Diversity of xerotolerant and xerophilic fungi in honey

RESEARCH Open Access Diversity of xerotolerant and xerophilic fungi in honey E. Rodríguez-Andrade 1 , A. M. Stchigel 1* , A. Terrab 2 , J. Guarro 1 and J. F. Cano-Lira 1 Abstract Fungi can colonize most of the substrata on Earth. Honey, a sugary food produced by bees (and other insects) has been studied little in terms of its fungal diversity. We have surveyed and evaluated the presence of xerotolerant and xerophilic fungi in a set of honey bee samples collected from across Spain. From 84 samples, a total of 104 fungal strains were isolated, and morphologically and phylogenetically characterized. We identified 32 species distributed across 16 genera, most of them belonging to the ascomycetous genera Aspergillus,Bettsia,Candida, Eremascus,Monascus,Oidiodendron,Penicillium,Skoua,Talaromyces and Zygosaccharomyces. As a result of this survey, eight new taxa are proposed: i.e. the new family Helicoarthrosporaceae, two new genera, Helicoarthrosporum and Strongyloarthrosporum in Onygenales; three new species of Eurotiales,Talaromyces affinitatimellis,T. basipetosporus, and T. brunneosporus; and two new species of Myxotrichaceae, Oidiodendron mellicola, and Skoua asexualis. Keywords: Eurotiales, Fungi, Honey, New taxa, Onygenales, Osmophiles, Xerophiles INTRODUCTION Honey is a natural sweetener produced by honey bees (insects of the genus Apis of the order Hymenoptera) from nectar (blossom honey or nectar honey) or from carbohydrate-rich secretions of living green parts of plants or excretions of plant-sucking phytophagous aphids (insects of the family Aphidida, order Hemiptera) (honeydew honey) after combination with the bee’s specific substances, placement, dehydration, and storage in the honey comb to ripen and mature. Honey is mostly composed of monosaccharides (dextrose and fructose), at a concentration of not lower than 60% and a much lesser amount of oligosaccharides, organic acids, enzymes (amylases and α-glucosidase) and solid particles. Due to its particular physicochemical nature and biological origin, honey should be an ideal substratum for the development of xerotolerant and xerophilic fungi. However, little information has been gathered about these fungi and their relationships with honey and honey products. Nonetheless, most of the fungal species from honey had been reported as new for science. Representative ascomycetous yeasts found in honey are Blastobotrys meliponae,Candida lundiana,C. magnoliae,C. sorbosivorans,C. suthepensis,Schizosaccharomyces octosporus,Trichosporon mucoides, Zygosaccharomyces favi,Z. mellis,Z. richteri,Z. rouxii, and Z. siamensis (Lochhead & Farrell 1931; RuizArgueso & Rodriguez-Navarro 1975; Carvalho et al. 2010; Saksinchai et al. 2012a,b;Čadežet al. 2015; Crous et al. 2016). The obligate xerophiles Ascosphaera apis and Bettsia alvei have been reported in honey, as well as several xerotolerant species of Alternaria,Aspergillus, Cladosporium and Penicillium and a few mucoralean fungi (Snowdon & Cliver 1996;Kačániová et al. 2009; Pettersson & Leong 2011;Kačániová et al. 2012; Sinacori et al. 2014; Grabowski & Klein 2015). Recently, Monascus mellicola, Penicillium apimei,P. meliponae,P. mellis, and Talaromyces brasiliensis were reported from honey produced by stingless bees (Melipona scutellaris, family Apidae, order Hymenoptera) inhabiting Brazilian forests (Barbosa et al. 2017,2018). Common environmental and plant pathogenic species of fungi have been reported in samples of honey collected in Spain (Pérez-Sánchez et al. 1997; Seijo et al. 2011; Magyar et al. 2016; Terrab et al. 2019) and Portugal (Martíns et al. 2003). In another study, the yeast Metschnikowia reukaufii was, © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: albertomigue[email protected] 1 Mycology Unit, Medical School and IISPV, Universitat Rovira i Virgili (URV), Sant Llorenç 21, 43201 Reus, Tarragona, Spain Full list of author information is available at the end of the article IM A Fun g u s Rodríguez-Andrade et al. IMA Fungus (2019) 10:20 https://doi.org/10.1186/s43008-019-0021-7 surprisingly, the only fungus reported for floral honey from Portugal and Spain (Magyar et al. 2005). Although honey should be a substratum amenable for the development of xerotolerant and xerophilic fungi, few studies have intentionally targeted these fungi. Therefore, the main objective of this study was to assess the diversity of honey-associated fungi, by employing a selective culture medium to a set of samples collected predominantly in Spain, and to characterize the morphology, physiology and phylogeny of new isolates and those considered of taxonomic interest. MATERIALS AND METHODS Fungal isolation A total of 83 samples of honeydew and blossom (nectar) honey from different locations in Spain (Fig. 1), and one from Argentina (San Martín, Buenos Aires province), have been processed. All samples were of the harvest in 2014, stored in settling tanks, and after a variable period of time clarified by filtration (with one exception, which was by centrifugation). Seventy-two of the Spanish samples corresponded to honeydew honeys, 45 from trading companies and 27 collected and processed by beekeepers. A few of the samples provided by commercial companies were categorized (according to the nature of the honeydew) as oak, holm oak and forest honey. The 11 samples of blossom honey were provided by beekeepers, and these were classified as multifloral. All samples provided by commercial companies were subjected to a thermal treatment, subjecting the honey at 45–55 °C for a few hours up to 2 days, or pasteurized (2 min at 80 °C). The samples provided by beekeepers have not undergone any heat treatment. For each sample, 10 g of honey was dissolved into 90 mL of sterile water in a sterile disposable plastic container, and 1 mL of such dilution (1:10) was aseptically plated onto two 90 mm diam. plastic Petri dishes and mixed with 15 mL of molten (at 50–55 °C) 18% glycerol agar (G18; DG18 [Hocking & Pitt 1980] without dichloran: 5 g peptone, 10 g dextrose, 1g KH 2 PO 4 , 0.5 g MgSO 4 ·7H 2 O, 15 g agar-agar, 110 g glycerol, 1 L tap water, and supplemented with 250 mg/L of L-chloramphenicol). Once the medium had solidified, one of the Petri dishes was incubated in darkness at 15 °C and the other at 25 °C for up to 2 months. The colonies developed were examined under a stereomicroscope. Fungal structures from selected (representative of Fig. 1 Locations of Spain where the samples were collected. Asturias (As), Ávila (Av), Badajoz (Ba), Burgos (Bu), Cáceres (Cac), Castellón (Cas), Ciudad Real (CR), Granada (Gra), León (Le), Ourense (Ou), Salamanca (Sa), Tarragona (Tarr), Toledo (To), Zamora (Zam), and Zaragoza (Zar) Rodríguez-Andrade et al. IMA Fungus (2019) 10:20 Page 2 of 30 all morphological variety) colonies were transferred to 50 mm diam. Petri dishes containing G18 by using a sterile insulin-type needle and incubated in the same conditions to obtain pure cultures. Phenotypic study For cultural characterization, suspensions of spores from the isolates were prepared in a semi-solid medium (0.2% agar; 0.05% Tween 80), and 0.5 μL of such suspension was inoculated onto malt extract agar (MEA; Difco, Detroit, USA; Samson et al. 2010), oatmeal agar (OA; 30 g of filtered oat flakes, 15 g agar-agar, 1 L tap water; Samson et al. 2010), Czapek yeast extract agar (CYA; 30 g sucrose, 3 g NaNO 3 , 5 g yeast extract, 1 g K 2 HPO 4 , 0.5 g KCl, 0.5 g MgSO 4 ·7H 2 O, 0.01 g FeSO 4 , 15 g agar-agar, 1 L tap water; Pitt 1979), yeast extract sucrose agar (YES; 20 g yeast extract, 150 g sucrose, 0.5 g MgSO 4 ·7H 2 O, 20 g agar-agar, 1 L tap water; Frisvad 1981), creatine sucrose agar (CREA; 3 g creatine, 30 g sucrose, 1.6 g K 3 PO 4 ·7H 2 O, 0.5 g MgSO 4 ·7H 2 O, 0.5 g KCl, 0.01 g FeSO 4 ·7H 2 O, 0.05 g bromocresol purple, 20 g agar-agar, 1 L tap water; Frisvad 1981), G18, potato dextrose agar (PDA; Pronadisa, Madrid, Spain; Hawksworth et al. 1995), 25% glycerol nitrate agar (G25 N; 7.5 g Czapek concentrate, 0.75 g K 2 HPO 4 , 3.7 g yeast extract, 250 mL glycerol, 12 g agar-agar, 1 L tap water; Pitt 1979), bromocresol purple milk solids glucose agar (BCP-MS-G; 80 g skim milk powder, 40 g glucose, 10 mL of 1.6% of bromocresol purple in 95% ethanol, 30 g agar-agar,1 L tap water; Kane & Smitka 1978), test opacity tween medium (TOTM; 10 g bacteriological peptone, 5 g NaCl, 1 g CaCl 2 , 5 mL Tween 80, 15 g agar-agar, 1 L tap water; Slifkin 2000), phytone yeast extract agar (PYE; Becton, Dickinson & Co., Sparks, MD, USA; Carmichael & Kraus 1959), malt extract yeast extract 70% fructose-glucose (MY70FG; 6 g malt extract, 6 g yeast extract, 10 g peptone, 350 g fructose, 350 g glucose, 12 g agar-agar, 1 L tap water; Beuchat & Hocking 1990), and blood agar (Becton, Dickinson & Co., Sparks, MD, USA). Colonies were characterized after three wk. at 25 °C in darkness. G18 medium was used to determine the minimum, optimal and maximum temperatures of growth. Christensen’s urea agar (EMD Millipore, Darmstadt, Germany; Christensen 1946) was inoculated and incubated during 4–7 days at 25 °C in darkness to detect the production of urease. Cycloheximide tolerance of the fungal strains was tested on Sabouraud dextrose agar (SDA; Pronadisa, Spain) supplemented with 0.2% of cycloheximide (Sigma, USA) after incubation at 30 °C for two wk. Fungal tolerance to NaCl was evaluated on SDA adding 3, 10 and 20% w/v NaCl, with the same incubation conditions as in the previous test. Colour notations were according to Kornerup & Wanscher (1978). The microscopic structures were characterized and measured from wet mountings of slide cultures, using water and 60% lactic acid. Photo micrographs were taken using a Zeiss AxioImager M1 light microscope (Oberkochen, Germany) with a DeltaPix Infinity X digital camera, using Nomarski differential interference contrast. The samples for scanning electron microscopy (SEM) were processed according to Figueras & Guarro (1988), and SEM micrographs were taken at 15 keV with a JEOL JSM 840 microscope. DNA extraction, amplification and sequencing Total deoxyribonucleic acid (DNA) was extracted according to Marimon et al. (2006), and a fragment of the 28S nrRNA gene (LSU) was amplified and sequenced using the primer pair LR0R (Rehner & Samuels 1994) and LR5 (Vilgalys & Hester 1990). For some isolates the following markers were amplified and sequenced: ribosomal internal transcribed spacers (ITS) (ITS5/ITS4; White et al. 1990); and fragments of the beta-tubulin (BenA) (Bt2a/Bt2b; Glass & Donaldson 1995), calmodulin (CaM) (Cmd5/Cmd6; Hong et al. 2005) and RNA polymerase II subunit 2 (rpb2) (RPB2-5F/RPB2-7cR; Liu et al. 1999) genes. Amplicons were sequenced at Macrogen Europe (Macrogen, Amsterdam, The Netherlands). Consensus sequences were obtained using the SeqMan software v. 7 (DNAStar Lasergene, Madison, WI, USA). Sequences we generated were deposited in GenBank (Table 1). Phylogenetic analysis A preliminary molecular identification of the isolates was carried out with LSU sequences using Basic Local Alignment Search Tool (BLAST; https://blast.ncbi. nlm.nih.gov/Blast.cgi) and only the type sequences or reliable reference strains from GenBank were considered for identification, and a maximum level of identity (MLI) of ≥98% was used for identification at the rank of species and < 98% at the rank of genus. BenA for to the genera Aspergillus,Penicillium, and Talaromyces, and ITS for the genera Monascus,Oidiodendron and Skoua were used for identification at the rank of species. An LSU tree was built to determine the phylogenetic relationships of all our isolates. Phylogenetic trees of ITS and a combination of ITSBenA-CaM-rpb2 were also built to distinguish the members of Myxotrichaceae and the genus Talaromyces, respectively. Cunninghamella bertholletiae (CBS 693.68), Mucor plumbeus (DAOM 220743), Mucor racemosus (ATCC 42647), and Rhizopus oryzae (CBS 112.07 and CBS 130146) were used as outgroup for the LSU tree; Aphanoascus keratinophilus (IMI 319010) for the Myxotrichaceae taxa tree; and Trichocoma paradoxa (CBS 247.57) for the Talaromyces tree. The sequence alignments and the maximumRodríguez-Andrade et al. IMA Fungus (2019) 10:20 Page 3 of 30 Table 1 Fungal taxa recovered with their nucleotide sequence accession number, and the geographic origin of the honey samples processed Taxon Culture collection accession number EMBL/GenBank nucleotide sequence accession number Geographic origin (province, community) BenA CaM rpb2 ITS LSU Alternaria multiformis FMR 16018 –––LT963545 LT963546 Salamanca, Castilla y León Ascosphaera atra FMR 16318 –––LT964944 LT984552 Cáceres, Extremadura Aspergillus asperescens FMR 16310 LT963510 –– LT986672 Zamora, Castilla y León Aspergillus montevidensis FMR 15994 LR027804 ––LT963466 LT984537 Castellón, Valencia Aspergillus pseudoglaucus FMR 9392 LT963512 –––LT984695 Castellón, Valencia Aspergillus pseudoglaucus FMR 15992 LT963513 –––LT984696 Castellón, Valencia Aspergillus pseudoglaucus FMR 15993 LT963514 –––LT984697 Castellón, Valencia Aspergillus pseudoglaucus FMR 16011 LT963518 –––LT984701 Salamanca, Castilla y León Aspergillus pseudoglaucus FMR 16112 LT963515 –––LT984698 Ciudad Real, Castilla-La Mancha Aspergillus pseudoglaucus FMR 16281 LT963516 –––LT984699 Ciudad Real, Castilla-La Mancha Aspergillus pseudoglaucus FMR 16317 LT963517 –––LT984700 Zamora, Castilla y León Bettsia alvei FMR 15670 ––––LT963566 Castellón, Valencia Bettsia alvei FMR 15672 ––––LT963567 Castellón, Valencia Bettsia alvei FMR 15678 ––––LT963568 Castellón, Valencia Bettsia alvei FMR 15681 ––––LT963569 Castellón, Valencia Bettsia alvei FMR 15685 ––––LT963570 Castellón, Valencia Bettsia alvei FMR 16111 ––––LT963571 Cáceres, Extremadura Bettsia alvei FMR 16115 ––––LT963572 Toledo, Castilla-La Mancha Bettsia alvei FMR 16305 ––––LT963574 Ourense, Galicia Bettsia alvei FMR 16313 ––––LT963575 Ourense, Galicia Bettsia alvei FMR 16568 ––––LT963573 Cáceres, Extremadura Bettsia alvei FMR 16570 ––––LT963576 Ourense, Galicia Candida magnoliae FMR 16311 ––––LT963487 Ourense, Galicia Candida magnoliae FMR 16314 ––––LT963488 Ourense, Galicia Candida magnoliae FMR 16496 ––––LT963486 Ourense, Galicia Candida sorbosivorans FMR 16278 ––––LT963489 Ourense, Galicia Cunninghamella bertholletiae FMR 16008 –––LT963490 LR215930 Salamanca, Castilla y León Eremascus albus FMR 16116 ––––LT964975 Cáceres, Extremadura Eremascus albus FMR 16118 ––––LT964976 Cáceres, Extremadura Eremascus albus FMR 16119 ––––LT964977 Toledo, Castilla-La Mancha Eremascus albus FMR 16493 ––––LT964978 Cáceres, Extremadura Helicoarthrosporum mellicola FMR 15673 ––––LT978462 Castellón, Valencia Helicoarthrosporum mellicola T FMR 15679 = CBS 143838 ––––LT906535 Castellón, Valencia Helicoarthrosporum mellicola FMR 16307 ––––LT978463 León, castilla y León Helicoarthrosporum mellicola FMR 16308 ––––LT906536 Zamora, Castilla y León Helicoarthrosporum mellicola FMR 16315 ––––LT906537 Cáceres, Extremadura Monascus pilosus FMR 16306 –––LT963491 LT984551 Zamora, Castilla y León Monascus purpureus FMR 16283 –––LT963492 LR215932 Ávila, Castilla y León Monascus purpureus FMR 16316 –––LT963493 LT984550 Cáceres, Extremadura Monascus purpureus FMR 16321 –––LT963494 LR215933 Cáceres, Extremadura Rodríguez-Andrade et al. IMA Fungus (2019) 10:20 Page 4 of 30 Table 1 Fungal taxa recovered with their nucleotide sequence accession number, and the geographic origin of the honey samples processed (Continued) Taxon Culture collection accession number EMBL/GenBank nucleotide sequence accession number Geographic origin (province, community) BenA CaM rpb2 ITS LSU Monascus ruber FMR 16284 –––LT963495 LT986673 Zamora, Castilla y León Mucor plumbeus FMR 16012 –––LT963539 LR215934 Ciudad Real, Castilla-La mancha Mucor plumbeus FMR 16013 –––LT963540 LT984540 Salamanca, Castilla y León Mucor plumbeus FMR 16017 –––LT963541 LT984548 Salamanca, Castilla y León Oidiodendron mellicola FMR 15680 –––LT906540 LT978465 Tarragona, Catalonia Oidiodendron mellicola T FMR 15683 = CBS 143839 –––LT906544 LT978464 Castellón, Valencia Oidiodendron mellicola FMR 16023 –––LT978506 LT978470 Salamanca, Castilla y León Oidiodendron mellicola FMR 16031 –––LT906541 LT978466 Ciudad Real, Castilla-La mancha Oidiodendron mellicola FMR 16117 –––LT978503 LT978467 Ciudad Real, Castilla-La Mancha Oidiodendron mellicola FMR 16120 –––LT978507 LT978471 Toledo, Castilla-La Mancha Oidiodendron mellicola FMR 16274 –––LT978509 LT978473 Burgos, Castilla y León Oidiodendron mellicola FMR 16282 –––LT978508 LT978472 Toledo, Castilla-La Mancha Oidiodendron mellicola FMR 16503 –––LT978504 LT978468 Ciudad Real, Castilla-La Mancha Oidiodendron mellicola FMR 16504 –––LT978505 LT978469 Ourense, Galicia Penicillium camemberti FMR 16016 LR027805 ––LT963578 LT984541 Salamanca, Castilla y León Penicillium citrinum FMR 16028 LT963451 –––LT984702 Salamanca, Castilla y León Penicillium corylophilum FMR 16010 LR027808 ––LT963581 LT984538 Asturias Penicillium corylophilum FMR 16027 LT963452 –––LT986674 Asturias Penicillium corylophilum FMR 16030 LR027809 ––LT963582 LT984547 Cáceres, Extremadura Penicillium cravenianum FMR 16019 LR027807 ––LT963580 LT984542 Salamanca, Castilla y León Penicillium cravenianum FMR 16020 LR027806 ––LT963579 LT984549 Cáceres, Extremadura Rhizopus oryzae FMR 16022 –––LT963543 LR215931 Cáceres, Extremadura Schizosaccharomyces octosporus FMR 16279 ––––LT963544 Ourense, Galicia Skoua asexualis FMR 16497 –––LT964664 LT964665 Cáceres, Extremadura Skoua asexualis FMR 16567 –––LT964666 LT964667 Cáceres, Extremadura Skoua asexualis T FMR 16572 = CBS 144072 –––LT964668 LT964669 León, castilla y León Skoua fertilis FMR 10812 LR585993 –LR586005 LR585979 LT965019 Castellón, Valencia Skoua fertilis FMR 10813 LR585994 –LR586006 LR585980 LT965023 Castellón, Valencia Skoua fertilis FMR 10814 LR585995 ––LR585981 LT965016 Castellón, Valencia Skoua fertilis FMR 10815 ––LR586007 LR585982 LT965015 Castellón, Valencia Skoua fertilis FMR 15671 LR585996 –LR586008 LR585983 LT965014 Castellón, Valencia Skoua fertilis FMR 15676 LR585997 –LR586009 LR585984 LT965017 Castellón, Valencia Skoua fertilis FMR 15682 LR585998 –LR586010 LR585985 LT965018 Castellón, Valencia Skoua fertilis FMR 15686 LR585999 –LR586011 LR585986 LT965020 Castellón, Valencia Skoua fertilis FMR 15687 LR586000 –LR586012 LR585987 LT965021 Castellón, Valencia Skoua fertilis FMR 15689 LR586001 ––LR585988 LT965022 Castellón, Valencia Skoua fertilis FMR 16032 –––LR585989 LT965024 Asturias Skoua fertilis FMR 16320 –––LR585990 LT965025 Zamora, Castilla y León Rodríguez-Andrade et al. IMA Fungus (2019) 10:20 Page 5 of 30 likelihood (ML) and Bayesian-inference (BI) phylogenetic analyses were performed as described previously (Valenzuela-Lopez et al. 2018). The final matrices used for the phylogenetic analysis were deposited in TreeBASE (www. treebase.org; accession number: S23122). Growth at different water activities (a w ) To test the capacity of growth in different water activities, media containing malt extract (1% w/w), yeast extract (0.25% w/w) and agar-agar (1% w/w) at pH 5.3 were adjusted at six different a w (0.97, 0.95, 0.93, 0.92, 0.88 and 0.82) by adding equal weights of fructose and glucose (corresponding to 22, 30, 40, 44, 48, and 55% w/ w of sugars, respectively) (Pitt & Hocking 1977). Water activity was measured in duplicate by a water activity meter (Aqualab, Decagon Devices CX3 02734) with an accuracy of ±0.002 at 25 °C. Triplicate plates were inoculated at their centre with 5 μL of spore suspension of selected fungi, and incubated at 25 °C in darkness, with the exception of FMR 15880, FMR 15883 and FMR 16031, which were at 15 °C (because of their poor growth at 25 °C). The colony diam. was measured after 21 days. RESULTS Fungal diversity All honey samples produced fungal colonies on G18 at 15 °C as well as at 25 °C. Table 1summarizes the fungal strains identified phenotypically and molecularly. With Table 1 Fungal taxa recovered with their nucleotide sequence accession number, and the geographic origin of the honey samples processed (Continued) Taxon Culture collection accession number EMBL/GenBank nucleotide sequence accession number Geographic origin (province, community) BenA CaM rpb2 ITS LSU Skoua fertilis FMR 16492 –––LR585991 LT965026 Cáceres, Extremadura Skoua fertilis FMR 16571 LR586002 –LR586013 LR585992 LT965027 Badajoz, Extremadura Strongyloarthrosporum catenulatum T FMR 16121 = CBS 143841 ––––LT906534 Toledo, Castilla-La Mancha Talaromyces affinitatimellis FMR 15674 LT965001 –––LT968852 Tarragona, Catalonia Talaromyces affinitatimellis FMR 15675 LT965002 –––LT968853 Tarragona, Catalonia Talaromyces affinitatimellis FMR 15677 LT965003 –––LT968854 Tarragona, Catalonia Talaromyces affinitatimellis FMR 15684 LT965004 –––LT968855 Castellón, Valencia Talaromyces affinitatimellis FMR 15688 LT906553 LT906550 LT906547 LT906538 LT964941 Castellón, Valencia Talaromyces affinitatimellis T FMR 15690 = CBS 143840 LT906552 LT906549 LT906546 LT906543 LT964939 Castellón, Valencia Talaromyces affinitatimellis FMR 16029 LT965005 –––LT968856 Cáceres, Extremadura Talaromyces affinitatimellis FMR 16033 LT906554 LT906551 LT906548 LT906539 LT964942 Salamanca, Castilla y León Talaromyces affinitatimellis FMR 16114 LT965006 –––LT968857 Salamanca, Castilla y León Talaromyces affinitatimellis FMR 16125 LT965009 –––LT968860 Zamora, Castilla y León Talaromyces affinitatimellis FMR 16126 LT965012 –––LT968861 Zamora, Castilla y León Talaromyces affinitatimellis FMR 16276 LT965010 –––LT968862 Zamora, Castilla y León Talaromyces affinitatimellis FMR 16494 LT965011 –––LT968863 Zamora, Castilla y León Talaromyces affinitatimellis FMR 16499 LT965007 –––LT968858 Cáceres, Extremadura Talaromyces affinitatimellis FMR 16501 LT965008 –––LT968859 Cáceres, Extremadura Talaromyces basipetosporus T FMR 9720 = CBS 143836 LT906563 –LT906545 LT906542 LT964940 Buenos Aires, Argentina Talaromyces brunneosporus T FMR 16566 = CBS 144320 LT962483 LT962488 LT962485 LT962487 LT964943 Salamanca, Castilla y León Xerochrysium xerophilum FMR 15669 –––LT986724 LT986675 Castellón, Valencia Zygosaccharomyces gambellarensis FMR 16277 ––––LT963549 Salamanca, Castilla y León Zygosaccharomyces gambellarensis FMR 16569 ––––LT963548 Cáceres, Extremadura Zygosaccharomyces mellis FMR 16280 ––––LT963550 Ourense, Galicia Zygosaccharomyces mellis FMR 16312 ––––LT963551 Ourense, Galicia Zygosaccharomyces siamensis FMR 16034 –––LT963547 LT984543 Salamanca, Castilla y León FMR = Faculty of Medicine of Reus culture collection; CBS = Westerdijk Fungal Biodiversity Institute (ex Centraalbureau voor Schimmelcultures). T = ex type Rodríguez-Andrade et al. IMA Fungus (2019) 10:20 Page 6 of 30 the exception of a few ascomycetous yeasts and of Mucorales, most of the fungi were filamentous Ascomycota. From the latter, the highest number of strains corresponded to Skoua (syn. Eremascus)fertilis,Bettsia alvei, and Oidiodendron sp., followed by an unknown arthrosporic fungus, Eremascus albus and Skoua sp. Alternaria multiformis,Ascosphaera atra, another unknown arthrospored fungus and Xerochrysium xerophilum were isolated only once. Obligate xerophilic species of Aspergillus were not found, but the xerotolerant A. pseudoglaucus, A. asperescens and A. montevidensis were isolated. Three species of Monascus were identified, i.e. M. pilosus, M. purpureus, and M. ruber.TheisolatesofPenicillium were classified as P. camemberti,P. citrinum,P. corylophilum, and P. cravenianum. Members of Talaromyces were classified at the rank of section, i.e. section Trachyspermi and section Purpurei. We only identified three species of Mucoromycota, viz. Cunninghamella bertholletiae,Mucor plumbeus, and Rhizopus oryzae. Regardless of their geographical origin, type of honey (nectar or honeydew) and if honey was or not thermally treated, S. fertilis and B. alvei were present in all honey samples. Molecular phylogeny Our first phylogenetic study included 206 LSU sequences with a total of 606 characters, including gaps, 352 of them being parsimony informative. The ML analysis was congruent with that obtained in the BI analysis, both displaying trees with similar topologies. The isolates were distributed across two main clades (Fig. 2a-c), the first (100% BS / 1 PP) corresponding to the Ascomycota and including 99 isolates, and the second (100% BS / 1 PP) involving the rest of the isolates and pertaining to the Mucoromycota. The first main clade was divided into six subclades: A (82% BS / 1 PP), which represents Onygenales; B (75% BS / 0.96 PP), Eurotiales; C (100% BS / 1 PP); Pleosporales, D (unssuported) as incertae sedis; E (100% BS / 1 PP), Schizosaccharomycetales, and F (94% BS / - PP), Saccharomycetales. Subclade A contains seven well-supported groups, six of which represent the known families of Onygenales, i.e. Gymnoascaceae (A1), Arthrodermataceae (A3), Nannizziopsiaceae (A4), Eremascaceae (A7), Ascosphaeriaceae (A8), and Spiromastigaceae (A9), and a seventh group (A5) composed of five of our strains probably representing a new family. The groups representing Ajellomycetaceae (A6) and Onygenaceae (A2) were unsupported. Strains in subclade A were distributed as follows: the five mentioned above into A5, FMR 16121 into a separate branch of the Ajellomycetaceae (A6), four strains conspecific with Eremascus albus (A7), and one (FMR 16318) identified as Ascosphaera atra (A8). Thirty-nine strains were placed in Eurotiales (Subclade B). One (FMR 16566) was placed together with Talaromyces flavus and T. kabodanensis in an unsupported branch, and 16 strains near to T. minioluteus into a well-supported sister clade (B1). Into B2 (unsupported), which includes species of Aspergillus, eight of the strains were placed in a branch (99% BS / 1 PP) together with A. glaucus, A. montevidensis and A. pseudoglaucus (sect. Aspergillus). For the final identification of these eight strains, we used BenA sequence comparison, which were found to be A. montevidensis (one strain) and A. pseudoglaucus (seven strains). FMR 16310 was placed in a branch together with the ex-type sequence of A. asperescens (sect. Nidulantes). Seven strains grouped into the sister clade B3 (unsupported), representing five species of Penicillium. FMR 15669 was identified as Xerochrysium xerophilum (B4), and five strains were initially identified as Monascus spp. Based on the comparison of ITS sequences, these five strains were finally identified as M. pilosus (one strain), M. purpureus (three strains), and M. ruber (one strain). Strain FMR 16018 was located together with Alternaria multiformis (Subclade C, Pleosporales). Subclade D (unsupported) was divided into three groups: D1, representing the Myxotrichaceae; D2, the genus Skoua; and D3, the Pseudeurotiaceae. This group had 38 strains, 10 among the genera Oidiodendron and Myxotrichum (D1), 17 together with Skoua fertilis (D2), and 11 within Bettsia alvei (D3). Subclade E (Schizosaccharomycetales), grouped FMR 16279 together with the ex-type sequence of Schizosaccharomyces octosporus. Subclade F (Saccharomycetales), had nine strains belonging to Zygosaccharomyces spp. (five strains) and Candida spp. (four strains). Clade G had 5 strains, Mucorales, divided into three groups that comprised Mucor spp. (three strains),Cunninghamella bertholletiae (FMR 16008) and Rhizopus oryzae (FMR 16022), respectively. Figures 3,4show the trees resulting from the phylogenetic analyses of Myxotrichaceae and Talaromyces, respectively. The phylogenetic tree based on the analysis of the ITS (Fig. 3), included 67 sequences belonging to Myxotrichaceae and Pseudeurotiaceae, whose alignments encompassed a total of 547 characters, including gaps, 204 of which were parsimony informative. The ML and BI analyses showed a similar tree topology. It comprised a main clade of Myxotrichaceae, where 20 strains were located, 17 of Skoua (14 identified as S. fertilis), and the remaining three in a separate branch that might represent a new species of the genus. Finally, three strains phylogenetically distant from the others appeared in a separate branch close to Myxotrichum setosum and Oidiodendron truncatum. The tree based on four concatenated loci (BenA,CaM, rpb2 and ITS; Table 2; Fig. 4) was built to resolve the phylogenetic relationships of the Talaromyces strains. The dataset contained 123 sequences with a total of 2265 characters, including gaps, (520 of them for ITS, 377 for BenA, 516 for CaM and 852 for rpb2), of which Rodríguez-Andrade et al. IMA Fungus (2019) 10:20 Page 7 of 30 Fig. 2 a-cML phylogenetic tree based on the analysis of LSU nucleotide sequences for all fungi isolated from honey. Members of Mucoromycota were chosen as out-group. Support in nodes is indicated above thick branches and is represented by posterior probabilities (BI analysis) of 0.95 and higher and/or bootstrap values (ML analysis) of 70% and higher. Fully supported branched (100% BS /1 PP) are indicated in bold. T = ex type. Alignment length 606 bp. The sequences generated by us are in Table 1 Rodríguez-Andrade et al. IMA Fungus (2019) 10:20 Page 8 of 30 Fig. 3 ML phylogenetic tree based on the analysis of ITS nucleotide sequences of representative taxa of the families Myxotrichaceae (in grey balckground) and Pseudeurotiaceae.Aphanoascus keratinophilus IMI 319010 was chosen as out-group. Support in nodes is indicated above thick branches and is represented by posterior probabilities (BI analysis) of 0.95 and higher and/or bootstrap values (ML analysis) of 70% and higher. Fully supported branched (100% BS /1 PP) are indicated in bold. T = ex type. Alignment length 544 bp Rodríguez-Andrade et al. IMA Fungus (2019) 10:20 Page 9 of 30 of a vegetative hyphae, sinuous to helical or in zig-zag, mostly simple, sometimes branched, 15–180 μm long, hyaline, disarticulating in conidia. Conidia mostly 1celled, sometimes up to 4-celled, mostly holoarthric, occasionally enteroarthric, in chains of up to 30, mostly barrel-shaped, prismatic or cuboid, sometimes triangular and “Y”-shaped, smooth-walled, thicker than the hyphae, thickener at the ends, 2–8×2–5μm, hyaline, disarticulating by schizolysis or rhexolysis from the conidiogenous hyphae. Chlamydospores produced on OA, terminally on or intercalary in the fertile hyphae hyaline, one to multicellular, smoothand thick-walled, globose, ovoid, pyriform, clavate or irregularly-shaped, truncate at the base or at both ends, to 10 μm long and 3–5μm wide. Colonies on G18 reaching 22–27 mm diam after 3 wk. at 15 °C, flat, velvety, yellowish-white (4A2), margins regular, sporulation sparse, exudate absent; reverse pale yellow (4A3), diffusible pigment absent; no growth on G18 over 35 °C; on PDA reaching 31–35 mm diam after 3 wk. at 25 °C, slightly elevated, velvety, slightly sulcate, yellowish (3A2) at the centre and white (3A1) at the edge, exudate absent; reverse reddish yellow (4A6) at the centre and pale orange (5A3) at the edge, diffusible pigment absent; on OA at 25 °C after 3 wk. very small, 7–8 mm diam, velvety, white (4A1), sporulation sparse, exudate absent; reverse pale orange (5A3), diffusible pigment absent. Minimum, optimal and maximum temperature of growth on G18 are 15 °C, 25 °C, and 30 °C, respectively; no hemolysis observed on blood agar at 25 °C, and on BCP-MS-G casein hydrolyzed without pH changes. Lipase negative, urease positive. Inhibited by cycloheximide and 20% NaCl, but tolerant to 3% and to 10% NaCl on Sabouraud dextrose agar. Other specimens examined: Spain: Valencia community: Castellón province, from decanted and filtered honey, 10 May 2014, A. Gómez Pajuelo (FMR 15673). Castilla y León community: León province, from decanted, filtered and thermally treated honey, 20 May 2014, A. Terrab (FMR 16307). Castilla y León community: Zamora province, from decanted and filtered honey, 5 Oct. 2014, A. Gómez Pajuelo (FMR 16308). Extremadura community: Cáceres province, from decanted, filtered and thermally treated honey, 16 May 2014, A. Terrab (FMR 16315). Strongyloarthrosporum Rodr.-Andr., Cano & Stchigel, gen. nov.MycoBank MB 823587. Etymology: From Greek στρογγυλός-, globose, −άρθρωση-, joint, and -σπορά, spore, referring to the morphology of the conidia. Diagnosis: Distinguished from other genera of Onygenales by the production of thick-walled globose arthroconidia, and because this fungus is an obligate xerophile. Type species: Strongyloarthrosporum catenulatum Rodr.- Andr. et al. 2019. Description:Mycelium of hyaline, septate hyphae. Conidiophores fertile lateral branches and part of the vegetative hyphae, disarticulating. Conidia enteroarthic, hyaline, mostly globose. Strongyloarthrosporum catenulatum Rodr.-Andr., Cano & Stchigel, sp. nov.Fig. 6. MycoBank MB 823588. Etymology: From Latin catenulatus, in chains, referring to the disposition of the conidia. Diagnosis:Strongyloarthrosporum catenulatum is phylogenetically close to the Ajellomycetaceae, a family of non-xerophilic fungi characterized by their thermally dimorphic nature and, consequently, pathogenic for animals. By contrast, S. catenulatum is an obligate xerophilic fungus with globose conidia sometimes disposed in chains. Type: Spain: Castilla-La Mancha community: Toledo province, from decanted, filtered and thermally treated honey, 12 May 2014, A. Terrab (CBS H23371 –holotype; CBS 143841 = FMR 16121 –ex-type cultures; LSU sequence GenBank LT906534). Description: Colonies on G18 reaching 20–21 mm diam after 3 wk. at 25 °C, elevated, velvety, sulcate, sporulation sparse, exudate absent, yellowish white (4A2) at the centre and white (3A1) at the edge; reverse orange-grey (5B2), diffusible pigment absent. Mycelium composed of hyaline, septate, smooth, thinto thick-walled, anastomosing hyphae, 1.5–4μm wide. Conidiophores reduced mostly to single fertile side branches and to the terminal part of the vegetative hyphae, 5–60 μm long, hyaline, disarticulating in conidia. Conidia hyaline, mostly onecelled, occasionally two-celled, holoand enteroarthric, solitary, disposed terminally, intercalary or sessile on the fertile hyphae, or produced in basipetal chains of up to ten conidia, smooth-walled, thicker than the hyphae, thickener at the ends, mostly globose, 3–6μm diam, flattened or not at one or both ends, disarticulating by rhexolytic secession from the conidiogenous hyphae. Chlamydospores and racquet hyphae absent. Colonies on G25 N reaching 19–20 mm diam after 3 wk. at 25 °C, elevated, velvety, sulcate, exudate absent, sporulation sparse, light orange (5A4) at the centre and grey (5B1) at the edge; reverse greyish orange (5B5), diffusible Rodríguez-Andrade et al. IMA Fungus (2019) 10:20 Page 16 of 30 pigment absent; on MY70FG reaching 29–30 mm diam after 3 wk. at 25 °C, flat, floccose, margins entire, sporulation sparse, white; reverse light yellow (4A4), diffusible pigments absent. Minimum, optimal and maximum temperature of growth on G18 are 15 °C, 25 °C, and 35 °C, respectively, does not grow on blood agar, BCP-MS-G, Sabouraud dextrose agar with different NaCl concentrations, TOTM, OA, PYE nor on Christensen’surea agar. Subclade B: Eurotiales Due to both LSU-based (Fig. 2; sister clade B1) and ITSBenA-CaM-rpb2-based (Fig. 4) phylogenetetic trees, four of our Talaromyces strains were placed in section Trachyspermi in a well-supported subclade divided in two branches, and one more strain was placed into the section Purpurei in a basal position (Fig. 4), phylogenetically distant and phenotypically different from other species of Talaromyces in this section, consequenly, we propose the recognition of three new species of the genus. Talaromyces basipetosporus Stchigel, Cano & Rodr.- Andr., sp. nov.Fig. 7. MycoBank MB 823589. Etymology: After the morphological similarity to the asexual morph of Basipetospora (formerly applied to the asexual morph of Monascus). Diagnosis: Differs from other species in sect. Trachyspermi in that the conidiogenesis is very similar to that of Monascus (syn. Basipetospora), characterized by retrogressively produced conidia, which have not been previously described in Talaromyces (see diagnosis of Talaromyces affinitatimellis). Type: Argentina: Buenos Aires province: San Martín, from decanted, filtered and thermally treated honey, 1 Oct. 2007, M. A. Álvarez (CBS H-23365 –holotype; CBS 143836 = FMR 9720 –ex-type cultures; LSU sequence GenBank LT964940). Description:Colonies on MEA reaching 10–11 mm diam after 3 wk. at 25 °C, slightly elevated, velvety to floccose, Fig. 6 Strongyloarthrosporum catenulatum CBS 143841 T .aColonies on G18, G25 N and MY70FG at 25 °C (from left to right), surface and reverse (from top to bottom). b–eConidiophores and conidia. Scale bar = 10 μm Rodríguez-Andrade et al. IMA Fungus (2019) 10:20 Page 17 of 30 margins entire, yellowish grey (4B2) at the centre and white (4A1) at the edge, exudate absent, sporulation sparse; reverse brownish red (8C8) at the centre and greyish orange (5B6) at the edge, diffusible pigments absent. Mycelium abundant, composed of subhyaline to pale brown, smooth to echinulate, thin-walled, septate, anastomosing hyphae, of 2–3μmwide.Conidiophores mostly reduced to a single conidiogenous cell, sometimes slender and with an additional conidiogenous locus near the base, arising alternately or oppositely at both sides of the vegetative hyphae, mostly separate from the vegetative hyphae by a basal septum. Conidiogenous cells smooth-walled to echinulate, mostly cylindrical and occasionally slightly slender towards the apex, sometimes broadening below the apex, but also flaskor barrel-shaped, very variable in length, 3–20(−45) × 1–2.5 μm, conidiogenesis retrogressive. Conidia one-celled, hyaline and echinulate when young, becoming brown to dark brown and nearly smooth-walled with the age, formed basipetally, in false chains of up to ten conidia, mostly globose, 3.0–5.0 μm diam. Sexual morph not observed. Colonies on DG18 reaching 13–14 mm diam after 3 wk. at 25 °C, colonies moderately elevated, texture floccose, Fig. 7 Talaromyces basipetosporus CBS 143836 T .aColonies from left to right (top row) CYA, MEA, DG18 and OA; (bottom row) CYA reverse, MEA reverse, YES and CREA. b–gConidiophores and conidia; the arrows shows the conidia formed basipetally. Scale bar = 10 μm Rodríguez-Andrade et al. IMA Fungus (2019) 10:20 Page 18 of 30 yellowish orange (4B7) with mycelium white (5A1) at edge, sporulation dense, exudate absent, diffusible pigments absent, reverse reddish golden (6C7) at centre and pale yellow (3A4) at edge; on G18 reaching 10–11 mm diam after 3 wk. at 25 °C, slightly elevated, velvety to floccose, margins regular, yellowish white (3A2), exudates uncolored, diffusible pigment absent, reverse pale orange (5A3) at the centre and white at the edge; on OA reaching 5–6 mm diam. After 3 wk. at 25 °C, flat, margins entire, mycelium grey, texture velvety to floccose, sporulation dense, diffusible pigments absent, exudate absent, colonies dark brown (5D4) at centre and grey with olive-brown (6B1-4E6) patches at edge; on PDA reaching 10–11 mm diam. After 3 wk. at 25 °C, elevated, velvety, brown (7E7) at the centre and brownish grey (4D2) at the edge, sporulation abundant, exudate absent, diffusible blackish olive (2G6) pigment present, reverse dark brown (7F4) at centre and brown (7E8) at the edge; on YES reaching 7–8 mm diam after 3 wk. at 25 °C, moderately elevated, sulcate, rough, sporulation strong, blackish brown (6G8), diffusible pigments absent, exudates absent, reverse yellowish brown (5E8). Minimum, optimal and maximum temperature of growth on G18 are 15, 25, and 30 °C, respectively; does not grow on CYA, Czapek 20% sucrose, CREA, Starch agar, or MY70FG. Talaromyces brunneosporus Rodr.-Andr., Cano & Stchigel, sp. nov. Figure 8. MycoBank MB 823590. Etymology: From Latin brunneus-, brown, and -sporarum, spore, in reference to the colour of the conidia. Diagnosis: Distinguished from other species in sect. Purpurei, with the exception of T. purpurei (the type species of the section), by the production of solitary phialides and monoverticillate conidiophores (biverticillate conidiophores in the other species of the section). However, T. brunneosporus can be differentiated from T. purpureus because lack of a sexual morph (present in the latter species), Fig. 8 Talaromyces brunneosporus CBS 144320 T .aColonies from left to right (top row) CYA, MEA, DG18 and OA; (bottom row) CYA reverse, MEA reverse, YES and CREA. b,cPoorly-developed (single phialide) and well-developed (monoverticillate) conidiophores; the arrows indicate the conspicuous collarette at the top of the phialides. dA chain of globose, dark brown, verrucose conidia. Scale bar = 10 μm Rodríguez-Andrade et al. IMA Fungus (2019) 10:20 Page 19 of 30 and produces penicillate conidiophores (having an aspergillate look in T. purpureus) and verrucose conidia (ornamented with spiral ridges in T. purpureus). Type: Spain: Castilla y León community: Salamanca province, from decanted, filtered and thermally treated honey, 1 Oct. 2014, A. Terrab (CBS H-23375 –holotype; CBS 144320 = FMR 16566 –ex-type cultures; LSU sequence GenBank LT964943). Description: Colonies on MEA reaching 13–14 mm diam after 3 wk. at 25 °C, slightly elevated, velvety to floccose, margins irregular, yellowish white (4A3), exudate absent, sporulation sparse, reverse light brown (6D8) at the centre and yellowish brown (5D6) at the edge, diffusible yellowish brown (5E6) pigment present. Mycelium abundant, composed of subhyaline, smoothand thin-walled, septate, anastomosing hyphae 2–3μm wide. Conidiophores mostly stalked, monoverticillate, smoothand thin-walled, bearing one to four conidiogenous cells at the top, frequently arising oppositely at both sides of the vegetative hyphae, sometimes reduced to a single conidiogenous cell, sessile or integrated to the vegetative hyphae (= adelophialides). Conidiogenous cells phialidic, smooth-walled, mostly slender towards the apex, flaskshaped, 8–12 × 2.5–3.5 μm, with a darkened apical area when the conidiogenous cells have produced several conidia, conidiogenesis enteroblastic. Conidia one-celled, globose, hyaline and smooth-walled when young, becoming brownish-green to dark brown and verrucose with the age, 3–4μm diam, in long false chains of up to 25 conidia. Sexual morph not observed. Colonies on CYA reaching 4–5 mm diam after 3 wk. at 25 °C, elevated, velvety, dark brown (8F4) at the centre and greyish-brown (7E3) at the edge, exudate absent, sporulation abundant, reverse dark brown (8F6) at the centre and reddish brown (8E5) at the edge, diffusible brown (6E7) pigment present; on DG18 reaching 10–11 mm diam after 3 wk. at 25 °C, moderately elevated, floccose, margins irregular, yellowish white (4A2) at the centre and olive-brown (4D6) at the edge, exudate absent, sporulation strong, reverse light brown (5D7), diffusible yellowish brown (5D5) soluble pigment present; on OA reaching 9–10 mm diam after 3 wk. at 25 °C, flat, floccose, margins entire, exudate absent, sporulation strong, colonies blackish olive (2G6) at the centre and brown (6E6) at the edge, diffusible olive brown (4E8) pigment present; on YES reaching 8–9 mm diam after 3 wk. at 25 °C, flat, floccose, black at the centre and yellowish-brown (5E6) at the edge, exudate absent, sporulation sparse, reverse dark violet (8E8), diffusible blackish brown (6G8) pigment present. Minimum, optimal and maximum temperature of growth on G18 are 15, 25, and 30 °C, respectively; no growth on CYA at 37 °C nor on CREA at 25 °C. Notes: Talaromyces brunneosporus and T. purpureus grow more slowly on CYA and MEA than other species of the section. However, T. brunneosporus produces dark brown colonies with a dark brown diffusible pigment on CYA, while the colonies of T. purpureus are pale beige and without diffusible pigments. Also, the colonies on OA and MEA are purplish in T. purpureus and pale coloured and dark brown in T. brunneosporus. Talaromyces affinitatimellis Rodr.-Andr., Stchigel & Cano, sp. nov.Fig. 9. MycoBank MB 823591. Etymology: From Latin affinitatis-, affinity, and -mellis, honey, after the substrate from which the fungus was isolated. Diagnosis: Differing from all other species in sect. Trachyspermi (with the exception of T. basipetosporus)by the production of conidia by retrogressive conidiogenesis. Talaromyces affinitatimellis differs from T. basipetosporus by the production cylindrical, smooth-walled to echinulate conidiogenous cells ending in a greenish brown, broad collarette-like structure (conidiogenous cells irregularly-shaped, smooth-walled, and without such apical structure in T. basipetosporus). Type: Spain: Valencia community: Castellón province, from decanted and filtered blossom honey, 10 May 2014, A. Gómez Pajuelo (CBS H23370 –holotype; CBS 143840 = FMR 15690 –ex-type cultures; LSU sequence GenBank LT964939). Description: Colonies on MEA reaching 29–30 mm diam. After 3 wk. at 25 °C, flat, floccose, not sulcate, margins entire, olive (3D3) at the centre and white (4A1) at edge, exudate absent, sporulation sparse; reverse pale orange (5A3) at centre and pale yellow (4A3) at edge, diffusible pigment absent. Mycelium abundant, composed of subhyaline to pale brown, smoothand thinwalled, septate, anastomosing hyphae, of 2–4μm wide. Conidiophores hyaline to pale brown, reduced to a single conidiogenous cell, occasionally with an additional conidiogenous locus near the base or lateraly disposed, or short-stalked and bearing two conidiogenous cells, sometimes with an additional lateral conidiogenous cell arising alternately at both sides of the vegetative hyphae, separate from them by a basal septum. Conidiogenous cells hyaline to pale brown, smooth-walled, mostly cylindrical and occasionally slightly slender towards the apex, sometimes ending in a greenish-brown, broad collarette-like structure, 3–20 × 1.5–3μm, conidiogenesis retrogressive but Rodríguez-Andrade et al. IMA Fungus (2019) 10:20 Page 20 of 30 enteroblastic. Conidia one-celled, hyaline and echinulate, becoming brown to dark brown and nearly smooth-walled with the age, produced basipetally in false chains of up to teninnumber,mostlyglobose,3.0–5.0 μmdiam.Sexual morph not observed. Colonies on DG18 reaching 13–14 mm diam after 3 wk. at 25 °C, moderately elevated, floccose, yellowish orange (4B7) with white (5A1) margins, exudates absent, sporulation strong; reverse reddish golden (6C7) at the centre and pale yellow (A4) at the edge, diffusible pigment absent; on G18 reaching 21–24 mm diam at 25 °C, slightly elevated, velvety to floccose, margins regular, yellowish white (4A4), exudates absent, sporulation abundant, reverse greyish orange (5B6), diffusible pigment absent; on OA reaching 12–13 mm diam after 3 wk. at 25 °C, flat, velvety to floccose, margins entire, black, exudates absent, sporulation abundant; colonies grey (7F1) at the centre and dark brown (6F4) to black at the edge, diffusible pigment absent; on PDA reaching 39–43 mm diam after 3 wk. at 25 °C, flat, velvety, margins slightly irregular, yellowishbrown (5F6) at the centre, grey (7F1) and yellowish brown (5E4) at the middle part, and light grey (5B1) at the edge, exudate absent, sporulation scarce, reverse dark brown (7F7) at the centre and brownish yellow (5C7) at the edge, diffusible pigment absent; on YES reaching 10–11 mm diam after 3 wk. at 25 °C, moderately elevated, floccose, white (4A1), exudate absent, sporulation sparse, reverse greyish orange (5B6), diffusible pigment absent. Minimum, optimal and maximum temperature of growth on G18 are 15, 25, and 35 °C, respectively; no growth on CYA, Czapek 20% or CREA, or at 40 °C on all tested media. Other specimens examined: Spain: Catalonia community: Tarragona province, from decanted and filtered blossom honey, 10 May 2014, A. Gómez Pajuelo (FMR 15674, FMR 15675, and FMR 15677); Valencia community: Castellón province, from decanted and filtered blossom honey, 10 May 2014, A. Gómez Pajuelo (FMR 15684 and FMR 15688); Extremadura community: Cáceres province, from decanted, filtered and thermally treated honeydew honey, 16 May 2014, A. Terrab (FMR 16029, FMR 16499, and FMR 16501); Castilla y León community: Salamanca province, from decanted, filtered and thermally treated Fig. 9 Talaromyces affinitatimellis CBS 143840 T .aColonies from left to right (top row) CYA, MEA, DG18 and OA; (bottom row) CYA reverse, MEA reverse, YES and CREA. b–dConidiophores and conidia; the arrows shows the conidia formed basipetally. Scale bar = 10 μm Rodríguez-Andrade et al. IMA Fungus (2019) 10:20 Page 21 of 30 honeydew honey, 01 Oct. 2014, A. Terrab (FMR 16033 and FMR 16114); Zamora province, from decanted, filtered and thermally treated honeydew honey, 05 Oct 2014, A. Terrab (FMR 16125, FMR 16126, FMR 16276, and FMR 16494). Subclade D: Incertae sedis Based on both LSU-based (Fig. 2; sister clade D1) and ITS-based (Fig. 3) phylogenetic trees, ten of our strains were located in a well-supported and separated branch related to species of the genera Oidiodendron and Myxotrichum, and phylogenetically distant from the most similar taxa included in the study, M. setosum and O. truncatum (Fig. 3). Recognition of all of these distinct strains was also supported by unique phenotypic characteristics; therefore, we propose the recognition of the new species Oidiodendron mellicola. Furthermore, because three of our strains were placed near Skoua fertilis in both LSU-based (Fig. 2; sister clade D2) and ITSbased (Fig. 3) phylogenies and because they showed different phenotypic features and enough phylogenetic distance relative to S. fertilis, we also propose the introduction of a further new species, Skoua asexualis. Oidiodendron mellicola Rodr.-Andr., Cano & Stchigel, sp. nov. Fig. 10. MycoBank MB 823586. Etymology: From Latin mellis-, honey, and -cola dwelling on, referring to the habitat. Diagnosis: Forming a terminal clade together with O. truncatum and M. setosum at a significant phylogenetic distance (5.3% from the other two species), and differing morphologically from other known species of Oidiodendron and the asexual morphs of Myxotrichum in the absence of well-differentiated conidiophores, and the slow growth. Type: Spain: Valencia community: Castellón province, from decanted and filtered blossom honey, 10 May 2014, A. Gómez Pajuelo (CBS H-23369 –holotype; CBS 143839 = FMR 15683 –ex-type cultures; ITS sequence GenBank LT906544). Description:Colonies on PDA at 15 °C reaching 15–16 mm diam after 3 wk., white (5A1), sporulation sparse Fig. 10 Oidiodendron mellicola CBS 143839 T .aColonies on PDA at 15 °C and at 25 °C, and on G18 at 25 °C (left to right), surface and reverse (from top to bottom). b–dConidiophores. eDisarticulating chains of conidia. Scale bar = 10 μm Rodríguez-Andrade et al. IMA Fungus (2019) 10:20 Page 22 of 30 (seen after 6 wk. of incubation), exudate absent, reverse orange-white (6A2) at the centre and orange-grey (6B2) at the edge, diffusible pigment absent. Mycelium composed of hyaline, septate, smoothand thin-walled hyphae, 1–3μm wide. Conidiophores reduced to fertile side branches and the terminal part of a vegetative hyphae, mostly simple or once branched near or at the base, 10– 40 μm long, pale olive, disarticulating in conidia. Conidia one-celled, mostly holoarthric, sometimes enteroarthric, mostly in chains of up to ten, occasionally solitary and sessile, mostly barrel-shaped, sometimes cylindrical, conical or “Y”-shaped, 5–14 × 2.5–5μm, pale olive, disarticulating by schizolytic or rhexolytic secession from the hyphae. Chlamydospores absent. Sexual morph absent. Colonies on PDA reaching 10–11 mm diam. After 3 wk. at 25 °C, elevated, compact, velvety, margins irregular, olive brown (4E3), exudates absent, sporulation abundant; reverse olive brown (4E5) at the center, grey (5D1) at the edge, diffusible pigment absent. Colonies on G18 reaching 11–12 mm diam after 3 wk. at 25 °C, elevated, velvety to floccose, yellowish white (4A2) at the centre and white (4A1) at the edge, margins regular, sporulation absent, reverse pale yellow (4A3), diffusible pigment absent; on G18 at 15 °C reaching 12–15 mm diam after 3 wk., similar in aspect than at 25 °C; on MY70FG and MEA 2% at 25 °C after 3 wk. reaching 1–3mm diam. Minimum, optimal and maximum temperature of growth on G18 are 5, 15, and 25 °C, respectively; no growth on OA or PCA at 25 °C. Other specimens examined: Spain: Catalonia community: Tarragona province, from decanted and filtered blossom honey, 10 May 2014, A. Gómez Pajuelo (FMR 15680); Castilla-La Mancha community, Ciudad Real province, from decanted, filtered and thermally treated honeydew honey, 10 May 2014, A. Terrab (FMR 16031, FMR 16117, and FMR 16503); Toledo province, from decanted, filtered and thermally treated honeydew honey, 12 May 2014, A. Terrab (FMR 16120 and FMR 16282); Galicia community: Ourense province, from decanted, filtered and thermally treated honeydew honey, 03 May 2014, A. Terrab (FMR 16504); Castilla y León community: Salamanca province, from decanted, filtered and thermally treated honeydew honey, 01 Oct. 2014, A. Terrab (FMR 16023); Burgos province, from decanted, filtered and thermally treated honeydew honey, 23 May 2014, A. Terrab (FMR 16274). Skoua asexualis Rodr.-Andr., Cano & Stchigel, sp. nov. Fig. 11. MycoBank MB 824092. Etymology: From Latin asexualis, without sex, because of lack of a known sexual morph. Diagnosis: Differing from the other known species of the genus, S. fertilis, in asexual reproduction, as the latter only produces ascospores within globose asci arising from the mycelium. Type: Spain: Castilla y León community: León province, from decanted, filtered and thermally treated honeydew honey, 1 Oct. 2014, A. Terrab (CBS H-23397 –holotype; CBS 144072 = FMR 16572 –ex-type cultures; ITS sequence GenBank LT964668). Description:Colonies on PDA reaching 6–7 mm diam after 3 wk. at 25 °C, elevated, velvety, sporulation abundant, exudates absent, diffusible pigment absent, colonies brown (7E6) at the centre and whitish at the edge, reverse brownish orange (6C5) at the centre and greyish orange (5B3) at the edge. Mycelium composed of hyaline, repeatedly septate, smoothand thin-walled hyphae, 2–6μm wide. Conidiophores absent. Conidia mostly one-celled, occasionally twoto three-celled, hyaline, solitary or in short chains, smoothand thick-walled, mostly globose, occasionally broadly ellipsoidal, pyriform, or irregular-shaped, truncate at one or both ends, 3–7μm diam, conidiogenesis holoblastic when sessile or terminal, and holothallic when intercalary, disarticulating by rhexolytic secession; the holoblastic and holothallic conidia produce a succession of secondary holoblastic conidia, forming a big, radiating mass of cells of up to 50 μm diam, which eventually detach as complex asexual propagules from the fertile hyphae. Chlamydospores similar to the conidia but thicker, mostly nonor occasionally one-septate, intercalary or terminal. Sexual morph unknown. Colonies on MEA reaching 3–4 mm diam after 3 wk. at 25 °C, colonies elevated, velvety to floccose, margins irregular, sporulation abundant, diffusible pigment absent, mycelium yellowish white (4A2), reverse pale yellow (4A3); on G18 reaching 4–5 mm diam after 3 wk. at 25 °C, elevated, floccose, margins irregular, sporulation sparse, diffusible pigment absent, exudates absent, colonies pale yellow (4A3) at the centre, reverse orange-grey (5B2). Minimum, optimal and maximum temperature of growth on G18 are 15, 25, and 30 °C, respectively; no growth on CYA, CREA, OA, or YES at 25 °C. Other specimens examined: Spain: Extremadura community: Cáceres province, from decanted, filtered and thermally treated honeydew honey, 16 May 2014, A. Terrab (FMR 16497 and FMR 16567). DISCUSSION This is the most comprehensive assessment of the diversity of the xerotolerant and xerophilic fungi of honey Rodríguez-Andrade et al. IMA Fungus (2019) 10:20 Page 23 of 30 intended for human consumption to date. We have isolated selectively and identified, by a polyphasic approach, six species of ascomycetous yeasts and 27 of filamentous ascomycetes, some representing new taxa, from honey samples. The yeasts, Candida magnoliae,C. sorbosivorans,Schizosaccharomyces octosporus,Zygosaccharomyces barkeri,Z.mellis, and Z. gambellarensis, had been reported from honey before, and C. magnoliae has also been associated with living honeybees (Gilliam et al. 1974b). All these yeasts have been described as osmophilic and able to grow at a w of 0.80 or lower (Tilbury 1967; van Eck et al. 1993; Ganthala et al. 1994; Erickson & McKenna 1999; Torriani et al. 2011). We found C. magnoliae and C. sorbosivorans were phylogenetically closely related (see Fig. 2), and it was reported that both differ only in a few physiological characteristics (James et al. 2001). To our knowledge, none of the species of Aspergillus that we isolated (A. asperescens,A. montevidensis, and A. pseudoglaucus) have previously been reported from honey. Aspergillus asperescens was originally isolated from soil and bat dung (Stolk 1954), but also from rotten wood and soybean seeds; however, most of the isolates were from cave soil (probably linked to bat dung). Aspergillus montevidensis and A. pseudoglaucus have been reported as the most important food-spoilage species of the genus (Pitt & Hocking 1977; Kozakiewicz 1989), but are known from extreme environments such as salterns (Butinar et al. 2005). Aspergillus montevidensis has been reported from various environmental samples (air, soil, etc.), and even on honeybees and bee larvae (http://gcm.wfcc.info/; Talice & Mackinnon 1931; Gilliam et al. 1974a); A. pseudoglaucus has been reported in air, paper and soil (http://gcm.wfcc.info/; Blochwitz 1929). Aspergillus montevidensis and A. pseudoglaucus are able to grow at a w values of 0.80 (Snow 1949; Armolik & Dickson 1956; Guynot et al. 2003). Monascus is a well-known genus with species (especially M. purpureus and M. ruber) of economic importance due to their use in production of foodstuffs, bioactive compounds, pigments and enzymes. Currently, Monascus is placed in Aspergillaceae (syn. Trichocomaceae) based on phylogenetic studies, and closely related to Leiothecium ellipsoideum and Xeromyces bisporus (Houbraken & Samson 2011; Pettersson et al. 2011). Recently, three new species Fig. 11 Skoua asexualis CBS 144072 T .aColonies on G18, MEA and PDA at 25 °C (left to right), surface and reverse (from top to bottom). b–d Conidiophores and conidia. Scale bar = 10 μm Rodríguez-Andrade et al. IMA Fungus (2019) 10:20 Page 24 of 30 were added, all of them associated with stingless bees: M. flavipigmentosus,M. mellicola, and M. recifensis (Barbosa et al. 2017). We found a small number of isolates, including M. pilosus,M. purpureus, and M. ruber. These species have been frequently reported in fermented and spoiled foods (van Tieghem 1884; Hesseltine 1965; Lin 1975; Hawksworth & Pitt 1983). Monascus ruber has also been found in soil and human clinical specimens (Hawksworth & Pitt 1983). Species of Monascus have been previously reported in honey by Snowdon & Cliver (1996) and by Barbosa et al. (2017). Monascus pilosus, M. purpureus, and M. ruber were reported previously (Hawksworth & Pitt 1983) as able to grow well on G25 N(a w = 0.93). The species of Penicillium we found in honey included P. camemberti,P. citrinum, P. corylophilum, and P. cravenianum. The most common source of isolation of P. camemberti is blue cheeses, but it can also be found on a wide variety of substrata (Thom 1906; http://gcm.wfcc.info/). Penicillium citrinum was originally reported in milk and bread in the USA (Thom 1910), but it is found globally and easy to recover from spoiled foods and diverse environmental sources (www. cabri.org/collections.html) including honey, pollen and bee nests (Barbosa et al. 2018). Penicillium corylophilum (Dierckx 1901) mostly occurs in damp buildings in North America and Western Europe, but also in foods and mosquitoes (Da Costa & De Oliveira 1998; McMullin et al. 2014), and honey (Sinacori et al. 2014). The minimum a w reported for the growth of P. camemberti, P. citrinum and P. corylophilum was around 0.80 (Abellana et al. 2001; Fontana 2008; Kalai et al. 2017). Penicillium cravenianum, a species moderately xerotolerant (grows on G25 N), has only been reported in soil (Visagie et al. 2016). Notably, all the isolates of Talaromyces that we found in honey belonged to three unrecognized species. Talaromyces basipetosporus was recovered from a honey sample in Buenos Aires province, Argentina, and is characterized by simple conidiophores that mimic those of the asexual morph of Monascus (syn. Basipetospora), which develops conidia by a retrogressive mode of conidiogenesis, a feature not previously reported in Talaromyces.Talaromyces affinitatimellis displays a similar conidiogenesis to T. basipetosporus and both species are phylogenetically closely related but phenotypically differentiated as T. affinitatimellis grows faster and produces more complex conidiophores. Talaromyces brunneosporus differs from the other species of sect. Purpurei, apart from T. purpureus, in having monophialidic and monoverticillate conidiophores (they are biverticillate in the other species). However, both species are distinguishable because T. brunneosporus produces penicillate conidiophores (not aspergillate as in T. purpureus), longer phialides, and verrucose conidia with a flattened base (T. purpureus conidia are ornamented by spiral ridges). Talaromyces basipetosporus has a high xerotolerance, with similar growth rates on MEA with sugars up to a w 0.82. Despite the decreasing growth rates of T. brunneosporus and T. affinitatimellis when sugar concentration increases, both fungi are able to grow at a w 0.82 (Fig. 12). Xerochrysium xerophilum (Pitt et al. 2013; syn. Chrysosporium xerophilum, Pitt 1966), is an extreme xerophile with a minimum a w for growth of 0.66 (Gock et al. 2003; Leong et al. 2011). This fungus, previously reported from chocolate, coconut, dried prunes, and stored corn (Pitt & Hocking 2009; Pitt et al. 2013), has not been found in honey until now. This species is phylogenetically close to Monascus (Pitt et al. 2013). Among the species of Onygenales,Ascosphaera atra and Eremascus albus were recovered once and four times, respectively. Ascosphaera atra (Skou & Hackett 1979) was originally reported from dead larvae of the alfalfa leafcutter bee covered in cysts of Ascosphaera aggregata (Skou 1975), and from pollen in the gut of healthy leafcutter larvae. This fungus was subsequently reported from grass silage (Skou 1986). Ascosphaera atra is homothallic and saprobic, probably being a common contaminant of pollen (Skou & Hackett 1979), which would explain its presence in honey samples. Eremascus albus is a well-known xerophilic fungus, with spores that can germinate at a w as low as 0.70 (Pitt 1965). This fungus has been reported to spoil malt extract (Eidam 1883), chocolate cake, dried fruits, and mustard powder (Harrold 1950), but never previously from honey. We identified several isolates belonging to the newly described family Helicoarthrosporaceae, which only includes the new monotypic genus Helicoarthrosporum, and a single strain belonging to the new monotypic genus Strongyloarthrosporum (Ajellomycetaceae). The morphology of Helicoarthrosporum mellicola resembles species of Scytalidium (S. cuboideum,S. ganodermophthorum, and S. sphaerosporum) because of the production of cuboid arthroconidia in long chains. However, Helicoarthrosporum is phylogenetically distant from Scytalidium, as the latter is related to Myxotrichaceae.Strongyloarthrosporum catenulatum was found to be phylogenetically close to Ajellomycetaceae, whose members are thermally dimorphic and pathogenic to animals (including the humans), and has never been reported as xerotolerant. However, having features not seen in that family, S. catenulatum is unequivocally a xerophilic fungus, only growing on G18, G25 N and MY70FG, and producing globose arthroconidia, either singly or in chains. The sole xerophilic fungus phylogenetically close to S. catenulatum is Eremascus albus (Eremascaceae), but it only develops a sexual morph. Regarding the family Myxotrichaceae,Skoua fertilis, which was detected in all honey samples, resembles Eremascus albus (Eidam 1883) in having naked asci arising Rodríguez-Andrade et al. IMA Fungus (2019) 10:20 Page 25 of 30