Re-Evaluation of the Taxonomy of Talaromyces minioluteus

Talaromyces minioluteus belongs to the section Trachyspermi, has a worldwide distribution and has been found on various substrates, especially on various (stored) food commodities and indoor environments. This species is phenotypically and phylogenetically closely related to T. chongqingensis and T. minnesotensis. The phylogenetic and morphological analyses of 37 strains previously identified as T. chongqingensis, T. minnesotensis and T. minioluteus revealed that this clade incudes eight species: the accepted species T. chongqingensis, T. minnesotensis and T. minioluteus, the newly proposed species T. calidominioluteus, T. africanus and T. germanicus, and the new combinations T. gaditanus (basionym Penicillium gaditanum) and T. samsonii (basionym Penicillium samsonii). In this study, we give insight of the phylogenetic relationships and provide detailed descriptions of the species belonging to this clade. Macromorphological features, especially colony growth rates, texture and conidial colors on agar media, are important characters for phenotypic differentiation between species.

In the past, based on a morphological approach, Pitt [5] introduced an infrageneric classification system in Penicillium with four subgenera. Penicillium subgenus Biverticillium species (most currently classified in Talaromyces) were characterized by its symmetrically, biverticillate conidiophores with acerose phialides and having slow growth on 25% glycerol nitrate agar. Early molecular analysis showed that these species were phylogenetically distinct from species classified in the other three subgenera [6,7]. Penicillium was polyphyletic with the inclusion of subgenus Biverticillium and it was thus suggested to be excluded from Penicillium [7]. Later multigene analyses confirmed this result and demonstrated that the majority of Talaromyces and subgenus Biverticillium species cluster together, forming a monophyletic clade within the Trichocomaceae [8,9]. Based on these results and in the light of the single name nomenclature concept, Talaromyces was redefined to also include the majority of species previously classified in subgenus Biverticillium [10] and for example, Penicillium minioluteum was given the new combination Talaromyces minioluteus.
Talaromyces minioluteus was originally described by Dierckx [11] as forming white colonies with grey-green spots and having a yellow-red reverse that turns brown-grey in

DNA Isolation, Amplification and Sequence Analysis
Total genomic DNA was extracted from colonies grown on malt extract agar (MEA) for 4 d with the DNeasy ® UltraClean ® Microbial Kit (Qiagen, Germany) according to manufacturer instructions. The genetic markers BenA, CaM, ITS and RPB2 were amplified using the primer pairs Bt2a/Bt2b [19], CMD5/CMD6 [20], V9G/LS266 [21,22] and RPB2-F1/RPB2-7CR_1 [9] (alternatively RPB2-5F_Eur/RPB2-7CR_Eur [23]), respectively. PCR reactions were performed in a 13 µL total volume containing 0.63 µL dimethylsulfoxide (DMSO, 5% w/w), in which 1 µL of template DNA was mixed with 0.98 µL dNTPs (1 mM), 0.25 µL of each primer (10 µM) and 0.05 µL of BioTaq DNA polymerase (5 U µL). The MgCl 2 concentration was 1.46 mM. The targeted loci were amplified with a cycle program comprising an initial denaturation step at 95 • C for 5 min, followed by 35 cycles of 30 s at 95 • C (denaturation), 40 s at 55 • C (primer annealing) for benA, CaM and ITS and 48 and 52 • C for RPB2, and 2 min at 72 • C (primer extension) and ending with a final extension of 7 min at 72 • C. Automated sequencing of both strands of the PCR amplicons was performed on a 3730xl DNA Sequencer (Thermo Fisher Scientific, Headquarters: Waltham, MA, USA) using the BigDye Terminator chemistry. The primers in the sequencing reactions were the same as those used for PCR amplifications. Consensus sequences for each locus were assembled using SeqMan Pro v.15 (DNASTAR, Madison, WI, USA). Novel sequences generated in this study were deposited in the NCBI nucleotide database (GenBank) under accession numbers depicted in Table 1.
Single gene alignments were generated in MAFFT v. 7 [24] and subsequently trimmed at both ends in MEGA7 [25]. Phylogenetic analysis was performed based on the individual BenA, CaM, ITS and RPB2 sequence datasets as well as on the combined BenA, CaM, ITS and RPB2 dataset. The individual loci in the combined dataset were analysed as separate partitions. Phylogenetic trees were constructed based on Maximum Likelihood (ML) using RAxML-HPC2 on XSEDE 8.2.10 [26] through the CIPRES Science Gateway v.3.3 [27] and Bayesian Inference (BI) with MrBayes v. 3.2 [28]. The substitution model used in both analyses was GTR with gamma distribution and invariable sites. For ML bootstrap supports were estimated by 1000 replicates. Bayesian analysis was run with four MCMC chains for 10,000,000 generations and stopped when the average standard deviation of split frequencies was <0.01. Burn in was set to 25% after which the likelihood values were stationary. Random trees were sampled every 100 generations. The resulting trees were visualized with TreeView v1.6.6 and edited in Adobe Illustrator CS5. Bayesian inference (BI) posterior probabilities (pp) values and bootstrap (bs) values are labelled on the nodes. Values less than 0.95 pp and 70% bs are not shown.
In order to get insight in the distribution of the investigated species, BenA and ITS sequences deposited in GenBank and belonging to the T. minioluteus-clade were re-analyzed. These sequences were merged in our dataset, aligned and a phylogram was constructed according to the methods described above.

Morphology
The selected strains were three-point inoculated on the agar media Czapek yeast autolysate agar (CYA), MEA (Oxoid), yeast extract sucrose agar (YES), creatine sucrose agar (CREA), CYA supplemented with 5% NaCl (CYAS), dichloran 18% glycerol agar (DG18) and oatmeal agar (OA). Agar media formulations are as described by Samson et al. (2014) [29]. Plates were incubated for 7 d at 25 • C in the dark. Additional CYA plates were incubated at 30 and 37 • C. Colony growth rate, colony texture, degree of sporulation, obverse and reverse colony colors, production of soluble pigments and exudates on all media were determined and recorded after 7 d of incubation. Color names and codes follow Kornerup and Wanscher [30]. Colonies were photographed with a Nikon D3200 camera. Microscopic observations were made in mounts prepared from colonies grown on MEA after the application of 96% ethanol to remove excess conidia and using lactic acid (60%) as mounting medium. Observations and images were made with a Nikon SMZ25 stereo-microscope and a Zeiss AxioImager.A2 with Differential Interference Contrast (DIC) microscopy, both equipped with Nikon DS-Ri2 cameras.

Phylogeny
Phylogenetic relationships within the T. minioluteus-clade were inferred using single gene sequence datasets of the four studied genetic markers (Figures 1 and 2), as well as the combined dataset containing BenA, CaM, ITS and RPB2 gene sequences ( Figure 3). In total, 37 strains were used for the BenA analysis and 35 in the other datasets. The total lengths of the aligned datasets were 491, 616, 621 and 954 base pairs for BenA, CaM, ITS and RPB2, respectively.

Phylogeny
Phylogenetic relationships within the T. minioluteus-clade were inferred using single gene sequence datasets of the four studied genetic markers (Figures 1 and 2), as well as the combined dataset containing BenA, CaM, ITS and RPB2 gene sequences ( Figure 3). In total, 37 strains were used for the BenA analysis and 35 in the other datasets. The total lengths of the aligned datasets were 491, 616, 621 and 954 base pairs for BenA, CaM, ITS and RPB2, respectively.       The phylogenies inferred on the BenA, CaM, ITS and RPB2 datasets were congruent. These data show that T. minioluteus comprise a clade with phylogenetically related, but distinct, species. Phylogenetic analysis revealed nine lineages (considered here as nine species), including the phenotypically distinct species T. udagawae. Four lineages are represented by the accepted species T. chongqingensis, T. minioluteus, T. minnesotensis and T. udagawae. The ex-type strains of P. gaditanum (CBS 169.81) and P. samsonii (CBS 137.84) are located in two distinct lineages and are combined in Talaromyces (as T. gaditanus and T. samsonii, see taxonomy). The new species T. africanus, T. calidominioluteus and T. germanicus are proposed for the three remaining lineages. The multi-gene phylogeny resolved the studied strains into four main clades. The segregation into these four clades is strongly supported by Bayesian Inference, while statistical support is lacking in the ML analysis. The most species rich clade includes T. minioluteus, T. gaditanus, T. chongqingensis and T. samsonii and is sister to the clade that includes the new species T. calidominioluteus and T. africanus. The sister to these is a clade that contains T. minnesotensis and T. germanicus. The phenotypically distinct species T. udagawae takes a basal position to the other three clades.

Morphology
Species from the T. minioluteus-clade can be segregated into three groups according to their growth rate on CYA at 30 • C. Talaromyces calidominioluteus and T. africanus grow faster than the others species (18-29 mm), T. minioluteus, T. samsonii and T. gaditanus grow moderately fast (9-16 mm) whereas T. germanicus, T. chongqingensis and T. udagawae grow the slowest (5-8 mm) ( Table 2). The mycelium and conidial color are also informative characters to distinguish species. Talaromyces minioluteus, T. samsonii, T. gaditanus, T. germanicus and T. chongqingensis produce yellow mycelium and conidia in green shades while T. calidominioluteus, T. africanus and T. minnesotensis have white mycelium and bluish green conidia. Furthermore, the colony texture on MEA is informative, for example T. africanus and T. germanicus produce velvety colonies while T. gaditanus and T. samsonii colonies have a floccose texture (Table 2, Figure 4). All species are well differentiated macro-morphologically, especially on CYA, MEA and OA (Figure 4), and generally produce reddish exudates or soluble pigments. The micromorphology of this group of species is highly similar and all produce symmetrically biverticillate conidiophores with (broadly) ellipsoidal or fusiform, smooth-walled conidia. The exception is T. africanus that produces subglobose, finely roughened conidia. Talaromyces udagawae is phenotypically distinct and the only species that produces a sexual morph.

Taxonomy
Three new species and two new combinations are introduced in this study. All species were compared with their morphologically and phylogenetically closest relatives. Diagnostic characters are summarized in Table 2 and an overview of growth rates on various diagnostic media is shown in Figure 4.
Notes: The ex-type strain was not available for examination during our study and the description above is based on CBS 270.35 (=CBS 147316). The phenotype of this old strain, isolated from Zea mays (corn) in 1912, differs significantly from the original description of T. chongqingensis [17]. We refer to Zhang et al. [17] for the description based on more recently isolated strains. CBS 270.35 produces densely cottony colonies, sporulates poorly and has greyish-blueish colored conidia on MEA. In contrast, the type of T. chongqingensis as a floccose texture on MEA, sporulates well and produces greyish green colored conidia [17]. Talaromyces chongqingensis and T. minioluteus are phylogenetically most closely related (Figures 1-3). CBS 270.35 grows slower (6-8 mm) on CYA incubated at 30 • C than T. minioluteus (9-14 mm). Furthermore, CBS 270.35 produces mono-and biverticillate conidiophores and ovoidal conidia, in contrast to the biverticillate conidiophores and broadly fusiform or ellipsoidal conidia produced by T. minioluteus. Unfortunately, no data is available for ex-type strain (CGMCC 3.20482) on the growth on CYA at 30 • C, a useful character for species identification in the T. minioluteus-clade. CBS 270.35 could be degenerated and might not be a good representative of the species. However, differences in the phenotype can also occur when a strain is grown at different labs. In order to make a more reliable comparison of the macromorphology of T. chongqingensis with the current data, the freshly isolated ex-type strain (CGMCC 3.20482) should be grown under the same conditions as the other strains in this study.
Notes: This species was originally described as Penicillium gaditanum. Based on morphological and chemical data, Penicillium gaditanum was treated by van Reenen-Hoekstra et al. [3] and Yilmaz et al. [4] as a synonym of T. minioluteus. A molecular treatment of this species was lacking and sequence analyses presented here shows that this species is related to T. minioluteus, but distinct based on the GCPSR concept. This species produces strongly floccose colonies on MEA, with bright yellow (pastel yellow) mycelium and fusiform, smooth-walled conidia. Phenotypically, this species resembles T. samsonii. Talaromyces samsonii has a violet brown to deep red reverse on CYA and (vivid) red soluble pigments, in contrast to the predominantly orange reverse and orange soluble pigments (if present) in T. gaditanus.
Etymology. Referring to Germany, the country where the type was isolated. Diagnosis: Colony diameter on CYA incubated at 30 • C 6-7 mm, colony texture on MEA velvety, conidial color en masse on MEA dull green to greyish green.
Notes: Talaromyces germanicus grows slower on CYA, MEA and YES than the phylogenetically related T. minnesotensis. Talaromyces germanicus, T. chongqingensis and T. udagawae have similar growth rates on CYA incubated at 30 • C. The first two species can be differentiated by their colony texture and conidial color on MEA: T. germanicus produces velvety colonies with dull green to greyish green conidia, in contrast to the floccose colonies with greyish turquoise conidia of T. chongqingensis. Talaromyces udagawae produces a sexual state, a feature not observed in the other T. minioluteus-clade species.
Notes: Talaromyces minioluteus is phylogenetically related to T. gaditanus, T. chongqingensis and T. samsonii. These species have, with the exception of T. chongqingensis, a similar growth rate on CYA 30 • C, CYA and MEA. Colonies of T. samsonii are more densely sporulating and have a floccose texture on MEA while T. minioluteus sporulates moderately dense and has a strongly funiculose texture. The colony reverse and soluble pigments produced by T. minioluteus are in shades of brown (light brown, dark brown, orange-brown), while the reverse of T. samsonii is violet-brown to deep red and the soluble pigments vivid red to red.
Notes: Talaromyces minnesotensis is phylogenetically related to T. germanicus. The former species grows faster on CYA incubated at 30 • C.
Notes: See also T. minioluteus. No holotype material of P. samsonii was deposited in a herbarium. A strain with a personal code (1032) is mentioned in the protologue and subcultures of this strain were deposited in CBS, IMI and CECT [15]. A subculture present in the CBS collection (CBS 137.84) is deposited in the CBS herbarium as CBS H-24877 and used as lectotype.
Notes: Talaromyces udagawae can be distinguished from other T. minioluteus-clade species by the production of ascomata and its distinctive ascospores, which are ornamented with three to five regularly transverse, nearly parallel ridges. It has restrictive growth on media examined and does not grow on CREA, DG18 or CYA incubated at 37 • C. The asexual morph was not observed during this study, but was described in Stolk and Samson [31].

Discussion
Visagie et al. [18] suggested that T. minioluteus could represent a species complex, and more recently, two species were described in this complex, T. chongqingensis and T. minnesotensis [16,17]. In addition to the ex-neotype strain of T. minioluteus (CBS 642.68), also the ex-type strains of P. samsonii (CBS 137.84) and P. purpurogenum var. rubisclerotium (CBS 270.35) belong to this clade [4]. Based on morphological similarities and sequence data (ITS and BenA), Yilmaz et al. [4] considered the latter two species synonyms of T. minioluteus. Penicillium gaditanum (CBS 169.81) was not included in their phylogenetic analyses and this species was treated as synonym of T. minioluteus based on morphological and chemical data [3]. In our study, we show that both P. gaditanum and P. samsonii are distinct species and we provide new Talaromyces combinations for these.
The taxonomic position of P. purpurogenum var. rubrisclerotium was uncertain. Yilmaz et al. [13] noted that many strains previously identified as P. purpurogenum var. rubrisclerotium resolved in a clade with T. amestolkiae, but that its presumed ex-type strain CBS 270.35 clustered with T. minioluteus [4]. Our results indicate that CBS 270.35 is in fact T. chongqingensis. Thom [32] reported strain Thom 2670 as representative of P. purpurogenum var. rubrisclerotium, producing sclerotia and able to grow at 37 • C. Both CBS 270.35 and CGMCC 3.20482 (ex-type of T. chongqingensis) deviate from the original description of P. purpurogenum var. rubrisclerotium [32] by lacking sclerotia production and their inability to grow at 37 • C [17]. Raper and Thom [12], Pitt [5] and Peterson and Jurjević [33] considered NRRL 1064 as the ex-type of P. purpurogenum var. rubrisclerotium. Raper and Thom [12] mentioned that there was little to suggest that NRRL 1064 was representative of the original concept of P. purpurogenum var. rubrisclerotium. This strain morphological deviates from the original description in being floccose, poorly sporulating and lacking production of dark-red sclerotia, which is a hallmark character of P. purpurogenum var. rubrisclerotium [5]. Based on morphology, Pitt [5] classified NRRL 1064 under Penicillium pinophilum. Using sequence data, Peterson and Jurjević [33] showed that NRRL 1064 phylogenetically rep-resents T. sayulitensis, a close relative of T. pinophilus. Both NRRL 1064 and CBS 138204 (ex-type of T. sayulitensis) grow well on CYA at 37 • C [5,18], while CBS 270.35 and CGMCC 3.20482 don't ( [4], this study). There is a chance that both NRRL 1064 and CBS 270.35 were contaminated in the past. Nevertheless, based on the ability to grow at 37 • C, we consider NRRL 1064 as the ex-type of P. purpurogenum var. rubrisclerotium and treat this variety as a synonym of T. sayulitensis (classified in sect Talaromyces), a species phylogenetically unrelated to T. minioluteus. Because a name has no priority outside the rank in which it is published, the name P. purpurogenum var. rubrisclerotium does not compete with T. sayulitensis (Figure 1).
The T. minioluteus-clade strains studied here share macro-and micromorphological characters. None of the species can grow on CYA incubated at 37 • C and all are able grow at 30 • C, though with different growth rates. They grow more restricted on CYA incubated at 25 • C than on MEA and generally form colonies having greyish green conidia with a velvety, floccose or funiculose texture. Most of the strains produce reddish pigments on CYA and/or MEA. The strains investigated in our study did not or poorly grew on CREA and they did not produce acid compounds, although Guevara-Suarez et al. [16] reported acid production in T. minnesotensis. The T. minioluteus-clade species can be differentiated using phenotypic characters and an overview of the most important characters is given in Table 2. The colony diameter on CYA incubated at 30 • C, colony texture on MEA, and conidial shape and ornamentation proved to be the most useful characters for identification. No sexual morph was observed in any of the T. minioluteus-clade strains. The closely related species T. udagawae forms ascomata that contain ascospores with almost parallel ridges [4,31,34]. The asexual morph was not observed in our study, but Stolk and Samson [31] observed this morph on hay-infusion agar. The conidiophores of this species are symmetrically biverticillate with long metulae.
In total, eight species are resolved in the T. minioluteus-clade based on this polyphasic approach combining morphology and physiology with sequence data of four loci (BenA, CaM, ITS and RPB2). The topologies of the single gene phylogenies are similar and largely adhere to the genealogical concordance phylogenetic species recognition (GCPSR) concept [35]. The exception is the phylogenetic relationship of CBS 147336 (=DTO 162-E5). This strain clusters with T. minnesotensis in the BenA, CaM and ITS phylogenies, but has a different position in the RPB2 phylogram, where it is a sister of T. calidominioluteus and T. africanus (80% BS, 0.98 pp). The morphology of CBS 147336 differs from the examined T. minnesotensis strains and may represent a new taxon ( Figure 4). Macromorphologically, CBS 147336 grows slower on all culture media and unlike T. minnesotensis, it does grow on CREA. It produces greenish grey colored conidia, white mycelium and red pigments whereas T. minnesotensis has deep green conidia, yellow mycelium and orange pigments. Talaromyces minnesotensis produces more compact conidiophores with more metulae per stipe and the conidia are slightly smaller in size. To date, there are no new Talaromyces species described that share BenA and CaM sequences with known species, and only differ in their RPB2 sequence [9]. The remarkable position of this strain could also be due to incomplete lineage sorting or occasional hybridization and introgression, which is also observed in the related genus Aspergillus (section Fumigati) [36]. Delimitation techniques based on coalescent theory and a multispecies coalescent analysis might be better to deal with this phenomenon than the relatively subjective evaluation and comparison of singlegene trees [36]. We defer with the description of this strain as a new species until more insight is obtained (e.g., by comparative genomics) and preferably, until more strains with this unique BenA/CaM/ITS/RPB2 sequence signature are obtained.
All T. minioluteus and related species can be identified using BenA, CaM, RPB2 or ITS sequences. In order to get insight in the distribution of the investigated species, reidentification of isolates/strains based on BenA and ITS sequences present in GenBank showed that 22 of 26 T. minioluteus strains/isolates actually represent other taxa in the clade (Supplementary Figure S1, Supplementary Table S1). The majority of sequences clustered with T. gaditanus (n = 7) followed by T. calidominioluteus (n = 6). In our study, we also found that these two species most common in the T. minioluteus-clade. Talaromyces minioluteus and related species have a worldwide distribution, but seem to be more frequently encountered in warmer climates [1,37,38]. Furthermore, these species occur on various substrates (e.g., indoor environment, wall paintings and fruits such as citrus, pear and quince). Based on our data and literature review, T. calidominioluteus is a postharvest pathogen of quince, tomato and orange fruits [1] and/or is associated with cacao, air/dust and grapes.
Re-evaluation of strains previously identified as T. minioluteus or T. minnesotensis showed that this clade consists of eight phenotypically similar species. Based on ITS sequences deposited in GenBank, it can be concluded that there is likely more diversity in this clade (Supplementary Figure S1) and a more robust sampling is needed. The present study provides a starting point for future studies on the ability of these species to produce (species-specific) extrolites (incl. mycotoxins). The results of such studies will also give insight in the possible mycotoxin production in foods, as these species are also known as post-harvest pathogens of fruits. Most importantly, this paper provides a more stable taxonomy for a complex that for a long time was problematic.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/jof7110993/s1, Figure S1: Maximum Likelihood (ML) tree inferred based on the ITS sequence dataset with GenBank sequences (indicated in red). The BI posterior probabilities and ML bootstrap percentages are presented at the nodes. The fully supported branches are thickened and the BS/PP values are indicated with asterisk. Values less than 70% bs (ML) or less than 0.95 pp (Bayesian analysis) are indicated with a hyphen or not shown. The phylogram is rooted with T. flavus (CBS 310.38 T ) and T. sayulitensis (CBS 138204). T = ex-type, Table S1: Re-identification of T. minioluteus sequences present in GenBank.