Introduction

Trichocomaceae species represent a diverse group of worldwide distributed fungi occurring in a diverse range of habitats, from soil to vegetation to air, indoor environments, and various food products1. Some species are associated with food spoilage2 and mycotoxin production (e.g., luteoskyrin, patulin, rubratoxins, viriditoxin)3,4; while others are being used or have the potential to be used in biotechnology for enzyme production 5,6,7,8. The family currently accommodates eight accepted genera: Acidotalaromyces, Ascospirella, Dendrosphaera, Rasamsonia, Sagenomella, Talaromyces, Thermomyces and Trichocoma1.

The genus Rasamsonia was established in 20129 and currently contains 14 accepted species10: Rasamsonia aegroticola, R. argillacea, R. brevistipitata, R. byssochlamydoides, R. columbiensis, R. composticola, R. cylindrospora, R. eburnea, R. emersonii, R. frigidotolerans, R. oblata, R. piperina, R. pulvericola and R. sabulosa. One of the hallmarks of Rasamsonia was its thermotolerant and thermophilic nature, a character used to distinguish it from the related genus Talaromyces. Recently, also the mesophilic species R. frigidotolerans and R. pulvericola were described in the genus, resulting in the genus’ expanded temperature growth range11,12. Nevertheless, the genus is morphologically distinct from phenotypically related genera (e.g., Paecilomyces, Talaromyces) by the production of olive-brown conidia, cylindrical phialides that usually gradually taper towards the apices and conidiophores with distinctly rough-walled stipes. Four species produce a sexual morph and these ascomata have a scanty covering1.

The genus Talaromyces was introduced for sexually reproducing Penicillium species, that produce soft-walled ascomata covered with interwoven hyphae1. Phylogenetic analysis revealed that Penicillium subgenus Biverticillium and Talaromyces together form a monophyletic clade. Currently, Talaromyces accommodates sexual and asexual reproducing species and is divided into eight sections, Bacillispori, Helici, Islandici, Purpurei, Subinflati, Talaromyces, Tenues and Trachyspermi13,14. Talaromyces sect. Bacillispori contains seven accepted species (Talaromyces bacillisporus, T. columbiensis, T. emodensis, T. hachijoensis, T. mimosinus, T. proteolyticus and T. unicus) and these species grow restrictedly on Czapek yeast extract agar (CYA), dichloran 18% glycerol agar (DG18), yeast extract sucrose agar (YES), creatine sucrose agar (CREA) and produce mono- to biverticillate conidiophores. With exception of T. proteolyticus, all species produce creamish white to yellow ascomata1.

In previous studies, microfungi associated with the upper parts (stems, leaves)15 and roots16 of Clematis L. plants were studied. During the analysis of the roots, a novel species named Paecilomyces clematidis was described, while several other isolates remained unidentified. In present study, we subjected those isolates to a detailed examination. Two putative new Trichocomaceae species were identified, accommodated in Rasamsonia and Talaromyces sect. Bacillispori. We introduce these new species here, and provide detailed descriptions, illustrations, and present data on their phylogenetic relationships with related species.

Materials and methods

Collection and isolation

Experimental research and field studies on plants, including the collection of plant material, was complied with relevant institutional, national, and international guidelines and legislation. During the spring of 2021 roots of Clematis L. were collected from the ornamental garden (48° 47′ 33.4″ N; 16° 47′ 55.7″ E) of Mendel University in Lednice, the Czech Republic. Collected roots were immediately transported to the laboratory for further processing. The roots were washed with running tap water to remove residual soil, washed again with running sterile distilled water and air-dried on sterile filter paper. Clean roots were sterilized in 1% sodium hypochlorite for one minute and then rinsed three times with sterile distilled water. The disinfected roots were cut into small segments of 2 × 2 × 10 mm and aseptically transferred onto potato dextrose agar (PDA, HiMedia, Mumbai, India) supplemented with 0.5 g L−1 streptomycin sulfate (Sigma–Aldrich, St. Louis, MO, USA). The plates were incubated at 25 °C in the dark for six weeks and checked for fungal growth every day. Newly developed colonies were immediately transferred onto new PDA plates and these initial cultures were subsequently purified by single spore isolation17. Reference strains and dry specimens are maintained in the CBS culture collection/fungarium and the working collection of the Food and Indoor Mycology research group (DTO), both housed at the Westerdijk Fungal Biodiversity Institute (Utrecht, the Netherlands), and at Fungal Culture Collection of Mendeleum (MEND-F), Mendel University in Brno (Lednice, Czech Republic). Taxonomic novelties were submitted to Mycobank (https://www.mycobank.org).

Morphology

Culture characteristics were determined after seven days of cultivation in darkness at 25 °C for the Talaromyces strains, and at 37 °C for the Rasamsonia strains. The strains were inoculated in three equidistant points on CREA, CYA, DG18, malt extract agar (MEA), oatmeal agar (OA) and CYA supplemented with 5% NaCl (CYAS). All media were prepared as previously described18. Colony diameters were measured after 7 d (both species) and 14 d (Talaromyces species only). The strains were also grown on MEA for 7 d and 14 d at 15, 18, 21, 24, 25, 27, 30, 33, 36, 37, 40, 45, 48, 50, 52 and 55 °C in darkness, to determine cardinal temperatures. Pictures of colonies were captured by Nikon D3200 camera equipped with Nikon 18–55 mm f/3.5–5.6 G AF-P DX VR optics. The picture processing and preparation of photographic plates was done in Adobe Photoshop CS 2018. From 7-day-old cultures grown at 25 °C (Talaromyces) and 37 °C (Rasamsonia) a conidiogenous layer with conidia were mounted in 60% lactic acid. Excess amounts of conidia were washed out with 70% EtOH. A compound ZEISS AxioSkop 2 microscope equipped with Nikon DS-Ri2 camera was used for bright-field digital images of the micromorphological features. Nikon NIS-Elements D software package was used for capturing pictures and taking measurements.

DNA extraction and amplification

Genomic DNA was extracted from 7-day-old mycelium grown on MEA at 25 °C (Talaromyces) and 37 °C (Rasamsonia) in darkness, using a NucleoSpin DNA extraction kit (Macherey–Nagel, Düren, Germany) following the manufacturer’s protocol. The internal transcribed spacer regions incl. 5.8S rDNA (ITS), and parts of the beta-tubulin (BenA), calmodulin (CaM), second largest subunit of nuclear RNA polymerase II (rpb2) and the large ribosomal subunit (LSU) were amplified by PCR using the primers and conditions previous described16,18. Sequencing was conducted in both directions with the same primer pair as the primers used for amplification at Eurofins Genomics Germany GmbH (Ebersberg, Germany). Sequences were edited and assembled in Geneious Prime 2022.1.1 (https://www.geneious.com). Newly generated sequences were deposited in GenBank.

Phylogenetic analysis

Additional sequences were downloaded from GenBank and subjected to phylogenetic analyses together with newly obtained sequences (Table 1). The dataset for each gene was aligned using the MAFFT v. 7 using the European Bioinformatics Institute platform (EMBL-EBI, https://www.ebi.ac.uk)19. Obtained alignments were manually checked, edited and combined using MEGA v.720. The combined ITS, BenA, CaM and rpb2 dataset was subjected to Maximum likelihood (ML) analyses. Phylogenetic trees were constructed using IQ-TREE 221, running 1000 bootstrap replicates. The best models for ML analyses were selected based on the Bayesian information criterion (BIC) calculated in IQ-TREE 2. Bayesian analyses (BI) employed MrBayes v. 3.2.722,23. The BI analyses included four parallel runs of 50 M generation starting from a random tree topology, every 1000 generations were sampled and the first 25% of the trees were discarded as the ‘burn-in’. The most suitable substitution model for BI analysis was determined separately for each loci using jModelTest v. 2.1.724. Trees were visualized in FigTree v. 1.4.4 and edited in Adobe Illustrator CC 2019. Resulted trees of both methods shared similar topology, thus we decided to present ML trees with support values of both methods –bootstrap (BS) and posterior probabilities (pp) labelled at the nodes. Values below 0.95 (pp) and 75% (BS) support are not shown or indicated with a hyphen. The alignments and corresponding trees are available at Figshare (https://doi.org/10.6084/m9.figshare.20490051).

Table 1 Fungal species and barcodes used in the phylogenetic analyses.
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Results

Phylogenetic analysis

The ML analyses based on the rpb2 dataset (Fig. S1) showed the phylogenetic placement of strains CBS 149229, CBS 148468, CBS 148467 and CBS 149228 within Trichocomaceae. Detailed statistics and the model selected for the ML analysis is given in Table 2. The phylogram was rooted with Paecilomyces brunneolus CBS 370.70 T. Briefly, the ML analysis grouped studied strains into 15 lineages, including outgroup. These lineages are in agreement with data published in studies on Trichocomaceae13,14,25. Based on these data, CBS 149229 and CBS 148468 can be classified in Rasamsonia, and CBS 148468 and CBS 148467 in Talaromyces sect. Bacillispori.

Table 2 Detailed characteristics of phylogeny datasets.
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The Rasamsonia dataset contained sequences from 17 strains, including the outgroup Trichocoma paradoxa (CBS 103.73 T). The combined dataset (ITS, BenA, and CaM) contained 1664 sites, including alignment gaps. Of these, 1007 were conserved and 435 parsimony-informative. Detailed results for each single gene dataset including corresponding models are given in Table 2. Strains CBS 148468 and CBS 149229 form a lineage within Rasamsonia (Fig. 1). This lineage is sister to a clade containing R. brevistipitata, R. columbiensis, R. frigidotolerans, R. oblata, R. pulvericola and R. sabulosa. Single locus trees of individual loci (ITS, BenA, CaM, rpb2) are shown in Fig. S2.

Figure 1
figure 1

Maximum likelihood tree generated from the combined analysis of ITS, BenA, and CaM sequence data. BS/pp values are given at the nodes. The tree was rooted to Trichocoma paradoxa (CBS 103.73 T). The new species Rasamsonia chlamydospora Spetik & Houbraken sp. nov. is highlighted in bold. Tex-type strain.

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The Talaromyces sect. Bacillispori dataset consisted of sequences from 10 strains, including the outgroups Talaromyces subinflatus (CBS 652.95 T) and Talaromyces flavus (CBS 310.38 T). The combined dataset (ITS, BenA, CaM and rpb2) contained 2512 sites, including alignment gaps. Of these, 1632 were conserved, 541 parsimony-informative and 817 unique. Detailed results for each single gene dataset including corresponding models are displayed in Table 2. The multilocus analysis resolved CBS 148467 and CBS 149228 in a fully supported clade, sister to T. bacillisporus CBS 296.48 T (84% BS, 1.00 pp) (Fig. 2). Additionally, a single locus ITS ML/BI analysis was performed (Fig. S3), showing the distant position of T. hachijoensis to the newly proposed T. clematidis.

Figure 2
figure 2

Maximum likelihood tree generated from the combined analysis of ITS, BenA, CaM and rpb2 sequence data. BS/pp values are given at the nodes. The tree was rooted to Talaromyces subinflatus (CBS 652.95 T) and Talaromyces flavus (CBS 310.38 T). The new species Talaromyces clematidis Spetik & Houbraken sp. nov. is highlighted in bold. Tex-type strain.

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Taxonomy

Based on the results of the phylogenetic analysis and the morphological examination (see below under Taxonomy and Discussion), we propose the names Rasamsonia chlamydospora for CBS 148468 and CBS 149229 and Talaromyces clematidis for CBS 148467 and CBS 149228.

Rasamsonia chlamydospora Spetik & Houbraken sp. nov. (Fig. 3).

Figure 3
figure 3

Rasamsonia chlamydospora (CBS 149229 T). Colonies 7 d, 37 °C from left to right (top row) CYA, DG18, MEA and OA; (bottom row) CYA reverse, DG18 reverse, YES and CREA. (A) In situ detail of the colony on MEA; (B–E) Conidiophores; (F, G) Chlamydospores; (H) Conidia. Scale bars: 500 μm (A); 10 μm (B–H); 5 μm (G).

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Mycobank number: MB 845365.

Etymology:—refers to chlamydospores, the globose to subglobose swollen cells produced by this species.

Type:—CZECH REPUBLIC, Břeclav: Lednice, university garden (48° 47′ 33.4″ N 16° 47′ 55.7″ E), isolated from the root of Clematis ´Snow Queen´ (Ranunculaceae), 2021, M. Spetik, Holotype: CBS H-25023, ex-type living culture CBS 149229 = DTO 473-E5 = MEND-F-0752.

Barcodes: ITS ON863770; LSU ON863795; BenA ON873765; CaM ON938198; rpb2 ON938202.

Colony diameter (7 d, in mm): 25 °C: CYA 10–13; CYAS No growth; DG18 2; MEA 25–28; OA 12–21; YES 10–12; CREA No growth. 37 °C: CYA 31–35; CYAS No growth; DG18 36–38; MEA > 65; OA > 65; YES 46–52; CREA 18–25.

Colony diameter at different temperatures (7 d in mm). On CYA: 15–21 °C No growth; 24 °C 5–7; 27 °C 16–18; 30 °C 20–23; 33 °C 27–30; 36 °C 28–33; 37 °C 31–35; 40 °C 26–29; 45 °C 15–16; 48 °C 7–8; > 50 °C No growth. On MEA: 15–18 °C No growth; 21 °C 9–11; 24 °C 25–27; 27 °C 45–46; 30 °C 56–58; 33 °C 66–71; 36 °C 72–76; 37 °C 70–74; 40 °C 75; 45 °C 39–45; 48 °C 21–25; 50 °C 12–13; 52 °C 5; 55 °C No growth. Cardinal growth temperatures: Minimum, on CYA between 21 and 24 °C and between 18 and 21 °C on MEA, optimum around 36 °C and maximum on CYA 48 °C, on MEA 52 °C. Colonies grown on CYA and MEA at various temperatures (15–45 °C) after 7 d are shown on Fig. S4.

Colony characters (37 °C, 7 d): CYA: Colonies low, plane; margin low, entire; mycelium light brown; texture floccose; sporulation moderately dense; soluble pigments absent; exudates absent; reverse white to light brown. MEA: Colonies moderately deep, radially sulcate; margin low, entire; mycelium olivaceous with white spots near the centre; texture velvety; sporulation dense; soluble pigments absent; exudates absent; reverse brown. DG18: Colonies raised, sulcate; margin low, entire; mycelium white to olivaceous; texture velvety to floccose; sporulation dense; soluble pigments absent; exudates absent; reverse brown. YES: Colonies flat, raised in centre; margin low, entire; mycelium olivaceous with white spots; texture velvety to floccose; sporulation dense; soluble pigments absent; exudates absent; reverse white. OA: Colonies flat; margin irregular; mycelium yellow to olivaceous; texture floccose; sporulation dense; soluble pigments absent; exudates absent; reverse brown. CYAS: No growth. CREA: Moderate growth, acid production absent.

Micromorphology: Mycelium 2–3.5 μm diam, distinctly rough-walled. Conidiophores monoverticillate, sometimes with subterminal branches. Stipes rough-walled, 15–70 × 2–3 μm. Phialides 3–6 per stipe, smooth, acerose to flask-shaped, 10–15 × 2–3 μm. Conidia smooth, cylindrical, 3.5–4.5 × 2.5–3 μm. Swollen cells resembling chlamydospores present, smooth, globose to subglobose, 6–8 μm. Ascomata not observed.

Notes: Most Rasamsonia species are thermotolerant or thermophilic, including R. chlamydospora. Thermophilic fungi are those with a maximum growth temperature of 50 °C or higher, and a minimum growth temperature of 20 °C or above26. The minimum growth temperature of R. chlamydospora depends on the growth medium, and the minimum growth temperature on MEA is between 18 and 21 °C. Comparison of microscopic features between all Rasamsonia species is given in Table 3. Rasamsonia chlamydospora shares the production of olive-brown conidia and cylindrical phialides that gradually taper towards the apices with other species but can be distinguished from other Rasamsonia species by the production of swollen cells that resemble chlamydospores. Comparing sequences obtained from the type strain with those of R. brevistipitata, the phylogenetically closest species, showed a pairwise nucleotide difference of 27 bp in ITS, 63 bp in BenA, 79 bp in CaM and 88 bp in rpb2.

Table 3 Morphological characteristics of Rasamsonia spp.
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Additional specimens examined:—CZECH REPUBLIC, Breclav: Lednice, university garden (48° 47′ 33.3″ N 16° 47′ 55.6″ E), isolated from the root of Clematis ´Snow Queen´ (Ranunculaceae), 2021, M. Spetik, living culture CBS 148468 = DTO 743-E4 = MEND-F-0751.

Talaromyces clematidis Spetik & Houbraken sp. nov. (Fig. 4).

Figure 4
figure 4

Talaromyces clematidis (CBS 149229 T). Colonies 7 d, 25 °C from left to right (top row), MEA, DG18, YES and OA; (bottom row) MEA reverse, DG18 reverse, YES reverse; MEA (14 d 25 °C). (A) Ascomata in situ on OA; (B, C) Conidiophores; (D) Conidia; (E) Asci and conidia; (F) Ascospores. Scale bars: 1000 μm (A), 10 μm (all others).

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Mycobank number: MB 845200.

Etymology:—refers to the host, Clematis.

Type:—CZECH REPUBLIC, Břeclav: Lednice, university garden (48°47′ 33.5″ N 16° 47′ 55.8″ E), isolated from the root of Clematis ´Snow Queen´ (Ranunculaceae), 2021, M. Spetik, Holotype: CBS H-25024, ex-type living culture CBS 149228 = DTO 473-E3 = MEND-F-0750.

Barcodes: ITS ON863768; LSU ON863793; BenA ON873763; CaM ON938196; rpb2 ON938200.

Colony diameter (25 °C, in mm), 7 d: CYA 2; CYAS No growth; DG18 3–5; MEA 8–11; OA 6–9; YES 4–6; CREA 25 °C No growth. 14 d: CYA 4–5; CYAS No growth; DG18 10–13; MEA 21–26; OA 15–18; YES 8–9; CREA No growth. Colonies grown on CYA, MEA, DG18, YES, OA at 25 °C after 7 d and 14 d are displayed in (Fig. S5).

Colony diameter at different temperatures (mm). On CYA, 7 d: 21 °C No growth; 24 °C 2; 27 °C 3–4; 2; 33 °C 2; 36 °C No growth. On MEA, 7 d: 15 °C 3–4; 18 °C 3–6; 21 °C 7; 24 °C 3–4; 27 °C 10–12; 30 °C 11–12; 33 °C 8; 36 °C 2; 37 °C No growth. On CYA 14 d: 15 °C 2; 18 °C 3–4; 21 °C 3; 24 °C 4; 27 °C 5; 30 °C 4–5; 33 °C 4–5; CYA 36 °C No growth. On MEA, 14 d: 15 °C 8; 18 °C 12–14; 21 °C 18–21; 24 °C 22–25; 27 °C 25–30; 30 °C 26–28; 33 °C 18–20; 36 °C 7; 37 °C No growth. Cardinal growth temperatures: Minimum 15 °C; optimum between 27 and 30 °C and maximum at 36 °C. Colonies grown on CYA and MEA at various temperatures (15–36 °C) after 14 d are displayed on (Fig. S6).

Colony characters (25 °C, 7 d): CYA: Growth restricted; colonies flat; margin plane, irregular; mycelium white; texture floccose; sporulation absent; soluble pigments absent; exudates absent; reverse white. MEA: Colonies raised in centre; margin low, entire (2 mm); mycelium white; texture floccose; sporulation dense; soluble pigments absent; exudates absent; reverse white. DG18: Colonies low; margin entire; mycelium white; sporulation absent; soluble pigments absent; exudates absent; reverse pale yellow. YES: Growth restricted; colonies medium raised; margin entire, circular; mycelium white; texture floccose; sporulation absent; soluble pigments absent; exudates absent; reverse white. OA: Colonies flat; margin entire; mycelium white at margin, olivaceous in centre; texture floccose; sporulation dense; soluble pigments absent; exudates absent; reverse white. CYAS: No growth. CREA: No growth.

Micromorphology: Conidiophores monoverticillate, sometimes with subtermal branches. Stipes smooth-walled, 15–30 × 2 μm. Phialides usually in groups of 3–6 per stipe, sometimes solitary, lateral of terminal on vegetative hyphae, smooth, acerose, 7–15 × 2–2.5 μm. Conidia smooth, ellipsoidal 3.5–5 × 2–3 μm. Ascomata yellow, subglobose to ovoidal, 100–600 μm; asci globose to subglobose 11–14 μm; ascospores smooth, ellipsoid, 6–7.5 × 4.5–5.5 μm.

Notes: Talaromyces clematidis forms a well-supported sister clade to T. bacillisporus. There are various characters (e.g., colony diameters on CYA at 25 °C and 37 °C, and ascospore ornamentation) that can be used to distinguish both species, and some of them are summarized in Table4. The pairwise nucleotide differences between the type strains of both species are 45 nucleotides in ITS, 73 bp in BenA, 94 bp in CaM and 112 bp in rpb2.

Additional specimens examined:—CZECH REPUBLIC, Břeclav: Lednice, university garden (48° 47′ 33.4″ N 16° 47′ 55.7″ E), isolated from the root of Clematis ´Snow Queen´ (Ranunculaceae), 2021, M. Spetik, living culture CBS 148467 = DTO 743-E2 = MEND-F-0749.

Discussion

Rasamsonia chlamydospora is characterized by production of swollen cells that resemble chlamydospores, which is a unique microscopic feature within Rasamsonia. Only R. byssochlamydoides was recorded rarely producing chlamydospores that are globose or subglobose, 4 µm diam29. In comparison, R. chlamydospora produces bigger chlamydospores (6–8 μm), grows faster on CYA and has smaller conidia (Table 3). The four-loci-based phylogeny delineated R. chlamydospora as a well-supported clade in Rasamsonia (Fig. 1; Fig. S2). Rasamsonia species have been reported from various countries, e.g. Belgium, Canada, Germany, Italy, Japan, Netherlands, UK, USA, Taiwan; and from various substrates including air, compost, cork, fruit concentrate, human tissues, house dust, peat, seeds, soil, sugar cane, urine or wood chips9,11,28. This is for the first-time reporting Rasamsonia species from a clematis plant.

Talaromyces clematidis is characterized by restricted growth on CYA, DG18 and YES and by production of yellow ascomata. These characters are shared with other Talaromyces species accommodated in sect. Bacillispori (Table 4). Despite the shared morphology with other Talaromyces species, T. clematidis forms a well-supported sister clade with T. bacillisporus (Fig. 2) employing a four gene (ITS, BenA, CaM, rpb2) dataset. Both species can be distinguished by different conidial shapes, being ellipsoidal in T. clematidis vs rod-shaped/ellipsoidal in T. bacillisporus. Both species produce ascomata which differ in color; yellow in T. clematidis, and white to orange in T. bacillisporus. Talaromyces clematidis doesn't grow at 37 °C on MEA and CYA while T. bacillisporus does. Species of Talaromyces sect. Bacillispori have been isolated from various substrates such as leaf, rye bread, sludge of anaerobic pasteurised organic household waste, soil, and from several countries including Colombia, Japan, Nepal, Netherlands, Sweden, Ukraine, UK, USA and Taiwan13,30. Only one Talaromyces strain was isolated from clematis before, in Boskoop, the Netherlands, and this strain (T. muroi, CBS 261.55) is accommodated in Talaromyces sect. Talaromyces13. This is the first time that a species of Talaromyces sect. Bacillispori is reported from clematis.

Table 4 Morphological comparison of Talaromyces sect. Bacillispori species.
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Conclusion

In this study, two novel Trichocomaceae species were introduced with comprehensive descriptions, illustrations and taxonomic placement using multi-locus (ITS, BenA, CaM, rpb2) sequence datasets. The proposed novelties are Rasamsonia chlamydospora and Talaromyces clematidis, accommodated in Talaromyces sect. Bacillispori, both isolated from a clematis root. Together with our previous study16, this study suggests potentially hidden fungal diversity within the root of clematis plants.