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Applied and Environmental Microbiology, February 2007, p. 1120-1125, Vol. 73, No. 4
0099-2240/07/$08.00+0     doi:10.1128/AEM.01432-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Biogeography, Host Specificity, and Molecular Phylogeny of the Basidiomycetous Yeast Phaffia rhodozyma and Its Sexual Form, Xanthophyllomyces dendrorhous

Laboratorio de Microbiología Aplicada y Biotecnología, Universidad Nacional del Comahue, Centro Regional Universitario Bariloche (CRUB)-Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET-UEb, CCT), Bariloche, Río Negro, Argentina,¹ Centro de Recursos Microbiológicos, Secção Autónoma de Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal²

Received 21 June 2006/ Accepted 11 December 2006

	ABSTRACT

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Phaffia rhodozyma (sexual form, Xanthophyllomyces dendrorhous) is a basidiomycetous yeast that has been found in tree exudates in the Northern Hemisphere at high altitudes and latitudes. This yeast produces astaxanthin, a carotenoid pigment with biotechnological importance because it is used in aquaculture for fish pigmentation. We isolated X. dendrorhous from the Southern Hemisphere (Patagonia, Argentina), where it was associated with fruiting bodies of Cyttaria hariotii, an ascomycetous parasite of Nothofagus trees. We compared internal transcribed spacer (ITS)-based phylogenies of P. rhodozyma and its tree host (Betulaceae, Corneaceae, Fagaceae, and Nothofagaceae) and found them to be generally concordant, suggesting that different yeast lineages colonize different trees and providing an explanation for the phylogenetic distance observed between the type strains of P. rhodozyma and X. dendrorhous. We hypothesize that the association of Xanthophyllomyces with Cyttaria derives from a previous association of the yeast with Nothofagus, and the sister relationship between Nothofagaceae and Betulaceae plus Fagaceae correlates with the phylogeny of X. dendrorhous strains originating from these three plant families. The two most basal strains of X. dendrorhous are those isolated from Cornus, an ancestral genus in the phylogenetic analysis of the host trees. Thus, we question previous conclusions that P. rhodozyma and X. dendrorhous represent different species since the polymorphisms detected in the ITS and intergenic spacer sequences can be attributed to intraspecific variation associated with host specificity. Our study provides a deeper understanding of Phaffia biogeography, ecology, and molecular phylogeny. Such knowledge is essential for the comprehension of many aspects of the biology of this organism and will facilitate the study of astaxanthin production within an evolutionary and ecological framework.

	INTRODUCTION

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Phaffia rhodozyma was originally isolated in the late 1960s, by Phaff and collaborators, who collected 10 strains from various broad-leafed trees in mountainous regions of Japan and Alaska (23). These strains had a red to orange color and could ferment glucose and produce amyloid compounds. Phaff et al. (23) referred to these strains as Rhodozyma montanae, but since a Latin diagnosis was not provided, the name was not valid. Later, this basidiomycetous yeast was described as Phaffia rhodozyma (20), and 67 additional strains were isolated from spring sap flows of Betula verrucosa in Russia (10).

The main carotenoid pigment produced by P. rhodozyma is astaxanthin, a compound that has not been found in other yeast species (3). Astaxanthin is economically important as an aquaculture feed component. This compound enhances pigmentation of fish and crustaceans (19) and is the most expensive feed ingredient in the aquaculture industry (12). Studies of the feasibility of P. rhodozyma as a dietary source of this pigment began almost 30 years ago (13). Such studies involved the development of methods for optimizing pigment production (24, 28), selection of hyperpigmented mutants (2), the use of chemical stimulants (11) or extracts of the filamentous fungus Epicoccum nigrum (5) in the growth medium, and genetic and metabolic engineering (21).

The sexual stage of P. rhodozyma was discovered in 1995, and its development is triggered when polyols are used as sole carbon sources (9). The teleomorphic state is characterized by the formation of a slender holobasidium with terminal basidiospores and was described as Xanthophyllomyces dendrorhous, with VKM Y-2786 (CBS 7918) as the type strain (9). Therefore, the name X. dendrorhous was used for the sexual form and P. rhodozyma was designated the asexual phase. The lack of sexual reproduction in the type strain of P. rhodozyma (CBS 5905) and differences in intergenic spacer (IGS) and internal transcribed spacer (ITS) sequences between CBS 5905 and VKM Y-2786 led Fell and Blatt (6) to propose that P. rhodozyma, represented solely by CBS 5905, was not conspecific with X. dendrorhous. The debate over this issue is not settled, and in another study isozyme analyses and restriction fragment length polymorphism and random amplified polymorphic DNA patterns suggested that CBS 5905 and five other strains of X. dendrorhous belong to a single species (27). The hypothesis that the anamorph P. rhodozyma is distinct, at the species level, from teleomorph X. dendrorhous also was favored by Kucsera et al. (14, 15), who based their arguments on the extensive chromosome length polymorphism seen in electrophoretic karyotypes (1, 22), the ability to produce respiration-deficient petite mutants spontaneously or following brief exposure to ethidium bromide, and a reexamination of the sexual form.

P. rhodozyma and X. dendrorhous have been found so far in tree exudates in the Northern Hemisphere at high altitudes and latitudes. No new strains of P. rhodozyma/X. dendrorhous have been isolated recently, and most of the current research is based on isolates first cultured in the 1960s and 1970s. This study is the first to document the occurrence of Phaffia yeasts in the Southern Hemisphere, and its main objective is to provide an adequate molecular and ecological characterization of the new isolates. Our working hypothesis is that Phaffia has a broader ecological distribution than the one presently known and that its occurrence in the Southern Hemisphere is linked to similar ecosystems in the Northern Hemisphere where Phaffia is known to occur. A deeper understanding of Phaffia biogeography, ecology, and molecular phylogeny is essential for the comprehension of many aspects of its biology and will facilitate the study of astaxanthin production within an evolutionary and ecological framework.

	MATERIALS AND METHODS

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Yeast isolation and reference cultures.
Water samples from Lake Ilon (71°56'S, 41°11'W), Nahuel Huapi National Park (Patagonia, Argentina), were collected during January 2003 in aseptic flasks and filtered in situ through a membrane with 0.45-µm pores with a sterile Nalgene (Rochester, NY) filtering apparatus. The filters were placed in sterile petri dishes and kept at 4°C for approximately 36 h, until arrival at the laboratory, where they were placed on MYP agar (components, g/liter: malt extract, 7; yeast extract, 0.5; peptone-soytone, 2.5; agar, 15 [pH 5]) plus 200 mg/liter chloramphenicol and incubated at 15 to 17°C until colonies emerged.

Stromata of Cyttaria hariotii, an ascomycetous fungal parasite of Nothofagus dombeyi ("Coihue"), were collected aseptically in November to December (spring season) of 2003 on the shore of Gutiérrez Lake (71°43'S, 41°18'W). Fifteen Cyttaria fruiting bodies were collected and stored individually in sterile flasks to avoid cross contamination or crushing. Stromata were divided into three groups based on their maturation status (immature, early in maturation, and mature) and checked for yeasts. Stromata were weighed (5 to 50 g), checked for developmental stage, cut into small pieces (2 x 2 cm), and placed in sterile plastic bags with a known volume of distilled sterile water in a water-to-fruiting-body ratio of 1 g/ml. Cyttaria fruiting bodies were manually crushed inside the plastic bags, which were then transferred to Erlenmeyer flasks and agitated at 20°C for 30 min at 300 rpm. The extract was diluted (10^–3 or 10^–4), and aliquots (100 to 200 µl) were spread on MYP agar plates. These plates were incubated at 15 to 17°C for 5 to 7 days, and every pigmented colony was subcultured on MYP agar for purification. The production of amyloid compounds and the ability to ferment glucose were evaluated by using the procedures of Yarrow (30). The two strains isolated from water samples plus 13 selected strains recovered from different Cyttaria fruiting bodies were used for further studies. The ability to form the sexual stage was investigated by using previously described procedures (14). All of the pigmented isolates were maintained on potato dextrose agar at 4°C. In addition, strains identified as X. dendrorhous were deep frozen in liquid nitrogen.

In addition to these newly collected cultures, reference cultures were obtained from the Centraalbureau voor Schimmelcultures (CBS); the Department of Soil Biology, Moscow State University (KBP); the Portuguese Yeast Culture Collection (PYCC); and the University of California at Davis (UCD) (Table 1).

Nucleotide sequence accession numbers.
The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this study are DQ661021 to DQ661034.

	RESULTS

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Patagonian isolates.
We isolated two orange-pigmented strains (CRUB 1149 and CRUB 1151) that formed amyloid compounds and gave positive results in the glucose fermentation test from high-altitude oligotrophic lakes of the Nahuel Huapi National Park (Patagonia). Based on sequences of the D1/D2 domains of large-subunit rDNA both strains were closely related to P. rhodozyma/X. dendrorhous, i.e., there were no sequence mismatches in the comparison with the type strain of Phaffia rhodozyma. The Phaffia/Xanthophyllomyces isolates from the Northern Hemisphere were found in slime fluxes of trees or tree stumps remaining after logging, so we hypothesized that the aquatic environment from which these two isolates were obtained was not their original habitat. Assuming that the yeast is primarily associated with ecological niches where fermentable sugars are present, we surveyed the plant biota surrounding the high-altitude lakes of the Nahuel Huapi National Park for the occurrence of slime fluxes. The main trees occurring in this region are Nothofagus dombeyi and Nothofagus pumilio, but trees of neither of these species had signs of active or dry slime fluxes.

While we were surveying the trees for slime fluxes, our attention was drawn to Cyttaria hariotii, an ascomycetous fungal parasite of Nothofagus spp., whose mycelium develops inside the trunk and the branches of the tree. As a reaction to the infection, persistent tumors are formed. Fructifications (stromata) are produced annually on the tumors. The stromata of C. hariotii are globose and yellow to orange, measure 2 to 6 cm in diameter, and mature in November-December (8). Yeasts were present at all maturation stages of the Cyttaria stromata. Mature fruiting bodies yielded the highest yeast counts (above 9 x 10⁴ CFU/g [wet weight]), while immature stromata and those in the first stages of maturation had 900 and 2,000 CFU/g, respectively. The proportion of pigmented yeasts in C. hariotii gradually declined with maturation. In late maturation stages, most of the yeast isolates were fermentative ascomycetes of the genera Candida and Saccharomyces and only 1 to 5% of the colonies on the isolation plates were pigmented (red to orange cultures). Overall, we obtained 40 pigmented isolates, of which 36 produced amyloid compounds. Glucose was fermented by 33 of the strains that formed amyloid compounds.

Based on MSP-PCR, the Patagonian isolates belonged to a single species, as the profiles of the various strains were basically identical (Fig. 1). The sequence of the D1/D2 domains of large-subunit rDNA of CRUB 0853 (DQ661033) also was identical to that of the aquatic strain CRUB 1149 and that of the type strain of Phaffia rhodozyma.

View larger version (68K): [in this window] [in a new window]   FIG. 1. MSP-PCR fingerprints of X. dendrorhous strains generated with microsatellite primer (GTG)5. A, B, and C indicate the three main fingerprints. The first and last lanes correspond to the molecular size marker ( DNA cleaved with HindIII and X174 DNA cleaved with HaeIII).

The proportion of X. dendrorhous strains recovered depended on the maturity of the stroma. Mature stromata yielded the most isolates (27 strains), none were recovered from immature stromata, and six were isolated from stromata in the first stages of maturation. N. dombeyi, the host tree of C. hariotii, was surveyed for Phaffia/Xanthophyllomyces, but no cultures were obtained from leaves, buds, or bark. Additional water samples from nearby lakes also were negative.

Molecular phylogeny and host specificity.
In spite of their overall similarity, the fingerprints obtained with primer (GTG)₅ (Fig. 1) could be used to subdivide the isolates into three groups. The first group (A) contained the type strains of P. rhodozyma and X. dendrorhous and several other strains from different geographic areas. The second group (B) included two strains of X. dendrorhous isolated by Phaff et al. (23), and the third group (C) included the Patagonian isolates. The same set of strains also was evaluated with primer M13 (data not shown), and the same three groups were identified.

We used ITS sequence data and a maximum parsimony analysis to evaluate the relatedness of the Phaffia/Xanthophyllomyces isolates (Fig. 2A). We expanded the ITS data set of Fell and Blatt (6) from 6 to 18 sequences. Four of the new sequences are from Patagonian isolates, and the remaining eight sequences are from Russian (two sequences) and Japanese (six sequences) strains isolated by W. I. Golubev and H. J. Phaff, respectively. The type strain of X. dendrorhous, which was isolated from Betula verrucosa in Russia, clustered with all of the other strains of this species originating from Betulaceae (Alnus and Betula) but not with the strains isolated from Cornus (Cornaceae) and Nothofagus (Nothofagaceae), the plant host of Cyttaria (Fig. 2A). The isolated position occupied by the type strain of P. rhodozyma CBS 5905 in the phylogenetic tree could be due to its association with a different tree (Fagus crenata, Fagaceae). Strains PYCC 4172 and CBS 9090 clustered together at the base of the clade composed of isolates from Betulaceae (Fig. 2A).

View larger version (30K): [in this window] [in a new window]   FIG. 2. Phylogenetic relationships between different isolates of X. dendrorhous and their plant hosts. The consensus trees were based on alignments of the complete ITS region (ITS 1, 5.8S rDNA, and ITS 2) and were generated via maximum parsimony analysis in PAUP*. The topology of the X. dendrorhous tree was rooted with Cryptococcus macerans, and that of the plant host tree was rooted with Fitzroya cupressoides. Names of plant families, localities, and host trees are indicated in the center of the figure. Numbers on the branches are bootstrap values (1,000 replicates; values below 50% are not shown). GenBank accession numbers of the sequences are indicated after strain numbers.

Given the apparent association of various ITS phylotypes of X. dendrorhous with different tree families, we tested the hypothesis that different populations of X. dendrorhous colonize different trees and analyzed the evolutionary relationships of the genera of trees that are known to harbor Xanthophyllomyces (Fig. 2B). We used complete ITS sequences, and in general, good agreement was observed between the fungal and the plant phylogenetic trees (Fig. 2).

Our ITS analysis was complemented with a phylogenetic analysis of the rDNA IGS region, a region previously evaluated by Fell and Blatt (6). They found that for X. dendrorhous, differences in sequences and the occurrence of gaps could be used to differentiate strains and for population-level studies. Our IGS alignment of 657 nucleotides differed substantially from the one presented by Fell and Blatt (6), with numerous short nucleotide repeats and gaps contributing to distinct alignment options. Similar difficulties also were reported by Fell and Blatt (6) for their IGS alignment. In spite of this we found, in our analysis, that the sequence of the Patagonian isolate was phylogenetically closer to that of CBS 5905 than to any of the already known sequences (Fig. 3).

View larger version (26K): [in this window] [in a new window]   FIG. 3. Unrooted tree depicting the phylogenetic relationships between different isolates of X. dendrorhous based on IGS sequences and maximum parsimony analysis performed in PAUP*. Numbers on the branches are bootstrap values (1,000 replicates; values of <50% are not shown). GenBank accession numbers of the sequences are indicated after strain numbers.

	DISCUSSION

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A peculiar characteristic of X. dendrorhous is its ability to ferment simple sugars. When mature, the fruiting bodies of C. hariotii have a sugar content of almost 10% (mainly D-glucose, fructose, and sucrose) and contain polyols such as glycerol, D-mannitol, and D-arabinitol (16). Although we recovered two isolates from freshwater in a high-altitude lake, surveys of pigmented yeasts in 15 other Patagonian aquatic environments did not yield any additional isolates of this species (17). We hypothesize that the primary habitat of the two aquatic isolates also was C. hariotii because Nothofagus spp. infected with the fungus surround the lake from which the two strains were obtained. Mature stromata normally fall to the ground where they decompose, but stromata falling from trees on the lake shore may fall in the water, where they float for some time.

South American populations.
MSP-PCR fingerprinting has been used to type fungal strains by several research groups. In most cases species-specific PCR patterns are obtained (17, 25, 29), but in some instances this approach results in intraspecific differentiation (4, 18). Our results suggest that primers (GTG)₅ and M13 are useful to discriminate populations of Phaffia/Xanthophyllomyces. The South American isolates formed a single, homogeneous cluster (group C) that did not include strains from other geographic areas. Thus, the Patagonian strains seem to represent a genetically uniform population even though they were isolated from different locations. Most of the studied strains from the Northern Hemisphere belonged to group A, which includes strains from Japan, Finland, and Russia. However, Japanese isolates also are found in group B, so the intraspecific polymorphism detected by MSP-PCR may not be strictly correlated with the geographic origin of the isolate.

Host specificity model.
The comparative analysis of ITS sequence data from Xanthophyllomyces/Phaffia and their tree hosts suggests that different yeast lineages colonize different trees. Specific (insect?) vectors colonize different trees and could contribute to the observed separation. Since the two phylogenetic trees (Fig. 2) have similar topologies, we hypothesize that the observed phylogenetic pattern of X. dendrorhous follows the speciation of its plant hosts. However, two unanswered questions arise from these findings: (i) are the isolation mechanisms stronger or weaker than those that contribute to the unity of X. dendrorhous as a species, and (ii) if the forces contributing to the isolation of the several Xanthophyllomyces lineages are dominant, is speciation currently in progress? Further studies are needed to elucidate the degree of gene flow between representatives of the different Xanthophyllomyces lineages.

From the evolutionary point of view one can postulate that the present association of Xanthophyllomyces with Cyttaria derived from an earlier association of the yeast with Nothofagus. The sister relationship between Nothofagaceae and Betulaceae plus Fagaceae (Fig. 2B) was also observed in studies of angiosperm phylogeny (Angiosperm Phylogeny Website, version 7, May 2006, P. F. Stevens [http://www.mobot.org/MOBOT/research/APweb]) and corresponds very well with the phylogeny of X. dendrorhous strains originating from these three plant families (Fig. 2A). The two most divergent and basal strains of X. dendrorhous are those originating from Cornus (Fig. 2A), a divergent and ancestral genus in the phylogenetic analysis of the host trees (Fig. 2B).

In spite of the considerable number of strains that were analyzed, the type strain of P. rhodozyma remains phylogenetically isolated. According to our model this result is not connected with the number of strains employed in the analysis but is rather a direct consequence of the habitat (tree species) from which each isolate was recovered. We anticipate that the isolated placement of the type strain of P. rhodozyma will change only if new isolates from Fagus are obtained. Thus, the isolated position of the type strain of P. rhodozyma does not necessarily mean that CBS 5905 is not conspecific with (and the asexual form of) X. dendrorhous.

Interpretations of previous studies with IGS sequence data suggested that this approach was adequate to reflect the biogeography of X. dendrorhous since ATCC 24228 and ATCC 24230, the two Pacific Rim strains, were separated from CBS 6938 and CBS 7918, the two European strains (6). Moreover, the phylogenetic differences were regarded as indications that X. dendrorhous includes two varieties, and the isolated position of CBS 5905 was interpreted as additional evidence that P. rhodozyma and X. dendrorhous are not conspecific. Our IGS analysis partially supports the conclusions of Fell and Blatt (6). We think that the IGS data are promising for a biogeographic study of isolates from Betulaceae since the Pacific Rim strains are distinct from the European isolates. However the association between the Patagonian isolate and CBS 5905 challenges the two-species concept. We anticipate that IGS sequence typing might be a useful approach for the characterization of isolates within a given host group.

Was the type strain of P. rhodozyma mislabeled? The relationship between CBS 5905, PYCC 4172, and CBS 9090 remains unresolved. According to the records from several culture collections, all of these strains are derived from UCD 67-210, the original type strain of P. rhodozyma, but the expected identity of these three strains is not seen with the molecular characters. The hypothesis that one or more of these strains has been mislabeled cannot yet be discarded, although we could not identify the strain that might represent the source of such mislabeling (in terms of either identical ITS sequences or, for CBS 5905, the absence of the sexual stage). Golubev (9) reported that VKM Y-2274 (type strain of P. rhodozyma) could form basidia and basidiospores. The VKM strain was obtained from the UCD collection in 1975 (W. I. Golubev, personal communication). However, Kucsera et al. (14) reported that CBS 5905 could not form a sexual stage. We confirmed that CBS 5905 is asexual and that PYCC 4172 and CBS 9090 are sexually fertile. Another possible explanation is that prolonged maintenance in culture collections resulted in a divergence of CBS 5905 from the other two strains. The oldest copy of the original culture is CBS 5905, which dates from 1968 and is the most divergent, while the VKM culture was obtained in 1975 and the PYCC culture in 1976 (via the NRRL culture collection). Such an extensive divergence (including loss of the ability to form sexual structures) has not previously been documented for yeasts. Further studies with additional molecular markers and the determination if there are multiple copies of the ITS region within each strain are needed to adequately clarify this situation.

P. rhodozyma or X. dendrorhous?
Our results are not consistent with the hypothesis that P. rhodozyma and X. dendrorhous represent different species (6, 15). Most of the differences in the sequence data can be attributed to intraspecific variation associated with host specificity. The issue of the relatedness of the type strains of P. rhodozyma (CBS 5905) and X. dendrorhous (CBS 7918) among themselves and with Patagonian isolate CRUB 0853 also was evaluated with genomic DNA-DNA reassociation experiments. These reassociation values all were high, which is consistent with the hypothesis that only a single species is present. For CBS 5905 x CBS 7918, DNA-DNA reassociation values ranged from 96 to 100%, values for CBS 5905 x CRUB 0853 ranged from 79 to 100%, and values for CBS 7918 x CRUB 0853 ranged from 79 to 90%.

In conclusion, we think that nomenclatural changes are premature and support the position that P. rhodozyma is the asexual stage of X. dendrorhous. We think that a better understanding of the processes underlying the observed differences within X. dendrorhous might have relevance since they could shed new light on the evolution of the astaxanthin biosynthetic pathway and on the ecological conditions that selected for this trait. Finally, we do not reject the hypothesis that speciation is in progress in X. dendrorhous. The driving force underlying such a process could be isolation due to host specificity of the different lineages of X. dendrorhous. We anticipate that multilocus sequence typing might be useful in assessing the degree of genetic isolation of the various lineages of X. dendrorhous identified in this report.

	ACKNOWLEDGMENTS

 
This work was partially funded by the Universidad Nacional del Comahue (project B121) and CONICET (project PIP6536) funds granted to M. van Broock and by ANPCYT (project PICT 22200). D. Libkind was supported by a CONICET Ph.D. fellowship. Bilateral cooperation between Argentina and Portugal was supported by a SECYT-GRICES cooperation agreement (PO/PA02-BI/002).

We thank the authorities of Parques Nacionales (Argentina), for providing permission for sample collection within the Nahuel Huapi National Park, and the following curators of culture collections for sending valuable cultures: Vincent Robert (CBS), Michael Vustin (KBP), and Kyria Boundy-Mills (UCD). We also thank Kyria Boundy-Mills, Jack Fell, Wladislav Golubev, and Gloria Scorzetti for their comments on the manuscript.

	FOOTNOTES

 
* Corresponding author. Mailing address: Laboratorio de Microbiología Aplicada y Biotecnología, Universidad Nacional del Comahue, Centro Regional Universitario Bariloche (CRUB), Quintral 1250, Bariloche, Río Negro, Argentina. Phone: 54 2944 423374. Fax: 54 2944 422111. E-mail: libkind{at}crub.uncoma.edu.ar.

Published ahead of print on 22 December 2006.

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