Abstract
Penicillium and Talaromyces species have a worldwide distribution and are isolated from various materials and hosts, including insects and their substrates. The aim of this study was to characterize the Penicillium and Talaromyces species obtained during a survey of honey, pollen and the inside of nests of Melipona scutellaris. A total of 100 isolates were obtained during the survey and 82% of those strains belonged to Penicillium and 18% to Talaromyces. Identification of these isolates was performed based on phenotypic characters and β-tubulin and ITS sequencing. Twenty-one species were identified in Penicillium and six in Talaromyces, including seven new species. These new species were studied in detail using a polyphasic approach combining phenotypic, molecular and extrolite data. The four new Penicillium species belong to sections Sclerotiora (Penicillium fernandesiae sp. nov., Penicillium mellis sp. nov., Penicillium meliponae sp. nov.) and Gracilenta (Penicillium apimei sp. nov.) and the three new Talaromyces species to sections Helici (Talaromyces pigmentosus sp. nov.), Talaromyces (Talaromyces mycothecae sp. nov.) and Trachyspermi (Talaromyces brasiliensis sp. nov.). The invalidly described species Penicillium echinulonalgiovense sp. nov. was also isolated during the survey and this species is validated here.
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Introduction
Stingless bees comprise a diverse group of highly eusocial insects occurring throughout the tropical regions in the world. They are important honey producers and pollinators of several plants (Ramírez et al. 2010; Brown and Oliveira 2014). An example of a stingless bee species is Melipona scutellaris (Hymenoptera: Apidae: Meliponini), an indigenous species occurring in the North-eastern part of Brazil and considered to be one of the first species to be domesticated in the Americas (Kerr 1996; Silva et al. 2013). In this part of Brazil, M. scutellaris is the main bee species in meliponiculture (stingless beekeeping). Meliponiculture in the rural areas is a sustainable activity and the honey from these bees is widely appreciated as a food source. The composition of the honey of the stingless bees differs from that of bees of the genus Apis (honey bees) (Vit et al. 2004). The honey of stingless bees contains, in comparison to honey of honey bees, a more complex mixture of carbohydrates and contains other types of organic acids, proteins, minerals, vitamins, pollen grains and enzymes (Almeida-Muradian et al. 2013). Recently, the interest in honey produced by stingless bees increased. Besides being a food source, also several other functionalities are linked to this type of honey, such as antiseptic, antimicrobial, anti-inflammatory and wound-healing properties (Silva et al. 2013; Rao et al. 2016).
Penicillium and Talaromyces are fungal genera classified in the order Eurotiales. In the dual nomenclature era (pre 2012), Talaromyces was known as a sexual genus related to Penicillium and other genera. In the last decade, the genera Talaromyces and Penicillium were re-defined due to new taxonomic insights and the introduction of single name nomenclature (Houbraken and Samson 2011; Samson et al. 2011; McNeill et al. 2012; Yilmaz et al. 2014). Currently, Penicillium and Talaromyces are separate genera that contain both sexual and asexual species. Visagie et al. (2014) accepted 354 Penicillium species and Yilmaz et al. (2014) 88 Talaromyces species, and these numbers are rapidly increasing (Houbraken et al. 2016a). Several of the new species that are being discovered are found during ecology and biodiversity studies of specific substrates or habitats (Houbraken et al. 2016a). Describing new species from poorly explored substrates and habitats, like those related to meliponiculture, will add to our knowledge on biodiversity. With this information, future studies will also be able to better understand the ecology of fungi in these type of environments.
Fungi, such as Penicillium and Talaromyces, can have a strong association to a specific substrate (Peterson et al. 2003; Kobayashi et al. 2008; Visagie 2012, Li et al. 2012; Rivera et al. 2012; Yilmaz et al. 2014). The genera Aspergillus, Penicillium, Monascus and Mucor are commonly associated with bees or their products (Egorova 1971; Gilliam et al. 1989; Eltz et al. 2002; Ferraz et al. 2008; Barbosa et al. 2017). Most fungi associated with bees and nests have a saprophytic lifestyle, but fungi can also have a mutualistic relationship with bees (Menezes et al. 2015). On the other hand, fungi are also reported to be pathogenic to many bee species and cause serious problems in honey bee (Apis mellifera) brood. Aspergillus flavus is the primary species responsible for stonebrood, a disease where dead and mummified larvae are present in the brood cells, but also other Aspergilli such as Aspergillus fumigatus and Aspergillus niger are reported as aetiological agents of this disease (Gilliam and Vandenberg 1988; Foley et al. 2014; Lopes et al. 2015; Sarwar 2016). Though it is generally accepted that infection only occurs in weakened colonies, the specific conditions predisposing the onset of disease are not fully understood (Shoreit and Bagy 1995).
Fungi play an important role in many ecosystems; however, only a limited number of studies dealt with the association between stingless bees in Brazil and filamentous fungi (e.g. Oliveira and Morato 2000; Ferraz et al. 2008; Góis et al. 2010) and yeasts (e.g. Teixeira et al. 2003; Rosa et al. 2003; Daniel et al. 2013; Barbosa et al. 2016). In the present study, we analysed three different substrates associated with M. scutellaris bees: bee pollen, nests and honey. In nature, the M. scutellaris bee nests are mainly located in tree hollows, and they are kept by beekeepers in artificial wooden hives. The bees use cerumen (a mixture of wax and floral resins) for the construction of their nests and this material is also used inside nests in storage pots, brood cells and entrance openings (Cortopassi-Laurino et al. 2006; Pianaro et al. 2007). The floral pollen is collected, packed into pollen pellets, and subsequently stored inside the nest by worker bees. This stored pollen is referred to as ‘bee bread’. The pollen spectrum has been studied in the past to get insight in the bee colony’s food requirements, pollinating functions and the plant species visited by the bees (Cortopassi-Laurino et al. 2007).
In this paper, we focus on the identification of Penicillium and Talaromyces species isolated from three different substrates (bee pollen, nests and honey) associated to M. scutellaris in the Atlantic Rainforest in Brazil. Phenotypic characters, combined with ITS and partial β-tubulin (BenA) sequences were applied to identify the isolates. Four Penicillium and three Talaromyces species could not be assigned to any known species and are described here as new. Those species are described using a polyphasic approach including morphology, ITS, BenA, calmodulin (CaM) and/or RNA polymerase II second largest subunit (RPB2) sequences and extrolites profiles.
Materials and methods
Strains
Six collections were performed between January and June 2014 in the tropical forest in Pernambuco, Brazil (8°7′30″S, 34°52′30″W and 8°4′36″S, 34°57′34″W). During each collection, four hives were sampled. Stingless bees process honey and pollen in cerumen pots. Per hive, four samples of the honey pots and four of the pollen pots were collected and combined, resulting in one mixed sample of each substrate. In the same hives, also the surface of brood cells and the pollen and honey pots were sampled using sterile cotton swabs (in total 48 swabs). Analysis of the samples was performed using dichloran 18% glycerol agar (DG18) and malt extract agar supplemented with chloramphenicol as described in Barbosa et al. (2017). The isolates were subsequently deposited in the Micoteca URM culture collection (Federal University of Pernambuco, Recife, Brazil) and ex-type strains in the CBS culture collection, housed at the Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands (under Material Transfer Agreement—MTA No. 01/2016/Micoteca URM) (Tables 1, 2). Holotype material (slide preparation) is deposited at Herbário Pe. Camille Torrend (Federal University of Pernambuco, Recife, Brazil). New species names and associated information were deposited in MycoBank.
Morphological analyses
For morphological analysis, the strains were three-point inoculated onto creatine agar (CREA), Czapek yeast extract agar (CYA), CYA supplemented with 5% NaCl (CYAS), dichloran 18% glycerol agar (DG18), malt extract agar (MEA, Oxoid), oatmeal agar (OA) and yeast extract sucrose agar (YES). All Petri dishes were incubated at 25 °C for 7 days and additional CYA and MEA plates were incubated at 15, 30 and 37 °C. Media preparation, inoculation and incubation were performed as described in Samson et al. (2010). Colony diameters were measured after 7 days of incubation and colony characteristics recorded (e.g. presence of soluble pigments, exudates, obverse and reverse colony colours, colour of mycelium). Microscopic observations of the asexual stage were made from colonies grown on MEA. The presence of a sexual stage was determined from cultures incubated on CYA, MEA and OA for at least 40 days at 25 °C. Lactic acid (60%) was used as mounting fluid and 96% ethanol was used to remove excess conidia. A Zeiss Stereo Discovery V20 dissecting microscope and a Zeiss AX10 Imager A2 light microscope, both equipped with Nikon DS-Ri2 cameras, were used to capture digital images using the software NIS-Elements D v4.50. The size, shape and pigmentation of microscopic features were recorded.
DNA isolation, PCR and sequencing
Genomic DNA extractions were made from 7 days old colonies grown on MEA using the UltraClean Microbial DNA kit (MoBio Laboratories, Solana Beach, CA, USA). Polymerase chain reaction (PCR) amplification of the ITS barcode (ITS1, 5.8S rDNA and ITS2), BenA, CaM and RPB2 gene regions were performed using methods described by Samson et al. (2010) and Houbraken et al. (2012). The PCR products were sequenced in both directions with the same primers using the BigDye® Terminator v. 3.1 Cycle Sequencing Kit (Applied Biosystems Life Technologies, Carlsbad, CA, USA) and purified with Sephadex, according to the manufacturers’ recommendations. Contigs were assembled in the SeqMan (v.10.0.1; Madison, WI, USA) program using the forward and reverse sequence. Newly generated sequences were deposited in the NCBI nucleotide database (GenBank) and the European Nucleotide Archive (Table 1).
Phylogenetic analysis
Sequence datasets were generated by combining the newly generated sequences with reference (preferably ex-type) sequences from NCBI (Visagie et al. 2014; Yilmaz et al. 2014; Taniwaki et al. 2015; Visagie et al. 2015; Chen et al. 2016; Laich and Andrade 2016; Luo et al. 2016; Romero et al. 2016; Rong et al. 2016; Visagie et al. 2016; Yilmaz et al. 2016; Guevara-Suarez et al. 2017; Wang et al. 2017a, b). The sequences were aligned using MAFFT v.7 (Katoh and Standley 2013) and manually optimized using MEGA v. 6.06 (Tamura et al. 2013). Individual alignments were concatenated by using Mesquite v. 3.04 (Maddison and Maddison 2016). The most suitable substitution model was determined using jModelTest v. 2.1.7 (Posada 2008). Phylogenetic trees were constructed using Maximum likelihood analyses (ML) using RAxML-HPC v. 8.2.8 (Stamatakis 2014) BlackBox with 1 000 rapid bootstrap inferences via the CIPRES science gateway (http://www.phylo.org/) (Miller et al 2010), while Bayesian inference (BI) analysis was performed in MrBayes 3.2.2 (Ronquist et al. 2012). In the Bayesian analyses, every 1 000 generations was sampled and the first 25% of the samples were discarded. Trees were visualized in FigTree v. 1.1.2 (Rambaut 2009) and edited in Adobe Illustrator v. 5.1. Bayesian inference (BI) posterior probabilities (pp) values and bootstrap (bs) values are labelled at the nodes. Values less than 0.95 pp and 70% bootstrap support are not shown. Branches with full support in Bayesian and RAxML analyses are thickened. Values below 0.95 pp and 70% are not shown and indicated with a hyphen. Aligned datasets and trees were uploaded to TreeBase (www.treebase.org) under submission number 21965.
Extrolite analysis
Extrolites were extracted from the Penicillium strains after growing them on CYA, YES and MEA at 25 °C for 7 days. The Talaromyces strains were inoculated on CYA, YES, MEA and OA, and incubated at 25 °C for 14 days. Three agar plugs of each medium were extracted as previously described (Smedsgaard 1997; Houbraken et al. 2012). After extraction, the liquid was transferred to a clean screw-cap vial and evaporated to dryness. The dried extracts were re-dissolved in methanol by ultrasonication and filtered through a 0.45 µm filter. The extracts were analysed by ultra-high performance liquid chromatography with diode-array detection (UHPLC-DAD) (Houbraken et al. 2012). The detected eluted compounds were identified by comparison of the retention time, retention index and the UV spectrum measured at 200–600 nm against UV spectra from made from standards and data from literature (Nielsen et al. 2011; Klitgaard et al. 2014).
Results
Isolation and identification
During this study on the fungal diversity of substrates related to stingless bees, isolates belonging to various genera [e.g. Aspergillus, Fusarium, Monascus (Barbosa et al. 2017), Penicillium, Talaromyces] were isolated. This study focusses on the identification of the detected Penicillium and Talaromyces diversity. The number of Talaromyces species (and isolates) detected during this study is low compared to Penicillium. Eighty-two Penicillium and 18 Talaromyces isolates were obtained during the survey on fungi present in honey, bee pollen and inside the nests of Melipona scutellaris bees. Phenotypic characters, combined with ITS and partial BenA sequences were used to identify isolates. In total, 21 Penicillium and six Talaromyces species were present among the investigated isolates. Among those, five Penicillium and three Talaromyces species displayed unique characters deviating from known species. Seven of those eight species are described here as new (see Taxonomy section), and one isolate (RB115), belonging to section Lanata-Divaricata, will be described elsewhere. Three new Penicillium species belong to section Sclerotiora and one to section Gracilenta; the three new Talaromyces are classified in sections Helici, Talaromyces and Trachyspermi. An overview of the species isolated during this study is given in Table 2. The highest Penicillium and Talaromyces occurrence frequency was observed in the samples collected from the inside of nests (66%). The majority of the isolated Penicillium species belonged to sections Sclerotiora (46%) and Citrina (30%). Penicillium brocae was most frequently isolated (22%), followed by Penicillium citrinum (16%), Penicillium rubens (6%) and Penicillium mellis sp. nov. (6%).
Phylogeny
The phylogenetic relationship of the new Penicillium and Talaromyces species with accepted species was determined by analysis of single and concatenated sequence datasets of three or four loci (ITS, BenA, CaM and/or RPB2). An overview of the length of each dataset and the most optimal substitution model is given Table 3. The multigene phylograms are show in the manuscript and the single gene trees in Supplementary data.
Penicillium section Gracilenta
Section Gracilenta contains four species, P. angustiporcatum, P. estinogenum, P. gracilentum and P. macrosclerotiorum. Penicillium apimei sp. nov. is in all phylogenies, with high statistical support (> 0.95 pp, > 70% bs), related to P. macrosclerotiorum (Fig. 1). ITS, BenA and CaM sequences can distinguish all species in this section.
Penicillium section Lanata-Divaricata
Isolates URM 7599, RB 217 and RB 218 cluster together in all phylograms, and always close to P. echinulonalgiovense CBS 328.59. The BenA phylogeny shows that these three isolates and P. echinulonalgiovense CBS 328.59 are related with full support to P. cataractum DAOMC 250534. The CaM, ITS, RPB2 and combined phylogenies could not resolve the phylogenetic relationship of these isolates (Fig. 2, Suppl. Figures 2, 3).
Penicillium section Sclerotiora
Isolate URM 7602T (Penicillium meliponae sp. nov.) resides in a well-supported clade with P. maximae NRRL 2060T and P. austrosinicum HMAS 248734T (ITS: 0.99 pp, 99% bs; BenA: 1.00 pp, 100% bs; CaM: 1.00 pp, 93% bs). Penicillium fernandesiae sp. nov. (URM 7600T) clusters with P. hirayamae CBS 229.60T in our ITS (< 0.95 pp, 91% bs) phylogram. Analysis of the BenA, ITS and combined dataset shows that this species belongs to a large clade containing e.g. P. sclerotiorum, P. maximae and P. hirayamae, the so-called P. sclerotiorum-clade. Isolates URM 7605T, URM 7611, RB 9, RB 69, RB 85 and RB 110 resolved in all analyses in a single, distinct, well-supported branch and are described here as P. mellis sp. nov. Analysis of BenA and ITS sequences could not resolve the phylogenetic position of P. mellis sp. nov. in section Sclerotiora. This species has, in the CaM phylogram, a basal position to a clade containing e.g. P. bilaiae, P. brocae and P. adametzioides. Penicillium mellis sp. nov. takes a basal position to P. bilaiae and related species in the phylogeny based on a combined dataset of ITS, BenA and CaM sequences (Fig. 3). This relationship is supported with a high posterior probability value (0.99), but a low bootstrap percentage (< 70%). A limited number of RPB2 sequences are available for section Sclerotiora and therefore no phylogenetic analysis was performed for this locus.
Talaromyces section Helici
Nine species are currently accepted in section Helici. The combined phylogenetic analysis (Fig. 4) revealed the presence of two well supported clades. One clade contained the species T. reverso-olivaceus, T. helicus, T. georgiensis, T. boninensis and T. varians (clade 1) and the other T. aerugineus, T. diversiformis, T. bohemicus and T. cinnabarinus (clade 2). Five strains isolated during this study clustered together in all (single gene) phylogenies and are here described as a new species named Talaromyces pigmentosus. Talaromyces pigmentosus sp. nov. clusters in clade 1 with T. reverso-olivaceus, T. helicus, T. boninensis and T. varians. The combined analysis showed, with high statistical support, that the T. pigmentosus sp. nov. isolates have a basal position to these clade 1 members (Fig. 4).
Talaromyces section Talaromyces
The phylogenetic relationship of T. mycothecae sp. nov. is difficult to determine based on the single gene phylogenies (Suppl. Figures 7, 8). In the BenA analysis, the species is close to T. neofusisporus, T. amestolkiae, T. ruber, T. stollii (0.99 pp, < 70% bs) and the species is in the CaM and RPB2 phylogenies close to T. ruber, T. amestolkiae and T. stolii, though with poor or no support (CaM < 0.95 pp, < 70% bs; RPB2 1.00 pp, < 70% bs). The phylogenetic relationship based on the BI analysis of the combined dataset indicated a relationship with T. neofusisporus, T. amestolkiae, T. ruber and T. stollii (1.00 pp); however, no statistical support in the ML analysis was found (< 70% bs) (Fig. 5).
Talaromyces section Trachyspermi
Isolates URM 7618T, URM 7619 and URM 7620 formed a clade together in all analyses. This set of isolates is described here as a new species named T. brasiliensis. The phylogenetic relationship of this species with other members of this section is unknown. The analysis of the combined dataset indicates that this species is basal to T. assistuensis, T. atroroseus, T. minioluteus, T. systylus, T. trachyspermus, T. ucrainicus and T. udagawae, but statistical support is lacking (< 0.95 pp, < 70% bs) (Fig. 6).
Extrolites
The majority of investigated Penicillium and Talaromyces species were producers of different kinds of extrolites. An overview of results is given in Table 4. Penicillium apimei sp. nov. produced spinulosin, four members of the geodin biosynthetic family (asterric acid, erdin, geodin, sulochrins) and an uncharacterized compound belonging to “biosynthetic family G”. The new species in section Sclerotiora produced sclerotiorins, patulodin (or similar) and kojic acid. Our fresh isolate of P. echinulonalgiovense (sect. Lanata-Divaricata) produced xanthoepocin and andrastin A. Talaromyces mycothecae produced duclauxin, a compound with a rubropunctatin chromophore and various extrolites also produced by other members of section Talaromyces. The new species T. pigmentosus (sect. Helici) and T. brasiliensis (sect. Trachyspermi) produced several uncharacterized extrolites that appear to be unique for the species.
Taxonomy
Penicillium apimei R.N. Barbosa, Souza-Motta, N.T. Oliveira & Houbraken sp. nov. (Figure 7)
MycoBank: MB 822208
Etymology: apimei refers to APIME, the stingless beekeeping association in Pernambuco, Brazil, which gave support for collecting samples used for this study.
Diagnosis: Penicillium apimei sp. nov. belongs to section Gracilenta and is phylogenetically unique. The species is strictly monoverticillate, grows well on MEA and CYA at 25 °C and is able to grow 37 °C.
Type: Brazil: Pernambuco: Recife, from honey of Melipona scutellaris, April 2014, R.N. Barbosa. Holotype (slide preparation) is deposited in the URM Mycology Herbarium (Recife, Brazil): URM 90489; ex-type strains URM 7591 = CBS 142502.
ITS barcode: MF278310. Alternative markers: BenA = LT854641; CaM = LT882717; RPB2 = LT854650.
Colony diam, 7 days (in mm): CYA 29–31; CYA 15 °C 10–12; CYA 30 °C 40–41; CYA 37 °C 7–9; MEA 25–27; MEA 15 °C 14–15; MEA 30 °C 39–40; MEA 37 °C 6–8; DG18 22–23; CYAS 20–23; OA 29–30; YES 38–40; CREA 22–23.
Colony characters: CYA, 25 °C, 7 days: Colonies moderately deep, radially sulcate; margins entire, low, narrow; mycelium white; colony texture velvety; sporulation moderate; conidial colour en masse greyish green; exudate clear to yellowish; soluble pigment yellow amber to brownish; reverse brown. MEA, 25 °C, 7 days: Colonies plane, slightly raised at centre, radially sulcate; margins entire, low, narrow; mycelium white sometimes inconspicuously grey; colony texture velvety to floccose; sporulation moderate to strong; conidial colour en masse greyish green; exudate present as small clear droplets; soluble pigment absent, reverse brownish. YES, 25 °C, 7 days: Colonies moderately deep, radially and concentrically sulcate; margins low, narrow, entire; mycelium white to grey; colony texture velvety; sporulation moderate to strong, conidia en masse greyish green; exudate absent; soluble pigment yellow; reverse yellow to brownish elsewhere. DG18, 25 °C, 7 days: Colonies plane, raised at centre; margins low, entire; mycelium white; colony texture velvety; sporulation moderate; conidial colour en masse greyish green; exudate absent; soluble pigment absent; reverse yellow, sometimes inconspicuously greenish. OA, 25 °C, 7 days: Colonies flat, entire; margins regular; mycelium white to inconspicuously yellow; colony texture velvety; sporulation sparse; conidial colour en masse greyish green; exudate absent; soluble pigment yellow; reverse yellowish to cream. CYAS 25 °C, 7 days: Colonies plane, raised at centre, radially and concentrically sulcate; margins low, narrow, entire; mycelium white; colony texture velvety; sporulation sparse, conidial colour en masse greyish; exudate absent; soluble pigment brownish; reverse brown. CREA, 25 °C, 7 days: good growth, acid production absent.
Micromorphology: Conidiophores strictly monoverticillate. Stipes smooth walled, 25–90 × 1–4.5 μm, vesiculate, up to 4 µm in diam. Phialides 4–10 per stipe, ampulliform, 6.5–9.5 × 2.0–3.0 μm. Conidia smooth walled, globose, 2.0–3.0 × 2.0–3.0 μm. Sclerotia or ascomata not observed.
Notes: Houbraken and Samson (2011) did not report any significant similarities shared between species belonging to section Gracilenta, except that all species weren’t able to grow at 37 °C and had brown reverses on Czapek agar or CYA. The reverse colony colour of P. apimei on CYA and YES is also in shades of brown, but the species is unique in for its ability to grow at 37 °C. This species is phylogenetically most closely related to P. macrosclerotiorum. Besides its ability to grow at 37 °C, it can further be differentiated from this species by the absence of sclerotia and slower growth on YES (38–40 vs 54–56 mm).
Penicillium echinulonalgiovense S. Abe ex Houbraken & R.N. Barbosa sp. nov.
MycoBank: MB822213
= Penicillium echinulonalgiovense S. Abe, Journal of General and Applied Microbiology 2: 80. 1956. [MB536546]. (nom. inval., Art. 39.1.).
Diagnosis: Penicillium echinulonalgiovense sp. nov. is phylogenetically unique. Colonies on CYA incubated at 25 °C for 7 days attain a diameter of 33–37 mm and on CYA 37 °C 8–12 mm. The growth on CREA is weak, the colony diameter 23–27 mm, and no acid compounds are produced. The conidiophore stipes are rough walled, and conidia are globose to subglobose and echinulate.
Type: Japan: unrecorded source, S. Abe. Holotype: CBS H–23172; ex-type strains CBS 328.59 = ATCC 18314 = FAT 907 = FRR 638 = IFO 6229 = IMI 068213 = QM 7301.
ITS barcode: GU981587. Alternative markers: BenA = GU981631; CaM = KX961269; RPB2 = KX961301.
Additional material examined. Australia, Atherton Tableland, Queensland, soil, R. van Leeuwen & J. Houbraken, DTO 030-D8; China, Hong Kong, soil, isol. by W. Gams & A. Aptroot, CBS 102417; Indonesia, Yogyakarta, storage room, DTO 232-C6; Netherlands, industrial installation, J. Houbraken, CBS 115322; Madagascar, Ifaty, forest soil, coll. F. Hagen, isol. J. Houbraken, DTO 088-A2; Malaysia, Langkawi, soil of rainforest, coll. R.A. Samson, isol. J. Houbraken, DTO 054-A1; USA, Florida, soil from citrus grove, R.A. Samson, DTO 010-A5. Brazil, Bee pollen of Melipona scutellaris URM 7599; inside of nests of Melipona scutellaris RB 217; RB 218 coll. R.N.Barbosa.
Notes: Penicillium echinulonalgiovense was described without a Latin diagnosis. To validate the species, an English diagnosis is given above, with the name of the original author maintained. The ITS and partial BenA and CaM sequence data had sufficient discriminatory power to differentiate P. echinulonalgiovense (CBS 328.59T) from P. simplicissimum and other described species in section Lanata-Divaricata. In the BenA analysis (Suppl. Figure 2), P. echinulonalgiovense is related to P. cataractum DAOMC 250534T and P. mariae-crucis (CBS 271.83T). Penicillium echinulonalgiovense can be differentiated from those species by its ability to grow on CYA incubated at 37 °C (8–12 mm). Furthermore, P. cataractum grows moderately well on CREA and produces high levels of acid compounds on this medium. Both P. echinulonalgiovense and P. mariae-crucis grow poorly on CREA and do not produce acid compounds. In addition, the reverse colours on CYA differ. The reverse colour of P. echinulonalgiovense on CYA is dark brown in the centre and beige towards the margins, the reverse colour of P. mariae-crucis is blackish brown and those of P. cataractum greyish yellow to greyish orange (Visagie et al. 2016).
Penicillium fernandesiae R.N. Barbosa, Souza-Motta, N.T. Oliveira & Houbraken sp. nov. (Figure 8)
MycoBank: MB822209
Etymology: Named in honour of prof. Maria José Fernandes, mycologist working with Aspergillus and Penicillium in the former Institute of Mycology of the University of Recife (IMUR), Pernambuco, Brazil.
Diagnosis: Red soluble pigments produced on CYA, no growth on MEA and CYA at 37 °C, restricted growth on CYA, MEA, YES, CYAS and no acid production on CREA.
Type: Brazil: Pernambuco: Recife, inside nests of Melipona scutellaris, May 2014, R.N. Barbosa. Holotype (slide preparation) is deposited in the URM Mycology Herbarium (Recife, Brazil): URM 90490; ex-type strains URM 7600 = CBS 142500.
ITS barcode: MF278314. Alternative markers: BenA = LT854645; CaM = LT854649; RPB2 = LT854654.
Colony diam, 7 days (in mm): CYA 15–18; CYA15 °C 5–6; CYA30 °C 20–22; CYA37 °C no growth; MEA 15–17; MEA 15 °C 4–5; MEA 30 °C 20–22; MEA 37 °C no growth; DG18 17–18; CYAS 15–17; OA 6–8; YES 21–22; CREA 3–4.
Colony characters: CYA, 25 °C, 7 days: Colonies moderately deep, gently radially sulcate; margins low, undulate, entire; mycelium yellow; colony texture velvety to floccose; sporulation absent; conidial colour en masse indeterminable; exudate orange; soluble pigment in shades of red; reverse orange to brownish at centre. MEA, 25 °C, 7 days: Colonies convex; margins low, narrow, entire; mycelium white, sometimes inconspicuously yellow; colony texture floccose; sporulation absent; conidial colour en masse indeterminable; exudate clear at centre and sometimes orange close the margins; soluble pigment absent; reverse orange. YES, 25 °C, 7 days: Colonies moderately deep, radially and concentrically sulcate; margins low, narrow, entire; mycelium white to slightly inconspicuously yellow; colony texture floccose; sporulation absent; conidial colour en masse indeterminable; exudate orange; soluble pigment absent; reverse brownish to orange. DG18, 25 °C, 7 days: Colonies moderately deep, gently radially sulcate; margins low, narrow, entire; mycelium yellow, texture velvety to floccose; sporulation absent; conidial colour en masse indeterminable; exudate orange; soluble pigment absent; reverse orange. OA, 25 °C, 7 days: Colonies flat, margins irregular; mycelium yellow; sporulation absent, conidial colour en masse indeterminable; exudate clear orange; soluble pigment absent; reverse orange. CYAS 25 °C, 7 days: Colonies moderately deep, radially and concentrically sulcate; margins low, narrow, entire; mycelium white; colony texture floccose; sporulation absent; conidial colour en masse indeterminable; exudate orange; soluble pigment absent; reverse brownish orange. CREA, 25 °C, 7 days: Very weak growth, acid production absent.
Micromorphology: Conidiophores strictly monoverticillate. Stipes smooth walled, 7.5–20 × 1.5–2.0 μm, non-vesiculate. Phialides 4–7 per stipe, ampulliform, 6–11 × 2.0–3.0 μm. Conidia smooth walled, globose, 2–3 μm. Sclerotia or ascomata not observed.
Notes: Penicillium fernandesiae sp. nov. belongs to the P. sclerotiorum-clade. The species produces sclerotiorins and these compounds are shared with P. hirayamae, P. meliponae and P. sclerotiorum and other species in the P. sclerotiorum-clade, which is in line with its phylogenetic placement. Penicillium fernandesiae produces red soluble pigments on CYA and these are not produced by the closely related species P. hirayamae. Red soluble pigment production is shared with P. adametzioides, a phylogenetically distant species (Visagie et al. 2013).
Penicillium meliponae R.N. Barbosa, Souza-Motta, N.T. Oliveira & Houbraken sp. nov. (Figure 9)
MycoBank: MB822210
Etymology: meliponae, refers to Melipona scutellaris, the stingless bee species investigated in this study.
Diagnosis: Penicillium meliponae sp. nov. have colony diameter on CYA, MEA, DG18, CYAS and CREA generally below to 32 mm. The species grows moderately well on CREA and has a strong acid production.
Type: Brazil: Pernambuco: Recife, honey of Melipona scutellaris, June 2014, R.N. Barbosa. Holotype (slide preparation) is deposited in the URM Mycology Herbarium (Recife, Brazil): URM 90491; ex-type strains URM 7602 = CBS 142495.
ITS barcode: MF278315. Alternative markers: BenA = LT854644; CaM = LT854648; RPB2 = LT854653.
Colony diam, 7 days (in mm): CYA 30–32; CYA15 °C 15–16; CYA30 °C 25–28; CYA37 °C no growth; MEA 30–31; MEA 15 °C 9–10; MEA 30 °C 24–25; MEA 37 °C no growth; DG18 25–26; CYAS 23–25; OA 26–28; YES 40–43; CREA 17–18.
Colony characters: CYA, 25 °C, 7 days: Colonies radially sulcate, slightly raised at centre; margins low, narrow, entire; mycelium white; colony texture floccose; sporulation absent at margin, strong in centre; conidial colour en masse greyish green; exudate orange; soluble pigment orange; reverse brown at centre, orange at the margins and yellow at the borders. MEA, 25 °C, 7 days: Colonies plane, moderately deep, lightly radially sulcate; margins entire, low, narrow, entire; mycelium white and slightly orange; colony texture floccose at centre somewhat velvety in some areas close the margins; sporulation strong in centre, weak at margins; conidial colour en masse greyish; exudate hyaline to pale orange; soluble pigment absent; reverse dull orange. YES, 25 °C, 7 days: Colonies moderately deep, raised at centre, randomly sulcate; margins low, narrow, entire; mycelium white, sometimes inconspicuously orange; colony texture floccose; sporulation sparse; conidial colour en masse greyish in some areas; exudate orange; soluble pigment absent; reverse reddish brown at centre fading to orange close to margin and yellow in the borders. DG18, 25 °C, 7 days: Colonies moderately deep, lightly sulcate; margins entire; mycelium white; colony texture floccose to velvety; sporulation sparse; conidial colour en masse indeterminable; exudate orange; soluble pigment absent; reverse orange at centre to yellow in the margins. OA, 25 °C, 7 days: Colonies plane, not sulcate; margins entire; mycelium yellow, sometimes white; colony texture velvety, sporulation sparse, conidial colour somewhat greyish, exudate orange, in small droplets; soluble pigment absent; reverse orange at centre to yellow at the margins. CYAS, 25 °C, 7 days: Colonies slightly raised, radially and concentrically sulcate; margins low, narrow, entire; mycelium white; colony texture floccose; sporulation absent to moderate at centre; conidial colour en masse greyish; exudate orange, soluble pigment absent; reverse reddish brown at centre fading to orange close to margin and yellow at the borders. CREA, 25 °C, 7 days: Moderate growth; acid produced.
Micromorphology: Conidiophores strictly monoverticillate. Stipes smooth walled 22.0–45 × 2.5–3.5 μm, vesiculate 4.0–6.5 μm. Phialides 4–12 per stipe, ampulliform, 6.0–9.0 × 2.5–4.0 μm. Conidia smooth walled, subglobose, 2.0–3.0 μm. Sclerotia not observed produced.
Notes: Penicillium meliponae sp. nov. is phylogenetically most closely related to P. maximae and P. austrosinicum. Penicillium meliponae sp. nov. produces smaller colonies on CYA, MEA, CYAS and CREA after 7 days incubation at 25 °C than P. austrosinicum and P. maximae. Furthermore, P. meliponae sp. nov. has a strong acid production on CREA, while P. maximae lacks acid production on CREA (Visagie et al. 2013). Penicillium austrosinicum produces subglobose, rough walled conidia, the conidia of P. meliponae sp. nov. are subglobose and smooth and those of P. maximae are ellipsoidal and smooth. Additionally, P. meliponae sp. nov. and P. maximae do not produce sclerotia, while P. austrosinicum does (Wang et al. 2017a).
Penicillium mellis R.N. Barbosa, Souza-Motta, N.T. Oliveira & Houbraken sp. nov. (Figure 10)
MycoBank: MB822211
Etymology: the species name refers to honey, the substrate from which the type species was isolated.
Diagnosis: This species is phylogenetically distinct from other sect. Sclerotiora members. The conidiophores are monoverticillate, stipes vesiculate, pale to brownish coloured sclerotia are produced and no acid production on CREA is observed.
Type: Brazil: Pernambuco: Recife, honey of Melipona scutellaris, May 2014, R.N. Barbosa. Holotype (slide preparation) is deposited in the URM Mycology Herbarium (Recife, Brazil): URM 90492; ex-type strain: URM 7605 = CBS 142499.
ITS barcode: MF278316. Alternative markers: BenA = LT854643; CaM = LT854647; RPB2 = LT854652.
Colony diam, 7 days (in mm): CYA 29–30; CYA15 °C 7–8; CYA30 °C 33–35; CYA37 °C 2–4; MEA 28–30; MEA 15 °C 9–10; MEA 30 °C 33–35; MEA 37 °C no growth; DG18 24–25; CYAS 26–27; OA 24–25; YES 34–36; CREA 10–11.
Colony characters: CYA, 25 °C, 7 days: Colony radially sulcate; margin entire, low; mycelium white sometimes inconspicuously green; colony texture velvety; sporulation absent at centre and sparse at margins; conidial colour en masse greyish green; exudates present as clear droplets; soluble pigment absent; reverse brown to pale. MEA, 25 °C, 7 days: Colonies low, plane; margins low, wide, entire; mycelium white; colony texture velvety; sporulation sparse, conidial colour en masse greyish green; sclerotia produced, inconspicuously brown, exudate present as clear droplets; soluble pigment absent; reverse cream. YES, 25 °C, 7 days: Colonies moderately deep, radially and concentrically sulcate; margins low, narrow, entire; mycelium white, inconspicuously grey; colony texture velvety; sporulation sparse to absent, conidial colour en masse indeterminable; exudate absent; soluble pigment absent; reverse pale yellow. DG18, 25 °C, 7 days: Colonies plane; margins low, entire; mycelium white; colony texture velvety; sporulation strong; conidial colour en masse greyish green; exudate absent; soluble pigment absent; reverse pale. OA, 25 °C, 7 days: Colonies flat, margins regular; mycelium white; colony texture velvety, sporulation dense, conidial colour en masse greyish; exudate present as clear droplets; soluble pigment absent; reverse white to pale. CYAS 25 °C, 7 days: Colonies radially and concentrically sulcate; margins low, entire; mycelium white; colony texture velvety; sporulation moderate to strong; conidial colour en masse greyish green; exudate absent; soluble pigment absent; reverse brownish. CREA, 25 °C, 7 days: moderate growth, no acid production.
Micromorphology: Conidiophores strictly monoverticillate. Stipes smooth walled, 25–40 × 2.0–3.5 μm, vesicilate 4.0–5.0 μm. Phialides 5–12 per stipe, ampulliform, 6.5–9.0 × 2.0–3.0 μm. Conidia smooth walled, globose to subglobose, 2.0–3.0 μm. Sclerotia present, 150–250 µm.
Additional material examined. Brazil, Pernambuco, Recife, Inside nest of Melipona scutellaris, R.N. Barbosa, URM 7611; RB 9; RB 85; RB 110; honey of Melipona scutellaris, R.N. Barbosa, RB 69.
Notes: Penicillium mellis sp. nov. is phylogenetically unique. It can be distinguished from other members in section Sclerotiora by its ability to produce pale to brownish coloured sclerotia on MEA, CYA and OA.
Talaromyces brasiliensis R.N. Barbosa, Souza-Motta, N.T. Oliveira & Houbraken sp. nov. (Figure 11)
MycoBank: MB822214
Etymology: Named after Brazil, the country of origin of the type strain.
Diagnosis: Talaromyces brasiliensis sp. nov. is phylogenetically unique. This species grows restricted on CYA and MEA at 25 °C and growth is absent to poor at 37 °C. The phialides of T. brasiliensis are ampulliform and the conidia globose and finely roughened.
Type: Brazil: Pernambuco: Recife, honey of Melipona scutellaris, June 2014, R.N. Barbosa. Holotype (slide preparation) is deposited in the URM Mycology Herbarium (Recife, Brazil): URM 90494; ex-type strains URM 7618 = CBS 142493.
ITS barcode: MF278323. Alternative markers: BenA = LT855560; CaM = LT855563; RPB2 = LT855566.
Colony diam, 7 days (in mm): CYA 5–6; CYA15 °C 3–4; CYA30 °C 5–6; CYA37 °C no growth; MEA 14–15; MEA 15 °C 6–7; MEA 30 °C 14–15; MEA 37 °C 4–5; DG18 10–11; CYAS no growth; OA 12–13; YES 6–8; CREA no growth.
Colony characters: CYA, 25 °C, 7 days: Colonies plane; margins entire; mycelium white; colony texture loosely floccose; sporulation poor; conidia en masse greyish green; exudates absent; soluble pigments absent; reverse cream to brownish. MEA, 25 °C, 7 days: Colonies plane; margins entire; mycelium white; colony texture loosely funiculose to floccose; sporulation strong; conidia en masse greyish; exudates absent; soluble pigments absent; reverse cream to yellow. YES, 25 °C, 7 days: Colonies loosely deep; margins entire; mycelium white; colony texture floccose; sporulation absent; conidia en masse indeterminable; exudates absent; soluble pigments absent; reverse cream to yellow. DG18, 25 °C, 7 days: Colonies raised at centre; margins entire, deep; mycelium white, occasionally inconspicuously grey; colony texture floccose; sporulation poor at centre, conidia en masse greyish; exudates absent; soluble pigments absent; reverse brown to pale. OA, 25 °C, 7 days: Colonies plane; margins entire; mycelium white, occasionally light yellow; colony texture velvety; sporulation strong at centre, week at margin; conidia en masse dull green; exudates present as small hyaline droplets; soluble pigments absent; reverse white to inconspicuously black. CREA 25 °C, 7 days: no growth.
Micromorphology: Conidiophores biverticillate, stipes smooth walled, 20–50 × 2.5–4 μm. Metulae 5–6, 8–11 × 2.5–3.5 μm. Phialides 3–4 per stipe, ampulliform tapering to very fine necks, 7–11 (–14) × 2.0–3 μm; conidia globose, finely roughened, 2–3 μm. Ascomata not observed.
Additional material examined. Brazil, Pernambuco, Recife, Inside nest of Melipona scutellaris, R.N. Barbosa, URM 7619; URM 7620.
Notes: Section Trachyspermi comprise species that normally grow slowly on CYA and slightly faster on MEA. Talaromyces brasiliensis sp. nov. also grows restricted on CYA (5–6 mm) and better on MEA (14–15 mm), confirming the phylogenetic results. Talaromyces brasiliensis sp. nov. is phylogenetically distinct (Fig. 6).
Talaromyces mycothecae R.N. Barbosa, Souza-Motta, N.T. Oliveira & Houbraken sp. nov. (Figure 12)
MycoBank: MB822215
Etymology: In honour of Micoteca URM (URM, University Recife Mycology), an important Latin-American Fungal Culture Collection founded by mycologist Augusto Chaves Batista.
Diagnosis: The reverse colour on MEA and OA is wine red. The species produces red coloured exudate droplets on YES and no acid compounds are produced on CREA. Furthermore, T. mycothecae sp. nov. grows well on CYA 37 °C and produces smooth walled, fusiform to ellipsoidal shaped conidia.
Type: Brazil: Pernambuco: Recife, inside nests of Melipona scutellaris, Feb 2014, R.N. Barbosa. Holotype (slide preparation) is deposited in the URM Mycology Herbarium (Recife, Brazil): URM 90495; ex-type strains URM 7622 = CBS 142494.
ITS barcode: MF278326. Alternative markers: BenA = LT855561; CaM = LT855564; RPB2 = LT855567.
Colony diam, 7 days (in mm): CYA 20–23; CYA15 °C 2–5; CYA 30 °C 28–30; CYA 37 °C 18–20; MEA 29–30; MEA 15 °C 3–6; MEA 30 °C 38–40; MEA 37 °C 20–22; DG18 10–12; CYAS no growth; OA 24–25; YES 25–26; CREA 4–5.
Colony characters: CYA, 25 °C, 7 days: Colonies plane, margins entire; mycelium white occasionally inconspicuously yellow; colony texture velvety to floccose; sporulation strong, conidia en masse greyish to dull green; exudates present as small clear droplets; soluble pigments absent; reverse yellow amber to dark brown at centre. MEA, 25 °C, 7 days: Colonies plane; margin entire, mycelium white; colony texture velvety; sporulation strong; conidia en masse greyish; exudates absent; soluble pigments absent; reverse yellow amber to wine-reddish. YES, 25 °C, 7 days: Colonies crateriform; margins entire; mycelium white; colony texture floccose; sporulation strong; conidia en masse greyish to dull green; exudates present as small red droplets; soluble pigments absent; reverse red near margins to wine-reddish in centre. DG18, 25 °C, 7 days: Colonies plane; margins entire; mycelium white; colony texture floccose; sporulation sparse; conidia en masse green; exudates present as small red droplets; soluble pigments absent; reverse cream at margins to reddish at centre. OA, 25 °C, 7 days: Colonies plane; margins low; mycelium white occasionally inconspicuously greenish; colony texture velvety to granular; sporulation abundant, conidia en masse dull green; exudates present as small clear droplets; soluble pigments absent; reverse reddish. CREA 25 °C, 7 days: Very weak growth, acid production absent.
Micromorphology: Conidiophores biverticillate; stipes smooth, 55–105 × 2–3 μm; metulae 3–4, 11.5–15.5 × 2.5–4 μm. Phialides 3–5 per stipe, acerose, 9.5–12.5 × 2.5–3.5 μm. Conidia smooth, fusiform to ellipsoidal, 2.5–4 × 3–3.5 μm. Ascomata not observed.
Additional material examined. Brazil, Pernambuco, Recife, isolated from inside nest of Melipona scutellaris, R.N. Barbosa, URM 7623; RB 95; RB 171.
Notes: Altough the relationship if Talaromyces mycothecae sp. nov. is difficult to determine, the species seems to be phylogenetically most closely related to T. neofusisporus, T. stollii, T. amestolkiae and T. ruber. Talaromyces neofusisporus produces synnemata on CYA and YES, and grows poorly at 37 °C (2–3 mm, CYA, 7 days) (Wang et al. 2016). In contrast, no synnemata and good growth at 37 °C (18–20 mm, CYA, 7 days) is observed for T. mycothecae. Yilmaz et al. (2012) used various characters, such as the ability to grow at 37 °C, the colony texture on MEA and CYA and the production of acid compounds on CREA to differentiate T. amestolkiae, T. ruber and T. stollii. No acid is produced on CREA by T. mycothecae and this is shared with T. ruber (T. amestolkiae and T. stollii are poor acid producers). Talaromyces mycothecae sp. nov. attains a diameter of 18–20 mm after 7 days on CYA at 37 °C and this is faster than T. amestolkiae (8–15 mm) and T. ruber (14–18 mm), but slower than T. stollii (25–35 mm) (Yilmaz et al. 2014). Based on the data above, T. mycothecae phenotypically resembles T. ruber. The characteristic yellow and red pigmented mycelium on YES of T. ruber is not observed in the T. mycothecae sp. nov. cultures.
Talaromyces pigmentosus R.N. Barbosa, Souza-Motta, N.T. Oliveira & Houbraken sp. nov. (Figure 13)
MycoBank: MB822216
Etymology: Referring to the brownish green pigmented conidiophores of the species.
Diagnosis: Talaromyces pigmentosus sp. nov. is phylogenetically unique. This species produces pigmented conidiophores and grows well on CYA and MEA at 37 °C. No sexual state is observed and the species has a cream reverse on MEA and cream (margins) to brownish (centre) reverse on CYA.
Type: Brazil: Pernambuco: Recife, inside nests of Melipona scutellaris, June 2014, R.N. Barbosa. Holotype (slide preparation) is deposited in the URM Mycology Herbarium (Recife, Brazil): URM 90496; ex-type strains URM 7624 = CBS 142805.
ITS barcode: MF278330. Alternative markers: BenA = LT855562; CaM = LT855565; RPB2 = LT855568.
Colony diam, 7 days (in mm): CYA 23–24; CYA 15 °C 5–7; CYA 30 °C 34–35; CYA 37 °C 35–36; MEA 23–24; MEA 15 °C 6–8; MEA 30 °C 34–35; MEA 37 °C 33–34; DG18 7–9; CYAS 2–3; OA 24–25; YES 23–24; CREA 2–4.
Colony characters: CYA, 25 °C, 7 days: Colonies moderately deep; margins entire; mycelium white sometimes inconspicuously green; colony texture velvety; sporulation absent; conidial colour en masse cannot be determinate; exudates absent; soluble pigments absent; reverse white to cream at margins to brownish in centre. MEA, 25 °C, 7 days: Colonies moderately deep, sunken at centre; margin entire; mycelium white; colony texture velvety; sporulation sparse; conidia en masse greyish; exudates present as small hyaline droplets; soluble pigments absent; reverse cream. YES, 25 °C, 7 days: Colonies moderately deep, sunken, raised at centre; margins entire; mycelium white; colony texture floccose; sporulation sparse to absent, conidia en masse greyish; soluble pigments absent; exudates absent; reverse cream to yellow. DG18, 25 °C, 7 days: Colonies raised at centre; margins low, plane; mycelium white; colony texture floccose; sporulation absent, conidia en masse indeterminable; exudates absent; soluble pigments absent; reverse brown at centre, light cream to white at margin. OA, 25 °C, 7 days: Colonies low, plane; margins low, plane; mycelium white; colony texture velvety; sporulation absent; conidia en masse indeterminable; exudates absent; soluble pigments absent; reverse light cream. CYAS, 25 °C, 7 days: Colonies low, plane; margins low, plane; mycelium white; colony texture velvety; sporulation absent; conidia en masse indeterminable; exudates absent; soluble pigments absent; reverse white. CREA 25 °C, 7 days: Very weak growth, acid production absent.
Micromorphology: Conidiophores biverticillate, brownish green pigmented. Stipes smooth walled, 17–65 × 2–4 μm. Metulae 3–4, divergent, 7–11 × 2–2.5 μm. Phialides, 3–6 per stipe, acerose, 7–11 × 2–3 μm; conidia smooth walled, globose to subglobose, 2.0 × 3.0 μm. Ascomata not observed.
Additional material examined. Brazil, Pernambuco, Recife, from bee pollen of Melipona scutellaris, R.N. Barbosa, URM 7625; RB 96, RB 171; from inside nest of Melipona scutellaris RB 30; RB 100.
Notes: Talaromyces pigmentosus sp. nov. is phylogenetically closely related to T. helicus, T. boninensis and T. reverso-olivaceus and shares the ability to grow well on CYA incubated at 37 °C (10–34 mm, 7 days). Talaromyces boninensis and T. helicus produce a sexual state and this is not observed T. reverso-olivaceus and T. pigmentosus sp. nov.. The new species can also be differentiated from T. reverso-olivaceus by its ability to produce brownish green pigmented stipes. The production of pigmented stipes is also shared with the phylogenetically more distant species T. varians (also in sect. Helici) and T. ptychoconidium (sect. Purpurei) (Yilmaz et al. 2014).
Discussion
Penicillium and Talaromyces species are well-known cosmopolitan filamentous fungi that play various roles in natural ecosystems, agriculture and biotechnology. Both genera have a sectional infrageneric classification system. Currently, 26 sections are accepted in Penicillium (Houbraken and Samson 2011; Houbraken et al. 2016b) and seven in Talaromyces (Yilmaz et al. 2014). A current monograph on Penicillium is lacking, but many Penicillium sections are studied using a polyphasic approach or multigene phylogenies (e.g. Houbraken et al. 2014; Peterson et al. 2015; Visagie et al. 2015; Houbraken et al. 2016b) and all Talaromyces sections were treated in detail in the monograph of Yilmaz et al. (2014). ITS and BenA sequences are proposed identification markers for Penicillium and Talaromyces and often are generated in taxonomic studies. In many of those studies it is shown that ITS sequencing is insufficient for Penicillium identifications as closely related species often share similar or identical sequences. On the other hand, this locus works relatively well to assign species to sections. Besides ITS sequences, we also generated BenA sequences of all isolated Penicillium and Talaromyces species. The BenA sequences are used for identification of Penicillium and Talaromyces species, as this is the recommended identification marker (Visagie et al. 2014; Yilmaz et al. 2014). With exception of the new species, all other isolates obtained during our survey could be reliably identified using BenA sequences.
Among the Penicillia, section Sclerotiora (46%) isolates were most frequently detected during this study. Penicillium brocae (27%) was predominantly present among the isolates belonging to section Sclerotiora, followed by P. mellis sp. nov. (7%), P. sclerotiorum (5%), P. mallochii (4%), P. sanshaense (1%), P. fernandesiae sp. nov. (1%) and P. meliponae sp. nov. (1%). Section Sclerotoria species generally produce monoverticillate conidiophores and exceptions are the biverticillate conidiophores observed in P. choerospondiatis, P. herquei, P. malachiteum, P. sanshaense and P. verrucisporum. They also have bright yellow or orange pigments, which may occur in the mycelium, sclerotia, ascocarps, soluble pigments and/or colony reverse pigmentation (Houbraken and Samson 2011; Rivera and Seifert 2011; Visagie et al. 2013). The species isolated during our survey also produced monoverticillate conidiophores, bright coloured colonies, sclerotia and/or mycelium, and none of the strains produced a sexual state. Interestingly, P. brocae was the most predominant Penicillium present in our study. This species was originally described from coffee berry borers (galleries, cuticle, feces and guts) in Mexico (Peterson et al. 2003), and more recently detected in faeces of another, unrelated beetle, Eufallia sp. (Wang and Chan 2015). Along with the description of P. brocae, Peterson et al. (2003) suggested that this species produces exogenous sterols necessary for the coffee berry borer’s development and thus is mutualistically associated with the insect. Similar to other insects, bees are unable to synthesize sterols and, thus, exogenous sterol is required (Ferreira-Caliman et al. 2012). A recent study showed that essential steroid precursors, needed for the development of Scaptotrigona depilis bees, are proved by a Zygosaccharomyces species (Paludo et al. 2018). Whether a symbiosis exists between steroids produced by Penicillium and Talaromyces species and Melipona scutellaris bees needs to be further investigated. Also P. mallochii and P. guanacastense, two species related to P. brocae, are associated with guts and faeces of leaf-eating caterpillars (Rivera et al. 2012) suggesting an association of other section Sclerotiora members with insects as well.
Isolates belonging to section Citrina made up for 30% of all Penicillia. Penicillium citrinum was most frequently detected (20%), followed by P. steckii (5%), P. shaerii (4%), P. paxilli (2%), and P. sumatrense (1%). These species are characterised by symmetrically biverticillate conidiophores, flask shaped phialides, small-sized conidia, and some species like P. shaerii produce greyish brown cleistothecia. These species are common in soils and have a preference for (sub)tropical soils. It’s unknown whether these species are saprotrophs or if they are associated with stingless bees. Section Citrina species produce various bioactive extrolites (mycotoxins, antibiotics) such as citrinin, curvularin, paspaline, paspalinine and paxillin (Houbraken et al. 2011a). The presence of these extrolites in honey and pollen samples wasn’t subject of this study; however, if present, they can affect the quality of the honey and pollen. How insects cope with mycotoxins has rarely been investigated (Gliński and Jarosz 2000; Traniello et al. 2001; Keller et al. 2014).
Another group of isolates obtained in this study belong to Lanata-Divaricata, a section re-established by Houbraken and Samson (2011). Most of the section Lanata-Divaricata species grow rapidly in culture. The conidiophores of these species are often strongly divaricate and have metulae that are born terminally, subterminally and in intercalary positions. Useful characters for identification are the shape and ornamentation of the conidia, growth on CYA incubated at 37 °C, colony diameters and morphology (e.g. reverse colours on CYA, YES), and growth rate on CREA. Three Lanata-Divaricata species were isolated: P. singorense, P. wotroi and P. echinulonalgiovense. One isolate (RB 115) represents a novel species in sect. Lanata-Divaricata, and this species will be described elsewhere (Y-Z Diao et al., in progress). Penicillium singorense and P. wotroi are known species and the former was originally described from house dust in Thailand. Recent collections show that P. singorense has a worldwide distribution and this species is isolated in USA (Florida), Korea and China (J. Houbraken, pers. observations). The distribution of P. wotroi seems to be restricted to South-America (Brazil, Argentina) (Houbraken et al. 2011b). The name P. echinulonalgiovense was invalidly published because it was described without a Latin description or diagnosis (Art. 39.1.; Melbourne Code). In subsequent treatments using morphological characters, this species was placed in synonymy with P. janthinellum (Smith 1963) and/or P. simplicissimum (Pitt 1979; Stolk and Samson 1983). However, molecular data shows that this species is distinct (Houbraken et al. 2011b) and we therefore reinstate this species as distinct. A search in the DTO and CBS collection shows that this species has a worldwide distribution (J. Houbraken, unpubl. results). Section Lanata-Divaricata species are usually found in soil and (decaying) leaves (Houbraken et al. 2011b), but various other substrates are listed in literature. These species probably have a broad ecological niche. For example P. excelsum was isolated from bees and ants, but also from other substrates such as flowers, leaves, Brazil nut kernels and shells (Taniwaki et al. 2015).
Insects are adapted to different ecosystems and have symbiotic and/or pathogenic associations with fungi and other microorganisms (Bode 2011; Mello et al. 2016). Studies investigating the fungi associated with bees (sensu lato) revealed that some fungi are common saprophytes in the environment of the beehive. Bees collect plant pollen and nectar from different kinds of plants and inevitably, also fungal fragments will be introduced to the bee pollen (e.g. Eltz et al. 2002; Barbosa et al. 2017; Paludo et al. 2018). The nutritional value of a fungal spore is lower than that of pollen; however, it is speculated that fungal spores can serve as a complement to the bee diet since the availability is high and harvest is relatively easy (Oliveira and Morato 2000; Eltz et al. 2002). Only a limited number of reports about fungi associated with stingless bees are found in literature, and the reports related to Penicillium and Talaromyces are even rarer (Ferraz et al. 2008). According to Pandey et al. (1983), pollen grains can secret substances that inhibit microbial spore germination. After collection, the pollen grains are processed by bees into bee bread and this product normally has low water activity. Many different fungal species can be found in honey, but these are probably latently present and will not grow due to the low water activity of the product. The natural introduction of fungi in the bee environment most likely occurs in the period between collection of the plant pollen, formation of bee pollen and drying and storage of the pollen in the nests.
An important characteristic of Penicillium and Talaromyces species is the production of a diverse range of bioactive extrolites (Nielsen et al. 2017). The extrolite production of the species that were isolated during this study was investigated. The (combination of) detected extrolites can potentially play a role in the interaction between different organisms (Frisvad 2008). Our results demonstrate that the detected species are able to produce several extrolites, including the mycotoxin citrinin. The occurrence of mycotoxins and mycotoxigenic fungi has been recorded in bee pollen (sensu lato) around the world (e.g. González et al. 2005; Kačániová et al. 2011; Rodríguez-Carrasco et al. 2013). Logically, the presence of this mycotoxins (e.g. citrinin) is unwanted and can negatively affect the quality of the bee pollen for human consumption. Extrolite function depends on their ecological interaction. Insects are well adapted to feed on plants that contain a broad spectrum of (chemical) compounds (Dowd 1992). They have a long evolutionary history of interacting with fungi and it is known that some insects can use fungi as feed (Dowd 1992). Often, mycotoxins co-occur with other fungal extrolites for which no function is known. Following the analogy with the situation in higher plants, it is possible that these co-occurring fungal extrolites can synergize (or antagonize) the toxicity of co-occurring mycotoxins (Dowd 1992). In nature, fungal metabolites can provide various fitness advantages ranging from protection to competition with other microbes for niche securement (Rohlfs and Churchill 2011). Penicillium apimei sp. nov. and Monascus are both isolated from stingless bees and are both producers of compounds belonging to the geodin biosynthetic family (Barbosa et al. 2017). It is tempting to speculate that this group of metabolites might have particular function in the bee habitat. Various extrolites could not be identified in our experimental conditions and could represent novel bioactive compounds. During this study, the quality of the nests and the health of the bees was followed over a long time and no disease was observed. If any of these (novel) compounds were secreted in the bee pollen or nests, then these compounds probably didn’t have an (large) effect on the bees health.
Data on the functional relationship between fungi and stingless bees are scarce. Further studies on fungi from honey, inside nests, bee body, stored pollen and native plants are needed to understand the relationships between these organisms in tropical ecosystems, and the benefits that such fungi can possibly confer on their hosts. This study could serve as the first step for more detailed studies on ecological interactions between stingless bees, fungi and their bioactive extrolites.
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Acknowledgements
We would like to thank National Council for Scientific and Technological Development (CNPq) (Process 201478/2015-3—SWE) and Coordination for the Improvement of Higher Education Personnel (CAPES) for financial support and scholarship for Renan N. Barbosa and Associação Pernambucana de Apicultores e Meliponicultores (APIME) is thanked for their help in collecting the honey/pollen samples. We would like to acknowledge the URM Culture Collection staff; Martin Meijer and Bart Kraak from the Westerdijk Fungal Biodiversity Institute for their support and Konstanze Bench for nomenclatural assistance. Renan N. Barbosa received scholarship and financial support from the National Council for Scientific and Technological Development (CNPq) (Process 201478/2015-3—SWE) and the Coordination for the Improvement of Higher Education Personnel (CAPES).
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Barbosa, R.N., Bezerra, J.D.P., Souza-Motta, C.M. et al. New Penicillium and Talaromyces species from honey, pollen and nests of stingless bees. Antonie van Leeuwenhoek 111, 1883–1912 (2018). https://doi.org/10.1007/s10482-018-1081-1
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DOI: https://doi.org/10.1007/s10482-018-1081-1