Sequence-based identification of species belonging to the genus Debaryomyces

Sequence-based identification of species belonging to the genus Debaryomyces

FEMS Yeast Research 5 (2005) 1157–1165 www.fems-microbiology.org Sequence-based identification of species belonging to the genus Debaryomyces Patricia...

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FEMS Yeast Research 5 (2005) 1157–1165 www.fems-microbiology.org

Sequence-based identification of species belonging to the genus Debaryomyces Patricia Martorell, M. Teresa Ferna´ndez-Espinar, Amparo Querol

*

Departamento de Biotecnologı´a de los Alimentos, Instituto de Agroquı´mica y Tecnologı´a de Alimentos (CSIC), P.O. Box 73, E-46100 Burjassot, Vale`ncia, Spain Received 1 July 2004; received in revised form 27 April 2005; accepted 4 May 2005 First published online 29 June 2005

Abstract In the present study we assessed the identification by sequence analysis of the 15 species belonging to the genus Debaryomyces. We found that the following species can be identified both quickly and correctly by direct sequence comparison of the ribosomal 5.8S-ITS region: D. carsonii, D. etchelsii, D. maramus, D. melissophilus, D. occidentalis and D. yamadae. In contrast, the species D. castellii, D. coudertii, D. hansenii, D. nepalensis, D. polymorphus, D. pseudopolymorphus, D. robertsiae, D. udenii and D. vanrijiae showed high sequence similarity in ribosomal regions with one or several species. In these cases, sequence comparison of the ACT1 gene is proposed to ensure unequivocal strain designation.  2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords: Debaryomyces; Identification; 5.8S-ITS region; 26S rRNA gene; ACT1 gene

1. Introduction The genus Debaryomyces comprises 15 species according to the last revision [1]. Many representatives can be isolated from natural habitats such as air, soil, pollen, tree exudates, plants, fruits, insects, and faeces and gut of vertebrates [2]. Nine of these Debaryomyces species, D. carsonii, D. etchellsii, D. hansenii, D. maramus, D. melissophilus, D. polymorphus, D. pseudopolymorphus, D. robertsiae and D. vanrijiae, have been found in a variety of processed foods, such as fruit juices and soft drinks, wine, beer, sugary products, bakery products, dairy products and meat or processed meats. The presence of Debaryomyces species in foods usually has no detrimental effects and in some cases is beneficial to the food. *

Corresponding author. Tel.: +34 96 390 0022; fax: +34 96 363 6301. E-mail address: [email protected] (A. Querol).

Some Debaryomyces species are important in the ripening of fermented foods like cheese and meat products [3,4]. In cheeses they metabolise lactic acid, thus raising the pH to allow the growth of proteolytic bacteria, and exhibit lipolytic activity that contributes to the development of cheese aromas. Proteolytic and lipolytic activities of Debaryomyces species have been described in curing of ham and ripening of sausages, and their presence in salami influences the red coloration and improves the quality of the product [3,4]. To a lesser extent, Debaryomyces species contribute to the ripening of pickles, where they oxidize the acids produced by lactic acid bacteria during fermentation [3,4]. Excessive growth of Debaryomyces species may nevertheless cause undesirable sensory changes due to the formation of off-odours and off-flavours. A high salt concentration favours the growth of Debaryomyces species in aged cheeses, dry salami and meat products [5]. These species have also been found as frequent

1567-1356/$22.00  2005 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.femsyr.2005.05.002

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contaminants in spoiled yoghurts, ice creams, fish, shellfish, etc. [3]. Identification of Debaryomyces yeasts is usually performed according to conventional methods, based mainly on the ability to ferment sugars and assimilate different carbon and nitrogen sources. Unfortunately, many of the results obtained with these tests are variable, thus making correct identification of Debaryomyces species difficult. As an example, serious difficulties have been experienced in identifying isolates assigned to Debaryomyces due to erroneous assimilation of D-xylose, and to a lesser extent, raffinose and L-arabinose [6]. To control the growth of these spoilage yeast species, it is essential to determine their occurrence and distribution in different food products. For this purpose, accurate and rapid methods for routine species identification and differentiation are necessary. Due to the difficulties in determining the precise species identity of Debaryomyces isolates using conventional methods, the application of molecular approaches seems to be a more suitable alternative. However, only a few studies have been devoted to the identification of Debaryomyces species using molecular tools. Thus, specific DNA probes for D. hansenii have been developed [7]. The disadvantages of this method are that it is time-consuming and that it cannot be applied for the identification of other Debaryomyces species. In the last decade, identification methods based on rDNA sequences have gained importance for yeast identification. Using the nucleotide information of the largesubunit (26S) rRNA, PCR primers have been designed for the specific identification of D. hansenii and its anamorph Candida famata for clinical purposes [8,9]. The differentiation of D. hansenii from other Debaryomyces species commonly present in foods was not indicated in these studies, and thus the usefulness of this method to identify food-related Debaryomyces isolates is not clear. The lack of studies with this purpose is probably due to the difficulty in finding species-specific signature nucleotides in the LSU sequences of these Debaryomyces species. Sequence analysis of this gene has shown that Debaryomyces species are closely related to each other, making their differentiation difficult [10,11]. Analysis of the small-subunit (18S) rRNA gene resulted in the same picture [11,12]. Usually, the non-coding internal transcribed spacer regions (ITS 1 + 2) exhibit greater interspecific differences than the 18S and 26S rRNA genes [12–15], thus allowing the differentiation of closely related species. One of the most successful methods to identify yeast species is based on the restriction fragment length polymorphism (RFLP) analysis of this 5.8S-ITS region. This method has been useful to delimit many yeast and fungal species [16–18] and it has been successfully extended to differentiate more than 130 yeast species belonging to 25 different genera [19]. The importance of this technique to identify yeast isolates of biotechnological inter-

est is evident, and led us to develop a database available online (http://yeast-id.com). Furthermore, the RFLP analysis of the 5.8S-ITS region has been applied to differentiate 10 out of 15 species known in the genus Debaryomyces [20]. Nevertheless, the ITS region showed great homogeneity. Giving the high discriminatory power for the differentiation of a great number of yeast species [19] we have tested seven additional enzymes to be sure whether or not the RFLP analysis of the 5.8S-ITS ribosomal region provides a reliable tool to identify species of the genus Debaryomyces. Moreover, to complete the aforementioned study [20], the approach was extended to all 15 species of the genus and thus D. carsonii, D. etchellsii, D. occidentalis, D. robertsiae and D. udenii were incorporated in the analysis. Moreover, a sequence analysis of the 5.8-ITS region from the type strain of each species, as well as of the nuclear gene ACT1 of some problematic species, were performed to provide a means for a rapid and accurate identification of Debaryomyces species.

2. Materials and methods 2.1. Yeast strains Thirty-eight strains representing all known species of the genus Debaryomyces were examined (Table 1). The strains were maintained in GYP medium (20 g l1 glucose, 10 g l1 peptone, 5 g l1 yeast extract and 20 g l1 agar, pH 6.0) at 4 C and growth in GYP at 28 C during 2 days. 2.2. 5.8S-ITS restriction analysis DNA was isolated according to Querol et al. [21] and diluted to approximately 50 ng ll1. The 5.8S-ITS region was amplified by PCR in a Progene thermocycler (Techne, Cambridge, UK). Primer pairs used in the PCR were its 1 (5 0 -TCCGTAGGTGAACCTGCGG3 0 ) and its 4 (5 0 -TCCTCCGCTTATTGATATGC-3 0 ) [22]. The thermal cycling parameters were an initial denaturation at 95 C for 5 min, followed by 40 cycles of denaturation at 94 C for 1 min, annealing at 55.5 C for 40 s, and extension at 72 C for 1 min, with a final extension at 72 C for 10 min. PCR products (10 ll or 0.5–1.0 lg) were digested without further purification with 13 restriction endonucleases (Roche Molecular Biochemicals, Mannheim, Germany) according to the supplierÕs instructions. Of these, 7 recognised 4-bp sequences (AluI, CfoI, HaeIII, HpaII, RsaI, Sau3AI, TaqI), 3 were 5-bp cutters (DdeI, HinfI, ScrFI), and the other 3 had a 6-bp target (HindIII, NdeI, ScaI). Restriction fragments were electrophoresed on 3%-agarose gels in 1 · TAE buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8), stained with ethidium bromide

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Table 1 Debaryomyces strains analysed in the present study Species

Strain designation

Isolation source

CECT

CBS

10227T 10230 10541 11407

2285T 810 5254 4050

Slime flux of Quercus Slimy bottled wine Alpechin Wine

Debaryomyces castellii

2923T

Soil

Debaryomyces coudertii

5167T

Droppings of bird

Debaryomyces carsonii

T

T

2011 6823 2012

Fermenting cucumbers Tinned butter Fermenting cucumbers

10026 10352 10360 11363 11364 11369T 11365 11370T

1792 1102 767T 4373 789T

Salt cod Tomato Cheese Chilled beef Beef and pork sausage Cherry Dry white wine Interdigital mycotic lesion

Debaryomyces maramus

11362T 11371

1958T 4264

Air Cider

Debaryomyces melissophilus

11409 11410T

6898 6344T

Soil Honey bee

Debaryomyces nepalensis

5921T

Soil

Debaryomyces occidentalis var. occidentalis var. persoonii

819T 2169T

Soil Soil

6741T

Soil Condensed milk Sweet pepper Soil

Debaryomyces etchellsii

Debaryomyces hansenii var. hansenii

var. fabryi

Debaryomyces polymorphus var. africanus var. polymorphus

Debaryomyces pseudopolymorphus

Debaryomyces robertsiae

11406 11408 11412

11361T 10099 10135 11359T 10056 10293 11360T T

10687

var. yarrowii

2008T

Frass on Populus belleana Frass on Rosa currina Tanning fluid, prepared from bark of sweet-chestnut

2934T

Larval feed of xylocopa caffra (bumble bee)

T

Debaryomyces udenii Debaryomyces vanrijiae var. vanrijiae

186T

10077 10519 11373T 10079T

Debaryomyces yamadae

and visualized under UV light. A 100-bp DNA ladder marker (Gibco BRL, Gaithersburg, MD) served as the size standard. 2.3. PCR amplification and DNA sequencing PCR products were cleaned with the UltraCleane PCR Clean-up Kit (Mo Bio Laboratories, Inc., Solana Beach, CA) and sequenced directly using the Taq DyeDeoxy terminator cycle sequencing kit (Perkin–Elmer,

7056

Soil

3024T 6246T

Frass on Syringa vulgaris Alpechin Soil Insect frass on Paulownia imperialis

7035T

Soil

Norwalk, CT), following the manufacturerÕs instructions, in an Applied Biosystems automatic DNA sequencer, Model 310 ABI PRISMe (Perkin–Elmer, Norwalk, CT.). To sequence the 5.8S-ITS region PCR primers its 1 and its 4 were used and two additional primers in order to obtain sequences from both strands, namely its 2 (5 0 -GCTGCGTTCTTCATCGATGC-3 0 ) and its 3 (5 0 -GCATCGATGAAGAACGCAG-3 0 ). Table 2 shows the GenBank accession numbers of the 5.8S-ITS

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Table 2 GenBank accession numbers of sequences used in this work for type strains of Debaryomyces Species

D. D. D. D. D. D. D. D. D. D. D. D. D. D. D. D. D.

carsonii castellii coudertii etchellsii hansenii (hansenii) hansenii (fabryii) maramus melissophilus nepalensis occidentalis (occid.) occidentalis (persoonii) polymorphus (polymor.) pseudopolymorphus robertsiae udenii vanrijiae yamadae

Strain designation

Accession Number

CECT

CBS

ITS-5.8S

10227T

2285T 2923T 5167T 2011T 767T 789T 1958T 6344T 5921T 819T 2169T 186T 2008T 2934T 7056T 3024T 7035T

AJ586521 AB054102 AB054018(1) AJ586528 AJ586526 AJ586530 AJ586525 AJ586529 AB053099(1) AB054023(1) AB054020(1) AJ586523 AJ586524 AJ586522 AB054098(1) AJ586527 AB054022(1)

11406T 11369T 11370T 11362T 11410T

11359T 11360T 10687T 11373T

D1/D2 of 26S rRNA(2)

Actin gene

U45841 U45846

AJ867052 AJ867051

U45808

AJ508505(3)

U45839

AJ867054

U45836 U45845 U45805 U45844 U45842

AJ604532 AJ604533 AJ867053 AJ868358 AJ604534

(1), (2), (3) (2) (3)

Sequences retrieved from GenBank. Sequences obtained by Kurtzman and Robnett [26]. Sequence obtained by Daniel and Meyer [22].

sequences of the type strain of the fifteen species and their varieties. Primers CA21 (5 0 -ATTGATAACGGTTCCGGTATGTG-3 0 ), CA22R (5 0 -TCGTCGTATTCTTGCTTTGAGATCCAC-3 0 ), CA1 (5 0 -GCCGGTGACGACGCTCCAAGAGCTG-3 0 ), CA15R (5 0 -TCGGTCAAATCTCTACCAGC-3 0 ), CA8 (5 0 -TGTACTCTTCTGGTAGAACTACCGG-3 0 ) and CA5R (5 0 -GTGAACAATGGATGGACCAGATTCGTCG-3 0 ) and the PCR conditions described in a previous study [23] were used to sequence the ACT1 gene. The universal primers CA21 and CA22R were used to amplify and subsequently sequence this gene in the type strains D. castellii CBS 2923T, D. nepalensis CBS 5121T, D. coudertii CBS 5167T, D. maramus CECT 11362T, D. pseudopolymorphus CECT 11360T and D. vanrijiae CECT 11373T. For the strains D. udenii CBS 7056T and D. polymorphus CECT 11359T, the combination of CA8/CA5R with CA1/ CA22R and CA1/CA15R with CA8/CA5R were used, respectively. GenBank accession numbers of these sequences are indicated in Table 2. Sequences of the D1/D2 domain of the 26S rRNA gene were retrieved from the GenBank Nucleotide Sequence Database (Table 2). 2.4. Genetic distances Sequences were aligned using ClustalX, a version of Clustal W [24] for Windows. All gene alignments were manually revised and edited in the insertion–deletion regions and areas of uncertain alignment. The model of sequence evolution that best fitted our sequence data was optimized using the hierarchical

model comparison by likelihood ratio tests, implemented in Modeltest version 3.6 [25]. The best-fitted model was HKY85 [26] with a gamma distribution (G) of substitution rates with a shape parameter a = 0.1347. Nucleotide distances were then estimated by maximum likelihood under the model HKY85 + G with a = 0.1347, a transition/transversion ratio of 2.337, and the following empirical nucleotide frequencies: (A = 0.3015; C = 0.1679; G = 0.1796; and T = 0.3510). These distances were used to obtain a Neighbor-Joining tree [27]. The tree reliability was assessed using nonparametric bootstrap re-sampling of 1000 pseudoreplicates. All these phylogenetic analyses were performed using PAUP* version 4.0b10 [28].

3. Results 3.1. RFLP analysis of the 5.8-ITS region Primers its1 and its4 were used to amplify the 5.8SITS region from 38 yeast strains belonging to 15 accepted Debaryomyces species [1]. PCR products showed a DNA fragment of approximately 650 bp. Subsequently, the amplicons were digested with thirteen restriction endonucleases in order to detect differences among the strains. AluI, HindIII, HpaII, NdeI and SacI did not recognize any restriction site in the ribosomal region of the 38 strains studied. HinfI, RsaI and Sau3A recognized only one site in the 5.8S-ITS region resulting in fragment sizes of 320 + 320, 490 + 190 and 400 + 225 bp, respectively. CfoI and HaeIII generated invariant restriction patterns

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of 310 + 310 + 50 and 420 + 140 + 90 bp, respectively. Only DdeI, ScrFI and TaqI yielded polymorphic patterns among the strains (Table 3). DdeI displayed a specific pattern for D. etchellsii with three bands of 327, 221 and 96 bp, and the other species exhibited the same pattern of 425 and 210 bp. ScrFI yielded three different patterns. One of them with a unique band of 650 bp (i.e. no restriction site), was shared by 10 species: D. carsonii, D. castellii, D. coudertii, D. hansenii, D. nepalensis, D. occidentalis, D. polymorphus, D. pseudopolymorphus, D. vanrijiae and D. yamadae. A different pattern was found in D. etchellsii, D. maramus, D. robertsiae and D. udenii, with two bands of 550 and 100 bp.

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Only the type strain of D. melissophilus, CECT 11410T, showed a distinctive pattern with 400 and 238 bp. Finally, TaqI yielded two patterns. The first pattern, with restriction fragments of 250, 210, 110 and 60 bp, was exhibited by all the strains belonging to D. carsonii, D. castellii, D. coudertii, D. nepalensis, D. maramus, D. pseudopolymorphus, D. vanrijiae and D. yamadae. The other group, with a restriction pattern of 325, 250 and 60 bp, appeared in all the strains of D. etchellsii, D. melissophilus, D. occidentalis and in the only strain analysed of D. robertsiae and D. udenii. The species D. hansenii and D. polymorphus contained strains exhibiting both TaqI restriction patterns.

Table 3 Restriction patterns obtained after digestion with three endonucleases that revealed some polymorphism at the 5.8S-ITS region of the Debaryomyces strains analysed Species

Strains CECT

D. carsonii

D. castelli D. coudertii D. etchellsii

D. hansenii var. hansenii

var. fabriyii D. maramus D. melissophilus D. nepalensis D. occidentalis var.occidentalis var.persoonii D. polymorphus var. africanus var. polymorphus

D. pseudopolymorphus

D. robertsiae D. udenii D. vanrijiae var. vanrijiae

var. yarrowii D. yamadae

Restriction patterns CBS

DdeI

ScrFI

TaqI

425 + 210 425 + 210 425 + 210 425 + 210 425 + 210 425 + 210 327 + 221 + 96 327 + 221 + 96 327 + 221 + 96

650 650 650 650 650 650 550 + 100 550 + 100 550 + 100

250 + 210 + 110 + 60 250 + 210 + 110 + 60 250 + 210 + 110 + 60 250 + 210 + 110 + 60 250 + 210 + 110 + 60 250 + 210 + 110 + 60 325 + 250 + 60 325 + 250 + 60 325 + 250 + 60

5921T

425 + 210 425 + 210 425 + 210 425 + 210 425 + 210 425 + 210 425 + 210 425 + 210 425 + 210 425 + 210 425 + 210 425 + 210 425 + 210

650 650 650 650 650 650 650 650 550 + 100 550 + 100 650 400 + 238 650

325 + 250 + 60 325 + 250 + 60 250 + 210 + 110 + 60 250 + 210 + 110 + 60 250 + 210 + 110 + 60 325 + 250 + 60 250 + 210 + 110 + 60 250 + 210 + 110 + 60 250 + 210 + 110 + 60 250 + 210 + 110 + 60 325 + 250 + 60 325 + 250 + 60 250 + 210 + 110 + 60

819T 2169T

425 + 210 425 + 210

650 650

325 + 250 + 60 325 + 250 + 60

7056T

425 + 210 425 + 210 425 + 210 425 + 210 425 + 210 425 + 210 425 + 210 425 + 210 425 + 210

650 650 650 650 650 650 650 550 + 110 550 + 110

250 + 210 + 110 + 60 250 + 210 + 110 + 60 250 + 210 + 110 + 60 325 + 250 + 60 250 + 210 + 110 + 60 250 + 210 + 110 + 60 250 + 210 + 110 + 60 325 + 250 + 60 325 + 250 + 60

7035T

425 + 210 425 + 210 425 + 210 425 + 210 425 + 210

650 650 650 650 650

250 + 210 + 110 + 60 250 + 210 + 110 + 60 250 + 210 + 110 + 60 250 + 210 + 110 + 60 250 + 210 + 110 + 60

T

10227 10230 10541 11407

2923T 5167T 11406T 11408 11412 10026 10352 10360 11363 11364 11369T 11365 11370T 11362T 11371 11409 11410T

11361T 10099 10135 11359T 10056 10293 11360T 10687T

10077 10519 11373T 10079T

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Table 4 Sequence similarities (above the diagonal) and nucleotide differences (below the diagonal) between 5.8S-ITS sequences of the type strains from species in the genus Debaryomyces (1) (1) D. hansenii (fabryi) (2) D. hansenii (hans.) (3) D. coudertii (4) D. nepalensis (5) D. maramus (6) D. robertsiae (7) D. udenii (8) D. etchelsii (9) D. castellii (10) D. yamadae (11) D. polymorphus (poly.) (12) D. pseudopolym. (13) D. vanrijiae (14) D. occidentalis (occid.) (15) D. occidentalis (perso.) (16) D. carsonii (17) D. melissophilus

1 4 6 10 17 16 51 31 37 33 32 34 33 32 51 62

T

(2)

(3)

(4)

(5)

(6)

(7)

(8)

(9)

(10)

(11)

(12)

(13)

(14)

(15)

(16)

(17)

99.8

99.3 99.4

98.9 99.1 98.9

98.2 98.3 97.8 97.4

96.8 97.0 96.4 96.4 96.4

97.0 97.2 96.6 96.6 96.6 99.1

89.9 90.1 89.9 90.7 89.9 89.5 89.7

94.0 94.2 93.8 93.6 93.6 91.6 92.4 88.6

92.8 93.0 92.6 92.1 92.4 90.5 91.3 87.2 98.3

93.6 93.8 93.4 93.2 93.2 91.6 92.4 88.6 99.3 97.6

93.8 94.0 93.6 93.4 93.4 91.4 92.2 88.4 99.8 98.1 99.4

93.4 93.6 93.2 93.0 93.0 91.4 91.8 88.0 99.3 97.9 99.3 99.1

93.6 93.8 93.4 93.8 92.8 93.0 93.4 89.9 95.0 95.2 94.6 94.8 94.5

93.8 94.0 93.6 94.0 93.3 93.6 94.0 90.1 95.2 95.0 94.8 95.0 94.6 99.4

89.8 90.0 89.4 89.8 88.7 89.2 89.2 85.1 90.3 90.5 89.9 90.5 90.1 93.0 93.2

87.4 87.6 87.4 87.4 86.3 86.8 87.2 83.0 88.3 88.3 87.9 88.6 87.9 90.5 90.7 91.8

3 5 9 16 15 50 30 36 32 31 33 32 31 50 61

6 12 19 18 51 32 38 34 33 35 34 33 53 62

14 19 18 47 33 40 35 34 36 32 31 51 62

19 18 55 33 39 35 34 36 37 36 56 67

5 53 43 48 43 44 44 36 33 54 65

T

52 39 44 39 40 42 34 31 54 65 T

57 63 57 58 60 51 50 54 63

9 4 1 4 26 25 72 81

13 10 11 25 26 48 58

3 4 28 27 51 60

5 27 26 48 57

29 28 50 60

T

3 36 48

35 47

42

T

(1) strain CECT 11370 , (2) strain CECT 11639 , (3) strain CBS 5167 , (4) strain CBS 5921 , (5) strain CECT 11362 , (6) strain CECT 10687T, (7) strain CBS 7056T, (8) strain CECT 11406T, (9) strain CBS 2923T, (10) strain CBS 7035T, (11) strain CECT 11359T, (12) strain CECT 11360T, (13) strain CECT 11373T, (14) strain CBS 819T, (15) strain CECT 2169T, (16) strain CECT 10227T, (17) strain CECT 11410T. Sequence similarities between species, higher than 99% are in bold. Table 5 DNA relatedness and sequence similarities in the 5.8S-ITS ribosomal region, the D1/D2 domain of the 26S rDNA and the ACT1 gene of the type strains of species pairs occurring in the genus Debaryomyces Species

Strain designation

D. polymorphus var. polymorphus D. pseudopolymorphus

CECT 11359T CECT 11360T

D. polymorphus var. polymorphus D. vanrijiae var. vanrijiae

nDNA relatedness (%)

% Similarity 5.8S-ITS

D1/D2

ACT1

21a

99.4F

99.5E

92.4A

CECT 11359T CECT 11373T

24a

99.3F

98.6E

93.2B

D. pseudopolymorphus D. vanrijiae var. vanrijiae

CECT 11360T CECT 11373T

NF

99.1F

98.8E

96.3B

D. hansenii var. hansenii D. coudertii

CECT 11369T CBS 5167T

16a

99.4F

99.4D

85.7a

D. hansenii var. hansenii D. nepalensis

T

CECT 11369 CBS 5921T

15a

99.1F

99.1E

90.7A

D. coudertii D. nepalensis

CBS 5167T CBS 5921T

NF

98.9F

98.8D

85.2A

D. robertsiae D. udenii

CECT10687T CBS 7056T

NF

99.1F

99.5E

82.8C

D. castellii D. polymorphus

CBS 2923T CECT 11359T

20a

99.3F

99.1

E

91.5A

T

D. castellii D. pseudopolymorphus

CBS 2923 CECT 11360T

NF

99.8F

98.9E

95.5A

D. castellii D. vanrijiae

CBS 2923T CECT 1137T

NF

99.3F

98.8E

95.6B

NF, data not found in the literature. A Based on ca. 959 nucleotides in actin gene. B Based on ca. 904 nucleotides in actin gene. C Based on ca. 658 nucleotides in actin gene. D Based on ca. 511 nucleotides in D1/D2. E Based on ca. 571 nucleotides in D1/D2. F Based on ca. 576 nucleotides in 5.8S-ITS region. a nDNA reassociation data are from Kurtzman and Robnett [32].

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3.2. Sequencing analysis of the 5.8-ITS region

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D. yamadae D.vanrijiae D. polymorphus var. polymorphus 77 D. pseudopolymorphus D. castelli D. occidentalisvar. occidentalis 72 D. occidentalis var.persoonii D. carsonii 79 32

20

A distinctive profile could not be assigned for each Debaryomyces species based on the 5.8S-ITS restriction patterns, and therefore the sequence of this region was analyzed for the 17 type strains belonging to the 15 Debaryomyces species and varieties (Table 2). The sequences, including the 19 and 20 bp of the its1 and its4 primers, varied from 635 to 644 nucleotides. The 5.8S-ITS region sequence similarities are shown in Table 4 above the diagonal and the absolute nucleotide differences below the diagonal. Similarity values among species varied from 85.1% for the pair D. etchelsii/D. carsonii to 99.8% for the pair D. castellii/ D. pseudopolymorphus, which corresponded to absolute nucleotide differences of between 54 and 1, respectively. Thus, as an alternative to the RFLP analysis, the identification of these species can be handled by the direct sequence analysis of this ribosomal region and the subsequent comparison with sequences in databases. However, in some cases, we obtained very high similarity values (98.9–99.8%), corresponding with nucleotide differences between 6 and 1. This is the case with 10 species pairs listed in Table 5, of which the corresponding similarity values of which are indicated in bold in Table 4. The phylogenetic tree based on the ITS and 5.8SrRNA sequences of the investigated strains is shown in Fig. 1. This tree shows that the Debaryomyces species form four significant clades according to bootstrap values. The first clade contains D. coudertii, D. hansenii (var. hansenii and var. fabryi), D. nepalensis, D. maramus, and the pair D. robertsiae and D. udenii. This group includes species with very low nucleotide divergences (Table 5), confirmed in the tree by the low bootstrap values (from 26% to 55%), indicating that there are few nucleotide differences supporting the within-clade relationships. The second lineage is formed by D. etchellsii which is closely related to the first clade, with a bootstrap value of 86%. The third group includes the species D. castellii, D. yamadae, D. vanrijiae, D, polymorphus and D. pseudopolymorphus, with very limited or no nucleotide divergences (Table 5). Finally, the fourth clade is the most heterogeneous, formed by two species pairs, D. carsonii and D. melissophilus (96% bootstrap), and D. occidentalis var. occidentalis and var. persoonii (54% bootstrap). 3.3. Sequence analysis of the D1/D2 region and the actin gene from species that are difficult to differentiate Two other gene regions were analyzed, namely the D1/D2 domains of the 26S rRNA [29,30] and the ACT1 gene coding for actin [23,30] from the species D. castellii, D. coudertii, D. hansenii, D. nepalensis,

79

91

D. melissophilus D. etchellsii

D. robertsiae D. udenii D. maramus D. hansenii var. fabryi 86 D. hansenii var. hansenii 54 D. coudertii 33 D. nepalensis 55 99

63

0.02

Fig. 1. Neighbor-joining tree based on 5.8S-ITS region sequences from the type strains of the species of Debaryomyces species. The tree has been rooted at the mid-point, located at the same average distance from each taxon. The numbers at the nodes indicate the level of bootstrap support based on 1000 re-samples. Branch lengths are proportional to the scale given in substitutions per nucleotide.

D. polymorphus, D. pseudopolymorphus, D. robertsiae, D. udenii and D. vanrijiae, that formed the 10 species pairs mentioned previously that are difficult to differentiate (Table 5). The 10 pairs of species showed a high sequence similarity in the D1/D2 domains, ranging from 98.6% for the pair D. polymorphus/D. vanrijiae to 99.5% for the pairs D. polymorphus/D. pseudopolymorphus and D. robertsiae/D. udenii (Table 5). These similarity values are either very close or within the values considered to indicate conspecificity (>99%) [29]. The partial sequences of the ACT1 gene gave lower similarity values, ranging from 82.8% for D. robertsiae/ D. udenii to 96.3% for D. pseudopolymorphus/ D. vanrijiae (Table 5). Therefore, the ACT1 gene sequences can be used as a tool to differentiate these species.

4. Discussion The 15 species included in the genus Debaryomyces form a group of yeast species that reproduce vegetatively by multilateral budding. They do not perform a true sexual process, as only heterogamous conjugation between nuclei in the mother cell and the bud and isogamous conjugation occurs. Fermentation is variable, from absent to weak or occasionally vigorous. Nitrate is not assimilated [1]. The identification of isolates at the genus level is facilitated by the presence of the typical asci and ascospores [20]. However, the variable results obtained in their physiological characterization and the considerable intraspecific variability occurring

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in some species (e.g., D. hansenii), complicate species identification. Other approaches, among them the analysis of the % G + C composition of the DNA, the ubiquinone system, proton magnetic resonance spectra of mannans, cell surface antigens, and physiological and biochemical characteristics, equally did not result in a correct identification of the various Debaryomyces species, [31]. Only nDNA–nDNA reassociation experiments discriminated successfully between these species [31], but this technique is too complex to be used for routine identification. Restriction analysis of the 5.8S-ITS has been applied previously to identify some species of the genus [20]. Restriction analysis with 10 different endonucleases showed a high homogeneity within Debaryomyces, despite the high resolution in the identification of species described in the literature for this technique [19,32–34]. In an attempt to understand this contradiction, in the present study we extended the RFLP analysis of this ribosomal region to the differentiation of all species known in the genus Debaryomyces and we used seven additional restriction enzymes, specifically DdeI, HindIII, HinfI, NdeI, Sau3A1, ScaI and ScrFI. In agreement with previous work [20], a lack of resolution was observed, as only a few different patterns, each shared by several species, were obtained. The sequence analysis of the 5.8S-ITS region demonstrated that, in general, this region is highly conserved within the genus Debaryomyces, thus explaining the limited polymorphisms observed in the restriction analysis. However, some of the species could be resolved by pair-wise comparison of these sequences, namely D. carsonii, D. etchelsii, D. maramus, D. melissophilus, D. occidentalis and D. yamadae. The differentiation of these species, can also be approached by sequence comparison of the D1/D2 LSU region, except for D. maramus and D. coudertii which showed a similarity value of 98.9%. The species D. castellii, D. coudertii, D. hansenii, D. nepalensis, D. polymorphus, D pseudopolymorphus, D. robertsiae, D. udenii and D. vanrijiae showed sequence similarities, above 99%, in the 5.8-ITS region, with one or several species. This is the case of D. castellii with D. vanrijiae (99.3%), D. coudertii with D. nepalensis (98.9%), D. robertsiae with D. udenii (99.1%), D. polymorphus with D. pseudopolymorphus, D. castellii and D. vanrijiae (99.4%, 99.3% and 99.3%, respectively), D. pseudopolymorphus with D. vanrijiae and D. castellii (99.1% and 99.8%, respectively) and D. hansenii var. hansenii with D. coudertii and D. nepalensis (99.4% and 99.1%, respectively). Sequence analysis of the D1/D2 domains of the 26S rRNA also indicated that these species pairs are closely related and difficult to differentiate. The low nucleotide divergences found to occur between these Debaryomyces species pairs is not congruent with the low nDNA reassociation values available in the literature for some of them (Table 5) [35]. Although

the ribosomal regions have proven to be a powerful tool for yeast species delimitation, this is not the first time that an incongruence is reported. Other examples are Candida fructus and C. musae, or C. zeylanoides and C. krissii [29,36], and Rhodosporidium glutinis and R. graminis [37], among others. In an attempt to find more polymorphic regions, which might be used to differentiate between closely related species, we sequenced the nuclear ACT1 gene [23,30]. As a result, the ACT1 gene showed higher levels of nucleotide divergence, ranging from 82.8% to 96.3%, thus allowing them to differentiate between closely related Debaryomyces species. The development of future molecular methods for their rapid and easy identification could be based on this molecular region. Unfortunately, repeated attempts to sequence the mitochondrial COX2 gene [38] were unsuccessful. The identification of the species currently accepted in the genus Debaryomyces cannot be achieved by applying RFLP analysis of the ribosomal 5.8S-ITS region, as has been proposed for many yeast species. Instead, we propose that their identification should be based on sequence analysis. In conclusion, the identification of the species D. carsonii, D. etchelsii, D. maramus, D. melissophilus, D. occidentalis and D. yamadae can be carried out by sequence comparison of the 5.8S-ITS region. In the case of the species D. castellii, D. coudertii, D. hansenii, D. nepalensis, D. polymorphus, D pseudopolymorphus, D. robertsiae, D. udenii and D. vanrijiae, a high sequence similarity was found in the ribosomal regions and sequence comparison of the ACT1 gene is proposed for unequivocal strain designation.

Acknowledgements This work was supported by two CICYT grants (ref. AGL2000-1492 and BIO2003-03793-C03-01) from the Spanish Ministerio de Ciencia y Tecnologı´a and a Generalitat Valencia grant (ref. GRUPOS03/012), all to A.Q. P.M. is recipient of a FPI predoctoral fellowship from the Ministerio de Ciencia y Tecnologı´a.

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