynbos1’ CBS139163T (JX157495) 0.98/- ‘P. fynbos2’ CBS139136T (JX140913) ‘P. fynbos1’ DAOM241105 (JX157508) T 0.99/83 T ‘P. fynbos3’ CBS139144 (JX140888) */88 P. aotearoae DAOMC250538 (KT887796) ‘P. fynbos5’ CBS139138T (JX140900) P. diabolicalicense DAOMC250542T (KT887762) T T ‘P. fynbos6’ CBS139160 (JX140893) */97 P. smithii CBS276.83 (KP016806) T */99 T ‘P. fynbos7’ CBS139157 (JX140895) */88 ‘P. fynbos9’ CBS139134 (JX157526) T T ‘P. fynbos8’ CBS139132 (FJ231006) 0.97/- P. maclennaniae CBS198.81 (KP016791) ‘P. fynbos4’ CBS139166T (JX140898) 0.99/80 P. raciborskii CBS224.28T (KP016800) P. corylophilum CBS312.48T (AF033450) P. atrosanguineum CBS380.75T (KP016771) P. rubefaciens CBS145.83T (KC411677) P. namyslowskii CBS353.48T (KP016795) P. fagi CBS689.77T (AF481124) */98 P. terrenum CBS313.67T (KP016808) P. atrosanguineum CBS380.75T (JN617706) */99 P. burgense CBS325.89T (KP016772) P. maclennaniae CBS198.81T (KC411689) P. lapidosum CBS343.48T (FJ530984) T T P. namyslowskii CBS353.48 (AF033463) */94 ‘P. fynbos5’ CBS139138 (JX157418) P. velutinum CBS250.32T (AF033448) ‘P. fynbos3’ CBS139144T (JX157453) P. diabolicalicense DAOMC250542T (KT887840) ‘P. fynbos4’ CBS139166T (JX157519) T T P. smithii CBS276.83 (KC411723) */99 P. rubefaciens CBS145.83 (KP016804) P. melinii CBS280.58T (KC411727) ‘P. fynbos6’ CBS139160T (JX157490) T P. melinii CBS218.30 (AF033449) 0.99/92 ‘P. fynbos8’ CBS139132T (JX157532) P. melinii CBS139143 (JX140920) ‘P. fynbos7’ CBS139157T (JX157479) P. melinii CBS139168 (JX140917) P. velutinum CBS250.32T (KP016810) T P. melinii CBS139142 (JX140919) */86 */* -/90 P. corylophilum CBS312.48 (KP016780) P. aotearoae DAOMC250538T (KT887874) P. fagi CBS689.77T (KP016784) P. melinii CBS139169 (JX140918) ‘P. fynbos2’ CBS139136T (JX157416) T P. burgense CBS325.89 (KC411736) 0.99/88 P. fundyense DAOMC250519T (KT887775) P. raciborskii CBS224.28T (AF033447) P. cinerascens NRRL748T (JX157405) ‘P. fynbos9’ CBS139134T (FJ231003) P. citreonigrum CBS321.59T (KP016769) T P. terrenum CBS313.67 (AM992111) -/85 P. citreonigrum CBS308.48T (KP016778) P. lapidosum CBS343.48T (AF033409) P. citreonigrum NRRL1187 (EF198626) T */* ‘P. fynbos1’ CBS139163 (JX140921) P. citreonigrum CBS258.29T (EF198628) ‘P. fynbos1’ DAOM241105 (JX140924) P. citreosulfuratum NRRL6172 (EF198631) T P. restrictum CBS367.48 (AF033457) P. citreosulfuratum CBS139165 (JX157537) T P. cinereoatrum CBS222.66 (KC411700) */* P. citreosulfuratum CBS139158 (JX157485) T P. meridianum CBS314.67 (AF033451) P. citreosulfuratum CBS139135 (JX157415) T P. katangense CBS247.67 (AF033458) P. citreosulfuratum CBS139140 (JX157441) T P. philippinense CBS623.72 (KC411770) P. citreosulfuratum CBS139162 (JX157492) T P. arabicum CBS414.69 (KC411758) P. citreosulfuratum IMI92228T (KP016777) T P. heteromorphum CBS226.89 (KC411702) P. citreosulfuratum NRRL31271 (EF198641) T P. albocinerascens CBS219.66 (KP016811) P. citreosulfuratum CBS139156 (JX157536) T P. glabrum CBS125543 (KM189530) P. glabrum CBS125543T (KM089152) 5.0 30.0 Fig. 5 Phylogenetic trees for ITS & CaM datasets of Penicillium sect. Exilicaulis. Branch support in nodes higher than 80 % bs and/or 0.95 pp are indicated above thickened branches (T = ex-type; * = 100 % bs or 1.00 pp; - = support lower than 80 % bs and/or 0.95 pp). New species are indicated by coloured blocks. GenBank accession numbers are provided between brackets. Taxon labels in grey, indicate unpublished new species from Visagie et al. (2016b).
P. griseolum CBS277.58T (EF506213) BenA Concat P. griseolum CBS277.58T P. canis NRRL62798T (KF900167) 0.98/- P. catenatum CBS352.67T P. striatisporum CBS705.68T (JX141156) P. nepalense CBS203.84T 0.98/- P. erubescens CBS318.67T (HQ646566) */97 T T 0.99/- P. erubescens CBS318.67 P. parvum CBS359.48 (HQ646568) T T P. vinaceum CBS389.48 P. menonorum NRRL50410 (HQ646573) T T */- P. guttulosum NRRL907 P. guttulosum NRRL907 (HQ646576) */98 T T P. rubidurum CBS609.73 P. pimiteouiense CBS102479 (HQ646569) */* 0.99/- P. pimiteouiense CBS102479T */95 P. rubidurum CBS609.73T (HQ646574) P. menonorum NRRL50410T P. vinaceum CBS389.48T (HQ646575) */97 */99 P. parvum CBS359.48T P. catenatum CBS352.67T (KJ834438) */97 P. striatisporum CBS705.68T P. nepalense CBS203.84T (KJ834474) */* P. canis NRRL62798T 0.99/99 P. dimorphosporum CBS456.70T (KJ834448) P. dimorphosporum CBS456.70T */82 P. laeve CBS136665T (KF667365) */* P. ovatum CBS136664T P. ovatum CBS136664T (KF667366) P. laeve CBS136665T P. cinerascens NRRL748T (JX141041) X4 X2 */* P. alutaceum CBS317.67T P. citreonigrum CBS258.29T (EF198621) -/82 P. decumbens CBS230.81T P. citreonigrum CBS308.48T (EF198623) */87 T -/83 P. cinerascens NRRL748 P. citreonigrum NRRL1187 (EF198622) T T P. fundyense DAOMC250519 P. citreonigrum CBS321.59 (KP016749) -/85 P. citreonigrum CBS258.29T P. fundyense DAOMC250519T (KT887814) 0.99/95 P. citreonigrum CBS308.48T P. citreosulfuratum IMI92228T (KP016753) */* P. citreonigrum NRRL1187 P. citreosulfuratum CBS139156 (JX141164) */* P. citreonigrum CBS321.59T P. citreosulfuratum CBS139140 (JX141163) P. citreosulfuratum NRRL6172 -/92 P. citreosulfuratum CBS139165 (JX141169) P. citreosulfuratum NRRL31271 P. citreosulfuratum CBS139158 (JX141167) */* P. citreosulfuratum IMI92228T P. citreosulfuratum CBS139135 (JX141162) */99 P. citreosulfuratum CBS139165 P. citreosulfuratum CBS139162 (JX141168) */* P. citreosulfuratum CBS139156 P. citreosulfuratum NRRL31271 (EF198614) -/91 P. citreosulfuratum CBS139158 */99 P. citreosulfuratum NRRL6172 (EF198620) X4 P. citreosulfuratum CBS139162 */* P. alutaceum CBS317.67T (KJ834430) X2 P. citreosulfuratum CBS139135 P. decumbens CBS230.81T (KJ834446) T P. citreosulfuratum CBS139140 ‘P. fynbos2’ CBS139136 (JX141077) T T ‘P. fynbos2’ CBS139136 P. corylophilum CBS312.48 (JX141042) 0.99/85 T T ‘P. fynbos3’ CBS139144 ‘P. fynbos4’ CBS139166 (JX141070) -/92 T T ‘P. fynbos6’ CBS139160 */- ‘P. fynbos6’ CBS139160 (JX141150) -/81 T T ‘P. fynbos8’ CBS139132 ‘P. fynbos8’ CBS139132 (JX141161) */84 T T ‘P. fynbos7’ CBS139157 */97 P. rubefaciens CBS145.83 (KJ834487) T 0.96/- T */98 P. rubefaciens CBS145.83 ‘P. fynbos7’ CBS139157 (JX141073) */* ‘P. fynbos4’ CBS139166T ‘P. fynbos3’ CBS139144T (JX141135) */* ‘P. fynbos5’ CBS139138T ‘P. fynbos5’ CBS139138T (JX141076) */86 P. corylophilum CBS312.48T P. fagi CBS689.77T (KJ834449) T T P. fagi CBS689.77 */86 P. atrosanguineum CBS380.75 (KJ834435) T T P. velutinum CBS250.32 P. raciborskii CBS224.28 (JX141069) */97 T T */* P. atrosanguineum CBS380.75 0.99/- ‘P. fynbos9’ CBS139134 (JX141048) */89 P. raciborskii CBS224.28T */89 T */* P. maclennaniae CBS198.81 (KJ834468) X4 T T P. namyslowskii CBS353.48 */97 P. velutinum CBS250.32 (JX141170) */98 T T P. burgense CBS325.89 P. smithii CBS276.83 (KJ834492) */* T X2 T P. terrenum CBS313.67 P. melinii CBS280.58 (KP016763) T */89 P. lapidosum CBS343.48 P. melinii CBS139168 (JX141052) */93 P. maclennaniae CBS198.81T */93 P. melinii CBS139169 (JX141053) */90 T T ‘P. fynbos9’ CBS139134 P. melinii CBS218.30 (KJ834471) P. smithii CBS276.83T P. melinii CBS139143 (JX141051) */94 */84 P. diabolicalicense DAOMC250542T */95 0.96/- P. melinii CBS139142 (JX141050) P. melinii CBS280.58T P. aotearoae DAOMC250538T (KT887835) -/87 T P. melinii CBS139168 */* ‘P. fynbos1’ CBS139163 (JX141120) */* */92 P. melinii CBS139169 ‘P. fynbos1’ DAOM241105 (JX141125) P. melinii CBS218.30T P. diabolicalicense DAOMC250542T (KT887801) */- P. melinii CBS139143 -/85 T P. burgense CBS325.89 (KJ834437) */98 */* T 0.99/- P. melinii CBS139142 P. terrenum CBS313.67 (KJ834496) T */* ‘P. fynbos1’ CBS139163 P. lapidosum CBS343.48T (KJ834465) T ‘P. fynbos1’ DAOM241105 P. namyslowskii CBS353.48 (JX141067) T T P. aotearoae DAOMC250538 P. philippinense CBS623.72 (KJ834482) T T P. cinereoatrum CBS222.66 0.99/* 0.98/97 P. arabicum CBS414.69 (KP016750) T T P. heteromorphum CBS226.89 P. restrictum CBS367.48 (KJ834486) T T P. arabicum CBS414.69 P. cinereoatrum CBS222.66 (KJ834442) T T P. philippinense CBS623.72 P. heteromorphum CBS226.89 (KJ834455) T 0.99/- T P. restrictum CBS367.48 P. katangense CBS247.67 (KP016757) T T P. meridianum CBS314.67 P. meridianum CBS314.67 (KJ834472) T T P. katangense CBS247.67 P. kurssanovii CBS625.67 (KP016758) T T P. kurssanovii CBS625.67 P. albocinerascens CBS219.66 (KP016748) T T P. albocinerascens CBS219.66 P. glabrum CBS125543 (KM088767) P. glabrum CBS125543T 20.0 40.0 Fig. 6 Phylogenetic trees for BenA & Concatenated (ITS, BenA & CaM) datasets of Penicillium sect. Exilicaulis. Branch support in nodes higher than 80 % bs and/or 0.95 pp are indicated above thickened branches (T = ex-type; * = 100 % bs or 1.00 pp; - = support lower than 80 % bs and/or 0.95 pp). New species are indicated by coloured blocks. GenBank accession numbers are provided between brackets. Taxon labels in grey, indicate unpublished new species from Visagie et al. (2016b). C.M. Visagie et al.: Fifteen new species of Penicillium 253
ITS P. amphipolaria DAOMC250549 (KT887838) CaM P. araracuaraense CBS113149T (KF296373) T -/80 P. amphipolaria DAOMC250550 (KT887839) P. wotroi CBS118171 (KF296369) T P. amphipolaria DAOMC250551 (KT887872) P. mariaecrucis CBS271.83T (KF296374) */89 T P. singorense CBS138214 (KJ775674) P. cataractum DAOMC250533 (KT887786) 0.96/- T */* P. penarojense CBS113178 (GU981570) P. cataractum DAOMC250534T (KT887769) P. vanderhammenii CBS126216T (GU981574) T P. cataractum DAOMC250535 (KT887787) P. oxalicum CBS219.30 (AF033438) -/91 P. onobense CBS174.81T (KF296371) T P. griseopurpureum CBS406.65 (KF296408) -/96 T T P. paraherquei CBS338.59 (KF296372) P. daleae CBS211.28 (GU981583) T T -/99 P. brasilianum CBS253.55 (AB667857) P. abidjanum CBS246.67 (GU981582) 0.95/88 P. skrjabinii CBS439.75T (KF296370) 0.98/- T P. lineolatum CBS188.77 (GU981579) */93 P. skrjabinii NRRL13055 (EU427280) P. ehrlichii CBS324.48T (AF033432) T P. skrjabinii DTO180I3 (JX141544) 0.99/92 P. brefeldianum CBS235.81 (AF033435) T T -/81 P. simplicissimum CBS372.48 (KF296368) P. limosum CBS339.97 (GU981568) */99 T P. panissanguineum DAOMC250561 (KT887783) P. zonatum CBS992.72 (GU981581) P. panissanguineum DAOMC250562T (KT887784) P. curticaule CBS135127T (FJ231021) */* P. infrabuccalum DAOMC250536 (KT887771) P. cluniae CBS326.89T (KF296406) P. infrabuccalum DAOMC250537T (KT887778) P. janthinellum CBS340.48T (GU981585) P. tanzanicum DAOMC250514T (KT887763) P. ortum CBS135669T (JX091427) */* P. tanzanicum DAOMC250515 (KT887764) P. ortum CBS135670 (JX091428) T P. tanzanicum DAOMC250516 (KT887785) P. cremeogriseum CBS223.66 (GU981586) P. penarojense CBS113178T (KF296381) P. ludwigii CBS417.68T (KF296409) */92 P. vanderhammenii CBS126216T (KF296382) P. koreense KACC47721T (KJ801939) */97 P. singorense CBS138214T (KJ775403) P. glaucoroseum NRRL908T (KF296407) */94 P. amphipolaria DAOMC250549 (KT887760) P. raperi CBS281.58T (AF033433) 0.99/- P. amphipolaria DAOMC250550 (KT887761) P. meloforme CBS445.74T (KC411762) 0.97/99 P. amphipolaria DAOMC250551T (KT887794) */83 P. coeruleum CBS141.45T (GU981606) 0.98/- 0.96/- P. daleae CBS211.28T (KF296385) P. levitum CBS345.48T (GU981607) 0.99/- P. griseopurpureum CBS406.65T (KF296384) P. javanicum CBS341.48T (GU981613) P. abidjanum CBS246.67T (KF296383) 0.99/84 P. javanicum CBS349.51 (KC411752) P. zonatum CBS992.72T (KF296380) P. caperatum CBS443.75T (KC411761) P. svalbardense CBS122416T (KC346338) P. reticulisporum CBS122.68T (AF033437) */* P. ochrochloron CBS133150 (KC346341) P. reticulisporum CBS513.74 (GU981618) P. ochrochloron CBS357.48T (KF296378) P. malacosphaerulum CBS135120T (FJ231026) P. malacosphaerulum CBS135121 (FJ231024) P. camponotum DAOMC250552 (KT887770) P. elleniae CBS118135T (GU981612) P. camponotum DAOMC250553 (KT887772) P. camponotum DAOMC250554 (KT887773) P. tanzanicum DAOMC250514T (KT887841) 0.98/86 P. camponotum DAOMC250555 (KT887774) P. tanzanicum DAOMC250515 (KT887842) P. camponotum DAOMC250556 (KT887776) P. tanzanicum DAOMC250516 (KT887863) P. camponotum DAOMC250557T (KT887777) P. subrubescens CBS129543 (KC797635) 0.99/* P. subrubescens CBS129617 (KC797636) P. camponotum DAOMC250558 (KT887779) P. subrubescens CBS130052 (KC797637) P. camponotum DAOMC250559 (KT887780) P. subrubescens CBS131061 (KC797639) P. camponotum DAOMC250560 (KT887781) P. subrubescens CBS134204 (KC797642) P. subrubescens CBS129543 (KC797590) -/81 P. subrubescens CBS129617 (KC797591) P. subrubescens CBS134205 (KC797643) T P. subrubescens CBS130205 (KC797638) P. subrubescens CBS132785 (KC346330) P. subrubescens CBS134204 (KC797597) P. subrubescens CBS131064 (KC797640) */* P. subrubescens CBS132785T (KC346350) P. subrubescens CBS134205 (KC797598) P. piscarium CBS362.48T (GU981600) */87 P. subrubescens CBS130052 (KC797592) P. camponotum DAOMC250552 (KT887848) P. subrubescens CBS131061 (KC797594) P. camponotum DAOMC250553 (KT887850) */95 P. subrubescens CBS130205 (KC797593) P. camponotum DAOMC250554 (KT887851) P. subrubescens CBS131064 (KC797595) */* P. camponotum DAOMC250555 (KT887852) P. pulvillorum CBS275.83 (KF296376) P. pulvillorum CBS280.39T (KF296377) 0.98/85 P. camponotum DAOMC250556 (KT887854) T P. camponotum DAOMC250557T (KT887855) P. rolfsii CBS368.48 (KF296375) 0.99/84 0.98/93 P. annulatum CBS135123 (JX141547) P. camponotum DAOMC250558 (KT887857) */99 T P. camponotum DAOMC250559 (KT887858) P. annulatum CBS135126 (JX141545) P. annulatum CBS135124 (JX141548) P. camponotum DAOMC250560 (KT887859) T T P. svalbardense CBS122416 (GU981603) 0.98/90 P. piscarium CBS362.48 (KF296379) P. excelsum ITAL7572T (KR815342) P. ochrochloron CBS133150 (KC346347) */* 0.97/- P. ochrochloron CBS357.48T (GU981604) P. excelsum ITAL7804 (KT749962) P. pulvillorum CBS275.83 (AF178513) P. excelsum ITAL7814 (KT749961) T P. pulvillorum CBS280.39T (AF178517) */* P. bissettii DAOM167011 (KT887767) P. vasconiae CBS339.79T (GU981599) P. bissettii DAOM167033 (KT887766) T P. annulatum CBS135123 (JX091423) P. vasconiae CBS339.79 (KF296386) T P. annulatum CBS135124 (JX091424) P. elleniae CBS118135 (KF296389) T P. annulatum CBS135126T (JX091426) 0.99/97 P. reticulisporum CBS122.68 (KF296391) P. rolfsii CBS368.48T (JN617705) -/98 P. reticulisporum CBS513.74 (KF296390) T */* P. malacosphaerulum CBS135120T (JX141542) */98 P. excelsum ITAL7572 (KR815341) P. excelsum ITAL7804 (KT749963) P. malacosphaerulum CBS135121 (JX141540) T P. excelsum ITAL7814 (KT749964) P. caperatum CBS443.75 (KF296392) T P. wotroi CBS118171T (GU981591) */* P. javanicum CBS341.48 (KF296387) P. infrabuccalum DAOMC250536 (KT887849) P. javanicum CBS349.51 (KF296388) T P. infrabuccalum DAOMC250537T (KT887856) P. curticaule CBS135127 (JX141536) P. araracuaraense CBS113149T (GU981597) -/98 0.99/ P. ortum CBS135669T (JX141551) P. mariaecrucis CBS271.83T (GU981593) P. ortum CBS135670 (JX141552) */* T P. simplicissimum CBS372.48T (GU981588) -/85 P. cremeogriseum CBS223.66 (KF296403) T P. cataractum DAOMC250533 (KT887864) */88 P. cluniae CBS326.89 (KF296402) */92 T P. cataractum DAOMC250534T (KT887847) 0.98/- P. janthinellum CBS340.48 (KF296401) P. glaucoroseum NRRL908T (KF296400) P. cataractum DAOMC250535 (KT887865) 0.99/81 -/* P. onobense CBS174.81T (GU981575) -/91 0.97/84 P. ludwigii CBS417.68T (KF296404) P. paraherquei CBS338.59T (AF178511) P. raperi CBS281.58T (KF296399) X4 T */* T -/86 P. brasilianum CBS253.55 (GU981577) P. brefeldianum CBS235.81 (EU021683) P. skrjabinii CBS439.75T (GU981576) P. limosum CBS339.97T (KF296398) P. skrjabinii DTO180I3 (JX091432) P. lineolatum CBS188.77T (KF296397) P. skrjabinii NRRL13055 (EU427287) */* P. coeruleum CBS141.45T (KF296393) P. panissanguineum DAOMC250561 (KT887861) P. levitum CBS345.48T (KF296394) P. panissanguineum DAOMC250562T (KT887862) P. meloforme CBS445.74T (KF296396) P. bissettii DAOM167011T (KT887845) P. ehrlichii CBS324.48T (KF296395) P. bissettii DAOM167033 (KT887844) P. oxalicum CBS219.30T (KF296367) P. glabrum CBS125543T (KM189530) P. glabrum CBS125543T (KM089152) 8.0 30.0 Fig. 7 Phylogenetic trees for ITS & CaM datasets of Penicillium sect. Lanata-Divaricata. Branch support in nodes higher than 80 % bs and/or 0.95 pp are indicated above thickened branches (T = ex-type; * = 100 % bs or 1.00 pp; - = support lower than 80 % bs and/or 0.95 pp). New species are indicated by coloured blocks. GenBank accession numbers are provided between brackets.
BenA P. cluniae CBS326.89T (KF296471) Concat P. janthinellum CBS340.48T (GU981625) P. cluniae CBS326.89T */85 P. ludwigii CBS417.68T (KF296468) */93 P. janthinellum CBS340.48T */99 P. glaucoroseum NRRL908T (KF296469) P. glaucoroseum NRRL908T T T */* P. ortum CBS135669 (JX091520) */* P. ludwigii CBS417.68 */- P. ortum CBS135670 (JX091521) */* P. ortum CBS135669T P. cremeogriseum CBS223.66T (GU981624) */- P. ortum CBS135670 */99 P. brefeldianum CBS235.81T (GU981623) P. cremeogriseum CBS223.66T P. limosum CBS339.97T (GU981621) */- */* P. brefeldianum CBS235.81T 0.99/- T */97 P. raperi CBS281.58 (GU981622) */99 P. limosum CBS339.97T T T */98 P. koreense KACC47721 (KM000846) */* P. raperi CBS281.58 P. curticaule CBS135127T (JX091526) P. curticaule CBS135127T P. lineolatum CBS188.77T (GU981620) P. lineolatum CBS188.77T */* P. reticulisporum CBS122.68T (GU981665) */* P. reticulisporum CBS122.68T P. reticulisporum CBS513.74 (GU981666) P. reticulisporum CBS513.74 */99 P. malacosphaerulum CBS135120T (JX091524) */* P. malacosphaerulum CBS135120T P. malacosphaerulum CBS135121 (JX091525) 0.97/- P. malacosphaerulum CBS135121 0.98/- P. caperatum CBS443.75T (GU981660) */* P. javanicum CBS341.48T */98 0.96/- */* P. javanicum CBS341.48T (GU981657) P. javanicum CBS349.51 -/88 P. javanicum CBS349.51 (KF296466) -/88 P. caperatum CBS443.75T P. elleniae CBS118135T (GU981663) P. elleniae CBS118135T */* P. coeruleum CBS141.45T (GU981655) */* P. coeruleum CBS141.45T P. levitum CBS345.48T (GU981654) P. levitum CBS345.48T P. ehrlichii CBS324.48T (KF296464) P. ehrlichii CBS324.48T P. meloforme CBS445.74T (GU981656) P. meloforme CBS445.74T 0.98/- P. amphipolaria DAOMC250550 (KT887800) P. amphipolaria DAOMC250550 */* P. amphipolaria DAOMC250551T (KT887833) */* P. amphipolaria DAOMC250551T */97 */99 P. amphipolaria DAOMC250549 (KT887799) P. amphipolaria DAOMC250549 */97 P. singorense CBS138214T (KJ775167) */99 P. singorense CBS138214T */97 P. penarojense CBS113178T (GU981646) */98 P. penarojense CBS113178T P. vanderhammenii CBS126216T (GU981647) P. vanderhammenii CBS126216T */99 P. daleae CBS211.28T (GU981649) */- */97 P. daleae CBS211.28T T T P. griseopurpureum CBS406.65 (KF296467) */82 P. griseopurpureum CBS406.65 P. abidjanum CBS246.67T (GU981650) P. abidjanum CBS246.67T P. zonatum CBS992.72T (GU981651) P. zonatum CBS992.72T */91 P. araracuaraense CBS113149T (GU981642) P. subrubescens CBS129543 P. wotroi CBS118171T (GU981637) P. subrubescens CBS132785T P. simplicissimum CBS372.48T (GU981632) P. subrubescens CBS129617 T P. subrubescens CBS134204 */* P. tanzanicum DAOMC250514 (KT887802) P. tanzanicum DAOMC250515 (KT887803) */* P. subrubescens CBS134205 P. tanzanicum DAOMC250516 (KT887824) */97 P. subrubescens CBS130052 P. subrubescens CBS131061 */* P. cataractum DAOMC250533 (KT887825) P. cataractum DAOMC250534T (KT887808) */- P. subrubescens CBS130205 P. cataractum DAOMC250535 (KT887826) P. subrubescens CBS131064 0.97/- P. mariaecrucis CBS271.83T (GU981630) P. camponotum DAOMC250552 */* P. infrabuccalum DAOMC250536 (KT887810) P. camponotum DAOMC250553 T P. infrabuccalum DAOMC250537 (KT887817) */87 P. camponotum DAOMC250554 */* P. panissanguineum DAOMC250561 (KT887822) P. camponotum DAOMC250555 0.97/- P. panissanguineum DAOMC250562T (KT887823) P. camponotum DAOMC250556 0.99/91 T T P. skrjabinii CBS439.75 (GU981626) */* P. camponotum DAOMC250557 */95 P. skrjabinii NRRL13055 (EU427271) P. camponotum DAOMC250558 P. skrjabinii DTO180I3 (JX091529) P. camponotum DAOMC250559 T */* P. onobense CBS174.81 (GU981627) P. camponotum DAOMC250560 P. paraherquei CBS338.59T (KF296465) */- */* P. ochrochloron CBS133150 P. brasilianum CBS253.55T (GU981629) */- P. ochrochloron CBS357.48T P. vasconiae CBS339.79T (GU981653) P. svalbardense CBS122416T */* P. bissettii DAOM167011T (KT887806) */* P. pulvillorum CBS275.83 P. bissettii DAOM167033 (KT887805) P. pulvillorum CBS280.39T P. annulatum CBS135124 (JX091517) P. piscarium CBS362.48T */* P. annulatum CBS135126T (JX091514) P. rolfsii CBS368.48T T */81 P. excelsum ITAL7572 P. annulatum CBS135123 (JX091516) */* T P. excelsum ITAL7804 */* P. excelsum ITAL7572 (KP691061) P. excelsum ITAL7804 (KT749959) P. excelsum ITAL7814 P. excelsum ITAL7814 (KT749957) */80 P. annulatum CBS135123 */* */* P. pulvillorum CBS275.83 (KF296463) P. annulatum CBS135126T P. pulvillorum CBS280.39T (GU981670) */- P. annulatum CBS135124 */* P. ochrochloron CBS133150 (KC346324) */* P. bissettii DAOM167011T P. ochrochloron CBS357.48T (GU981672) P. bissettii DAOM167033 P. subrubescens CBS129543 (KC797610) P. vasconiae CBS339.79T T 0.96/- P. subrubescens CBS130205 (KC797613) */* P. tanzanicum DAOMC250514 P. subrubescens CBS131064 (KC797615) P. tanzanicum DAOMC250515 T P. tanzanicum DAOMC250516 0.99/- P. subrubescens CBS132785 (KC346327) P. subrubescens CBS130052 (KC797612) */* P. panissanguineum DAOMC250561 */* P. subrubescens CBS131061 (KC797614) P. panissanguineum DAOMC250562T 0.97/- P. subrubescens CBS129617 (KC797611) P. simplicissimum CBS372.48T P. subrubescens CBS134204 (KC797617) */94 P. onobense CBS174.81T */93 P. subrubescens CBS134205 (KC797618) P. paraherquei CBS338.59T T P. camponotum DAOMC250552 (KT887809) */* P. brasilianum CBS253.55 T P. camponotum DAOMC250553 (KT887811) */98 P. skrjabinii CBS439.75 0.95/- */* P. camponotum DAOMC250554 (KT887812) P. skrjabinii NRRL13055 P. camponotum DAOMC250555 (KT887813) P. skrjabinii DTO180I3 T P. camponotum DAOMC250556 (KT887815) */95 P. araracuaraense CBS113149 T P. wotroi CBS118171T */98 P. camponotum DAOMC250557 (KT887816) P. camponotum DAOMC250558 (KT887818) */* P. cataractum DAOMC250533 P. camponotum DAOMC250559 (KT887819) P. cataractum DAOMC250534T P. camponotum DAOMC250560 (KT887820) P. cataractum DAOMC250535 P. svalbardense CBS122416T (KC346325) P. mariaecrucis CBS271.83T P. piscarium CBS362.48T (GU981668) */* P. infrabuccalum DAOMC250536 P. rolfsii CBS368.48T (GU981667) P. infrabuccalum DAOMC250537T X4 T -/97 P. oxalicum CBS219.30T (KF296462) -/* P. oxalicum CBS219.30 P. glabrum CBS125543T (KM088767) P. glabrum CBS125543T 30.0 X2 70.0 Fig. 8 Phylogenetic trees for BenA & Concatenated (ITS, BenA & CaM) datasets of Penicillium sect. Lanata-Divaricata. Branch support in nodes higher than 80 % bs and/or 0.95 pp are indicated above thickened branches (T = ex-type; * = 100 % bs or 1.00 pp; - = support lower than 80 % bs and/or 0.95 pp). New species are indicated by coloured blocks. GenBank accession numbers are provided between brackets. 254 Persoonia – Volume 36, 2016
Section Exilicaulis — Fig. 5, 6 having unique sequences. BenA and CaM also easily distin- Three new species are introduced in section Exilicaulis. guish all species of the section. The concatenated phylogeny Sequence datasets contain some undescribed species from the resolved P. amphipolaria as a close relative of P. singorense; fynbos, South Africa (in grey, Visagie et al. 2016b). ITS generally P. camponotum as closely related to P. subrubescens; while has poor resolution in this section. Of the new species, P. dia- P. tanzanicum, P. bissettii, P. cataractum, P. infrabuccalum and bolicalicense and P. aotearoae share identical sequences with P. panissanguineum resolved in several clades on a poorly sup- previously known species, whereas P. fundyense has a unique ported back bone typically having species with rough walled ITS sequence. The BenA and CaM genes distinguish the new conidiophores such as P. simplicissimum. species from its close relatives. Penicillium fundyense sits in a clade with P. cinerascens, P. citreonigrum and P. citreo Extrolites sulfuratum (= P. toxicarium fide Serra et al. 2008), recently An overview of the extrolites detected during this study and reviewed in Visagie et al. (2016b). The single strain DAOMC their product ions are summarised in Table 2, while Table 3 250519 was sufficiently distinct from other strains in the sec- summarises the extrolites produced by each species analysed. tion to propose a new species. Penicillium aotearoae and We also list extrolites produced by each species with their P. diabolicalicense is resolved in the P. melinii clade closely respective descriptions in the taxonomy section. related to P. melinii and P. xanthomelinii. Six of the Penicillium species studied here produced andrastin Section Lanata-Divaricata — Fig. 7, 8 metabolites in varying amounts. Penicillium cataractum and ITS performs remarkably well for identification of the seven P. infrabuccalum were both major producers of andrastin A–D. new species we introduce in section Lanata-Divaricata, with all Penicillium nucicola, P. panissanguineum and two strains of
Table 2 Overview of the major extrolites detected and product ions.
Code m/z Formula Product ion m/z
andrastin A 487.2687 C28H38O7 243.1743 395.2229 427.1995 349.2160 225.1637 andrastin B* 487.2702 C28H40O7 427.2486 455.2455 457.2603 419.0410 andrastin C 473.2898 C28H40O6 203.1431 179.0702 441.2272 381.2057 335.2004 andrastin D 429.2636 C26H36O5 397.2374 369.2425 379.2267 245.1899 351.2318 aurantioclavine 227.1543 C15H18N2 210.1277 171.0916 58.0660 130.0651 154.0651 b austin 501.2481 C27H32O9 381.2056 363.1950 409.2004 391.1901 335.2000 b bisdechlorogeodin 331.0812 C17H14O7 287.0911 299.0549 272.0677 255.0649 317.0653 citrinin 251.0915 C13H14O5 233.0808 205.0860 dehydrohistidyltryptophanyldiketopiperazine 322.1299 C17H15N5O2 193.0720 130.0652 162.0295 fiscalin A 474.2137 C26H27N5O4 258.1237 199.0865 171.0916 fiscalin C 488.2290 C27H29N5O4 213.1021 258.1236 185.1072 201.1018 fumitremorgin B 480.2523 C27H33N3O5 383.1967 355.2018 406.1764 424.1865 462.2385 fusaperazine E 361.1580 C19H24N2O3S 245.0920 217.0970 153.0367 293.0953 69.0707 fusaperazine related 406.1795 C20H27N3O4S 107.0495 216.1382 359.1836 290.1133 291.1210 glandicoline B 420.1667 C22H21N5O4 289.0716 335.1018 261.0767 334.0935 307.1086 marcfortine A 478.2699 C28H35N3O4 450.2748 419.2314 348.1232 marcfortine B 464.2544 C27H33N3O4 436.2593 419.2327 405.2178 meleagrin 434.1820 C23H23N5O4 334.0931 403.1639 318.0741 289.0718 306.0985 b mevastatin 391.2477 C23H34O5 185.1324 159.1167 211.1480 229.1585 235.1482 b N,N-dimethylrestricticin 366.2639 C21H35NO4 115.0757 149.1326 231.1743 263.2004 83.0499
N6-formyl-roquefortine C 418.1874 C23H23O3N5 333.0977 349.1164 332.0900 350.1243 neoxaline 436.1978 C23H25N5O4 336.1087 405.1794 280.1192 263.1187 145.0760 oxalicine A 504.2379 C30H33NO6 148.0392 216.0291 122.0602 234.0396 363.1601 oxalicine B 520.2332 C30H33NO7 148.0394 216.0292 122.0603 234.0398 218.0449 oxaline 448.1981 C24H25N5O4 348.1090 417.1794 332.0894 349.1168 386.1612 penicillic acid 171.0652 C8H10O4 125.0598 112.0522 97.0652 111.0444 83.0498 penitrem A 634.2930 C37H44ClNO6 Confirmed by RT and isotope pattern, CYA media only pulvilloric acid* 295.1186 C15H20O6 195.0293 277.1083 177.0185 151.0389 251.1286 b restricticin 338.2324 C19H31NO4 231.1742 170.0811 161.1324 263.2004 306.2063 isofumigaclavine A 299.1753 C18H22N2O2 239.1542 196.1122 197.1077 144.0808 168.0806 isofumigaclavine B 257.1647 C16H20N2O 239.1542 roquefortine C 390.1925 C22H23N5O2 322.1296 193.0719 198.1280 334.1296 358.1661 roquefortine M 422.1821 C22H23O4N5 192.0642 205.0722 353.1120 roquefortine N 440.1928 C22H25O5N5 192.0642 154.0611 210.0747 422.1823 b trichodermamide A (penicillazine) 433.1242 C20H20N2O9 248.0552 220.0603 192.0653 176.0343 b trichodermamide C 447.1397 C21H22N2O9 262.0709 234.0761 206.0812 219.0764 176.0342
* Analyzed as [M-H]-1 in negative mode. All other ions are [M+H]+. b Putative assignments based interpretation of MS2 fragmentation pathways. C.M. Visagie et al.: Fifteen new species of Penicillium 255
P. camponotum also produced andrastin, while only lower not produce this pulvilloric acid related compound. Penicillium levels of andrastin A and C were produced by P. costaricense. tanzanicum also produced this compound. There was a correlation between the production of andrastins Although all P. camponotum strains produced the indole alkaloid and a pulvilloric acid related compound. As explained in the marcfortine A and B, their metabolite profiles could be separated Discussion, this compound is either an unreported analogue into three groups that partly correlated with geographical origin. of pulvilloric acid or the structure of pulvilloric acid has been Penicillium amphipolaria was the only species that produced mischaracterised in the literature. Of all the andrastin produc- fumitremorgin compounds. The compounds produced on YES ers, only P. camponotum, P. costaricense and P. nucicola did and CYA agar were fumitremorgin A, fumitremorgin C, dihydro xyfumitremorgin C and oxofumitremorgin B. When grown in
Table 3 Overview of the extrolites produced by the strains of this study. P. alogum P. amphipolaria P. aotearoae P. bissettii P. camponatum P. cataractum P. costaricense P. corvianum P. diabolicalicense P. fundyense P. improvisum P. infrabuccalum P. nucicola P. panissanguineum P. tanzanicum P. andrastin A – – – – + + + – – – – + + + – andrastin B – – – – + + – – – – – + + + – andrastin C – – – – + + + – – – – + + + – andrastin D – – – – + + – – – – – + + + – aurantioclavine – – – + – – – – – – – – – – – austin – – – – – – – – – – – – + – – bisdechlorogeodin – – – – – – – – – – – – – – + citrinin c – – – – – – – – – – + – – – – dehydrohistidyl-tryptophanyl-diketopiperazine – – – + – – – – – + – – – – – fiscalin A – – – – – – – – – – – – – – + fiscalin C – – – – – – – + – – – – – – + fumitremorgin B – + – – – – – – – – – – – – – fusaperazine E – + – – – – – – – – – – – – – fusaperazine related – + – – – – – – – – – – – – – glandicoline B – – – + – – – – – – – – – – – marcfortine A – – – – + – – – – – – – – – – marcfortine B – – – – + – – – – – – – – – – meleagrinc – – – + – – – – – + – – – – – mevastatin – – – – – – – + – – – – – – – N,N-dimethylrestricticin – – – – – – – – + – – – – – – N6-formyl-roquefortine C – – – – – – – – – + – – – – – neoxaline – – – + – – – – – + – – – – – oxalicine A – – – – – – – – – – – + – – – oxalicine B – – – + – – – – – – – + – – – oxaline – + – – – – – – – + – – – – – penicillic acid – – – + – – – – – – – – – – – penitrem Ac – – – – – – – – + – – – – – – pulvilloric acid – – – – – + – – – – – + – + + restricticin – – – – – – – – + – – – – – – isofumigaclavine Ac – – – – – – – – – – + – – – – isofumigaclavine B – – – – – – – – – – + – – – – roquefortine Cc – – – + – – – – – + – – – – – roquefortine M – – – + – – – – – + – – – – – roquefortine N – – – + – – – – – – – – – – – trichodermamide C – – – – – + – + – – – – – – – trichodermamide A (penicillazine) – – – – – + – – – – – – – – –
C11H15O3N – – – – – – + + – – – – – – –
C18H22O5 – – – – – – – + – – – – – – –
C29H20O7S2 – – – – – + – + – – – – – – –
C20H30O6N2 – – – – – + – – – – – – – – –
C22H23O4N3 – + – – – – – – – – – – – – –
C27H31O7N3 – + – – – – – – – – – – – – –
C14H19O4N – – – + – – – – – – – – – – –
C21H28O8 + – – – – – – – – – – – – – –
C19H24O8 – – + – – – – – – – – – – – –
C15H22O2 – – – – + – – – – – – – – – –
C20H20O6 – – – – – – – – – – – – + – – c Confirmed by comparison to authentic standard. 256 Persoonia – Volume 36, 2016
Fig. 9 Mass spectra of pulvilloric acid analyzed in ESI+ and ESI-. The intact ESI+ [M-H]- ion is observed in the negative electrospray ionization mode. The intact [M+H]+ ion is absent in positive electrospray ionization mode and the major 100 + 279.1233 ion species is [M-H2O+H] . +1 [C 15H 18O 5 + H] 80 2.11 ppm
ce O O H n a 60 H O O
Abun d H
e O 40 ti v
Rel a 20 280.12637
281.12866 0 295.11838 100 ESI- 295.1184 -1 [C 15H 20O 6 – H] 80 -0.9 2 ppm
60 O O H O H O O 40 H O 20 296.12152 277.10791 297.12360 0 275 280 285 290 295 300 305 m/z
CYA broth, other uncharacterised fumitremorgins were the tion moderately dense, conidia en masse greyish turquoise to extrolites produced. Very few extrolites were detected in cul- greyish green (24D4–E5–25E5); soluble pigments absent; tures of P. diabolicalicense, but the fungal neurotoxin penitrem exudates absent; reverse greyish yellow (2B3), greenish grey A was detected. to dull green (1B2–29D4). YES 25 °C, 7 d: Colonies moder- The two strains of P. bissettii produced significantly different ately deep, radially and concentrically sulcate, crateriform; extrolites. DAOMC 167033 produced the mycotoxin penicillic margins low, narrow, entire; mycelia white; texture velutinous; acid. DAOMC 167011 produced meleagrin, neoxaline and sporulation moderately dense, conidia en masse greyish tur- aurantioclavine along with other uncharacterised metabolites. quoise (24D5–E5); soluble pigments absent; exudates absent; reverse greyish yellow (3B6), pale yellow (3A3). OA 25 °C, 7 d: Colonies low, plane; margins low, wide, entire; mycelia white; Taxonomy texture velutinous; sporulation moderately dense, conidia en masse greyish turquoise (24D4–E6); soluble pigments absent; Penicillium section Aspergilloides — Fig. 1, 2 exudates absent. CREA 25 °C, 7 d: Weak growth, acid weakly produced. Penicillium improvisum Visagie, Malloch & Seifert, sp. nov. — Conidiophores monoverticillate, sometimes divaricate; stipes MycoBank MB815769; Fig. 10 smooth, some roughened hyphae present, 40–200(–330) × In: subgenus Aspergilloides, section Aspergilloides. 2.5–3.5 μm; vesicle 4.5–6.5 μm; branches 13–32.5 μm; phia- lides ampulliform, (3–)5–12 per stipe, 7.5–10.5 × 2.5–3.5 μm ITS barcode: KT887867. Alternative identification markers: BenA = (8.9 ± 0.8 × 3.0 ± 0.2); conidia smooth walled, globose to subglo- KT887828, CaM = KT887789. bose, 2.5–3 × 2.5–3 μm (2.8 ± 0.1 × 2.8 ± 0.1), average width/ Etymology. Latin, improvisum, meaning unexpected/unforeseen, named length = 0.96, n = 43. for the conidiophores, which have randomly arranged branches. Extrolites — citrinin, isofumigaclavine A, isofumigaclavine B.
Type specimen. Canada, Ontario, Niagara falls, soil from oak/maple/ash Additional material examined. Canada, Niagara falls, soil from oak/maple/ forest, 28 Sept. 1996, D. Malloch (holotype DAOM 695768, culture ex-type ash forest, 28 Sept. 1996, D. Malloch, culture DAOMC 250546 (= CBS 140993 DAOMC 250547 = CBS 140994 = KAS 2386 = W 156). = KAS 2381 = W 155), DAOMC 250548 (= CBS 140995 = KAS 2393 = W 154). Colony morphology — Colony diam, 7 d, in mm: CYA 21–24; Notes — Phylogenetic analyses resolved P. improvisum, CYA 37 °C no growth; MEAbl 24–30; YES 28–31; OA 18–22; classified in section Aspergilloides, in a unique clade distinct CREA 14–15. from the 12 clades recognised by Houbraken et al. (2014) CYA 25 °C, 7 d: Colonies moderately deep, raised at centre, (Fig. 1, 2). Section Aspergilloides species are typically mono- radially sulcate; margins low, narrow, entire; mycelia white; verticillate, but some do produce extra subterminal branches texture velutinous; sporulation moderately dense, conidia en as observed in some conidiophores of P. glabrum, P. armarii, masse greyish turquoise (24D5–E5); soluble pigments absent; P. athertonense and P. bussumense. In addition, more complex exudates absent; reverse greyish yellow (3B4), pale to yellowish patterns occur in P. saturniforme (biverticillate), P. verhagenii grey (2A2–B2). MEA 25 °C, 7 d: Colonies low, plane; margins (biverticillate and divaricate) and P. ranomafanaense (divari- low, wide, irregular; mycelia white; texture velutinous; sporula- cate). In common with the latter two, P. improvisum produces C.M. Visagie et al.: Fifteen new species of Penicillium 257
Fig. 10 Morphological characters of Penicillium improvisum. a. Colonies from left to right (top row) CYA, MEA, YES; (bottom row) CYA reverse, MEA reverse, YES reverse; b. colony texture on CYA; c. colony texture on MEA; d–j. conidiophores; k. conidia. — Scale bar: d–k = 10 μm. 258 Persoonia – Volume 36, 2016
Fig. 11 Morphological characters of Penicillium corvianum. a. Colonies from left to right (top row) CYA, MEA, YES; (bottom row) CYA reverse, MEA reverse, YES reverse; b. colony texture on CYA; c. colony texture on MEA; d. sclerotia; e–l. conidiophores; m. conidia. — Scale bar: e–k = 10 μm. C.M. Visagie et al.: Fifteen new species of Penicillium 259 divaricate conidiophores. However, P. ranomafanaense and feature and are only known from P. coralligerum and P. novae- P. verhagenii produce rough-walled conidia in contrast to the zeelandiae. However, sclerotia generally do not dominate smooth-walled conidia of P. improvisum. Additionally, P. ver- colony appearance in the latter two species. Also, their conidio hagenii produces larger conidia (3.5–4 µm) than P. improvisum, phores are consistently symmetrically biverticillate compared while P. ranomafanaense has more ellipsoidal conidia in com- to the more diverse range of conidiophore branching observed parison to the globose conidia of the new species. in P. corvianum.
Penicillium section Canescentia — Fig. 3 Penicillium nucicola Visagie, Malloch & Seifert, sp. nov. — MycoBank MB815771; Fig. 12
Penicillium corvianum Visagie & Seifert, sp. nov. — Myco- In: subgenus Penicillium, section Canescentia. Bank MB815770; Fig. 11 ITS barcode: KT887860. Alternative identification markers: BenA = In: subgenus Penicillium, section Canescentia. KT887821, CaM = KT887782. ITS barcode: KT887875. Alternative identification markers: BenA = Etymology. Latin, nucicola (from nux), meaning living on nuts. KT887836, CaM = KT887797. Type specimen. Canada, Ontario, Niagara falls, fallen nuts of Carya ovata, Etymology. Latin, corvianum, named after Corviano, Italy, where the May 1996, D. Malloch (holotype DAOM 695770, culture ex-type DAOMC isolates originate. 250522 = CBS 140987 = W 59 = KAS 2203). Type specimen. Italy, Tuscany, Natural Monument Corviano, soil, 27 Colony morphology — Colony diam, 7 d, in mm: CYA 20–23; Aug. 2008, K.A. Seifert (holotype DAOM 695764, culture ex-type DAOMC CYA 37 °C no growth; MEAbl 12–14; YES 37–40; OA 17–20; 250517 = CBS 141000 = KAS 3618 = IT-2008-4-D). CREA 13–15. Colony morphology — Colony diam, 7 d, in mm: CYA 30–35; CYA 25 °C, 7 d: Colonies moderately deep, radially sulcate; CYA 37 °C no growth; MEAbl (17–)28–32; YES 36–40; OA margins moderately deep, wide, entire; mycelia white, in- 30–32; CREA 21–23. conspicuously yellow; texture floccose; sporulation moder- CYA 25 °C, 7 d: Colonies moderately deep, radially sulcate, ately dense, conidia en masse greyish green (26D5–E5–E6); brown to brownish yellow sclerotia present; margins moderately soluble pigments reddish pink, yellow halo close to colonies, deep, wide, entire; mycelia white; texture floccose; sporulation sometimes absent; exudates clear; reverse greyish yellow to absent to sparse, conidia en masse dull green (26D4); soluble olive (4B4–D7), pale yellow (4A3), sometimes deep green pigments absent; exudates clear; reverse yellowish white to (27E8) at centre. MEA 25 °C, 7 d: Colonies low, plane; margins pale yellow (2A2–3), orange (5A5). MEA 25 °C, 7 d: Colonies low, narrow, entire; mycelia white; texture floccose; sporulation low, plane, creamish to brown sclerotia; margins low, narrow, moderately dense, conidia en masse greyish green (26D5–E5– entire; mycelia white; texture floccose; sporulation absent, E6); soluble pigments deep yellow, inconspicuous sometimes; sparse in some colonies, conidia en masse dull green (26D4); exudates absent; reverse olive brown (4D6–E6), light yellow to soluble pigments absent; exudates absent; reverse pale yel- greyish yellow (4A4–B6). YES 25 °C, 7 d: Colonies moderately low to light orange (4A3–5A4), orange (6B7) in some colony deep, radially and concentrically sulcate; margins low, narrow, areas. YES 25 °C, 7 d: Colonies moderately deep, radially and entire; mycelia white; texture floccose; sporulation moder- concentrically sulcate, crateriform, brownish sclerotia sparsely ately dense, conidia en masse greyish green (26D5–E5–E6); produced; margins low, narrow, entire; mycelia white; texture soluble pigments reddish brown, yellow halo close to colonies, floccose; sporulation absent to sparse, conidia en masse absent sometimes; exudates absent; reverse light yellow (3A5), greenish grey (26B2); soluble pigments absent; exudates clear; greyish yellow (4C4). OA 25 °C, 7 d: Colonies low, plane; mar- reverse brownish orange to light brown (5C4–7C8–D8), pale gins low, wide, entire; mycelia white, yellow; texture floccose; yellow to light yellow (4A3–4). OA 25 °C, 7 d: Colonies low, sporulation moderately dense, conidia en masse greyish green plane, cream to brown sclerotia produced; margins low, wide, (26D5–E5–E6); soluble pigments yellow close to colonies, entire; mycelia white; texture floccose; sporulation absent to deep green surrounding yellow pigment; exudates absent. sparse, conidia en masse greenish grey (26B2); soluble pig- CREA 25 °C, 7 d: Weak growth, acid moderately produced. ments absent; exudates clear. CREA 25 °C, 7 d: Weak growth, Conidiophores biverticillate; stipes rough, some warted, 180– acid not produced. 890 × 2.5–4 μm; metulae divergent, 2–6 per stipe, 10.5–15 Conidiophores on MEA very sparse, monoverticillate, minor × 3–4 μm; phialides ampulliform, 4–10 per metula, 7.5–10.5 proportion biverticillate and also phialides born directly on hy- × 2.5–3.5 μm (8.9 ± 0.8 × 2.9 ± 0.2); average length metula/ phae, on CYA biverticilllate, sometimes monoverticillate; stipes phialide 1.4; conidia rough walled, some finely rough, globose smooth, (20–)100–400 × 1.5–2.5 μm; metulae divergent, 2–4 to subglobose, 2.5–3 × 2.5–3 μm (2.8 ± 0.2 × 2.7 ± 0.2), aver- per stipe, (7.5–)9.5–15 × 2–3(–3.5) μm; phialides ampulliform, age width/length = 0.97, n = 56. when not monophialidic 3–8 per metula/stipe, 6–9 × 2–3 μm Extrolites — andrastin A, andrastin B, andrastin C, andrastin
(7.3 ± 0.7 × 2.7 ± 0.3); average length metula/phialide 1.6; D, austin, uncharacterised C20H20O6. conidia smooth walled, globose to subglobose, 2–3 × 2–3 μm Additional material examined. Canada, Ontario, 5 km NW of Milton, fallen (2.1 ± 0.2 × 2.1 ± 0.2), average width/length = 0.96, n = 41. nuts of Fagus grandifolia, 24 Oct. 1995, D. Malloch, DAOMC 250521 (= CBS Sclerotia 130–265 × 110–230 μm. 140973 = KAS 2101 = W 109). Extrolites — fiscalin C, trichodermamide C, mevastatin, un characterised C H O , C H O S , C H O N. Notes — Classified in section Canescentia, P. nucicola sits 18 22 5 29 20 7 2 11 15 3 on a well-supported branch distinct from all other species (Fig. Additional material examined. Italy, Tuscany, Natural Monument Corviano, 3). Morphologically it resembles P. atrovenetum, P. antarcticum, soil, 27 Aug. 2008, K.A. Seifert, culture DAOMC 250518 (= CBS 141001 = P. coralligerum, P. novae-zeelandiae and P. herquei (the latter KAS 3622 = IT-2008-4-A). classified in section Sclerotiora). These species commonly pro- Notes — Penicillium corvianum is classified in section Ca duce broad, symmetrical biverticillate conidiophores with bright nescentia. Phylogenies (Fig. 3) resolve it in a larger clade with yellow and green colours often evident in colonies, as observed P. canescens, P. jensenii and P. janczewskii. Colonies of the for P. nucicola on OA (Fig. 12). However, the restricted growth new species are dominated by the presence of abundant cream on MEA (12–14 mm diam) easily distinguishes P. nucicola from to brown sclerotia. Sclerotia in the section are not a common all of these morphologically similar species. 260 Persoonia – Volume 36, 2016
Fig. 12 Morphological characters of Penicillium nucicola. a. Colonies from left to right (top row) CYA, MEA, YES; (bottom row) CYA reverse, MEA reverse, YES reverse; b. colony texture on CYA; c. colony texture on MEA; d–j. conidiophores; k. conidia. — Scale bar: d–k = 10 μm. C.M. Visagie et al.: Fifteen new species of Penicillium 261
Fig. 13 Morphological characters of Penicillium alogum. a. Colonies from left to right (top row) CYA, MEA, YES; (bottom row) CYA reverse, MEA reverse, YES reverse; b. colony texture on CYA; c. colony texture on MEA; d–k. conidiophores; l. conidia. — Scale bar: d–l = 10 μm. 262 Persoonia – Volume 36, 2016
Fig. 14 Morphological characters of Penicillium costaricense. a. Colonies from left to right (top row) CYA, MEA, YES; (bottom row) CYA reverse, MEA reverse, YES reverse; b. colony texture on CYA; c. colony texture on MEA; d–j. conidiophores; k. conidia. — Scale bar: d–k = 10 μm. C.M. Visagie et al.: Fifteen new species of Penicillium 263
Penicillium section Charlesia & Stolkia — Fig. 4 new species (3–4 μm) (Peterson & Sigler 2002). From a mor- phological perspective, P. alogum is most similar to P. stolkiae, Penicillium alogum Visagie, Frisvad & Seifert, sp. nov. — Myco having similar colony colours, growth rates and conidiophore Bank MB815772; Fig. 13 branching patterns. However, P. stolkiae produces longer In: subgenus Aspergilloides, section Stolkia. phialides (7–10 μm) and slightly larger conidia (2.8–3.5 µm) fide Pitt (1980). ITS barcode: KT887869. Alternative identification markers: BenA = KT887830, CaM = KT887791. Penicillium costaricense Visagie, M. Urb & Seifert, sp. nov. — Etymology. Latin, alogum, meaning irrational, irregular, nonsensical. MycoBank MB815773; Fig. 14 Named for the irregular conidiophore branching observed in the species. In: subgenus Aspergilloides, section Charlesia. Type specimen. USA, Wyoming, Middle Mountain, Wind River Range, Tundra wetland soil, June 2001, M. Christensen (holotype DAOM 695759, ITS barcode: KT887873. Alternative identification markers: BenA = culture ex-type DAOMC 250543 = CBS 140996 = IBT 23947= KAS 2475). KT887834, CaM = KT887795. Colony morphology — Colony diam, 7 d, in mm: CYA (15–)19– Etymology. Latin, costaricense, named after Costa Rica, the origin of the 22; CYA 37 °C no growth; MEAbl 31–35; YES 21–22(–29); OA isolate.
22–25; CREA 12–14. Type specimen. Costa Rica, Area de Conservación Guanacaste, Santa CYA 25 °C, 7 d: Colonies deep, radially and concentrically sul- Rosa, intestines of Rothschildia lebeau (01-CACG-589) feeding on Spondias cate, crateriform in some isolates; margins low, narrow, entire; mombin, 19 Oct. 2001, J. Diaz (holotype DAOM 695765, culture ex-type DAOMC 250520 = CBS 140998 = KAS 2597 = 01-RGTHC-294). mycelia white; texture floccose; sporulation sparse to mode rately dense, conidia en masse greenish grey to dull green Colony morphology — Colony diam, 7 d, in mm: CYA 15–17; (29D2–3); soluble pigments absent; exudates absent, after CYA 37 °C no growth; MEAbl 19–21; YES 21–22; OA 11–12; two weeks inconspicuously red present; reverse dull to greyish CREA 6–7. yellow (3B4–C4), yellowish white (3A2). MEA 25 °C, 7 d: Colo- CYA 25 °C, 7 d: Colonies moderately deep, radially sulcate, nies low, plane, sometimes slightly raised centrally; margins crateriform; margins low, narrow, entire; mycelia white; texture low, wide, entire; mycelia white; texture velutinous; sporulation velutinous; sporulation sparse to moderately dense, conidia moderately dense to sparse, conidia en masse greyish green to en masse greyish turquoise to dull green (25C4–D3); soluble dark green (29E5–F5), in some a lighter greyish green (29D4); pigments absent; exudates absent; reverse greyish green to soluble pigments absent; exudates absent, after two weeks in- olive (1D4–2D4), yellowish white to pale yellow (3A2–3). MEA conspicuously red present; reverse greyish green (29E5), pale 25 °C, 7 d: Colonies low, plane; margins low, wide, entire; my- yellow (2A3), yellowish grey (2B2). YES 25 °C, 7 d: Colonies celia white; texture velutinous; sporulation moderately dense, deep, plane; margins low, narrow, entire; mycelia white, orange conidia en masse greyish green to dark green (25E5–F5); white (5A2–6A2); texture floccose; sporulation absent, conidia soluble pigments absent; exudates absent; reverse greyish en masse not determined; soluble pigments absent; exudates yellow (2B3–C4–3C5). YES 25 °C, 7 d: Colonies moderately absent; reverse greyish orange (5B5), yellowish white (4A2). deep, radially and concentrically sulcate, crateriform; margins OA 25 °C, 7 d: Colonies low, plane; margins low, wide, entire; low, narrow, entire; mycelia white; texture velutinous; sporu- mycelia white; texture velutinous; sporulation moderately dense lation sparse to moderately dense, conidia en masse grey- near centre, conidia en masse greyish green (30E6); soluble ish green to dull green (25C4–D4); soluble pigments absent; pigments absent; exudates absent. CREA 25 °C, 7 d: Weak exudates absent; reverse olive (2D3–E3), light yellow to dull growth, acid not produced. yellow (3A4–B3). OA 25 °C, 7 d: Colonies low, plane; margins Conidiophores monoverticillate and divaricate, sometimes soli- low, narrow, entire; mycelia white; texture velutinous; sporula- tary phialides present; stipes smooth, inconspicuous green tion moderately dense, conidia en masse greyish green to dark ish brown pigment, 10–43 × 2–3 μm; metulae divergent, 2 green (25E5–F5); soluble pigments absent; exudates absent. when present, of unequal length on the same conidiophore, CREA 25 °C, 7 d: Weak growth, acid not produced. 7–12(–20) × 2–3 μm; phialides ampulliform, sometimes soli- Conidiophores monoverticillate; stipes smooth, 50–280 × 2–3 tary and elongated to almost acerose, mostly 3–5 per metula, μm; vesicle 4–6.5 μm; phialides ampulliform, 10–20 per stipe, 5.5–7(–8) × 2.5–3 μm (6.4 ± 0.5 × 2.7 ± 0.2); average length 8.5–11.5 × 2–3.5 μm (9.7 ± 0.7 × 2.7 ± 0.3); conidia smooth metula/phialide 1.7; conidia thick walled, finely rough walled, walled, subglobose, 2.5–3 × 2–3 μm (2.6 ± 0.1 × 2.4 ± 0.1), globose, 2.5–3 × 2.5–3 μm (2.7 ± 0.1 × 2.7 ± 0.1), average average width/length = 0.94, n = 72. width/length = 0.96, n = 64. Extrolites — andrastin A, andrastin C, uncharacterised
Extrolite — uncharacterised C21H28O8. C11H15O3N. Additional material examined. USA, Wind River Mountain, Wyoming, Notes — Penicillium costaricense is known from only the one Tundra wetland soil, 1998, M. Christensen, culture DAOMC 250544 (= CBS isolate and forms a subbasal branch distinct from the main body 126173 = IBT 23948 = KAS 2476); Middle Mountain, Wind River Mountain, of section Charlesia and P. georgiense, the basal species in Wyoming, Tundra wetland soil, 1998, M. Christensen, culture DAOMC 250545 (= CBS 126150 = IBT 23950 = KAS 2477). the section (Fig. 4). The new species produces monoverticillate conidiophores, readily distinguished from the biverticillate to Notes — Penicillium alogum belong to section Stolkia and is divaricate conidiophores of P. georgiense. The same character closely related to P. stolkiae, P. subarcticum, P. boreae, P. pul- also distinguishes it from P. charlesii and P. fellutanum. The lum and P. canariense (Fig. 4). These species all produce coni- remaining species, P. coffeae, P. phoeniceum and P. indicum diophores with some brown pigmentation. Penicillium boreae all produce monoverticillate conidiophores. However, P. indicum and P. canariense typically produce biverticillate conidiophores, (20–30 mm diam) and P. phoeniceum (5–8 mm diam) are able while the others mainly have monoverticillate/simple conidio to grow on CYA at 37 °C, in contrast to P. costaricense, which phore branching patterns. Penicillium pullum produces large, does not grow at 37 °C. Microscopically, P. costaricense most conspicuously rough walled conidia (3.5–4.5 μm) that turn dark closely resembles P. coffeae, but the latter grows much slower with age, which easily distinguish it from P. alogum. On the on CYA and MEA (11–17 mm, 11–13 mm diam) than the new other hand, P. subarcticum produces bigger conidia than the species. 264 Persoonia – Volume 36, 2016
Fig. 15 Morphological characters of Penicillium aotearoae. a. Colonies from left to right (top row) CYA, MEA, YES; (bottom row) CYA reverse, MEA reverse, YES reverse; b. colony texture on CYA; c. colony texture on MEA; d–i. conidiophores; j. conidia. — Scale bar: d–j = 10 μm. C.M. Visagie et al.: Fifteen new species of Penicillium 265
Penicillium section Exilicaulis — Fig. 5, 6 Type specimen. New Zealand, Arthurs Pass National Park, Devil’s Punch- bowl, light yellow brown, beneath moss and Nothofagus, 18 Feb. 2003, Penicillium aotearoae Visagie & Seifert, sp. nov. — MycoBank Keith Seifert (holotype PDD 107542, culture ex-type DAOMC 250542 = CBS MB815774; Fig. 15 140967 = KAS 1726).
In: subgenus Aspergilloides, section Exilicaulis. Colony morphology — Colony diam, 7 d, in mm: CYA 20–25; CYA 37 °C no growth; MEAbl 36–40; YES 31–34; OA 30–31; ITS barcode: KT887874. Alternative identification markers: BenA = CREA 20–21. KT887835, CaM = KT887796. CYA 25 °C, 7 d: Colonies moderately deep, radially sulcate; Etymology. Latin, aotearoae, from the Maori name for New Zealand, the margins low, narrow, entire; mycelia white, deep yellow; texture origin of the isolate. floccose; sporulation absent to sparse, conidia en masse not Type specimen. New Zealand, South Island, Arthurs Pass National Park, determined; soluble pigments absent; exudates absent; reverse Devil’s Punchbowl, isol. ex clay, light yellow brown, beneath moss and brown (5F6), light brown (5D3), pale yellow to greyish yellow Nothofagus, 18 Feb. 2003, K.A. Seifert (holotype PDD 107543, culture ex- (4A3–B3). MEA 25 °C, 7 d: Colonies moderately deep, plane; type DAOMC 250538 = CBS 140999 = KAS 3088). margins low, wide, entire; mycelia white; texture floccose; Colony morphology — Colony diam, 7 d, in mm: CYA 30–32; sporulation moderately dense, conidia en masse dark green CYA 37 °C no growth; MEAbl 29–30; YES 35–37; OA 33–35; (25F6–26F6); soluble pigments absent; exudates absent; re- CREA 18–19. verse dull yellow to greyish green (3B3–29B3–C3), yellowish white (2A2–3A2). YES 25 °C, 7 d: Colonies moderately deep, CYA 25 °C, 7 d: Colonies low to moderately deep, radially and radially sulcate; margins moderately deep, narrow, entire; concentrically sulcate; margins low, narrow, entire; mycelia mycelia white; texture floccose; sporulation sparse to moder- white; texture floccose; sporulation sparse to moderately dense, ately dense, conidia en masse greenish grey to greyish green conidia en masse greyish turquoise to greyish green (24D4– (25B2–D4); soluble pigments absent; exudates absent; reverse 25D4); soluble pigments absent; exudates clear to absent; greyish orange (5B6), pale yellow to light yellow to yellowish reverse brown (6D7), greyish orange (5B6), light yellow (4A5), orange (4A3–6). OA 25 °C, 7 d: Colonies low, plane; margins greyish green (28C4). MEA 25 °C, 7 d: Colonies low, plane; low, wide, entire; mycelia white; texture velutinous; sporulation margins low, wide, entire; mycelia white; texture velutinous; sparse to moderately dense, conidia en masse greyish green sporulation moderately dense, conidia en masse greyish (25E5); soluble pigments absent; exudates absent. CREA turquoise to greyish green (24D4–25D4); soluble pigments 25 °C, 7 d: Strong growth, acid strongly produced. inconspicuously yellow; exudates absent; reverse dull yellow to Conidiophores biverticillate, often divaricate; stipes rough, olive yellow (3B4–D6). YES 25 °C, 7 d: Colonies deep, radially 200–800 × 2–3 μm; branches 20–68 μm; metulae diver- and concentrically sulcate, crateriform; margins low, narrow, gent, 2–4 per stipe, unequal length on same conidiophore, entire; mycelia white; texture floccose; sporulation sparse to 12–26 × 2–3 μm; phialides ampulliform, 4–8 per metula, moderately dense, conidia en masse greyish green (25B3–C4); (5.5–)6.5–8(–9) × 2.5–3 μm (7.2 ± 0.7 × 2.7 ± 0.2); average soluble pigments inconspicuously yellow; exudates absent; length metula/phialide 2.1; conidia rough walled, subglobose, reverse brown (7E7), orange to brownish orange (6B8–C8), minor proportion globose, 2–3 × 2–2.5 μm (2.6 ± 0.2 × 2.5 ± yellowish orange (4A6). OA 25 °C, 7 d: Colonies low, plane; 0.0), average width/length = 0.96, n = 41. margins low, wide, entire; mycelia white; texture velutinous; Extrolites — N,N-dimethylrestricticin (CYA), penitrem A (CYA), sporulation sparse to moderately dense, conidia en masse dull restricticin. green (26E4); soluble pigments absent; exudates absent. CREA 25 °C, 7 d: Strong growth, acid weakly produced. Notes — Penicillium diabolicalicense is classified in sec- Conidiophores biverticillate, divaricate, some monoverticillate; tion Exilicaulis and is closely related to P. melinii, P. ‘fynbos1’ stipes rough to finely rough, 115–180 × 2–3 μm; branches (Visagie et al. 2016b) and P. aotearoae described above (Fig. 16.5–46 μm; metulae divergent, 2–5 per stipe, 9.5–24 × 2–3 5, 6). The new species can easily be distinguished from its μm; phialides ampulliform, 6–20 per metula, 6.5–9 × 2–3 μm close relatives based on its more restricted growth on CYA at (7.5 ± 0.6 × 2.4 ± 0.2); average length metula/phialide 2.1; 25 °C compared to the fynbos species, faster growth on MEA conidia finely rough walled, globose, 2–3 × 2–3 μm (2.5 ± 0.1 compared to P. melinii, and growth slower on CYA but faster × 2.4 ± 0.1), average width/length = 0.96, n = 67. growth on MEA compared to P. aotearoae.
Extrolites — uncharacterised C19H24O8. Notes — Penicillium aotearoae is classified in section Exili- Penicillium fundyense Visagie, David Clark & Seifert, sp. nov. caulis and is closely related to P. melinii, P. ‘fynbos1’ (an — MycoBank MB815776; Fig. 17 undescribed species from the fynbos in South Africa, Visagie In: subgenus Aspergilloides, section Exilicaulis. et al. 2016b) and P. diabolicalicense described below (Fig. ITS barcode: KT887853. Alternative identification markers: BenA = 5, 6). The new species can be distinguished from the others KT887814, CaM = KT887775. based on colony growth rates. It grows more restrictedly than the fynbos species on MEA, but faster on MEA compared to Etymology. Latin, fundyense, named after the Bay of Fundy, origin of the strain. P. melinii, and faster on CYA but more restricted on MEA com- pared to P. diabolicalicense. Type specimen. Canada, New Brunswick, St. Andrews, Carpenter ants (Camponotus pennsylvanicus), Aug. 2000, D. Clark (holotype DAOM 695767, culture ex-type DAOMC 250519 = CBS 140980 = NBBR-2-3 = W466 = KAS Penicillium diabolicalicense Visagie & Seifert, sp. nov. — 2174). MycoBank MB815775; Fig. 16 Colony morphology — Colony diam, 7 d, in mm: CYA 20–21; In: subgenus Aspergilloides, section Exilicaulis. CYA 37 °C no growth; MEAbl 22–24; YES 28–30; OA 25–26; ITS barcode: KT887840. Alternative identification markers: BenA = CREA 14–15. KT887801, CaM = KT887762. CYA 25 °C, 7 d: Colonies moderately deep, radially sulcate, Etymology. Latin, diabolicalicense, meaning Devil’s Punchbowl, the origin crateriform; margins low, narrow, entire; mycelia white; texture of type strain – diabolus, diaboli = devil; calix, calicis = drinking bowl. velutinous; sporulation moderately dense, conidia en masse 266 Persoonia – Volume 36, 2016
Fig. 16 Morphological characters of Penicillium diabolicalicense. a. Colonies from left to right (top row) CYA, MEA, YES; (bottom row) CYA reverse, MEA reverse, YES reverse; b. colony texture on CYA; c. colony texture on MEA; d–i. conidiophores; j. conidia. — Scale bar: d = 50 µm, e–j = 10 μm. C.M. Visagie et al.: Fifteen new species of Penicillium 267
Fig. 17 Morphological characters of Penicillium fundyense. a. Colonies from left to right (top row) CYA, MEA, YES; (bottom row) CYA reverse, MEA reverse, YES reverse; b. colony texture on CYA; c. colony texture on MEA; d–j. conidiophores; k. conidia. — Scale bar: d–k = 10 μm. 268 Persoonia – Volume 36, 2016
Fig. 18 Morphological characters of Penicillium amphipolaria. a. Colonies from left to right (top row) CYA, MEA, YES; (bottom row) CYA reverse, MEA reverse, YES reverse; b. colony texture on CYA; c. colony texture on MEA; d–l. conidiophores; m. conidia. — Scale bar: d–m = 10 μm. C.M. Visagie et al.: Fifteen new species of Penicillium 269 dull green to greyish green (25E4–5); soluble pigments absent; texture floccose and velutinous; sporulation moderately dense, exudates absent; reverse olive (3D4), yellowish white (2A2). conidia en masse dull green (26D3–4); soluble pigments MEA 25 °C, 7 d: Colonies low, lightly sulcate centrally; margins deep yellow, absent in DAOMC 250550; exudates absent; re- low, narrow, entire; mycelia white; texture velutinous; sporula- verse yellow to olive yellow (3B6–C6), greenish grey (1B2) in tion moderately dense, conidia en masse dull green to greyish KAS1396. YES 25 °C, 7 d: Colonies moderately deep, radially green (25E4–5); soluble pigments absent; exudates absent; and concentrically sulcate, raised at centre; margins low, wide, reverse olive (3E4), greyish yellow (3B4–C4). YES 25 °C, 7 d: entire; mycelia white, inconspicuously yellow; texture floccose; Colonies moderately deep, radially and concentrically sulcate, sporulation very sparse, moderately dense in the ex-type, co- crateriform; margins low, narrow, entire; mycelia white; texture nidia en masse greenish grey to dull green (26B3–D4); soluble velutinous; sporulation sparse to moderately dense, conidia en pigments absent; exudates absent; reverse greyish yellow to masse greyish green (25B3–C4); soluble pigments absent; olive brown (4C6–D6), light yellow (2A4), pale yellow (3A3) in exudates deep yellow, inconspicuous; reverse brownish orange DAOMC 250550. OA 25 °C, 7 d: Colonies low, plane; margins (5C6), light yellow to greyish yellow (4A4–B5). OA 25 °C, 7 d: low, wide, entire; mycelia white; texture floccose and velutinous; Colonies low, plane; margins low, wide, entire; mycelia white; sporulation moderately dense, conidia en masse greenish white texture velutinous; sporulation sparse to moderately dense, to dull green (26B2–D3–E3); soluble pigments yellow, absent conidia en masse greyish green to dull green (26C3–D3); in DAOMC 250550; exudates absent. CREA 25 °C, 7 d: Strong soluble pigments absent; exudates absent. CREA 25 °C, 7 d: growth, acid moderately produced. Strong growth, acid not produced. Conidiophores biverticillate and divaricate, minor proportion Conidiophores monoverticillate, biverticillate and divaricate both monoverticillate; stipes smooth, 240–460 × 2–2.5 μm; branch- common, often difficult to distinguish between branches and es 12–42 μm; metulae divergent, 2–4 per stipe, 9.5–21 × 2–3 metulae; stipes smooth, 15–200(–400) × 2–2.5 μm; branches μm; phialides ampulliform, 3–6 per metula, 6.5–10 × 2–3 μm 16–30(–47) μm; metulae divergent, 2–4 per stipe, 16–30 × (7.4 ± 0.8 × 2.5 ± 0.8); average length metula/phialide 2.1; 2–2.5 μm; phialides ampulliform, 8–16 per metula, 6–8.5 × conidia smooth walled, mainly broadly ellipsoidal, some glo- 2–2.5 μm (7.4 ± 0.7 × 2.4 ± 0.2); average length metula/phialide bose, 2–3 × 2–2.5 μm (2.4 ± 0.1 × 2.2 ± 0.1), average width/ 3.3; conidia smooth walled, globose, 2–3 × 2–3 μm (2.4 ± 0.2 length = 0.9, n = 78. × 2.4 ± 0.2), average width/length = 0.96, n = 46. Extrolites — fumitremorgin B, fusaperazine E, fusaperazine
Extrolites — dehydrohistidyltryptophanyldiketopiperazine, related (C20H27N3O4S), oxaline, uncharacterised C22H23O4N3, meleagrin, N6-formyl-roquefortine C, neoxaline, oxaline, roque- C27H31O7N3. fortine C, roquefortine M. Additional material examined. Canada, Quebec, peat moss factory, 2001, Notes — Penicillium fundyense is classified in section Exili C. Duchaine, cultures DAOMC 250549 (= CBS 140965 = KAS 1392 = R-273), DAOMC 250550 (= CBS 140966 = KAS 1396 = R-293). caulis, with phylogenies resolving it in a clade with P. cineras- cens, P. citreonigrum and P. citreosulfuratum. The species of Notes — Penicillium amphipolaria is classified in section this complex are almost impossible to distinguish morpho- Lanata-Divaricata and is a close relative of P. singorense (Fig. logically, but chemically they represent distinct species, which 7, 8). The new species can easily be distinguished from its resulted in Visagie et al. (2016b) suggesting the use of ITS and close relatives by its poor growth on CYA at 37 °C, in contrast BenA sequences for identifications of strains in this clade. This to the strong growth of P. singorense (40–43 mm diam) (Visagie approach is followed here, because P. fundyense shows only et al. 2014a). Also, P. amphipolaria has a more complex con- minor morphological differences from its close relatives, but is idiophore branching pattern compared to the generally simple phylogenetically distinct from the other three. conidiophores of P. singorense (Visagie et al. 2014a).
Penicillium section Lanata-Divaricata — Fig. 7, 8 Penicillium bissettii Visagie & Seifert, sp. nov. — MycoBank MB815778; Fig. 19
Penicillium amphipolaria Visagie, Malloch & Seifert, sp. nov. In: subgenus Aspergilloides, section Lanata-Divaricata. — MycoBank MB815777; Fig. 18 ITS barcode: KT887845. Alternative identification markers: BenA = In: subgenus Aspergilloides, section Lanata-Divaricata. KT887806, CaM = KT887767.
ITS barcode: KT887872. Alternative identification markers: BenA = Etymology. Latin, bissettii, named after John Bissett, recently retired KT887833, CaM = KT887794. mycologist, AAFC Ottawa, who collected and isolated the strains, in honour Etymology. Latin, amphipolaria, meaning both poles, named to indicate of his contributions to soil fungus ecology. the origin of the two strains on opposite sides of the Earth, close to the north Type specimen. Canada, Quebec, near Lacolle, soil from Spruce forest, and south poles. 5 Apr. 1977, J. Bissett (holotype DAOM 695761, culture ex-type DAOMC 167011 = CBS 140972 = KAS 1951). Type specimen. Antarctica, Quartermain Mountains of Antarctic Dry Val- leys (-77.842741° 161.122229°), soil, Jan. 1998, W.C. Mahaney (holotype Colony morphology — Colony diam, 7 d, in mm: CYA 42–43; DAOM 695760, culture ex-type DAOMC 250551 = CBS 140997 = W 284 = CYA 37 °C no growth; MEAbl 35–43; YES 47–52; OA 45–48; KAS 2555). CREA 23–26. Colony morphology — Colony diam, 7 d, in mm: CYA (30–)36– CYA 25 °C, 7 d: Colonies moderately deep, radially sulcate; 39; CYA 37 °C 5–7; MEAbl 50–52; YES 48–51; OA 45–50; margins low, wide, entire; mycelia white; texture floccose; sporu- CREA 24–29. lation moderately dense, sparse in DAOMC 167033, conidia en CYA 25 °C, 7 d: Colonies moderately deep, radially sulcate; masse greyish green to dull green (26C3–D4), greenish grey margins low, wide, entire; mycelia white, inconspicuously (26B2); soluble pigments absent; exudates absent; reverse yellow; texture floccose; sporulation very sparse, moderate brown to dark brown (7E6–F6), yellowish white to greyish yel- in the ex-type, conidia en masse dull green (26D4) in the low (4A2–B4). MEA 25 °C, 7 d: Colonies low, plane, pinkish red ex-type; soluble pigments absent; exudates clear to absent; underneath mycelial and sporulating areas; margins low, wide, reverse greyish yellow to olive brown (4C6–D6), light yellow entire; mycelia white, red submerged in media; texture floccose (2A4), pale yellow (3A3) in DAOMC 250550. MEA 25 °C, 7 d: and velutinous; sporulation absent, sparse in some colonies, Colonies low, plane; margins low, wide, entire; mycelia white; conidia en masse greyish green to dull green (26D4–E5), 270 Persoonia – Volume 36, 2016
Fig. 19 Morphological characters of Penicillium bissettii. a. Colonies from left to right (top row) CYA, MEA, YES; (bottom row) CYA reverse, MEA reverse, YES reverse; b. colony texture on CYA; c. colony texture on MEA; d–j. conidiophores; k. conidia. — Scale bar: d–k = 10 μm. C.M. Visagie et al.: Fifteen new species of Penicillium 271 greenish grey (26B2); soluble pigments absent; exudates dates absent; reverse greenish grey to greyish green (30B2– absent; reverse greyish red to reddish brown (8B6–D6). YES B3), yellowish white (2A2). YES 25 °C, 7 d: Colonies moder- 25 °C, 7 d: Colonies moderately deep, radially and concentri- ately deep, radially and concentrically sulcate; margins low, cally sulcate, some strains sporulating in concentrical rings; wide, entire; mycelia white, inconspicuously pinkish grey; tex- margins low, wide, entire; mycelia white; texture floccose; ture floccose; sporulation moderately dense, conidia en masse sporulation moderately dense, absent in some strains, conidia dull green to greyish green (25D4–26D4–E5); soluble pigments en masse dull green (26D4–E4), greenish white (26B2); soluble absent; exudates absent; reverse brownish orange (5C3), pigments absent; exudates absent; reverse brownish orange greyish orange (5B4–5), pale yellow to light yellow (4B3–5). (6C5), light yellow to greyish yellow (4A3–B5). OA 25 °C, 7 d: OA 25 °C, 7 d: Colonies low, plane; margins low, wide, entire; Colonies low, plane; margins low, wide, entire; mycelia white; mycelia white; texture floccose; sporulation absent to sparse to texture velutinous and floccose; sporulation sparse, conidia moderately dense, conidia en masse greyish green to dull green en masse greenish grey to greyish green (26B2–3); soluble (26C3–D3–4); soluble pigments absent; exudates absent. pigments pink, absent in DAOMC 167033; exudates absent. CREA 25 °C, 7 d: Weak growth, acid moderately produced. CREA 25 °C, 7 d: Weak growth, acid not produced. Conidiophores biverticillate, minor proportion divaricate; stipes Conidiophores biverticillate, terverticillate, minor proportion di- rough, 220–620 × 2.5–3.5 μm; branches 13–44 μm; metulae varicate; stipes rough, 190–670 × 2.5–3.5 μm; branches 14–25 divergent, 2–4 per stipe, sometimes of unequal length on same μm; metulae divergent, 3–4 per stipe or branch, 11–15.5 × conidiophore, 12–26 × 2.5–4 μm; phialides ampulliform, 4–8 2.5–3.5 μm; phialides ampulliform, 3–6 per metula, 6.5–9 × per metula, 8–11.5 × 2.5–3.5 μm (9.5 ± 0.8 × 2.9 ± 0.2); aver- 2.5–3 μm (7.5 ± 0.7 × 2.6 ± 0.2); average length metula/phialide age length metula/phialide 1.7; conidia rough to finely rough 1.8; conidia smooth walled, globose to subglobose, 2–3 × 2–3 walled, globose, 2.5–3.5 × 2.5–3.5 μm (2.9 ± 0.1 × 2.9 ± 0.1), μm (2.6 ± 0.2 × 2.4 ± 0.1), average width/length = 0.92, n = 72. average width/length = 0.97, n = 72. Extrolites — DAOMC 167033: penicillic acid, uncharacteri Extrolites — andrastin B, andrastin C, andrastin D, unchar- sed C14H19O4N. DAOMC 167011: aurantioclavine, dehydrohis- acterised C15H22O2 (only New Brunswick strains), andrastin A tidyltryptophanyldiketopiperazine, glandicoline B, meleagrin, (only German strains), marcfortine A, marcfortine B (all strains). oxalicine B, roquefortine C, roquefortine M, roquefortine N. Additional material examined. Canada, New Brunswick, St. Andrews, Additional material examined. Canada, Quebec near Lacolle, soil from Carpenter ants (Camponotus pennsylvanicus), Aug. 2000, D. Clark, culture Spruce forest, 5 Apr. 1977, J. Bissett, culture DAOMC 167033 (= CBS 140971 DAOMC 250552 (= CBS 140975 = NBCP-17-4 = W 447 = KAS 2166), = KAS 1949). DAOMC 250553 (= CBS 140977 = NBCP-9-1 = W 451 = KAS 2169), DAOMC 250554 (= CBS 140978 = NBCP-1-1 = W 452 = KAS 2170), DAOMC 250555 Notes — Penicillium bissettii is classified in section Lanata- (= CBS 140979 = NBCP-17-4B = W 463 = KAS 2172), DAOMC 250556 (= Divaricata in a clade distantly related to all other species in the CBS 140981 = NBCP-23-1 = W 469 = KAS 2176), DAOMC 250558 (= CBS section. The multigene phylogeny places it closest to P. vas 140984 = NBCP-5-1 = W 477 = KAS 2182). – Germany, Grosse-Pfahl, Bay- coniae and P. annulatum (Fig. 7, 8), but on a poorly supported ern, ant nest in Picea abies, Aug. 2000, D. Clark, DAOMC 250559 (= CBS 140985 = TP-4-1= W 498 = KAS 2193); Pfahl Viechtach, Bayern, Carpenter backbone. Penicillium bissettii and P. vasconiae share the ants (Camponotus herculeanus), Aug. 2000, D. Malloch, culture DAOMC production of red mycelia submerged in the media (Ramírez 250560 (= CBS 140986 = TPF-8-3 = W 512 = KAS 2199). & Martinez 1980). However, P. vasconiae has smooth walled conidiophores, in comparison with the roughened conidiophores Notes — Penicillium camponotum is classified in section of the new species. Penicillium annulatum grows well on CYA Lanata-Divaricata as a close relative to P. subrubescens (Fig. at 37 °C in contrast to P. bissettii, which does not grow at this 7, 8). However, the new species produces weak acid on CREA, temperature. which is absent in P. subrubescens (Mansouri et al. 2013). Also, P. camponotum has larger globose conidia compared to the smaller subglobose to broadly ellipsoidal conidia of Penicillium camponotum Visagie, David Clark & Seifert, sp. nov. P. subrubescens. — MycoBank MB815779; Fig. 20
In: subgenus Aspergilloides, section Lanata-Divaricata. Penicillium cataractum Visagie, Malloch & Seifert, sp. nov. — ITS barcode: KT887855. Alternative identification markers: BenA = MycoBank MB815780; Fig. 21 KT887816, CaM = KT887777. In: subgenus Aspergilloides, section Lanata-Divaricata. Etymology. Latin, camponotum , named in relation to genus of Carpenter ants that the species was isolated from. ITS barcode: KT887847. Alternative identification markers: BenA = KT887808, CaM = KT887769. Type specimen. Canada, New Brunswick, St. Andrews, Carpenter ants (Camponotus pennsylvanicus), Aug. 2000, D. Clark (holotype DAOM 695762, Etymology. Latin, cataractum, meaning waterfall, reflecting the origin of culture ex-type DAOMC 250557 = CBS 140982 = NBBR-2-1 = W 471 = KAS the strains, Niagara Falls, Ontario. 2177). Type specimen. Canada, Niagara falls, on fallen nuts of Carya cordiformis, Colony morphology — Colony diam, 7 d, in mm: CYA (23–)37– May 1996, D. Malloch (holotype DAOM 695763, culture ex-type DAOMC 250534 = CBS 140974 = W 4 = KAS 2145). 45; CYA 37 °C no growth to microcolonies; MEAbl 48–55(–60); YES 53–60; OA 49–52; CREA 20–25. Colony morphology — Colony diam, 7 d, in mm: CYA 34–42; CYA 25 °C, 7 d: Colonies moderately deep, lightly radially CYA 37 °C no growth; MEAbl 15–25; YES 45–60; OA 28–37; sulcate; margins low, narrow, entire; mycelia white; texture CREA 13–15. floccose; sporulation sparse to moderately dense, absent in CYA 25 °C, 7 d: Colonies moderately deep, radially sulcate; some strains, conidia en masse greyish green to dull green margins low, wide, entire; mycelia white; texture floccose; (25C4–E4–26E4); soluble pigments reddish brown, absent in sporulation sparse to moderately dense, conidia en masse some isolates; exudates clear; reverse greyish yellow (4B4–5), dull green (26E4), greyish green (25C3); soluble pigments ab- yellowish white to pale yellow (3A2–3), brownish orange sent; exudates clear; reverse greyish yellow to greyish orange (5C5–6C5) in isolates with soluble pigments. MEA 25 °C, 7 d: (4B4–5B4), pale yellow (4A2). MEA 25 °C, 7 d: Colonies low to Colonies low, plane; margins low, wide, entire; mycelia white; moderately deep, plane; margins low, wide, irregular; mycelia texture floccose; sporulation moderately dense, conidia en white; texture floccose; sporulation moderately dense, conidia masse dull green (26D3–4–E4); soluble pigments absent; exu- en masse dull green (25D4–E4); soluble pigments absent; 272 Persoonia – Volume 36, 2016
Fig. 20 Morphological characters of Penicillium camponotum. a. Colonies from left to right (top row) CYA, MEA, YES; (bottom row) CYA reverse, MEA reverse, YES reverse; b. colony texture on CYA; c. colony texture on MEA; d–j. conidiophores; k. conidia. — Scale bar: d–k = 10 μm. C.M. Visagie et al.: Fifteen new species of Penicillium 273
Fig. 21 Morphological characters of Penicillium cataractum. a. Colonies from left to right (top row) CYA, MEA, YES; (bottom row) CYA reverse, MEA reverse, YES reverse; b. colony texture on CYA; c. colony texture on MEA; d–j. conidiophores; k. conidia. — Scale bar: d–k = 10 μm. 274 Persoonia – Volume 36, 2016
Fig. 22 Morphological characters of Penicillium infrabuccalum. a. Colonies from left to right (top row) CYA, MEA, YES; (bottom row) CYA reverse, MEA reverse, YES reverse; b. colony texture on CYA; c. colony texture on MEA; d–k. conidiophores; l. conidia. — Scale bar: d–l = 10 μm. C.M. Visagie et al.: Fifteen new species of Penicillium 275 exudates absent; reverse greyish yellow to greyish orange white; texture floccose; sporulation sparse to moderately dense, (4B4–5B4), pale yellow (4A2), yellowish grey (4B2). YES 25 °C, conidia en masse greyish green (25B3–4–26B3–4); soluble 7 d: Colonies moderately deep, radially and concentrically pigments absent; exudates absent; reverse brownish orange sulcate, raised at centre; margins low, wide, entire; mycelia (5C6), greyish yellow to greyish orange (4C3–5C3). OA 25 °C, white; texture floccose; sporulation moderately dense, conidia 7 d: Colonies low, plane; margins low, wide, irregular; mycelia en masse greyish green to dull green (25C4–E5); soluble pig- white; texture floccose; sporulation moderately dense, conidia ments absent; exudates absent; reverse greyish yellow (4B5), en masse greyish green to dull green (26C3–D4); soluble pale yellow to light yellow (4A3–4). OA 25 °C, 7 d: Colonies low, pigments absent; exudates absent. CREA 25 °C, 7 d: Weak plane, orange colour in some isolates underneath sporulation; growth, acid not produced. margins low, wide, entire; mycelia white; texture floccose and Conidiophores biverticillate, terverticillate, minor proportion velutinous; sporulation sparse to moderately dense, conidia divaricate; stipes rough, 210–640 × 2–3 μm; branches 20– en masse dull green (26D4–E4); soluble pigments absent; 30(–48) μm; metulae divergent, 2–5 per stipe or branch, 10–19 exudates absent. CREA 25 °C, 7 d: Moderate growth, acid × 2.5–3.5 μm; phialides ampulliform, 5–10 per metula, 7.5–9.5 strongly produced. × 2–3 μm (8.3 ± 0.5 × 2.4 ± 0.2); average length metula/phialide Conidiophores biverticillate, terverticillate, minor proportion 1.5; conidia finely rough walled, subglobose to broadly ellip- divaricate; stipes rough, (120–)200–650 × 3–4 μm; branches soidal, 2.5–3 × 2–3 μm (2.7 ± 0.2 × 2.4 ± 0.1), average width/ 13–30 μm; metulae divergent, 2–5 per stipe or branch, 11–20 length = 0.89, n = 49. × 3–4 μm; phialides ampulliform, 5–10 per metula, 8–10.5 × Extrolites — andrastin A, andrastin B, andrastin C, andrastin 3–3.5 μm (9.5 ± 0.7 × 3.1 ± 0.2); average length metula/phialide D, oxalicine A, oxalicine B, pulvilloric acid. 1.4; conidia rough walled, globose to minor proportion broadly ellipsoidal, 3–3.5 × 3–3.5 μm (3.3 ± 0.1 × 3.3 ± 0.1), average Additional material examined. Canada, New Brunswick, St. Andrews, width/length = 0.95, n = 60. Carpenter ants (Camponotus pennsylvanicus), Aug. 2000, D. Clark, culture DAOMC 250536 (= CBS 140976 = NBSM-4-4 = W 449 = KAS 2168). Extrolites — andrastin A, andrastin B, andrastin C, andras- tin D, pulvilloric acid, trichodermamide A, trichodermamide C, Notes — Penicillium infrabuccalum is classified in section uncharacterised C29H20O7S2, C20H30O6N2. Lanata-Divaricata. The single gene phylogenies resolved it in a clade with P. mariae-crucis and the new species P. cataractum Additional material examined. Canada, Niagara falls, on fallen nuts of Carya ovata, May 1996, D. Malloch, culture DAOMC 250533 (= CBS 140991 (Fig. 7, 8). However, P. mariae-crucis produces sclerotia and = W 6 = KAS 2270); Niagara falls, on fallen nuts of Carya cordiformis, May reddish brown colonies, which are absent in both of the closely 1996, D. Malloch, culture DAOMC 250535 (= CBS 140992 = W 7 = KAS related new species. Penicillium infrabuccalum grows faster on 2271). MEA and has smaller conidia than P. cataractum. Notes — Penicillium cataractum belongs to section Lanata- Divaricata and is a close relative of P. mariae-crucis and the Penicillium panissanguineum Visagie, Ketch & Seifert, sp. nov. new species P. infrabuccalum (Fig. 7, 8). Penicillium mariae- — MycoBank MB815783; Fig. 23 crucis produces sclerotia and reddish brown colonies, which are In: subgenus Aspergilloides, section Lanata-Divaricata. absent in both of the closely related new species. Penicillium cataractum has more restricted growth on MEA and has larger ITS barcode: KT887862. Alternative identification markers: BenA = conidia than P. infrabuccalum. KT887823, CaM = KT887784. Etymology. Latin, panissanguineum, meaning blood of a chimpanzee, Penicillium infrabuccalum Visagie, David Clark & Seifert, sp. named in relation to chimpanzees that ate the soil from where strains were isolated and the red colony reverse on MEA. nov. — MycoBank MB815782; Fig. 22 Type specimen. Tanzania, Kigoma region, soil related with termite mounds, In: subgenus Aspergilloides, section Lanata-Divaricata. Oct. 1996, coll. M.A. Huffman, isol. L. Ketch (holotype DAOM 695771, culture ITS barcode: KT887856. Alternative identification markers: BenA = ex-type DAOMC 250562 = CBS 140989 = W 93 = KAS 2209). KT887817, CaM = KT887778. Colony morphology — Colony diam, 7 d, in mm: CYA 41–44; Etymology. Latin, infrabuccalum, meaning from infrabuccal pockets of CYA 37 °C no growth; MEAbl 30–35; YES 48–55; OA 40–45; the Carpenter ant Camponotus pennsylvanicus, the niche of this fungus. CREA 20–21.
Type specimen. Canada, New Brunswick, St. Andrews, Carpenter ants CYA 25 °C, 7 d: Colonies moderately deep, radially sulcate; (Camponotus pennsylvanicus), Aug. 2000, D. Clark (holotype DAOM 695769, margins low, wide, entire; mycelia white; texture floccose; sporu- culture ex-type DAOMC 250537 = CBS 140983 = NBSM-6-2 = W 475 = KAS lation moderately dense, conidia en masse greyish green to 2181). dull green (26C3–D4), greenish grey (26B2); soluble pigments Colony morphology — Colony diam, 7 d, in mm: CYA 37–45; absent; exudates absent; reverse brown to dark brown (7E6– CYA 37 °C no growth; MEAbl 29–31; YES 54–58; OA 35–37; F6), yellowish white to greyish yellow (4A2–B4). MEA 25 °C, CREA 28–30. 7 d: Colonies low, plane, pinkish red underneath mycelial and CYA 25 °C, 7 d: Colonies moderately deep, radially sulcate; sporulating areas; margins low, wide, irregular; mycelia white; margins low, wide, entire; mycelia white; texture floccose; texture floccose and velutinous; sporulation moderately dense, sporulation sparse to moderately dense, conidia en masse conidia en masse greyish green to dull green (26D4–E5), dull green (26E4–27E3–4), greenish white (25A2); soluble greenish grey (26B2); soluble pigments pink; exudates absent; pigments absent; exudates clear; reverse greyish orange reverse greyish red to deep red (10B5–C8). YES 25 °C, 7 d: (5B3), greyish yellow (4B3), pale yellow (3A2). MEA 25 °C, Colonies moderately deep, radially and concentrically sulcate, 7 d: Colonies low to moderately deep, plane; margins low, raised at centre; margins low, wide, entire; mycelia white, pink- narrow, irregular; mycelia white; texture floccose; sporula- ish areas present; texture floccose; sporulation very sparse, tion moderately dense to dense, conidia en masse dull green conidia en masse light grey; soluble pigments absent; exudates (26D3–27D3–4); soluble pigments absent; exudates absent; absent; reverse greyish red to red (10B5–7), pale yellow (4A3). reverse greenish white (30A2), greenish grey to greyish green OA 25 °C, 7 d: Colonies low, plane; margins low, wide, entire; (30B2–3). YES 25 °C, 7 d: Colonies moderately deep, radially mycelia white; texture floccose; sporulation moderately dense, and concentrically sulcate; margins low, wide, entire; mycelia conidia en masse greenish white to dull green (26B2–D3–E3); 276 Persoonia – Volume 36, 2016
Fig. 23 Morphological characters of Penicillium panissanguineum. a. Colonies from left to right (top row) CYA, MEA, YES; (bottom row) CYA reverse, MEA reverse, YES reverse; b. colony texture on CYA; c. colony texture on MEA; d–j. conidiophores; k. conidia. — Scale bar: d–k = 10 μm. C.M. Visagie et al.: Fifteen new species of Penicillium 277
Fig. 24 Morphological characters of Penicillium tanzanicum. a. Colonies from left to right (top row) CYA, MEA, YES; (bottom row) CYA reverse, MEA reverse, YES reverse; b. colony texture on CYA; c. colony texture on MEA; d. sclerotia; e–i. conidiophores; j. conidia. — Scale bar: d = 50 µm, e–j = 10 μm. 278 Persoonia – Volume 36, 2016 soluble pigments pink and absent; exudates absent. CREA to subglobose, often pointed on one side with connective still 25 °C, 7 d: Weak growth, acid not produced. visible, 2.5–3.5 × 2.5–3.5 μm (3.1 ± 0.2 × 3.0 ± 0.1), average Conidiophores biverticillate, minor proportion terverticillate width/length = 0.96, n = 55; sclerotia 33–105 × 32–96 μm. and divaricate, solitary phialides sometimes present; stipes Extrolites — bisdechlorogeodin, fiscalin A, fiscalin C, pulvil- rough, 200–730 × 2–2.5 μm; branches 19.5–34 μm; metulae loric acid. divergent, 2–4 per stipe or branch,10–16.5(–21) × 2.5–3 μm; Additional material examined. Tanzania, Kigoma region, soil related phialides ampulliform, 3–7 per metula, 6.5–10 × 2.5–3.5 μm with termite mounds, Oct. 1996, coll. M.A. Huffman, isol. L. Ketch, cultures (8.2 ± 0.8 × 2.8 ± 0.2); average length metula/phialide 1.6; co- DAOMC 250515 (= CBS 140969 = 51.92 = KAS 1947), DAOMC 250516 (= nidia smooth walled, globose to broadly ellipsoidal, 2.5–3.5 × CBS 140990 = 50.11 = KAS 2256). 2.5–3.5 μm (3.1 ± 0.2 × 3 ± 0.1), average width/length = 0.95, Notes — Penicillium tanzanicum is classified in section La n = 76. nata-Divaricata. The phylogeny assigns it to a clade of species Extrolites — andrastin A, andrastin B, andrastin C, andrastin typically having roughened conidiophores, i.e. P. simplicissi D, pulvilloric acid. mum, P. araracuaraense and P. wotroi (Fig. 7, 8). Penicillium Additional material examined. Tanzania, Kigoma region, soil related with tanzanicum does not grow on CYA at 37 °C, in contrast with termite mounds, Oct. 1996, coll. M.A. Huffman, isol. L. Ketch, culture DAOMC P. wotroi and P. simplicissimum, which grow at 37 °C. It should 250561 (= CBS 140988 = W 85 = KAS 2208). be noted that this character has been considered unstable Notes — Penicillium panissanguineum is classified in sec- for delineating species in the P. simplicissimum complex (Pitt tion Lanata-Divaricata and is in a clade of species that typically 1980, Houbraken et al. 2011b). Additionally, P. tanzanicum also produce rough-walled conidiophores, such as P. simplicissi grows more restrictedly than P. simplicissimum. Penicillium mum. However, the inability of the new species to grow on CYA araracuaraense produces a weak acid on CREA, whereas at 37 °C and its bright red colony reverse colour on MEA dis- acid production is absent in P. tanzanicum. The main character tinguish it from all other species in this clade. distinguishing the new species from all three of its close rela- tives, is the abundant yellowish sclerotia on MEA, which are absent in close relatives. Penicillium tanzanicum Visagie, Ketch & Seifert, sp. nov. — MycoBank MB815781; Fig. 24 DISCUSSION In: subgenus Aspergilloides, section Lanata-Divaricata. Penicillium species are reported often in ecological surveys. If ITS barcode: KT887841. Alternative identification markers: BenA = identified to the species rank, they are usually determined using KT887802, CaM = KT887763. micromorphological and colony characters. Although there are Etymology. Latin, tanzanicum, named after Tanzania, origin of the strains. some recent comprehensive monographs of some subgenera or sections of Penicillium (Frisvad & Samson 2004, Houbraken Type specimen. Tanzania, Kigoma region, soil related with termite mounds, Oct. 1996, coll. M.A. Huffman, isol. L. Ketch (holotype DAOM 695766, culture et al. 2011a, b, 2014, Visagie et al. 2013, 2016a, b, Peterson et ex-type DAOMC 250514 = CBS 140968 = 50.118 = KAS 1946). al. 2015), the available generic monographs are now 35 years out of date and the species concepts employed were biased Colony morphology — Colony diam, 7 d, in mm: CYA 36–40; heavily towards isolates from food or other niches of particular CYA 37 °C no growth; MEAbl 41–43; YES 47–50; OA 39–40; interest to humans. Species with apparently diverse ecologies, CREA 28–31. such as e.g. P. citrinum (according to Pitt 1980 one of the most CYA 25 °C, 7 d: Colonies moderately deep, radially sulcate; common eukaryotes on Earth), P. glabrum, P. janthinellum and margins low, wide, entire; mycelia white, inconspicuously yel- P. simplicissimum, incorporate great phenotypic diversity. Vari- lowish grey; texture floccose; sporulation sparse, conidia en ability in growth, form and ecology made reliable identification masse greenish grey (27B2–D2); soluble pigments absent; of new isolates very difficult. The consequence is that before exudates absent; reverse brownish orange (6C4–7C4), yel- the development of a standardised molecular approach and lowish white to orange white (4A2–5A2). MEA 25 °C, 7 d: extrolite profiling, species with more restricted ecological niches Colonies low, plane, yellow to greyish yellow colour due to that do not intrude on the human economy tended to be lumped abundant sclerotia dominating colony appearance; margins into other species, their unique genetic or biochemical status low, wide, entire; mycelia white, inconspicuously yellow; texture unrecognised. Many of the strains described as new in this floccose; sporulation absent to very sparse, conidia en masse paper were identified originally as known species using micro- indeterminable; soluble pigments absent; exudates absent; morphology before molecular data indicated the significance of reverse yellowish brown (5D5), pale yellow to greyish yellow some of the deviating or inconspicuous characters. (4A2–B3–5). YES 25 °C, 7 d: Colonies moderately deep, radi- There is increasing evidence of ecological specialisation in ally and concentrically sulcate, raised at centre; margins low, Penicillium. Several of the new species described in this pa- wide, entire; mycelia white; texture floccose; sporulation very per come from studies of esoteric ecological niches, such as sparse, conidia en masse not determined; soluble pigments soil eaten by chimpanzees, infrabuccal cavities of carpenter absent; exudates absent; reverse yellowish brown (5D5), pale ants, caterpillar intestines, nuts in temperate forests, or leaf yellow to greyish yellow (4A2–B3–5). OA 25 °C, 7 d: Colonies surfaces in tropical rain forests. It seems likely that several low, plane, yellow to greyish yellow colour due to abundant of our new species are actual functional components and not sclerotia; margins low, wide, entire; mycelia white, inconspicu- incidental contaminants of the ecosystem samples. There was ously yellow; texture floccose; sporulation very sparse, conidia no particular effort to target Penicillium specifically in most of en masse not determined; soluble pigments absent; exudates these studies, so the strains were isolated among other fungi absent. CREA 25 °C, 7 d: Weak growth, acid not produced. at differing frequencies. The Tanzanian soil eaten by chimpan- Conidiophores biverticillate, divaricate; stipes rough, (80–)200– zees, for example, yielded a broad diversity of hyphomycetes, 875 × 2.5–3.5 μm; branches 12.5–13.5; metulae divergent, 2–4 including many Penicillium species that were a significant part per stipe, unequal length on same conidiophore, 11.5–26 × of the mycota (Ketch 1998). Even though it is likely that some 2.5–3.5 μm; phialides ampulliform, 4–8 per metula, 8–11 × penicillia are actually true soil fungi or associates of roots, 2.5–3.5 μm (9.5 ± 0.0.7 × 3.0 ± 0.3); average length metula/ we speculate that many species have other primary habitats phialide 1.9; conidia rough walled, some finely rough, globose where they may perform various functions, similar to the close C.M. Visagie et al.: Fifteen new species of Penicillium 279 association that a number of subgenus Penicillium species whereas these extrolites were absent in DAOMC 167033, which have with food related products (Frisvad & Samson 2004) such produced penicillic acid. as grains, dairy products or fruits. This may explain why it is Pulvilloric acid (C15H18O5) was first identified in cultures of often difficult to obtain numerous strains of one species when P. pulvillorum (Brian et al. 1957), and its structure was eluci- making isolations from soil. dated (Barber et al. 1986, Barret et al. 1969, McOmie et al. Previous studies hint at possible interactions between Penicil- 1966) following isolation under acidic conditions (pH = 1). lium and insect hosts or habitats. Penicillium brocae has a very Recently, pulvilloric acid was identified in the related P. wotroi close association with coffee-berry borers (Peterson et al. 2003) and P. araracuaraense (Houbraken et al. 2011b). Using LC-high while P. mallochii and P. guanacastense are known only from resolution mass spectrometry (LC-HRMS) in negative ionization the intestines of Costa Rican caterpillars (Rivera et al. 2012). mode, we’ve identified a similar compound of chemical formula
Penicillium herquei and the leave-rolling weevil (Euops chin- C15H20O6 (m/z 295.1186) as an extrolite of P. panissanguineum, ensis) have a close association (Kobayashi et al. 2008, Li et P. cataractum, P. infrabuccalum and P. tanzanicum. An isochro- al. 2012), and mites are suspected dispersal vectors for some man similar to pulvilloric acid, with an identical chemical formula Penicillium species in Protea repens infructescences (Visagie to that reported in this study, was characterised in cultures of the 2012). The dominant fungi in buccal cavities of carpenter ants phylogenetically related species P. simplicissimum and patented were species of Mortierella (Clark 2002), but our new species as an amyloid aggregation inhibitor (Hirai et al. 2001). Inter- P. camponotum seems to be a consistent inhabitant of this estingly, when these strains were screened by positive mode niche, and occurred in both Canada and Germany. The other LC-HRMS, the m/z of the major peak was 279.1226, which cor- two species, P. infrabuccalum with two isolates and P. fundy- responds to the chemical formula of pulvilloric acid (C15H18O5) ense with one, are known only from this niche but with so few (Fig. 9). The LC conditions were adjusted to ensure that the strains available, it is difficult to interpret the significance of two contradictory signals observed in positive and negative the association. ionization modes were indeed from the same compound. Whatever the actual ecological roles of the species described Non-aqueous extraction solutions were also used to determine in this paper, it seems likely that the extrolites they produce if the extraction method was causing an undesired chemical will have crucial ecological functions. From the 15 species reaction. We determined that during ionization in positive described here, 36 known extrolites were identified and 11 mode, protonation of this compound causes it to immediately + uncharacterised compounds detected (Table 3). As shown re- dissociate by neutral loss of water, producing a [C15H18O5+H] peatedly in Penicillium, extrolite profiling is a valuable addition product ion that can be mistaken as the molecular ion. This is to morphological characterisation and gene comparisons for a charge-directed fragmentation phenomenon known to occur species delimitation (e.g. Frisvad & Filtenborg 1990, Sonjak when the protonated, gas-phase ion is highly unstable (Ren- et al. 2007, Tuthill et al. 2001), although chemotaxonomic aud et al. 2013). In negative mode ionization, the molecular comparisons with close relatives were not possible for most of charge is obtained through the deprotonation of the carboxylic our new species because of the lack of data for sister spe- acid and the compound does not undergo extensive in-source cies. Among the species we studied, P. diabolicalicense, fragmentation, exemplifying the benefits of LC-MS screening P. improvisum, P. alogum and P. aotearoae produced unique, with both polarities. Our results suggest that ‘pulvilloric acid’ previously unknown extrolites. Isofumigaclavine A and citrinin which was previously identified in cultures of P. wotroi and were extrolites of P. improvisum. Restricticins are broad spec- P. araracuaraense and quite possibly P. pulvillorum itself, is trum antifungal polyenes previously only identified in cultures not the structure as it exists on artificial solid and liquid growth of P. restrictum (Hensens et al. 1991, Schwartz et al. 1991) and media; it is the patented isochroman isolated from cultures of Penicillium sp. NR6564 (Matsukuma et al. 1992). Penicillium P. simplicissimum. The originally reported pulvilloric acid struc- diabolicalicense also produces these compounds, but the less ture is known to be unstable and require careful extraction and active N,N-dimethylrestricticin occurred in greater concentra- work-up methodology, to avoid rearrangement and decomposi- tions. In this study, P. diabolicalicense (section Exilicaulis) tion reactions from occurring during isolation (Tanenbaum & was the only producer of the neurotoxin penitrem A (on CYA), Nakajima 1969). Therefore, it is reasonable to believe that the which is otherwise well-known among species of subgenus two structures could be inter-convertible due to an increased Penicillium. Penicillium alogum and P. aotearoae produced stability of the molecule provided by the aromatisation of the uncharacterised extrolites of formula C21H28O8 and C19H24O8, originally proposed structure of pulvilloric acid to its isochro- respectively. Penicillium amphipolaria was the only producer of man. However, the true form of pulvilloric acid within the natural fusaperazine E, fumitremorgin B and several related, uncharac- environments of its producers remains unknown. In solid and terised metabolites. Andrastins were the extrolites of P. nucicola, liquid growth media, the formula of this extrolite is C15H20O6 and P. cataractum, P. infrabuccalum, P. panissanguineum and some we will simply maintain the name of pulvilloric acid. P. camponotum. Despite these similarities, all species produced unique combinations of additional extrolites (Table 3). Acknowledgements This paper is warmly dedicated to Dr Robert A. Infraspecific variation of extrolite production was observed for Samson on the occasion of his 70th birthday. Rob is a teacher, mentor and P. camponotum, P. cataractum and P. bissettii. All strains of colleague to the senior authors of this paper and we celebrate the remark- P. camponotum produced marcfortine A and marcfortine B, yet able achievements of his career, in particular his contributions to Penicillium taxonomy and phylogeny, with this paper. KAS thanks Peter Johnston, My- the predominant extrolites produced by Canadian and Ger- cology & Bacteriology, Landcare Research, Auckland, New Zealand, for his man strains differed significantly. The seven Canadian strains assistance in arranging a collecting permit for Arthur’s Pass National Park, isolated in New Brunswick from carpenter ants (Camponotus where two of the new species in this paper were collected in 2003. Walter pennsylvanicus) produced an uncharacterised compound of Gams assisted with collection of soil samples in Italy in 2008. Walter Gams and Shaun Pennycook assisted with the derivation of appropriate epithets for formula C15H22O2 that was absent in the German strains isolated from Camponotus herculeanus, which produced andrastin A. some of our species. KAS, RA, GL-S, JR and MS received operating support The greatest infraspecific variation of extrolites occurred within from several competitive grants offered by Agriculture & Agri-Food Canada strains of P. bissettii, both of which were isolated from soil Science and Technology Branch. DWM acknowledges nearly three decades of support from the Natural Sciences and Engineering Research Council of samples collected from a spruce forest in Quebec, Canada Canada (grants OGP0000145 and STR0149534). LK was supported by the on the same date. DAOMC 167011 produced high amounts of Natural Science and Engineering Research Council of Canada (NSERC) meleagrin and aurantioclavine in all three growth media tested postgraduate scholarship program. MU acknowledges support from NSERC 280 Persoonia – Volume 36, 2016 and the US National Science Foundation (NSF) to R. Greg Thorn, from NSF Mansouri S, Houbraken J, Samson RA, et al. 2013. Penicillium subrubescens, to Daniel H. Janzen and from CR-USA to Costa Rican parataxonomists at the a new species efficiently producing inulinase. Antonie van Leeuwenhoek Area de Conservación Guanacaste who collected and reared the caterpillar 103: 1343–1357. host of P. costaricense and isolated and sent the fungi to the Thorn lab in Matsukuma S, Ohtsuka T, Kotaki H, et al. 1992. A new series of natural London, Ontario. We are grateful to the Molecular Microbiology Technology antifungals that inhibit P450 lanosterol C-14 demethylase. I. Taxonomy, Laboratory group at ORDC for their support with DNA sequencing. fermentation, isolation and structural elucidation. The Journal of Antibiot- ics 45: 151–159. McOmie JWF, Turner AB, Tute MS. 1966. The structure of pulvilloric acid. References Journal of the Chemical Society C 1966: 1608–1613. Nylander AJJ, Ronquist F, Huelsenbeck JP, et al. 2004. Bayesian phylogenetic Barber J, Bradley SM, Pope SM. 1986. Isolation, purification and nuclear mag- analysis of combined data. Systematic Biology 53: 47–67. netic resonance spectra of pulvilloric acid. Mycotoxin Research 2: 25–32. Peterson SW, Jurjević Ž, Frisvad JC. 2015. Expanding the species and Barret GC, McOmie JWF, Nakajima S, et al. 1969. Absolute configuration of chemical diversity of Penicillium section Cinnamopurpurea. PLoSOne pulvilloric acid 1969: 1068–1069. 10: e0121987. Blakeslee AF. 1915. Lindner’s roll tube method of separation cultures. Phyto Peterson SW, Pérez J, Vega F, et al. 2003. Penicillium brocae, a new spe- pathology 5: 68–69. cies associated with the coffee berry borer in Chiapas, Mexico. Mycologia Bragulat MR, Abarca ML, Cabanes FJ. 2001. An easy screening method for 95: 141–147. fungi producing ochratoxin A in pure culture. International Journal of Food Peterson SW, Sigler L. 2002. Four new Penicillium species having Thy- Microbiology 71: 139–144. sanophora-like melanized conidiophores. Mycological Research 106: Brian PW, Curtis PJ, Hemming HG, et al. 1957. Pulvilloric acid, an antibiotic 1109–1118. obtained from cultures of Penicillium pulvillorum. Transactions British Myco Pitt JI. 1980. The genus Penicillium and its teleomorphic states Eupenicillium logical Society 40: 369–374. and Talaromyces. Academic Press, London. Clark DW. 2002. Fungi associated with the infrabuccal pockets of Cam- Ramírez C, Martinez C. 1980. Some new species of Penicillium recovered ponotus pennsylvanticus and other formicine ants. MSc thesis, University from the atmosphere in Madrid and from other substrata. Mycopathologia of Toronto, Department of Botany, Canada. 72: 181–191. Filtenborg O, Frisvad JC, Thrane U. 1990. The significance of yeast extract Renaud JB, Overton S, Mayer PM. 2013. Energy and entropy at play in com- composition on metabolite production in Penicillium. Modern concepts in petitive dissociations: The case of uneven positional dissociation of ionized Penicillium and Aspergillus classification: 433–441. triacylglycerides. International Journal of Mass Spectrometry 352: 77–86. Frisvad JC. 1981. Physiological criteria and mycotoxin production as aids in Rivera KG, Díaz J, Chavarría-Díaz F, et al. 2012. Penicillium mallochii and identification of common asymmetric Penicillia. Applied and Environmental P. guanacastense, two new species isolated from Costa Rican caterpillars. Microbiology 41: 568–579. Mycotaxon 119: 315–328. Frisvad JC, Filtenborg O. 1990. Revision of Penicillium subgenus Furca- Ronquist F, Huelsenbeck JP. 2003. MrBayes 3: Bayesian phylogenetic infer- tum based on secondary metabolites and conventional characters. In: ence under mixed models. Bioinformatics 19: 1572–1574. Samson RA, Pitt JI (eds), Modern concepts in Penicillium and Aspergillus Schoch CL, Seifert KA, Huhndorf S, et al. 2012. Nuclear ribosomal internal classification: 159–172. Plenum Press, New York. transcribed spacer (ITS) region as a universal DNA barcode marker for Frisvad JC, Samson RA. 2004. Polyphasic taxonomy of Penicillium subgenus Fungi. Proceedings of the National Academy of Sciences of the United Penicillium. A guide to identification of food and air-borne terverticillate States of America 109: 6241–6246. Penicillia and their mycotoxins. Studies in Mycology 49: 1–174. Schwartz RE, Dufresne C, Flor JE, et al. 1991. Restricticin, a novel glycine- Hensens OD, Wichmann CF, Liesch JM, et al. 1991. Structure elucidation containing antifungal agent. The Journal of Antibiotics 44: 463–471. of restricticin, a novel antifungal agent from Penicillium restrictum. Tetra- Serra R, Peterson S, CTCOR, et al. 2008. Multilocus sequence identification hedron 47: 3915–3924. of Penicillium species in cork bark during plank preparation for the manu- Hirai H, Ichiba T, Tonai H. 2001. Isochroman compounds and their production facture of stoppers. Research in Microbiology 159: 178–186. process. Pfizer Inc, New York (patent CA2291931). Sonjak S, Uršič V, Frisvad JC, et al. 2007. Penicillium svalbardense, a new Houbraken J, Frisvad JC, Samson RA. 2011a. Taxonomy of Penicillium species from Arctic glacial ice. Antonie van Leeuwenhoek 92: 43–51. section Citrina. Studies in Mycology 70: 53–138. Swofford DL. 2003. PAUP*. Phylogenetic analysis using parsimony (*and Houbraken J, Lopez-Quintero CA, Frisvad JC, et al. 2011b. Penicillium other methods). Version 4. Sinauer Associates, Massachusetts. araracuarense sp. nov., Penicillium elleniae sp. nov., Penicillium penaro- Tanenbaum SW, Nakajima S. 1969. The biosynthesis of pulvilloric acid. I. jense sp. nov., Penicillium vanderhammenii sp. nov. and Penicillium wotroi Production, isolation, and degradation. Biochemistry 8: 4622–4626. sp. nov., isolated from leaf litter. International Journal of Systematic and Tuthill D, Frisvad JC, Christensen M. 2001. Systematics of Penicillium sim- Evolutionary Microbiology 61: 1462–1475. plicissimum based on rDNA sequences, morphology and secondary me- Houbraken J, Samson RA. 2011. Phylogeny of Penicillium and the segrega- tabolites. Mycologia 93: 298–308. tion of Trichocomaceae into three families. Studies in Mycology 70: 1–51. Visagie CM. 2012. The polyphasic taxonomy of Penicillium and Talaromyces Houbraken J, Visagie CM, Meijer M, et al. 2014. A taxonomic and phyloge- spp. isolated from the diverse fynbos biome. PhD thesis, Stellenbosch netic revision of Penicillium section Aspergilloides. Studies in Mycology University, Department of Microbiology, South Africa. 78: 373–451. Visagie CM, Hirooka Y, Tanney JB, et al. 2014a. Aspergillus, Penicillium and Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software Talaromyces isolated from house dust samples collected around the world. version 7: improvements in performance and usability. Molecular Biology Studies in Mycology 78: 63–139. and Evolution 30: 772–780. Visagie CM, Houbraken J, Dijksterhuis J, et al. 2016a. A taxonomic review Ketch LA. 1998. Microbiological investigations of geophage in chimpanzees. of Penicillium species producing conidiophores with solitary phialides, MSc thesis, Univeristy of Toronto, Canada. classified in section Torulomyces. Persoonia 36: 134–155. Kobayashi C, Fukasawa Y, Hirose D, et al. 2008. Contribution of symbiotic Visagie CM, Houbraken J, Frisvad JC, et al. 2014b. Identification and nomen- mycangial fungi to larval nutrition of a leaf-rolling weevil. Evolutionary clature of the genus Penicillium. Studies in Mycology 78: 343–371. Ecology 22: 711–722. Visagie CM, Houbraken J, Rodriques C, et al. 2013. Five new Penicillium Kornerup A, Wanscher JH. 1967. Methuen handbook of colour. Methuen & species in section Sclerotiora: a tribute to the Dutch Royal family. Per- Co Ltd, Copenhagen. soonia 31: 42–62. Li X, Guo W, Ding J. 2012. Mycangial fungus benefits the development Visagie CM, Seifert KA, Houbraken J, et al. 2016b. A phylogenetic revision of a leaf-rolling weevil, Euops chinesis. Journal of Insect Physiology 58: of Penicillium sect. Exilicaulis, including nine new species from fynbos in 867–873. South Africa. IMA fungus: accepted manuscript.