Species Diversity in Penicillium and Talaromyces from Herbivore Dung, and the Proposal of Two New Genera of Penicillium-Like Fungi in Aspergillaceae

VOLUME 5 JUNE 2020 Fungal Systematics and Evolution PAGES 39–75

doi.org/10.3114/fuse.2020.05.03

Species diversity in Penicillium and Talaromyces from herbivore dung, and the proposal of two new genera of penicillium-like fungi in Aspergillaceae

M. Guevara-Suarez1,2, D. García1, J.F. Cano-Lira1, J. Guarro1, J. Gené1*

1Unitat de Micologia, Facultat de Medicina i Ciències de la Salut and IISPV, Universitat Rovira i Virgili, Reus, Spain 2Laboratorio de Micología y Fitopatología (LAMFU), Vicerrectoría de Investigaciones, Universidad de los Andes, Bogotá, Colombia

*Corresponding author: [email protected]

Key words: Abstract: Coprophilous fungi are saprotrophic organisms that show great diversity, mainly on herbivore dung. The Aspergillaceae physico-chemical characteristics of this peculiar substrate combined with the high level of fungal adaptation to different coprophilous fungi environmental conditions offer the perfect setting for discovering new taxa. This study focused on the species diversity of Eurotiales penicillium-like fungi isolated mainly from herbivore dung collected at different Spanish locations. From 130 samples, a phylogeny total of 104 isolates were obtained, and 48 species were identified. Preliminary identifications were based on morphology Trichocomaceae and partial β-tubulin (tub2) gene sequences. Putative new taxa were characterized by a multi-gene sequencing analysis testing the tub2, the internal transcribed spacer rDNA (ITS), calmodulin (cmdA), and RNA polymerase II second largest subunit (rpb2) genes, and a detailed phenotypic study. Using this polyphasic approach and following the genealogical concordance phylogenetic species recognition (GCPSR) method, we propose the new genera Penicillago (for Penicillium nodositatum) and Pseudopenicillium (for Penicillium megasporum and P. giganteum) in the family Aspergillaceae, and 11 new species, including seven Penicillium, three Talaromyces and one Pseudopenicillium. A lectotype and epitype are designed for Penicillium nodositatum. Our results show that the species diversity of penicillium-like fungi on herbivore dung has not been widely studied and that this substrate seems to be a good reservoir of interesting Eurotialean fungi. Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] Effectively published online: 21 August 2019

INTRODUCTION (Seifert & Samson 1985, Frisvad & Samson 2004, Wang & Zhuang 2005). Currently, the new concept of the genus, which Herbivore dung is a complex substrate that has a high amount is based on molecular taxonomy, coprophilous penicillia belongs of readily available carbohydrates with high nitrogen content, in section Robsamsonia (Houbraken et al. 2016), which also water-soluble vitamins, growth factors and mineral ions (Bell includes other species previously assigned to series Urticicolae 1983). Due to the nutritional properties, together with its (Frisvad & Samson 2004). Furthermore, other species of physical structure, pH, and the varying moisture content, it is Penicillium previously found on dung currently belong to the an excellent substrate for a great diversity of fungi to grow (Bell genus Talaromyces (Houbraken et al. 2016). Coprophilous 1983, Richardson 2001, Herrera et al. 2011). species reported in this latter genus areT. atroroseus, T. helicus, Ascomycetes accounts for the largest number of species T. flavus, T. muroii and T. trachyspermus, and two others T. found on dung from herbivorous animals; genera such as dupontii and T. emersonii, which have been transferred to Ascobolus, Lasiobolus, Podospora or Thelebolus practically only respectively, Thermomyces and Rasamsonia (Masunga et al. occur on this substrate (Webster 1970, Bell 1983, Richardson 2006, Frisvad et al. 2013, Yilmaz et al. 2014). 2001). On the contrary, other ascomycetes like Eurotialean The taxonomy of penicillium-like fungi has changed penicillium-like fungi are not considered predominantly dramatically due to phylogenetic data. Houbraken & Samson coprophilous (Krug et al. 2004), but they can be commonly found (2011) demonstrated that Penicillium is phylogenetically more- on this substrate when its soluble materials are exhausted, and closely related to Aspergillus (family Aspergillaceae) than to its relative humidity has reduced to below 85 % (Kuthubutheen Talaromyces (family Trichochomaceae). In the last decade, the & Webster 1986). However, species diversity of this latter group species of these genera have been delineated on the basis of has been poorly studied on herbivore dung. Although Seifert et a polyphasic approach that has mainly included the evaluation al. (1983) provided a list of near 20 Penicillium species isolated of morphological and multi-locus sequence analyses testing from animal faeces, their identification was based exclusively on with the internal transcribed spacer region rDNA (ITS), and phenotypic features. the β-tubulin (tub2), calmodulin (cmdA), and/or the DNA- Traditionally, based on similarities in ecology, morphology dependent RNA polymerase II largest subunit (rpb2) genes and extrolite production, most of the Penicillium species (Peterson 2008, Houbraken et al. 2014a, Samson et al. 2014, considered coprophilous (i.e. P. brevistipitatum, P. clavigerum, P. Visagie et al. 2014b, Yilmaz et al. 2014). In addition, tub2 concentricum, P. coprobium, P. coprophilum, P. formosanum, P. was recommended as an identification marker in Penicillium glandicola and P. vulpinum) were classified in series Claviformia (Visagie et al. 2014b) and Talaromyces (Yilmaz et al. 2014).

Fungal Systematics and Evolution is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License © 2020 Westerdijk Fungal Biodiversity Institute 39 Guevara-Suarez et al.

The use of this already generalised approach has proven to Molecular identification and phylogenetic analysis be able to discriminate very close species and to discover and characterise new taxa (Visagie et al. 2014b, Guevara-Suarez et Isolates were cultured on PDA or malt extract agar (MEA; Difco, al. 2017, Barbosa et al. 2018). Detroit, USA) for 7─14 d at 25 °C. DNA was extracted using the Given the scarcity of data on species isolated from dung and FastDNA® kit protocol (MP Biomedicals, Solon, OH) and for the identified by modern techniques, we consider this the perfect lysis step done with a FastPrep® FP120 cell disrupter (Thermo setting for discovering new taxa. Therefore, the present work Savant, Holbrook, NY).

focuses on the study of the species diversity of penicillium- Preliminary species identifications were carried out BLASTing Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] like fungi from herbivore dung samples collected across tub2 DNA sequences using GenBank. In the case of putative Spain. Identification was made using both morphology and by new species, the ITS region, including the 5.8S rRNA gene, and comparing multi-locus sequences with a database of ex-type fragments of cmdA and/or rpb2 genes were also amplified and verified reference strains. For multisequence analyses, the and sequenced. The primer pairs used were: ITS5/ITS4 for ITS Genealogical Phylogenetic Species Recognition (GCPSR) method (White et al. 1990), Bt2a/Bt2b for tub2 (Glass & Donaldson was applied to data in support of discovering putative new 1995), CMD5/CMD6 for cmdA (Hong et al. 2006), and RPB2-5F/ species (Taylor et al. 2000). RPB2-7Cr for rpb2 (Liu et al. 1999). The amplification protocol and PCR conditions were performed using methods and primers previously described (Peterson 2008, Houbraken & Samson MATERIALS AND METHODS 2011, Samson et al. 2014, Visagie et al. 2014b, Yilmaz et al. 2014). The amplified products were purified and sequenced Sampling and fungal isolation at Macrogen Corp. Europe (Amsterdam, the Netherlands) with a 3730xl DNA analyzer (Applied Biosystems, Foster City, CA). Dung samples were collected in 2016 and 2017 from Spanish Consensus sequences were obtained using SeqMan v. 7.0.0 regions that have different climates and biodiversity, such as (DNASTAR, Madison, WI). Newly generated sequences and their Andalusia, the Balearic and Canary Islands, Cantabria, Castilla- GenBank/EMBL accession numbers are summarized in Table 1. Leon, Catalonia, Extremadura and Galicia. The dung collected Sequences were retrieved from GenBank taking into was mostly from herbivorous animals, such as rabbits, sheep, account the last update of the database of the International deer, goats, cattle and horses. However, some samples from Commission of Penicillium and Aspergillus (http://www. other animals, such as wild pigs or foxes, and even soil mixed aspergilluspenicillium.org), which includes all the accepted with dung was also studied. The samples were placed in species for Penicillium and Talaromyces (Samson et al. 2014, individual paper or plastic bags and processed no later than Visagie et al. 2014b, Yilmaz et al. 2014). Single and concatenated 3 d after collection. Individual samples were divided into phylogenetic analyses were performed to delineate putative two parts: one processed using moist chambers (Richardson new species and the phylogenies corresponding to each section 2001) and the other by a modified Waksman (1922) dilution of those genera were properly reconstructed. series method. For moist chambers, pieces of the sample Data sets for each locus were aligned individually using were placed on moist filter paper with sterile distilled water ClustalW (Thompson et al. 1994), in MEGA v. 6.0 software in individual Petri dishes and incubated at room temperature (Tamura et al. 2013), refined with MUSCLE (Edgar 2004) under (22–25 °C) for up to 30 d. For the dilution series, approximately the same platform, and manually adjusted when needed. one gram of dung or associated soil was diluted 1:10 (w/v) in Larger alignments including different sections of each genus sterile water and shaken for approximately 10 min. Aliquots was performed using the MAFFT tool in the EMBL-EBI Web of the suspensions were pipetted into Petri dishes and mixed Services portal (https://www.ebi.ac.uk/Tools/msa/mafft/) and with 20 mL of melted cooled agar medium. Culture media manually adjusted in MEGA v. 6.0. Phylogenetic reconstructions used for isolation were potato dextrose agar (PDA; Pronadisa, by maximum likelihood (ML) and Bayesian inference (BI) were Madrid, Spain), potato carrot agar (PCA; 20 g potatoes, 20 g carried out using MEGA v. 6.0 and MrBayes v. 3.1.2 (Huelsenbeck carrot, 20 g agar, 1000 mL distilled water), both supplemented & Ronquist 2001, Ronquist & Huelsenbeck 2003, Tamura et al. with chloramphenicol (200 mg/L), and dichloran rose-bengal 2013), respectively. For ML analyses, the trees were inferred chloramphenicol agar (DRBC; 5 g peptone, 10 g glucose, 1 g using Nearest-Neighbour-Interchange as a heuristic method and

KH2PO4, 0.5 g MgSO4, 25 mg rose-bengal, 2 mg dichloran, 200 gaps were treated as partial deletion with a 95 % site coverage mg chloramphenicol, 20 g agar, 1 000 mL distilled water). All cut-off. Phylogenetic support for internal branches was assessed media were supplemented with dieldrin in dimethyl-ketone (1 by 1 000 ML bootstrapped pseudoreplicates and bootstrap %). Petri dishes were incubated at room temperature for up support (bs) ≥ 70 % was considered significant. The BI analyses to 30 d. The moist chambers and Petri dishes were examined were performed using five million Markov chain Monte Carlo at regular intervals with the aid of a stereo-microscope, and (MCMC) generations, with two runs (one cold and three heated conidia from sporulating colonies were transferred to PDA chains) and samples were stored every 1 000 generations. The supplemented with chloramphenicol. 50 % majority-rule consensus tree and posterior probability Isolates identified morphologically as penicillium-like were values (pp) were calculated after discarding the first 25 % of the preserved and deposited in the culture collection of the Faculty samples. A pp value ≥ 0.95 was considered significant. The best of Medicine, Reus (FMR). Cultures of interesting species as well substitution models for each data partition were estimated using as type material and cultures of the new species were deposited jModelTest v. 2.1.3 according to the Akaike criterion (Darriba at the Westerdijk Fungal Biodiversity Institute (CBS, Utrecht, the et al. 2012, Guindon & Gascuel 2003). The length, number of Netherlands). Nomenclatural novelties and descriptions were variable and phylogenetic informative sites, and substitution deposited in MycoBank (Crous et al. 2004). models for each data partition are summarized in Table 2. The resulting trees were plotted using FigTree v. 1.3.1 (http://tree.

Table 1. Isolates of Penicillium, Talaromyces and related genera included in the study and their GenBank/EMBL accession numbers. Genus/Species1 Section Collection Substrate and Origin GenBank/EMBL accession numbers 2 numbers ITS tub2 cmdA rpb2 P. arabicum Exilicaulis FMR 15298 Dung, Castile and Leon – LT898226 – – FMR 15095 Dung, Catalonia – LT898225 – – P. atramentosum Paradoxa FMR 15309 Dung, Castile and Leon – LT898224 – –

Editor-in-Chief FMR 15046 Dung, Catalonia – LT898221 – – Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected]

FMR 15092 Dung, Catalonia – LT898222 – – FMR 15102 Dung, Catalonia – LT898223 – – P. balearicum Paradoxa FMR 15191T = Dung, Balearic Islands LT899762 LT898227 LT899758 LT899760 CBS 143044 FMR 15196 Dung, Balearic Islands LT899763 LT898228 LT899759 LT899761 P. beceitense Ramosa FMR 15038T = Dung, Catalonia LT899780 LT898229 LT899764 LT899798 CBS 142989 P. biforme Fasciculata FMR 15312 Dung, Castile and Leon – LT898230 – – FMR 15313 Dung, Castile and Leon – LT898231 – – P. brasilianum Lanata-Divaricata FMR 15483 Dung, Galicia – LT898232 – – P. brevicompactum Brevicompacta FMR 15105 Dung, Catalonia – LT898233 – – P. brevistipitatum Robsamsonia FMR 15103 Dung, Catalonia – LT898234 – – P. burguense Exilicaulis FMR 15493 Dung, Galicia – LT898235 – – P. canariense Stolkia FMR 15838 Dung, Canary Islands – LT898236 – – P. canescens Canescentia FMR 15028 Dung, Catalonia – LT898237 – – P. caprifimosum Turbata FMR 15041T = Dung, Catalonia LT899781 LT898238 LT899765 LT899799 CBS 142990 P. chrysogenum Chrysogena FMR 15100 Dung, Catalonia – LT898244 – – P. cinereoatrum Exilicaulis FMR 15033 Dung, Catalonia – LT898284 – – P. citrinum Citrina FMR 15646 Dung, Castile and Leon – LT898242 – – FMR 15647 Dung, Castile and Leon – LT898243 – – FMR 15094 Dung, Catalonia – LT898239 – – FMR 15486 Dung, Galicia – LT898240 – – FMR 15520 Dung, Galicia – LT898241 – – P. concentricum Robsamsonia FMR 15195 Dung, Balearic Islands – LT898245 – – FMR 15840 Dung, Castile and Leon – LT898246 – – P. coprobium Robsamsonia FMR 15201 Dung, Balearic Islands – LT898247 – – FMR 15311 Dung, Castile and Leon – LT898248 – – P. coprophilum Robsamsonia FMR 15187 Dung, Balearic Islands – LT898249 – – P. cremeogriseum Lanata-Divaricata FMR 15487 Dung, Galicia – LT898250 – – FMR 15488 Dung, Galicia – LT898251 – – P. crustosum Fasciculata FMR 15185 Dung, Balearic Islands – LT898259 – – FMR 15186 Dung, Balearic Islands – LT898260 – – FMR 15189 Dung, Balearic Islands – LT898261 – – FMR 15194 Dung, Balearic Islands – LT898262 – – FMR 15197 Dung, Balearic Islands – LT898263 – – FMR 15200 Dung, Balearic Islands – LT898264 – – FMR 15213 Dung, Balearic Islands – LT898265 – – FMR 15034 Dung, Catalonia – LT898252 – – FMR 15036 Dung, Catalonia – LT898253 – – FMR 15037 Dung, Catalonia – LT898254 – – FMR 15042 Dung, Catalonia – LT898255 – – FMR 15043 Dung, Catalonia – LT898256 – – FMR 15045 Dung, Catalonia – LT898257 – –

Table 1. (Continued). GenBank/EMBL accession numbers 1 Collection Genus/Species Section 2 Substrate and Origin numbers ITS tub2 cmdA rpb2 FMR 15098 Dung, Catalonia – LT898258 – – FMR 15494 Dung, Galicia – LT898266 – – P. cvjetkovicii Cinnamopurpurea FMR 15310 Dung, Castile and Leon – LT898267 – –

Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] P. expansum Penicillium FMR 15097 Dung, Catalonia – LT898268 – – FMR 15484 Dung, Galicia – LT898269 – – P. fimosum Paradoxa FMR 15104T = Dung, Catalonia LT970836 LT898273 LT970837 CBS 142991 P. flavigenum Chrysogena FMR 15096 Dung, Catalonia – LT898270 – – P. frequentans Aspergilloides FMR 15193 Dung, Balearic Islands – LT898271 – – FMR 15212 Dung, Balearic Islands – LT898272 – – P. glabrum Aspergilloides FMR 15184 Dung, Balearic Islands – LT898274 – – FMR 15190 Dung, Balearic Islands – LT898275 – – FMR 15209 Dung, Balearic Islands – LT898276 – – P. griseofulvum Robsamsonia FMR 15203 Dung, Balearic Islands – LT898280 – – FMR 15204 Dung, Balearic Islands – LT898281 – – FMR 15207 Dung, Balearic Islands – LT898282 – – FMR 15314 Dung, Castile and Leon – LT898283 – – FMR 15029 Dung, Catalonia – LT898277 – – FMR 15030 Dung, Catalonia – LT898278 – – FMR 15093 Dung, Catalonia – LT898279 – – P. ibericum Paradoxa FMR 15040T = Dung, Catalonia LT899782 LT898285 LT899766 LT899800 CBS 142992 FMR 15107 Soil, Galicia LT899783 LT898286 LT899767 LT899801 P. lilacinoechinulatum Sclerotiora FMR 15492 Dung, Galicia – LT898287 – – P. magnielliptisporum Paradoxa FMR 15044 Dung, Catalonia – LT898288 – – P. mediterraneum Roquefortorum FMR 15188T = Dung, Balearic Islands LT899784 LT898291 LT899768 LT899802 CBS 142754 FMR 15031 = Dung, Catalonia LT899785 LT898289 LT899769 LT899803 CBS 142755 FMR 15032 Dung, Catalonia LT899786 LT898290 LT899770 LT899804 P. momoii Exilicaulis FMR 15208 Dung, Balearic Islands – LT898292 – – P. murcianum Canescentia FMR 15304 Dung, Andalusia – LT898293 – – FMR 15305 Dung, Andalusia – LT898294 – – FMR 15308 Dung, Andalusia – LT898295 – – FMR 15491 Dung, Galicia – LT898296 – – FMR 15845 Dung, Galicia – LT898297 – – P. polonicum Fasciculata FMR 15099 Dung, Catalonia – LT898298 – – P. radiolubatum Canescentia FMR 15485 Dung, Canary Islands – LT898299 – – P. roseoviride Aspergilloides FMR 15645 Dung, Castile and Leon – LT898300 – – P. rubefaciens Exilicaulis FMR 15202 Dung, Balearic Islands – LT898301 – – FMR 15297 Dung, Castile and Leon – LT898302 – – P. rudallense Aspergilloides FMR 15843 Dung, Canary Islands – LT898303 – – P. sizovae Citrina FMR 15300 Dung, Castile and Leon – LT898304 – – FMR 15521 Dung, Galicia – LT898305 – – P. synnematicola Robsamsonia FMR 15192T = Dung, Balearic Islands LT898167 LT898172 LT898137 LT898142 CBS 142669 FMR 15210 Dung, Balearic Islands LT898168 LT898173 LT898138 LT898143 FMR 15211 Dung, Balearic Islands LT898169 LT898174 LT898139 LT898144

Table 1. (Continued). GenBank/EMBL accession numbers 1 Collection Genus/Species Section 2 Substrate and Origin numbers ITS tub2 cmdA rpb2 FMR 16481 = Soil, Catalonia LT898170 LT898175 LT898140 LT898145 CBS 143045 FMR 16491 = Dung, Catalonia LT898171 LT898176 LT898141 LT898146

CBS 143046 Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected]

Penicillium sp. Exilicaulis FMR 15841 Dung, Castile and Leon LT898311 – – Pgo. nodositata - FMR 15296 = Dung, Balearic Islands LT899787 LT898312 LT899771 LT899805 CBS 142988 - FMR 16442 Dung, Extremadura LT899788 LT898313 LT899772 LT899806 Pse. cervifimosum - FMR 15299T = Dung, Castile and Leon LT899789 LT898315 – LT899807 CBS 142670 Pse. giganteum - FMR 14718 Soil, unknown LT899790 LT898314 – LT899808 T. angelicus Talaromyces FMR 15489 Dung, Galicia LT899791 LT898316 LT899773 LT899809 FMR 15490 Dung, Galicia LT899792 LT898317 LT899774 LT899810 T. catalonicus Trachyspermi FMR 16441T = Dung, Catalonia LT899793 LT898318 LT899775 LT899811 CBS 143039 T. coprophilus Talaromyces FMR 15199T = Dung, Balearic Islands LT899794 LT898319 LT899776 LT899812 CBS 142756 T. muroii Talaromyces FMR 15496 Dung, Galicia – LT898321 – – T. pseudofuniculosus Talaromyces FMR 15307T = Dung, Andalusia LT899796 LT898323 LT899778 LT899814 CBS 143041 FMR 15035 Dung, Catalonia LT899797 LT898322 LT899779 LT899815 T. ruber Talaromyces FMR 15839 Dung, Castile and Leon – LT898324 – – T. sayulitensis Talaromyces FMR 15842 Dung, Canary Islands – LT898325 – – 1P. = Penicillium; Pgo. = Penicillago; Pse. = Pseudopenicillium; T. = Talaromyces; new taxa proposed in this study are in bold; T = ex-type strain. 2CBS: Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; FMR: Facultat de Medicina i Ciències de la Salut, Reus, Spain. bio.ed.ac.uk/software/figtree/) and edited in Adobe Illustrator RESULTS CS3. The alignments and trees were deposited in TreeBASE (www.treebase.org) under the submission number 21345. During the study, 130 dung samples were processed and 104 isolates of penicillium-like fungi were recovered, including Phenotypic characterisation three from soil (Table 1). Preliminary identification based on morphology and tub2 sequences showed that most of the isolates Phenotypic characterization was carried out using standard belonged to Penicillium (n = 91; 87.5 %) and a few to Talaromyces growth conditions suggested by Visagie et al. (2014b) and (n = 9; 8.65 %), while four of them (3.85 %) could not be assigned Yilmaz et al. (2014). Briefly, isolates were cultured onto MEA to any genus despite exhibiting a penicillium-like morphology. 2 % (Samson et al. 2010), oatmeal agar (OA; Samson et al. To maximize the quality of the analyses, three separate tub2 2010), Czapek yeast autolysate agar (CYA; Pitt 1979), yeast alignments were done for the different genera studied. We also extract sucrose agar (YES; Frisvad 1981), creatine sucrose agar carried out additional analyses of single-gene ITS, cmdA and rpb2 CREA; Frisvad 1981) and dichloran 18 % glycerol agar (DG18; and combined datasets from the diferent genes, corresponding to) Hocking & Pitt 1980), incubated at 25 °C for 7 d in darkness. the genera or sections where putative new species were included Colony growth rates were also measured after 7 d at 30 and 37 (Table 2). The topology of the trees was similar for both methods, °C on MEA, CYA, YES and OA. Colours used for descriptions refer with ML trees used to represent the results. Bootstrap and BI to Kornerup & Wanscher (1978). Microscopic features were posterior probability values are indicated on relevant branches. examined on colonies grown on MEA after 1─2 wk, mounted Final identification of the isolates, resulting from the phylogenetic on slides with Shear’s solution or 60 % lactic acid, and excess and phenotypic characterization, is shown in Table 1. conidia removed with 70 % ethanol. Microscopic features were captured with a Zeiss Axio-Imager M1 light microscope Penicillium phylogeny using Nomarski differential interference contrast and phase- contrast optics (Zeiss, Oberkochen, Germany) with a DeltaPix The phylogenetic tree based on the tub2 locus with the 91 Infinity x digital camera and DeltaPix InSight v. 5.3.11 software. isolates of Penicillium is shown in Fig. 1. The aligned dataset Photoplates were assembled from separate photographs using was 404 bp long, with 257 variable sites and 237 phylogenetic PhotoShop CS3.1. informative. The best substitution model for ML was K2+G, and for BI it was GTR+G+I. In general, the topology of the phylogenetic tree showed well-delimitated sections.

P. crustosum CBS 115503T (AY674353) FMR 15213 FMR 15098 FMR 15197 FMR 15494 FMR 15034 FMR 15036 97/1 FMR 15042 FMR 15043 FMR 15200 FMR 15186

Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. FMR 15037 E-mail: [email protected] 71/1 FMR 15045 FMR 15189 FMR 15194 FMR 15185 P. biforme CBS 29748T (FJ930944) FMR 15313 99/1 --/0.99 FMR 15312 97/1 P. polonicum CBS 22228T (AY674305) Sect. FMR 15099 P. gladioli CBS 33248T (AY674287) Fasciculata 96/1 FMR 15031 FMR 15032 88/1 FMR 15188 95/1 P. roqueforti CBS 221.30T (AF000303) P. psychrosexualis CBS 128137T (HQ442356) Sect. T 92/1 97/1 P. carneum CBS 112297 (AY674386) P. paneum CBS 101032T (AY674387) Roquefortorum 80/0.99 99/1 P. flavigenum CBS 419.89T (AY495993) FMR 15096 P. chrysogenum CBS 306.48T (AY495981) FMR 15100 Sect. 74/-- P. rubens CBS 129667T (JF909949) Chrysogena P. halotolerans CBS 131537T (JX996816) T 91/-- 87/1 P. expansum CBS 32548 (AY674400 ) FMR 15097 Sect. --/1 81/-- FMR 15484 P. marinum CBS 109550T (AY674392) Penicillium 94/0.96 FMR 15192 82/0.99 FMR 15210 70/-- FMR 15211 88/0.97 FMR 16481 FMR 16491 P. glandicola CBS 49875T (AY674415) P. griseofulvum CBS 18527T (AY674432) FMR 15029 FMR 15093 --/1 FMR 15207 FMR 15314 --/1 FMR 15204 FMR 15203 FMR 15030 P. dipodomyicola CBS 17387T (AY674409) P. concentricum CBS 47775T (AY674413) 99/1 FMR 15840 FMR 15195 90/1 P. brevistipitatum AS3.6887T (DQ221695) Subgenus 96/1 FMR 15103 Penicillium P. coprophilum CBS 110760T (AY674421) 99/1 FMR 15187 92/1 P. robsamsonii CBS 140573T (KT698885) P. fimorum CBS 140576T (KT698889) T 84/-- P. compactum CBS 138918 (KM973203) P. coprobium CBS 56190T (AY674425) Sect. 96/1 FMR 15201 Robsamsonia 96/1 FMR 15311 82/0.99 FMR 15041 P. bovifimosum CBS 102825T (KJ834436) Sect. 95/1 P. turbatum CBS 383.48T (KJ834499) Turbata 92/0.96 P magnielliptisporum CBS 138225T (KJ775179) FMR 15044 90/0.99 FMR 15104 P. atramentosum CBS 291.48T (AY674402) 94/-- FMR 15102 98/0.95 FMR 15046 91/-- FMR 15309 FMR 15092 FMR 15107 97/1 FMR 15040 Sect. FMR 15196 Paradoxa 99/1 FMR 15191

Fig. 1. ML tree of Penicillium inferred from tub2 including the sections recovered from dung in this work. Branch lengths are proportional to phylogenetic distance. Some of the larger branches were condensed, with the proportions showed above the parallel diagonal lines. Bootstrap support values/Bayesian posterior probability scores above 69 %/0.94 are indicated on the nodes. Isolates corresponding to new species are shown in bold. Between parentheses, GenBank accession numbers of tub2 sequences. The tree is rooted to Talaromyces flavus CBS 310.38 and Talaromyces duclauxii CBS 322.48. T = ex-type strain.

T 98/0.99 P. kojigenum CBS 345.61 (KJ834463) P. lanosum NRRL 2009T (DQ285627) 81/0.97 P. swiecickii CBS 119391T (KJ834494) T P. jamesonlandense CBS 102888 (DQ309448) Sect. 97/0.98 FMR 15038 P. ribium CBS 127809T (DQ285625) Ramosa 96/1 P. brevicompactum CBS 25729T (AY674437) Sect. 99/1 FMR 15105 T 2X P. kongii AS3.15329 (KC427171) Brevicompacta T 89/1 P. canescens CBS 300.48 (JX140946) FMR 15028 T Editor-in-Chief P. radiatolobatum CBS 340.79 (KP016920) Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] 99/1 FMR 15485 FMR 15305 98/1 FMR 15308 2X P. murcianum CBS 161.81T (KP016924) FMR 15304 Sect. FMR 15845 FMR 15491 Canescentia P. restrictum CBS 367.48T (KJ834486) P. cinereoatrum CBS 222.66T (KJ834442) FMR 15033 T P. philippinense CBS 623.72 (KJ834482) P. restrictum- clade 82/0.97 P. meridianum CBS 314.67T (KJ834472) FMR 15841 P. arabicum CBS 414.69T (KP016750) FMR 15298 99/1 FMR 15095 FMR 15202 FMR 15297 2X P. rubefaciens CBS 145.83T (KJ834487) 96/1 P. corylophilum CBS 312.48T (JX141042) P. momoii CV 1015T (JX141073) 75/-- FMR 15208 Sect. 99/1 P. burgense CBS 325.89T (KJ834437) FMR 15493 Exilicaulis P. cremeogriseum CBS 223.66T (GU981624) 93/0.98 FMR 15488 FMR 15487 93/1 P. brefeldianum CBS 235.81T (GU981623) 2X T 92/1 --/1 P. limosum CBS 339.97 (GU981621) Sect. 81/1 P. brasilianum CBS 253.55T (GU981629) 77/1 FMR 15483 Lanata- 72/0.99 84/1 P. onobense CBS 174.81T (GU981627) 98/1 P. skrjabinii CBS 439.75T (GU981626) Divaricata 4X P. boreae CBS 111717T (JN617715) T 99/1 P. stolkiae CBS 315.67 (JN617717) Sect. 2X P. canariense CBS 111720T (JN617714) 86/-- FMR 15838 Stolkia P. sizovae CBS 413.69T (GU944535) Subgenus 97/1 FMR 15521 Aspergilloides FMR 15300 P. citrinum CBS 139.45T (GU944545) 2X 98/1 FMR 15094 FMR 15646 99/0.99 FMR 15647 Sect. FMR 15520 FMR 15486 Citrina 96/1 P. lilacinoechinulatum CBS 454.93T (KC773790) --/0.97 FMR 15492 76/-- T 77/-- P. adametzii CBS 209.28 (JN625957) Sect. P. brocae CBS 116113T (KC773787) 78/0.98 4X P. sclerotiorum NRRL 2074T (JN626001) Sclerotiora 98/1 P. cvjetkovicii NRR 35841T (KF932931) 4X 96/-- 3X FMR 15310 Sect. P. parvulum NRRL 35504T (EF506218) 5X T Cinnamopurpurea 4X P. cinnamopurpureum CBS 429.65 (EF626948) 5X 99/1 P. roseoviride CBS 267.35T (KM088787) 96/1 FMR 15645 --/1 T 94/0.98 P. aurantioviolaceum CBS 13777 (KM089005) P. fusisporum CBS 137463T (KF769400) P. valentinum CBS 172.81T (KM088788) P. glabrum CBS 125543T (GU981619) FMR 15184 4X 99/1 77/-- FMR 15209 FMR 15190 P. frequentans CBS 105.11T (GKM088762) 95/1 FMR 15212 FMR 15193 Sect. P. rudallense CBS 138162T (KM088741) 99/1 FMR 15843 Aspergilloides T. flavus CBS 310.38T (JX494302) T 2X T. duclauxii CBS 322.48 (JX091384) 0.02 Fig. 1. (Continued).

The analysis distributed the Penicillium isolates in at least 38 species, belonging to 17 sections. The two major clades coincided with the two subgenera currently accepted – – – – 666

810 Penicillium i.e. Aspergilloides 956 463 375 187 223 401 124 104 440 in , and 1797 TrN+I+G TrN+I+G HKY+I+G Penicillium (Houbraken & Samson 2011). Miscellaneous Aspergillaceae Subgenus Penicillium (84 % bs/-- pp) is

represented by the sections that according Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] to the numbers of isolates obtained are: Fasciculata and Robsamsonia with 18 isolates (19.78 %) each, followed by Paradoxa (n = 10; 11 %), Canescentia (n = – – – – 7; 7.69 %), Roquefortorum (n = 3; 3.29 %), 86 176 366 247 524 482 104 158 398 119 474 1354 Chrysogena (n = 2; 2.19 %), Penicillium (n TIM2+I+G TPM1+I+G TPM3uf+G

Trachyspermi = 2; 2.19 %), and Brevicompacta, Ramosa, and Turbata each with one isolate (1.09 %). Subgenus Aspergilloides (72 % bs/0.99 pp) Talaromyces is represented by the sectionsExilicaulis (n – – – – 45 72 205 395 250 503 475 140 178 370 360 = 8; 8.79 %), Aspergilloides (n = 7; 7.69 %), 1205 TrN+I+G HKY+I+G HKY+I+G Citrina (n = 7; 7.69 %), Lanata-Divaricata Talaromyces (n = 3; 3.29 %), and Sclerotiora and Stolkia each with one isolate (1.09 %). Isolates of section Fasciculata (--% bs/0.99 pp) were identified as P. crustosum (n = 15), P. biforme (n = 2), and 8 50 55 29 68 13 24 105 915 248 106 501 409 506 P. polonicum (n = 1), the former being the 2331 TIM1ef TPM1+G TIM3ef+I TPM2uf+I most prevalent species (14.42 %) found in

Roquefortorum the study. In fact, P. crustosum is a relatively common species commonly isolated from nuts, meat, cheese, feeds, vegetables, and pomaceous and stone fruits (Sonjak et al. 96 67 18 303 804 574 164 243 486 127 122 352 207 428 2068 GTR+I GTR+I K80+G GTR+G 2005).

Robsamsonia In section Roquefortorum (92 % bs/1

, and related taxa with taxonomic novelties proposed in the study. in the proposed novelties with taxonomic taxa , and related Talaromyces pp) three isolates (FMR 15031, FMR 15032, FMR 15188) were clustered in and the same terminal clade. This is a small – – – – 92 64 16 47 section of closely related species (i.e. P. 172 382 223 544 112 387 485 Penicillium 1416 GTR+I TIM2+G Ramosa carneum, P. paneum, P. psychrosexualis TIM2ef+G Penicillium and P. roqueforti) commonly associated with the cheese industry (Houbraken et al. 2010, 2016). Our isolates were related to P. roqueforti, but formed an independent and distant branch that

Paradoxa could represent an undescribed species. 88 18 29 915 365 239 614 195 159 492 100 151 412 504 2323

K80+G In order to evaluate possible intra- and TIM2+G TrN+I+G TIM2ef+G inter-specific variability within the species and confirm the phylogenetic position of and Turbata our putative new species, we performed an additional analysis with tub2 gene (see Fig. S1 in supplemental material) with more sequences of the species available in GenBank. This demonstrated that P. Pi Pi Pi Pi Pi Pvar Pvar Pvar Pvar Pvar roqueforti is divided in two clades with Model* Model* Model* Model* Length (bp) Length Length (bp) Length Length (bp) Length Length (bp) Length Length (bp) Length an intra-specific variability of around 0.08 %, with our isolates forming a separate branch from the P. roqueforti complex. The concatenated analysis using ITS, tub2, cmdA and rpb2 (Fig. 2) supported

Overview and details used for phylogenetic analyses in sections of analyses phylogenetic used for and details Overview the novelty of these three isolates, ITS rpb2 tub2 cmdA dataset dataset dataset dataset dataset being therefore proposed as the new

Concatenated species P. mediterraneum. Although, P. Pvar = variable sites; Pi = phylogenetic informative sites; * = substitution model for Bayesian inference Bayesian for model * = substitution sites; informative Pi = phylogenetic sites; = variable Pvar Table 2. Table mediterraneum and P. roqueforti have

P. mediterraneum FMR 15188T sp. nov. 100/1 P. mediterraneum FMR 15032 99/1 P. mediterraneum FMR 15031

100/1 P. roqueforti CBS 221.30T P. carneum CBS 112297T 100/1 --/1 T P. psychrosexualis CBS 128137 Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected]

P. paneum CBS 101032T P. samsonianum AS 3.15403T P. osmophilum CBS 462.72T

0.01 Fig. 2. ML tree of Penicillium section Roquefortorum inferred from the combined ITS, tub2, cmdA, and rpb2 loci. Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores above 70 %/0.95 are indicated on the nodes. The tree is rooted to P. samsonianum AS 3.15403 and P. osmophilum CBS 462.72. The name in red is the new species described in this study. T = ex-type strain. identical ITS barcodes, both species have uniquetub2 , cmdA and the concatenate phylogeny (Fig. 3) including the currently rpb2 sequences. accepted species in the section, showed that the five isolates In section Chrysogena (74 % bs/-- pp), which includes well- grouped together in a supported and undescribed lineage known species usually isolated from soil and also from indoor closely related to the ex-type strain of P. glandicola. An environments (Houbraken et al. 2012), two dung isolates were additional tub2 analysis (see Fig. S2 in supplemental material) identified as P. chrysogenum and P. flavigenum, respectively. with more sequences of P. glandicola showed that our isolates Whereas the two isolates of the sectionPenicillium (--% bs/1 pp), and P. glandicola were 97.4 % similar. The genetic differences a group that currently mainly includes plant pathogenic penicillia and morphological peculiarities observed in this group of dung (Houbraken et al. 2016), were both identified as P. expansum. isolates justify the description of a new species, namely P. Isolates of section Robsamsonia (88 % bs/0.97 pp) were synnematicola. identified as P. brevistipitatum (n = 1), P. concentricum (n = 2), The only isolate (FMR 15041) of section Turbata (95 % bs/1 P. coprobium (n = 2), P. coprophilum (n = 1), and P. griseofulvum pp) did not fit with any species of the section, although it was (n = 7); however, five isolates (FMR 15192, FMR 15210, FMR closely related to P. bovifimosum. In Paradoxa (94 % bs/-- pp) 10 15211, FMR 16481 and FMR 16491) could not be assigned to isolates were included; four were identified asP. atramentosum, any known species. The preliminary tub2 analysis, but also one as P. magnielliptisporum, and the rest (FMR 15040, FMR

P. synnematicola FMR 15192T sp. nov. 85/0.98 P. synnematicola FMR 15210

100/1 P. synnematicola FMR 15211 P. synnematicola FMR 16481 99/1 P. synnematicola FMR 16491 77/0.96 P. glandicola CBS 498.75T P. vulpinum CBS 126.23T T 100/1 P. brevistipitatum AS 3.6887 P. coprophilum CBS 110760T T 100/1 P. compactum AS3.15411 94/1 P. coprobium CBS 561.90T --/1 71/-- P. concentricum CBS 477.75T 79/-- P. fimorum CBS 140575T 100/1 P. robsamsonii CBS 140573T P. dipodomyicola CBS 173.87T 97/0.99 P. griseofulvum CBS 185.27T P. brevicompactum CBS 257.29T

0.02

Fig. 3. ML tree of Penicillium section Robsamsonia inferred from the combined ITS, tub2, cmdA, and rpb2 loci. Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores above 70 %/0.95 are indicated on the nodes. The tree is rooted to P. brevicompactum CBS 257.29. The name in red is the new species described in this study. T = ex-type strain.

T 99/1 P.ibericum FMR 15040 sp. nov. 92/1 P.ibericum FMR 15107

P. atramentosum CBS 291.48T

P. fimosum FMR 15104T sp. nov.

P. balearicum FMR 15191T sp. nov. 77/0.96

Editor-in-Chief 100/1 Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] P. balearicum FMR 15196

100/1 P. magnielliptisporum CBS 138225T

--/1 P. mexicanum CBS 138227T

P. paradoxum CBS 527.65T

100/1 P. crystallinum CBS 479.65T

79/1 P. malodoratum CBS 490.65T

P. madriti CBS 347.61T

T 100/1 P. turbatum CBS 383.48

99/1 P. caprifimosum FMR 15041T sp. nov. 0.02 98/1 P. bovifimosum CBS 102825T

Fig. 4. ML tree of Penicillium sections Paradoxa and Turbata inferred from the combined ITS, tub2 and cmdA loci. Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores above 70 %/0.95 are indicated on the nodes. The names in red are the new species described in this study. T = ex-type strain

15104, FMR 15107, FMR 15191 and FMR 15196) were allocated swiecickii. The similarity among our isolate and its phylogenetic in three single branches which represent three putative new sisters was 98.3 %, proving that our isolate is an undescribed species for the genus. The concatenated phylogeny of sections species, which is proposed below as P. beceitense. Paradoxa and Turbata with the six unidentified isolates is shown The only isolate of sectionBrevicompacta (99 % bs/1 pp) was in Fig. 4. In the former section, the isolates FMR 15107 and identified asP. brevicompactum, a species commonly inhabiting FMR 15040 formed a terminal clade separated from other one in soil and decaying vegetation, but also described from food, formed by FMR 15191 and 15196. The lineages of these two cereals, textiles, clinical specimens and faeces of snake (Pitt clades were in turn distant from the two terminal branches 1979, Guevara-Suarez et al. 2016). The seven isolates classified where FMR 15104 and the ex-type strain of P. atramentosum in section Canescentia (98 % bs/1 pp) were identified as P. were located, respectively. Genetic differences of the three canescens (n = 1), P. murcianum (n = 3) and P. radiatolobatum undescribed lineages with respect to P. atramentosum (97.07 % (n = 3). Despite tub2 being unable to discriminate between P. and 98.23 % similar with tub2 and concatenate dataset for FMR radiatolobatum and P. murcianum, these two species can be 15107 and FMR 15040, respectively; 96.2 % and 96.2 % withtub2 recognized with the cmdA and rpb2 barcodes. and concatenate dataset for FMR 15191 and FMR 15196; 94.68 Section Exilicaulis (99 % bs/1 pp) included eight of our % and 95 % with tub2 and concatenate dataset for FMR 15104) isolates. It is noteworthy that the ML general tree based justifies them as distinct taxa. The three species are proposed only on tub2 presented some doubtful results in the species below as P. ibericum, P. balearicum and P. fimosum.An additional identification of some isolates, especially those belonging to the phylogenetic analysis with tub2 including more sequences of P. restrictum-clade. However, when we carried out a restricted the most closely related species confirms our proposal (see Fig. alignment with only members of the section and reconstructing S3 in supplemental material). In sectionTurbata, P. bovifimosum the ML tree, most of them were satisfactory identified (see Fig. and FMR 15041 were located in the same clade, but the latter S4 in supplemental material). These were P. arabicum (n = 2), formed a long terminal branch that proved to be a distinct P. burgense (n = 1), P. cinereoatrum (n = 1), P. momoii (n = 1), species, described below as P. caprifimosum. and P. rubefaciens (n = 2). The only isolate that could not be Section Ramosa (97 % bs/0.98 pp) only included the identified was FMR 15841, precisely located in theP. restrictum- unidentified isolate FMR 15038. Currently, the section clade. The most recent review of the section by Visagie et al. comprises 13 species (Visagie et al. 2014a, 2016a, Rong et al. (2016b) indicates that such a clade needs further revision due 2016), although two of them, P. lanosum and P. kojigenum, to the high variability between strains currently belonging to the were considered conspecific by Samson & Pitt (2000). The clade. Thus, the identification of FMR 15841 remains uncertain concatenate analysis performed with ITS, tub2, and cmdA (Fig. until its taxonomy is resolved. 5), rpb2 was not included due to the lack of sequences for all Section Lanata-Divaricata (92 % bs/1 pp) included three species in the section, showed that FMR 15038 was placed in an dung isolates. One of them was identified as P. brasilianum, a independent branch between two clades, one with P. kojigenum widespread species commonly found in soil (Pitt 1979) and and P. lanosum, and the other with P. jamesonlandense and P. recently also reported from human clinical specimens (Guevara-

T 100/1 P. kojigenum CBS 345.61 76/0.98 P. lanosum CBS 106.11T

81/1 P. beceitense FMR 15038T sp. nov. P. jamesonlandense CBS 102888T 96/1 P.swiecickii CBS 119391T

89/1 P. ribium CBS 127809T

Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] P. scabrosum CBS 683.89T --/1 P. simile CBS 129191T 94/1 77/-- P. raistrickii CBS 261.33T P. sajarovii CBS 277.83T

T --/1 P. soppii CBS 226.28 99/1 P. chroogomphum CBS 136204T 96/0.99 P. lenticrescens CBS 138215T P. virgatum CBS 114838T P. brevicompactum CBS 257.29

0.02

Fig. 5. ML tree of Penicillium section Ramosa inferred from the combined ITS, tub2 and cmdA loci. Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores above 70 %/0.95 are indicated on the nodes. The tree is rooted to P. brevicompactum CBS 257.29. The name in red is the new species described in this study. T = ex-type strain.

Suarez et al. 2016). The other two isolates were identified as generally the most common species in the section, being isolated P. cremeogriseum, a species previously found in forest soil in from a wide range of substrates, including soil, food, bark, and Ukraine (Houbraken & Samson 2011, Visagie et al. 2016a). To indoor environments (Houbraken et al. 2014b). our knowledge, this is the first report of P. brasilianum and P. cremeogriseum associated with herbivore dung. Talaromyces phylogeny The only species identified in our study belonging to section Stolkia (99 % bs/1 pp) was P. canariense. This species was The phylogenetic tree based on the tub2 locus (Fig. 6) shows described from soil in Canary Islands (Peterson & Sigler 2002), the relationships of the nine Talaromyces isolates included in the same geographical origin as our isolate. This is the second the study. The aligned dataset was 388 bp long, from which 217 isolate of the species found so far. were variable sites and 170 phylogenetic informative. The ML Isolates of section Citrina (98 % bs/1 pp) were identified as substitution model was K2+G+I, while for BI it was HKY+G+I. Two P. citrinum (n = 5) and P. sizovae (n = 2). Although we have not main clades were formed, representing sections Talaromyces found previous records of these species from dung, they have (98 % bs/1 pp) and Trachyspermi (90 % bs/1 pp). Only three a worldwide distribution, being isolated from soil, foodstuff, sect. Talaromyces species were identified, namely T. muroii and many other types of substrates (Houbraken et al. 2011). (FMR 15496), T. ruber (FMR 15839) and T. sayulitensis (FMR This section comprises nearly 40 species, but only those 15842); the rest (FMR 15489, FMR15490, FMR 15199, FMR identified here were included in the analysis to simplify thetub2 15035 and FMR 15307) could not be identified with this single phylogenetic tree (Fig. 1). marker, just like the only isolate (FMR 16441) included in section In sectionsSclerotiora (77 % bs/-- pp) and Cinnamopurpurea Trachyspermi. (96 % bs/-- pp) P. lilacinoechinulatum (n = 1) and P. cvjetkovicii To resolve the taxonomy of these unidentified isolates, we (n = 1) were respectively identified. Although the former species calculated a phylogeny for each section using sequences of the was previously considered synonym of P. bilaiae, they are ITS, tub2 and cmdA genes from the accepted species in both currently recognised as distinct penicillia (Visagie et al. 2013). sections, except for T. amyrossmaniae in section Trachyspermi. P. lilacinoechinulatum is an uncommon species which type was This is a recently described species that morphologically differs collected from Japanese soil (Abe 1956). Penicillium cvjetkovicii from our isolates and species of Trachyspermi by producing is a recently described from indoor air samples in the USA short determinate synnemata on both the natural substrate and (Peterson et al. 2015). in vitro (Rajeshkumar et al. 2019). Although rpb2 was sequenced Aspergilloides (99 % bs/1 pp) was the last section in for all our isolates (Table 1), this marker was not incorporated Penicillium that included dung isolates. The species identified in the concatenated analysis due to the lack of sequences for were P. glabrum (n = 3), P. frequentans (n = 2), P. roseoviride all ex-type strains. The combined phylogeny of the members of (n = 1), and P. rudallense (n = 1). Although the former two are sectionTalaromyces is shown in Fig. 7. This analysis showed that currently accepted as distinct species, they were considered the unidentified isolates FMR 15489 and FMR 15490 exhibited synonyms for a long time (Houbraken et al. 2014b). Penicillium both a similarity of 99.42 % with respect to the ex-type strain frequentans together with P. spinulosum and P. glabrum are of T. angelicus, a monotypic species described by Sang et al.

T. siamensis CBS 475.88T (JX091379) T. cnidii KACC 46617T (KF183641) T. flavovirens CBS 102801T (JX091376) T. xishaensis HMAS 248732T (KU644581) T. aculeatus CBS 289.48T (KF741929) T. apiculatus CBS 312.59T (KF741916) T. beijingensis CBS140617T (KU866837) T. dimorphus NN072337T (KY007111) T. fuscoviridis CBS 193.69T (KU866843) T. angelicus KACC 46611T (KF183640) 92/-- Editor-in-Chief FMR 15489 Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. 95/0.97 E-mail: [email protected] FMR 15490 T. veerkampii CBS 500.78T (KF741918) FMR 15199 T. pinophilus CBS 631.66T (JX091381) T. liani CBS 225.66T (JX091380) 86/-- T. mae NN071328T (KY007106) T. lentulus NN071323T (KY007104) 87/1 T. adpressus CBS 140620T (KU866844) 93/0.97 T. sayulitensis CBS 138204T (KJ775206) FMR 15842 T. purgamentorum CBS 113145T (KX011487) T. cucurbitiradicus ACCC 39155T (KY053228) 95/1 T. funiculosus CBS 272.86T (JX091383) FMR 15035 FMR 15307 T. macrosporus CBS 317.63T (JX091382) T. rapidus CBS 142382T (LT559087) T. flavus CBS 310.38T (JX494302) 90/1 T. marneffei CBS 388.87T (JX091389) T. calidicanius CBS 112002T (HQ156944) 93/1 T. duclauxii CBS 322.48T (JX091384) T. viridis CBS 114.72T (JX494310) T. euchlorocarpius DTO176I3T (KJ865733) 86/-- T. primulinus CBS 321.48T (JX494305) T. kabodanensis CBS 139564T (KP851986) T. viridulus CBS 252.87T (JX091385) T. oumae-annae CBS 138208T (KJ775213) --/0.98 T. australis CBS 137102T (KF741922) T. stellenboschiensis CBS 135665T (JX091605) 87/1 T. verruculosus CBS 388.48T (KF741928) 99/-- T. muroii CBS 756.96T (KJ865727) FMR 15496 T. galapagensis CBS 751.74T (JX091388) T. indigoticus CBS 100534T (JX494308) 98/1 T. rubicundus CBS 342.59T (JX494309) T. neofusisporus CBS 139516T (KP765381) 80/0.99 T. mycothecae URM 7622T (LT855561) T. amazonensis CBS 140373T (KX011490) T --/1 --/1 T. stollii CBS 408.93 (JX315633) T. amestolkiae CBS 132696T (JX315623) 97/-- T. ruber CBS 132704T (JX315629) 98/1 FMR 15839 T. derxii CBS 412.89T (JX494306) T. fusiformis CBS 140637T (KU866843) 70/1 T 98/1 T. alveolaris CBS 142379 ( LT559086) T. aurantiacus CBS 314.59T (KF741917) T. intermedius CBS 152.65T (JX091387) 76/1 T. panamensis CBS 128.89T (HQ156948) T. francoae CBS 113134T (KX011489) 70/-- T. thailandensis CBS 133147T (JX494294) 97/-- T. qii CBS 139515T (KP765380) 73/1 T. mangshanicus HMAS 248733T (KX447530) 89/1 T. kendrickii CBS 136666T (KF741921 ) Sect. 82/0.97 T. purpureogenus CBS 286.36T (JX315639) T Talaromyces 98/1 T. stipitatus CBS 375.48 (KM111288) 88/0.98 T. minesotensis CBS 142381T (LT559083) 75/1 T. minioluteus CBS 642.68T (KF114799) T. assiutensis CBS 147.78T (KJ865720) T. atroroseus CBS 133442T (KF114789) T. diversus CBS 320.48T (KJ865723) T. brasiliensis CBS142493T (LT855560) --/0.98 FMR 16441 90/1 T. heiheensis HMAS 248789T (KX447525) --/0.99 T. albobiverticillius CBS 140498 (ex-type of T. rubrifaciens) (KR855648) T. albobiverticillius CBS 133440T (KF114778) 86/-- T. erythromellis CBS 644.80T (HQ156945) T. solicola CBS 133445T (GU385731) Sect. T. austrocalifornicus CBS 644.95T (KJ865732) T Trachyspermi 100/1 T. convolutus CBS 100537 (KF114773) T. dendriticus CBS 660.80T (JX091391) T. purpureus CBS 475.71T (GU385739) 0.02

Fig. 6. ML tree of Talaromyces inferred from tub2 including the sections recovered from dung in this work (Talaromyces and Trachyspermi). Branch lengths are proportional to phylogenetic distance. Some of the larger branches were condensed, with the proportions showed above the parallel diagonal lines. Bootstrap support values/Bayesian posterior probability scores above 69 %/0.95 are indicated on the nodes. Isolates corresponding to new species are shown in bold. Between parentheses, GenBank accession numbers of tub2 sequences. The tree is rooted to T. dendriticusCBS 660.80 and T. purpureus CBS 475.71 (Section Purpurei). T = ex-type strain.

99/1 T. apiculatus CBS 312.59T T. aculeatus CBS 289.48T T. fuscoviridis CBS 193.69T T. angelicus KACC 46611T 100/1 82/1 T. angelicus FMR 15489 99/1 T. angelicus FMR 15490 T. liani CBS 225.66T T. sayulitensis CBS 138204T T. beijingensis CBS140617T

T T. dimorphus NN072337 Editor-in-Chief 100/1 Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] T. lentulus NN071323T T. coprophilus FMR 15199T sp. nov. T. flavovirens CBS 102801T T. xishaensis HMAS 248732T T 85/1 T. cnidii KACC 46617 99/1 T. siamensis CBS 475.88T T. galapagensis CBS 751.74T T. veerkampii CBS 500.78T T 100/1 T. pinophilus CBS 631.66 T. adpressus CBS 140620T 76/-- T. mae NN071328T 100/1 T. pseudofuniculosus FMR 15307T sp. nov. T. pseudofuniculosus FMR 15035T 100/1 T. cucurbitiradicus ACCC 39155T T. funiculosus CBS 272.86T T. macrosporus CBS 317.63T T. rapidus CBS 142382T 94/0.99 100/1 T. indigoticus CBS 100534T T. rubicundus CBS 342.59T T. muroii CBS 756.96T T. mycothecae URM 7622T T. neofusisporus CBS 139516T T. amazonensis CBS 140373T T 73/1 T. stollii CBS 408.93 T 92/1 T. amestolkiae CBS 132696 100/1 T. ruber CBS 132704T T. intermedius CBS 152.65T T. viridis CBS 114.72T 99/1 T. calidicanius CBS 112002T 99/1 T. duclauxii CBS 322.48T T. marneffei CBS 388.87T T. flavus CBS 310.38T T. euchlorocarpius DTO176I3T T. kabodanensis CBS 139564T 76/-- 76/-- T. primulinus CBS 321.48T T. purgamentorum CBS 113145T T. oumae-annae CBS 138208T --/0.97 T. viridulus CBS 252.87T T. australis CBS 137102T T 99/1 T. stellenboschiensis CBS 135665 99/1 T. verruculosus CBS 388.48T 89/0.96 T. panamensis CBS 128.89T 100/1 T. qii CBS 139515T T. thailandensis CBS 133147T 100/1 T. francoae CBS 113134T 74/0.99 80/-- T. kendrickii CBS 136666T 100/1 97/1 T. mangshanicus HMAS 248733T T 92/1 T. derxii CBS 412.89 T 100/1 T. fusiformis CBS 140637 T. alveolaris CBS 142379T 86/-- T. aurantiacus CBS 314.59T 89/1 T. purpureogenus CBS 286.36T T. stipitatus CBS 375.48T T. dendriticus CBS 660.80T T. purpureus CBS 475.71T

0.02

Fig. 7. ML tree of Talaromyces sectionTalaromyces inferred from the combined ITS, tub2 and cmdA loci. Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores above 70 %/0.95 are indicated on the nodes. The tree is rooted to T. dendriticus CBS 660.80 and T. purpureus CBS 475.71 (Section Purpurei). The names in red are the new species described in this study. T = ex-type strain.

(2013). Although most of their morphological features matched penicillia currently excluded from the genus Penicillium (i.e. P. with those of the protologue of T. angelicus, we observed some giganteum, P. megasporum and P. nodositatum), but belonging phenotypic variation with respect to the description of the to the family Aspergillaceae (Fig. 9). Previous molecular data on species by Yilmaz et al. (2014); i.e, production of diffusible red these fungi suggested that they could represent distinct genera pigment in one of our isolates (FMR 15490), the colonies of our in the family (Peterson et al. 2010, Visagie et al. 2013). The isolates were deep turquoise and pastel red rather than yellow aligned dataset of our analysis was 393 bp long, from which 242 to white on MEA, they grew faster at 37 °C (on CYA 34–37 mm were variable sites and 216 phylogenetic informative. The best

diam in 7 d vs 25–27 mm in Yilmaz et al. 2014), and exhibited substitution model for ML was K2 +G+I and for BI it was GTR Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] stipes up to 100 μm long (up to 120 μm in Yilmazet al. 2014). On +G+I. While FMR 15296 and FMR 16442 were located in a well- the other hand, while FMR 15199 was located in an independent supported clade along with the ex-type strain of P. nodositatum branch clearly separated from the other species of the section; (CBS 333.90), the other two (FMR 14718 and FMR 15299) were FMR 15035 and FMR 15307 clustered together and were closely in a fully supported distant clade together with the ex-type related to T. curcubitiradicus and T. funiculosus, but with enough strains of P. giganteum (NRRL 3553) and P. megasporum (NRRL genetic difference to be considered a distinct species (see also 2232). The phylogeny inferred with sequences of the ITS, tub2 Fig. S5 in supplemental material). These genetic differences and rpb2 genes (Fig. 10), including the isolates under study and the particular phenotypic features observed in the three and members of different genera of Aspergillaceae, confirms FMR isolates allow us to propose the following new species, T. the proposal of two novel genera, which are described below coprophilus (FMR 15199) and T. pseudofuniculosus (FMR 15035 as Penicillago (Pgo.), typified by Penicillium nodositatum, and FMR 15307). Both can be easily identified with the tub2 and Pseudopenicillium (Pse.) with Penicillium giganteum and marker. Penicillium megasporum, this latter species here selected as The combined phylogenetic analysis of the section the type. The former genus is phylogenetically more related to Trachyspermi (Fig. 8) showed that the isolate FMR 16441 Penicillium, whereas the latter to the genus Hamigera. was located in a single branch within the same clade as T. The analysis of ITS and tub2 showed that the ex-type strain albobiverticillius, T. erythromellis, T. heiheensis, and T. solicola of P. nodositatum (CBS 333.90) and the isolates FMR 15296 and (88 % bs/0.99 pp). Considering the phylogenetic position of our FMR 16442 have practically identical sequences. However, CBS isolate and the morphological differences observed, the new 333.90 was not included in the concatenated phylogeny with the species T. catalonicus is proposed. three markers (Fig. 10) since its rpb2 sequence was not available for comparison. Despite this, we observed some cultural Phylogeny of miscellaneous Aspergillaceae difference between our isolates respect to the protologue of P. nodositatum (Valla et al. 1989), microscopically they fit with the According to the tub2 phylogeny, isolates FMR 15296, FMR features of the species. Differences include better growth for 16442, FMR 14718 and FMR 15299 were related to some FMR 15296 and FMR 16442 on CYA and MEA at 25 °C (40–44 mm

T 100/1 T. assiutensis CBS 147.78 99/1 T. trachyspermus CBS 373.48T 100/1 T. systylus BAFCcult 3419T T. ucrainicus CBS 162.67T

T 99/1 T. atroroseus CBS 133442 T. udagawae CBS 579.72T 100/1 97/0.95 T. minioluteus CBS 642.68T 94/0.98 T. minesotensis CBS 142381T T. diversus CBS 320.48T T. austrocalifornicus CBS 644.95T T. solicola CBS 133445T T. catalonicus FMR 16441T sp. nov. 88/0.99 --/1 T 89/1 T. albobiverticillius CBS 133440 T. erythromellis CBS 644.80T T. heiheensis HMAS 248789T T. aerius CBS 140611T T. purpureogenus CBS 286.36T

0.05 Fig. 8. ML tree of selected of Talaromyces sectionTrachyspermi inferred from the combined ITS, tub2 and cmdA loci. Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores above 70 %/0.94 are indicated on the nodes. The tree is rooted to T. purpureogenus CBS 286.36 (Section Talaromyces). The name in red is the new species described in this study. T = ex-type strain.

As 80/0.98 Aspergillus 99/1 Sub. Penicillium pe 98/1 Penicillium 99/1 Sub. Aspergilloides Penicillium nodositatum CBS 333.90T (KC790399) r 100/1 FMR 15296 gilla 100/1 FMR 16442 Aspergillago clavatoflavus NRRL 5113T (EF669686)

Editor-in-Chief T Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] Penicilliopsis clavariiformis NRRL 2482 (EF669687) Penicilliopsis zonata NRRL 5079 (EF669679) 98/1 Penicillium giganteum NRRL 3553T (GU092779) c 97/1 100/1 FMR 14718 eae Penicillium megasporum NRRL 2232T (GU092757) FMR15299 100/1 Hamigera T 100/1 Talaromyces flavus CBS 310.38 (JX494302) Talaromyces purpureogenus CBS 286.36T (JX315639) 0.02

Fig. 9. ML tree of selected members of Aspergillaceae family inferred from tub2. Branch lengths are proportional to phylogenetic distance. Some clades were collapsed to facilitate layout and illustrate the phylogenetic relationships between the shown genera. Bootstrap support values/Bayesian posterior probability scores above 70 %/0.95 are indicated on the nodes. Isolates corresponding to new or interesting taxa are shown in bold. Between parentheses, GenBank accession numbers of tub2 sequences. The tree is rooted to T. flavusCBS 310.38 and T. purpureogenus CBS 286.36. T = ex-type strain.

T 100/1 A. glaucus NRRL 116 100/1 A. montevidensis NRRL 108T Aspergillus A.restrictus NRRL 154T T 100/1 P. canescens NRRL 910 99/1 P. chrysogenum NRRL 807T Penicillium P. citrinum NRRL 1841T 79/0.98 Pgo. nodositata FMR 15296 Penicillago gen. nov. & comb. nov. 100/1 Pgo. nodositata FMR 16442 100/1 Ago. clavatoflavus NRRL 5113T Aspergillago Pen. clavariiformis NRRL 2482T Penicilliopsis Pen. zonata NRRL 5079T T 100/1 Pse. giganteum NRRL 3553 comb. nov. Pse. giganteum FMR 14718 97/1 Pseudopenicillium gen. nov. 100/1 Pse. megasporum NRRL 2232T comb. nov. Pse. cervifimosum FMR15299T sp. nov. T 99/0.99 H. paravellanea NRRL 35720 99/0.99 H. avellanea NRRL 1938T 98/1 H. inflata NRRL 58014T 100/1 H. insecticola NRRL 35386T Hamigera 100/1 H. pallida NRRL 35718T T 97/1 H. terricola NRRL 29055 H. fusca NRRL 35601T Talaromyces flavus CBS 310.38T Talaromyces purpureogenus CBS 286.36T 0.02

Fig. 10. ML tree of members of Aspergillaceae family inferred from the combined ITS, tub2, and rpb2 loci. Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores above 70 %/0.95 are indicated on the nodes. The tree is rooted to Talaromyces flavus CBS 310.38 and Talaromyces purpureogenus CBS 286.36. The names in red are the new taxa proposed in this study. T = ex-type strain.

and 34–36 mm diam in 7 d, respectively, vs 13–19 mm and 16– smaller conidia (up to 6 μm) than those of the other two species 30 mm in CBS 333.90) and showed the ability to grow at 37 °C. in the genus (conidia up to 12 μm in Pse. giganteum, and up Considering the phylogenetic position of our isolates and their to 10 μm in Pse. megasporum), and the genetic differences morphological similarity with P. nodositatum, they were the obtained (98 % similar to Pse. giganteum, and 98.16 % similar base to propose the new combination Pgo. nodositata. Other to Pse. megasporum) allow us to propose the new species Pse. fungus excluded from Penicillium s. str. and closely related to cervifimosum. Pgo. nodositata was P. kabunicum (Visagie et al. 2013). Since only

the ITS barcode was available for the ex-type strain of the latter Taxonomy Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] species (CBS 575.90) in the GenBank database (MH862240), we constructed an ITS phylogenetic tree with different genera in Penicillium balearicum Guevara-Suarez, Cano & Gené, sp. nov. Aspergillaceae showing that P. kabunicum clustered with Pgo. MycoBank MB822061. Fig. 11. nodositata (see Fig. S6 in supplemental material). The isolate FMR 14718 matched morphologically and Etymology: Name refers to the geographical area (the Balearic molecularly with P. giganteum, whereas FMR 15299 was Islands) where the species was found. placed in a new lineage into the Pseudopenicillium clade (Figs 9, 10). The morphological differences observed, such as having In: Section Paradoxa.

Fig. 11. Morphological characters of Penicillium balearicum (FMR 15191T). A. Colonies from left to right (top row) CYA, MEA, DG18 and OA; (bottom row) CYA reverse, MEA reverse, YES, and CREA. B–E. Conidiophores. F. Conidia. Scale bars = 10 μm.

Typus: Spain, Balearic Islands, Mallorca, Pollença, from Typus: Spain, Aragon, Beceite, from herbivore dung, Feb. 2016, herbivore dung, Feb. 2016, J. Guarro, M. Guevara-Suarez & J.P.Z. J. Guarro, M. Guevara-Suarez, E. Rosas (holotype CBS H-23183; Siqueira (holotype CBS H-23215; culture ex-type FMR 15191 = cultures ex-type FMR 15038 = CBS 142989; ITS barcode CBS 143044; ITS barcode LT899762, alternative markers: tub2 LT899780, alternative markers:tub2 LT898229, cmdA LT899764, LT898227, cmdA LT899758, rpb2 LT899760). rpb2 LT899798).

Additional material examined: Spain, Balearic Islands, Mallorca, Colony diameter in 7 d (mm): On CYA: 25 °C 25–27, 30 °C no Pollença, from herbivore dung, Feb. 2016, J. Guarro, M. Guevara-Suarez growth, 37 °C no growth; on MEA: 25 °C 23–25; on YES: 25 °C

Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] & J.P.Z. Siqueira (FMR 15196). 26–28; on OA: 25 °C 26–30; on DG18: 25 °C 19–20, on CREA: 25 °C 3–5. Colony diameter in 7 d (mm): On CYA: 25 °C 25–36, 30 °C 5–6, 37 °C no growth; on MEA: 25 °C 14–17, 30 °C 4–6; on YES: 25 °C Colony characters at 25 °C in 7 d: On CYA, colonies sunken at 36–45, 30 °C 4–6; on OA: 25 °C 26–30, 30 °C 2–6; on DG18: 25 °C the centre, slightly radially sulcate, velvety, mycelium white, 14–27, on CREA: 25 °C 14–20. sporulation dense, with conidial masses dull green (25E4), margin entire; reverse greyish green (28C5); exudate and soluble Colony characters at 25 °C in 7 d: On CYA, colonies velvety, flat, pigment absent. On MEA, colonies flat, velvety, mycelium white, mycelium white, sporulation dense, conidial masses dark dull sporulation dense, with conidial masses conidial masses dull green (28E3), margin dentate; reverse pale orange (5A3); exudate green (25E4), margin entire; reverse orange (5A6) to greenish present, with pale droplets; soluble pigment absent. On MEA, grey (30C2); exudate and soluble pigment absent. On DG18, colonies velvety, elevated, sporulation dense, conidial masses colonies flat, velvety, mycelium white, sporulation dense, dark dull green (28E3), margin lobate; reverse light orange (5A4); conidial masses dull green (26E4), margin entire; reverse exudate and soluble pigment absent. On DG18, colonies flat and greyish green (30B5). On YES, colonies radially sulcate, velvety, velvety, mycelium white, sporulation dense, conidial masses dull mycelium white, sporulation dense, with conidial masses dark green (27D4); reverse greenish white (27B2). On YES, colonies green (27F14), margin lobate; reverse greyish yellow (4B5); cerebriform, raised at the centre, radially sulcate, mycelium white, exudate and soluble pigment absent. On OA, colonies flat, sporulation dense at the centre, with conidial masses greenish white velvety, sporulation dense, conidial masses dark dull green (27C2), margin lobate; reverse bright orange-red (8A8); exudate (28D4), margin entire; reverse greenish grey (30C2); exudate present, small brown (7E6-8) droplets; soluble pigment absent. On and soluble pigment absent. On CREA, acid production absent. OA, colonies fasciculate, mycelium white, sporulation dense, with conidial masses greenish grey (28B2); exudate and soluble pigment Micromorphology: On MEA, conidiophores predominantly absent. On CREA, acid production moderately strong. terverticillate, minor proportion biverticillate; stipes 190–250 × 3.5–4 μm, smooth-walled; branches 30–40 μm; metula 3–5 per Micromorphology: On MEA, conidiophores typically terverticil- branch, divergent, cylindrical, 10–12(–15) × 2.5–3 μm; phialides late, minor proportion biverticillate and quaterverticillate; stipes 4–6 per metula, ampulliform, 8–10 × 2.5–3 μm; conidia globose, smooth, 250–500 × 2–2.5 μm; metula 3–4 per branch, divergent, 2.5–3 × 2.5–3 μm, smooth to finely roughened, greenish. 10–14 × 2–3 μm; phialides 3–4 per metula, ampulliform, 7–10 × 2–2.5 μm; conidia globose to subglobose, 2.5–3 × 2–3 μm, Distinguishing characters: Penicillium beceitense is closely smooth-walled, brownish yellow. related to P. lanosum and P. kojigenum, two species that are indistinguishable according to our phylogeny of the section Distinguishing characters: Penicillium balearicum is resolved Ramosa (Fig. 5). To date, there is no updated description in the P. atramentosum-clade containing six species, P. for either P. lanosum or P. kojigenum. The protologue of P. atramentosum, P. balearicum, P. fimosum, P. ibericum (the latter kojigenum described conidia that are rough-walled, measuring three described here), P. magnielliptisporum and P. mexicanum 2.2–2.6 μm (Smith 1961). The absence of growth at 30 °C is the (Visagie et al. 2014a). Penicillium atramentosum, P. balearicum, relevant feature to distinguish P. beceitense from other species P. magnielliptisporum and P. mexicanum have smooth-walled of the section, such asP. chroogomphum, P. jamesonlandense, P. stipes and conidia, and the conidia of the latter two are larger lanosum, P. ribium, and P. soppii (Rong et al. 2016). (5 × 4 μm in P. magnielliptisporum, 4 × 3.5 μm in P. mexicanum); while P. atramentosum can be differentiated by its production Penicillium caprifimosum Guevara-Suarez, Dania García & Cano, of red soluble pigment on CYA and by the lack of growth at sp. nov. MycoBank MB822064. Fig. 13. 30 °C (Pitt 1979, Frisvad & Samson 2004).Penicillium balearicum differs from P. fimosum and P. ibericum mainly by having longer Etymology: From the Latincapra = “goat”, and fimosum = “dung- stipes (up to 500 μm long), by the production of abundant dwelling”, describing the substrate from where the species was brown exudate droplets on YES, and by the moderately strong isolated. acid production on CREA. In: Section Turbata. Penicillium beceitense Guevara-Suarez, Gené & Guarro, sp. nov. MycoBank MB822063. Fig. 12. Typus: Spain, Catalonia, Els Ports Natural Park, from goat dung, Feb. 2016, J. Guarro, M. Guevara-Suarez & E. Rosas (holotype Etymology: Name refers to Beceite, the vilage where the fungus CBS H-23184; culture ex-type FMR 15041 = CBS 142990; ITS was found. barcode: LT899781, alternative markers: tub2 LT898238, cmdA LT899765, rpb2 LT899799). In: Section Ramosa.

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Fig. 12. Morphological characters of Penicillium beceitense (FMR 15038T). A. Colonies from left to right (top row) CYA, MEA, DG18 and OA; (bottom row) CYA reverse, MEA reverse, YES, and CREA. B–F. Conidiophores. G. Conidia. Scale bars = 10 μm.

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

Fig. 13. Morphological characters of Penicillium caprifimosum(FMR 15041T). A. Colonies from left to right (top row) CYA, MEA, DG18 and OA; (bottom row) CYA reverse, MEA reverse, YES, and CREA. B–F. Conidiophores. G. Conidia. Scale bars = 10 μm.

Colony diameter in 7 d (mm): On CYA: 25 °C 30–35, 30 °C 35–38, and soluble pigment absent. On MEA, colonies flat, velvety, 37 °C 18–20; on MEA: 25 °C 24–26, 30 °C 19–21, 37 °C 8–10; on sporulation dense, conidial masses dark dull green (28E3), YES: 25 °C 25–24, 30 °C 30–31; on OA: 25 °C 23–25, 30 °C 28–30, irregular, margin slightly crenate; reverse light orange (5A4) 37 °C 9–11; on DG18: 25 °C 16–21, on CREA: 25 °C 3–6. to dull green (28D4); exudate and soluble pigment absent. On DG18, colonies velvety, flat, mycelium white, sporulation Colony characters at 25 °C in 7 d: On CYA, colonies flat, velvety, dense, conidial masses dull green (27D4); reverse greenish mycelium white, sporulation dense, with conidial masses dull white (27B2). On YES, colonies irregularly sulcate, slightly

green (28E5), margin entire; reverse purplish grey (14B2); raised at the centre, mycelium white, sporulation dense, with Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] exudate absent; soluble pigment pinkish (12A2). On MEA, conidial masses dull green (26E5), margin slightly crenate; colonies flat, velvety, sporulation dense, conidial masses dark reverse bright orange-red (8A8) at the centre; exudate and dull green (28E3), margin lobate; reverse pale yellow (4A3) to soluble pigment absent. On OA, colonies slightly granulose, greenish grey (26B3); exudate and soluble pigment absent. mycelium white, sporulation dense, conidial masses greyish On DG18, colonies velvety, flat, mycelium white, sporulation green (28E7); exudate and soluble pigment absent. On CREA, dense, conidial masses dull green (27D4); reverse greyish moderate acid production. green (27B5). On YES, colonies velvety, raised at the centre, cerebriform, mycelium greenish white (27A2), sporulation Micromorphology: On MEA, conidiophores predominantly sparse, margin lobate; reverse light brown (7D5); exudate biverticillate, minor proportion terverticillate and some and soluble pigment absent. On OA, colonies granulose and irregularly branched; stipes 70–130 × 2.5–3(–4) μm, smooth; flat, radially sulcate towards the periphery, mycelium white, metula 3–4 per branch, rather divergent, 8–14 × 2.5–3(–4) μm; sporulation dense, conidial masses dull green (28E2), margin phialides 3–4 per metula, ampulliform, (6–)8–10 × 2.5–3 μm; entire; exudate and soluble pigment absent. On CREA, acid not conidia globose to subglobose, 2.5–3 × 2.5–3(–4) μm, smooth, produced. brownish yellow.

Micromorphology: On MEA, conidiophores biverticillate, Distinguishing characters: Penicillium fimosum is closely related sometimes with subterminal branches; stipes smooth-walled, to P. atramentosum and P. ibericum (described below). Penicillium 100–250 × 3–3.5 μm; metula 3–4 per branch, appressed, 10–12 fimosum and P. atramentosum differ from P. ibericum by the × 2–3 μm; phialides 3–4 per metula, ampulliform, 8–10 × 2–2.5 absence or limited growth at 30 °C. Penicilium atramentosum μm; conidia globose to subglobose, 2.5–3 × 2–2.5 μm, smooth- can be distinguished by the production of reddish brown walled, brownish yellow. exudate, soluble pigment and colony reverse on CYA, features absent in P. fimosum. Moreover, P. fimosum has shorter stipes Distinguishing characters: Penicillium caprifimosum can be (70–130 μm) than P. atramentosum (300–500 μm) (Pitt 1979). differentiated from its closest relative,P. bovifimosumdescribed from dry cow manure (Tuthill & Frisvad 2002), by its better Penicillium ibericum Guevara-Suarez, Cano & Dania García, sp. growth at 25 and 37 °C on MEA and CYA. The maximum colony nov. MycoBank MB822070. Fig. 15. diameter reported for P. bovifimosum is 16–21 mm on MEA and 25–29 mm on CYA in 7 d at 25 °C and 6 mm on CYA at 37 °C (Tuthill Etymology: Name refers to the occurrence of the species on the & Frisvad 2002). In addition, this species produces cleistothecia Iberian Peninsula. in culture, which are absent in P. caprifimosum. In: Section Paradoxa. Penicillium fimosum Guevara-Suarez, Guarro & Dania García, sp. nov. MycoBank MB822069. Fig. 14. Typus: Spain, Catalonia, Els Ports Natural Park, from herbivore dung, Feb. 2016, J. Guarro, M. Guevara-Suarez & E. Rosas Etymology: Name refers to the substrate from where the species (holotype CBS H-23186; culture ex-type FMR 15040 = CBS was isolated. 142992; ITS barcode: LT899782, alternative markers: tub2 LT898285, cmdA LT899766, rpb2 LT899800). In: Section Paradoxa. Additional material examined: Spain, Galicia, Las Dunas de Corrubedo Typus: Spain, Catalonia, Pratdip, from hervibore dung, Feb. 2016, Natural Park, from soil, Feb. 2016, D. García (FMR 15107). D. García (holotype CBS H-23185; culture ex-type FMR 15104 = CBS 142991; ITS barcode: LT970836, alternative markers: tub2 Colony diameter in 7 d (mm): On CYA: 25 °C 31–35, 30 °C 18–21, LT898273, cmdA LT970837). 37 °C no growth; on MEA: 25 °C 18–20, 30 °C 8–9; on YES: 25 °C 33–35, 30 °C 18–20; on OA: 25 °C 21–23, 30 °C no growth; on Colony diameter in 7 d (mm): On CYA: 25 °C 31–34, 30 °C 3–4, DG18: 25 °C 5–6; on CREA: 25 °C 21–22. 37 °C no growth; on MEA: 25 °C 25–30, 30 °C no growth; on YES: 25 °C 50–55, 30 °C 5–7; on OA: 25 °C 25–27, 30 °C no growth; on Colony characters at 25 °C in 7 d: On CYA, colonies flat, velvety, DG18: 25 °C 16–20, on CREA: 25 °C 20–23. slightly radially sulcate, mycelium white, sporulation dense, conidial masses dark dull green (26E4), margin slightly lobate; Colony characters at 25 °C in 7 d: On CYA, colonies velvety, slightly reverse reddish brown (9E5) to reddish (9A2); exudate present, radially sulcate at the centre, mycelium white, sporulation with pale red (10A3) droplets; soluble pigment reddish brown. dense, conidial masses dark dull green (26E4), margin slightly On MEA, colonies, velvety and flat, sporulation dense, conidial lobate and fimbriate; reverse greyish orange (6B4) at the masses dark dull green (28E3); reverse light orange (5A4), centre, greyish green (28C3) towards the periphery; exudate margin slightly irregular; exudate and soluble pigment absent.

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

Fig. 14. Morphological characters of Penicillium fimosum(FMR 15104T). A. Colonies from left to right (top row) CYA, MEA, DG18 and OA; (bottom row) CYA reverse, MEA reverse, YES, and CREA. B, D–F. Conidiophores. C. Conidia. Scale bars = 10 μm.

On DG18, colonies sunken in the middle, velvety, mycelium 150 × 2–3(–4) μm, smooth; metula 2–4 per branch, divergent, white, sporulation moderate, conidial masses dull green 8–12 × 2–2.5 μm; phialides 3–4 per metula, ampulliform, 8–10 × (27D4); reverse greenish white (27B2). On YES, velvety, sulcate, 2.5–3 μm; conidia globose, 2.5–3 × 2.5–3 μm, smooth to finely mycelium white, sporulation dense, conidial masses dull green roughened, subhyaline to pale green. (26E5), margin crenate; reverse orange-grey (5B3); exudate and soluble pigment absent. On OA, colonies irregular, granulose, Distinguishing characters: Penicillium ibericum is closely related mycelium white, sporulation dense, conidial masses greyish to P. atramentosum and P. fimosum. It can be distinguished by green (28E7); exudate and soluble pigment absent. On CREA, having shorter stipes (70–150 μm) than P. atramentosum (300– moderate acid production. 500 μm) (Pitt 1979), and by its more restricted growth on YES (33–35 mm diam) and DG18 (5–6 mm) compared to P. fimosum Micromorphology: On MEA, conidiophores predominantly (YES 50–55 mm, DG18 16–20 mm). terverticillate, minor proportion quarterverticillate; stipes 70–

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

Fig. 15. Morphological characters of Penicillium ibericum (FMR 15040T). A. Colonies from left to right (top row) CYA, MEA, DG18 and OA; (bottom row) CYA reverse, MEA reverse, YES, and CREA. B. Colony texture on CYA at 25 °C after 1 wk incubation.C. Conidia. D–G. Conidiophores. Scale bars = 10 μm.

Fig. 16. Morphological characters of Penicillium mediterraneum (FMR 15188T). A. Colonies from left to right (top row) CYA, MEA, DG18 and OA; (bottom row) CYA reverse, MEA reverse, YES, and CREA. B–G. Conidiophores. H. Conidia. Scale bars = 10 μm.

Penicillium mediterraneum Guevara-Suarez, Gené & Cano, sp. Etymology: Name refers to the geographical area where the nov. MycoBank MB822071. Fig. 16. species was found.

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

In: Section Roquefortorum. Typus: Spain, Balearic Islands, Mallorca, Camí Vell d’Orient, from goat dung, Feb. 2016, J. Guarro, M. Guevara-Suarez & J.P.Z. Typus: Spain, Balearic Islands, Mallorca, Puigpuñent, from Siqueira (holotype CBS H-23132, culture ex-type FMR 15192 = herbivore dung, Feb. 2016, J. Guarro, M. Guevara-Suarez & J.P.Z. CBS 142669; ITS barcode: LT898167, alternative markers: tub2 Siqueira (holotype CBS H-23143; culture ex-type FMR 15188 = LT898172, cmdA LT898137, rpb2 LT898142). CBS 142754; ITS barcode: LT899784, alternative markers: tub2 LT898291, cmdA LT899768, rpb2 LT899802). Additional materials examined: Spain, Mallorca, Escorca, from goat J. Guarro, M. Guevara-Suarez & J.P.Z. Siqueira dung, Feb. 2016, (FMR Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] Additional materials examined: Spain, Catalonia, Els Ports Natural Park, 15210); Mallorca, Camí Vell d’Orient, from goat dung, Feb. 2016, J. herbivore dung, Feb. 2016, J. Guarro, M. Guevara-Suarez & E. Rosas Guarro, M. Guevara-Suarez & J.P.Z. Siqueira (FMR 15211); Catalonia, (FMR 15031 = CBS 142755); Catalonia, Els Ports Natural Park, herbivore Barcelona, Montseny Natural Park, from herbivore dung, Apr. 2017, dung, Feb. 2016, J. Guarro, M. Guevara-Suarez & E. Rosas (FMR 15032). J. Gené, M. Guevara-Suarez & I. Iturrieta-González (FMR 16481 = CBS 143045); Catalonia, Poblet, from herbivore dung, Mar. 2017, J. Guarro, Colony diameter in 7 d (mm): On CYA: 25 °C 45–60, 30 °C 24–26, Guevara-Suarez & I. Iturrieta-González (FMR 16491 = CBS 143046). 37 °C no growth; on MEA: 25 °C 45–60, 30 °C 23–26; on YES: 25 °C >60, 30 °C 35–40; on OA: 25 °C 31–38, 30 °C 8–15; on Colony diameter in 7 d (mm): On CYA: 25 °C 33–37, 30 °C 9–10, DG18: 25 °C 43–45; on CREA: 25 °C 22–25. 37 °C no growth; on MEA: 25 °C 11–13, 30 °C 5–7; on YES: 25 °C 30–34, 30 °C 9–17; on OA: 25 °C 31–38, 30 °C 8–15; on DG18: Colony characters at 25 °C in 7 d: On CYA, colonies flat, velvety, 25 °C 17–19; on CREA: 25 °C 6–9. mycelium white, sporulation dense, conidial masses dull green (25E4), margin entire; reverse greyish green (28C5); exudate and Colony characters at 25 °C in 7 d: On CYA, colonies granular, soluble pigment absent. On MEA, colonies flat, velvety, mycelium raised at the centre, mycelium white, sporulation dense, white, margin crenate, sporulation dense, with conidial masses conidial masses dark dull green (25E4), margin dentate; reverse conidial masses dull green (25E4), margin irregular, slightly light orange (5A5); exudate present, consisting of small hyaline lobulate, reverse greyish green (28E5); exudate and soluble droplets; soluble pigment absent. On MEA, colonies fasciculate pigment absent. On DG18, colonies flat, velvety, mycelium due to the presence of small feathery synnemata, mycelium white, sporulation dense, conidial masses dull green (26E4), white, sporulation dense, with conidial masses dark green margin entire; reverse greyish green (30C6). On YES, colonies (28F5), margin crenate; reverse orange (6B8); exudate absent velvety, raised at the centre, irregularly sulcate, mycelium white, and soluble pigment deep orange (5A8). On DG18, colonies sporulation dense, conidial masses dull green (26E4), margin velvety, mycelium white, sporulation dense, conidial masses entire and fimbriate; reverse greyish green (28C6); exudate and dull green (27E4); reverse greyish orange (5B3). On YES, colonies soluble pigment absent. On OA, colony cottony, mycelium white, granular, raised at the centre, radially sulcate, mycelium white, sporulation dense, conidial masses dark dull green (28D4), sporulation dense, conidial masses dull green (27E4), margin margin entire; exudate and soluble pigment absent. On CREA, dentate; reverse brownish orange (6C8); exudate and soluble moderate acid production. pigment absent. On OA, colony strongly fasciculate, mycelium white, sporulation dense, conidial masses dark dull green Micromorphology: On MEA, conidiophores bi- and terverticillate; (27E4), margin crenate; reverse yellowish white (4A); exudate stipes rough-walled, 50–100 × 2.5–3 μm; metula 2–3 per branch, and soluble pigment absent. On CREA, weak acid production. appressed, cylindrical, 10–14 × 2–3 μm; phialides 2–4 per metula, ampulliform, 10–13 × 2–3 μm; conidia globose, 2.5–4 Micromorphology: On MEA, synnemata present, up to 1 mm (–5) × 2–4 μm, smooth-walled, greenish. long; conidiophores ter- to quaterverticillate, minor proportion biverticillate; stipes 100–218 × 3–4 μm (up to 1 mm long in Distinguishing characters: All the species in section synnemata), coarsely roughened; metula 3–4 per branch, rather Roquefortorum show fast growth on CYA and MEA and produce appressed, cylindrical, smooth to conspicuously roughened, rough-walled conidiophores. Penicillium roquefortiis the closest- 10–14 × 2–3 μm; phialides 2–4 per metula, ampulliform, 10–13 related species to P. mediterraneum, and it can be distinguished × 2–3 μm; conidia globose, subglobose to broadly ellipsoidal, by a more restricted growth on CYA at 30 °C (5–15 mm diam) and 2.5–3 × 2–2.5 μm, smooth-walled, subhyaline to pale green. by its blackish green colony reverse on YES (Frisvad & Samson 2004, Houbraken et al. 2010). Distinguishing characters: Section Robsamsonia includes some species that produce synnemata, e.g. P. coprophilum, Penicillium synnematicola Guevara-Suarez, Dania García & P. glandicola and P. vulpinum. Penicillium coprophilum and P. Guarro, sp. nov. MycoBank MB822072. Fig. 17. vulpinum can be differentiated from our new species by their smooth-walled stipes (Houbraken et al. 2016). Penicillium Etymology: Name refers to the formation of synnemata. synnematicola is closely related to P. glandicola, a species with world-wide distribution isolated from oak trees and forest In: Section Robsamsonia. soil, including dungy soil and mouse dung (Houbraken et al. 2016), and both produce coarsely roughened synnematous

Fig. 17. Morphological characters of Penicillium synnematicola (FMR 15192T). A. Colonies from left to right (top row) CYA, MEA, DG18 and OA; (bottom row) CYA reverse, MEA reverse, YES, and CREA. B. Colony texture on CYA at 25 °C after 1 wk incubation. C. Colony texture on OA at 25 °C after 1 wk incubation. D–G. Conidiophores. H. Conidia. Scale bars = 10 μm.

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

conidiophores. However, P. glandicola can be distinguished sequence data of that strain showed that P. nodositaum was by having shorter phialides (7.5–10.5 µm) and ellipsoidal and closely related to P. kabunicum, and neither of them belonged in yellow-green conidia. In addition, the colonies of P. glandicola Penicillium s. str. (Houbraken & Samson 2011, Visagie et al. 2013). show orange-brown to brown and bright orange-red reverse on We observed that the only sequence available for P. kabunicum CYA and YES, respectively, and tend to have a more restricted (CBS 574.90, ITS sequence GenBank MH862240) showed a growth on both media (17–30 mm on CYA; 19–36 mm on YES) 99.64 % of similarity with that of P. nodositatum (CBS 333.90, (Houbraken et al. 2016). ITS sequence GenBank NR_103703) (Fig. S6 in supplemental

material), suggesting they could be conspecific. However, the Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] Penicillago Guevara-Suarez, Gené & Dania García, gen. nov. protologue of P. kabunicum (Baghdadi 1968) disagree with that MycoBank MB822073. of P. nodosotatum (Valla et al. 1989) in features such as the conidial morphology (globose, smooth-walled and hyaline in P. Etymology: Name refers to the phylogenetic closeness to kabunicum vs ellipsoid to subglobose, echinulate and brown in Penicillium (Fig. 10). P. nodosotatum). Therefore, further comparative studies will be needed to determine whether both species should be treated In: Family Aspergillaceae. as synonym. The possibility of examining two Spanish isolates that fit Diagnosis: Morphologically differs from Penicillium by its morphologically with P. nodositatum and their DNA sequences symmetrically biverticillate conidiophores, ampulliform to coincide with those of the ex-type strain of this species, has acerose phialides with a fine long neck, and conidia with allowed us to delineate a new genus of penicillium-like fungi conspicuous disjunctors. Conidiophore structure resembles in the family Aspergillaceae (Figs 9, 10). Because herbarium Talaromyces, but phylogenetically distant. type material for P. nodositatum is lost according to Visagie et al. (2013), the illustrations included in the protologue of the Mycelium superficial and immersed, composed of septate, species (Valla et al. 1989) are selected here as lectotype, and the branched, canary yellow to chrome yellow hyphae. Conidiophores metabolically inactive culture CBS 333.90 as epitype deposited symmetrically branched, penicillium-like, composed of long, in the Westerdijk Fungal Biodiversity Institute. septate, hyaline stipes, often terminating in a small vesicle giving rise to a verticil of metulae, occasionally irregularly branched; Penicillago nodositata (Valla) Guevara-Suarez, Gené & Dania metulae appressed to divergent, cylindrical to somewhat García, comb. nov. MycoBank MB822074. Fig. 18. obpyriform, bearing a compact verticil of conidiogenous cells. Basionym: Penicillium nodositatum Valla, Pl. Soil 114: 142. 1989. Conidiogenous cells phialidic, ampulliform to acerose, with a fine and long neck. Conidia dry, catenate, with conspicuous Typus: Lectotype designed here: figs 1–4 in Valla et al. disjunctors, subglobose to ellipsoid, coarsely echinulate, (1989), Penicillium nodositatum Valla, a new species inducing subhyaline to brown; conidial chains short to moderately long. myconodules on Alnus roots, Plant & Soil 114: 142–146, Sexual morph unknown. MBT388228; Epitype designed here: metabolically inactive culture CBS 333.90, France, Isère, Col d’Ornon, from myconodules Type species: Penicillago nodositata (Valla) Guevara-Suarez, in roots of Alnus incana, 1986, G. Valla, MBT388229; culture ex- Gené & Dania García. epitype CBS 333.90.

Notes: The genus Penicillago is introduced to accommodate Additional materials examined: Spain, Balearic Islands, Mallorca, from Penicillium nodositatum. It is noteworthy that this species has wild pig dung, Feb. 2016, J. Guarro, M. Guevara-Suarez & J.P.Z. Siqueira had a confusing taxonomic history. Initially, Valla et al. (1989) (FMR 15296 = CBS 142988; ITS barcode: LT899787, alternative markers: classified P. nodositatum in the subgenus Biverticillium from tub2 LT898312, cmdA LT899771, rpb2 LT899805); Extremadura, its morphological affinity with members of the series Islandica Badajoz, Granja de Torrehermosa, from herbivore dung, Dec. 2016, J. (Pitt 1979). Later, based on molecular data, it was considered a Cano-Lira (FMR 16442). member of the Penicillium section Sclerotiora, and tentatively placed in synonymy with P. bilaiae by Houbraken & Samson Colony diameter in 7 d (mm): On CYA: 25 °C 40–44, 30 °C 34–36, (2011), despite morphological differences in the conidiophore 37 °C 7–8; on MEA: 25 °C 34–36, 30 °C 24–27, 37 °C 0–3; on YES: structure (biverticillate in P. nodositatum vs monoverticillate 25 °C 50–55, 30 °C 45–55, 37 °C 11–10; on OA: 25 °C 24–26, in P. bilaiae). However, Visagie et al. (2013) noticed that in the 30 °C 22–24, 37 °C 4–6; on DG18: 25 °C 2–3; on CREA: 25 °C previous study that synonymy was based on a culture of P. 22–30. bilaiae (CBS 330.90) mislabeled as P. nodositatum. The origin of the problem was probably introduced by Pitt & Samson (2000) Colony characters at 25 °C in 7 d: On CYA, colonies cottony, since they erroneously designated on pag. 66 "No. 330.90 slightly sulcate, mycelium light yellow (3A5) to white, sporulation (CBS)" as neotype of P. nodositatum. In fact, the International sparse after 7d, conidial masses yellowish brown (5E8) after Commission of Penicillium and Aspergillus (http://www. 14 d, margin fimbriate; reverse greyish orange (5B4) at the aspergilluspenicillium.org/) included the living ex-type strain of centre, white to pale yellow towards the periphery; exudate and the herbarium specimen of P. nodositatum as CBS 333.90, this soluble pigment absent. On MEA, colonies slightly cottony, flat, therefore being the correct ex-type strain for the species. Newly mycelium light yellow (3A5) to white, sporulation dense after

Fig. 18. Morphological characters of Penicillago nodositata (FMR 15296). A. Colonies from left to right (top row) CYA, MEA, DG18 and OA; (bottom row) CYA reverse, MEA reverse, YES, and CREA. B–F. Conidiophores. G. Conidia. Scale bars = 10 μm.

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the 14 d, with conidial masses dark green (28F5), margin slightly megasporum, the latter being selected as the type since it was fimbriate; reverse light yellow (4A4); exudate and soluble the first described. Penicillium giganteum and P. megasporum pigment absent. On DG18, colonies restricted, flat, mycelium were recovered from soil samples in India and the UK, white, sporulation absent, margin entire; reverse light yellow respectively. Despite their different geographical origin, both (4A4); exudate and soluble pigment absent. On YES, colonies were considered conspecific and classified in seriesMegasporus velvety, raised at the centre, irregularly sulcate, mycelium yellow (Pitt 1979). Penicillium asperosporum, also included in this (3A7) to white, sporulation sparse, margin fimbriate; reverse series, was later shown to be a synonym of P. montanense in

greyish orange (5B5); exudate and soluble pigment absent. On section Aspergilloides (Houbraken et al. 2014b). Multi-gene Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] OA, colony slightly cottony, mycelium white, sporulation sparse, phylogenies (Peterson et al. 2010, Houbraken & Samson 2011) margin entire; reverse yellowish white (4A2); exudate and demonstrated that P. megasporum and P. giganteum were soluble pigment absent. On CREA, acid production absent. distinct species, closely related to the genus Hamigera. Our phylogeny confirms this relationship and shows thatHamigera Micromorphology: On MEA, conidiophores symmetrically and the afore-mentioned Penicillium species comprise two biverticillate, sometimes irregularly branched; stipes smooth, sister clades with enough genetic differences to be considered hyaline, often with a small vesicle at the apex, 3.5–5.5 μm wide, distinct genera (Figs 9, 10). 200–300 × 2.5–3 μm; metulae divergent, cylindrical to somewhat obpyriform, 9–10(–15) × 3–4(–5) μm; phialides four to seven per Pseudopenicillium cervifimosum Guevara-Suarez, Dania García metula, ampulliform or tending towards acerose, often tapering & Gené, sp. nov. MycoBank MB822079. Fig. 19. abruptly to a long, slender neck, 6–8(–9) × 2.5–3.5 μm; conidia in short to moderately long disorder chains, subglobose to broadly Etymology: From the Latin cervus = “deer”, and fimosum = ellipsoidal, echinulate, 2.5–4 × 3–4(–5) μm, pale brown. “dung-dwelling”, describing the substrate from where the species was isolated. Pseudopenicillium Guevara-Suarez, Cano & Guarro, gen. nov. MycoBank MB822076. Typus: Spain, Castile and Leon, Palencia, Monte el Viejo, Deer Reserve Park, from deer dung, Mar. 2016, J. Guarro & Etymology: Pseudo- meaning “false”-, name refers to the M. Guevara-Suarez (holotype CBS H-23133; culture ex-type morphological similarity but not belonging to Penicillium. FMR 15299 = CBS 142670; ITS barcode: LT899789, alternative markers: tub2 LT898315, rpb2 LT899807). In: Family Aspergillaceae. Colony diameter in 7 d (mm): On CYA: 25 °C 26–30, 30 °C 30–32, Diagnosis: Phylogenetic closeness to Hamigera, but differs 37 °C 6–9; on MEA: 25 °C 13–17, 30 °C 22–24, 37 °C 3–5; on YES: in the lack of sexual morph and by its brown globose conidia 25 °C 24–26, 30 °C 35–38, 37 °C 23–25; on OA: 24–26, 30 °C –34, with conspicuous disjunctors. Morphologically differs from 36 °C 14–16; on DG18: 25 °C 16–17; on CREA: 25 °C 18–20. Penicillium and Penicillago by its short and often irregularly branched conidiophores producing large conidia in short chains. Colony characters at 25 °C in 7 d: On CYA, colonies flat, velvety, mycelium white, sporulation dense, central area with conidial Mycelium superficial and immersed, composed of septate, masses dark green (29F3), margin entire; reverse pale yellow branched, hyaline to subhyaline hyphae. Conidiophores (4A3); exudate and soluble pigment absent. On MEA, colonies undifferentiated, reduced to conidiogenous cells arising directly growing moderately, velvety, slightly umbonate, mycelium from the main hyphae, or differentiated composed of short to white, sporulation dense, with conidial masses olive-yellow moderately long, aseptate or few septate, hyaline to brown (3C8) to olive (3F8), margin lobate; exudate and soluble pigment stipes, terminating in an irregular penicilli; penicilli varying absent; reverse greyish yellow (2B4). On YES, colonies dome- from monoverticillate to partly biverticillate, with few metulae shaped, velvety, mycelium white, sporulation absent, margin bearing each a verticil of conidiogenous cells; metulae terminal entire; reverse pale yellow (4A3); exudate and soluble pigment or subterminal, generally divergent, cylindrical or apically absent. DG18, colonies velvety, slightly sulcate, mycelium white, inflated.Conidiogenous cells phialidic, ampulliform, with swollen sporulation dense, with conidial masses dull green (28E3), margin base and tapering abruptly to a slender neck of variable length. entire; reverse yellowish white (4A3). On OA, colonies cottony, Conidia dry, catenate, with conspicuous disjunctors, globose mycelium white, sporulation dense, with conidial masses dark to subglobose, with spinulose walls, brown, dark in masses; green (29F3), margin fimbriate; exudate and soluble pigment conidial chains short. Sexual morph unknown. absent. On CREA, strong acid production.

Type species: Pseudopenicillium megasporum (P.A. Orpurt & D.I. Micromorphology: On MEA, conidiophores short, arising Fennell) Guevara-Suarez, Cano & Gené as lateral branches from hyphae, biverticillate, sometimes monoverticillate; stipes 10–35 × 2.5–3 μm, cylindrical, without Notes: The genus Pseudopenicillium is proposed to vesicle, smooth to finely verruculose, hyaline; metulae accommodate a new species and two species previously divergent, cylindrical, 9.5–10 × 2.5–3 μm; phialides in verticils classified in the genus Penicillium, P. giganteum and P. of 2–4, ampulliform with swollen base, tapering abruptly to a

Fig. 19. Morphological characters of Pseudopenicillium cervifimosum (FMR 15299T). A. Colonies from left to right (top row) CYA, MEA, DG18 and OA reverse; (bottom row) CYA reverse, MEA reverse, YES, and CREA. B. Colony texture on OA at 25 °C after 1 wk incubation. C–H. Conidiophores. I. Conidia. Scale bars = 10 μm.

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slender neck, (6–)7.5–9 × 2.5–3 μm; conidia globose, 5–5.5 × 6 on YES: 25 °C 45–50, 30 °C 50–55, 37 °C 25–30; on OA: 25 °C 40– μm, spinulose, thick-walled, pale brown to brown. 45, 30 °C 40–42, 37 °C 12–14; on DG18: 25 °C 20–22, on CREA: 25 °C 17–19. Distinguishing characters: Pseudopenicillium cervifimosum is closely related to Pse. giganteum and Pse. megasporum. Colony characters at 25 °C in 7 d: On CYA, colonies velvety, radially This group of species have a similar conidiogenous apparatus sulcate, mycelium white, sporulation dense, conidial masses dull and produce globose spinulose conidia. However, they can be green (27E4), margin lobate; reverse greyish orange (5B4); exudate

distinguished by their conidial sizes (i.e., up to 6 μm in Pse. absent, soluble pigment only at 30 and 37 °C, light yellow (3A5) Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] cervifimosum, up to 10 μm in Pse. megasporum and up to 12 to light orange (5A6). On MEA, colonies velvety, flat, sporulation μm in Pse. giganteum). In addition, Pse. megasporum has a dense, with conidial masses greyish green (27E5), margin entire; less abundant growth on CYA at 25 °C (15–25 mm) and shows reverse yellow (2B8); exudate and soluble pigment absent. On vesiculate stipes (Pitt 1979, Peterson et al. 2010). DG18, colonies flat, slightly cotton at the centre, velvety in the periphery, mycelium white, sporulation dense, conidial masses Pseudopenicillium giganteum (R.Y. Roy & G.N. Singh) Guevara- dull green (26E5), margin entire; reverse greyish green (28B4). On Suarez, Gené & Cano, comb. nov. MycoBank MB822077. YES, colonies raised at the centre, irregularly sulcate, mycelium Basionym: Penicillium giganteum R.Y. Roy & G.N. Singh, Trans. white, sporulation dense, with conidial masses dark green (30F3), Br. Mycol. Soc. 51: 805. 1968. margin entire; reverse orange-yellow (4B8); exudate and soluble pigment absent. On OA, colonies velvety, flat, mycelium white, Typus: India, West Bengal, Varanasi, Banaras Hindu University, sporulation dense, conidial masses dark green (27F3), margin Botanical Garden, from soil, Nov. 1967, G.N. Singh (holotype IMI entire; reverse greenish grey (29C2); exudate and soluble pigment 132774; cultures ex-type NRRL 3553 = ATCC 48996 = CBS 144.69 absent. On CREA, weak acid production. = FRR 535 = IMI 132774). Micromorphology: On MEA, conidiophores biverticillate, Descriptions and illustrations: See Roy & Singh (1968) and sometimes irregularly branched, stipes (40–)100–130 × 2–2.5 Peterson et al. (2010). μm, smooth-walled, hyaline; metulae divergent, 10–11 × 2–2.5 μm; phialides 4–6 per metula, ampulliform to acerose, (7–)8–10 Additional material examined: Spain, unknown geographic region, from × 2–2.5 μm; conidia globose to subglobose, 2–2.5 × 1.8–2 μm, soil (FMR 14718). smooth-walled, brownish yellow. Ascomata not observed.

Notes: The new isolate is the second specimen known for this Distinguishing characters: The most closely related species to T. taxon. In general, its morphological features are consistent with catalonicus are T. albobiverticillius, T. erythromellis, T. heiheensis, those of the species as described by Roy & Singh (1968). and T. solicola (Fig. 8). Talaromyces catalonicus is characterised by its ability to grow at 37 °C; in contrast, the above-mentioned Pseudopenicillium megasporum (P.A. Orpurt & D.I. Fennell) species do not grow or grow very restricted at this temperature Guevara-Suarez, Cano & Gené, comb. nov. MycoBank MB822078. (Samson et al. 2011, Yilmaz et al. 2014). Basionym: Penicillium megasporum P.A. Orpurt & D.I. Fennell, Mycologia 47: 233. 1955. Talaromyces coprophilus Guevara-Suarez, Cano & Dania García, sp. nov. MycoBank MB822088. Fig. 21. Typus: UK, England, Suffolk, Lakenheath Warren, from heath soil, J.H. Warcup (holotype IMI 216904; cultures ex-type NRRL Etymology: Name refers to the source of isolation of the fungus. 2232 = ATCC 12322 = CBS 256.55 = FRR 2232 = MUCL 38804 = QM 6879 = WE 2232 = IMI 216904). In: Section Talaromyces.

Descriptions and illustrations: See Orpurt & Fennell (1955), Pitt Typus: Spain, Balearic Islands, Mallorca, Escorca, from herbivore (1979) and Peterson et al. (2010). dung, Feb. 2016, J. Guarro, M. Guevara-Suarez & J.P.Z. Siqueira (holotype CBS H-23144; culture ex-type FMR 15199 = CBS Talaromyces catalonicus Guevara-Suarez, Gené & Guarro, sp. 142756; ITS barcode: LT899794, alternative identification nov. MycoBank MB822080. Fig. 20. markers: tub2 LT898319, cmdA LT899776, rpb2 LT899812).

Etymology: Name refers to the region (Catalonia) from where Colony diameter in 7 d (mm): On CYA: 25 °C 38–40, 30 °C 40–45, the fungus was isolated. 37 °C 30–35; on MEA: 25 °C 33–35, 30 °C 30–35, 37 °C 21–24; on YES: 25 °C 45–50, 30 °C 50–55, 37 °C 38–42; on OA: 25 °C 35–40, In: Section Trachyspermi. 30 °C 40–45, 37 °C 25–26; on DG18: 25 °C no growth, on CREA: 25 °C 8–10. Typus: Spain, Catalonia, Poblet, from herbivore dung, Feb. 2017, J. Guarro, M. Guevara-Suarez & I. Iturrieta-González (holotype Colony characters at 25 °C in 7 d: On CYA, colonies slightly raised CBS H-23212; culture ex-type FMR 16441 = CBS 143039; ITS at the centre, velvety, mycelium light yellow (4A5) to white, barcode: LT899793, alternative identification markers: tub2 sporulation absent to sparse, with margin entire and fimbriate; LT898318, cmdA LT899775, rpb2 LT899811). reverse light orange (5A4); soluble pigment and exudate absent. On MEA, colonies slightly raised at centre, velvety, flat, mycelium Colony diameter in 7 d (mm): On CYA: 25 °C 35–40, 30 °C 38–40, light yellow (4A5) to white, conidial sporulation absent to 37 °C 15–18; on MEA: 25 °C 17–19, 30 °C 19–20, 37 °C 16–18; sparse, young ascomata visible, margin fimbriate; reverse light

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Fig. 20. Morphological characters of Talaromyces catalonicus (FMR 16441T). A. Colonies from left to right (top row) CYA, MEA, DG18 and OA reverse; (bottom row) CYA reverse, MEA reverse, YES, and CREA. B–E. Conidiophores. F. Conidia. Scales bars = 10 μm. orange (5A4); soluble pigment and exudate absent. On YES, Distinguishing characters: Talaromyces coprophilus is colonies raised at centre, velvety, mycelium pinkish (7A3) to characterised by the production of orange-red ascomata with white, sporulation absent, with margin entire; reverse brownish spiny ellipsoidal ascospores, the lack of growth on DG18, and orange (7C6); exudate absent and soluble pigment brownish red a good growth on other culture media tested. Phylogenetically, (10C7). On OA, colonies granular, flat, with young bright orange it forms an independent and distant branch in an unsupported ascomata visible, conidial sporulation sparse, margin fimbriate; clade where the ex-type strains of T. cnidii, T. flavovirens, T. reverse greenish grey (28C3); exudate and soluble pigment siamensis, and T. xishaensis are found (Fig. 7). Morphologically, absent. On CREA, strong acid production. T. coprophilus resembles T. flavovirens in the production of a sexual morph; however, the latter differs in slower growth on Micromorphology: On MEA, conidiophores mono- to CYA at 25 °C (19–20 mm after 7d), and larger ascospores (4–7 × biverticillate, stipes 40–70 × 2–2.5 μm, smooth, hyaline; metulae 3–4 μm) (Yilmaz et al. 2014). rather appressed, 10–12 × 2.5–3 μm; phialides 3–4 per metula, acerose, 9–12(–14) × 2–2.5 μm; conidia ellipsoidal, 2–3 × 2.5–3 Talaromyces pseudofuniculosus Guevara-Suarez, Dania García μm, smooth-walled, brownish yellow. Ascomata after 1–2 wk of & Gené, sp. nov. MycoBank MB822090. Fig. 22. incubation on OA and MEA at 25 °C, bright orange to orange- red, globose, 200–310 μm diam, with peridial hyphae branched Etymology: –pseudo, meaning “false”-, name refers to its and verruculose; asci globose to subglobose, 6–7 × 7–9(–10) phylogenetic relationship with T. funiculosus. μm; ascospores ellipsoidal, 3.5–4 × 3–4 μm, spiny, thick-walled, golden yellow. In: Section Talaromyces.

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Fig. 21. Morphological characters of Talaromyces coprophilus (FMR 15199T). A. Colonies from left to right (top row) CYA, MEA, DG18 and OA reverse; (bottom row) CYA reverse, MEA reverse, YES, and CREA.B. Detail of the colony texture with ascomata on MEA after 2 wk incubation.C. Ascoma. D, E. Part of an ascoma and peridial hyphae. F. Asci. G. Ascospores. H, I. Conidiophores. J. Conidia. Scales bars C, D = 100 μm, E–J = 10 μm.

Typus: Spain, Andalusia, La Rocina, Doñana National Park, from LT899796, alternative markers:tub2 LT898323, cmdA LT899778, herbivore dung, Mar. 2016, D. García (holotype CBS H-23214; rpb2 LT899814). culture ex-type FMR 15307 = CBS 143041; ITS barcode:

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Fig. 22. Morphological characters of Talaromyces pseudofuniculosus (FMR 15307T). A. Colonies from left to right (top row) CYA, MEA, DG18 and OA reverse; (bottom row) CYA reverse, MEA reverse, YES, and CREA. B–E. Conidiophores. F. Conidia. Scale bars = 10 μm.

Additional material examined: Spain, Catalonia, Els Ports Natural Park, absent. On MEA, colonies velvety to cottony, flat, sporulation from herbivore dung, Feb. 2016, J. Guarro, M. Guevara-Suarez & E. dense, conidial masses greyish green (28D5), margin entire to Rosas (FMR 15035). slightly crenate; reverse deep yellow (5A4) to greyish green (30C5); exudate and soluble pigment absent. On YES, colonies Colony diameter in 7 d (mm): On CYA: 25 °C 36–43, 30 °C 45–40, slightly raised at the centre, sulcate, cottony, mycelium white, 37 °C 31–35; on MEA: 25 °C 24–29, 30 °C 43–45, 37 °C 23–26; on sporulation dense in the centre, conidial masses greyish green YES: 25 °C 34–35, 30 °C 40–44, 37 °C 32–34; on OA: 25 °C 33–38, 30 (27D4), margin entire; reverse olive yellow (3C7); exudate and °C 42–45, 37 °C 27–30; on DG18: 25 °C 12–15, on CREA: 25 °C 2–4. soluble pigments absent. On DG18, colonies flat and velvety, mycelium white; moderate sporulation, conidial masses dull Colony characters at 25 °C in 7 d: On CYA, colonies slightly raised green (27D4); reverse greenish white (27B2). On OA, colonies at centre, radially sulcate, cottony, mycelium white, sporulation slightly granular, sporulation dense, conidial masses greenish dense, conidial masses greyish green (29D5), with margin grey (28B2), margin entre; reverse greyish green (30C5); exudate entire; reverse dark orange (5D8); soluble pigments and exudate and soluble pigment absent. On CREA, acid production strong.

Micromorphology: On MEA, conidiophores biverticillate; stipes Among Penicillium isolates, those of the sectionsFasciculata 40–70 × 2.5–3 μm, smooth-walled, brownish yellow; metulae and Robsamsonia were the most frequently isolated, accounting 3–4 per branch, appressed, 8–10 × 2–3 μm; phialides 3–4 per nearly 20 % of the isolates in each section. It is of note, metula, acerose, 8–10(–11) × 2–3 μm; conidia mostly ellipsoidal, however, that in Fasciculata most isolates identified were of P. 2–3 × 1.5–2(–2.5) μm, smooth-walled. Ascomata not observed. crustosum, while in Robsamsonia, as expected, we found the most species diversity among the sections included here. This Distinguishing characters: Talaromyces cucurbitiradicus, latter section currently comprises 11 species (Houbraken et al.

recently described by Su & Niu (2018), and T. funiculosus are 2016) and we identified five of these taxa, in addition tothe Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] the closest related species to T. pseudofuniculosus. The former new P. synnematicola. This species would seem to be a common can be distinguished from the other two by the production of coprophilous fungus in the Mediterranean area, as its isolates chlamydospores and absence of growth at 37 °C. Talaromyces were recovered exclusively from samples collected in different funiculosus differs from the new species by its divergent metulae locations from Mallorca and Catalonia. As mentioned before, (appressed in T. pseodofuniculosus) and by the pigmentation Houbraken et al. (2016) restricted the majority of coprophilous of the conidiophores, being olivaceous in T. funiculosus Penicillium species in sectionRobsamsonia . However, our results and brownish yellow in T. pseudofuniculosus. Moreover, T. clearly show that the coprophilous penicillia are distributed pseudofuniculosus has a restricted growth on CREA (2–4 mm across different and phylogenetically distant sections of the diam; 20–30 mm diam in T. funiculosus) and does not produce genus (Fig. 1). In sections such as Aspergilloides, Exilicatus funiculose colonies, a typical feature of T. funiculosus mainly on and Paradoxa, we even found a similar diversity to the section MEA and OA (Yilmaz et al. 2014). Robsamsonia. Of particular note is Paradoxa with the highest number of new penicillia proposed in this study (P. ibericum, P. fimosum and P. balearicum). However, we also identified P. DISCUSSION atramentosum and P. magnielliptisporum as first records on dung. The former species is commonly associated to cheese In recent years, the phylogeny of the genera Penicillium, and P. magnielliptisporum was described from house dust Talaromyces and other members of family Trichocomaceae (Visagie et sal. 2014a). The only species in the section reported sensu lato was studied in order to clarify their taxonomic previously from animal faeces was P. paradoxum, an aspergillus- relationships. Subsequently, many species were re-classified and like Penicillium collected in different countries such as Denmark, new taxonomic approaches proposed for species delineation and India, New Zealand, the Netherlands and UK (Houbraken et identification. Considering that several of these fungi have been al. 2014b, Rajeshkumar et al. 2016). Paradoxa is a very small discovered from specific and poorly studied substrates using section (nine species, including our new proposal) in comparison modern taxonomic techniques (Houbraken et al. 2014b, Barbosa to sectionsAspergilloides and Exillicaulis, which have 50 species et al. 2018), we explored the species diversity of penicillium-like each, but without any species reported from dung (Houbraken fungi from herbivore dung collected in different Spanish regions et al. 2014d, Visagie et al. 2016). (Andalusia, Balearic and Canary Islands, Castile and Leon, The most Talaromyces isolates found in our survey were Catalonia and Galicia) using the gene markers recommended for from section Talaromyces. This is the largest section in the their study (Visagie et al. 2014b, Yilmaz et al. 2014). genus, with nearly 60 species, 22 of them described in the last Our results agree with other studies in which a large set of four years from different substrates, including human clinical isolates of penicillium-like fungi could be identified with the tub2 specimens (Visagie et al. 2015, Crous et al. 2016, Cheng et al. sequence analysis (Guevara-Suarez et al. 2016, Visagie et al. 2016, Wang et al. 2016a, b, 2017, Yilmaz et al. 2016, Guevara- 2014a, Chen et al. 2016, Barbosa et al. 2018). It is a good marker, Suarez et al. 2017, Barbosa et al. 2018, Jiang et al. 2018). We easy to amplify and sequence, and useful for the classification at added two more new taxa in the section (T. coprophilus and the sectional and species level, and even for detection of putative T. pseudofuniculosus), but also identified several interesting undescribed species. However, in addition to the ITS and tub2 species firstly reported from dung, such asT. angelicus, T. muroii, barcodes, the characterization of penicillia requires multi-gene T. ruber, and T. sayulitensis. Unlike T. ruber, which is a world- analyses along with other markers such as fragments of cmdA and/ wide species isolated from soil, air and fuel (Yilmaz et al. 2012), or rpb2 loci. Based on this molecular approach, we were able to the other three are considered uncommon fungi previously find a remarkable diversity of penicillium-like fungi that have not found in the roots of Angelica gigas in Korea (Sang et al. 2013), been described on herbivore dung so far. Among the 104 isolates in soil in Taiwan (Chen et al. 2016), and in house dust in Mexico recovered, we identified a total of 48 species, 38 Penicillium (Visagie et al. 2014a), respectively. The other new Talaromyces including seven new taxa (i.e. P. balearicum, P. beceitense, P. species described is T. catalonicus in section Trachyspermi. This caprifimosum, P. fimosum, P. ibericum, P. mediterraneum, and is a much smaller section than Talaromyces, with 19 species P. synnematicola), seven Talaromyces including three new taxa including our new one (Yaguchi et al. 1996, Yilmaz et al. 2014, (T. catalonicus, T. coprophilus, and T. pseudofuniculosus), and Chen et al. 2016, Luo et al. 2016, Wang et al. 2016b, Guevara- three species in the newly prosposed genera Penicilago (Pgo. Suarez et al. 2017, Barbosa et al. 2018, Rajeshkumar et al. 2019). nodositata) and Pseudopenicillium (Pse. giganteum, and the The most recently described are T. aerius from indoor air in new species Pse. cervifimosum). In addition, of note is that the China (Chen et al. 2016), T. amyrossmaniae from decaying fruits highest number of species identified (34 out of 48) and novel taxa in India (Rajeshkumar et al. 2019), T. brasiliensis from honey described (9 out of 11) were recovered from samples collected in and nest of bee in Brazil (Barbosa et al. 2018), T. heiheensi from the Mediterranean area, namely Catalonia and the Balearic island rotten wood in China (Wang et al. 2017), T. minnesotensis from of Mallorca (Table 1). This is not surprising if we consider that human specimens in the USA (Guevara-Suarez et al. 2017), and this area is recognized as one of the most species rich regions in T. rubrifaciens from air outlets of public buildings also in China Southern Europe (Cuttellot et al. 2008, Rundel et al. 2018). (Luo et al. 2016), although this latter was considered a synonym

of T. albobiverticillius by Chen et al. (2016). To date, however, Frisvad JC (1981). Physiological criteria and mycotoxin production as there are no reports of species in either of these two sections aids in identification of common asymmetric penicillia.Applied and isolated from dung samples. Environmental Microbiology 41: 568–579. Penicilago nodositata seems to be a common fungus usually Frisvad JC, Samson RA (2004). Polyphasic taxonomy of Penicillium associated with the roots of Alnus in Europe (Valla 1989, subgenus Penicillium. A guide to identification of the food and Sequerra et al. 1997); it is not surprising therefore to recover air-borne terverticillate Penicillia and their mycotoxins. Studies in this fungus from animal dung, in our case from a wild pig, Mycology 49: 1–173. which feeds on a wide variety of plant material including roots, Frisvad JC, Yilmaz N, Thrane U, et al. (2013). Talaromyces atroroseus, a

Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] shoots and fruit. Conversely, Pse. giganteum is a rare species new species efficiently producing industrially relevant red pigments. only known from its type collection, a garden soil sample from PLoS ONE 8: e84102. India (Roy & Singh 1968). In addition to the latter species, we Glass NL, Donaldson GC (1995). Development of premier sets designed also recovered from herbivore dung other uncommon species for use with the PCR to amplify conserved genes from filamentous of Penicillium and Talaromyces, which are monotypic or Ascomycetes. Applied and Environmental Microbiology 61: 1323– have very few isolates available for investigation. These are P. 1330. canariense (section Stolkia), P. cremeogriseum (section Lanata- Guevara-Suarez M, Sutton DA, Cano-Lira JF, et al. (2016). Identification Divaricata), P. momoii (section Exilicaulis), and P. roseoviride and antifungal susceptibility of penicillium-like fungi from clinical (sectionAspergilloides ), including the three Talaromyces species samples in the United States. Journal of Clinical Microbiology 54: mentioned before from section Talaromyces (i.e. T. angelicus, T. 2155–2161. muroii and T. sayulitensis). Therefore, our study highlights that Guevara‐Suarez M, Sutton DA, Gené J,et al. (2017). Four new species of herbivore dung is a good substrate for recovering interesting Talaromyces from clinical sources. Mycoses 60: 651–662. species of penicillium-like fungi, although their ecological role Guindon S, Gascuel O (2003). A simple, fast and accurate method to on that substrate remains unclear and would merit further estimate large phylogenies by maximum-likelihood. Systematic investigation. Biology 52: 696–704. Herrera J, Poudel R, Khidir HH (2011). 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Wang XC, Chen K, Xia YW, et al. (2016b) A new species of Talaromyces Fig. S3. ML tree of selected Penicillium section Paradoxa species, (Trichocomaceae) from the Xisha Islands, Hainan, China. Phytotaxa inferred from tub2. Branch lengths are proportional to phylogenetic 267: 187–200. distance. Bootstrap support values/Bayesian posterior probability Wang XC, Chen K, Qin WT, et al. (2017). Talaromyces heiheensis and T. scores above 70 %/0.95 are indicated on the nodes. The tree is rooted mangshanicus, two new species from China. Mycological Progress to species of section Tubata. The names in red are the new species 16: 73–81. described in this study. T = ex-type strain. Webster J (1970). Presidential address: coprophilous fungi.Transactions of the British Mycological Society54 : 161–180. Fig. S4. ML tree of selected Penicillium section Exilicaulis species,

Editor-in-Chief Prof. dr P.W. Crous, Westerdijk Fungal Biodiversity Institute, P.O. Box 85167, 3508 AD Utrecht, The Netherlands. E-mail: [email protected] White TJ, Bruns T, Lee S, et al. (1990). Amplification and direct sequencing inferred from tub2, including the isolates belonging to this section of fungal ribosomal RNA genes for phylogenetics. In:PCR protocols: recovered in this work. Branch lengths are proportional to phylogenetic a guide to methods and applications (Innis MA, Gelfand DH, Sninsky distance. Bootstrap support values/Bayesian posterior probability JJ, White TJ, eds). Academic Press, USA: 315–322. scores above 70 %/0.95 are indicated on the nodes. The tree is rooted Yaguchi T, Someya A, Udagawa SI (1996). A reappraisal of intrageneric to P. trzebinskii CBS 382.48. T = ex-type strain. classification of Talaromyces based on the ubiquinone systems. Mycoscience 37: 55–60. Fig. S5. ML tree of selected Talaromyces section Talaromyces species, Yilmaz N, Houbraken J, Hoekstra ES, et al. (2012). Delimitation and inferred from tub2, including the isolates belonging to this section characterization of Talaromyces purpurogenus and related species. recovered in this work. Branch lengths are proportional to phylogenetic Persoonia 29: 39–54. distance. Bootstrap support values/Bayesian posterior probability Yilmaz N, López-Quintero CA, Vasco-Palacios AM, et al. (2016). Four scores above 70 %/0.95 are indicated on the nodes. The tree is rooted novel Talaromyces species isolated from leaf litter from Colombian to T. dendriticusCBS 660.80 and T. purpureus CBS 475.71. The names in Amazon rain forests. Mycological Progress 15: 1041–1056. red are the new species described in this study. T = ex-type strain. Yilmaz N, Visagie CM, Houbraken J, et al. (2014). Polyphasic taxonomy of the genus Talaromyces. Studies in Mycology 78: 175–341. Fig. S6. ML tree of selected members of Aspergillaceae family inferred from ITS, including the ex-type strain of Penicillium kabuniccum (CBS Supplementary Material: http://fuse-journal.org/ 575.90). Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores above Fig. S1. ML tree of selected Penicillium section Roquefortorum species, 70 %/0.95 are indicated on the nodes. Isolates corresponding to novel inferred from tub2. Branch lengths are proportional to phylogenetic taxa are shown in red. Between parentheses, GenBank accession distance. Bootstrap support values/Bayesian posterior probability numbers of ITS sequences. The tree is rooted to T. flavus CBS 310.38 scores above 70 %/0.95 are indicated on the nodes. The tree is rooted and T. purpureogenus CBS 286.36. T = ex-type strain. to P. sansonianum AS 3.5403 and P. osmophilum CBS 462.72. The name in red is the new species described in this study. T = ex-type strain.

P. roqueforti CBS 221.30T (AF000303) P. roqueforti MUCL 18048 (KM503654) 88/0.96 P. roqueforti CBS 47984 (AY674382) P. roqueforti CBS 304.97 (KM503657) P. roqueforti MUCL 35036 (KM503655) 99/1 P. roqueforti CBS 112579 (KM503656)

P. roqueforti CBS 498.73 (HQ442359) P. roqueforti CBS 23438 (AY674381) P. roqueforti CBS 13567 (AY674380) 98/1 89/0.98 P. roqueforti DI16-81 (LT559021) P. roqueforti DI16-80 (LT559020) P. roqueforti DI16-79 (LT5590199)

P. mediterraneum FMR 15188T sp. nov. 99/1 99 P. mediterraneum FMR 15031 P. mediterraneum FMR 15032 P. psychrosexualis CBS 128136 (HQ442357) 90/0.98 P. psychrosexualis CBS 128035 (HQ442358) P. psychrosexualis CBS 128137T (HQ442356) P. carneum CBS 44978 (AY674384) 99/1 100/1 P. carneum CBS 46695 (AY674385) P. carneum CBS 468.95 (JN097814 ) 88/0.98 P. carneum CBS 112297T (AY674386) P. carneum CBS 467.95 (HQ442352) P. paneum CBS 112296 (HQ442354)

P. paneum CBS 112294 (HQ442355) 96/1 P. paneum CBS 463.95 (HQ442353) P. paneum CBS 46595 (AY674388) 100/1 P. paneum CBS 46495 (AY674389) P. paneum CBS 109601 (KJ866977) P. paneum CBS 101032T (AY674387)

P. samsonianum AS 3.5403T (KJ668582) P. osmophilum CBS 462.72T (AY674376)

0.005 Fig. S1. ML tree of selected Penicillium section Roquefortorum species, inferred from tub2. Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores above 70 %/0.95 are indicated on the nodes. The tree is rooted to P. sansonianum AS 3.5403 and P. osmophilum CBS 462.72. The name in red is the new species described in this study. T = ex-type strain. Fig. S2

P. glandicola CBS 498.75T (AY674373) 86/-- P. glandicola NRRL 35685 (EF198565) P. glandicola CBS 49875 (AY674415) 81/-- P. glandicola ATHUM 3001 (FJ004413)

74/-- P. glandicola CBS 111218 (AY674414) 89/-- P.glandicola NG p10 (KX359621)

P. synnematicola FMR 15192T sp. nov. P. synnematicola FMR 15210 P. synnematicola FMR 15211 99/1 P. synnematicola FMR 16481 P. synnematicola FMR 16491 P. vulpinum CBS 126.23T (KJ834501)

100/1 P. dipodomyicola CBS 173.87T (AY674409) 91/-- P. griseofulvum CBS 185.27T (AY674432) T 97/1 P. brevistipitatum AS 3.6887 (DQ221695) P. coprophilum CBS 110760T (AY674421)

T 99/1 P. compactum AS3.15411 (KM973203) P. coprobium CBS 561.90T (AY674421) P. concentricum CBS 477.75T (AY674413) P. fimorum CBS 140575T (KT698889) 90/-- P. robsamsonii CBS 140573T (KT698885) P. brevicompactum CBS 257.29T (AY674437)

0.05

Fig. S2. ML tree of selected Penicillium section Robsamsonia species, inferred from tub2. Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores above 70 %/0.95 are indicated on the nodes. The tree is rooted to P. brevicompactum CBS 257.29. The name in red is the new species described in this study. T = ex-type strain. Fig. S3 P. atramentosum DTO 178G2 (KJ775095) 99/1 P atramentosum CBS 291.48T (AY674402) P. atramentosum CBS 490.84 (KJ866975) 89/-- P. atramentosum CBS 109611 (KJ866972)

P. ibericum FMR 15040T sp. nov. 92/0.96 99/1 P. ibericum FMR 15107

P. balearicum FMR 15191T sp. nov. 100/1 P. balearicum FMR 15196 P. fimosum FMR 15104T sp. nov. 99/1 P. magnielliptisporum CBS 138225T (KJ775179) 89/0.98 99/1 P. magnielliptisporum DTO 128I1 (KJ775180) --/1 P. atramentosum CBS 109588 (KJ866976) 99/1 P. mexicanum CBS 138227T (KJ775178) P. malodoratum CBS 490.65T (EF669681) P. crystallinum CBS 479.65T (EF669681) 91/0.97 P. paradoxum CBS 527.65T (EF669682) P. madriti CBS 347.61T (KJ834470) T 99/1 P. turbatum CBS 383.48 (KJ834499) T 88/0.96 P. caprifimosum FMR 15041 sp. nov. 0.02 75/-- P. bovifimosum CBS 102825T (KJ834436)

Fig. S3. ML tree of selected Penicillium section Paradoxa species, inferred from tub2. Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores above 70 %/0.95 are indicated on the nodes. The tree is rooted to species of section Tubata. The names in red are the new species described in this study. T = ex-type strain. Fig. S4

99/1 P. velutinum CBS 250.32T (JX141170) P. maclennaniae CBS 198.81T (KJ834468) P. hemitrachum CBS 139134T (JX141048) P. smithii CBS 276.83T (KJ834492) P. diabolicalicense CBS 140967T (KT887801) P. xanthomelinii CBS 139163T (JX141120) P. melinii CBS 218.30T (KJ834471) P. aotearoae KAS 3088T (KT887835) P. namyslowskii CBS 353.48T (JX141067) 97/1 P. raciborskii CBS 224.28T (JX141069) P. atrosanguineum CBS 380.75T (KJ834435) T 99/1 P. lapidosum CBS 343.48 (KJ834465) 96/0.98 P. terrenum CBS 313.67T (KJ834496) FMR 15493 86/-- T 99/1 P. burgense CBS 325.89 (KJ834437) P. atrolazulinum CBS 139136T (JX141077) 98/1 P. fagi CBS 689.77T (KJ834449) P. cravenianum CBS 139138T (JX141076) P. corylophilum CBS 312.48T (JX141042) P. pagulum CBS 139166T (JX141070) P. consobrinum CBS 139144T (JX141135) P. repensicola CBS 139160T (JX141150) P. subturcoseum CBS 139132T (JX141161) FMR 15208 P. momoii CBS 139157T (JX141073) 97/0.98 P. rubefaciens CBS 145.83T (KJ834487) 88/-- FMR 15202 90/-- FMR 15297 99/-- P. alutaceum CBS 317.67T (KJ834430) P. decumbens CBS 230.81T (KJ834446) P. citreosulfuratum IMI 92228T (KP016753) 95/-- P. fundyense CBS 140980T (KT887814) 98/0.99 P. citreonigrum CBS 258.29T (EF198621) P. cinerascens NRRL 748T (JX141041) P. restrictum CBS 367.48T (KJ834486) P. chalabudae CBS 219.66T (KP016748) P. kurssanovii CBS 625.67T (KP016758) FMR 15841 87/0.96 P. philippinense CBS 623.72T (KJ834482) P. katangense CBS 247.67T (KP016757) --/1 P. meridianum CBS 314.67T (KJ834472) P. heteromorphum CBS 226.89T (KJ834455) P. cinereoatrum CBS 222.66T (KJ834442) FMR 15033 P. arabicum CBS 414.69T (KP016750) FMR 15298 FMR 15095 P. laeve CBS 136665T (KF667365) T 78/-- P. ovatum CBS 136664 (KF667366) P. dimorphosporum CBS 456.70T (KJ834448) 95/-- P. vinaceum CBS 389.48T (HQ646575) P. nepalense CBS 203.84T (KJ834474) P. catenatum CBS 352.67T (KJ834438) P. parvum NRRL 2095T (HQ646568) T 97/0.96 P. menonorum NRRL 50410 (HQ646573) 73/-- P. erubescens NRRL 6223T (HQ646566) P. striatisporum CBS705.68T (JX141156) P. canis NRRL 62798T (KF900167) P. rubidurum NRRL 6033T (HQ646574) T 91/0.95 P. pimiteouiense NRRL 25542 (HQ646569) P. guttulosum NRRL 907T (HQ646576) P. trzebinskii CBS 382.48T (KM089034) X2 0.02

Fig. S4. ML tree of selected Penicillium section Exilicaulis species, inferred from tub2, including the isolates belonging to this section recovered in this work. Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores above 70 %/0.95 are indicated on the nodes. The tree is rooted to P. trzebinskii CBS 382.48. T = ex-type strain. Fig. S5 T. cucurbitiradicus ACCC 39153 88 T. cucurbitiradicus ACCC 39152 T. cucurbitiradicus ACCC 39155T T. pseudofuniculosus FMR 15307T sp. nov. T. pseudofuniculosus FMR 15035 T. funiculosus CABI IMI193019 95/1 T. funiculosus CBS 885.71 T. funiculosus CBS 884.70 T. funiculosus CBS 883.70 T. funiculosus CBS 171.91 T. funiculosus CBS 272.86 T. flavovirens CBS 102801T T. cnidii KACC 46617T T. siamensis CBS 475.88T T. xishaensis HMAS 248732T T. aculeatus CBS 289.48T T. apiculatus CBS 312.59T 82/1 T. dimorphus NN072337T T. fuscoviridis CBS 193.69T T 97/1 T. angelicus KACC 46611 T. angelicus FMR 15489 71/0.96 95/1 T. angelicus FMR 15490 T. pinophilus CBS 631.66T T. liani CBS 225.66T T. purgamentorum CBS 113145T 100/1 T. coprophilus FMR 15199T sp. nov. T. veerkampii CBS 500.78T T. mae NN071328T T. lentulus NN071323T T. adpressus CBS 140620T 77/-- T. sayulitensis CBS 138204T T. sayulitensis FMR 15842 T. fusiformis CBS 140637T 99/1 T. aurantiacus CBS 314.59T T. alveolaris CBS 142379T T. galapagensis CBS 751.74T T. muroii CBS 756.96T T. neofusisporus CBS 139516T 79/0.96 T. mycothecae URM 7622T T. amazonensis CBS 140373T T 84/-- T. stollii CBS 408.93 T 94/1 T. amestolkiae CBS 132696 86/1 T. ruber CBS 132704T T. beijingensis CBS140617T 94/1 T. indigoticus CBS 100534T T. rubicundus CBS 342.59T T. viridis CBS 114.72T T. derxii CBS 412.89T T. primulinus CBS 321.48T T. flavus CBS 310.38T T. kabodanensis CBS 139564T T. oumae-annae CBS 138208T T. panamensis CBS 128.89T T. rapidus CBS 142382T T. macrosporus CBS 317.63T T. dendriticus CBS 660.80T T. purpureus CBS 475.71T 0.05 Fig. S5. ML tree of selected Talaromyces sectionTalaromyces species, inferred from tub2, including the isolates belonging to this section recovered in this work. Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores above 70 %/0.95 are indicated on the nodes. The tree is rooted to T. dendriticus CBS 660.80 and T. purpureus CBS 475.71. The names in red are the new species described in this study. T = ex-type strain. Fig. S6

95/1 A. glaucus NRRL 116T ( EF652052) 92/0.98 A. montevidensis NRRL 108T (EF652077) Aspergillus A. restrictus NRRL 154T (EF652042) P. chrysogenum NRRL 807T (AF033465) 77/-- T Penicillium 100/1 P. canescens NRRL 910 (AF033493) Pen. zonata NRRL 507T (EF6697129) Pen. clavariiformis NRRL 2482T (AF033391) Penicilliopsis 93/1 Pse. giganteum NRRL 3553T (GU092966) Pse. giganteum FMR 14718 95/1 Pse. cervifimosum FMR 15299T Pseudopenicillium gen. nov. Pse. megasporum NRRL 2232T (AF033494) H. avellanea NRRL 1938 (AF454075) 84/0.97 H. paravellanea NRRL 35720 (GU092952) H. insecticola NRRL 35386 (EF634410) 73/-- H. inflata NRRL 58014 (GU092949) Hamigera 100/1 H. pallida NRRL 35718 (GU092950) H. fusca NRRL 35601 (GU092938) H. terricola NRRL 29055 (GU092946) Penicillium kabunicum CBS 575.90T (MH862240) Pgo. nodositata CBS 333.90T (NR103703) Penicillago gen. nov. 100/1 Pgo. nodositata FMR 15296 Pgo. nodositata FMR 16442 Talaromyces flavus CBS 310.38 (JN899360) Talaromyces purpureogenus CBS 286.36 (JN899372)

0.02

Fig. S6. ML tree of selected members of Aspergillaceae family inferred from ITS, including the ex-type strain of Penicillium kabuniccum (CBS 575.90). Branch lengths are proportional to phylogenetic distance. Bootstrap support values/Bayesian posterior probability scores above 70 %/0.95 are indicated on the nodes. Isolates corresponding to novel taxa are shown in red. Between parentheses, GenBank accession numbers of ITS sequences. The tree is rooted to T. flavus CBS 310.38 and T. purpureogenus CBS 286.36. T = ex-type strain.