Description of Crinitomyces reliqui gen. nov., sp. nov. and Reassignment of Trichosporiella flavificans and Candida ghanaensis to the Genus Crinitomyces

The systematic position of 16 yeast strains isolated from Thailand, Hungary, The Netherlands, and the Republic of Poland were evaluated using morphological, physiological, and phylogenetic analyses. Based on the similarity of the D1/D2 domain of the LSU rRNA gene, the strains were assigned to two distinct species, Trichosporiella flavificans and representatives of a new yeast species. Phylogenetic analyses revealed that Candida ghanaensis CBS 8798T showed a strong relationship with the aforementioned two species. The more fascinating issue is that Candida and Trichosporiella genera have been placed in different subphyla, Saccharomycotina and Pezizomycotina, respectively. The close relationship between Trichosporiella flavificans, Candida ghanaensis and the undescribed species was unexpected and needed to be clarified. As for morphological and physiological characteristics, the three yeast species shared a hairy colony appearance and an ability to assimilate 18 carbon sources. Based on phylogenetic analyses carried out in the present study, Crinitomyces gen. nov. was proposed to accommodate the new yeast species, Crinitomyces reliqui sp. nov. (Holotype: TBRC 15054, Isotypes: DMKU-FW23-23 and PYCC 9001). In addition, the two species Trichosporiella flavificans and Candida ghanaensis were reassigned to the genus Crinitomyces as, Crinitomyces flavificans (Type: CBS 760.79) comb. nov. and Crinitomyces ghanaensis (Type: CBS 8798) comb. nov., respectively.


Introduction
Several species concepts have been applied for yeast identification. A phenotypic species concept and growth profiles were initially used, while a biological species concept including data from mating experiments was later employed [1]. However, the phenotypic concept is limited due to the simplicity of fungal features, such as spore characters, which may lead to phenotypically cryptic taxa [2], and the lack of phenotypic divergence may also occur from the failure of accurate diagnosis [3][4][5]. With the introduction of sequencing technology, the sequence-based species concepts, including the phylogenetic analysis, became broadly applied and extensively employed in fungal taxonomy [1]. As a result, a more accurate classification of yeasts has been obtained.
A number of yeasts have been identified by phenotypic approach, often leading to misidentification or incorrect taxonomic assignment. One of these species-rich and polyphyletic yeast genera within Saccharomycetales order is the genus Candida. In the past, asexual yeasts with multilateral budding but showing nondistinctive cellular morphology were temporarily assigned to the genus Candida [6]. Currently, the placement of many Candida species is still unclear although sequencing technology has been frequently employed in yeast identification and classification. Phylogenetic analysis led to the recognition that various Candida species are distributed throughout the subphylum Saccharomycotina [7]. This situation is aggravated by the observation that even now some novel species are described in the Candida genus as a temporary placement based on short sequences and/or a small number of genes in phylogenetic analyses and, in addition, the lack of taxonomic characters needed to classify them elsewhere [8]. Fortunately, due to the availability of sufficient DNA sequence datasets, various Candida species have been transferred to new or already existing genera such as Scheffersomyces [9], Danielozyma, Deakozyma, Middelhovenomyces [10], Diutina [11], Saturnispora [12], Groenewaldozyma [13], Teunomyces [7] and Limtongozyma [14]. However, many Candida species are awaiting more analysis and accurate classification.
During our investigation of yeast communities in food waste, the strain DMKU-FW23-23 was found. The initial search of the GenBank using BLASTn search of the D1/D2 domain of the large subunit (LSU) of ribosomal RNA (rRNA) gene revealed that this yeast strain was distinct from the described yeast species in the database, but related with Trichosporiella flavificans CBS 760.79 T and Candida ghanaensis CBS 8798 T . It is surprising that Trichosporiella and Candida are placed in different subphyla i.e., Pezizomycotina and Saccharomycotina, therefore it is unlikely that they are closely related to each other. Obtaining the correct placement and description of the new yeast species represented by strain DMKU-FW23-23 based on an integrative (polyphasic) taxonomic approach and reassignment of Trichosporiella flavificans and Candida ghanaensis were accomplished in this study. Table 1 presents the list of strains considered in this study. The strains from Thailand were isolated by the direct isolation method as described by Sakpuntoon et al. [15]. Yeast extract peptone dextrose (YPD) agar supplemented with 0.025% (w/v) sodium propionate and 0.02% (w/v) chloramphenicol was used as yeast isolation medium. The inoculated agar plates were incubated at 30 ± 2 • C until colonies appeared. Yeast colonies were selected based on different colony morphologies and then purified by cross streaking on YPD agar without antibiotics. The strains CBS 15,014 and CBS 142,641 were isolated from soil and sediment from wastewater treatment facility in The Netherlands, respectively, while the strain CBS 161.94 was isolated from sewage sludge in Katowice, the Republic of Poland. All Hungarian strains were isolated from Danube water. The water samples were taken from the surface of the river from the riverbank by sterile wide mouth screw capped bottles. The samples were kept in a refrigerator until they were processed within 24 h. Hundred milliliter aliquots of the samples were enriched in 500 mL yeast nitrogen base (YNB) medium supplemented with 0.5% (v/v) carbon-source (methanol or hexadecane) and incubated on a horizontal shaker for seven days (25 • C, 100 rpm), then 0.1 mL of each culture was transferred to a 16 mm culture tube containing 5 mL of liquid medium with the same composition. Following an additional week of incubation on a rotary shaker (25 • C, 30 rpm) the enriched cultures were serially diluted and surface plated on Rose-Bengal Chloramphenicol (RBC) agar. Representative strains were isolated on glucose (2%)-peptoneyeast extract (GPY) agar after 7 days of incubation at 25 • C in darkness and purified by repeated streaking. For preservation of the isolated strains, a single yeast colony was cultured in a yeast extract malt extract (YM) broth for 18-24 h. Cell pellets were then collected by centrifugation, washed twice with sterile distilled water, and resuspended in fresh YM medium. The active yeast was then preserved in a metabolically inactive state by storing at −80 • C in YM broth supplemented with 30% (v/v) glycerol for long-term preservation.

DNA Sequencing and Phylogenetic Analysis
Yeasts were grown in YM broth for 18-24 h. The cell pellets were then collected and used for DNA extraction by enzymatic method [16]. The small subunit (SSU) rRNA gene, internal transcribed spacer (ITS) region and the D1/D2 domain of the large subunit (LSU) rRNA gene were amplified with the primer pairs SSU1f/SSU4r [17], SSU3f/SSU2r [17], NL5A/NS7A [18] and NL1/NL4 [19] respectively. The PCR products were purified with a FavorPrep TM Gel/PCR Purification Mini Kit (Favorgen, Austria) and were then sent for DNA sequencing to First BASE Laboratories located at Seri Kembangan in Selangor state, Malaysia. Sequence assembly and alignment were conducted by the BioEdit version 7.0.5.3 program [20]. Aligned sequences were compared with the sequences in the GenBank database (http://www.ncbi.nlm.nih.gov/, accessed on 20 January 2022) using a BLASTn search. Phylogenetic trees were constructed based on the neighbor-joining method with the MEGA version 7.0.26 program [21]. Bootstrap analysis for the estimation of confidence levels of the clades was performed on 1000 bootstrap replications [22], and only values greater than 50% were shown. Table 2 shows the accession numbers of reference sequences retrieved from the GenBank database.
T : Type strain of species.

Phenotypic Characterization
The investigated yeasts were morphologically and physiologically characterized by standard methods described by Kurtzman et al. [23]. Yeasts were grown for three days in YM broth and YM agar at 25 • C for morphological study. Pseudo-hyphae and true hyphae formation were investigated on corn meal agar slide cultures at 25 • C for three days. Growth at different temperatures (15,25,30,35,37,40,42 and 45 • C) was determined in YM broth. The strains were examined individually or mixed in pairs for ascospore formation using different media including PDA, YM agar, YPD agar, corn meal agar, 5% malt extract agar, Gorodkowa agar, V8 agar, Fowell's acetate agar [24] and yeast carbon base ammonium sulfate (YCBAS) agar [25] at 25 • C for up to twelve weeks with periodic microscopic inspection. Carbon and nitrogen source assimilation, carbohydrate fermentation, starchlike compounds production, and cycloheximide resistance tests were conducted in liquid media. A urea hydrolysis test was performed on a urea slant medium. Acid production and Diazonium Blue B (DBB) tests were conducted on solid medium in Petri dishes. All experiments were carried out with three replicates.

Species Delineation and Molecular Phylogeny
BLASTn search analysis of the D1/D2 domain of the LSU rRNA gene against the GenBank database was performed to identify the yeast strain DMKU-FW23-23 found during a study of yeast community in food waste. The result showed that the top two results from a BLASTn search hit with the currently recognized species Trichosporiella flavificans CBS 760.79 T and Candida ghanaensis CBS 8798 T , respectively. Surprisingly, these two species are described in different subphyla i.e., T. flavificans was placed in the subphylum Pezizomycotina and C. ghanaensis in the subphylum Saccharomycotina. The relationship between these two species was unexpected and needed to be clarified. Thus, a placement of the strains DMKU-FW23-23, T. flavificans CBS 760.79 T and C. ghanaensis CBS 8798 T was thoroughly investigated in this study. Seven additional strains, CBS 15240, CBS 15241, CBS 15242, CBS 15243, CBS 15014, CBS 161.94, and CBS 142641, that were similar to the strain DMKU-FW23-23, were found from BLASTn search analysis. Pairwise alignment revealed that the strain DMKU-FW23-23 and its companions differed from each other at 0-2 nucleotide substitutions without gaps in the D1/D2 domain of the LSU rRNA gene, while their ITS region showed no nucleotide substitutions and 0-1 gap (Table 3.). Table 3. Pairwise DNA sequence comparisons between the strain DMKU-FW23-23 and its related strains.  Table 4. Identical sequences (0 nucleotide substitution with 0-1 gap) were found in the D1/D2 domain of the LSU rRNA gene and 0-3 nucleotide substitutions with 0-6 gaps were found in the ITS region among T. flavificans CBS 760.79 T and its related strains. To find the accurate taxonomic placement of the strain DMKU-FW23-23 and its companions, T. flavificans CBS 760.79 T and C. ghanaensis CBS 8798 T , a phylogenetic tree based on the D1/D2 domain of the LSU rRNA gene was constructed. In addition to the abovenoted strains, related species were included in the analysis from the subphyla Saccharomycotina and Pezizomycotina. The results revealed that the strain DMKU-FW23-23 clustered with T. flavificans CBS 760.79 T and C. ghanaensis CBS 8798 T and their placements were found within the subphylum Saccharomycotina (Figure 1).  This result suggested that the assignment of T. flavificans to the genus Trichosporiella, which is nested in the subphylum Pezizomycotina, must be revised, and it should be transferred to the subphylum Saccharomycotina. In order to find its accurate placement within the subphylum Saccharomycotina, a phylogenetic tree based on a concatenated sequence of three genes including the small subunit (SSU) rRNA gene, the ITS region and the D1/D2 domain of the LSU rRNA gene was constructed (Figures 2 and S1). The result demonstrated that the strain DMKU-FW23-23 and its companions formed a single lineage and were placed next to T. flavificans CBS 760.79 T and also grouped together with C. ghanaensis CBS 8798 T . These three species formed a distinct monophyletic clade that is clearly separated from other described yeast species. Hence, a novel yeast genus namely Crinitomyces is proposed to accommodate T. flavificans and C. ghanaensis which are reassigned as Crinitomyces flavificans and Crinitomyces ghanaensis, respectively. Moreover, the strain DMKU-FW23-23 and its companion strains are also proposed as a novel yeast species within this novel genus and the name Crinitomyces reliqui sp. nov. is proposed.

Yeasts
transferred to the subphylum Saccharomycotina. In order to find its accurate placement within the subphylum Saccharomycotina, a phylogenetic tree based on a concatenated sequence of three genes including the small subunit (SSU) rRNA gene, the ITS region and the D1/D2 domain of the LSU rRNA gene was constructed (Figures 2 and S1). The result demonstrated that the strain DMKU-FW23-23 and its companions formed a single lineage and were placed next to T. flavificans CBS 760.79 T and also grouped together with C. ghanaensis CBS 8798 T . These three species formed a distinct monophyletic clade that is clearly separated from other described yeast species. Hence, a novel yeast genus namely Crinitomyces is proposed to accommodate T. flavificans and C. ghanaensis which are reassigned as Crinitomyces flavificans and Crinitomyces ghanaensis, respectively. Moreover, the strain DMKU-FW23-23 and its companion strains are also proposed as a novel yeast species within this novel genus and the name Crinitomyces reliqui sp. nov. is proposed. Phenotypic characters of the three yeast species: Crinitomyces reliqui sp. nov., Crinitomyces flavificans comb. nov. and Crinitomyces ghanaensis comb. nov. were compared and are summarized in Table 5. Table 5. Physiological characteristics of Crinitomyces reliqui in comparison to its closely related species.

Physiological Characteristics 1 2 3
Fermentation A broad range of carbon sources was found to be assimilated, namely glucose, galactose, sorbose, cellobiose, maltose, sucrose, trehalose, melezitose, L-arabinose, D-xylose, erythritol, glucitol, mannitol, glycerol, ethanol, succinic acid, salicin and N-acetyl-glucosamine and only two carbon sources, inulin, and citric acid, were not assimilated by any of these yeasts. However, Crinitomyces ghanaensis CBS 8798 T did not show fermentation ability. It should be noted that, by all strains of Crinitomyces flavificans, a yellow pigment was exuded onto the agar medium during cultivation. For morphological characteristics, yeast colonies of the three species are white to cream, convex and butyrous, with a dull surface. All of the three yeast species showed a hairy colony morphology (Figure 3) which is the origin of the genus name, Crinitomyces.

Ecology
The type strain of Crinitomyces reliqui DMKU-FW23-23 T was isolated from domestic food waste in Thailand while the two related strains, CBS 161.94 and CBS 142641, were isolated from similar types of habitats, sewage sludge and wastewater in Republic of Poland and The Netherlands, respectively. However, the strain CBS 15,014 was isolated from soil and the four related strains were isolated from water surface of a river in The Netherlands and Hungary, respectively. These habitats showed a contrast in terms of amount and type of nutrients available. The occurrence of this species in food waste, sewage sludge and rivers raised the possibility that the water of the rivers might have been polluted at the time of sampling. Crinitomyces reliqui is suggested to be a cosmopolitan species because all eight strains of this species were found from four different countries. mine and only two carbon sources, inulin, and citric acid, were not assimilated by any of these yeasts. However, Crinitomyces ghanaensis CBS 8798 T did not show fermentation ability. It should be noted that, by all strains of Crinitomyces flavificans, a yellow pigment was exuded onto the agar medium during cultivation. For morphological characteristics, yeast colonies of the three species are white to cream, convex and butyrous, with a dull surface. All of the three yeast species showed a hairy colony morphology (Figure 3) which is the origin of the genus name, Crinitomyces.

Ecology
The type strain of Crinitomyces reliqui DMKU-FW23-23 T was isolated from domestic food waste in Thailand while the two related strains, CBS 161.94 and CBS 142641, were isolated from similar types of habitats, sewage sludge and wastewater in Republic of Poland and The Netherlands, respectively. However, the strain CBS 15,014 was isolated from soil and the four related strains were isolated from water surface of a river in The Netherlands and Hungary, respectively. These habitats showed a contrast in terms of amount and type of nutrients available. The occurrence of this species in food waste, sewage sludge and rivers raised the possibility that the water of the rivers might have been polluted at the time of sampling. Crinitomyces reliqui is suggested to be a cosmopolitan species because all eight strains of this species were found from four different countries.
The yeast Crinitomyces flavificans CBS 760.79 T was isolated from washings of ion-exchange resin in a guanosine monophosphate manufacturing plant in Japan whereas its seven companion strains were isolated from food waste and water in different countries. Therefore, C. flavificans should also be claimed as a cosmopolitan species.
The isolation sources and geographical origin of all investigated strains are summarized in Table 1. Cells are spherical or ellipsoidal and asexual reproduction proceeds by multilateral budding. Septate hyphae are produced. Ascospore formation is not observed. DBB reaction is negative, starch-like compounds are not produced, and urea hydrolysis is negative. The yeast Crinitomyces flavificans CBS 760.79 T was isolated from washings of ionexchange resin in a guanosine monophosphate manufacturing plant in Japan whereas its seven companion strains were isolated from food waste and water in different countries. Therefore, C. flavificans should also be claimed as a cosmopolitan species.

Taxonomy
The isolation sources and geographical origin of all investigated strains are summarized in Table 1. Cells are spherical or ellipsoidal and asexual reproduction proceeds by multilateral budding. Septate hyphae are produced. Ascospore formation is not observed. DBB reaction is negative, starch-like compounds are not produced, and urea hydrolysis is negative.
Based on the phylogenetic analyses carried out in this study, T. flavificans is reclassified here in Saccharomycotina as Crinitomyces flavificans. All phenotypic characters of the type strain, CBS 760.79, were re-examined in this study and results were found consistent with those of the first report. Nevertheless, some characters were differed among the companion strains and were then reported as "variable" as shown in the Table 5.

Conclusions and Discussion
Candida flavificans CBS 760.79 T was isolated from washing of the ion-exchange resin in a guanosine monophosphate manufacturing plant and classified based on DNA base composition, proton-magnetic-resonance spectrum of polysaccharide, and serological characteristics [27]. However, in 1985, it was reclassified as Trichosporiella flavificans CBS 760.79 T due to a stronger coherence between hyphal cells and an absence of arthroconidia that split Trichosporiella from Candida [28]. Sequence analysis of this yeast genus has not been accomplished since then. In the era in which molecular study and sequencing technology play an important role in taxonomic study, yeast classification by the aforementioned analyses may not be sufficient and may also cause misidentifications.
Even if molecular methods and phylogenetic analyses have been used to identify yeast, unstable placement in the evolution line may occur, since they may be characterized based on short sequences and/or a small number of genes in phylogenetic analysis. Candida ghanaensis CBS 8798 T isolated from soil in Ghana, was first described with a weakly supported phylogenetic placement based on the D1/D2 domain of the LSU of rRNA gene by Kurtzman [26]. Subsequent analysis of the SSU of the rRNA gene sequence revealed that C. ghanaensis had a weak and probably insignificant affinity with C. incommunis, but the highest matches in the GenBank database were found with members of the Dipodascus/Geotrichum clade [8]. However, a well-supported placement of this yeast species has not yet established and a phylogenetic reconstruction from additional data was required.
Accurate classification of yeasts may not be achieved by a single method. Although molecular methods are irreplaceable, yeast identification requires combined application of several approaches (polyphasic taxonomy). Similarly, multiple conspecific strains are more reliable to propose a new yeast species. Nevertheless, it will also be more reliable if conspecific strains are isolated from different samples and/or geographical regions.
In this study we described a new yeast species, Crinitomyces reliqui, which is closely related to T. flavificans and C. ghanaensis as they formed a well-supported clade in the phylogenetic trees and they also shared morphological and some physiological characteristics. We proposed here a novel yeast genus, Crinitomyces, to accommodate the novel species Crinitomyces reliqui as well as T. flavificans and C. ghanaensis, which were reassigned as Crinitomyces flavificans and Crinitomyces ghanaensis, respectively.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/jof8030224/s1, Figure S1: Phylogenetic tree based on concatenated sequences of the SSU rRNA gene, the ITS region and the D1/D2 domain of the LSU rRNA gene showing the placement of Trichosporiella flavificans, Candida ghanaensis and Crinitomyces reliqui within the subphylum Saccharomycotina. The phylogenetic tree was constructed using the maximum likelihood (ML) method with optimized models for 3 partitions (SSU, ITS region and LSU) in IQ-TREE. The numbers provided on branches are frequencies with which a given branch appeared in 1000 bootstrap replications. Bootstrap values of less than 50% are not shown. Deakozyma indianensis CBS 12903 T was served as an outgroup species. Bar, 0.1 substitutions per site.