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6 Running title
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D. hansenii: a rare fungal pathogen in humans
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Marie Desnos-Ollivier1, Marie Ragon1**, Vincent Robert2, Dorothée Raoux1,
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Jean-Charles Gantier1 and Françoise Dromer1*
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12 Institut Pasteur, Centre National de Référence Mycologie et Antifongiques, Unité de
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The Netherlands 2
*
Corresponding author: Centre National de Référence Mycologie et Antifongiques, Unité de Mycologie
Moléculaire, CNRS URA 3012, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, France. Phone : 33 1 40
61 33 89. Fax : 33 1 45 68 84 20. Email : dromer@pasteur.fr
**Present address: Département Génétique et Ecologie Evolutives, Laboratoire Ecologie Systématique et
Evolution, CNRS UMR8079,Université de Paris-Sud, Orsay, France
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1 Abstract
3 and in various types of cheese. Pichia guilliermondii is widely distributed in nature and is a
4 common constituent of the normal human microflora. Both species have been described in
5 human infections but are extremely difficult to differentiate phenotypically. Thus, frequent
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errors in the identification occur. The 62 clinical and environmental isolates sent between
2000 and 2007 to the French National Reference Center for Mycoses and Antifungals as D.
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8 hansenii or P. guilliermondii were analysed using carbon assimilation pattern, presence of
9 pseudohyphae and sequencing of ITS and D1/D2 regions of the rRNA gene. The objective of
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the study was to assess using nucleotide sequences whether phenotypic identification was
correct and whether phenotypic characteristics could be used to differentiate the two species
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12 when sequencing was not available. We found that 58% of the isolates were misidentified and
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palmioleophila, C. haemulonii type II and Clavispora lusitaniae. In conclusion, D. hansenii
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may not be as frequent a human pathogen as previously thought. Sequencing of either ITS or
D1/D2 regions is a good tool to differentiate the species more frequently confused with D.
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2
1 Introduction
3 and in various types of cheese (3, 26). It has been described in human infections (11, 31, 35).
4 However, its incidence during candidemia is low based on data surveillance implemented by
5 the Centers for Diseases Control (9) whereas one review on worldwide-collected isolates
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states that D. hansenii accounts for 0.08 to 0.5% of isolates recovered during invasive
candidiasis (22). Pichia guilliermondii is widely distributed in nature (routinely isolated from
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8 insects, soil, plants, atmosphere, sea water, exudates of various trees, and processed foods)
9 and is a common constituent of the normal human microflora (20). Globally, this species and
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its anamorphic state Candida guilliermondii accounts for 1 to 2% of all candidemia (14, 23).
However, the species D. hansenii (Candida famata) and P. guilliermondii are extremely
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12 difficult to differentiate phenotypically (17, 18). Thus, frequent errors in the identification
13 occur. We undertook the retrospective analysis of all isolates, mostly recovered from clinical
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specimens sent to the French National Reference Center for Mycoses and Antifungals
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(NRCMA) as D. hansenii or P. guilliermondii. The objective of the study was to assess using
nucleotide sequences whether phenotypic identification was correct and whether phenotypic
characteristics could be used to differentiate the two species when sequencing was not
18 available.
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21 Strains
22 All the epidemiologically unrelated clinical or environmental isolates (n = 41) sent between
23 September 2000 and April 2007 as D. hansenii or P. guilliermondii to the NRCMA for
25 clinical or environmental isolates were collected for this specific study (Table 1). Type strains
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1 for anamorph of each species were obtained from the Centraalbureau voor Schimmelcultures
2 (CBS, Utrecht, the Netherlands) (Table S1). All isolates were stored frozen in 40% glycerol at
3 -80°C.
5 Carbon assimilation patterns were obtained with the commercialized strips (ID32C,
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bioMérieux, Marcy-l’Etoile, France). The presence of pseudohyphae was determined after
growth for 24 h and 5 d of incubation at 30°C in potato-carrot-ox bile medium (used routinely
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8 in France instead of cornmeal agar or rice agar, (5)). Minimal inhibitory concentrations
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determined using the EUCAST microdilution method (7). The concentrations corresponding
to the MIC that inhibited 50% (MIC50) and 90% (MIC90) of the isolates were determined.
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12 Molecular characterization
13 After 24 h of incubation at 27°C on Sabouraud dextrose agar plates, single colonies were
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transferred to 1 ml of distilled water in a microcentrifuge tube and DNA extraction was
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performed using the High Pure PCR Template Preparation Kit (Roche Applied Science,
were used for the amplification of the ITS1-5.8S-ITS2 (primers V9D (8) and LS266 (15)),
18 and the 26S (primers NL1 and NL4, (19)) rDNA regions. Reaction volumes of 50 µl
19 contained 3µl of genomic DNA, 2.5 U of AmpliTaq Gold, 5 µl of PCR Buffer 10X, 5 µl of
20 MgCl2 25 mM, 5 µl of 2.5 mM dNTP (Roche) and 1.25 µl of 20 µM primers. The PCR
22 France) set up with a first cycle of denaturation for 10 min at 95°C, followed by 30 cycles of
23 denaturation at 94°C for 30 s, 30 s at 58°C and elongation at 72°C for 30 s, with a final
24 extension step of 10 min at 72°C. Both strands of purified amplified fragments were
25 sequenced at the Genopole of the Pasteur Institute, on an ABI Prism 3700 DNA Analyzer
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1 (Applied Biosystems, Courtaboeuf, France), with the same primers that were used in the PCR
2 step. Sequences were edited with Chromas Pro version 1.33 (Technelysium Pty Ltd.,
3 Australia).
4 The sequences of the ITS1-5.8S-ITS2 regions were delimited by the sequences of the primers
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the same way, the sequences of the D1/D2 region of the 26S subunit were delimited by the
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8 (GTGAAATTGTTGAAAGGGAA, (30)). Sequences were compared with those of the type
9 strains to confirm species identification. The D1/D2 and ITS bounded sequences, including
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the sequences of the type strains, were aligned using the ClustalW software (32).
Phylogenetic analyses were performed using the Bayesian Markov chain Monte Carlo method
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12 based on MrBayes software (28) and were based on a concatenated alignment of the ITS and
13 D1/D2 datasets.
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Results
confirmed for 23 isolates, 12 were identified as Pichia caribbica based on the comparison of
18 their ITS and 26S sequences with the type strain sequences (CBS 2022), and one was
19 identified as Pichia jadinii based on both carbon assimilation profile and nucleotide
22 2 belong to the currently recognized species Candida haemulonii type II and 2 to Clavispora
24 A Bayesian tree was constructed by using the bounded sequences of the ITS and 26S regions
25 of all isolates, including the type strains (Figure 1). The isolates of P. guilliermondii are
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1 divided into two closely related groups, one including the type strain (CBS 566) and 20/33
2 isolates, the second contained 13 isolates. Pichia caribbica isolates were clustered in three
3 groups, one including the type strain (CBS 2022) and 15/18 isolates, the second and the third
4 composed respectively of 2 and 1 isolate. Both species were closely related. The isolates of D.
5 hansenii, including the type strain CBS 1795 were closely related to those of C.
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palmioleophila. Isolates of P. jadinii, C. lusitaniae and C. haemulonii type II are well
separated from the above species, and would have been using only one of the nucleotide
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8 sequences.
9 Analysis of carbon assimilation patterns by each species showed that all three isolates of D.
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hansenii assimilated DL-Lactate, whereas none of the P. guilliermondii and P. caribbica
isolates did (Table 2). None of the D. hansenii isolates was resistant to 0.01 % cycloheximide
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12 whereas >90% of P. guilliermondii and P. caribbica isolates were. For C. palmioleophila, the
13 carbon assimilation patterns were ambiguous with D. hansenii profiles. For C. haemulonii
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type II, C. lusitaniae and P. jadinii the majority of the codes were lacking in the ID32C
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database or show a low discrimination with D. hansenii. None of the D. hansenii isolates was
able to produce pseudohyphae, whereas 27/33 and 17/18 isolates of P. guilliermondii and P.
18 = 33) or P. caribbica (n =18), different carbon assimilation profiles were obtained (8 and 9,
19 respectively), some of which were shared by both species, one being a frequent code for both
20 (Table S2). Each isolate of D. hansenii had a unique profile that was not found for any of the
21 P. guilliermondii and P. caribbica isolates. An UPGMA tree was constructed by using the
22 significant carbon sources of the ID32C assimilation pattern for the 62 clinical and
24 MIC values of amphotericin B and flucytosine were similar for all P. guilliermondii, P.
25 caribbica and D. hansenii isolates (data not shown). MIC values of fluconazole (range=0.124-
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1 2 µg/ml), and to a lesser degree those of voriconazole (<0.015 µg/ml) and caspofungin
3 (MIC50/MIC90 [range]= 8/64 µg/ml [2-≥64] for fluconazole, 0.06/0.5 µg/ml [0.03-2] for
4 voriconazole, and 0.125/1 µg/ml [0.014-2] for caspofungin) and on P. caribbica (4/64 µg/ml [1-
5 ≥64] for fluconazole, 0.125/0.25 µg/ml [0.06-≥8] for voriconazole, and 0.125/0.25 µg/ml [0.03-0.5]
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for caspofungin) isolates.
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8 Discussion
9 Debaryomyces hansenii (teleomorph of Candida famata) has been repeatedly associated with
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catheter-related bloodstream infection and rarely with other infections (4, 12, 24, 25, 27, 31).
However, in most of the reported cases, the species was identified using phenotypic
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12 parameters. Based on our results and on previous reports, one may wonder if some of these
13 cases could not have been misidentified. In our study, 36/62 (58%) of the isolates classified as
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D. hansenii or P. guilliermondii were misidentified. Isolates initially identified as D. hansenii
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belong to 6 different species (D. hansenii, P. guilliermondii, P. caribbica, C. palmioleophila,
guilliermondii, 12 belong to the very closely related species P. caribbica and one was P.
18 jadinii (Table 1). Debaryomyces hansenii may thus be a frequent species of the gut microflora
19 in individuals who like cheese. It does not seem to represent a frequent cause of fungemia.
20 Whether nucleotide sequencing was used for the identification of the isolates to the species
21 level in the ARTEMIS study reporting up to 0.5% and 1% of C. famata and C. guilliermondii
22 among isolates responsible for invasive candidiasis is unknown. In the current active
23 surveillance program on yeasts fungemia in the Paris area, none was due to D. hansenii
25 the 1975 cases recorded so far over the 5-years survey (YEASTS program, unpublished data).
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1 Debaryomyces hansenii is an uncommon yeast characterized by its cryo- and halotolerance
2 (6) but these criteria as well as production of ascospores are not used in routine yeast
4 found for the majority of P. guilliermondii and P. caribbica isolates, all isolates of D.
5 hansenii assimilated DL-Lactate, but were unable to produce pseudohyphae and were
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susceptible to 0.01% cycloheximide ((3), Table 2). No single D. hansenii carbon assimilation
profile was shared with the other species studied here. Furthermore, there was a trend towards
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8 lower azoles and caspofungin MICs than for P. guilliermondii, a finding not reported for
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Pichia guilliermondii is a genetically heterogenous complex comprising several
phenotypically indistinguishable taxa which have been brought into synonymy (3, 13, 34) or
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12 assigned new names (29). One of these synonymous species is Candida fermentati described
13 by Bai in 1996 (1). Changes in nomenclature could also explain additional mistakes such as
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P. jadinii synonym of C. guilliermondii var. nitratophila. The teleomorphic state of C.
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fermentati has been recently described by Vaughan-Martini in 2005 based on molecular data
and is named Pichia caribbica (34). Since P. caribbica has been described, many analyses
18 DNA banding profiles, D1/D2 sequences) reclassified clinical and environmental isolates
20 but also P. caribbica (2, 14, 29, 34). In our hands, some carbon assimilation profiles were
21 shared by the two species, one of the codes being a frequent code for both (Table S2) and
23 In 1997, Nishikawa designed specific primers in the LSU region to differentiate D. hansenii
24 and P. guilliermondii (17). Lan in 2006 proposed the sequencing of the RIBO gene to
25 differentiate P. caribbica and P. guilliermondii but the sequences seem too divergent within
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1 the same species (14). Recently, Tsui et al. (33) demonstrated that a polygenic sequencing
2 method is best able to differentiate closely related species such as P. guilliermondii and P.
3 caribbica. Here, sequencing of the ITS and 26S regions was sufficient, but using both
5 We also want to underline another pitfall related to these species’ identification. Sequences
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recorded before 2004 and the complete sequencing of D. hansenii genome by Dujon et al.
(10) in the GenBank database are sometimes erroneous. For example, the ITS sequence of
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8 strain CBS8417 (accession #AF209874) deposited in 2001 as D. hansenii var. fabryii has
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guilliermondii in 2006. In the same way, the ITS sequence of strain BPY-01 recorded in
GenBank as D. hansenii var. fabryii in 2002 (accession # AY125962) is homologous with the
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12 ITS sequence of P. guilliermondii type strain CBS566. This underlines the importance of
13 using databases such as that of the CBS where taxonomy is more reliable than in public
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repositories as pointed out by Nilsson and colleagues (16).
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In conclusion, D. hansenii may not be as frequent a human pathogen as previously thought.
Sequencing of either the ITS or the D1/D2 regions is a good tool to differentiate the species
more frequently confused with D. hansenii keeping in mind that reliable databases should be
18 used.
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21 Acknowledgments
22 The authors thank the Institut de Veille Sanitaire for its financial support. The technical help
23 of the sequencing facilities and specifically that of Isabelle Iteman, Anne-Sophie Delannoy,
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32. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997.
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Wagner, D., A. Sander, H. Bertz, J. Finke, and W. V. Kern. 2005. Breakthrough
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Figure legend
Figure 1: Bayesian phylogenetic majority rule consensus tree of the combined D1/D2 and ITS
datasets for the 62 clinical and environmental isolates and the type strains of D. hansenii, P.
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T=Type strain. Shown in brackets are the Genbank accession numbers D1/D2 and ITS. Scale bar
P T
C E
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Table 1: Molecular identification, ID32C code and production of pseudohyphae for the 62 clinical and environmental
isolates sent to the NRCMA between September 2000 and April 2007 as Debaryomyces hansenii or Pichia
guilliermondii.
strain site of year of City, Country first identification ID32C code Pseudo
D
200600362 sputum 2006 Paris, France D. hansenii D. hansenii 5777174137 E+ -
200600935 table surface 2006 Fort de France, Martinique D. hansenii D. hansenii 5777751137 E- -
E
200501145 sputum 2005 Paris, France D. hansenii P. caribbica 7577752117 E+ +
T
200600033 blood 2006 Reims, France D. hansenii P. caribbica 5577370117 E+ +
P
200700175 blood 2006 Paris, France D. hansenii P. caribbica 7577350117 E+ +
E
200500086 blood 2005 Paris, France D. hansenii P.guilliermondii 7577350117 E+ +
C
200500821 environment 2002 Dreux, France D. hansenii P.guilliermondii 7577350117 E+ -
C
200501142 urines 2001 Paris, France D. hansenii P.guilliermondii 7577350117 E+ +
A
200501305 blood 2005 Pontoise, France D. hansenii P.guilliermondii 7577350117 E+ +
200400824 sputum 2004 Fort de France, Martinique D. hansenii C. haemulonii type II 7167370317 E+ -
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Table 1 (Cont’d)
site of year of Pseudo
Strain isolation isolation City, Country first identification identification sequence ID32C code hyphae
D
200600368 BAL 2006 Paris, France P.guilliermondii P. caribbica 7577370117 E+ -
E
200601194 blood 2006 Paris, France P.guilliermondii P. caribbica 7577750117 E+ +
T
200500719 blood 2005 Paris, France P.guilliermondii P.guilliermondii 7577352117 E+ +
P
200500861 blood 2003 Paris, France P.guilliermondii P.guilliermondii 7577372137 E+ +
E
200500864 spleen 2004 Marseille, France P.guilliermondii P.guilliermondii 7577352117 E+ +
C
200501202 blood 2005 Paris, France P.guilliermondii P.guilliermondii 7577350117 E+ -
C
200501319 throat 2002 Paris, France P.guilliermondii P.guilliermondii 7577350117 E+ +
A
200501343 urines 2002 Paris, France P.guilliermondii P.guilliermondii 7577352117 E+ +
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Table 2: Percentage of the clinical and environmental isolates of Pichia guilliermondii, P.
caribbica and D. hansenii forming pseudohyphae and exhibiting growth in the presence of
(n = 33) (n = 18) (n = 3)
Cycloheximide 0.01%
DL-Lactate
D-xylose
97
100
94
100
0
100
66
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Ribose
Ramnose
6
9
33
22
P T 33
66
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Erythritol 0 0 66
Melibiose 74 22 0
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Glucuronate 0 0 66
Gluconate 0 0 33
AC
Lactose
Sorbose
Esculine
Pseudohyphae
3
94
91
83
0
100
89
94
66
100
66
18
CBS 621 (EU568908;EU568909)
100
Pichia jadinii
CNRMA 200500811 (EU568926; EU568927)
67 CNRMA 200501223 (EU568922; EU568923)
99 CNRMA 200500823 (EU568924 ;EU568925) Clavispora lusitaniae
CBS 4413 (EU568906; EU568907)
98 CNRMA 200600079 (EU568918; EU568919)
Candida haemulonii type II
99 CNRMA 200400824 (EU568920; EU568921)
CNRMA 200600130 (EU568964; EU568965)
CNRMA 200600032 (EU568966; EU568967)
CNRMA 200600335 (EU568962; EU568963)
CNRMA 200600561 (EU568960; EU568961)
CNRMA 200601010 (EU568956; EU568957)
CNRMA 200600938 (EU568958; EU568959)
CNRMA 200600938 (EU568958; EU568959)
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CNRMA 200700041 (EU568952; EU568953)
CNRMA 200700329 (EU568948; EU568949)
CNRMA 200700261 (EU568950; EU568951)
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CNRMA 200500030 (EU568946; EU568947)
CNRMA 200500086 (EU568944; EU568945)
CNRMA 200500861 (EU568940; EU568941)
T
CNRMA 200500816 (EU568942; EU568943)
CNRMA 200501141 (EU568938; EU568939)
CNRMA 200501142 (EU568936; EU568937)
CNRMA 200501144 (EU568934; EU568935)
P
CNRMA 200501305 (EU568932; EU568933) Pichia guilliermondii
CNRMA 200501314 (EU568930; EU568931)
97 CNRMA 200501320 (EU568928; EU568929)
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CBS 566 (EU568910; EU568911)
CNRMA 200500864 (EU568970; EU568971)
CNRMA 200500863 (EU568972; EU568973)
CNRMA 200500821 (EU568974; EU568975)
C
CNRMA 200500809 (EU568976; EU568977)
CNRMA 200700040 (EU568978; EU568979)
CNRMA 200601191 (EU568980; EU568981)
CNRMA 200500719 (EU568982; EU568983)
C
CNRMA 200601042 (EU568984; EU568985)
CNRMA 200600803 (EU568986; EU568987)
CNRMA 200501343 (EU568988; EU568989)
A
CNRMA 200501319 (EU568990; EU568991)
CNRMA 200500950 (EU568992; EU568993)
CNRMA 200501202 (EU568968; EU568969)
CBS 2022 (EU568912; EU568913)
CNRMA 200700593 (EU569000; EU569001)
CNRMA 200700035 (EU569002; EU569003)
92 CNRMA 200601082 (EU569004; EU569005)
CNRMA 200600368 (EU569006; EU569007)
CNRMA 200600196 (EU569008; EU569009)
CNRMA 200501316 (EU569010; EU569011)
CNRMA 200600033 (EU569012; EU569013)
CNRMA 200501317 (EU569014; EU569015) Pichia caribbica
CNRMA 200500949 (EU569016; EU569017)
69 CNRMA 200501201 (EU569018; EU569019)
CNRMA 200501145 (EU569020; EU569021)
CNRMA 200501146 (EU569022; EU569023)
CNRMA 200501000 (EU569024; EU569025)
CNRMA 200500862 (EU569026; EU569027)
CNRMA 200500812 (EU569028; EU569029)
CNRMA 200700175 (EU568994; EU568995)
97 CNRMA 200601194 (EU568996; EU568997)
CBS 1795 (EU568914; EU568915)
98 99 CNRMA 200600362 (EU569038; EU569039)
Debaryomyces hansenii
CNRMA 200500815 (EU569036; EU569037)
CNRMA 200600935 (EU569040; EU569041)
99 CBS 7418 (EU568916; EU568917)
CNRMA 200500840 (EU569030; EU569031)
Candida palmioleophila
99 CNRMA 200500825 (EU569032; EU569033)
CNRMA 200500813 (EU569034; EU569035)
CNRMA 200500808 (EU568998; EU568999) Pichia caribbica
0.01
Figure 1