Candida metrosideri pro tempore sp. nov. and Candida ohialehuae pro tempore sp. nov., two antifungal-resistant yeasts associated with Metrosideros polymorpha flowers in Hawaii

Joon Klaps; Clara de Vega; Carlos M. Herrera; Robert R. Junker; Bart Lievens; Sergio Álvarez-Pérez

doi:10.1371/journal.pone.0240093

Abstract

Flowers produce an array of nutrient-rich exudates in which microbes can thrive, making them hotspots for microbial abundance and diversity. During a diversity study of yeasts inhabiting the flowers of Metrosideros polymorpha (Myrtaceae) in the Hawai’i Volcanoes National Park (HI, USA), five isolates were found to represent two novel species. Morphological and physiological characterization, and sequence analysis of the small subunit ribosomal RNA (rRNA) genes, the D1/D2 domains of the large subunit rRNA genes, the internal transcribed spacer (ITS) regions, and the genes encoding the largest and second largest subunits of the RNA polymerase II (RPB1 and RPB2, respectively), classified both species in the family Metschnikowiaceae, and we propose the names Candida metrosideri pro tempore sp. nov. (JK22^T = CBS 16091 = MUCL 57821) and Candida ohialehuae pro tempore sp. nov. (JK58.2^T = CBS 16092 = MUCL 57822) for such new taxa. Both novel Candida species form a well-supported subclade in the Metschnikowiaceae containing species associated with insects, flowers, and a few species of clinical importance. The ascosporic state of the novel species was not observed. The two novel yeast species showed elevated minimum inhibitory concentrations to the antifungal drug amphotericin B (>4 μg/mL). The ecology and phylogenetic relationships of C. metrosideri and C. ohialehuae are also discussed.

Citation: Klaps J, de Vega C, Herrera CM, Junker RR, Lievens B, Álvarez-Pérez S (2020) Candida metrosideri pro tempore sp. nov. and Candida ohialehuae pro tempore sp. nov., two antifungal-resistant yeasts associated with Metrosideros polymorpha flowers in Hawaii. PLoS ONE 15(10): e0240093. https://doi.org/10.1371/journal.pone.0240093

Editor: Andrey M. Yurkov, Leibniz Institut - Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, GERMANY

Received: March 20, 2020; Accepted: September 21, 2020; Published: October 8, 2020

Copyright: © 2020 Klaps et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: RRJ was supported by the Deutsche Forschungsgemeinschaft (DFG, JU2856/2–2) https://www.dfg.de/. SAP acknowledges a ‘Ramón y Cajal’ contract funded by the Spanish Ministry of Science [RYC2018-023847-I] https://www.ciencia.gob.es. The funders had no tole in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Flowers have been increasingly recognized as unexplored reservoirs of yeast diversity due to the production of nutrient-rich exudates, such as floral nectar or stigmatic secretions, in which microbes can thrive [1–3]. Study of yeast diversity in flowers has led during the last decade to the discovery of more than 50 new species of ascomycetes yeasts, dispersed across different continents [2, 4–14]. Additionally, the taxonomic status of several flower-inhabiting yeast species still remains unclear because their phylogenetic affiliation is not yet satisfactorily resolved by available multilocus sequences, and/or they seem unable to produce sexual structures under standard laboratory conditions, making accurate classification challenging. This group of species with uncertain taxonomic placement includes several representatives from the family Metschnikowiaceae currently classified within the anamorphic genus Candida [11], such as Candida auris and the members of the Candida haemulonis species complex, which are closely related to the genus Clavispora. Notably, C. auris and some members of the C. haemulonis species complex are currently receiving increased attention because of their human pathogenic potential [15–17]. Additionally, the aforementioned species often show elevated minimum inhibitory concentrations (MICs) to multiple antifungals [15–21].

Recent mycological surveying of the flowers of the endemic Hawaiian plant species Metrosideros polymorpha (called ʻōhiʻa lehua in the vernacular language) [22] resulted in the cultivation of several isolates of uncertain identity. Sequence analysis of the D1/D2 regions of the large subunit (LSU) rRNA gene and the internal transcribed spacer (ITS) regions showed that the isolates represent two potential novel species from the family Metschnikowiaceae which are genetically distinct from any currently recognized species. The aim of this study was to characterize these novel yeast taxa, including their antifungal susceptibility profile, and to formally describe them as Candida metrosideri pro tempore sp. nov. (JK22^T = CBS 16091 = MUCL 57821) and Candida ohialehuae pro tempore sp. nov. (JK58.2^T = CBS 16092 = MUCL 57822). In addition, we discuss the phylogenetic relationships, antifungal susceptibility, and ecology of these new species.

Materials and methods

Isolates

The flower samples used in this study were collected in 2013 at Hawai’i Volcanoes National Park (Hawaii Island, HI, USA). These samples were part of a larger sample collection from a previous study that aimed to assess the bacterial diversity and community composition associated with the plant species Metrosideros polymorpha Gaud. (Myrtaceae) along an environmental and elevational gradient (see details in Junker and Keller [22]). Metrosideros polymorpha is found in all major islands of the Hawaiian archipelago, where it is distributed in a variety of habitats ranging from near the sea level to approx. 2500 m of altitude. Furthermore, M. polymorpha can grow on a broad range of substrates including recent lava flows and exhibits high resistance to different environmental stressors such as freezing, volcanic vapors, excessive moisture or dryness [23]. On the island of Hawaii, there are six varieties of M. polymorpha that differ in the morphology of vegetative and reproductive organs [24, 25], but our sampling focused exclusively on M. polymorpha var. polymorpha. Peak flowering occurs between February and July, although flowers can be found at any time of the year [26]. Floral visitors of this plant species are both native and introduced birds and insects, including bees, ants, and wasps [27–29], but these were not documented for the present study. As typical for bird-pollinated plants, the flowers of this species produce large amounts of nectar that is presented in cup-like receptacles. The average sugar concentration of M. polymorpha nectar is around 30% w/w [27].

A total of 91 floral samples were used in the present study, including nectar (n = 30), stamens (n = 31) and styles (n = 30) of 33 individual M. polymorpha trees from 14 populations. Five yeast isolates were characterized in this study: i) isolate JK22^T, retrieved from the stamens of a flower collected in a first plant population (GPS coordinates: 19.67, -155.34; 1590 m above the sea level); and ii) isolates JK58.2^T, JK58.4, JK58.5, and JK58.6, obtained from the styles of a single flower collected in a different population (19.37, -155.22; 999 m).

Phylogenetic analyses

Genomic DNA was isolated from yeast colonies as described in Álvarez-Pérez et al. [30]. PCR amplification of the D1/D2 region of the LSU rRNA gene was done using the NL1 and NL4 primers [31], and sequences coding for the small-subunit (SSU) rRNA gene, ITS1, 5.8S rRNA gene and ITS2 were amplified with primers Fungi-18S-up and ITS4 [13]. In addition, the protein-encoding genes RPB1 (largest subunit of RNA polymerase II) and RPB2 (second largest subunit of RNA polymerase II) were amplified with primers RPB1-Af and RPB1-Cr and RBP2-5F and RPB2-7Cr, respectively [32]. In all cases, reaction mixtures contained 5 μL of Titanium Buffer (10X, Clontech), 0.25 mM MgCl₂ (Thermo Scientific), 0.25 mM of each primer (Sigma-Aldrich), 0.5 U Titanium Taq polymerase (50X, Clontech), and 50 ng of template DNA in a final volume of 50 μL. All PCR amplifications were carried out in a BioRad T100 thermocycler and consisted of a denaturation step of 2 min at 95°C, followed by 35 cycles of 30 s at 94°C, 30 s at 52°C and 2 min at 72°C, and a final extension of 10 min at 72°C. PCR products were purified using Thermo Scientific’s GeneJET PCR Purification Kit and sent for two-way Sanger sequencing to Macrogen Europe (Amsterdam, The Netherlands). Sequencing primers were the same as those used for PCR amplification in all cases except for RPB2, for which the large amplicon length made necessary the use of four sequencing primers: RBP2-5F, RPB2-7Cr, RPB2-6F, and RPB2-6R [16]. Upon sequencing, DNA sequences were assembled and manually edited with the program SeqTrace [33]. Next, DNA sequences were aligned using Muscle [34] and the resulting alignments were used to build neighbor joining (NJ) and maximum likelihood (ML) phylogenetic trees, using MEGA X [35] and the online version of PhyML v3.0 (http://www.atgc-montpellier.fr/phyml/ [36]), respectively. NJ analyses were performed using Kimura’s two parameter model, whereas ML analyses were based on the best available model selected by jModeltest v2.1.10 [37]. In all cases, bootstrap support values were obtained from 1000 random resamplings. Consensus trees were visualized and edited in MEGA X.

Phenotypic characterization

Yeast isolates were phenotypically characterized according to the standard procedures described by Kurtzman et al. [38]. Assays testing for fermentations and growth on nitrogen sources were performed in liquid media, and incubated at 25°C for up to 33 and 15 days, respectively. Assimilation of carbon compounds was assessed on agar plates by replica plating and incubated at 25°C for up to 7 days. Determination of the hemolytic activity of isolates was performed by streaking a single colony of each isolate on Columbia blood agar (VWR) and incubating the plates for 3 days at 25°C. The Gram-positive hemolytic bacterium Staphylococcus aureus ATCC 29213^T was used as a positive control in these tests, and the presence of a halo of blood digestion around the colonies was interpreted as a positive result. Growth under microaerobiosis was determined by culturing all isolates on potato dextrose agar (PDA, Neogen) and incubating the plates at 25°C for 5 days in a candle jar. Additionally, growth under anaerobic conditions was evaluated by streaking isolates on PDA and incubating the plates into a jar containing an anaerobiosis generator sachet and anaerobiosis indicator (Microbiology Anaerocult, Merck) for 4 days at 25°C. In both tests, the appearance of yeast colonies on the plates was recorded as a positive result. Further, colony morphology was determined using plate cultures grown on yeast malt agar (YM; 2.0% agar (Oxoid), 1.0% glucose (Sigma-Aldrich), 0.5% Bacto Peptone (Becton, Dickinson and Company, BD), 0.3% malt extract (Oxoid), 0.3% yeast extract (Oxoid), pH 6.2 ± 0.2) or PDA for 48–72 h at 25°C. Single colonies were examined with an SZX10 stereomicroscope (Olympus). Microscopic inspection of cells was performed with an optical microscope (DP74 Microscope Digital Camera, Olympus) using a phase contrast objective. Ascospore formation was analyzed on YM agar, 5% malt extract agar, diluted (1:9 and 1:19) V8 agar, restriction growth agar, Gorodkowa agar, and Custer’s chalk agar [38]. All these media were inoculated by spreading portions of yeast colonies on the agar surface using sterile plastic loops (VWR). Evaluation of mating compatibility was performed by mixing conspecific yeast isolates in every possible pair-wise combination [38], and inoculated plates were incubated for 7 weeks at 20 and 25°C. Since no conspecific isolates were available for JK22^T, mating tests were not performed for this isolate.

Finally, given the close phylogenetic relatedness of the studied isolates with multi-drug resistant fungal pathogens such as Candida auris and C. haemulonis (see Results and discussion), we determined the in vitro antifungal susceptibility of such isolates by the commercially prepared Sensititre YeastOne YO10 colorimetric antifungal panel (Thermo Fisher). Antifungals included in this test were anidulafungin, amphotericin B, micafungin, 5-flucytosine, and a number of azoles including fluconazole, itraconazole, posaconazole and voriconazole. On the day of the assay, suspensions of 1.5–8×10³ cells/mL were prepared in YeastOne inoculum broth, and the dried panels were rehydrated by dispensing 100 μL of yeast suspension into each well. Assay plates were covered with adhesive seals and incubated at 25°C for 48 h. The MIC endpoints were defined as the lowest concentration of the antifungal drugs preventing the development of a red color (i.e. first blue or purple well). All isolates were tested twice on different days.

Ethics

Ethical approval for collecting plant samples at Hawaii Volcanoes National Park was granted by the USA Department of the Interior -National Park Service on March 2014 (HAVO-2013-SCI-003). Accessed lands are protected.

Nomenclature

The electronic version of this article in Portable Document Format (PDF) in a work with an ISSN or ISBN will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies.

In addition, new names contained in this work have been submitted to MycoBank from where they will be made available to the Global Names Index. The unique MycoBank number can be resolved and the associated information viewed through any standard web browser by appending the MycoBank number contained in this publication to the prefix http://www.mycobank.org/MB/. The online version of this work is archived and available from the following digital repositories: PubMed Central and Zenodo.

Results and discussion

The overall frequency of yeasts occurrence in the samples analyzed (n = 91) was 55%. In total, 80 yeast isolates were obtained, out of which five isolates were representative of two undescribed species. Other yeasts retrieved from the flowers of M. polymorpha were identified as Metschnikowia rancensis (n = 21 isolates), Aureobasidium melanogenum (n = 15), Starmerella bombicola (n = 8), Debaryomyces vindobonensis (n = 7), Rhizosphaera macrospora (n = 5), Wickerhamiella azyma (n = 4), Metschnikowia hawaiiana (n = 2), Papiliotrema terrestris (n = 2), Priceomyces melissophilus (n = 2), Curvibasidium pallidicorallinum (n = 2), Starmerella apicola (n = 1), Starmerella bombi (n = 1), Starmerella sorbosivorans (n = 1), Dimennazyma cisti-albidi (n = 1), Filobasidium globisporum (n = 1), Kodamaea ohmeri (n = 1) and Rhodotorula mucilaginosa (n = 1).

Analysis of the D1/D2 region of the LSU rRNA gene (JK22^T = 522 bp, JK58.2^T = 515 bp) from the studied isolates revealed that all of them had a low sequence identity with the sequences included in GenBank (<93.5%). One of these isolates is described in the present article as Candida metrosideri sp. nov. (JK22^T = CBS 16091 = MUCL 57821), and the other four isolates as Candida ohialehuae sp. nov. (type strain: JK58.2^T = CBS 16092 = MUCL 57822) (Table 1). All isolates belonging to C. ohialehuae were retrieved from the styles of the same M. polymorpha flower, while the isolate representing C. metrosideri was found in the stamens of a different M. polymorpha flower. Furthermore, C. metrosideri was co-isolated with Metschnikowia rancensis, whereas C. ohialehuae was found to co-occur with M. rancensis and Debaryomyces hansenii.

Download:

Table 1. Isolation sources and GenBank accession numbers of the yeast isolates characterized in this study.

https://doi.org/10.1371/journal.pone.0240093.t001

Phylogenetic affiliation

Candida ohialehuae showed three D1/D2 intraspecific sequence variants. JK58.2^T differed in the D1/D2 sequence by one nucleotide substitution from isolates JK58.4, JK58.5, which have identical sequences, and isolate JK58.6 diverged by one gap. No polymorphism was detected among C. ohialehuae isolates for the ITS, RPB1, and RPB2 sequences. Comparison of the D1/D2 region of the LSU rRNA gene obtained for C. metrosideri and C. ohialehuae revealed a total of 68 substitutions and 15 gaps (15.9% variation).

Phylogenetic analysis of the D1/D2 region of the LSU domain obtained by NJ and ML methods revealed that C. metrosideri clusters with Candida duobushaemulonis, Candida vulturna, and Candida pseudohaemulonis, whereas C. ohialehuae forms a well-supported sister clade with Candida konsanensis (42 substitutions and 10 indels, 9.7% variation), Candida heveicola (45 substitutions and 8 indels, 10.3% variation), and Candida chantaburiensis (46 substitutions and 10 indels, 10.8% variation) (Fig 1 and Table 2). The clade including both new species and the aforementioned close relatives was found to be well supported (88% and 98.6% bootstrap in the NJ and ML trees, respectively).

Download:

Fig 1. Phylogenetic placement of Candida metrosideri and Candida ohialehuae based on DNA sequence analysis of the D1/D2 domains of the large subunit (LSU) rRNA gene, as obtained by maximum likelihood analysis.

Evolutionary distances were computed using the Hasegawa, Kishino and Yano (HKY85) model and are in the units of the number of base substitutions per site (see the scale). The rate variation among sites was modeled with a gamma distribution (shape parameter = 0.69) and a proportion of invariant sites (0.46). There was a total of 561 positions in the final alignment. Bootstrap node support values, based on 1000 replicates, ≥70% are shown next to the branches. Additionally, NJ (Kimura-2-parameter model, gamma distribution’s shape parameter = 0.69) bootstrap node support values ≥70% are marked by asterisks.

https://doi.org/10.1371/journal.pone.0240093.g001

Download:

Table 2. Sequence divergence from Candida metrosideri JK22^T to closely related species based on nucleotide sequences obtained from selected loci^{^a}.

https://doi.org/10.1371/journal.pone.0240093.t002

Previous studies have shown that strains belonging to the same yeast species usually differ by less than 1% mismatches in the D1/D2 domain [31, 39, 40] (but see Lachance et al. [41] for a description of unusual intra-species polymorphism in this gene region in Clavispora lusitaniae). The significant sequence divergence in the D1/D2 region displayed by C. ohialehuae and C. metrosideri between them and with respect to previously described species suggests that these might deserve the consideration of new yeast taxa. Nevertheless, in order to confirm our taxonomic proposal and the phylogenetic placement of C. metrosideri and C. ohialehuae, we built additional phylogenies based on the ITS region (ITS1 + 5.8S + ITS2: JK22^T, 242 bp; JK58.2^T, 243 bp; S1 Fig), the RPB1 (JK22^T, 657 bp; JK58.2^T, 657 bp; S2 Fig) and RPB2 (JK22^T, 1055 bp; JK58.2^T, 1076 bp; S3 Fig) genes, and a concatenation of LSU rRNA + ITS + RPB1 + RPB2 sequences (Fig 2). Unfortunately, most clades observed in the ITS tree were only poorly supported (bootstrap values <70%; S1 Fig), and the absence of RPB1 and RPB2 sequences in public databases for several species closely related to the genus Clavispora hindered further confirmation of the phylogenetic placement of most taxa. Notably, the clustering of C. metrosideri, and C. ohialehuae with C. doubushaemulonis, C. haemulonis and C. pseudohaemulonis was confirmed by phylogenetic analysis of the LSU rRNA + ITS + RPB1 + RPB2 concatamer (Fig 2).

Download:

Fig 2. Phylogenetic placement of Candida metrosideri and Candida ohialehuae based on the analysis of concatenated LSU rRNA + ITS + RPB1 + RPB2 (2069 bp in total), as obtained by maximum likelihood.

Evolutionary distances were computed using the general time-reversible (GTR) model and are in the units of the number of base substitutions per site (see the scale). The rate variation among sites was modeled with a gamma distribution (shape parameter = 0.39) and a proportion of invariant sites (0.14). Bootstrap node support values, based on 1000 replicates, ≥70% are shown next to the branches. Additionally, NJ (Kimura-2-parameter model, gamma distribution’s shape parameter = 0.28) bootstrap node support values ≥70% are marked by asterisks. The tree was rooted on Clavispora opuntiae CBS 7068.

https://doi.org/10.1371/journal.pone.0240093.g002

Although the phylogenetic analyses performed suggested that the new species described here belong to the family Metschnikowiaceae and are closely related to Clavispora, studied isolates cannot be unequivocally assigned to this genus. Additionally, previous studies have demonstrated that C. auris and C. haemulonis are distant from Clavispora lusitaniae and do not constitute a monophyletic group [20, 42]. Therefore, we propose to refer to these novel species as Candida metrosideri pro tempore sp. nov. and Candida ohialehuae pro tempore sp. nov. The term ‘pro tempore’ was proposed to indicate lineages that are temporary generic assignments in absence of further taxonomic evidence [20, 43].

Phenotypic characterization

The results of the physiological tests for the novel species C. metrosideri and C. ohialehuae, and close phylogenetic relatives are shown in Table 3 and S1 Table, and the micromorphology of some of the studied isolates is shown in Fig 3. Candida ohialehuae ferments both glucose and sucrose as do most of its closest relatives, including C. heveicola, C. konsanensis, C. vulturna, and C. duobushaemulonis. In contrast, C. metrosideri and C. chantaburiensis can ferment glucose but not sucrose. Fermentation of fructose, which together with sucrose and glucose is a sugar commonly found in flowers (e.g. in floral nectar), was not observed for C. metrosideri, whereas C. ohialehuae showed a delayed positive reaction. Notably, C. metrosideri is unable to assimilate galactose, whereas the type strains of close phylogenetic relatives can assimilate this sugar. On the other hand, C. ohialehuae and C. auris cannot assimilate citric acid and succinic acid. Additionally, C. ohialehuae and C. metrosideri together with C. vulturna can assimilate potassium nitrate and sodium nitrite, whereas all other closely-related species cannot. Ethanol, which in floral microhabitats can be released by yeasts as a fermentation by-product [44], is not assimilated by C. vulturna, C. ohialehuae and C. chantaburiensis, but their closest relatives can. Both C. metrosideri and C. ohialehuae can endure strong osmotic pressures (up to 60% w/w for some tested isolatess) and high saline solutions (10% NaCl), traits which are shared by most of the closely related species. These traits allow the species to be well adapted to environments with low water activity. Finally, C. metrosideri, C. ohialehuae and their sister species C. vulturna, C. heveicola and C. konsanensis are able to grow in vitamin-free medium. Finally, ascospore production was not observed for any of the pure or mixed cultures of C. ohialehuae on common sporulation media (see Materials and methods) after eight weeks of incubation at 20 or 25°C.

Download:

Fig 3. Micromorphology of Candida metrosideri and Candida ohialehuae.

Multipolar budding and pseudohyphae of Candida ohialehuae JK58.2^T (A, C and D, respectively), and budding cells of Candida metrosideri JK22^T (B).

https://doi.org/10.1371/journal.pone.0240093.g003

Download:

Table 3. Phenotypic characteristics of Candida metrosideri, Candida ohialehuae and their close phylogenetic relatives.

A complete version of this table is presented as S1 Table.

https://doi.org/10.1371/journal.pone.0240093.t003

Thermotolerance and hemolytic activity are considered important virulence factors that help mammal pathogens to survive inside their hosts [45]. Tested isolates of C. metrosideri and C. ohialehuae did not display any hemolytic activity and were unable to grow at 37°C. In contrast, closely-related species such as C. auris, C. ruelliae, C. haemulonis, C. duobushaemulonis, C. pseudohaemulonis, C. vulturna, C. heveicola, C. chantaburiensis, and C. konsanensis can grow at 37°C, and most of them have been repeatedly isolated from clinical samples [16, 19–21, 46–49]. However, based on the study performed we cannot discard the possibility that C. ohialehuae and C. metrosideri might act as opportunistic pathogens of humans and/or animals that have niches outside clinical and endothermic environments in which they can thrive [50]. Such possibility should be tested in the future, e.g. by using vertebrate and/or invertebrate animal models of fungal infection [51, 52].

Due to the close phylogenetic relatedness of C. metrosideri and C. ohialehuae to other yeast species whose members often display resistance to multiple antifungals [16–21, 49], we analyzed the in vitro susceptibility of our isolates to amphotericin B, 5-fluorocytosine, and a number of azoles and echinocandins. The results of these tests (Table 4) revealed that C. metrosideri and C. ohialehuae are sensitive to azoles and 5-fluorocytosine. Anidulafungin and micafungin MICs were also relatively low for C. metrosideri (0.25 and 0.06 μg/mL, respectively), but moderately elevated for C. ohialehuae (1–2 and 0.5 μg/mL). In contrast, both species showed elevated MIC values for amphotericin B (≥4 μg/mL in all cases). Amphotericin B binds to ergosterol, inserts into the cytoplasmic membrane and forms pore-like structures resulting in osmotic instability, loss of membrane integrity, metabolic disruption, and ultimately cell death [53, 54]. Decreased susceptibility to amphotericin B is often caused by a decrease of the ergosterol content in the plasmalemma or a change in the target lipid as a consequence of mutations in the genes encoding the ergosterol biosynthesis pathway [55]. Nevertheless, resistance to amphotericin B often results in a decreased fitness and ability of strains to survive in animal hosts [56, 57].

Download:

Table 4. Antifungal susceptibility profile of the Candida metrosideri and Candida ohialehuae isolates characterized in this study.

https://doi.org/10.1371/journal.pone.0240093.t004

Adaptation to plants

As both C. metrosideri and C. ohialehuae were isolated from floral parts (stamens and styles, respectively) and most of their close phylogenetic relatives were originally found in plant material of tropical regions near the Pacific ocean (e.g. C. ruelliae [19], C. heveicola [49], C. chantaburiensis [47], C. konsanensis [48], and C. vulturna [20]), it can be hypothesized that these yeasts may be adapted to efficient growth on plant surfaces. In this regard, the phenotypic assays carried out in this study confirmed that both C. metrosideri and C. ohialehuae can assimilate the three main simple sugars present in floral nectar and other plant secretions (i.e. glucose, fructose, sucrose), and also other carbohydrates that are less abundant but fairly common in those habitats (e.g. xylose and maltose). Furthermore, both C. metrosideri and C. ohialehuae together with other closely-related species can grow in high osmotic (up to 60% w/w of glucose in most cases) and microaerophilic conditions, confirming that these yeasts can withstand some of the prevailing selective pressures of floral microhabitats [58–61]. Interestingly, none of these yeasts have ever been detected in nectar despite their capabilities of enduring nectar’s selective pressures, whereas other Metschnikowiaceae members such as Metschnikowia reukaufii, M. gruessii and M. rancensis have been identified as the main fungal inhabitants of floral nectar [62]. In this regard, it should be noticed that microhabitat heterogeneity can have an impact on the microbiota hosted by different flower organs [22]. Besides, the nectar of insect- and bird-pollinated plants often has different properties that select for specific microbial species [63]. As M. polymorpha flowers are visited by both birds and insects, it should be investigated if the relative frequency of visitation by each of these groups has any impact on its nectar yeast communities. Finally, in order to be dispersed from flower to flower, flower microbes need to be capable of surviving in/on the body of the animal visitors of flowers, which remains to be studied in most cases (but see Pozo et al., [64]). Therefore, the ability of C. metrosideri and C. ohialehuae to colonize and thrive in nectar, floral surfaces, other plant-associated habitats and the body of the animal visitors of flowers should be further assessed in future studies.

Taxonomy

The description of the two new species presented in this paper is based on a single isolate, case of Candida metrosideri, or on multiple isolates retrieved from the same sample, case of C. ohialehuae (but note that, nevertheless, these C. ohialehuae isolates does not seem to be strictly clonal, as they presented D1/D2 intraspecific sequence variations and some phenotypic differences; see Tables 3 and 4 and S1 Table). Despite detailed inspection of the yeast isolates obtained from other flower samples, no additional isolates conspecific with C. metrosideri or C. ohialehuae were found. BLAST searches of D1/D2 LSU rRNA gene sequences obtained for the studied isolates demonstrated these diverged significantly from any other know species (6.5% for C. metrosideri and 9.7% for C. ohialehuae). Moreover, by analyzing the phylogenetic affiliation, it can be seen that both new species are close relatives of several emerging multi-drug resistant pathogens. Given the (phylo)genetic differentiation of C. metrosideri and C. ohialehuae with respect to other Metschnikowiaceae members and the decreased susceptibility of studied isolates to amphotericin B and some echinocandins, we consider that our taxonomic proposal is relevant enough.

Description of Candida metrosideri pro tempore Klaps, de Vega, Herrera, Junker, Lievens & Álvarez-Pérez 2020 sp. nov. [Mycobank accession: MB 832536].

Etymology: Specific epithet metrosideri [me.tro.si’de.ri. L. fem. s. Metrosideros -i], referring to the genus name of the host plant from which the yeast species was first isolated.

Standard description. This description is based on the characteristics of a single isolate (JK22^T). On YM agar at 25°C after 48h colonies are white to cream, soft and butyrous, with an entire margin and a size ranging from 0.5 mm to 1 mm. Pseudohyphae or true mycelium are not formed. Cells are rounded to slightly ovoid (5–8 × 7–8 μm) and occur singly or in parent-bud pairs. The presence of vacuoles of different sizes is common, and cell budding is typically monopolar on a narrow base. Glucose and trehalose are weakly fermented. Galactose, sucrose, maltose, lactose, raffinose, melezitose, fructose, cellobiose, and melibiose are not fermented. Glucose, fructose, inulin, sucrose, trehalose, maltose, melezitose, methyl-α-D-glucoside (slowly), cellobiose, salicin, starch, L-sorbose (slowly), D-xylose (slowly), D-ribose, ethanol, glycerol, ribitol (slowly), xylitol, D-mannitol, D-glucitol, gluconolactone, 2-keto-D-gluconate, succinate, citrate (slowly), D-gluconate, D-glucosamine, N-acetyl-D-glucosamine, nitrate, nitrite (weakly), ethylamine, lysine, cadaverine, ammonium, L-ornithine, D-tryptophan, and urea are assimilated. Raffinose, melibiose, galactose, lactose, L-rhamnose, L-arabinose, D-arabinose, meso-erythritol, galactitol, myo-inositol, 5-keto-D-gluconate, DL-lactate, methanol, and hexadecane are not assimilated. Growth occurs in media containing 10% NaCl, 50% glucose, 0.01% cycloheximide, and 0.1% cycloheximide, but not 1% (v/v) acetic acid or 60% glucose. Growth is positive in vitamin-free medium (slowly), microaerobiosis, anaerobiosis (weakly), and at 10°C (slowly), 20°C, 25°C, 30°C, and negative at 4°C and 37°C. Tween 80 is hydrolyzed and arbutin is split. Acid production from glucose is weak. Gelatin and urea are not hydrolyzed, and no hemolytic activity is displayed.

The holotype JK22^T was isolated from the stamens of a Metrosideros polymorpha flower collected in the Hawai’i Volcanoes National Park (Hawaii Island, HI, USA), and is permanently preserved as metabolically inactive cultures, isotypes, in the CBS yeast collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands (CBS 16091) and the Mycothèque de l’Université Catholique de Louvain (BCCM/MUCL), Louvain-la-Neuve, Belgium (MUCL 57821).

Description of Candida ohialehuae pro tempore Klaps, de Vega, Herrera, Junker, Lievens & Álvarez-Pérez 2020 sp. nov. [Mycobank accession: MB 832537].

Etymology: Specific epithet ohialehuae [o.hia.le.hua.’e. L. fem. s. ʻōhiʻa lehua -e], referring to the vernacular name of the host plant from which this yeast species was first isolated.

Standard description. This description is based on the characteristics of four isolates (JK58.2^T, JK58.4, JK58.5, and JK58.6). On YM agar at 25°C after 48h colonies are white, soft and butyrous, flat or slightly domed, with a smooth or slightly wrinkled surface and entire margin. Colony size ranges from 1 mm to 2 mm. True mycelium is not formed. Cells are rounded to slightly ovoid (3–5 × 3–5 μm) and occur singly or in small groups. The presence of vacuoles is common, and cell budding is generally multipolar on a narrow base. Some isolates (JK58.2^T, JK58.4, but not JK58.5 and JK58.6) form pseudohyphae after 8 weeks at 25°C on Custer’s chalk agar, but not on any other tested medium. Ascospores have not been observed in pure or mixed cultures on common sporulation media after eight weeks of incubation at 20 or 25°C. Glucose, fructose (latent), raffinose (weakly), sucrose (weakly), and trehalose (weakly) are fermented. Galactose, maltose, lactose, melezitose, cellobiose, and melibiose are not fermented. Glucose, fructose, inulin, sucrose, raffinose (slowly), galactose (slowly), trehalose, maltose, melezitose (weakly), starch, L-sorbose, D-xylose (slowly), glycerol, ribitol (slowly), galactitol (slowly), D-mannitol, D-glucitol, gluconolactone, 2-keto-D-gluconate, D-gluconate, D-glucosamine, N-acetyl-D-glucosamine, nitrate, nitrite (weakly), ethylamine, lysine, cadaverine, ammonium, L-ornithine, D-tryptophan, and urea are assimilated. Melibiose, lactose, methyl-α-D-glucoside, cellobiose, salicin, L-rhamnose, L-arabinose, D-arabinose, D-ribose, ethanol, meso-erythritol, xylitol, myo-inositol, 5-keto-D-gluconate, succinate, citrate, DL-lactate, methanol, hexadecane, are not assimilated. Growth occurs in media containing 10% NaCl, 50% glucose, 60% glucose (slowly), 0.01% cycloheximide, and 0.1% cycloheximide. All tested isolates split arbutin, produce acids from glucose, and can grow in microaerobiosis, anaerobiosis and vitamin-free medium (slowly), but not on media containing 1% (v/v) acetic acid. Growth is positive at 10°C (slowly), 20 °C, 25°C, 30°C, and negative at 4°C and 37°C. Gelatin, urea, and Tween 80 are not hydrolyzed, and no isolate shows hemolytic activity.

The holotype JK58.2^T was isolated from the styles of a Metrosideros polymorpha flower collected in the Hawai’i Volcanoes National Park (Hawaii Island, HI, USA), and is permanently preserved as metabolically inactive cultures, isotypes, in the CBS yeast collection of the Westerdijk Fungal Biodiversity Institute, Utrecht, the Netherlands (CBS 16092) and the Mycothèque de l’Université Catholique de Louvain (BCCM/MUCL), Louvain-la-Neuve, Belgium (MUCL 57822).

Conclusion

The phylogenetic and phenotypic evidence provided in this study confirms that the studied isolates belong to the Candida haemulonis clade in the family Metschnikowiaceae, representing two undescribed species for which we propose the names Candida metrosideri pro tempore sp. nov. and Candida ohialehuae pro tempore sp. nov. The ecology and clinical relevance of these novel species should be further explored in future studies, as well as the causes and possible consequences of their decreased susceptibility to amphotericin B.

Supporting information

S1 Fig. Phylogenetic placement of Candida metrosideri and Candida ohialehuae based on DNA sequence analysis of the ITS1-5.8S-ITS2 rDNA region, as obtained by maximum likelihood analysis.

Evolutionary distances were computed using the general time-reversible (GTR) model and are in the units of the number of base substitutions per site (see the scale). There was a total of 374 positions in the final alignment. The rate variation among sites was modeled with a gamma distribution (shape parameter = 0.18). Bootstrap node support values, based on 1000 replicates, ≥70% are shown next to the branches. Additionally, NJ (Kimura-2-parameter model, gamma distribution’s shape parameter = 0.18) bootstrap node support values ≥70% are marked by asterisks.

https://doi.org/10.1371/journal.pone.0240093.s001

(TIFF)

S2 Fig. Phylogenetic placement of Candida metrosideri and Candida ohialehuae based on DNA sequence analysis of the RPB1 gene, as obtained by maximum likelihood analysis.

Evolutionary distances were computed using the general time-reversible (GTR) model and are in the units of the number of base substitutions per site (see the scale). There was a total of 416 positions in the final alignment. The rate variation among sites was modeled with a gamma distribution (shape parameter = 0.27). Bootstrap node support values, based on 1000 replicates, ≥70% are shown next to the branches. Additionally, NJ (Kimura-2-parameter model, gamma distribution’s shape parameter = 0.69) bootstrap node support values ≥70% are marked by asterisks.

https://doi.org/10.1371/journal.pone.0240093.s002

(TIFF)

S3 Fig. Phylogenetic placement of Candida metrosideri and Candida ohialehuae based on DNA sequence analysis of the RPB2 gene, as obtained by maximum likelihood analysis.

Evolutionary distances were computed using the general time-reversible (GTR) model and are in the units of the number of base substitutions per site (see the scale). There was a total of 576 positions in the final alignment. The rate variation among sites was modeled with a gamma distribution (shape parameter = 1.26) and a proportion of invariant sites (0.51). Bootstrap node support values, based on 1000 replicates, ≥70% are shown next to the branches. Additionally, NJ (Kimura-2-parameter model, gamma distribution’s shape parameter = 1.26) bootstrap node support values ≥70% are marked by asterisks.

https://doi.org/10.1371/journal.pone.0240093.s003

(TIFF)

S1 Table. Complete phenotypic characteristics of Candida metrosideri, Candida ohialehuae and their close phylogenetic relatives.

https://doi.org/10.1371/journal.pone.0240093.s004

(DOCX)

Acknowledgments

The authors thank Robin Gottlieb for assistance in the field, Curtis C. Daehler for organizational support and the United States Department of the Interior (National Park Service) and Rhonda Loh for the scientific research and collecting permit (HAVO-2013-SCI-0003). The authors also thank Dr. Sofie Houtmeyer’s assistance with the optical microscope at KU Leuven Campus De Nayer.

References

1. Aleklett K, Hart M, Shade A. The microbial ecology of flowers: an emerging frontier in phyllosphere research. Botany 2014;92: 253–266.
- View Article
- Google Scholar
2. de Vega C, Albaladejo RG, Guzmán B, Steenhuisen SL, Johnson SD, Herrera CM, et al. Flowers as a reservoir of yeast diversity: description of Wickerhamiella nectarea f.a. sp. nov., and Wickerhamiella natalensis f.a. sp. nov. from South African flowers and pollinators, and transfer of related Candida species to the genus Wickerhamiella as new combinations. FEMS Yeast Res. 2017;17(5): pmid:28810705
- View Article
- PubMed/NCBI
- Google Scholar
3. Klaps J, Lievens B, Álvarez-Pérez S. Towards a better understanding of the role of fungi in plant-animal interactions. Fungal Biol Biotechnol. 2020;7:1. pmid:31921433
- View Article
- PubMed/NCBI
- Google Scholar
4. Alimadadi N, Soudi MR, Wang SA, Wang QM, Talebpour Z, Bai FY. Starmerella orientalis f.a., sp. nov., an ascomycetous yeast species isolated from flowers. Int J Syst Evol Microbiol. 2016;66(3): 1476–1481. pmid:26780917
- View Article
- PubMed/NCBI
- Google Scholar
5. Amoikon TLS, Grondin C, Djéni TN, Jacques N, Casaregola S. Starmerella reginensis f.a., sp. nov. and Starmerella kourouensis f.a., sp. nov., isolated from flowers in French Guiana. Int J Syst Evol Microbiol. 2018;68(7):2299–2305. pmid:29781796
- View Article
- PubMed/NCBI
- Google Scholar
6. de Vega C, Albaladejo RG, Lachance MA. Metschnikowia maroccana f.a., sp. nov., a new yeast species associated with floral nectar from Morocco. Int J Syst Evol Microbiol. 2018;68(6):2028–2035. pmid:29688165
- View Article
- PubMed/NCBI
- Google Scholar
7. de Vega C, Guzmán B, Lachance MA, Steenhuisen SL, Johnson SD, Herrera CM. Metschnikowia proteae sp. nov., a nectarivorous insect-associated yeast species from Africa. Int J Syst Evol Microbiol. 2012;62(Pt 10):2538–2545. pmid:22407789
- View Article
- PubMed/NCBI
- Google Scholar
8. de Vega C, Guzmán B, Steenhuisen SL, Johnson SD, Herrera CM, Lachance MA. Metschnikowia drakensbergensis sp. nov. and Metschnikowia caudata sp. nov., endemic yeasts associated with Protea flowers in South Africa. Int J Syst Evol Microbiol. 2014;64(Pt 11):3724–3732. pmid:25106927
- View Article
- PubMed/NCBI
- Google Scholar
9. Lachance MA, Bowles JM, Starmer WT. Geography and niche occupancy as determinants of yeast biodiversity: the yeast–insect–morning glory ecosystem of Kípuka Puaulu, Hawai’i. FEMS Yeast Res. 2003;4(1):105–111. pmid:14554202
- View Article
- PubMed/NCBI
- Google Scholar
10. Lachance MA, Ewing CP, Bowles JM, Starmer WT. Metschnikowia hamakuensis sp. nov., Metschnikowia kamakouana sp. nov. and Metschnikowia mauinuiana sp. nov., three endemic yeasts from Hawaiian nitidulid beetles. Int J Syst Evol Microbiol. 2005;55(Pt 3):1369–1377. pmid:15879284
- View Article
- PubMed/NCBI
- Google Scholar
11. Lachance MA. Metschnikowia: half tetrads, a regicide and the fountain of youth. Yeast. 2016;33(11):563–574. pmid:27599462
- View Article
- PubMed/NCBI
- Google Scholar
12. Li SL, Li ZY, Yang LY, Zhou XL, Dong MH, Zhou P, et al. Starmerella jinningensis sp. nov., a yeast species isolated from flowers of Erianthus rufipilus. Int J Syst Evol Microbiol. 2013;63(Pt 1):388–92. pmid:23104359
- View Article
- PubMed/NCBI
- Google Scholar
13. Sipiczki M, Kajdacsi E. Jaminaea angkorensis gen. nov., sp. nov., a novel anamorphic fungus containing an S943 nuclear small-subunit rRNA group IB intron represents a basal branch of Microstromatales. Int J Syst Evol Microbiol. 2009;59(Pt 4):914–920. pmid:19329628
- View Article
- PubMed/NCBI
- Google Scholar
14. Sipiczki M. Starmerella syriaca f.a., sp. nov., an osmotolerant yeast species isolated from flowers in Syria. Antonie Van Leeuwenhoek. 2015;107(4):847–856. pmid:25583140
- View Article
- PubMed/NCBI
- Google Scholar
15. Casadevall A, Kontoyiannis DP, Robert V. On the Emergence of Candida auris: climate change, azoles, swamps, and birds. mBio. 2019;10(4):e01397–19. pmid:31337723
- View Article
- PubMed/NCBI
- Google Scholar
16. Cendejas-Bueno E, Kolecka A, Alastruey-Izquierdo A, Theelen B, Groenewald M, Kostrzewa M, et al. Reclassification of the Candida haemulonii complex as Candida haemulonii (C. haemulonii group I), C. duobushaemulonii sp. nov. (C. haemulonii group II), and C. haemulonii var. vulnera var. nov.: three multiresistant human pathogenic yeasts. J Clin Microbiol. 2012;50(11):3641–3651. pmid:22952266
- View Article
- PubMed/NCBI
- Google Scholar
17. Spivak ES, Hanson KE. Candida auris: an emerging fungal pathogen. J Clin Microbiol. 2018;56(2):e01588–17. pmid:29167291
- View Article
- PubMed/NCBI
- Google Scholar
18. Chowdhary A, Prakash A, Sharma C, Kordalewska M, Kumar A, Sarma S, et al. A multicenter study of antifungal susceptibility patterns among 350 Candida auris isolates (2009–17) in India: role of the ERG11 and FKS1 genes in azole and echinocandin resistance. J Antimicrob Chemother. 2018;73(4):891–899. pmid:29325167
- View Article
- PubMed/NCBI
- Google Scholar
19. Saluja P, Prasad GS. Candida ruellia sp. nov., a novel yeast species isolated from flowers of Ruellia sp. (Acanthaceae). FEMS Yeast Res. 2008;8(4):660–666. pmid:18422614
- View Article
- PubMed/NCBI
- Google Scholar
20. Sipiczki M, Tap RM. Candida vulturna pro tempore sp. nov., a dimorphic yeast species related to the Candida haemulonis species complex isolated from flowers and clinical sample. Int J Syst Evol Microbiol. 2016;66(10):4009–4015. pmid:27411802
- View Article
- PubMed/NCBI
- Google Scholar
21. Sugita T, Takashima M, Poonwan N, Mekha N. Candida pseudohaemulonii sp. nov., an amphotericin B- and azole-resistant yeast species, isolated from the blood of a patient from Thailand. Microbiol Immunol. 2006;50(6):469–473. pmid:16785719
- View Article
- PubMed/NCBI
- Google Scholar
22. Junker RR, Keller A. Microhabitat heterogeneity across leaves and flower organs promotes bacterial diversity. FEMS Microbiol Ecol. 2015;91(9):fiv097. pmid:26253507
- View Article
- PubMed/NCBI
- Google Scholar
23. Mueller-Dombois D, Fosberg FR. Vegetation of the Tropical Pacific Islands. New York NY USA: Springer-Verlag; 1998.
24. Dawson JW, Stemmermann RL. Metrosideros Banks ex Gaertn. In: Wagner WL, Herbst DR, Sohmer S. Manual of the flowering plants of Hawai'i. Honolulu HI USA: University of Hawai'i Press; 1999. pp. 964–970.
25. James SA, Puttock CF, Cordell S, Adams RP. Morphological and genetic variation within Metrosideros polymorpha (Myrtaceae) on Hawai'i. N Z J Bot. 2004;42(2):263–270.
- View Article
- Google Scholar
26. Ralph CJ, Fancy SG. Demography and movements of Apapane and Iiwi in Hawaii. Condor. 1995;97:729–742.
- View Article
- Google Scholar
27. Junker RR, Bleil R, Daehler CC, Bluthgen N. Intra-floral resource partitioning between endemic and invasive flower visitors: consequences for pollinator effectiveness. Ecological Entomology. 2010;35:760–767.
- View Article
- Google Scholar
28. Hanna C, Foote D, Kremen C. Invasive species management restores a plant–pollinator mutualism in Hawaii. J Appl Ecol. 2013;50(1):147–155.
- View Article
- Google Scholar
29. Hanna C, Foote D, Kremen C. Competitive impacts of an invasive nectar thief on plant–pollinator mutualisms. Ecology, 2014;95(6):1622–1632. pmid:25039226
- View Article
- PubMed/NCBI
- Google Scholar
30. Alvarez-Perez S, Blanco JL, Alba P, Garcia ME. Mating type and invasiveness are significantly associated in Aspergillus fumigatus. Med Mycol. 2010;48(2): 273–277. pmid:19634068
- View Article
- PubMed/NCBI
- Google Scholar
31. Kurtzman CP, Robnett CJ. Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie Van Leeuwenhoek. 1998;73(4):331–371. pmid:9850420
- View Article
- PubMed/NCBI
- Google Scholar
32. Liu YJ, Whelen S, Hall BD. Phylogenetic relationships among ascomycetes: evidence from an RNA polymerase II subunit. Mol Biol Evol. 1999;16(12):1799–1808. pmid:10605121
- View Article
- PubMed/NCBI
- Google Scholar
33. Stucky JB. SeqTrace: a graphical tool for rapidly processing dna sequencing chromatograms. J Biomol Tech. 2012;23(3):90–93. pmid:22942788
- View Article
- PubMed/NCBI
- Google Scholar
34. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32(5):1792–1797. pmid:15034147
- View Article
- PubMed/NCBI
- Google Scholar
35. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018 Jun 1;35(6):1547–1549. pmid:29722887
- View Article
- PubMed/NCBI
- Google Scholar
36. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol. 2010;59(3):307–21. pmid:20525638
- View Article
- PubMed/NCBI
- Google Scholar
37. Darriba D, Taboada GL, Doallo R, Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nat Methods. 2012;9(8):772. pmid:22847109
- View Article
- PubMed/NCBI
- Google Scholar
38. Kurtzman CP, Fell J, Boekhout T. Methods for isolation, phenotypic characterization and maintenance of yeasts. In: Kurtzman CP, Fell J, Boekhout T, editors. The Yeasts, a Taxonomic Study. San Diego: Elsevier; 2011. pp. 87–110.
39. Fell JW, Boekhout T, Fonseca A, Scorzetti G, Statzell-Tallman A. Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis. Int J Syst Evol Microbiol. 2000;50 Pt 3:1351–1371. pmid:10843082
- View Article
- PubMed/NCBI
- Google Scholar
40. Vu D, Groenewald M, Szöke S, Cardinali G, Eberhardt U, Stielow B, et al. DNA barcoding analysis of more than 9000 yeast isolates contributes to quantitative thresholds for yeast species and genera delimitation. Stud Mycol. 2016;85:91–105. pmid:28050055
- View Article
- PubMed/NCBI
- Google Scholar
41. Lachance MA, Daniel HM, Meyer W, et al. The D1/D2 domain of the large-subunit rDNA of the yeast species Clavispora lusitaniae is unusually polymorphic. FEMS Yeast Res. 2003;4(3):253–258. pmid:14654429
- View Article
- PubMed/NCBI
- Google Scholar
42. Shen XX, Opulente DA, Kominek J, et al. Tempo and Mode of Genome Evolution in the Budding Yeast Subphylum Cell. 2018;175(6):1533–1545.e20. pmid:30415838
- View Article
- PubMed/NCBI
- Google Scholar
43. Wang QM, Begerow D, Groenewald M, Liu XZ, Theelen B, Bai FY, et al. Multigene phylogeny and taxonomic revision of yeasts and related fungi in the Ustilaginomycotina. Stud Mycol. 2015;81:55–83. pmid:26955198
- View Article
- PubMed/NCBI
- Google Scholar
44. Wiens F, Zitzmann A, Lachance MA, Yegles M, Pragst F, Wurst FM, et al. Chronic intake of fermented floral nectar by wild treeshrews. Proc Natl Acad Sci U S A. 2008;105(30):10426–31. pmid:18663222
- View Article
- PubMed/NCBI
- Google Scholar
45. Wan L, Luo G, Lu H, Xuan D, Cao H, Zhang J. Changes in the hemolytic activity of Candida species by common electrolytes. BMC Microbiol. 2015;15:171. pmid:26296996
- View Article
- PubMed/NCBI
- Google Scholar
46. Ben-Ami R, Berman J, Novikov A, Bash E, Shachor-Meyouhas Y, Zakin S, et al. Multidrug-Resistant Candida haemulonii and C. auris, Tel Aviv, Israel. Emerg Infect Dis. 2017;23(1):195–203. pmid:28098529
- View Article
- PubMed/NCBI
- Google Scholar
47. Limtong S, Yonngmanitchai W. Candida chanthaburiensis sp. nov., Candida kungkrabaensis sp. nov. and Candida suratensis sp. nov., three novel yeast species from decaying plant materials submerged in water of mangrove forests. Antonie Van Leeuwenhoek. 2010;98(3):379–388. pmid:20467812
- View Article
- PubMed/NCBI
- Google Scholar
48. Sarawan S, Mahakhan P, Jindamorakot S, Vichitphan K, Vichitphan S, Sawaengkaew J. Candida konsanensis sp. nov., a new yeast species isolated from Jasminum adenophyllum in Thailand with potentially carboxymethyl cellulase-producing capability. World J Microbiol Biotechnol. 2013;29(8):1481–1486. pmid:23504189
- View Article
- PubMed/NCBI
- Google Scholar
49. Wang SA, Jia JH, Bai FY. Candida alocasiicola sp. nov., Candida hainanensis sp. nov., Candida heveicola sp. nov. and Candida musiphila sp. nov., novel anamorphic, ascomycetous yeast species isolated from plants. Antonie Van Leeuwenhoek. 2008;94(2):257–265. pmid:18344072
- View Article
- PubMed/NCBI
- Google Scholar
50. Opulente DA, Langdon QK, Buh KV, et al. Pathogenic budding yeasts isolated outside of clinical settings. FEMS Yeast Res. 2019;19(3):foz032. pmid:31076749
- View Article
- PubMed/NCBI
- Google Scholar
51. Arvanitis M, Glavis-Bloom J, Mylonakis E. Invertebrate models of fungal infection. Biochim Biophys Acta. 2013;1832(9):1378–1383. pmid:23517918
- View Article
- PubMed/NCBI
- Google Scholar
52. Hohl TM. Overview of vertebrate animal models of fungal infection. J Immunol Methods. 2014;410:100–112. pmid:24709390
- View Article
- PubMed/NCBI
- Google Scholar
53. Brajtburg J, Powderly WG, Kobayashi GS, Medoff G. Amphotericin B: current understanding of mechanisms of action. Antimicrob Agents Chemother. 1990;34(2):183–188. pmid:2183713
- View Article
- PubMed/NCBI
- Google Scholar
54. Gray KC, Palacios DS, Dailey I, Endo MM, Uno BE, Wilcock BC, et al. Amphotericin primarily kills yeast by simply binding ergosterol. Proc Natl Acad Sci U S A. 2012;109(7):2234–9. pmid:22308411
- View Article
- PubMed/NCBI
- Google Scholar
55. Ellis D. Amphotericin B: spectrum and resistance. J Antimicrob Chemother. 2002;49 Suppl 1:7–10. pmid:11801575
- View Article
- PubMed/NCBI
- Google Scholar
56. Mesa-Arango AC, Rueda C, Román E, Quintin J, Terrón MC, Luque D, et al. Cell wall changes in amphotericin B-resistant strains from Candida tropicalis and relationship with the immune responses elicited by the host. Antimicrob Agents Chemother. 2016;60(4):2326–35. pmid:26833156
- View Article
- PubMed/NCBI
- Google Scholar
57. Vincent BM, Lancaster AK, Scherz-Shouval R, Whitesell L, Lindquist S. Fitness trade-offs restrict the evolution of resistance to amphotericin B. PLoS Biol. 2013;11(10):e1001692. pmid:24204207
- View Article
- PubMed/NCBI
- Google Scholar
58. Álvarez-Pérez S, Herrera CM, de Vega C. Zooming-in on floral nectar: a first exploration of nectar-associated bacteria in wild plant communities. FEMS Microbiol Ecol. 2012; 80(3):519–602. pmid:22324904
- View Article
- PubMed/NCBI
- Google Scholar
59. Álvarez-Pérez S, Lievens B, Fukami T. Yeast-bacterium interactions: the next frontier in nectar research. Trends Plant Sci. 2019;24(5):393–401. pmid:30792076
- View Article
- PubMed/NCBI
- Google Scholar
60. Herrera CM, Canto A, Pozo M, Bazaga P. Inhospitable sweetness: nectar filtering of pollinator-borne inocula leads to impoverished, phylogenetically clustered yeast communities. Proc Biol Sci. 2010;277(1682):747–754. pmid:19889702
- View Article
- PubMed/NCBI
- Google Scholar
61. Lievens B, Hallsworth JE, Pozo MI, Belgacem ZB, Stevenson A, Willems KA, et al. Microbiology of sugar-rich environments: diversity, ecology and system constraints. Environ Microbiol. 2015;17(2):278–98. pmid:25041632
- View Article
- PubMed/NCBI
- Google Scholar
62. Guzmán B, Lachance MA, Herrera C. Phylogenetic analysis of the angiosperm-floricolous insect–yeast association: Have yeast and angiosperm lineages co-diversified? Mol Phylogenet Evol. 2013;68(2):161–175. pmid:23583418
- View Article
- PubMed/NCBI
- Google Scholar
63. Mittelbach M, Yurkov AM, Nocentini D, Nepi M, Weigend M, Begerow D. Nectar sugars and bird visitation define a floral niche for basidiomycetous yeast on the Canary Islands. BMC Ecol. 2015;15(1):2. pmid:25638173
- View Article
- PubMed/NCBI
- Google Scholar
64. Pozo MI, Bartlewicz J, van Oystaeyen A, et al. Surviving in the absence of flowers: do nectar yeasts rely on overwintering bumblebee queens to complete their annual life cycle?. FEMS Microbiol Ecol. 2018;94(12): pmid:30285114
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Aleklett K, Hart M, Shade A. The microbial ecology of flowers: an emerging frontier in phyllosphere research. Botany 2014;92: 253–266.
View Article
Google Scholar

[2] View Article

[3] Google Scholar

[ref2] 2. de Vega C, Albaladejo RG, Guzmán B, Steenhuisen SL, Johnson SD, Herrera CM, et al. Flowers as a reservoir of yeast diversity: description of Wickerhamiella nectarea f.a. sp. nov., and Wickerhamiella natalensis f.a. sp. nov. from South African flowers and pollinators, and transfer of related Candida species to the genus Wickerhamiella as new combinations. FEMS Yeast Res. 2017;17(5): pmid:28810705
View Article
PubMed/NCBI
Google Scholar

[5] View Article

[6] PubMed/NCBI

[7] Google Scholar

[ref3] 3. Klaps J, Lievens B, Álvarez-Pérez S. Towards a better understanding of the role of fungi in plant-animal interactions. Fungal Biol Biotechnol. 2020;7:1. pmid:31921433
View Article
PubMed/NCBI
Google Scholar

[9] View Article

[10] PubMed/NCBI

[11] Google Scholar

[ref4] 4. Alimadadi N, Soudi MR, Wang SA, Wang QM, Talebpour Z, Bai FY. Starmerella orientalis f.a., sp. nov., an ascomycetous yeast species isolated from flowers. Int J Syst Evol Microbiol. 2016;66(3): 1476–1481. pmid:26780917
View Article
PubMed/NCBI
Google Scholar

[13] View Article

[14] PubMed/NCBI

[15] Google Scholar

[ref5] 5. Amoikon TLS, Grondin C, Djéni TN, Jacques N, Casaregola S. Starmerella reginensis f.a., sp. nov. and Starmerella kourouensis f.a., sp. nov., isolated from flowers in French Guiana. Int J Syst Evol Microbiol. 2018;68(7):2299–2305. pmid:29781796
View Article
PubMed/NCBI
Google Scholar

[17] View Article

[18] PubMed/NCBI

[19] Google Scholar

[ref6] 6. de Vega C, Albaladejo RG, Lachance MA. Metschnikowia maroccana f.a., sp. nov., a new yeast species associated with floral nectar from Morocco. Int J Syst Evol Microbiol. 2018;68(6):2028–2035. pmid:29688165
View Article
PubMed/NCBI
Google Scholar

[21] View Article

[22] PubMed/NCBI

[23] Google Scholar

[ref7] 7. de Vega C, Guzmán B, Lachance MA, Steenhuisen SL, Johnson SD, Herrera CM. Metschnikowia proteae sp. nov., a nectarivorous insect-associated yeast species from Africa. Int J Syst Evol Microbiol. 2012;62(Pt 10):2538–2545. pmid:22407789
View Article
PubMed/NCBI
Google Scholar

[25] View Article

[26] PubMed/NCBI

[27] Google Scholar

[ref8] 8. de Vega C, Guzmán B, Steenhuisen SL, Johnson SD, Herrera CM, Lachance MA. Metschnikowia drakensbergensis sp. nov. and Metschnikowia caudata sp. nov., endemic yeasts associated with Protea flowers in South Africa. Int J Syst Evol Microbiol. 2014;64(Pt 11):3724–3732. pmid:25106927
View Article
PubMed/NCBI
Google Scholar

[29] View Article

[30] PubMed/NCBI

[31] Google Scholar

[ref9] 9. Lachance MA, Bowles JM, Starmer WT. Geography and niche occupancy as determinants of yeast biodiversity: the yeast–insect–morning glory ecosystem of Kípuka Puaulu, Hawai’i. FEMS Yeast Res. 2003;4(1):105–111. pmid:14554202
View Article
PubMed/NCBI
Google Scholar

[33] View Article

[34] PubMed/NCBI

[35] Google Scholar

[ref10] 10. Lachance MA, Ewing CP, Bowles JM, Starmer WT. Metschnikowia hamakuensis sp. nov., Metschnikowia kamakouana sp. nov. and Metschnikowia mauinuiana sp. nov., three endemic yeasts from Hawaiian nitidulid beetles. Int J Syst Evol Microbiol. 2005;55(Pt 3):1369–1377. pmid:15879284
View Article
PubMed/NCBI
Google Scholar

[37] View Article

[38] PubMed/NCBI

[39] Google Scholar

[ref11] 11. Lachance MA. Metschnikowia: half tetrads, a regicide and the fountain of youth. Yeast. 2016;33(11):563–574. pmid:27599462
View Article
PubMed/NCBI
Google Scholar

[41] View Article

[42] PubMed/NCBI

[43] Google Scholar

[ref12] 12. Li SL, Li ZY, Yang LY, Zhou XL, Dong MH, Zhou P, et al. Starmerella jinningensis sp. nov., a yeast species isolated from flowers of Erianthus rufipilus. Int J Syst Evol Microbiol. 2013;63(Pt 1):388–92. pmid:23104359
View Article
PubMed/NCBI
Google Scholar

[45] View Article

[46] PubMed/NCBI

[47] Google Scholar

[ref13] 13. Sipiczki M, Kajdacsi E. Jaminaea angkorensis gen. nov., sp. nov., a novel anamorphic fungus containing an S943 nuclear small-subunit rRNA group IB intron represents a basal branch of Microstromatales. Int J Syst Evol Microbiol. 2009;59(Pt 4):914–920. pmid:19329628
View Article
PubMed/NCBI
Google Scholar

[49] View Article

[50] PubMed/NCBI

[51] Google Scholar

[ref14] 14. Sipiczki M. Starmerella syriaca f.a., sp. nov., an osmotolerant yeast species isolated from flowers in Syria. Antonie Van Leeuwenhoek. 2015;107(4):847–856. pmid:25583140
View Article
PubMed/NCBI
Google Scholar

[53] View Article

[54] PubMed/NCBI

[55] Google Scholar

[ref15] 15. Casadevall A, Kontoyiannis DP, Robert V. On the Emergence of Candida auris: climate change, azoles, swamps, and birds. mBio. 2019;10(4):e01397–19. pmid:31337723
View Article
PubMed/NCBI
Google Scholar

[57] View Article

[58] PubMed/NCBI

[59] Google Scholar

[ref16] 16. Cendejas-Bueno E, Kolecka A, Alastruey-Izquierdo A, Theelen B, Groenewald M, Kostrzewa M, et al. Reclassification of the Candida haemulonii complex as Candida haemulonii (C. haemulonii group I), C. duobushaemulonii sp. nov. (C. haemulonii group II), and C. haemulonii var. vulnera var. nov.: three multiresistant human pathogenic yeasts. J Clin Microbiol. 2012;50(11):3641–3651. pmid:22952266
View Article
PubMed/NCBI
Google Scholar

[61] View Article

[62] PubMed/NCBI

[63] Google Scholar

[ref17] 17. Spivak ES, Hanson KE. Candida auris: an emerging fungal pathogen. J Clin Microbiol. 2018;56(2):e01588–17. pmid:29167291
View Article
PubMed/NCBI
Google Scholar

[65] View Article

[66] PubMed/NCBI

[67] Google Scholar

[ref18] 18. Chowdhary A, Prakash A, Sharma C, Kordalewska M, Kumar A, Sarma S, et al. A multicenter study of antifungal susceptibility patterns among 350 Candida auris isolates (2009–17) in India: role of the ERG11 and FKS1 genes in azole and echinocandin resistance. J Antimicrob Chemother. 2018;73(4):891–899. pmid:29325167
View Article
PubMed/NCBI
Google Scholar

[69] View Article

[70] PubMed/NCBI

[71] Google Scholar

[ref19] 19. Saluja P, Prasad GS. Candida ruellia sp. nov., a novel yeast species isolated from flowers of Ruellia sp. (Acanthaceae). FEMS Yeast Res. 2008;8(4):660–666. pmid:18422614
View Article
PubMed/NCBI
Google Scholar

[73] View Article

[74] PubMed/NCBI

[75] Google Scholar

[ref20] 20. Sipiczki M, Tap RM. Candida vulturna pro tempore sp. nov., a dimorphic yeast species related to the Candida haemulonis species complex isolated from flowers and clinical sample. Int J Syst Evol Microbiol. 2016;66(10):4009–4015. pmid:27411802
View Article
PubMed/NCBI
Google Scholar

[77] View Article

[78] PubMed/NCBI

[79] Google Scholar

[ref21] 21. Sugita T, Takashima M, Poonwan N, Mekha N. Candida pseudohaemulonii sp. nov., an amphotericin B- and azole-resistant yeast species, isolated from the blood of a patient from Thailand. Microbiol Immunol. 2006;50(6):469–473. pmid:16785719
View Article
PubMed/NCBI
Google Scholar

[81] View Article

[82] PubMed/NCBI

[83] Google Scholar

[ref22] 22. Junker RR, Keller A. Microhabitat heterogeneity across leaves and flower organs promotes bacterial diversity. FEMS Microbiol Ecol. 2015;91(9):fiv097. pmid:26253507
View Article
PubMed/NCBI
Google Scholar

[85] View Article

[86] PubMed/NCBI

[87] Google Scholar

[ref23] 23. Mueller-Dombois D, Fosberg FR. Vegetation of the Tropical Pacific Islands. New York NY USA: Springer-Verlag; 1998.

[ref24] 24. Dawson JW, Stemmermann RL. Metrosideros Banks ex Gaertn. In: Wagner WL, Herbst DR, Sohmer S. Manual of the flowering plants of Hawai'i. Honolulu HI USA: University of Hawai'i Press; 1999. pp. 964–970.

[ref25] 25. James SA, Puttock CF, Cordell S, Adams RP. Morphological and genetic variation within Metrosideros polymorpha (Myrtaceae) on Hawai'i. N Z J Bot. 2004;42(2):263–270.
View Article
Google Scholar

[91] View Article

[92] Google Scholar

[ref26] 26. Ralph CJ, Fancy SG. Demography and movements of Apapane and Iiwi in Hawaii. Condor. 1995;97:729–742.
View Article
Google Scholar

[94] View Article

[95] Google Scholar

[ref27] 27. Junker RR, Bleil R, Daehler CC, Bluthgen N. Intra-floral resource partitioning between endemic and invasive flower visitors: consequences for pollinator effectiveness. Ecological Entomology. 2010;35:760–767.
View Article
Google Scholar

[97] View Article

[98] Google Scholar

[ref28] 28. Hanna C, Foote D, Kremen C. Invasive species management restores a plant–pollinator mutualism in Hawaii. J Appl Ecol. 2013;50(1):147–155.
View Article
Google Scholar

[100] View Article

[101] Google Scholar

[ref29] 29. Hanna C, Foote D, Kremen C. Competitive impacts of an invasive nectar thief on plant–pollinator mutualisms. Ecology, 2014;95(6):1622–1632. pmid:25039226
View Article
PubMed/NCBI
Google Scholar

[103] View Article

[104] PubMed/NCBI

[105] Google Scholar

[ref30] 30. Alvarez-Perez S, Blanco JL, Alba P, Garcia ME. Mating type and invasiveness are significantly associated in Aspergillus fumigatus. Med Mycol. 2010;48(2): 273–277. pmid:19634068
View Article
PubMed/NCBI
Google Scholar

[107] View Article

[108] PubMed/NCBI

[109] Google Scholar

[ref31] 31. Kurtzman CP, Robnett CJ. Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie Van Leeuwenhoek. 1998;73(4):331–371. pmid:9850420
View Article
PubMed/NCBI
Google Scholar

[111] View Article

[112] PubMed/NCBI

[113] Google Scholar

[ref32] 32. Liu YJ, Whelen S, Hall BD. Phylogenetic relationships among ascomycetes: evidence from an RNA polymerase II subunit. Mol Biol Evol. 1999;16(12):1799–1808. pmid:10605121
View Article
PubMed/NCBI
Google Scholar

[115] View Article

[116] PubMed/NCBI

[117] Google Scholar

[ref33] 33. Stucky JB. SeqTrace: a graphical tool for rapidly processing dna sequencing chromatograms. J Biomol Tech. 2012;23(3):90–93. pmid:22942788
View Article
PubMed/NCBI
Google Scholar

[119] View Article

[120] PubMed/NCBI

[121] Google Scholar

[ref34] 34. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32(5):1792–1797. pmid:15034147
View Article
PubMed/NCBI
Google Scholar

[123] View Article

[124] PubMed/NCBI

[125] Google Scholar

[ref35] 35. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018 Jun 1;35(6):1547–1549. pmid:29722887
View Article
PubMed/NCBI
Google Scholar

[127] View Article

[128] PubMed/NCBI

[129] Google Scholar

[ref36] 36. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol. 2010;59(3):307–21. pmid:20525638
View Article
PubMed/NCBI
Google Scholar

[131] View Article

[132] PubMed/NCBI

[133] Google Scholar

[ref37] 37. Darriba D, Taboada GL, Doallo R, Posada D. jModelTest 2: more models, new heuristics and parallel computing. Nat Methods. 2012;9(8):772. pmid:22847109
View Article
PubMed/NCBI
Google Scholar

[135] View Article

[136] PubMed/NCBI

[137] Google Scholar

[ref38] 38. Kurtzman CP, Fell J, Boekhout T. Methods for isolation, phenotypic characterization and maintenance of yeasts. In: Kurtzman CP, Fell J, Boekhout T, editors. The Yeasts, a Taxonomic Study. San Diego: Elsevier; 2011. pp. 87–110.

[ref39] 39. Fell JW, Boekhout T, Fonseca A, Scorzetti G, Statzell-Tallman A. Biodiversity and systematics of basidiomycetous yeasts as determined by large-subunit rDNA D1/D2 domain sequence analysis. Int J Syst Evol Microbiol. 2000;50 Pt 3:1351–1371. pmid:10843082
View Article
PubMed/NCBI
Google Scholar

[140] View Article

[141] PubMed/NCBI

[142] Google Scholar

[ref40] 40. Vu D, Groenewald M, Szöke S, Cardinali G, Eberhardt U, Stielow B, et al. DNA barcoding analysis of more than 9000 yeast isolates contributes to quantitative thresholds for yeast species and genera delimitation. Stud Mycol. 2016;85:91–105. pmid:28050055
View Article
PubMed/NCBI
Google Scholar

[144] View Article

[145] PubMed/NCBI

[146] Google Scholar

[ref41] 41. Lachance MA, Daniel HM, Meyer W, et al. The D1/D2 domain of the large-subunit rDNA of the yeast species Clavispora lusitaniae is unusually polymorphic. FEMS Yeast Res. 2003;4(3):253–258. pmid:14654429
View Article
PubMed/NCBI
Google Scholar

[148] View Article

[149] PubMed/NCBI

[150] Google Scholar

[ref42] 42. Shen XX, Opulente DA, Kominek J, et al. Tempo and Mode of Genome Evolution in the Budding Yeast Subphylum Cell. 2018;175(6):1533–1545.e20. pmid:30415838
View Article
PubMed/NCBI
Google Scholar

[152] View Article

[153] PubMed/NCBI

[154] Google Scholar

[ref43] 43. Wang QM, Begerow D, Groenewald M, Liu XZ, Theelen B, Bai FY, et al. Multigene phylogeny and taxonomic revision of yeasts and related fungi in the Ustilaginomycotina. Stud Mycol. 2015;81:55–83. pmid:26955198
View Article
PubMed/NCBI
Google Scholar

[156] View Article

[157] PubMed/NCBI

[158] Google Scholar

[ref44] 44. Wiens F, Zitzmann A, Lachance MA, Yegles M, Pragst F, Wurst FM, et al. Chronic intake of fermented floral nectar by wild treeshrews. Proc Natl Acad Sci U S A. 2008;105(30):10426–31. pmid:18663222
View Article
PubMed/NCBI
Google Scholar

[160] View Article

[161] PubMed/NCBI

[162] Google Scholar

[ref45] 45. Wan L, Luo G, Lu H, Xuan D, Cao H, Zhang J. Changes in the hemolytic activity of Candida species by common electrolytes. BMC Microbiol. 2015;15:171. pmid:26296996
View Article
PubMed/NCBI
Google Scholar

[164] View Article

[165] PubMed/NCBI

[166] Google Scholar

[ref46] 46. Ben-Ami R, Berman J, Novikov A, Bash E, Shachor-Meyouhas Y, Zakin S, et al. Multidrug-Resistant Candida haemulonii and C. auris, Tel Aviv, Israel. Emerg Infect Dis. 2017;23(1):195–203. pmid:28098529
View Article
PubMed/NCBI
Google Scholar

[168] View Article

[169] PubMed/NCBI

[170] Google Scholar

[ref47] 47. Limtong S, Yonngmanitchai W. Candida chanthaburiensis sp. nov., Candida kungkrabaensis sp. nov. and Candida suratensis sp. nov., three novel yeast species from decaying plant materials submerged in water of mangrove forests. Antonie Van Leeuwenhoek. 2010;98(3):379–388. pmid:20467812
View Article
PubMed/NCBI
Google Scholar

[172] View Article

[173] PubMed/NCBI

[174] Google Scholar

[ref48] 48. Sarawan S, Mahakhan P, Jindamorakot S, Vichitphan K, Vichitphan S, Sawaengkaew J. Candida konsanensis sp. nov., a new yeast species isolated from Jasminum adenophyllum in Thailand with potentially carboxymethyl cellulase-producing capability. World J Microbiol Biotechnol. 2013;29(8):1481–1486. pmid:23504189
View Article
PubMed/NCBI
Google Scholar

[176] View Article

[177] PubMed/NCBI

[178] Google Scholar

[ref49] 49. Wang SA, Jia JH, Bai FY. Candida alocasiicola sp. nov., Candida hainanensis sp. nov., Candida heveicola sp. nov. and Candida musiphila sp. nov., novel anamorphic, ascomycetous yeast species isolated from plants. Antonie Van Leeuwenhoek. 2008;94(2):257–265. pmid:18344072
View Article
PubMed/NCBI
Google Scholar

[180] View Article

[181] PubMed/NCBI

[182] Google Scholar

[ref50] 50. Opulente DA, Langdon QK, Buh KV, et al. Pathogenic budding yeasts isolated outside of clinical settings. FEMS Yeast Res. 2019;19(3):foz032. pmid:31076749
View Article
PubMed/NCBI
Google Scholar

[184] View Article

[185] PubMed/NCBI

[186] Google Scholar

[ref51] 51. Arvanitis M, Glavis-Bloom J, Mylonakis E. Invertebrate models of fungal infection. Biochim Biophys Acta. 2013;1832(9):1378–1383. pmid:23517918
View Article
PubMed/NCBI
Google Scholar

[188] View Article

[189] PubMed/NCBI

[190] Google Scholar

[ref52] 52. Hohl TM. Overview of vertebrate animal models of fungal infection. J Immunol Methods. 2014;410:100–112. pmid:24709390
View Article
PubMed/NCBI
Google Scholar

[192] View Article

[193] PubMed/NCBI

[194] Google Scholar

[ref53] 53. Brajtburg J, Powderly WG, Kobayashi GS, Medoff G. Amphotericin B: current understanding of mechanisms of action. Antimicrob Agents Chemother. 1990;34(2):183–188. pmid:2183713
View Article
PubMed/NCBI
Google Scholar

[196] View Article

[197] PubMed/NCBI

[198] Google Scholar

[ref54] 54. Gray KC, Palacios DS, Dailey I, Endo MM, Uno BE, Wilcock BC, et al. Amphotericin primarily kills yeast by simply binding ergosterol. Proc Natl Acad Sci U S A. 2012;109(7):2234–9. pmid:22308411
View Article
PubMed/NCBI
Google Scholar

[200] View Article

[201] PubMed/NCBI

[202] Google Scholar

[ref55] 55. Ellis D. Amphotericin B: spectrum and resistance. J Antimicrob Chemother. 2002;49 Suppl 1:7–10. pmid:11801575
View Article
PubMed/NCBI
Google Scholar

[204] View Article

[205] PubMed/NCBI

[206] Google Scholar

[ref56] 56. Mesa-Arango AC, Rueda C, Román E, Quintin J, Terrón MC, Luque D, et al. Cell wall changes in amphotericin B-resistant strains from Candida tropicalis and relationship with the immune responses elicited by the host. Antimicrob Agents Chemother. 2016;60(4):2326–35. pmid:26833156
View Article
PubMed/NCBI
Google Scholar

[208] View Article

[209] PubMed/NCBI

[210] Google Scholar

[ref57] 57. Vincent BM, Lancaster AK, Scherz-Shouval R, Whitesell L, Lindquist S. Fitness trade-offs restrict the evolution of resistance to amphotericin B. PLoS Biol. 2013;11(10):e1001692. pmid:24204207
View Article
PubMed/NCBI
Google Scholar

[212] View Article

[213] PubMed/NCBI

[214] Google Scholar

[ref58] 58. Álvarez-Pérez S, Herrera CM, de Vega C. Zooming-in on floral nectar: a first exploration of nectar-associated bacteria in wild plant communities. FEMS Microbiol Ecol. 2012; 80(3):519–602. pmid:22324904
View Article
PubMed/NCBI
Google Scholar

[216] View Article

[217] PubMed/NCBI

[218] Google Scholar

[ref59] 59. Álvarez-Pérez S, Lievens B, Fukami T. Yeast-bacterium interactions: the next frontier in nectar research. Trends Plant Sci. 2019;24(5):393–401. pmid:30792076
View Article
PubMed/NCBI
Google Scholar

[220] View Article

[221] PubMed/NCBI

[222] Google Scholar

[ref60] 60. Herrera CM, Canto A, Pozo M, Bazaga P. Inhospitable sweetness: nectar filtering of pollinator-borne inocula leads to impoverished, phylogenetically clustered yeast communities. Proc Biol Sci. 2010;277(1682):747–754. pmid:19889702
View Article
PubMed/NCBI
Google Scholar

[224] View Article

[225] PubMed/NCBI

[226] Google Scholar

[ref61] 61. Lievens B, Hallsworth JE, Pozo MI, Belgacem ZB, Stevenson A, Willems KA, et al. Microbiology of sugar-rich environments: diversity, ecology and system constraints. Environ Microbiol. 2015;17(2):278–98. pmid:25041632
View Article
PubMed/NCBI
Google Scholar

[228] View Article

[229] PubMed/NCBI

[230] Google Scholar

[ref62] 62. Guzmán B, Lachance MA, Herrera C. Phylogenetic analysis of the angiosperm-floricolous insect–yeast association: Have yeast and angiosperm lineages co-diversified? Mol Phylogenet Evol. 2013;68(2):161–175. pmid:23583418
View Article
PubMed/NCBI
Google Scholar

[232] View Article

[233] PubMed/NCBI

[234] Google Scholar

[ref63] 63. Mittelbach M, Yurkov AM, Nocentini D, Nepi M, Weigend M, Begerow D. Nectar sugars and bird visitation define a floral niche for basidiomycetous yeast on the Canary Islands. BMC Ecol. 2015;15(1):2. pmid:25638173
View Article
PubMed/NCBI
Google Scholar

[236] View Article

[237] PubMed/NCBI

[238] Google Scholar

[ref64] 64. Pozo MI, Bartlewicz J, van Oystaeyen A, et al. Surviving in the absence of flowers: do nectar yeasts rely on overwintering bumblebee queens to complete their annual life cycle?. FEMS Microbiol Ecol. 2018;94(12): pmid:30285114
View Article
PubMed/NCBI
Google Scholar

[240] View Article

[241] PubMed/NCBI

[242] Google Scholar

Figures

Abstract

Introduction

Materials and methods

Isolates

Phylogenetic analyses

Phenotypic characterization

Ethics

Nomenclature

Results and discussion

Phylogenetic affiliation

Phenotypic characterization

Adaptation to plants

Taxonomy

Description of Candida metrosideri pro tempore Klaps, de Vega, Herrera, Junker, Lievens & Álvarez-Pérez 2020 sp. nov. [Mycobank accession: MB 832536].

Description of Candida ohialehuae pro tempore Klaps, de Vega, Herrera, Junker, Lievens & Álvarez-Pérez 2020 sp. nov. [Mycobank accession: MB 832537].

Conclusion

Supporting information

S1 Fig. Phylogenetic placement of Candida metrosideri and Candida ohialehuae based on DNA sequence analysis of the ITS1-5.8S-ITS2 rDNA region, as obtained by maximum likelihood analysis.

S2 Fig. Phylogenetic placement of Candida metrosideri and Candida ohialehuae based on DNA sequence analysis of the RPB1 gene, as obtained by maximum likelihood analysis.

S3 Fig. Phylogenetic placement of Candida metrosideri and Candida ohialehuae based on DNA sequence analysis of the RPB2 gene, as obtained by maximum likelihood analysis.

S1 Table. Complete phenotypic characteristics of Candida metrosideri, Candida ohialehuae and their close phylogenetic relatives.

Acknowledgments

References