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ISSN 1022-7954, Russian Journal of Genetics, 2017, Vol. 53, No. 5, pp. 561–567. © Pleiades Publishing, Inc., 2017. Original Russian Text © E.S. Naumova, Ch.-Fu Lee, V.I. Kondratieva, A.Zh. Sadykova, G.I. Naumov, 2017, published in Genetika, 2017, Vol. 53, No. 5, pp. 562–569.

GENETICS OF MICROORGANISMS

Molecular Genetic Polymorphism of Soil Yeasts of the Genus Williopsis from Taiwan Island E. S. Naumovaa, Ch.-Fu Leeb, V. I. Kondratievaa, A. Zh. Sadykovaa, and G. I. Naumova, * a

State Research Institute for Genetics and Selection of Industrial Microorganisms, Moscow, 117545 Russia bDepartment of Applied Science, National Tsing Hua University, Hsinchu, 30014 Taiwan *e-mail: [email protected] Received June 30, 2016

Abstract⎯Comparative molecular genetic study of Williopsis yeasts isolated in different world regions reveals some peculiarities of species content in Taiwan. Some Williopsis yeasts may represent novel species. In Taiwan, four of the five known Williopsis species are documented: W. saturnus, W. suaveolens, W. mrakii, and W. subsufficiens. The W. saturnus yeasts predominate in Taiwanese soils, while W. suaveolens is more frequently isolated in Europe. Keywords: soil yeast Williopsis, phylogenetic analysis, biological species, Taiwan, killer toxins DOI: 10.1134/S1022795417040111

INTRODUCTION Over the last decade, the gene pool of yeast used in fundamental and applied research was considerably expanded. In modern science and practice, great attention is paid to unconventional non-Saccharomyces yeasts that are able to produce various physiologically active substances and to function as biological agents suppressing the development of harmful fungi. In this connection Taiwan, which possesses endemic flora and fauna, is of relevance for studies of yeast biodiversity. Geographical isolation and tropical climate are obviously important factors that affect evolution of Taiwanese yeasts. Ascomycetous yeasts Williopsis are commonly found in soils and other natural environments in Taiwan [1]. Strains capable of producing broad-spectrum killer toxins (mycocins) are common among these yeasts [2–5]. Williopsis yeasts can be used in agriculture as biocontrolling microorganisms for protection of plants and damp grain from fungi infections and in medicine and veterinary science for mycosis treatment [6, 7]. The modern classification of ascomycetous yeasts is based on phylogenetic analysis of a number of molecular markers, most importantly the D1/D2 domain of the 26S rRNA gene [8, 9]. Additionally, analysis of the 5.8S-ITS fragment that comprises the 5.8S rRNA gene and internal transcribed spacers ITS1 and ITS2 is used to identify the phylogenetic relationship of closely related species. This region is characterized by a significant interspecific divergence and a low level of intraspecific polymorphism. The length of the ITS region is constant among strains of the same species [10]; however, the sequence can vary [11, 12]. The

following Williopsis species were identified by genetic and molecular analysis: W. saturnus, W. mrakii, W. sargentensis, W. suaveolens, and W. subsufficiens [13–15]. W. beijerinckii, which was identified via genetic analysis, was assigned as synonym of W. saturnus on the basis of a high level of total DNA-DNA reassociation [16]. The goal of the present study was to perform a phylogenetic analysis of Williopsis yeasts isolated in Taiwan and other regions of the world, as well as a genetic hybridization analysis of putative novel species. MATERIALS AND METHODS Media and Strains The studied strains of genus Williopsis and their origins are listed in Table 1. The yeasts were cultivated at 28°С on complete agar medium YPD of the following composition (g/L): glucose, 20; peptone, 10; yeast extract, 10; agar, 20. The composition of the minimal medium (g/L): yeast nitrogen base without amino acids (Difco, United States), 6.7; glucose, 20; agar, 20. The composition of the sporulation medium (g/L): sodium acetate, 10; potassium chloride, 5; agar, 20. Polymerase Chain Reaction (PCR) PCR was performed on a Tercyc DNA thermocycler (DNA-Technology, Russia) directly on yeast cells using NL-1 (5'-GCATATCAATAAGCGGAGGAAAG-3') and NL-4 (5'-GGTCCGTGTTTCAAGACGG-3') primers for the D1/D2 domain or ITS1 (5'-TCCGTAGGTGAACCTGCGG) and ITS4 (5'-TССTCCGCTTATTGA-

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Table 1. Origin of the studied strains of Williopsis Strain

Source and place of isolation

NN11S10 GY29S04 TJ12S73 EN8S15 NU4M05 TJ13S72 ES13S01 ES27S03 SC4S03 EN2S10 EN6S07 EN7S13

Soil, Jianshih, Hsinchu Soil, Sinyi, Nantou Mycena sp., Sioulin, Hualein Soil, Meishan, Chia-I Fungus, Beinan, Taitung Soil, Sioulin, Hualein Soil, Sinyi, Nantou Soil, Sinyi, Nantou Soil, Dasi, Taoyuan Soil, Meishan, Chia-I Soil, Meishan, Chia-I Soil, Meishan, Chia-I

EN3S04 EN5S03 FN2S02 SJ7S10 SA5S09 GY5S03 EU6S01

Soil, Meishan, Chia-I Soil, Meishan, Chia-I Soil, Taian, Miaoli Soil, Yuchih, Nanto Soil, Sanyi, Miaoli Soil, Taoyuan, Kaohsiung Soil, Lujhu Township, Taoyuan

NF1M01 NF3S12 NF7S03 EN26S06 GY12S05 GE5S03

Pluteus sp., Wulai, Taipei Soil, Datong, Ilan Soil, Datong, Ilan Soil, Sinyi, Nantou Soil, Taoyuan, Kaohsiung Soil, Rueisuei, Hualein

GE7S02

Soil, Rueisuei, Hualein

Strain Taiwan strains Williopsis saturnus FN10S01 FN20S01 EN12S03 EN17S10 EN22S15 SM7S10 GY8S06 SY3S03 SU10S07 SU16S01 SG1S01 ND6S71 W. mrakii GE4S14 GE7S01 GE16S03 GE19S10 EU5S06 SG5S06 W. suaveolens TJ1S71 TJ10S73 GJ3S05 NF18L15 NF19S06 EJ4M05


Soil, Taian, Miaoli Soil, Wufeng, Hsinchu Soil, Sinyi, Nantou Soil, Sinyi, Nantou Soil, Sinyi, Nantou Soil, Jianshih, Hsinchu Soil, Taoyuan, Kaohsiung Soil, Yuanshan, Yilan Soil, Renai, Nantou Soil, Renai, Nantou Soil, Renai, Nantou Soil, Taian, Miaoli Soil, Wanrong, Hualein Soil, Rueisuei, Hualein Soil, Changbin, Taitung Soil, Fongbin, Hualein Soil, Lujhu Township, Taoyuan Soil, Renai, Nantou

Soil, Sioulin, Hualein Soil, Sioulin, Hualein Soil, Wutai, Pingtung Polytrichum commune, Wulai, Taipei Soil, Wulai, Taipei Pleuroteus sp., Jianshih, Hsinchu

W. subsufficiens EU5S02

Soil, Lujhu Township, Taoyuan County

Williopsis sp. FN14S01

Soil, Wufeng, Hsinchu

GY3S10

Soil, Taoyuan, Kaohsiung

SA20S03

Soil, Emei, Hsinchu

GY13S02

Soil, Taoyuan, Kaohsiung

Strains from other regions of the world W. saturnus CBS 254 (T)

Soil, Himalayas

CBS 5761

Soil, USA

CBS 8880

Soil, Alps, Slovenia

CBS 8882

Soil, Krasnodar krai, Russia

W. mrakii CBS 1707 (T)

Soil, Papua New Guinea

NRRL YB-3257

Soil, India

W. suaveolens CBS 255 (T)

Soil, Denmark

CBS 8878

Soil, Tula oblast, Russia

VKM Y-838

Unknown

CBS 8879

Soil, Rostov oblast, Russia

W. sargentensis RUSSIAN JOURNAL OF GENETICS

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Table 1. (Contd.) Strain


CBS 6342 CBS 5763 (T) CBS 6291 CBS 1669 CBS 1670

Strain

Lake water, New Hampshire, USA W. subsufficiens Soil, Liberia NRRL YB-1718 Soil, Nigeria Williopsis sp. Soil, Minnesota, USA NBRC 1776 Soil, Minnesota, USA


Soil, Liberia

Soil, Japan

T, type culture. CBS 8879 = KBP 2709, CBS 8878 = KBP 2706, CBS 8882 = VKM Y-1638, CBS 8880 = KBP 3655.

TATGC) primers for the 5.8S-ITS fragment. A small portion of biomass of the yeast (on the tip of an inoculation loop) was suspended in 30 μL of buffer solution comprising 3 μM MgCl2, 0.03 μM dNTP, and 50 pmol of each primer. The resulting mixture was heated at 95°С for 15 min to lyse the cells, and then 2.5 units of Taq polymerase (Syntol, Russia) was added. Amplification of the D1/D2 region was performed as follows: initial denaturation at 94°С for 1 min; then 36 cycles as follows: denaturation of DNA at 94°С for 1 min, annealing at 52°С for 1 min, DNA synthesis at 72°С for 1 min; and final elongation at 72°С for 1 min. For amplification of 5.8S-ITS fragments, initial denaturation was at 94°С for 3 min; then 30 cycles as follows: denaturation at 94°С for 30 s, annealing at 56°С for 30 s, DNA synthesis at 72°С for 60 s; and final elongation at 72°С for 10 min. The amplified products were then subjected to electrophoresis in 1% agarose gel at a voltage of 60–65 V in 0.5× TBE buffer solution (45 μM Tris, 10 μM EDTA, and 45 μM boric acid) for two hours and stained with ethidium bromide. Determination of Nucleotide Sequences and Phylogenetic Analysis Nucleotide sequences of the D1/D2 domain and internal transcribed spacers ITS1 and ITS2 were determined by two strands using direct sequencing by the Sanger method on a Beckman-Coulter automated sequencer (USA). The search for homology with known nucleotide sequences was carried out using BLAST (http://www.ncbi.nlm.nih.gov). The multiple alignment of nucleotide ITS sequences was performed manually using the BioEdit software program (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). The cluster analysis was carried out using the UPGMA method in MEGA 6 [17]. Genetic Hybridization Analysis Highly fertile monosporic cultures of the studied strains were marked with auxotrophic mutations via UV irradiation. Hybrids were obtained by mass crossRUSSIAN JOURNAL OF GENETICS

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ing of sporulating cultures with complementary auxotrophic mutations [18]. Sporulating strains obtained by the hybridization were treated with digestive juice of garden snail Helix pomatia (DJS). After the cloning on the minimal medium and subsequent reinoculations onto the complete medium and then onto the sporulation medium, we isolated ascospores from the resulting hybrids using a micromanipulator. Further, we studied the monosporic progeny. The ascus walls were destroyed by DJS treatment. In the case of low viability of the hybrid ascospores, a random spore analysis was used by treatment of spores with ethanol. RESULTS We conducted a molecular genetic study of 55 strains of Williopsis found in Taiwan and 17 strains that were isolated in other regions of the world (Table 1). The latter were obtained from the following collections: CBS, Centraalbureau voor Schimmelcultures, Utrecht, Netherlands; NBRC (=IFO), NITE Biological Resource Center, Osaka, Japan; NRRL, Northern Region Research Center, Peoria, USA; VKM, AllRussian Collection of Microorganisms, Moscow; KBP, Collection of Department of Soil Biology, Moscow State University, Moscow. The species belonging of those strains was previously determined ([14]; http://www.cbs.knaw.nl). First of all, the species affiliation of 55 Taiwanese strains were determined using sequencing of the D1/D2 domain of the 26S rRNA gene. We compared the resulting nucleotide sequences to the sequences of the D1/D2 domain of the type cultures of five known Williopsis species present in the GenBank database: W. saturnus CBS 254, W. suaveolens CBS 255, W. mrakii CBS 1707, W. sargentensis CBS 6342, and W. subsufficiens CBS 5763. According to the analysis, all Taiwanese strains belong to genus Williopsis. Using molecular analysis, we identified the studied strains as four species of genus Williopsis of (Table 1): 24 strains were identified as W. saturnus, 12 strains as W. suaveolens, and 13 strains as W. mrakii. GE7S02 and EU5S02 belong to W. subsufficiens. We were unable to find W. sargentensis yeasts among the studied Taiwanese 2017

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strains. According to the analysis, FN14S01, SA20S03, GY3S10, and GY13S02 strains do not belong to any of the five known species. Out of the 17 strains isolated in other regions of the world, three do not belong to any known species of Williopsis: the North American strains (CBS 1669 and CBS 1670) and the Japanese strain NBRC 1776. The North American strains have identical D1/D2 sequences. Previously, we showed that CBS 1669, CBS 1670, and NBRC 1776 are characterized by unique PCR profiles with M13 microsatellite primer [14]. To determine the phylogenetic relationship of the Williopsis strains isolated in Taiwan and other regions of the world, we performed a comparative analysis of nucleotide sequences of the 5.8S-ITS region of rDNA that comprises the 5.8S rRNA gene and internal transcribed spacers ITS1 and ITS2. We amplified 5.8SITS fragments of 72 analyzed strains, including five species testers. The size of amplified fragments was the same in all the studied and control strains and was approximately 650 bp, which confirms that all strains belong to the genus Williopsis. A phylogenetic tree (figure) was reconstructed on the basis of the obtained nucleotide sequences of 5.8S-ITS regions. The 72 studied strains formed four main clusters. The first one included the type culture W. saturnus CBS 254, 24 Taiwanese isolates, and strains CBS 5761, CBS 8880, and CBS 8882 isolated in the United States, Slovenia, and Russia, respectively. The type culture of W. suaveolens CBS 255, 12 Taiwanese strains, and strains CBS 8878, CBS 8879, and VKM Y-838 isolated from the soil in the European part of Russia were in the second cluster. The type culture W. mrakii CBS 1707, 13 Taiwanese strains, and the NRRL YB-3257 strain isolated in India formed the third cluster. The fourth cluster comprised two Taiwanese strains (GE7S02, EU5S02) and two strains from Liberia, including the type culture of W. subsufficiens CBS 5763. Four Taiwanese strains (FN14S01, SA20S03, GY3S10, and GY13S02), two North American strains (CBS 1669 and CBS 1670), and Japanese strain NBRC 1776 were not included in any of the four clusters. FN14S01 and SA20S03 have identical nucleotide sequences of the D1/D2 domain and 5.8S-ITS fragment of rDNA. CBS 1669 and CBS 1670 also have identical D1/D2 and 5.8S-ITS sequences. Thus, the results of the phylogenetic analysis of nucleotide sequences of the D1/D2 domain and 5.8S-ITS fragments indicate the existence of five novel species: Williopsis sp. 1 (CBS 1669 and CBS 1670), Williopsis sp. 2 (NBRC 1776), Williopsis sp. 3 (FN14S01 and SA20S03), Williopsis sp. 4 (GY3S10), and Williopsis sp. 5 (GY13S02). To determine taxonomic status of divergent strains, we performed a genetic hybridization analysis of two of them: CBS 1669 and NBRC 1776. We used the type culture of Williopsis sargentensis CBS 6342 as a test strain. It should be noted that the Williopsis yeasts are

diplonts and have a homothallic life cycle. CBS 1669, NBRC 1776, and CBS 6342 sporulated well on the acetate medium, and the viability of their ascospores in the first generation was 83, 48, and 66%, respectively. Single-spore cloning in the second generation increased the viability of the ascospores of the studied strains to 100, 99, and 98%, respectively. Using UV irradiation, we marked highly fertile single-spore clones of the studied strains with auxotrophic mutations. We obtained the ade1 and lys8 mutations in CBS 1669, his4 and ura2 in NBRC 1776, and arg25 and met26 in CBS 6342. First, control intrastrain crosses were carried out. All of the hybrids obtained had rather high viability of ascospores: 84% in CBS 1669 and reduced viability in strains NBRC 1776 and CBS 6342: 64 and 66%, respectively. In all cases, we observed normal digenic meiotic segregation of the auxotrophic markers (Table 2, hybrids nos. 1–3). To determine the species belonging of CBS 1669 and NBRC 1776, they were crossed with each other and with the tester W. sargentensis CBS 6342. Hybrids were obtained in all combinations, confirming that the strains belong to the same genus. The viability of ascospores of the CBS 1669 × NBRC 1776 hybrids was 0 to 1% (Table 2, hybrids nos. 4a and 4b). The only surviving segregant was revealed to be prototrophic after tetrad analysis. Random spore analysis revealed the abnormal segregation of control markers, which is characteristic of the interspecific hybrids. The vast majority of the monosporic clones were prototrophic, single-auxotrophic segregants were extremely rare, and recombinant double auxotrophs were not found. The results of the genetic hybridization analysis point to the lack of normal meiotic division, which is typical of interspecific crosses. The hybrids of CBS 1669 and NBRC 1776 with the W. sargentensis tester CBS 6342 (Table 2, hybrids nos. 5, 6a, and 6b) were also sterile or had low viability of ascospores: 17–24%. Additionally, all hybrids were characterized by abnormal segregation of the control auxotrophic markers. A great majority of the single-spore clones obtained using a micromanipulator and a random spore analysis were prototrophic. According to the results of the genetic analysis, hybrids nos. 5 and 6 can also be classified as interspecific ones with clearly abnormal meiosis. DISCUSSION In the present study, we performed a molecular analysis of 72 strains of the Williopsis yeasts of various geographical origins. Most of the studied strains were isolated from soil in different regions of Taiwan. The degree of divergence of the Taiwanese Williopsis yeasts from the strains isolated in other parts of the world was determined on the basis of a comparative analysis of nucleotide sequences of the internal transcribed spacers ITS1 and ITS2. Among the 55 studied Taiwanese strains, we found four out of the five known Williopsis species: W. saturnus, W. suaveolens, W. mrakii, and

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CBS 8880 FN10S01 EN12S03 EN2S10 SG1S01 CBS 5761 ND6S71 SU16S01 EN7S13 GY8S06 SM7S10 SY3S03 NN11S10 TJ13S72 W. saturnus SC4S03 NU4M05 GY29S04 EN22S15 ES13S01 EN17S10T CBS 254 TJ12S73 CBS 8882 SU10S07 EN8S15 EN6S07 ES27S03 FN20S01 FN14S01 Williopsis sp. 3 SA20S03 Williopsis sp. 4 GY3S10 CBS 1670 Williopsis sp. l CBS 1669 GJ3S05 EJ4M05 TJ1S71 NF19S06 NF18L15 GE5S03 CBS 255T TJ10S73 W. suaveolens EN26S06 NF3S12 GY12S05 BKM Y-838 CBS 8879 CBS 8878 NF1M01 NE7S03 CBS 1707T EN5S03 SG5S06 NRRL YB-3257 EU6S01 EU5S06 GY5S03 W. mrakii GE19S10 EN3S04 SA5S09 GE4S14 FN2S02 GE7S01 SJ7S10 GE16S03 NBRC 1776 Williopsis sp. 2 GE7S02 T CBS 5763 NRRL YB1718 W. subsufficiens EU5S02 CBS 6291 GY13S02 T Williopsis sp. 5 CBS 6342 W. sargentensis 0.005

0.004

0.003

0.002

0.001

0

Molecular differentiation of the Williopsis yeasts according to phylogenetic analysis of nucleotide sequences of the 5.8S-ITS region of rDNA. The tree is constructed by the UPGMA method in MEGA6 software [17]. T, type culture. RUSSIAN JOURNAL OF GENETICS

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Table 2. Genetic analysis of intrastrain and interstrain hybrids of Williopsis sp. yeasts CBS 1669 and NBRC 1776 and W. sargentensis yeasts CBS 6342 No. of hybrid

Origin of hybrids, genotypes

Number of isolated spores

Viability of spores, %

Segregation of control markers AB:aB:Ab:ab*

Intrastrain hybrids** 1 2 3

1669 × 1669 ade1/lys8

132

84

5P:2N:9T

1776 × 1776 his4/ura2

100

64

1P:3N:7T

6342 × 6342 met26/arg25

100

66

4P:4N:8T

Interstrain hybrids*** 4 4a 4b

1669 × 1776 ade1/his4 ade1/ura2

92 100

0 1

5

1669 × 6342 ade1/met26

100

17

11:6:0:0 (110:26:0:0)

1776 × 6342 his4/met26 his4/arg25

62 80

0 24

(115:3:0:0) 19:0:0:0 (103:2:0:0)

6 6a 6b

(125:4:0:0) 1:0:0:0 (105:3:0:0)

* a, b, auxotrophy of the first (to the left of the cross sign) and the second parent, respectively; A, B, prototrophy. ** P:N:T, tetrads of parental and nonparental ditypes and tetratype. *** Results of the random spore analysis are listed in parentheses.

W. subsufficiens. Only the W. sargentensis yeasts were not found. The results obtained indicated the peculiarity of the gene pool of the Williopsis yeasts in Taiwan. The W. saturnus yeasts, isolated from soil, occurs most commonly in Taiwan (Table 1). However, in the temperate zone of the European part of the former Soviet Union, genus Williopsis is mostly represented by populations of W. suaveolens species [19, 20]. This species is associated with the alluvial boggy soils in the river floodplains of the temperate climate zone [21]. In Russia, the W. saturnus yeasts occur only in specific types of soil. This species is dominant (frequency of occurrence exceeding 50%) in soils of the rice fields of Krasnodar krai [20, 22]. The presence of W. mrakii yeasts is another unique characteristic of the species composition of Williopsis yeasts in Taiwan. The 13 found strains of this species were isolated in various regions of Taiwan (Table 1). It should be noted that W. mrakii yeasts are rarely found in other regions of the world. Currently, only single isolates of this species obtained from soil in Japan, the United States, India, and Papua New Guinea are known [9]. There are only two strains of W. mrakii at the CBS yeast collection: the type culture CBS 1707 and CBS 7843 (http://www.cbs.knaw.nl). It is known that W. mrakii yeasts are producers of killer toxins with a broad range of antimicrobial activity and can suppress the growth

of pathogenic microflora, including the Candida albicans yeast that causes mycosis [3]. We showed that all the 13 strains of W. mrakii can produce killer toxins, and some strains are superactive: EN5S03, FN2S02, GY5S03, GE19S10, and SG5S06 [5]. The comparative molecular genetic study of Taiwanese Williopsis yeasts and related strains from other regions of the world allowed us to identify five divergent populations of this genus: three in Taiwan, one in Japan, and one in North America. The data of the phylogenetic analysis are consistent with the results of the genetic analysis of CBS 1669 (North America) and NBRC 1776 (Japan). These strains crossed well with each other and the tester strain W. sargentensis CBS 6342, which confirms that they belong to the same genus. However, low viability of ascospores of the resulting hybrids and the lack of normal digenic meiotic segregation of the control markers suggest the genetic isolation of CBS 1669 and NBRC 1776 and their belonging to two novel biological species of genus the Williopsis. We plan to perform the genetic study of the Taiwanese strains FN14S01, SA20S03, GY3S10, and GY13S02 to determine their taxonomic status. Formal description of novel species of genus Williopsis is a subject for a separate study.


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ACKNOWLEDGMENTS This study was supported by a Russian-Taiwanese grant from the Russian Foundation for Basic Research (no. 14-04-92001-ННС_a) and the NSC (no. 1032923-B-134-001-MY3). REFERENCES 1. Liu, Y.-R., Liu, C.-H., Young, S.-S., and Lee, C.-F., New records of Williopsis saturnus and Zygowilliopsis californica in Taiwan, Fung. Sci., 2007, vol. 22, nos. 3–4, pp. 79–86. 2. Nomoto, H., Kitano, K., Shimazaki, T., et al., Distribution of killer yeasts in the genus Hansenula, Agric. Biol. Chem., 1984, vol. 48, no. 3, pp. 807–809.

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22. Golubev, V.I. and Vdovina, N.V., The yeast flora of the soil of rice fields cultivated with herbicides, Povedenie, prevrashchenie i analiz pestitsidov i ikh metabolitov v pochve (Behavior, Transformation, and Analysis of Pesticides and Their Metabolites in Soil) (Proc. 1st AllUnion Conf.), Pushchino, 1973, pp. 66–73.

11. James, S.A., Collins, M.D., and Roberts, I.N., Use of an rRNA internal transcribed spacer region to distin-

Translated by A. Lisenkova


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